Isolating, Selecting And Optimizing Culture Condition For The EPA-rich Marine Microalgae

In this study, phytoplankton net was used to collect water which derived from offshoresea, Zhejiang, in April to June,2012, and plate separation and water separation and so on wasutilized to separate marine microalgae. EPA (Eicosapentaenoic acid)-rich microalgae wasselected, and effects of the ecological factors (temperature, light intensity and salinity),nutrient factors (nitrogen, phosphorus, iron, silicon) and different growth phases on thegrowth, total lipids and fatty acid composition of rich-EPA strains were studied to optimizeculture condition for high EPA and PUFA (polyunsaturated fatty acid) yields and to providetheoretical basis for the mass producting and practicing. Main results are as follows:Ten species of marine microalgae were separated, and five strains that cultivated easilyand grew faster were selected. They were respectively Tropidoneis maxima, Amphoracoffeaeformis, Skeletonema.munzelii SM-1, Skeletonema.munzelii SM-2, Thalassiosira binata.They were cultured by using MAV medium, and the culture condition was water temperature17℃to25℃, salinity25, natural illumination, no air inflation. At the same time, growth rate,total lipids and fatty acid composition were determined. Finally, two rich-EPA strains, theywere respectively Skeletonema.munzelii SM-1(24.83%) and Skeletonema.munzelii SM-2(27.74%).Under the culture condition of pH8.05, light/darkness12h:12h, MAV medium, no airinflation, single factor containing temperature, light intensity and salinity was studied. Settingrespectively temperature10℃,15℃,20℃,25℃,30℃; light intensity20μ mol m-2s-1、40μmol m-2s-1、60μ mol m-2s-1、80μ mol m-2s-1、100μ mol m-2s-1、120μ mol m-2s-1; salinity10、15、20、25、30、35、40, the results showed that the optimum growth condition forSM-1and SM-2were respectively temperature20～25℃,20℃; light intensity60μ mol m-2s-1; salinity30. The optimum condition to accumulate total lipids for SM-1and SM-2wastemperature10℃; light intensity20μ mol m-2s-1; salinity15. The optimum condition toaccumulate PUFA for both temperature15℃; light intensity20μ mol m-2s-1; salinity15.Maximum level of PUFA for SM-1and SM-2were respectively46.92%,53.01%, and thehighest content of EPA respectively19.39%,21.31%. Under the culture condition of temperature25℃, light intensity60μ mol m-2s-1, salinity25, pH8.05, light/darkness12h:12h, air inflation, single factor was researched. In the research,we selected KNO3, NH4Cl,(NH4)2SO4, CO(NH2)2as nitrogen sources, and took FeC6H5O7,FeSO4, FeCl3as iron sources. This research indicated that KNO3and CO(NH2)2weresuitable nitrogen sources for SM-1and SM-2, and FeSO4was suitable iron source for both.Setting respectively nitrogen concentrations0mg/L、5mg/L、10mg/L、15mg/L、20mg/L、25mg/L、30mg/L, phosphorus concentrations0mg/L、0.5mg/L、1mg/L、1.5mg/L、2mg/L、2.5mg/L、3mg/L, iron concentrations0mg/L、0.1mg/L、0.25mg/L、0.5mg/L、1mg/L, silicon concentrations0mg/L、0.5mg/L、1mg/L、2mg/L、4mg/L, the resultsshowed that the optimum growth condition for SM-1and SM-2were respectively nitrogenconcentration20mg/L to25mg/L,25mg/L to30mg/L; phosphorus concentration1.5mg/L,2mg/L; silicon concentration0.5mg/L, silicon concentration2mg/L to4mg/L,1mg/L. ForSM-1and SM-2, the nutritional conditions to accumulate total lipids were respectivelynitrogen stress; phosphorus deficiency; lack of silicon; and iron concentration0.25mg/L,1mg/L, and the nutritional conditions to accumulate PUFA were silicon deficiency, nitrogenconcentration10mg/L,30mg/L; phosphorus concentration3mg/L,2.5mg/L to3mg/L; ironconcentration0.25mg/L,0.5mg/L. The highest content of PUFA and EPA were respectively49.11%,50.48%, meanwhile the maximum level of EPA were respectively17.12%,18.39%.Under the culture condition of temperature25℃, light intensity60μ mol m-2s-1, salinity25, light/darkness12h:12h, pH8.05, MAV medium, no air inflation, early phase of index, latephase of index and stationary phase of SM-1and SM-2were respectively3rd day,5th dayand7th day. The growth phase to accumulate total lipids for both was late phase of index,meanwhile the growth phase to accumulate to accumulate EPA and PUFA was early phase ofindex. The maximum level of PUFA were respectively17.40%,18.43%, and the highestcontent of PUFA were respectively32.71%,34.84%.