한수지 49(1), , 2016 Original Article Korean J Fish Aquat Sci 49(1), ,2016 배양방법에따른 Euglena gracilis 의성장및지방산조성 정우철 최종국 강창민 1 최병대 강석중 * 경상대학교해

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1 한수지 49(), 38-44, 26 riginal Article Korean J Fish Aquat Sci 49(),38-44,26 배양방법에따른 Euglena gracilis 의성장및지방산조성 정우철 최종국 강창민 최병대 강석중 * 경상대학교해양식품생명의학과, 안전성평가연구소경남환경독성본부 Effects of Culture Methods on the Growth Rates and Fatty Acid Profiles of Euglena gracilis U-Cheol Jeong, Jong-Kuk Choi, Chang-Min Kang, Byeong-Dae Choi and Seok-Joong Kang* Department of Seafood and Aquaculture Science, Gyeongsang National University, Tongyeong 5364, Korea Institute of Toxicology, Jinju 52834, Korea The quality and quantity of live food sources strongly influence the success of fish production in farming operations. Thus, critical studies of live forage species are a crucial element for progress in fish aquaculture. The fat content of food is an especially important determinant of growth in marine fish. mega-3 highly unsaturated fatty acids (HUFA) are essential components of diet that determine the nutritional value of larval fish. Euglena is a protist that has potential as a forage species. These single-celled organisms have plant and animal characteristics they are motile, elliptical in shape and 5 5 µm in diameter. Their nutritional content is excellent, but most studies have focused on cells raised in autotrophic culture. We therefore examined differences in the lipid and fatty acid contents, and the growth of Euglena cells grown under autotrophic, heterotrophic, and mixotrophic conditions. Biomass production reached 5.3 g/l, 2.28 g/l, and 3.66 g/l under mixotrophy, heterotrophy, and autotrophy, respectively. The proportional n-3 HUFA content differed among culture methods:.4%, 5.8% and.% in mixotrophic, heterotrophic and autotrophic cultures, respectively. Mixotrophy was to be the best form of cultivation for improving the growth and nutritional content of Euglena. Key words: Euglena gracilis, Fatty acid, Heterotrophic, Autotrophic, Mixotrophic 서론 (euglenoids) 5-5 µm,, (Rodríguez-Zavala et al., 2).,, (Ruiz et al., 24).., (Hayashi et al., 994; Chisti and Yan, 2), (Choi et al., 24),, (Ishikawa et al., 28; Lira-Silva et al., 2). Bio-fuel (Navarro et al., 997; Ramalho et al., 998; Tucci et al., 26; Jasso-Cha vez et al., 2)., Korean J Fish Aquat Sci 49() 38-44, February 26 This is an pen Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 6 January 26; Revised 6 February 26; Accepted 7 February 26 *Corresponding author: Tel: Fax: address: sjkang@gnu.ac.kr Copyright 26 The Korean Society of Fisheries and Aquatic Science 38 pissn:374-8, eissn:

2 유글레나성장및지방산조성 39 (autotrophic) (Chae et al., 26)., (Dos et al., 27)., (Regnault et al., 995)., -,3 glucan (paramylon) (Barsanti et al., 2ab; Choi et al., 23). Vitamin E, 5-24% (dry weight) (Navarro et al., 997). (Biomass) (Food), (Fiber), (Feed), (Fertilizer), (Fuel) (Rocchetta et al., 26; Courchesne et al., 29). E. gracilis 2% -,3 glucan, (5-24%), EPA, DHA, (Harwood, 988; Hayashi et al., 994; James and Browse, 999; Choi et al., 23). (Hayashi et al., 992). (E. gracilis).. 실험주및배지 재료및방법 Euglena gracilis KMMCC-35, Table Cramer-Myers medium (Cramer and Myers, 952). Cramer-Myers medium Glucose 5 g/l, Sodium glutamate 5 g/l, 2 5. 배양방법 (Autotrophic culture), (Heterotrophic culture) (Mixotrophic culture). L Table. Composition of Cramer-Myers (C&M) medium KH 2 P 4 (NH 4 ) 2 S 4 MgS 4 7H 2 CaCl 2 2H 2 5 ml, 8L:6D, 3, lx, Air.2 µm sterilized air filter (Advantec mfs, Inc., Japan). 5 ml 2 ml, L:24D, (KSI-2L, Koencon Co., Ltd) 2 rpm. 5 ml 2 ml, 8L:6D, 3, lx cells/ml 7 3, 2 / Haemacytometer 3, 4 (UNIN 32R, Hanil Science Industrial Co., Ltd. Korea) 3, rpm 25,. 총지질 Components EDTA-3Na 2H 2 Fe(S 4 ) 2 6H 2 MnCl 2 4H 2 CoS 4 7H 2 ZnS 4 7H 2 Na 2 Mo 4 2H 2 CuS 4 5H 2 Concentration. g. g.2 g.2 g.8 g 3. mg.8 mg.5 mg.4 mg.2 mg.2 mg Vitamin B. mg Vitamin B 2.5 µg Distilled water final volume Bligh and Dyer (959). 5 g (homogenizer AM-2, Nihonseiki Kaisha Co. Ltd., Tokyo, Japan) 5, rpm 5, Chloroform Methanol 2: 2 chloroform, Na 2 S 4 chloroform. chloroform (Rotavapor L Final ph ph 3.5 Cramer-Myers (C&M) medium (Cramer and Myers, 952).

