June Dec.

Microb. Resour. Syst. 33June（1）：19 2017─ 29, 2017 Vol. 33, No. 1 Fatty acid composition profiles of 235 strains of three microalgal divisions within the NIES Microbial Culture Collection

Eri Mitani1), Fumiyo Nakayama1) , Izumi Matsuwaki1) , Ikuyo Ichi1, 2) , Atsushi Kawabata3) , Masanobu Kawachi3)＊ and Misako Kato1, 2)

1)Graduate School of Humanities and Sciences, 2)Institute for Human Life Innovation, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan 3)National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305-8506, Japan

To evaluate the occurrence of polyunsaturated fatty acids as phenotypic markers, we analyzed the fatty acid compositions of 235 strains of microalgae within the divisions Heterokontophyta, Cryptophyta, and Haptophyta held in the Microbial Culture Collection at the National Institute for Environmental Studies. Eicosapentaenoic acid (EPA, 20:5) was present at >10% of the total fatty acid composition in 119 strains. Docosahexaenoic acid (DHA, 22:6) DHA was present at >10% of the total in 16 strains, most in the Haptophyta, in addition to two unidentified strains in the (Heterokontophyta). Strains of the Heterokontophyta other than the Chrysophyceae and the Pelagophyceae and the strains of Cryptophyta typically contained more EPA than DHA. The Heterokontophyta, with 11 classes, including the newly recognized Aurearenophyceae, Pelagophyceae, and Schizocladiophyceae, showed diverse fatty acid profiles, although clear phenotypic results defining each taxonomic group were not confirmed.

Key words: Cryptophyta, docosahexaenoic acid, eicosapentaenoic acid, fatty acid, Haptophyta, Heterokontophyta, microalgae

INTRODUCTION ing commercial attention (Guschina & Harwood, The fatty acid compositions of a large number of 2006). PUFAs have important nutritional value for microalgae have been reported. Of particular inter- human health (Abedi & Sahari, 2014). Selection and est from a chemotaxonomic viewpoint is the occur- screening of PUFA-producing species are considered rence of polyunsaturated fatty acids (PUFAs). Cul- important to increasing the commercial use of algae. ture strains of microalgae are maintained in the Here we show the fatty acid composition of 235 Microbial Culture Collection at the National Institute strains within the divisions Heterokontophyta, Cryp- for Environmental Studies, Tsukuba, Japan (MCC- tophyta, and Haptophyta held in the MCC-NIES. NIES). An exhaustive analysis of fatty acid composi- tion using this culture collection will provide useful MATERIALS AND METHODS information for investigators. Differential segrega- Microalgae were supplied by MCC-NIES and were tion of microalgae by their fatty acid profiles indi- grown under light at 20℃ in the culture media des- cates the potential utility of fatty acids as biomark- ignated by MCC-NIES. Stationary growing cells ers (Sahu et al., 2013). We hope to establish the fatty were harvested by centrifugation and then lyophi- acid compositions of algae in the MCC-NIES in order lized. to distinguish them. Microalgae are a potential Lipids were extracted by the method of Bligh & source of bioactive compounds with pharmaceutical, Dyer (1959). The dried lipid fraction was mixed with biomedical, and nutraceutical prospects. In particu- 2 ml of 5% (w/w) HCl-methanol and heated at 90℃ lar, their production of PUFAs, such as eicosapentae- for 2 h to obtain fatty acid methyl esters. The solu- noic acid (EPA, 20:5) and docosahexaenoic acid (DHA, tion was extracted twice with 2 ml n-hexane. The n- 22:6), is the subject of intensive research and increas- hexane fraction was concentrated for use in gas chromatographic analysis. Gas chromatography was performed with a fused-silica capillary column (0.25 ＊ Corresponding author mm internal diameter ×50 m; Chrompack Cp-Sil 88, E-mail: [email protected] Agilent Technologies Inc., USA), with the oven tem-

─ 19 ─ Fatty acid composition of microalgae Mitani et al. perature programmed to increase from 150 to 210℃ 2012; Boelen et al., 2013; Wang et al., 2014), we did not at 5℃ min−1. The fatty acid composition was calcu- detect any in three strains (NIES-377, 553, 586). Six- lated by a Chromatopac C-R8A data processor (Shi- teen strains in the genus Skeletonema (NIES-17, 223, madzu Corp., Kyoto, Japan). To identify the fatty 323, 324, 2533, 2534, 2535, 2536, 2537, 2837, 2839, 2840, acid methyl esters, we performed gas chromato- 2841, 2842, 2843, 2844) had similar fatty acid composi- graphic – mass spectrometric analysis with a GCMS- tions to those of S. costatum (Berge et al., 1995; Chen, QP2010 Ultra (Shimadzu) equipped with a Varian 2012) and S. marinoi (Chandrasekaran et al., 2014). capillary column (0.25 mm internal diameter ×60 m; In addition to fatty acid production, biodiesel pro- Agilent Technologies) at an ionization energy of 70 duction by several genera of has been re- eV. The oven temperature was initiated at 120℃ for ported (Graham et al., 2012). Some, such as Cyclotel- 1 min and then raised to 175℃ at 25℃ min−1, then to la, are capable of heterotrophic growth, so a high 235℃ at 5℃ min−1, and then held for 5 min. The in- yield of biomass is expected (Glaude & Maxey, 1994; jection and interface temperatures were 250℃, and Pahl et al., 2010). The content of 20:5 in four strains the ion source temperature was 200℃. of Cyclotella was 8.3% to 15.4%. Fistulifera saprophila (NIES-2722) had a very high content of 20:5 (25.7%). RESULTS AND DISCUSSION The closely related F. solaris was shown to be a Fatty acid compositions are shown in Table 1. powerful candidate for production of 20:5 and bio- diesel because of its high lipid productivity (Liang et Heterokontophyta al., 2014). Through the analysis of nuclear-encoded SSU In the Chrysophyceae, Ochromonas danica is one rRNA and plastid-encoded rbcL, psaA, psbB, and of the most studied species with respect to fatty acid psbC, Yang et al. (2012) reported three major clades and lipid biosynthesis (Vogel & Eichenberger, 1992). of the Heterokontophyta. SI clade comprised the The structure of two algal betaine lipids —1,2-diacyl- Schizocladiophyceae, Phaeophyceae, Aurearenophy- glyceryl-O-4′-(N,N,N-trimethyl)homoserine and 1,2-di- ceae, , Phaeophyceae, Raphido- acylglyceryl-O-2′-hydroxymethyl-(N,N,N-trimethyl) phyceae, and Xanthophyceae. In particular, Schizo- β-alanine— was first determined from their produc- cladiophyceae and the Phaeophyceae formed a sub- tion in O. danica (Nichols & Appleby, 1969; Vogel et clade within SI clade. The Chrysophyceae, al., 1990). The algae in the genus Synura, sometimes Eustigmatophyceae, Pinguiophyceae, Synchromophy- classified in the class Synurophyceae (Siver et al., ceae and Synurophyceae formed the SII clade. The 2015), are characterized by distinctive siliceous sur- Bacillariophyceae, , face scales. In Synura, 14:0, 16:0, and 18:4 were abun- and Pelagophyceae formed the SIII clade. dant, and 22:6 was more abundant than 20:5. The major fatty acids in the Heterokontophyta Nannochloropsis oculata (NIES-2145) is the most were 14:0, 16:0, and 16:1, and most Bacillariophyceae studied alga in the Eustigmatophyceae (Tonon et al., species contained 20:5. The highest content of 20:5 2002; Olofsson et al., 2012). A high 20:5 content appears was found in Asterionellopsis glacialis (NIES-265, to be a characteristic feature of Nannochloropsis 417), in which it accounted for 40% of the total fatty (Huerlimann et al., 2010; Lim et al., 2012). Contents of acids. One of the most well-known marine diatoms 16:0, 16:1, 18:0, and 20:5 were >10% in N. oculata, and that produce 20:5 is Phaeodactylum tricornutum, the high contents of 16:0 and 16:1 were similar to those with 20:5 constituting up to 40% of its total (Yong- in Vischeria sp. (NIES-2147, 2148). manitchai & Ward, 1991; Fernández et al., 2000; The picoplankton Pelagomonas calceolata was de- Tonon et al., 2002; Liang et al., 2006). The yield of scribed as a new species, in a new genus, family, or- 20:5 in A. glacialis was equivalent to that in P. tri- der, and class, the Pelagophyceae, by Andersen et al. cornutum. The content of 22:6 was 0% to 4% in the (1993). We detected 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, Bacillariophyceae. The fatty acid composition of 18:2, 18:3, 18:4, and 22:6 in all Pelagophyceae strains, spp., which are food for the larvae of but detected 20:5 in only five strains. The fatty acid crustaceans and fish, has been reported (Liang et al., and polar lipid compositions in the Pelagophyceae 2006; Chen, 2012; Boelen et al., 2013; Wang et al., have not previously been reported. 2014). Although trace amounts of 22:6 have been re- The major fatty acids in 24 strains in the Raphido- ported in Chaetoceros spp. (Liang et al., 2006; Chen, phyceae were 14:0, 16:0, and 20:5. marina

