| Nannochloropsis oculata, which is rich in eicosapentaenoic acid（EPA）, has high healthy and medicinal value. This study is aimed at improving the relative EPA content of fatty acid products from N. oculata by optimizing cultivation strategy of N. oculata and extraction and purification methods.Based on the light intensity experiment, the specific growth rate of N. oculata increased with ascending light intensity under this experimental conditions. The specific growth rate, which increased from 0.25 d-1 to 0.54 d-1, increased by 116% when the light intensity increased from 1000 lux to 9000 lux. The increasing light intensity reduced the contents of chlorophyll a and enhanced the accumulation of total oil and carbohydrate, but had little effects on total protein content of N. oculata. The relative content of saturated fatty acids increased with the increasing light intensity, while EPA content and its yield decreased from 25.5% to 13.1% and 5.1 mg·L-1 to 2.7 mg·L-1 respectively.Through the experiment of nitrogen sources, ammonium acetate was found to be the best for the growth of N. oculata, and its biomass could reach 0.41 g·L-1, which was higher than sodium nitrate, ammonium chloride, urea and ammonium nitrate group by 17.1%, 5.1%, 7.9% and 10.8%. The cellular contents of chlorophyll a were affected by nitrogen sources. The cellular content of chlorophyll a of the nitrate group was higher than ammonium chloride, ammonium acetate, urea and ammonium nitrate group by 23.1%, 17.1%, 20% and 11.6%. The cellular contents of total oil and protein of ammonium acetate group were higher than other groups, and nitrogen source had little effects on the cellular content of total carbohydrate. Nitrate group has the highest relative EPA content, being 27.1%, while ammonium acetate group has the highest EPA yield, being 15.6 mg·L-1.Through the two stages of nitrogen source feeding experiments, EPA yield and relative EPA content were improved simultaneously by adding ammonium acetate in the batch cultivation. The optimum feeding time of ammonium acetate was the 11 th day. The EPA yield and its relative content of N. oculata reached 26.8 mg·L-1 and 24.3% respectively, which were higher than the control group by 22.4% and 7.5% respectively.By comparing the effects of 3 kinds of fatty acid extraction methods on the yield rate of EPA and fatty acid composition of microalgae, it was found that the yield rate of EPA could reach 39.4% by direct saponification method of non-grinding powder of N. oculata, and was higher than the direct saponification extraction of grinding powder of N. oculata, and B-D extraction method and supercritical extraction method of grinding and non-grinding powder of N. oculata by 10.4%、6.8%、82.4%、275.2% and 579.3%.The intermediate responses of three elements were decided by using single factor experiment: the ratio of urea to fatty acid was 3, the ratio of 95% ethanol to urea was 5, urea inclusion temperature was 0 oC. 17 experimental groups were generated by using Design-expert 8.0.6. By using the model prediction analysis, the optimum conditions for urea inclusion was generated by setting the ratio of urea to fatty acid 2.3, 95% ethanol to urea 4.7, and temperature 1.1 oC. The predicted relative EPA content was 57.0%, and the predicted fatty acid yield was 79.8%. The test value of relative EPA content and fatty acid yield was 55.6% and 80.4% under the optimum condition, which were in consistent with the predicted values, proving that the model could better reflect the urea purification conditions of EPA. |
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