Study On The Construction Of Heterotrophic And High-Containing EPA/DHA Microalgae By Cell Fusion Technique

Polyunsaturated fatty acids(PUFAs), notably eicosapentaenoic acid(EPA) and docosahexaenoic acid(DHA), have been reported to have many beneficial physiological activities. These PUFAs are considered to be essential to many marine animals such as fish, brine shrimp and shellfish. The use of EPA and DHA for reducing the risk of heart disease and atherosclerosis, as well as the treatment of other diseases including cancer, inflammatory problem, rheumatoid, arthritis asthma and diabetes, has shown encouraging results. In order to obtain high-quality EPA/DHA, study on the construction of heterotrophic and high-containing EPA/DHA microalgae by cell fusion technique was made.The present studies report the methods and results.1 Selection of original species of marine microalgae In order to prepare material for cell fusion, growth rate, total lipid content, EPA/DHA composition and contents of 11 species of marine microalgae [Dicrateria inornate, Isochrysis galbana, Pavlova viridis (phototrophic microalgae), Pyramimonas sp., Tetraselmis sp. (mixotrophic microalgae), Nannochloropsis oculata, Platymonas sp., Chlorella sp., Nitzschia closterium, Chaetoceros sp., Berkeleya rutilans], which often used in marine aquaculture, were investigated. The resultsare as follow. Tetraselmis sp. grows more quickly than other microalgae, its growth rate is 0.140g/(L·d); B. rutilans is in the second place, its growth rate is 0.074g/(L?d). Total lipids contents of D. inornate and I. galbana, which are higher than that of other microalgae, are 13.1% and 12.1 % of cell dried weight, respectively. The contents of EPA and DHA of P. viridis are 25% and 6%, respectively. The content of EPA of Chlorella sp. is 28% and that of B. rutilans is 19%, and their contents of DHA are all not determinated. As mentioned above, Tetraselmis sp. and P. viridis are selected as the original species for cell fusion.2 Product i on and regeneration of protoplasts of the original species of Tetraselmis sp. and P. viridisTetraselmis sp. was selected as the object for the determination of degradation conditions, because the cell wall of the other original species, P. viridis, is very weak and easy to produce protoplast. Effects of complex cell wall-lytic enzymes, growth phase, temperature and pH on the cell wall degradation of Tetraselmis sp. were studied in the second part of this paper. The results are as follow. Complex enzyme is the most efficient enzyme system for cell wall degradation. The composition and content of enzyme solution are cellulase R-10, 2.0%; macerozyme R-10, 1.5%; semicellulase, 0.5%. Cells of original species in logarithmic phase are degraded more easily. The optimal temperature and pH are 29 °C and 6.0, respectively. The enzymolysis curves of Tetraselmis sp. and P. viridis, and the regeneration ratio of Tetraselmis sp. were also investigated. The results show that after Tetraselmis sp. cells are incubated in complex enzyme solution about 100 minutes, 80% of cells are turned into protolasts, and for P. viridis, it needs 60 minutes. The regeneration ratio of protoplasts isolated from Tetraselmis sp. on solid regeneration medium is about 11.17%. Protoplasts for cell fusion would be produced under the...