─ 20 ─ Microb. Resour. Syst. June 2017 Vol. 33, No. 1 0.0 0.0 2.8 0.0 1.2 2.2 1.5 3.2 6.5 1.6 1.2 4.4 0.0 0.3 0.3 0.7 1.9 0.0 1.3 0.7 3.3 1.1 3.0 1.3 0.0 0.5 0.4 0.0 0.8 0.0 6.8 2.8 6.0 0.0 0.0 1.8 0.9 0.0 2.2 0.0 0.0 3.5 1.8 1.0 0.3 2.6 0.9 4.6 Others 0.0 0.0 1.2 0.0 0.3 0.5 0.7 0.0 0.0 0.3 2.9 5.5 0.0 0.6 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 1.3 0.0 3.2 0.3 0.0 0.0 0.0 0.0 2.0 1.1 1.2 0.9 3.1 0.0 0.4 0.0 7.7 0.0 0.0 0.0 0.0 2.7 0.4 0.5 0.0 0.1 24:1 0.0 0.0 0.0 1.3 2.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.6 0.9 0.0 0.0 0.0 1.7 0.7 0.0 0.0 1.0 1.2 0.0 0.0 0.0 0.6 1.0 0.7 0.6 3.1 0.7 1.6 1.4 0.0 0.6 0.0 0.0 3.3 2.9 1.1 1.0 0.9 1.3 1.7 0.4 0.0 22:6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.8 0.0 0.0 0.7 0.8 0.9 1.2 1.0 0.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 22:5 8.8 0.0 4.3 7.4 6.5 4.4 9.2 6.8 5.0 0.0 3.4 9.2 0.0 9.2 8.3 9.6 5.0 0.0 0.0 7.4 28.1 46.1 41.5 12.7 14.4 11.2 11.7 10.3 21.0 13.8 13.8 10.3 20:5 14.9 10.1 14.6 25.7 10.4 17.4 15.4 14.7 12.4 16.0 30.6 19.8 21.3 26.6 14.2 13.5 0.0 1.2 0.0 0.0 0.0 5.0 2.7 1.7 1.7 1.0 3.1 2.1 0.0 4.3 0.4 4.5 6.2 1.3 0.0 2.3 1.9 1.6 1.0 1.4 0.5 0.0 5.2 3.4 1.3 0.0 0.0 0.0 0.0 0.0 3.4 2.6 0.0 1.0 1.6 1.8 0.9 0.9 1.6 7.0 20:4 12.2 14.8 10.5 12.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.3 0.0 0.0 0.2 0.2 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 18:5 0.0 0.0 0.0 1.1 1.5 0.0 1.2 0.5 0.2 0.0 0.4 0.6 0.0 1.4 0.4 8.8 0.0 1.2 1.0 1.0 0.0 0.2 0.0 1.4 2.4 0.0 0.0 2.7 1.4 1.2 0.0 0.3 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.4 0.2 0.0 1.0 1.2 0.4 0.4 20.9 17.0 18:4 0.4 0.0 0.0 0.4 0.0 0.9 0.8 0.6 1.1 0.0 0.5 0.9 0.0 0.4 0.9 9.3 0.6 0.0 0.9 0.5 0.0 2.0 0.4 0.0 2.2 0.7 0.0 0.0 2.3 0.0 1.1 0.0 0.4 0.0 0.0 0.4 0.0 0.0 0.5 0.0 2.3 2.0 0.5 1.1 0.5 0.9 16.9 16.4 18:3 0.0 1.4 7.1 0.0 1.4 4.1 3.4 2.5 1.8 2.2 0.6 2.2 3.0 1.1 1.7 5.3 5.6 2.7 0.6 5.9 4.9 4.9 0.5 3.4 0.0 4.7 0.0 1.5 3.8 2.3 1.5 0.8 2.2 2.2 2.1 0.8 2.2 0.8 0.0 0.4 0.8 1.2 1.4 0.0 0.0 3.9 18:2 28.2 11.5 4.0 0.0 2.8 0.6 0.0 3.8 2.3 1.3 3.1 5.2 2.9 1.1 3.7 8.1 6.5 0.2 2.9 4.1 1.6 4.1 3.4 2.2 2.2 2.3 2.0 2.7 6.5 6.7 3.1 0.6 2.2 2.5 2.4 8.4 1.2 1.4 0.0 1.7 2.0 2.0 1.9 4.7 0.9 0.4 5.3 10.6 13.4 11.1 18:1 2.4 0.5 1.6 1.8 0.8 0.5 1.1 0.7 0.6 4.5 0.5 2.5 0.9 0.8 0.4 7.0 0.8 1.6 1.1 0.6 0.9 2.4 9.3 0.6 6.1 2.3 0.8 6.0 2.1 0.7 0.8 9.6 5.0 0.4 1.1 0.9 8.4 0.5 0.7 2.1 0.6 0.0 1.1 13.3 18.1 10.8 18:0 11.7 20.4 4.2 0.0 9.4 2.6 0.0 0.0 0.6 1.4 0.0 0.3 0.7 0.0 5.1 0.8 0.0 0.0 0.0 0.6 0.0 0.0 3.7 2.9 1.4 5.3 0.0 2.9 0.0 0.0 0.7 0.7 1.6 1.5 0.5 0.5 0.6 0.0 0.7 0.0 0.0 8.0 5.5 1.4 0.4 1.0 3.2 3.3 0.9 10.5 16:4 0.9 1.6 0.0 1.0 0.0 3.6 3.0 1.6 3.6 5.9 2.3 1.9 2.7 1.0 4.7 0.0 0.0 0.0 6.1 2.9 1.4 2.0 2.2 2.6 5.2 0.0 1.7 3.7 2.4 4.7 3.3 3.7 3.9 0.0 4.5 0.0 3.0 3.1 2.1 4.1 1.0 1.0 2.2 3.4 16:3 21.6 12.6 10.4 13.4 6.1 6.9 0.0 3.0 4.5 1.7 2.1 2.0 2.3 4.5 2.5 0.8 0.0 2.5 7.8 0.6 0.6 0.0 7.2 4.9 1.7 5.1 2.8 4.0 8.4 0.4 3.7 7.5 7.6 4.4 1.6 1.8 7.8 4.8 3.8 4.3 0.0 1.1 3.4 1.3 3.8 3.9 3.5 3.4 3.3 2.7 7.0 0.9 16:2 8.6 0.0 1.1 1.0 1.3 7.0 1.3 0.6 7.6 12.7 11.4 21.2 40.5 48.3 58.7 50.5 21.3 53.2 54.6 33.5 31.0 22.2 16:1 40.9 51.1 39.0 44.7 35.5 23.5 20.1 53.2 19.4 22.0 23.2 14.9 15.5 34.8 28.0 19.7 38.3 52.8 45.3 14.9 24.0 22.7 49.6 38.4 43.5 19.6 7.7 3.6 8.1 9.9 6.6 22.3 24.6 18.1 14.2 14.2 13.8 25.1 16.3 17.0 21.3 14.1 32.9 24.4 26.7 43.7 16:0 20.4 17.9 21.5 25.3 21.6 13.8 54.0 17.4 10.7 24.2 17.3 15.8 15.2 12.0 15.1 20.4 17.5 21.2 28.0 17.3 32.6 25.1 32.4 28.4 23.0 11.5 15.9 30.5 4.7 2.5 1.8 3.0 9.5 4.9 2.5 4.7 1.3 5.0 1.2 1.5 3.3 4.8 0.9 5.6 5.1 6.6 4.5 7.8 5.5 7.9 6.3 5.1 6.1 9.4 5.1 3.8 4.3 1.8 1.8 5.2 5.3 21.4 27.4 26.0 14.6 13.4 14:0 26.8 21.1 16.9 22.2 11.9 25.0 18.7 10.5 20.6 15.8 4.0 4.3 3.0 1.9 1.6 1.6 0.6 1.5 3.7 3.0 0.4 3.3 1.3 1.4 2.0 2.1 5.1 2.3 4.4 0.6 1.1 1.4 0.8 3.0 0.5 2.8 0.9 2.4 5.5 2.9 2.7 1.8 1.4 5.7 3.3 0.3 1.9 9.4 3.3 2.1 1.5 2.2 1.7 1.7 1.6 0.6 21.4 12:0 16.0 Strain NIES-553 NIES-377 NIES-333 NIES-417 NIES-265 NIES-414 NIES-413 NIES-409 NIES-408 NIES-407 NIES-372 NIES-71 NIES-2717 NIES-330 NIES-1349 NIES-1865 NIES-1864 NIES-1863 NIES-2725 NIES-588 NIES-587 NIES-2723 NIES-466 NIES-391 NIES-2722 NIES-461 NIES-2668 NIES-2720 NIES-350 NIES-1047 NIES-1046 NIES-1045 NIES-2365 NIES-2364 NIES-2363 NIES-805 NIES-2718 NIES-1383 NIES-2731 NIES-2730 NIES-590 NIES-589 NIES-1340 NIES-1339 NIES-2729 NIES-2726 NIES-487 NIES-2351 Table 1 Relative proportions (%) of fatty acid in crude lipids from mic roalgal strains the MCC-NIES Chaetoceros sociale Chaetoceros sociale Aulacoseira granulata Asterionellopsis glacialis Asterionellopsis glacialis Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium minutissimum Achnanthidium convergens Achnanthes subconstricta Achnanthes kuwaitensis Aurearena cruciata Aurearena cruciata Aurearena cruciata Species Mayamaea atomus Lithodesmium variabile Hantzschia amphioxys Gomphonema parvulum Gomphonema parvulum capucina Fistulifera saprophila Eunotia pectinalis Eucampia sp. Eolimna minima Ditylum brightwellii Cylindrotheca sp. Cylindrotheca fusiformis Cylindrotheca closterium Cyclotella meneghiniana Cyclotella meneghiniana Cyclotella meneghiniana Cyclotella meneghiniana Craticula molestiformis Pseudonitzschia sp. Planothidium lanceolatumt Planothidium frequentissimum Odontella longicruris Odontella aurita Nitzschia sp. Nitzschia sp. Nitzschia palea Nitzschia palea Nitzschia palea Nitzschia closterium Bacillariophyceae Aurearenophyceae Heterokontophyta

