NTH Ha et al., 6th ACM meeting – Hanoi 2009 39 ISOLATION AND SELECTION OF TETRASELMIS STRAINS FOR FEEDING GEO-DUCK LARVAE AT VANDON, QUANG NINH Nguyen Thi Hoai Ha 1 , Le Thi Phuong Hoa 2 , Pham Thi Bich Dao 1 , Lưu Thị Thùy Giang 1 , Tran Thi Diep 1 . 1 Institute of Microbiology and Biotechnology, Vietnam National University, Hanoi 2 Faculty of Biology, Hanoi National University of Education Abstract Tetraselmis species are widely used as food for aquatic animals such crustaceans, bivalves and fish especially in their larvae stage. Three Tetraselmis strains were successfully isolated from the shrimp farming at Vandon, Quangninh. Among them, Tetraselmis strain T1 showed best growth. Medium f/2 was selected as best medium for its growth. Fatty acid profile of Tetraselmis strain T1 consisted of C 16 and C 18 acids as most abundant, which is typical for most green algae. Essential fatty acids, -linolenic acid and linoleic acid accounted for 34.2% of total fatty acids, half of total unsaturated fatty acids. Eicosapentaenoic acid (EPA) was also in high concentration. The result suggested the high quality of as food for aquatic animals like geo-duck. On the way to apply Tetraselmis strain T1 in feeding geo-duck, outline for its biomass production was structured. Key words: microalage, Tetraselmis, geo-duck, EPA. 1. Introduction Microalgae are the base of the entire aquatic food chain which has an important role in the aquaculture. They are major source of nutrients for all stages of bivalves, for larval stages of crustaceans and fish, and as food for zooplankton which are fed to late larval and juvenile fish and crustaceans [10, 11]. Among them, many Tetraselmis species are widely used especially at the larval stage of aquatic animals [11]. Geo-duck is a bivalve species, which inhabits commonly in Quang Ninh and is served as a specialty with high nutrition [3]. However, geo-duck resource is decreasing seriously. In a NTH Ha et al., 6th ACM meeting – Hanoi 2009 40 way to find suitable food for sustainable development and conservation of this resource we carried out the research “Isolation and selection of Tetraselmis strains for feeding Geo-Duck larvae at Vandon, Quangninh”. 2. Materials and methods 2.1. Isolation and classification of Tetraselmis strains Seawater was collected from the shrimp farming at Vandon, Quangninh. Tetraselmis strains were isolated based on their morphological properties using micropipettes [5,9]. Algal strains were grown at room temperature and illuminated under neon light (Philips daylight tubes) on 10: 14 h light: dark cycles. 2.2. Selection conditions and nutrition component analysis Media f/2 [5, 13], ASW (artificial sea water) and ESM (modified Erd-Schreiber's and Schreiber's medium) were used to select culture medium for the growth and development of each strain. Cells were cultured in 250-ml conical flask and harvested every two days. Cell density was determined with a Neubauer haemocytometer in three replicates. In order to survey the influence of temperature on photosynthetic system of Tetraselmis strains, florescence indexes were measured using Plant Efficiency Analyser (PEA), Hansatech Intruments Ltd., Englanh, from which Fv/Fm ratio was calculated [1,2]. Fatty acid composition was determined according to Krienitz et al. [8] by gas chromatography (Finnigan Trace GC) using an ultra-column BPX70. Fatty acids were identified by comparing retention times with those of a calibration standard solution. 3. Results and discussion 3.1. Isolation of Tetraselmis strains Tetraselmis strains were isolated from the seawater at the shrimp farming in Vandon, Quangninh and signified as T1, T2, T3. They were grown in non-aerated f/2 medium. Cell density was determined every 2 days until 6 days of cultivation. Data were shown in Figure 1. Among selected Tetraselmis species, T1 showed best growth, especially at 6-day culture and would be used in following experiments. NTH Ha et al., 6th ACM meeting – Hanoi 2009 41 0 20 40 60 80 100 120 T1 T2 T3 Time (days) Cell density (x104/ml) culture day 2 culture day 4 culture day 6 Figure 1. The growth of three Tetraselmis strains T1, T2, T3 3.2. Taxanomic properties of Tetraselmis T1 species Observation on the