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A research study on the ultrastructural investigation of lipid formation and docosahexaenoic acid (dha) production in isochrysis sp. Marine microalgae. The study explores the effects of culture media and conditions on pufa yields and total fatty acid contents. The presence of lipid bodies in isochrysis sp. Is confirmed through staining techniques and microscopic observations.
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*Corresponding author. Fax: 886-2-23626455; E-mail: m046@ccms.ntu.edu.tw
(Received May 22, 2000; Accepted December 12, 2000)
Abstract. This study investigates methods for extracting lipids from microalgae and analyzes the effects of culture media as well as culture conditions on PUFA yields and total fatty acid contents. Experimental results of an optimal culturing of Isochrysis spp. were based on a 3.2% salinity culture medium. These microalgae were cultured in a 1- 2 L Roux’s flat-flask and a 5 L jar fermentor. The optimum culture temperature and initial pH for DHA production were 25°C and 8.0, respectively. Pigments included chlorophylls a and c. The DHA yield increased with cultivation time until the eighth day. Optimum DHA amounts in the cells were reached under aeration with 10% CO 2 and with continuous illumination of 10 klux. The biomass dry weight reached 4 g per liter of culture, and the DHA produc- tion reached 16 mg per liter of culture. Lipid bodies in Isochrysis spp. and related genera were observed during culture by light and transmission electron microscopy; 0.5~3.0 μm sized lipid bodies were confirmed by staining with Sudan Black B in cells from log stage to stationary stage cultures. These results demonstrated that DHA- containing lipid bodies in cells can be produced and accumulated in marine Isochrysis spp.
Keywords: Docosahexaenoic acid; Isochrysis sp.; Lipid formation; Polyunsaturated fatty acids (PUFA); Ultrastructure.
Cell Growth
Light and Electron Microscopies
Total Lipid and Fatty Acid Analysis
Results
Table 1. The fatty acid composition of several marine microalgae (% total fatty acids).
Fatty Pavlova Isochrysis sp. Isochrysis sp. Isochrysis Isochrysis Pavlova lutheri Nannochloropsis Chlorella
acid salina CCMP 463 CCMP 1324 galbana
galbana CCMP 1325 oculata
minutussima TK1 TK2 UTEX
14:0 10.1±0.2 10.4±0.9 10.2±0.5 17.5±1.0 16.3±0.9 10.3±0.5 5.1±0.4 4.5±0. 16:0 23.4±0.4 14.9±0.7 17.6±0.6 14.3±0.4 12.9±0.7 20.8±1.2 32.1±1.4 33.9±1. 16:1n-7 6.2±0.1 4.5±0.2 3.9±0.1 6.3±0.5 4.0±0.1 18.4±0.4 24.9±1.7 23.2±1. 18:0 0.9±0.1 N.D. N.D. N.D. N.D. 0.4±0.1 2.7±0.2 2.9±0. 18:1n-9 16.9±0.3 29.8±1.2 32.0±1.4 15.1±0.7 28.1±1.0 3.3±0.5 16.5±0.9 20.4±1. 18:2n-6 7.8±0.4 5.7±0.4 4.1±0.3 8.8±0.4 3.0±0.1 1.9±0.1 1.9±0.3 3.4±0. 18:3n-3 3.1±0.1 6.4±0.5 6.4±0.3 8.2±0.3 5.5±0.2 1.5±0.2 N.D. N.D. 18:4n-3 5.9±0.1 17.5±0.9 15.1±0.6 24.9±1.4 18.9±0.3 6.8±0.5 N.D. N.D. 20:4n-6 1.6±0.1 N.D. N.D. N.D. N.D. N.D. 2.8±0.2 1.9±0. 20:5n-3 11.8±0.3 N.D. N.D. N.D. N.D. 21.0±0.5 9.4±0.7 8.7±0. 22:6n-3 4.4±0.4 10.7±0.5 10.9±0.3 8.2±0.6 11.1±0.4 6.2±0.3 N.D. N.D. ΣUn-3 25.2 34.6 32.3 41.4 31.8 35.5 12.2 8. ΣUn-6 9.4 5.7 4.1 8.8 3.0 1.9 4.7 5. n-3/n-6 2.7 6.1 7.9 4.7 10.6 18.7 2.6 1.
N.D. = None detected.
Table 2. Influence of different salinities on the fatty acid composition of Isochrysis sp. CCMP 1324 (% total fatty acids).
Fatty acids
NaCl conc
0.8% 1.6% 2.4% 3.2%
14:0 18.4±0.9 18.6±0.9 16.9±1.2 16.3±0. 16:0 15.5±0.7 13.4±0.7 13.4±0.7 12.9±0. 16:1n-7 5.6±0.3 5.5±0.4 4.3±0.2 4.0±0. 18:0 N.D. N.D. N.D. N.D. 18:1n-9 27.7±1.1 27.9±1.7 28.0±1.4 28.1±1. 18:2n-6 4.3±0.2 6.2±0.7 5.0±0.4 3.0±0. 18:3n-3 5.1±0.2 4.8±0.2 4.9±0.3 5.5±0. 18:4n-3 14.9±0.9 14.6±0.7 17.1±0.8 18.9±1. 20:5n-3 N.D. N.D. N.D. N.D. 22:6n-3 9.4±0.9 8.9±0.7 10.5±0.9 11.2±0. Total n-3 29.4 28.4 32.4 35. PUFA 33.6 34.9 37.4 38.
N.D. = None detected.
Figure 2. Transmission electron micrographs of Isochrysis sp. CCMP 1324, showing the lipid body (arrow) formation in chloro- plast (A~C), their size from small to large and finally rounded in spherical form (D) at early log phase. Scale bar = 200 nm.
Figure 3. Transmission electron micrographs of Isochrysis sp. CCMP 1324 vegetative cell. After the fourth day of growth, no oil drop was observed (A), but oil droplets (arrows) could be observed in the stationary phase (6~11th day) (B~D). Scale bar = 500 nm.
Table 3. Variation in fatty acid composition of Isochrysis sp. CCMP 1324 in different sodium acetate concentration (% total fatty acids).
Fatty acids
CH 3 COONa
10 mM 20 mM 30 mM 40 mM 50 mM 14:0 17.8±0.9 16.5±0. 8 16.4±0.4 15.9±0.7 16.9±0. 16:0 12.8±0.6 11.9±0.5 12.0±0.4 11.7±0.4 12.7±0. 16:1n-7 4.7±0.2 4.5±0.2 3.7±0.1 4.6±0.1 4.8±0. 18:0 N.D. N.D. N.D. N.D. N.D. 18:1n-9 30.7±1.2 31.8±1.4 32.9±1.2 32.4±1.5 33.5±1. 18:2n-6 5.7±0.2 4.8±0.1 4.7±0.2 4.9±0.2 3.8±0. 18:3n-3 5.1±0.3 4.4±0.2 4.7±0.1 4.7±0.4 3.9±0. 18:4n-3 12.8±0.5 13.1±0.4 13.6±0.7 13.0±0.5 12.7±0. 20:5n-3 N.D. N.D. N.D. N.D. N.D. 22:6n-3 10.3±0.6 13.1±0.5 11.9±0.5 12.7±0.8 12.1±0. Total n-3 28.3 30.8 30.2 30.4 28. PUFA 34.0 35.4 34.9 35.3 32. N.D. = None detected.
Acknowledgements. We are especially grateful to Ms. Ji, S. J. for technical assistance. The material in this paper is part of the dissertation submitted to the National Taiwan University, Taipei, Taiwan, in partial fulfillment of the requirements for the degree of Doctor in Agricultural Chemistry.
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