─ 21 ─ Fatty acid composition of microalgae Mitani et al. 2.6 0.0 2.3 3.4 3.5 2.5 2.0 1.2 1.9 0.6 3.4 2.9 0.0 2.4 2.0 1.3 1.0 0.0 0.0 0.0 0.0 0.0 0.9 0.0 0.0 1.3 0.8 2.2 0.0 0.0 0.0 1.5 0.0 2.4 3.8 0.0 0.0 0.0 0.0 0.0 0.0 0.6 1.0 0.2 0.2 5.6 0.0 28.1 Others 1.9 0.9 2.1 0.6 0.9 0.8 0.4 0.0 1.2 0.6 1.7 0.5 0.0 1.0 1.5 0.0 1.9 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 3.6 0.0 2.5 0.0 0.0 1.2 0.0 0.0 0.0 0.0 1.1 0.6 1.5 0.0 0.0 0.0 24:1 0.0 0.0 1.8 2.1 1.1 2.4 2.3 2.0 1.8 0.0 2.8 0.7 1.3 1.6 1.2 1.9 0.0 0.0 6.4 5.2 3.7 2.6 2.4 1.3 2.0 2.7 7.3 0.0 0.7 2.4 0.0 0.0 0.0 0.2 2.5 1.1 8.0 7.7 3.6 7.0 3.8 7.2 2.9 0.0 0.0 0.0 0.0 4.8 22:6 0.0 0.0 0.0 0.0 1.1 1.7 1.0 0.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.5 3.5 7.6 3.4 6.2 6.6 7.6 8.6 4.1 0.0 5.1 1.8 0.0 0.0 0.0 0.0 0.4 0.0 2.3 0.0 0.8 2.0 1.6 1.9 0.0 0.0 0.0 2.3 3.1 22:5 14.0 1.0 9.8 8.2 2.8 5.3 6.7 7.7 8.2 8.5 5.9 4.0 1.4 0.7 3.1 4.2 0.0 6.9 8.6 0.0 0.3 0.0 0.0 0.0 6.9 0.0 0.3 0.4 1.0 0.0 0.0 0.0 4.8 1.6 1.0 10.5 12.3 11.8 18.2 16.6 17.7 19.2 18.5 20:5 11.3 12.5 16.3 10.1 12.4 13.1 0.0 0.0 0.0 0.3 0.0 0.0 0.7 0.5 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.5 8.4 7.0 0.0 0.0 1.2 0.0 2.9 5.4 3.0 7.0 3.1 0.0 0.8 0.0 0.0 0.7 5.4 1.7 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.1 1.4 3.4 2.0 2.9 20:4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.8 0.0 0.0 1.6 0.0 0.0 0.6 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 5.0 9.8 2.8 3.9 5.5 0.0 1.2 0.3 0.3 0.5 0.8 0.0 18:5 0.0 0.0 1.9 1.8 1.6 2.5 3.6 2.0 2.4 0.7 2.5 0.0 2.2 2.4 1.8 0.6 0.0 0.0 2.1 0.0 0.0 0.0 7.5 7.9 0.0 0.0 7.0 0.0 0.4 0.3 9.2 2.3 1.6 0.2 0.3 0.1 0.0 18:4 29.1 11.9 14.8 29.3 14.3 18.9 15.6 21.6 21.4 12.4 16.7 0.0 0.0 0.0 0.0 0.4 0.5 0.6 0.6 1.4 0.0 0.0 0.0 0.0 0.9 0.0 0.0 1.3 0.7 5.3 5.7 0.0 6.4 0.0 0.0 7.0 0.3 0.4 0.0 6.3 9.7 3.6 6.9 4.1 0.9 3.8 0.5 0.3 1.8 18:3 10.7 13.1 16.6 10.2 13.3 34.8 21.7 15.4 12.0 10.5 0.0 1.6 2.2 0.9 0.4 0.5 0.3 9.3 2.1 4.0 0.0 1.7 3.0 0.0 0.6 3.6 1.4 0.5 4.8 9.4 5.9 2.4 6.0 8.9 9.5 7.5 0.8 6.3 2.5 0.8 0.6 5.6 1.2 0.0 2.0 3.1 1.6 3.2 4.5 5.9 3.7 1.4 4.3 3.4 2.0 1.6 18:2 11.1 11.8 9.1 8.7 0.0 0.0 2.0 0.0 6.7 7.1 0.8 9.3 2.8 2.9 7.9 3.7 2.0 3.4 7.7 5.5 2.7 4.0 7.9 3.9 4.6 5.6 6.1 0.0 4.1 4.6 2.3 0.0 5.7 2.7 1.9 2.1 8.7 1.7 5.5 2.9 7.9 7.9 4.4 14.4 19.1 14.1 18:1 12.2 18.1 13.9 14.4 3.1 3.7 2.1 0.5 1.1 1.1 0.8 4.5 1.3 3.4 2.9 2.2 3.5 1.8 0.9 0.6 1.9 3.5 5.8 2.1 4.6 4.3 2.5 6.7 9.7 9.3 2.4 4.9 2.9 6.5 3.5 6.5 1.9 7.2 9.5 0.9 0.7 24.4 10.0 18:0 12.1 10.3 13.9 19.5 15.2 16.4 11.0 18.0 15.5 0.0 0.0 4.7 0.9 6.6 8.1 8.9 1.0 0.0 8.5 0.0 3.0 9.3 6.9 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 4.1 2.5 0.0 0.0 0.0 0.0 0.0 0.0 1.5 0.0 0.0 0.0 0.0 10.7 16:4 2.1 1.1 4.6 9.7 5.2 7.4 1.7 7.7 5.6 2.7 6.4 5.7 3.5 6.8 3.