morphology of Tetraselmis T1 species (Fig. 2) showed Tetraselmis cells are usually solitary, free-swimming, thecate cells with four flagella and approximately 12-20 micrometers long. The four flagella are slightly shorter than the cell body. Each cell contains a single pyrenoid, located more or less in the middle of the cell. Division: Prasinophyta Class: Prasinophyceae Order: Dunalliellales Family: Polyblepharidaceae Genus: Tetraselmis Figure 2. Morphology of Tetraselmis strain T1 3.3. Selection of culture medium Tetraselmis strains are photoautotrophic microorganisms. However, the biophysical properties of microalgae may vary with respect to the alteration of environment [10,11]. Therefore, the selection of suitable culture medium is essential for the optimal development of the microalgae. In this study, f/2, ESM and ASW medium were used for Tetraselmis strain T1. NTH Ha et al., 6th ACM meeting – Hanoi 2009 42 Table 1. Cell density of Tetraselmis T1 grown on different media Culture time (days) Cell density (10 4 / ml) f/2 medium ESM medium ASW medium 0 12,5 12,5 12,5 2 44 37,5 22 4 75 75 31 6 100 87,5 12,5 8 122 115 6,3 10 100 107 0 12 89 87 0 12.5 44 75 100 122 100 89 12.5 37.5 75 87.5 115 107 87 12.5 22 31 12.5 6.3 0 0 0 20 40 60 80 100 120 140 0 2 4 6 8 10 12 Time (days) Cell density (x10 4 cell/ml) F/2 medium ESM medium ASW medium Figure 3. The growth of Tetraselmis T1 on different media Data showed the population density of Tetraselmis T1 increase steadily on f/2 medium and reach the maximum (122x10 4 cells/ml) on the eighth day of culture. In the meantime, the value was lower in case of ESM medium and much lower for ASW medium (Table 1, Fig. 3). It is suggested that f/2 is the most suitable for culturing Tetraselmis strain T1 and can be applied for biomass production in aquaculture. 3.4. The effect of temperature on photosystem II of Tetraselmis T1 Temperature has effects on the structure and function of photosystems in microalgae and higher plants, thereby on the photosynthesis productivity and their growth. In this study, we investigated the change of Fv/Fm ratio according to the temperature (Fig. 4). The data suggested low heat-tolerance ability of Tetraselmis T1. Fv/Fm ratio was reduced immediately when the temperature increased 1 o C and more seriously when it is over 34 o C and especially 40 o C. However, Tetraselmis T1 microalgae grown on f/2 medium seemed to be most stable. NTH Ha et al., 6th ACM meeting – Hanoi 2009 43 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 25 26 28 30 32 34 36 38 40 42 44 46 Temperature (oC) Fv/Fm F2 ESM ASW Figure 4. Variation of Fv/Fm ratio related to temperature on different media 3.5. Analysis of fatty acid components of Tetraselmis T1 Lipid has its own specificity in each species of living organisms. Fatty acids are mostly located in the cell membrane, which is much enough to apply to taxonomic characterization. Furthermore, most animals cannot synthesize essential fatty acids (linoleic and linolenic acid) thus have to depend on the diet [4,6]. Fatty acid components of Tetraselmis strain T1 were analysed. The fatty acid profile of Tetraselmis strain T1 (Table 2) consisted of huge range of fatty acids from 12C to 23C with C 16 and C 18 being most abundant. This distribution is typical of most green algae [6, 10]. The unsaturated fatty acids accounted for 68.5%, among which C 18 polyunsaturated fatty acids (PUFA) were the major. Alpha-linolenic acid was found in highest concentration (27.1%) and higher Tetraselmis sp. (-linolenic acid 16.7%) which have been reported by Pratoomyot [7]. Linoleic acid and - linoleic acid are essential fatty acids that can be converted into long-chain PUFA in algae, which many marine animals showed limited ability [8]. Furthermore, oleic acid and eicosapentaenoic acid (EPA) have remarkably high content 13.2% and 9.3%, respectively which was comparable to previous reports for which was comparable to previous reports for T. suecica with small amount of NTH Ha et al., 6th ACM meeting – Hanoi 2009 44 EPA (4.5%)[15]. EPA of the membrane phospholipids is a precursor in prostaglandin synthesis, which leads to synthesis of a number of other tissue hormones [6,8]. However, this