1. Feedstocks Cull Potato  Underutilized agricultural biomass with low commercial value  Can provide both carbon and nitrogen sources for algae’s growth Biodiesel Waste Glycerol  Negative value by-product of biodiesel industry  Difficult to be purified, due to many impurities  However, it’s good carbon source for algae’s growth Converting them to high value DHA could provide a great potential market for them The Health Benefit of DHA (C22:6,  -3)  Component of the photoreceptor cells of infant retinas  Involved in the development of infant brains  Supplemental DHA in infant formula is strongly recommended by WHO  Reduced risk of age-related neurological disorders, such as Alzheimer’s and dementia Zhanyou Chi, Yan Liu, Bo Hu, Craig Frear, Shulin Chen Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164-6120 Production of DHA from High Cell Density Culture of Schizochytrium with a “Shift” Strategy (Picture from Martek Annual Report) Oil Crops Algal Fermentation Process Cow Stomach “Extractor” Cull Potatoes Omega-3 food Biodiesel Crude Glycerol Feed additives to fish Omega-3 Algal cells before shifting Algal biomass enriched with lipids Waste Water Nutraceuticals and foods with omega-3 (Picture from www.wikipedia.com) DHA Producing Alga Strain: Schizochytrium limacinum SR21  Heterotrophic algae growing on glucose and glycerol as carbon source  High lipids content in algae biomass (more than 50%)  High ratio of DHA to total fatty acids (more than 30%)  Fast growing Two-stage Growth of Schizochytrium  cell number increasing stage: cell reproduction and rapid cell number increase with little increase in size and weight of each cell  cell size increasing stage: cells stop reproduction but enlarge due to lipids accumulation Hypothesis Optimizing culture conditions of these two stages separately and using a “shifting strategy” will increase the production rate Omega-3 Used in Aquaculture  1 million tons of fish oil produced globally per year, 70~80% of it used in aquaculture as fish meal  Aquaculture feed demand increases while ocean fishery resources decline, using fish meal to support aquaculture growth becomes non-sustainable  Organic fish movement requires an omega-3 source that is not originated from fish meal  The omega-3 enriched in the algae biomass produced in this process will be a better source Potential DHA Market in U.S. $ ( millions) Infant Formula200 Dairy beverages820 Cheese500 Beverages (non-dairy)770 Snacks/candy/cookies/crackers625 Bread510 Cereal/Breakfast food465 Yogurt70 Other1,500 Total5,460 (UBS Global Life Sciences Conference, September 27, 2006) BACKGROUND RESULTS 1. Verification of the two-stage growth 2. High oxygen concentration culture in the reproduction stage 3. Oxygen consumption in the cell increasing stage5. Enhancing biomass production with fed batch culture 6. High cell density culture  The cell number stopped increasing after 24 hours, but the dry cell weight kept increasing  The only explanation is that the “body weight” of each cell was increasing  Therefore, to improve biomass production: 1. produce more cells in the first stage 2. grow bigger cells in the second stage  The specific oxygen uptake rate (SOUR) reached its maximum at 8th hr, and decreased to a very low level after 24th hour  Large amount of oxygen was consumed in the cell reproduction stage, but only a little oxygen was consumed in the lipid accumulation stage  Shifting strategy is necessary, in that it produce high cell density at first, and provide optimal condition for lipids accumulation  The bottle neck of the algae biomass production in previous study is low cell density produced in the cell reproduction stage  High oxygen concentration in the reproduction stage produced much more cells  Algae biomass production could be greatly enhanced with this high cell density 4. Biomass production with shift strategy  Culture the cell at high oxygen and nitrogen source concentration at high temperature, then shift the culture to low oxygen and nitrogen concentration at low temperature  Biomass production was greatly enhanced with shift strategy  The cell body weight suppose to be further enhanced, if feed more nutrients in the culture to the high cell density Shift time (hr) Dry cell weight (g/L) cell density (10 6 cells/ml) cell body weight (mg/10 6 cells) Control (no shifting) 21.5510.42 1824.61060.23 2425.31180.21 3029.31240.24 4037.91400.27 4836.21620.22  To obtain high biomass production with this high cell density culture, more nutrients and carbon source need to be supplemented in the shifted culture, to make the algae cells accumulate more lipids inside cells  With the feeding, the cell body weight was enhanced to 0.38 mg/10 6 cells, 56 g/L algae biomass was obtained  Culture with 360× 10 6 cells/ml at initial was conducted, 102.4 g/L algae biomass was obtained  DHA Production efficiency was greatly enhanced with this high cell density culture  This process is very promising to be industrialized ACKNOWLEDGMENTS This research is supported by the Washington State University IMPACT Center and the Washington State Potato Commission