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 4.8 5.1 0.0 0.0 0.0 0.0 0.0 0.0 1.2 0.0 0.0 0.0 0.0 14.0 20.2 10.2 16:3 3.6 0.0 8.2 6.0 9.8 9.3 6.8 7.1 5.0 4.1 6.3 6.4 9.8 9.5 5.7 5.0 3.6 2.5 0.0 0.0 3.1 1.6 0.0 0.0 0.0 0.0 0.0 1.5 0.6 1.5 0.0 0.0 0.9 4.7 6.8 8.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 1.6 0.7 0.7 0.0 10.4 16:2 5.5 2.4 0.0 0.0 2.4 1.2 0.0 0.0 0.0 0.0 0.0 1.4 1.7 4.4 0.0 0.0 3.4 8.7 9.0 5.0 9.0 0.0 1.7 4.1 17.7 16.9 20.9 18.9 13.3 12.0 17.6 17.7 14.9 21.0 16.0 24.7 22.7 22.2 25.5 42.8 42.1 16:1 34.7 17.1 37.8 14.0 32.9 47.4 21.5 9.7 9.0 6.7 9.8 6.1 6.0 9.8 5.8 7.0 9.7 9.4 14.8 25.2 12.0 16.9 10.1 19.6 12.5 13.8 12.3 15.6 14.4 22.1 16:0 11.6 10.1 20.7 17.2 14.0 43.7 25.2 29.9 19.8 24.1 26.1 26.8 15.3 14.9 16.6 15.4 18.1 17.9 16.1 40.0 49.6 22.2 18.1 27.5 20.9 9.9 9.1 3.5 2.6 6.0 9.7 8.9 1.4 5.1 3.9 27.8 19.2 11.2 18.8 18.5 14.2 17.2 22.1 14.8 29.9 13.0 14.7 18.5 18.9 14:0 19.4 20.1 16.2 20.2 27.7 11.7 14.4 14.5 19.4 15.9 30.5 23.2 23.2 22.4 23.7 11.9 10.5 22.7 14.1 18.5 12.1 10.9 10.3 22.7 Table 1 Continued Table 2.2 7.7 5.0 1.8 4.9 6.1 2.0 2.1 3.8 9.9 3.7 4.2 2.4 2.7 3.8 1.8 1.5 0.7 4.2 4.9 7.2 3.6 4.0 3.5 6.9 4.6 2.6 1.8 2.8 5.7 6.4 5.0 0.8 2.4 2.0 3.5 2.7 1.5 6.1 5.4 5.2 2.4 1.2 0.9 12:0 13.8 39.5 16.1 16.7 Strain NIES-2844 NIES-2537 NIES-2843 NIES-2842 NIES-324 NIES-323 NIES-223 NIES-17 NIES-2841 NIES-2536 NIES-2535 NIES-2840 NIES-2839 NIES-2534 NIES-2533 NIES-2837 NIES-2732 NIES-1353 NIES-234 NIES-696 NIES-695 NIES-1007 NIES-233 NIES-2144 NIES-2300 NIES-2143 NIES-2142 NIES-3494 NIES-1376 NIES-1827 NIES-1826 NIES-284 NIES-2890 NIES-1862 NIES-556 NIES-534 NIES-225 NIES-3391 NIES-2691 NIES-2690 NIES-2689 NIES-1003 NIES-1700 NIES-1699 NIES-2148 NIES-2147 NIES-2145 NIES-395 Skeletonema tropicum Skeletonema tropicum Skeletonema pseudocostatum Skeletonema menzelii Skeletonema marinoi-dohrnii complex Skeletonema marinoi-dohrnii complex Skeletonema marinoi-dohrnii complex Skeletonema marinoi-dohrnii complex Skeletonema japonicum Skeletonema japonicum Skeletonema japonicum Skeletonema grevillei Skeletonema dohrnii Skeletonema dohrnii Skeletonema dohrnii Skeletonema ardens Sellaphora seminulum Sellaphora seminulum Species Synura spinosa Synura sphagnicola Synura sphagnicola Synura petersenii Synura petersenii Poterioochromonas malhamensis Ochromonas sp. Ochromonas minuta Ochromonas danica Mallomonas sp. Mallomonas sp. Lagynion subglobosum Epipyxis glabra Dinobryon divergens Chrysamoeba radians Giraudyopsis sp. Triceratium dubium Thalassionema nitzschioides Tabellaria flocculosa Pelagomonas calceolata Pelagomonas calceolata Pelagomonas calceolata Pelagomonas calceolata Pelagomonas calceolata Chrysophaeum taylorii Chrysophaeum taylorii Vischeria stellata Vischeria punctata Nannochloropsis oculata Uroglena americana Bacillariophyceae Heterokontophyta Chrysophyceae Chrysomerophyceae Pelagophyceae Eustigmatophyceae