microalgae was deficient of DHA which was similar to T. chui and T. suecica but contrasts to Tetraselmis spp. [7]. This result suggested high quality of Tetraselmis T1, which can be applied in aquatic food webs such as for feeding geo-duck. Table 2. Percentage composition of fatty acids in Tetraselmis strain T1 Fatty acids Quantities (%) C12:0 Lauric Acid 0.8 C15:0 Pentadecanoic Acid 1.9 C16:0 Palmitic Acid 10.8 C16:1 Palmitoleic Acid 1.0 C17:0 Heptadecanoic Acid 0.8 C17:1 Heptadecenoic Acid 0.3 C18:0 Stearic Acid 0.6 C18:1 Oleic Acid 13.2 C:18:2 n6 Linoleic Acid 7.1 C:18:3 n3 -linolenic Acid 27.1 C:20:1 Eicosenoic Acid 2.1 C:20:2 n6 Eicosadienoic Acid 6.1 C:20:4 n6 Arachidonic acid (ARA or AA) 0.4 C:20:5 n3 Eicosapentaenoic Acid (EPA) 9.3 C:22:2 Docosadienoic Acid 0.9 C:22:6 n3 DocosahexaenoicAcid (DHA) 0 C:23 Tricosanoic Acid 0.9 NTH Ha et al., 6th ACM meeting – Hanoi 2009 45 3.6. Biomass collecting of Tetraselmis strain T1 In order to collect biomass from Tetraselmis strain T1 for feeding geoduck larvae 8 day age, Tetraselmis strain T1 was cultured in f/2 medium, grown at room temperature and illuminated under neon light (Philips daylight tubes) 4000 - 5000 Lux as the following scheme with illustrated pictures. Microalgae volume was used depending on geo-duck larvae density in the container. At Van Don, Quang Ninh, we have been fed geoduck larvae with 120 – 180 liters microalage with cell density 12.2-12.5 10 5 cells/ml for one tank about 2.510 5 – 16.610 5 geo duck larvae/m 3 per day . Stock culture Cell density 2.25-3.310 5 cells/ml 2-6 litre flask After 5-7 days, cell density 5,610 5 cells/ml 30–60 litre plastic bag Cell density 8.8- 12.2  10 5 cells/ml after 4-6 days 160 litre tank After 4-6 days, cell density 12.2 – 12.5  10 5 cells/ml 10 litre container After 2-3 days, cell density 8.5-1110 5 cells/ml NTH Ha et al., 6th ACM meeting – Hanoi 2009 46 4. Conclusions Three Tetraselmis strains were successfully isolated from the shrimp farming at Vandon, Quangninh and annotated as T1, T2, T3. Among them, Tetraselmis strain T1 showed best growth and well cultured in f/2 medium. Fatty acid composition of Tetraselmis strain T1 was typical of most green algae with high content of C 16 and C 18 acids. Approximately 68.5% of total fatty acids were unsaturated, among which -linolenic acid and linoleic acid were major components. There was also high content of EPA (9.3%), a precursor in prostaglandin synthesis. The data suggested the signification of using Tetraselmis strain T1 in feeding geo- duck as high quality food. The outline of biomass production of Tetraselmis strain T1 for aquaculture of geo-duck larvae in Van Don, Quang Ninh was established. Acknowledgements The authors wish to thank the Asia Research Center, Vietnam National University, Hanoi for suport. Many thanks also to go the Center of Aquaculture Breeding farm, Vandon district for supplying marine microalgae. References 1. Dang Dinh Kim, Dang Hoang Phuoc Hien. Microalgae biotechnology, (1999), Agricultural publishing house, 203. 2. 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Microbiol. 1962.pp 229-239. 13. R.R.L. Guillard, Culture of phytoplankton for feeding marine invertebrates In Smith W.L. and Chanley M.H (Eds.) Culture of Marine Invertebrate Animals. Plenum Press, New York, USA. 1975. pp 26-60. 14. S.S. An, T. Friedl, E. Hegewald, “Phynogenetic relationships of Scenedesmus and Scenedesmus–like coccoid green algae as inferred from ITS–2 rDNA sequence comparisons”, Plant Biology, Vol. 1, No 4, (1999), pp. 418 – 428. 15. Volkman et al., 1991. Fatty acids from Microalgae of the genus Pavlova. Phytochem., pp: 1855-1859. . food for sustainable development and conservation of this resource we carried out the research Isolation and selection of Tetraselmis strains for feeding Geo-Duck larvae at Vandon, Quangninh” Quangninh”. 2. Materials and methods 2.1. Isolation and classification of Tetraselmis strains Seawater was collected from the shrimp farming at Vandon, Quangninh. Tetraselmis strains were isolated based. Ha et al., 6th ACM meeting – Hanoi 2009 39 ISOLATION AND SELECTION OF TETRASELMIS STRAINS FOR FEEDING GEO-DUCK LARVAE AT VANDON, QUANG NINH Nguyen Thi Hoai Ha 1 , Le Thi Phuong Hoa 2 ,