4 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 R-4, BUCHI) 4,.. 지질 classes TLC/FID Iatrorecorder TC-2 intergrator Iatroscan New MK-5 (Iatron Laboratory Inc., Tokyo, Japan). 2 L/min detector 6 ml/min, scanning speed.3 cm/s.

n-hexane : diethyl ether : acetic acid = 97:3: (v/v) cm, Rod Rod-Dryer 5 Iatroscan. cholesterol ester, free fatty acid, triglyceride, cholesterol phospholipid.

Iso-octane (4 ml), Iso-octane ml methyl ester. GLC megawax TM -32 fused-silica capillary column (3 m.32 mm.25 µm, i.d., Supelco Co., Bellefonte, PA, USA) Clarus 6 (Perkin Elmer Co. Ltd., USA). Column 85 8 3 /min 23,.

) (one-way ANVA) (Regression Analysis) Duncan's multiple range test (Duncan, 955) (P<.5). 배양방법에따른성장 결과및고찰 E. gracilis, Fig.

3 4 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 R-4, BUCHI) 4,.. 지질 classes TLC/FID Iatrorecorder TC-2 intergrator Iatroscan New MK-5 (Iatron Laboratory Inc., Tokyo, Japan). 2 L/min detector 6 ml/min, scanning speed.3 cm/s. Rod S- (.9 5 mm, ) 5, ml ml 5 Rod-Dryer (TK-5 Iatron Lab. Inc., Tokyo, Japan) 5 Iatroscan 3. Rod L Microdispenser (Drummond Scientific Co., Bromall, PA, USA) (NaCl ). n-hexane : diethyl ether : acetic acid = 97:3: (v/v) cm, Rod Rod-Dryer 5 Iatroscan. cholesterol ester, free fatty acid, triglyceride, cholesterol phospholipid. 지방산 methyl ester (C 23: methyl ester) ml ( mg) cap tube,.5 N NaH-methanol.5 ml, 8. 2% BF 3 - methanol 2 ml tube, methyl. 3 Iso-octane ml 3 vortex mixer. 3 ml iso-octane. Iso-octane (4 ml), Iso-octane ml methyl ester. GLC megawax TM -32 fused-silica capillary column (3 m.32 mm.25 µm, i.d., Supelco Co., Bellefonte, PA, USA) Clarus 6 (Perkin Elmer Co. Ltd., USA). Column /min 23,. 25, 27 carrier gas He (. kg/cm 2 ). ECL, 4:, 6:, 8:, 8:2, 8:3, 2:, 22:, 24: (Sigma Chemical Co., St. Louis, M, USA) GC-MS menhaden oil. 통계처리 SPSS (6.) (one-way ANVA) (Regression Analysis) Duncan's multiple range test (Duncan, 955) (P<.5). 배양방법에따른성장 결과및고찰 E. gracilis, Fig.. Biomass (x 4 cells ml - ) %, Total fatty acid composition Autotrophic Heterotrophic 6 Mixotrophic Cultivation time (h) Fig.. The effect of cultivation methods on the cell growth of Euglena gracilis. Data were means±s.d. of triplicate. Autotrophic Heterotrophic Mixotrophic C4: C6: C8:2n-6 C8:3n-3 C2:5n-3 C22:6n-3 Fatty acid Fig. 2. Fatty acid profile of Euglena gracilis grown in different culture conditions. Data were means±s.d. of triplicate. Cell concentration (w/w, g/l)