─ 22 ─ Microb. Resour. Syst. June 2017 Vol. 33, No. 1 4.6 5.0 3.0 2.6 0.8 1.6 2.1 3.1 0.0 0.0 6.4 1.3 0.0 1.2 0.0 0.0 0.0 0.0 7.2 5.0 4.5 3.8 0.0 2.0 1.4 1.2 0.6 0.0 7.1 4.2 5.9 4.6 2.9 2.6 3.7 0.0 1.0 1.0 2.2 0.0 1.0 0.0 0.0 0.5 0.9 2.7 1.3 Others 0.2 0.1 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.9 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 1.6 0.0 3.4 0.0 0.0 0.3 0.0 0.0 0.0 24:1 2.1 3.0 0.5 1.3 2.6 0.8 0.0 0.0 3.7 9.3 1.3 8.0 2.9 1.8 5.9 0.0 2.1 0.5 0.7 0.4 0.0 0.0 0.0 0.0 0.0 0.0 3.6 3.8 1.2 2.0 0.9 1.6 2.5 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 11.9 11.6 22:6 0.9 2.7 1.9 1.5 0.0 1.1 0.0 0.0 0.0 1.7 0.0 2.8 4.0 2.8 3.1 3.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.9 2.7 1.3 2.4 1.8 2.1 2.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 22:5 3.4 6.9 0.0 0.0 0.0 0.0 2.8 0.6 0.0 0.0 0.0 6.2 1.5 5.4 7.8 6.5 9.3 0.0 0.0 7.8 38.9 24.7 21.8 14.5 14.8 11.5 20:5 16.3 21.1 25.1 16.1 16.8 34.7 16.8 19.0 20.9 16.1 17.4 21.8 13.3 20.9 27.3 11.1 23.6 15.9 12.7 14.0 35.1 3.4 3.2 9.2 3.7 0.7 6.3 2.9 4.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.6 2.5 2.3 1.4 1.1 0.0 1.6 4.9 7.1 5.0 6.0 4.4 2.7 3.2 4.8 3.2 6.3 2.6 9.6 6.2 9.7 2.7 3.7 3.5 0.0 0.0 4.9 8.7 20:4 11.7 11.8 0.3 0.2 1.2 0.4 0.0 1.9 0.0 0.2 0.6 3.3 2.9 1.0 2.3 5.3 6.0 2.1 6.0 0.2 0.5 2.1 1.3 2.0 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.9 0.3 0.3 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.3 0.8 18:5 5.4 9.2 0.0 0.0 3.7 7.3 0.0 8.5 8.7 0.0 0.2 9.4 6.6 2.5 6.0 7.0 8.4 6.4 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.5 5.6 0.3 11.5 13.6 10.1 13.3 17.3 11.0 23.0 18:4 13.9 15.4 11.6 10.6 12.3 13.2 10.8 11.0 14.4 11.9 12.8 5.0 4.2 5.0 4.3 0.0 0.5 0.0 9.8 6.7 6.6 5.8 7.0 4.4 5.9 2.4 0.0 3.8 1.2 2.7 3.2 6.7 5.6 3.8 3.4 4.5 5.4 4.9 2.4 2.7 5.0 2.4 3.4 1.6 2.9 7.6 4.9 0.0 0.6 1.5 0.0 0.0 0.0 0.0 2.6 1.5 18:3 11.7 13.9 3.0 2.9 4.4 3.3 3.0 4.3 1.8 9.7 2.6 2.7 2.3 3.9 2.9 1.4 2.4 2.5 3.7 6.3 1.0 1.2 2.6 3.1 2.6 2.2 4.0 3.1 4.9 2.9 4.8 4.0 3.3 4.7 1.9 6.6 2.0 5.3 1.9 1.0 1.2 1.2 2.5 1.5 7.3 5.5 4.3 10.2 18:2 38.0 2.3 2.0 1.4 4.0 3.0 4.5 4.8 5.3 6.8 3.1 4.7 6.8 5.3 7.6 0.7 0.5 1.0 4.0 7.9 7.4 9.2 4.5 5.1 8.4 7.0 5.9 2.5 3.0 1.2 2.2 2.0 1.7 3.1 3.1 3.8 0.0 4.7 0.0 8.1 14.6 12.0 11.7 12.7 23.4 18:1 21.7 11.6 12.1 1.4 1.2 1.2 4.1 6.8 2.2 4.2 3.5 7.9 3.0 3.2 4.5 3.7 0.6 0.9 0.5 0.5 2.1 7.7 0.5 0.5 0.5 2.4 2.6 2.1 1.6 1.4 1.8 3.1 1.2 1.4 1.9 1.2 3.1 5.0 7.4 1.9 3.7 4.4 1.3 2.3 3.6 14.6 17.8 19.6 18:0 12.8 12.5 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 16:4 0.0 0.2 0.2 0.2 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.4 0.0 0.0 0.0 0.4 0.1 0.0 0.0 0.0 0.0 0.2 1.0 0.6 0.4 0.0 0.0 0.0 2.3 3.3 5.7 0.0 1.0 7.2 0.0 0.0 0.0 0.6 16:3 0.5 1.5 2.2 1.2 0.7 0.2 0.0 0.9 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 2.0 0.0 1.5 5.2 1.5 0.0 1.6 2.3 1.1 1.2 0.9 1.4 0.9 1.2 3.3 2.5 2.6 0.5 1.5 1.3 4.3 7.0 6.8 1.9 8.3 0.0 1.7 0.5 1.1 16:2 11.4 4.7 8.0 8.3 6.0 3.5 5.0 5.5 1.9 0.0 9.1 4.5 1.5 0.0 3.2 3.9 0.0 4.9 7.1 7.4 4.9 3.2 4.7 3.8 4.3 7.1 9.1 8.2 5.8 7.1 0.0 2.4 12.6 10.5 16:1 11.1 15.1 11.7 13.8 11.7 41.7 31.4 24.7 17.3 20.6 38.8 35.8 11.6 14.7 16.9 19.7 18.7 22.0 12.2 29.1 25.6 28.7 33.1 19.5 15.6 23.7 15.4 15.8 25.0 29.8 16:0 19.6 17.3 27.0 25.4 22.2 53.4 54.9 39.0 22.2 14.6 10.3 15.6 15.6 20.4 29.6 22.6 21.7 28.5 17.9 15.9 24.4 22.2 17.9 19.8 34.2 23.5 21.0 34.0 17.4 37.8 28.0 8.4 8.2 7.4 9.4 5.3 6.6 9.9 5.5 3.7 4.0 6.3 7.7 4.4 8.7 8.5 9.3 2.9 3.2 3.6 5.9 6.8 3.2 1.0 7.0 5.7 12.1 43.4 19.8 37.3 18.0 13.5 29.0 22.0 15.8 13.1 14:0 15.0 14.8 10.6 18.8 23.0 14.1 23.3 22.4 12.9 11.1 12.7 26.4 Table 1 Continued Table 2.2 2.0 4.0 2.0 7.7 8.2 1.1 1.5 3.2 5.7 5.1 6.3 2.8 4.2 3.6 9.8 5.0 1.0 1.3 1.9 1.2 1.7 6.8 3.0 3.1 2.9 5.1 4.4 2.3 2.5 2.6 2.8 2.7 3.5 3.4 1.2 4.0 5.1 3.3 0.9 3.1 8.1 1.3 1.4 1.8 14.0 11.6 12:0 Strain NIES-558 NIES-557 NIES-115 NIES-14 NIES-1 NIES-2693 NIES-1302 NIES-1002 NIES-548 NIES-2772 NIES-2771 NIES-2692 NIES-1387 NIES-1386 NIES-2770 NIES-3691 NIES-3690 NIES-3689 NIES-1379 NIES-15 NIES-293 NIES-145 NIES-9 NIES-716 NIES-1009 NIES-1303 NIES-605 NIES-560 NIES-462 NIES-136 NIES-2633 NIES-1976 NIES-1975 NIES-1974 NIES-603 NIES-559 NIES-2859 NIES-1385 NIES-1384 NIES-1011 NIES-1963 NIES-3333 NIES-622 NIES-2899 NIES-621 NIES-1044 NIES-1831 Chattonella marina var. Chattonella marina var. Chattonella marina var. Chattonella marina var. Chattonella marina var. antiqua Phaeomonas sp. Glossomastix chrysoplasta Glossomastix chrysoplasta Acinetospora crinita Unidentified pelagophyte Unidentified pelagophyte Unidentified pelagophyte Unidentified pelagophyte Unidentified pelagophyte Pelagomonas sp. Pelagomonas calceolata Pelagomonas calceolata Species Pelagomonas calceolata Olisthodiscus luteus Olisthodiscus luteus Heterosigma akashiwo Heterosigma akashiwo Haramonas dimorpha Gonyostomum semen Fibrocapsa japonica Fibrocapsa japonica Fibrocapsa japonica Fibrocapsa japonica Fibrocapsa japonica Chattonella subsalsa Chattonella marina var. antiqua Chattonella marina var. antiqua Chattonella marina var. antiqua Chattonella marina var. ovata Chattonella marina var. Ophiocytium sp. Ophiocytium parvulum Ophiocytium capitatum Ophiocytium capitatum Mischococcus sp. Heterothrix sp. Botrydium granulatum Botrydiopsis sp. Botrydiopsis arrhiza Schizocladia ischiensis Olisthodiscus luteus Raphidophyceae Pinguiophyceae Phaeophyceae Heterokontophyta Pelagophyceae Xanthophyceae Schizocladiophyceae

