Cultivation And Enzymolysis For Lipid Production Of Nannochloropis Oculata Rich In EPA

Nannochloropis oculata (N. oculata) is one of the most potential raw materials for biodiesel production because of its high rate of growth and high contents of lipid and EPA. The work studied the effects of the nutritional factors-N, EM agents and environmental factor-light on the growth, lipid accumulation and fatty acid of composition of N. oculata by single factor experimental method. Because N. oculata’s cell is small, cell wall is tenacity and has complicated structure, the broken of cell wall is difficult. It is important for lipid extraction to choose a suitable method to break the cell wall of algae. The work also studied the process of enzymolysis for lipid extraction, and optimized the process conditions.(1) CH3COONH4was better for N. oculata than NaNO3, CO(NH2)2and NH4Cl, could promote the growth and lipid accumulation. The increase of the nitrogen concentration was beneficial for the growth and fatty acids desaturation, against lipid accumulation. The biomass, lipid yield and EPA content of N. oculata reached the maximum, and was2.215g/L,58.2mg/(L·d) and12.8%respectively, when the concentration of CH3COONH4was408mg/L. (2) EM agents not only promoted the growth and shorten the culture cycle from14days to12days, but also increased lipid accumulation and EPA synthesis. The biomass, lipid and EPA content of N. oculata increased then decreased with the increase of the inoculation quantity of EM agents. When6mL EM agents was inoculed into1L culture, the biomass, lipid and EPA content of N. oculata reached the maximum, and was2.5709g/L,40.53%and16.74%respectively.(3) Effects of light quality, such as white, red, blue and green, on the growth and lipid accumulation of N. oculata were studied. The results showed that N. oculata preferred red light to white, blue and green light. The biomass, lipid and EPA content of N. oculata increased then decreased with the increase of the light intensity. The biomass, lipid and EPA content of N. oculata was2.2889g/L,39.55%and20.04%respectively when the intensity of red light was2987Lux.(4) The composition of N. oculata’s cell wall was42.22%cellulose,11.25%hemicellulose,32.32%pectin and14.21%other substances, so cellulase was chosen to degrade cell walls of N. oculata to extract algae lipid and EPA. The enzymolysis catalyzed by cellulase was used to break algal cell wall for lipid and EPA extraction, and was compared to the acidolysis catalyzed by HC1and the solvent extraction. The lipid extraction yield was38.60%by enzymolysis, which was near to the acidolysis, and outclass the solvent extraction. The EPA extraction yield was9.23%by enzymolysis, and was1.76times as much as the acidolysis and2.43times as the solvent extraction. Furthermore, the conditions of enzymolysis was mild, enzymolysis was an appropriate extraction method for lipid and EPA.(5) The effects of enzyme dosage, reaction time, pH and temperature on the broken of cell wall were investigated, and the suitable scope of enzymatic hydrolysis were preliminary determined by single factor experimental method:enzyme dosage1.0-2.0g/(kg dry power), reaction time1.5-2.5h, temperature35～45℃and pH3.0-5.0.(6) Response surface method was used to analyze the effects of enzyme dosage, reaction time, pH and temperature on the extraction yield of the lipid and EPA. The sequences of factors influencing the lipid and EPA extraction yield were temperature>pH>enzyme dosage>recation time and temperature>reaction time>pH>enzyme dosage. Furthermore, the process conditions were optimized by Response Surface. The highest lipid extraction yield was achieved at the condtions:enzyme dosage1.59g/(kg dry power), reaction time2.11h, temperature39.3℃and pH4.17. The predicted lipid extraction yield was38.94%, the experimental value was38.60%±0.15%under this condition, the difference between the two values was very small, so the model was reliable. But the highest EPA extraction yield was achieved when enzyme dosage, reaction time, temperature and pH were1.60g/(kg dry power),1.86h,37.9℃and4.10respectively. The predicted EPA extraction yield was9.62%, the experimental value was 9.59%±0.12%under this condition, the predicted value was near to the experimental value, thus the model equations could analysis and forecast for experimental result replaced of the real points of the experiment.