4 유글레나성장및지방산조성 4 4 cells/ml..2 4 cell/ml cell/ml, cell/ml, cell/ml, cell/ml, cell/ml, cell/ ml cell/ml, 4. g/ L..2 4 cell/ml cell/ml, cell/ml, cell/ml, cell/ml, cell/ ml, cell/ml cell/ml cell/ml, 2. g/l, (P<.5) cell/ml, cell/ml, cell/ml, cell/ml, cell/ml, cell/ml, cell/ ml cell/ml cell/ml (P<.5), 5. g/l. 4 cells/ml., cells/ml (P<.5)., cells/ml cells/ ml (P<.5) cells/ml, cells/ml (P<.5). E. gracilis,. E. gracilis. 총지질및지질종류의변화 Table %, 2.2%, 9.6% (P<.5). E. gracilis class Table %. 9.48%, 83.72%, 68.5% (P<.5).. El-Sheekh and Fathy (29) Chlorella vulgaris chlorophyll 2. chlorophyll,,. triglycerols (TG) 24.93%, 2.4%, 5.74% (P<.5).. TG. Cholesteryl ester (CHL) Table 2. Lipid classes in cells cultivated under different culture conditions Lipid class Culture conditions AC HC 2 MC 3 Total lipids (%) 8.36±.8 2.2±.2 9.6±.5 FFA 4.87±.6.99±.8.9±.2 TG ± ±.6 2.4±.35 CHL 6.9± ± ±.2 PL ± ± ±.23 AC: Autotrophic culture, 2 HC: Heterotrophic culture, 3 MC: Mixitrophic culture, 4 FFA: free fatty acids, 5 TG: triacylglycerols, 6 CHL: cholesteryl ester, 7 PL: polar lipids and chlorophylls. The values are mean±s.d. (n=3). Different superscript letters within rows represent significant differences between treatments (P<.5).

5 42 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중.9% (P<.5), 2.79%, 4.93% (P<.5). TG. Free fatty acids (FFA).87% (P<.5),.9%,.99% (P<.5).,. 지방산변화 E. gracilis Table 3., (Saturated fatty acid), 5.54%, 7.9% 34.78%, (P<.5)., 6:, 8:.. (unsaturated fatty acid) 48.46%, 29.9% 65.22%, (P<.5). 8:3n % 2.3%,.73% (P<.5). 8:2n % 7.6%,.42% (P<.5). 2:5n-3(EPA), 4.79%, 2.22% 3.88%, (P<.5), 22:6n-3(DHA).74%,.56% 2.47% (P<.5), (P<.5). n-3 HUFA (highly unsaturated fatty acid),.%, 5.8%.4% (P<.5), (P<.5), n-6 HUFA 8.88%, 8.%.3% (P<.5), Table 3. Fatty acid compositions of Euglena gracilis grown in different culture conditions (% of total fatty acids) Fatty acid Culture Conditions AC HC 2 MC 3 4: 2.3±.2 3.5±.8 8.9±.6 5: 2.28± ± ±.3 Iso6: 5.8± ±.8.9±. 6: 27.65±.8 6.5±.6.25±.2 6:n-5 3.8± ± ±.6 6:2n-4.47±. 2.±.4.36±.4 7:.9±. 4.9± ±.2 6:3n-4.26±.3 3.5±. 9.±.8 6:3n-.±..2±..45±. 6:4n-.±..35±..54±. 8: 2.45±.2.44±.3.45±. 8:n-9.35±..79± ±.3 8:n-7.±..4±..7±.2 8:2n ±.6.42±. 7.6±. 8:2n-4.25±..5±..23±.8 8:3n ±..73±. 2.3±.6 8:4n-.±..65±.3.4±.2 2:.6±..54±..23±. 2:n-7.±. 2.±.6.43±. 2:2n ±.3.9±.2.7±. 2:3n-6.6±..69±..6±. 2:4n ± ±. 3.64±.3 2:3n-3.38±.2.5±..77±. 2:4n-3.77±.6.74±..54±.2 2:5n ± ± ±.5 22:n-7.±..95±.2.98±. 22:4n-6.23±..99±..3±.3 22:5n-6 2.±.6.84± ±.8 22:4n-3.5±..8±..7±. 22:5n-3.28±..6±..2±.3 22:6n-3.74±.2.56± ±.4 SFA USFA n-3 HUFA n-6 HUFA n-6/n AC: Autotrophic culture, 2 HC: Heterotrophic culture, 3 MC: Mixitrophic culture, 4 SFA: Saturated fatty acid, 5 USFA: Unsaturated fatty acid, 6 HUFA: Highly unsaturated fatty acid. The values are mean±s.d. (n=3). a-f Different superscript letters within rows represent significant differences between treatments (P<.5).