─ 23 ─ Fatty acid composition of microalgae Mitani et al. 0.0 0.0 5.9 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 3.9 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1 2.2 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 0.9 0.0 0.0 0.0 0.0 0.0 Others 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 24:1 5.6 5.3 3.7 0.7 0.0 0.0 1.0 0.8 1.3 0.5 0.0 2.2 0.8 0.0 0.0 0.0 0.0 0.0 4.1 2.8 5.4 3.7 4.3 2.5 1.4 4.1 3.4 1.6 4.0 4.1 2.8 4.0 3.0 1.8 1.8 0.6 5.2 8.7 6.5 5.6 7.9 7.4 4.4 8.2 7.1 9.1 3.4 7.5 22:6 1.3 0.0 0.0 0.0 1.4 7.3 7.3 8.3 7.1 4.4 6.2 4.0 4.3 4.3 5.3 6.2 5.1 0.0 2.8 3.3 3.2 4.2 3.5 2.4 3.1 4.3 3.4 3.4 3.8 4.2 2.8 2.5 3.1 7.9 7.1 2.3 0.0 0.0 0.9 0.0 1.9 0.0 0.0 0.3 0.0 0.0 0.0 22:5 13.6 5.6 5.4 9.4 7.1 6.9 7.2 16.4 10.4 13.7 13.4 16.8 21.7 14.3 11.6 16.5 13.6 10.3 15.0 14.7 10.5 14.6 20:5 15.9 13.9 19.4 22.7 19.7 15.8 13.6 21.2 14.1 13.3 18.1 21.0 14.2 14.1 12.4 17.9 12.6 11.9 15.0 11.2 13.4 14.3 12.3 10.0 10.3 13.3 11.8 2.3 0.0 0.0 0.0 0.7 0.0 0.0 0.6 0.0 0.7 1.1 0.0 0.0 0.0 0.5 0.0 0.0 0.8 0.0 3.1 1.8 1.2 1.2 0.0 1.3 1.4 0.0 1.1 2.4 1.8 0.0 0.7 0.0 0.0 1.0 0.3 3.3 0.6 9.5 0.0 0.0 0.5 0.0 0.6 0.0 1.3 0.0 2.1 20:4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.9 4.9 0.0 0.0 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18:5 9.8 0.0 0.0 0.4 21.4 25.6 24.5 25.2 13.7 33.8 30.4 35.3 18.2 27.7 20.0 20.4 23.0 22.8 28.9 17.6 18:4 13.1 24.3 28.1 18.6 28.6 30.3 19.3 22.3 21.1 20.5 21.5 23.8 21.0 21.9 19.0 12.1 21.3 29.1 14.9 23.1 30.9 21.1 24.3 24.5 22.7 21.6 15.5 30.8 0.0 2.0 9.1 7.5 9.4 5.9 25.5 14.7 13.9 20.1 17.3 12.2 15.6 11.5 16.5 16.1 16.3 13.5 12.4 14.3 13.8 11.9 18:3 12.2 15.4 17.4 11.5 14.9 25.8 19.1 17.6 13.5 19.6 19.2 16.4 34.4 32.1 21.2 27.6 24.0 24.3 14.2 13.2 17.8 14.1 11.7 17.7 12.3 10.6 4.0 4.9 4.1 3.9 9.2 7.3 2.3 5.8 4.6 4.3 3.1 2.4 2.1 1.9 3.5 1.7 0.0 1.7 0.0 0.6 2.1 1.6 0.6 2.1 1.2 1.1 8.6 5.5 3.2 0.7 2.3 5.0 3.5 4.5 6.1 4.3 3.5 1.6 1.8 0.7 3.1 0.6 4.0 6.8 1.6 2.6 2.3 1.8 18:2 0.0 4.4 1.4 3.4 1.8 7.4 5.1 4.4 7.9 4.8 3.9 8.2 6.1 2.6 2.0 2.5 0.0 0.3 2.5 1.4 1.1 3.1 4.0 4.8 1.1 0.0 1.5 3.9 7.5 9.6 5.9 0.0 7.2 5.5 8.0 9.0 3.0 8.2 8.5 5.1 7.9 8.1 11.7 13.4 20.1 10.3 18:1 10.3 12.4 2.1 6.7 7.9 1.7 1.7 8.9 1.9 2.5 1.8 5.2 2.0 4.1 3.9 3.3 1.8 4.9 7.8 9.4 4.6 1.5 1.5 1.8 1.3 4.3 8.4 2.1 4.0 5.5 1.4 2.1 4.1 0.9 2.8 3.7 5.3 4.2 3.5 1.8 3.0 3.0 2.9 4.8 7.1 3.4 8.7 3.3 5.6 2.9 18:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16:4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16:3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.4 16:2 0.0 2.7 0.0 2.3 3.8 3.2 3.4 3.0 2.1 3.5 1.2 2.8 3.6 0.0 2.9 3.2 2.5 0.0 5.2 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 2.3 0.8 0.0 2.5 5.9 2.4 2.5 2.2 3.8 2.3 1.5 3.2 1.9 10.3 16:1 10.6 9.0 9.3 16.3 10.1 14.4 14.2 24.0 13.9 13.9 18.9 12.3 18.1 12.9 16.1 24.5 24.0 18.4 13.3 22.8 16:0 19.6 22.3 12.5 19.1 16.0 15.3 19.1 29.1 12.5 16.8 17.7 13.8 12.8 27.9 11.5 21.9 19.0 18.0 17.3 16.0 15.2 17.9 19.4 16.3 12.8 10.4 14.2 12.0 13.6 2.7 4.5 6.5 4.1 4.4 5.2 0.0 1.6 3.2 4.4 4.5 5.2 1.9 3.8 0.8 4.6 2.6 6.2 7.0 4.6 1.5 5.1 3.1 3.7 6.1 3.9 5.0 1.9 5.8 4.9 1.4 2.6 4.7 4.2 2.1 5.8 4.3 6.1 7.4 5.5 6.4 8.3 9.4 4.2 14:0 15.2 10.9 12.4 11.8 Table 1 Continued Table 2.4 3.4 6.0 3.8 0.7 4.6 2.6 3.0 4.7 3.4 3.6 8.9 1.9 2.6 2.3 5.0 4.8 5.0 4.1 2.4 5.4 3.5 3.1 3.9 5.9 4.6 7.9 3.2 6.2 2.7 3.3 2.6 5.0 1.4 3.5 2.1 2.3 5.9 5.5 9.3 3.6 6.0 5.9 4.0 4.3 12:0 12.1 11.3 16.0 Strain NIES-274 NIES-698 NIES-697 NIES-2331 NIES-705 NIES-711 NIES-710 NIES-709 NIES-708 NIES-707 NIES-706 NIES-1370 NIES-704 NIES-703 NIES-1004 NIES-714 NIES-713 NIES-712 NIES-2716 NIES-2964 NIES-348 NIES-347 NIES-346 NIES-282 NIES-281 NIES-280 NIES-279 NIES-1327 NIES-345 NIES-278 NIES-277 NIES-344 NIES-276 NIES-767 NIES-766 NIES-715 NIES-275 NIES-2332 NIES-1730 NIES-1005 NIES-3015 NIES-1375 NIES-1006 NIES-702 NIES-765 NIES-701 NIES-700 NIES-699 ovata Cryptomonas irregularis Cryptomonas acuta sp. Chroomonas placoidea Chroomonas nordstedtii Chroomonas nordstedtii Chroomonas nordstedtii Chroomonas nordstedtii Chroomonas nordstedtii Chroomonas nordstedtii Chroomonas mesostigmatica Chroomonas dispersa Chroomonas collegionis Chroomonas coerulea Chroomonas coerulea Chroomonas coerulea Chroomonas caudata Species Hemiselmis andersenii Cryptomonas sp. Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas tetrapyrenoidosa Cryptomonas rostratiformis Cryptomonas rostratiformis Cryptomonas rostratiformis Cryptomonas rostratiformis Cryptomonas platyuris Cryptomonas platyuris Cryptomonas paramaecium Cryptomonas paramaecium Cryptomonas paramaecium Cryptomonas ovata sp. Rhodomonas sp. Rhodomonas sp. Rhodomonas salina Rhodomonas salina Rhodomonas salina Rhodomonas falcata Rhodomonas duplex Rhodomonas chrysoidea Rhodomonas baltica Rhodomonas atrorosea Cryptophyta