6 유글레나성장및지방산조성 43 (P<.5).. biomarker (Volkman et al., 998),. (stigma),, (Walne 98),. (Coleman et al., 988; Girotti 2), starvation (Kim and Boo, 998; Watanabe and Suzuki, 22). n-3 HUFA (P<.5). 8:2n-6..,.. (Thompson et al.,992), (Jiang and Chen, 2). (Jiang and chen, 992), (Honda et al., 998). -3.,. 사사 25 ( ). References Barsanti LR, Bastianini A, Passarelli V, Tredici MR and Gualtieri P. 2a. Fatty acid content in wild type and WZSL mutant of Euglena gracilis. J Appl Phycol 2, Barsanti LR, Vismara R, Passarelli V and Gualtieri P. 2b. Paramylon(b-,3- glucan) contentin wild type and WZSL mutant of Euglena gracilis. Effectsof growth conditions. J Appl Phycol 3, Bligh EG and Dyer WJ A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, Chae SR, Hwang EJ and Shin HS. 26. Single cell protein production of Euglena Gracilis and carbondioxide fixation in an innovative photo-bioractor. Bioresour Technol 97, Choi JA, h TH, Choi JS, Chang DJ and Joo CK. 23. Impact of beta-,3-glucanisolated from Euglena gracilis on corneal epithelial cell migration and on wound healing in a rat alkali burn model. Curr Eye Res 38, Chisti Y and Yan J. 2. Energy from algae: current status and future trends algal biofuels a status report. Appl Energy 88, Choi SW, Park IK and Park BS. 24. Effect of dietary supplementation of fresh water algae euglena on the performance and egg quality and fatty acid composition of egg yolk in laying hens. Korean J Poult Sci 3, Coleman LW, Rosen BH and Schwartzbach SD Environmental-control of carbohydrate and lipid-synthesis in Euglena. Plant Cell Physiol 29, Courchesne NMD, Parisien A, Wang B and Lan CQ. 29. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J Biotechnol 4, 3-4. Cramer M and Myers J Growth and photosynthetic characteristics of Euglenagracilis. Arch Microbiol 7, Dos SFV, Rocchetta I, Conforti V, Bench S, Feldman R and Levin M. 27. Gene expression patterns in Euglena gracilis: insights into the cellular response to environmental stress. Gene 389, Duncan DB Multiple range and multiple F test. Biometric, -42. El-Sheekh MM and Fathy A. 29. Variation of some nutritional constituents and fatty acid profiles of chlorella vulgaris beijerinck grown under auto and heterotrophic conditions. In-

7 44 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 tern J Botany 5, Girotti AW. 2. Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms. J Photochem Photobiol 63, 3-3. Harwood JL Fatty acid metabolism. Ann Rev Plant Physiol Plant Mol Biol 39, -38. Hayashi M, Toda K, Yoneji T, Sato and Kitaoka S Dietary value of rotifer and artemia enriched with Euglena gracilis for red sea bream. Nippon Suisan Gakkaishi 59, Hayashi M, Kyoji T, Hiroto I, Reiko K and Shozaburo K Effects of shifting ph in the stationary phase of growth on the chemical composition of Euglena gracilis. Biosci Biotech Biochem 58, Honda D, Yokochi T, Nakahara T, Erata M and Higashihara T Schizochytrium limacinum sp. nov., a new thraustochytrid from a mangrove area in West Pacific cean. Mycol Res 2, Ishikawa T, Nishikawa H, Gao Y, Sawa Y, Shibata H, Yabuta Y, Maruta T and Shigeoka S. 28. The pathwayvia D- galacturonate/l-galactonate is significant for ascorbatebiosynthesis in Euglena gracilis. J Biol Chem 283, James GW and Browse J 999. The Δ8-Desaturase of Euglena gracilis: An alternate pathway for synthsis of 2-carbon polyunsaturated fatty acids. Archiv Biochem Biophys 365, Jasso-Cha vez R, Pacheco-Rosales A, Lira-Silva E, Gallardo- Pe rez JC, Garcı a N and Moreno-Sa nchez R. 2. Toxic effects of Cr(VI) and Cr(III) on energy metabolism of heterotrophic Euglena gracilis. Aquat Toxicol, Jiang Y and Chen F. 2. Effect of temperature and temperature shift on docosahexaenoic acid production by the marine microalgae Crypthecodiniumcohnii. JACS, 77, Kim JT and Boo SM Morphology, population size and environmental factors of two morphotypes in Euglena geniculate (Euglenophyceae) in Korea. Algological Studies 9, Lira-Silva E, Ramı rez-lima IS, lı n-sandoval V, Garcı a- Garcı a JD, Garcı a-contreras R, Moreno-Sa nchez R and Jasso-Cha vez R. 2. Removal, accumulation and resistance to chromium in heterotrophic Euglena gracilis. J Hazard Mater 93, Navarro L, Torres-Marquez ME, Gonza lez-moreno S, Devars S, Herna ndez R and Moreno-Sa nchez R Comparison of physiological changes in Euglena gracilis during exposure to heavy metals of heterotrophic and autotrophic cells. Comp Biochem Physiol 6C, Ramalho JC, Campos PC, Teixeira M and Nunes MA Nitrogen dependent changes in antioxidant system and in fatty acid composition of chloroplast membranes from Coffea arabica L. plants submitted to high irradiance. Plant Sci 35, Regnault A, Chervin D, Chammal A, Piton F, Calvayrac R and Mazliak P Lipid composition of Euglena gracilis in relation to carbon-nitrogen balance. Phytochemistry 4, Rocchetta I, Conforti V, Mazzuca M, Balzaretti V and Rı os demolina MC. 26. Effect of chromium on the fatty acid composition of two strains of Euglena gracilis. Environ Pollut 4, Rodríguez-Zavala JS, rtiz-cruz MA, Mendoza-Hernández G and Moreno-Sánchez R. 2. Increased synthesis of a-tocopherol, paramylon and tyrosine by Euglena gracilis under conditions of high biomass production. J Appl Microbiol 96, , Ruiz LB, Rocchetta I, dos Santos Ferreira V and Conforti VTD. 24. Isolation, culture and characterization of a new strain of Euglena gracilis. New strain of Euglena gracilis. Phycol Res 52, Thompson PA, Guo M, Harrison PJ and Whyte JNC Effects of variation in temperature: ωⅡ. n the fatty acid composition of eight species of marine phytoplankton. J Phycol 28, Tucci S, Proksch P and Martin W. 26. Fatty acid biosynthesis in mitochondria of Euglena gracilis. In: Benning, C., hlrogge, J. (Eds.), Advances in Plant Lipid Research: Proceedings of the 7th International Symposium on Plant Lipids, East Lansing, Michigan, July 26. Michigan State University Press, pp Volkman JK, Barrett SM, Blackburn SI, Mansoup MP, Siwes EL and Gelin F Microalgal biomaekers: A review of recent research developments. rg Geochem 29, Walne PL.98. In phytoflagellates. Elsevier, north Holland. Euglenoid flagellates pp Watanabe M and Suzuki T. 22. Involvement of reactive oxygen stress in damium-induced cellular damage in Euglena gracilis. Comp Biochem Physiol Part C 3, 49-5.

Mixotrophic 배양조건에서유글레나성장및지질변화 31 실험균주및배양방법 재료및방법 Euglena gracilis KMMCC-131 (KMMCC), Table 1 Hutner medium (Leticia et al., 1997). 121,.62 g/l 7 3, (U

Mixotrophic 배양조건에서유글레나성장및지질변화 31 실험균주및배양방법 재료및방법 Euglena gracilis KMMCC-131 (KMMCC), Table 1 Hutner medium (Leticia et al., 1997). 121,.62 g/l 7 3, (U 한수지 49(1), 3-37, 216 riginal Article Korean J Fish Aquat Sci 49(1),3-37,216 Mixotrophic 배양조건에따른 Euglena gracilis 의성장과지질에미치는영향 정우철 최종국 강창민 1 최병대 강석중 * 경상대학교해양식품생명의학과, 1 안전성평가연구소경남환경독성본부 Effect of Growth