─ 24 ─ Microb. Resour. Syst. June 2017 Vol. 33, No. 1 3.1 1.9 1.3 0.0 0.0 0.0 0.5 0.7 5.2 1.3 0.0 1.1 0.8 0.9 2.0 0.7 1.2 0.7 2.8 0.0 0.0 0.0 2.8 0.6 0.0 0.0 8.7 5.9 3.0 0.0 6.3 8.5 1.0 0.0 0.0 0.0 2.0 1.6 2.2 0.0 0.0 3.6 0.0 0.0 Others 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 3.4 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 2.2 0.0 0.0 0.0 0.0 4.4 4.7 2.6 0.0 0.4 2.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 0.0 0.0 0.0 0.0 24:1 7.2 3.5 0.0 6.0 5.0 1.3 5.4 9.0 8.9 8.6 4.0 3.4 7.4 2.1 5.2 3.2 2.3 1.5 8.7 9.0 7.9 5.1 9.9 1.7 0.0 5.2 8.9 1.1 8.9 8.6 15.5 17.4 12.6 11.4 22:6 14.2 19.0 13.4 16.0 18.5 22.2 16.6 11.4 12.6 12.5 0.0 0.5 5.6 0.0 0.0 0.0 0.6 0.0 5.2 8.8 8.0 2.1 8.6 7.1 6.9 8.5 4.2 0.9 0.0 0.7 0.0 1.5 1.1 0.0 0.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 1.2 0.0 0.0 0.0 0.0 6.1 1.4 0.0 0.0 22:5 0.0 2.7 3.0 3.0 0.0 1.1 0.0 4.7 8.0 0.0 3.0 3.4 0.0 1.2 4.6 1.0 1.0 0.0 0.8 0.0 0.0 0.0 1.4 1.9 6.3 4.0 3.8 0.0 4.2 0.0 0.0 0.5 0.0 2.3 1.2 24.7 22.7 26.2 20.0 16.9 11.9 25.0 20:5 24.8 11.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1 1.3 1.3 1.0 0.0 1.2 1.5 0.3 0.0 0.0 0.9 0.2 0.0 0.0 0.8 0.0 1.3 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:4 5.2 8.0 0.0 0.8 3.8 9.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9.2 0.8 0.0 4.7 2.6 0.9 8.2 4.7 5.0 4.5 1.8 6.8 1.3 8.3 1.6 5.4 0.0 8.0 0.0 5.0 3.7 2.5 4.0 15.6 13.8 18:5 10.2 10.1 22.5 5.4 5.5 5.0 5.8 0.0 2.5 2.7 4.5 7.3 4.6 9.6 9.3 3.2 3.8 9.7 7.6 3.5 4.8 4.7 5.4 5.1 2.0 6.5 4.2 5.0 8.5 0.0 9.4 0.0 3.0 8.8 14.9 12.1 18:4 11.5 11.2 10.4 18.3 25.8 11.6 11.6 10.5 15.2 12.5 19.7 3.1 2.4 2.1 6.0 0.0 3.1 2.4 4.0 0.0 1.2 1.0 1.0 0.5 0.9 0.3 0.0 0.9 2.5 5.4 6.8 8.6 1.4 2.9 3.5 3.0 3.3 2.8 5.7 0.7 3.2 1.8 4.7 1.4 6.2 0.0 2.3 0.0 2.1 6.7 0.7 18:3 13.4 11.6 10.1 13.1 4.7 6.5 2.9 0.0 2.6 2.6 5.7 1.3 1.2 2.4 1.3 2.5 2.0 1.4 0.0 0.5 1.6 0.0 6.9 8.7 2.8 5.7 4.0 3.9 2.8 0.0 3.4 1.5 0.0 2.4 1.3 3.5 3.2 4.5 2.4 0.0 9.0 0.0 2.2 1.2 0.4 6.8 10.8 18:2 19.1 7.2 4.9 0.0 0.0 6.1 9.9 1.6 1.1 0.0 0.4 3.0 2.9 0.0 0.3 7.9 8.6 4.2 7.3 0.0 9.8 6.0 8.2 11.7 28.2 12.1 18:1 12.0 10.7 30.0 34.4 23.8 14.4 18.9 15.4 12.3 10.6 21.1 10.5 20.8 13.9 23.9 10.1 10.0 13.7 11.4 5.3 4.3 7.1 6.3 0.6 5.4 6.2 2.1 1.0 1.5 0.5 1.7 2.9 0.6 0.7 7.0 3.2 3.3 1.3 1.5 7.1 1.8 2.6 6.3 3.4 3.2 1.2 0.8 1.6 8.8 6.0 4.7 9.1 0.5 2.8 2.8 1.9 14.6 16.2 18:0 11.5 12.2 11.8 20.6 23.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16:4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.3 0.0 0.0 2.7 0.0 0.0 0.0 2.6 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 2.1 0.0 0.0 0.0 0.0 0.2 0.0 0.0 16:3 0.0 0.0 0.0 0.0 0.0 0.0 1.1 0.0 0.0 2.7 1.4 0.0 3.4 0.6 0.0 0.0 5.7 2.4 4.6 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.0 0.0 0.7 2.6 2.5 0.0 0.0 0.0 0.0 1.0 0.0 0.0 16:2 0.0 0.9 4.5 2.1 0.0 0.0 1.3 3.5 0.0 9.8 9.5 9.0 9.8 9.3 1.5 0.8 5.4 0.6 1.3 4.0 4.7 0.0 0.0 0.9 0.0 0.0 0.0 0.0 3.8 1.2 1.4 6.5 6.6 0.0 1.9 0.0 0.0 2.6 1.0 3.3 13.8 13.4 25.3 13.8 16:1 9.7 8.9 8.8 11.7 11.6 12.0 18.9 12.7 33.2 33.3 34.4 28.5 24.3 12.8 14.7 10.7 12.2 12.5 19.7 12.0 16:0 12.3 24.3 25.5 26.5 28.9 16.6 18.3 20.2 16.8 37.6 12.1 12.7 12.0 14.1 23.3 38.9 18.3 28.2 36.2 27.7 51.2 26.9 20.6 20.2 6.3 6.3 0.7 0.4 0.9 0.5 1.7 8.7 8.7 1.2 20.5 23.7 32.7 24.1 23.7 43.2 12.7 14.5 20.9 17.9 19.1 19.5 20.3 16.8 21.0 14:0 22.8 32.5 15.1 12.2 29.1 33.6 11.9 23.9 20.2 18.9 16.3 18.2 20.3 14.1 16.0 29.3 15.2 17.9 45.8 Table 1 Continued Table 7.0 3.9 2.9 3.6 8.4 9.0 0.7 2.9 4.5 7.6 3.5 2.2 2.8 1.1 3.4 1.5 1.4 1.0 2.2 3.9 2.1 0.7 0.5 1.5 6.6 1.6 1.2 3.2 4.5 1.7 7.8 6.9 3.9 1.1 0.5 2.0 2.8 2.7 4.7 7.3 1.9 1.9 3.4 12:0 19.3 Strain NIES-2506 NIES-1391 NIES-1392 NIES-562 NIES-741 NIES-2904 NIES-997 NIES-2872 NIES-2694 NIES-2773 NIES-1965 NIES-1816 NIES-1815 NIES-1401 NIES-1400 NIES-1399 NIES-1398 NIES-623 NIES-1813 NIES-2703 NIES-388 NIES-1964 NIES-1395 NIES-2590 NIES-1001 NIES-1016 NIES-1393 NIES-2376 NIES-353 NIES-3121 NIES-2698 NIES-2697 NIES-2370 NIES-837 NIES-2369 NIES-2696 NIES-1874 NIES-1324 NIES-2707 NIES-2878 NIES-2705 NIES-1017 NIES-1330 NIES-8 Chrysochromulina sp. Chrysochromulina sp. Chrysochromulina simplex Chrysochromulina parva Chrysochromulina hirta Chrysochromulina brevifilum Calyptrosphaera sphaeroide Calyptrosphaera oblonga Calcidiscus leptoporus Pavlova sp. Pavlova sp. Pavlova sp. Pavlova sp. Pavlova sp. Pavlova sp. Pavlova sp. Pavlova pinguis Species Pavlova gyrans Pleurochrysis haptonemofera Platychrysis sp. Phaeocystis globosa Ochrosphaera neapolitana Ochrosphaera neapolitana Isochrysis galbana Imantonia rotunda Hymenomonas coronata Hyalolithus neolepis Helicosphaera walichii Gephyrocapsa oceanica Gephyrocapsa ericsonii Emiliania huxleyi Emiliania huxleyi Emiliania huxleyi Emiliania huxleyi Corisphaera gracilis Coccolithus braarudii Chrysoculter rhomboideus Umbilicosphaera sibogae Umbilicosphaera hulburtiana Umbilicosphaera foliosa Syracosphaera pulchra Prymnesium parvum Prymnesium calathiferum Pleurochrysis roscoffensis Prymnesiophyceae Haptophyta Pavlovophyceae 16:1 is mixture of isomers 16:1 (9) and 16:1 (3t); 18:1 is a mixture of isomers 18:1 (9) and 18:1 (11); 18:3 is a mixture of isomers 18:3 (6, 9, 12) and 18:3 (9,12,15); 20:4 is a mixture of isomers 20:4 (5, 8, 11, 14) and 20:4 (8, 14, 17).

─ 25 ─ Fatty acid composition of microalgae Mitani et al.

(NIES-1, 14, 115, 557, 558, 559, 603, 1974, 1975, 1976), contain well-known species of the Prymnesiales and Fibrocapsa japonica (NIES-136, 462, 560, 605, 1303), coccolithophorids. The major fatty acids in the Hap- and Heterosigma akashiwo (NIES-9, 145, 293) kill fish tophyta were 14:0, 16:0, 16:1, 18:4, and 22:6. The con- in Japanese aquaculture through algal blooms (Giner tent of 20:5 also was high in the Pavlovophyceae. In et al., 2008). These strains also contained a higher addition, 18:5 was detected in some strains in the proportion of 18:4 than other . Other Prymnesiophyceae. Emiliania huxleyi is a model studies of the content of 18:4 in the Raphidophyceae coccolithophorid, and its fatty acid composition has support our results (Mostaert et al., 1998; Nichols et been reported (Bell & Pond, 1996; Boelen et al., 2013; al., 1987; Marshall et al., 2002; Giner et al., 2008; Kotajima et al., 2014). Four Emiliania strains and the Dorantes-Aranda et al., 2015). 16:0 and 16:1 were closely related species Gephyrocapsa oceanica had abundant in the Xanthophyceae, and 20:5 was de- similar fatty acid compositions to those previously tected in all strains except those of Botrydiopsis reported (Bendif et al., 2016). In the Pavlovophyceae, (NIES-621, 2899). PUFA production has been investigated most inten- The Pinguiophyceae and the Aurearenophyceae sively in Pavlova lutheri (Kato et al., 1995; Meireles et were identified as new classes in 2002 and 2008, re- al., 2003; Guihéneuf & Stengel, 2013). This species is spectively (Kawachi et al., 2002; Kai et al., 2008). widely used in aquaculture as live feed for marine High EPA content (23.5% to 56.0% of total fatty acids) invertebrates (Rico-Villa et al., 2006; Ponis et al., 2008). is a characteristic feature of five genera in the Pin- Fatty acid compositions of MCC-NIES strains in the guiophyceae (Kawachi et al., 2002). We found 20:5 in Pavlovophyceae were similar to that of P. lutheri three strains at 3% to 15% of the total. Aurearena (Kato et al., 1995). In addition, two unidentified cruciata (NIES-1863, 1864, 1865) had >20% of 16:0 and strains of Pavlova, NIES-1815 and 1816, had good 4% to 14% of 20:5. quantities of both 20:5 and 22:6, suggesting advan- Most of the Phaeophyceae are macroalgae, with tages in aquaculture nutrition. only one filamentous unialgal strain in the MCC- NIES (NIES-548). The major fatty acids in Acinetos- CONCLUSION pora crinita (NIES-548) were 16:0, 18:1, 18:2, 18:3, 18:4, The fatty acid profiles presented here provide use- and 20:5. ful information in the search for microalgae as sourc- The Schizocladiophyceae were classified in 2003 es of valuable compounds. Microalgae as sources of (Kawai et al., 2003). The major fatty acids in the fila- PUFAs such as 20:5 and 22:6, with beneficial effects mentous marine alga Schizocladia ischiensis (NIES- on human health, are attracting increasing attention. 1044) were 16:0, 16:1, 18:1, and 20:5. The major fatty Microalgae offer sources of many other useful com- acid composition of Giraudyopsis sp. (NIES-1862), in pounds, such as antioxidants, pigments, biofuel, and the Chrysomerophyceae, was similar to that of A. bioplastics. We hope to enrich the information avail- crinita (NIES-548), in the Phaeophyceae. able to microalga researchers through the MCC- NIES. Cryptophyta The Cryptophyceae, the only class in the Cryp- REFERENCES tophyta, are marine or freshwater flagellated unicells, Abedi, E. & Sahari, M.A. 2014. Long-chain and most are photosynthetic. The major fatty acids polyunsaturated fatty acid sources and evaluation in most strains were 16:0, 18:3, 18:4, and 20:5. In par- of their nutritional and functional properties. Food ticular, most strains contained high proportions of Sci. Nutr. 2: 443-463. 18:3+18:4 (15.1%−53.4%). Furthermore, 22:6 was Andersen, R.A., Saunders, G.W., Paskind, M.P. & found in all strains of Cryptomonas, Rhodomonas, and Sexton, J.P. 1993. Ultrastructure and 18S rRNA Hemiselmis except for NIES-3185. The results in gene sequence for Pelagomonas calceolata gen. et Cryptomonas and Rhodomonas support previous re- sp. nov. and the description of a new algal class, ports (Dunstan et al., 2005; Wacker et al., 2016). the Pelagophyceae classis nov. J. Phycol. 29: 701- 715. Haptophyta Bell, M.V. & Pond, D. 1996. Lipid composition during The Haptophyta are divided into two classes, the growth of motile and coccolith forms of Emiliania Pavlovophyceae and the Prymnesiophyceae, which huxleyi. Phytochemistry 41: 465-471.

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NIES 藻類コレクションに属する 3 植物門 235 株の脂肪酸組成プロファイル

三谷英李1），中山史葉1），松脇いずみ1），市 育代1, 2），川端篤志3），河地正伸3），加藤美砂子1, 2）

1）お茶の水女子大学大学院人間文化創成科学研究科， 2）お茶の水女子大学ヒューマンライフイノベーション研究所，3）国立環境研究所

表現型的マーカーとしての高度不飽和脂肪酸の有無について調査するために，国立環境研究所（NIES）の藻類コレクショ ンから，不等毛植物門，クリプト植物門，ハプト植物門について，合計 235 株の脂肪酸分析を行った．エイコサペンタエン 酸（EPA，20:5）は分析した 119 株において，総脂肪酸の 10%以上を占めていた． ドコサヘキサエン酸（DHA, 22:6）は 16 株のハプト植物と未同定の 2 株のペラゴ藻綱（不等毛植物門）において，総脂肪酸の 10% 以上を占めていた．黄金色藻綱や ペラゴ藻綱以外の不等毛植物の株やクリプト植物の株では，一般に DHA よりも EPA のほうが，多く含まれていた．近年新 しく設立され，本研究で初めて脂肪酸分析が行われたアウレアレナ藻綱，ペラゴ藻綱，シゾクラディア藻綱を含む 11 綱の不 等毛植物を調査した結果，多様な脂肪酸組成プロファイルが明らかになったものの，表現型として分類群を特徴づけるよう な明瞭な結果は確認されなかった．

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