High density culture of Aurantiochytrium limacinum SR21 for DHA production with crude glycerol from biodiesel industry Zhanyou Chi, Craig Frear, Shulin Chen Department of Biological Systems Engineering, Washington State University World Congress on Industrial Biotechnology & Bioprocessing, 6/29/10

-3 Polyunsaturated Fatty Acids ---- Chemical Structure EPA (20:5 -3) DHA (22:6 -3)

The Health Benefit of DHA (C22:6, -3) Component of the photoreceptor cells of infant retinas Involved in the development of infant brain Supplement of DHA in infant formula is strongly recommended by WHO Reduced risk of age related neurological disorders, such as Alzheimer’s and dementia

Example Products with -3 Fatty Acids

Functional Food Development with ω-3 Fatty Acids Infant Formula Dairy drinks Cheese Beverage (ex dairy) Snacks/candy/cookies/crackers Bread Cereal/Breakfast food Yogurt

Sources of -3 Fatty Acids ----- algae and fish oil Phototrophic Algae Heterotrophic Algae Original Producers !

-3 Fatty Acids and Aquaculture 1 million tons of fish oil produced globally per year, 70~80% of them used in aquaculture Aquaculture feed demand increases and ocean fishery resources exhausting, using fish meal to support aquaculture growth becomes not sustainable Organic fish movement requires an -3 fatty acids source that is not originated from fish meal Production of -3 fatty acids from algae culture is the best choice

Development of Biodiesel Cited from: http://www.biodiesel.org/pdf_files/fuelfactsheets/Production_Graph_Slide.pdf

Utilization of Crude Glycerol from Biodiesel Industry Oil Crops Oil Biodiesel 1 Gallon 1 Lbs Crude Glycerol

Utilization of Crude Glycerol Category Products Bio-Polymer Feedstock 1,3-Propanedieol Propylene glycol Dichloropropanol (DCP) Acrolein Succinic acids Glycerol carbonate Biofuel Butanol Ethanol Hydrogen Other Dihydroxyacetone Fuel additives High Value Product ω-3 Fatty Acids

Two-Stage Culture Strategy Development Traditional fed-batch culture can’t enhance dry cell weight No possible inhibitor determined in the fermentation broth Biomass Concentration (g/L) = Cell density (cells/L) × Cell body weight (g/cell) Hypothesis: either not enough cell produced in the cell reproduction stage, or the cell produced didn’t accumulate enough lipid

“Shifting” Culture Strategy Development New Produced Cells Lipid Accumulated Cells

The Reproduction of Aurantiochytrium Vegetative Cells Zoosporangium Zoospore Amoeboid cells Cell Cluster Picture cited from Honda et al, 1998

Effect of D.O. Level Cell reproduction requests high D.O. level Lipid accumulation requests low D.O. level

Hypothesized Mechanism of Oxygen Effect to Fatty Acid Synthesis Cytosol Mitochondrion Glucose Pyruvate Pyruvate Oxidative Phosphorylation NADPH NADP+ Acetyl -CoA Oxalo-acetate Malate NADH CO2 Lipid Synthesis Oxaloacetate Citrate Acetyl-CoA TCA cycle ATP + CoA Citrate Isocitrate 2-Oxo-glutarate

D.O. Shifting Culture Culture method Dry cell weight (g/L) Cell density (106 cells /ml) Cell body weight (mg/106 cells) DHA yield (g/L) DHA content (%) Fermentor to flask 36.2 162 0.22 6.35 17.5 Flask 32.1 67 0.48 5.97 18.6 The improved cell density produced compromised biomass concentration than expected Culture condition for cell size growth stage need to be improved

Effect of C/N ratio in Feeding Medium The glycerol daily feeding rate was 25% of the amount in original medium Different nitrogen daily feeding rates were investigated

High Density Process Development ---- Feeding Strategy

Oxygen Limitation at Lipid Accumulation Stage in the High Density Culture

Effect of C/N Ratio to Cell Density Growth

Effect of Temperature to Cell Density Growth

Effect of Feeding to Cell Density Growth

Optimized Parameters in the Three-Stage Fed-Batch Culture Process Hours 0~24 24~80 80~120 DO 50~20% 5~3% 0.5~0% Temperature (°C) 30 20 Carbon source concentration (g/L) 30~50 Nitrogen concentration (g/L) 1.5~1.0 1.0~0.5 0.5~0

High Density and High Productivity Three-Stage Fed-Batch Culture Process

Summary Optimized condition for cell reproduction stage culture improved cell density from 59*106 cells /ml to 683*106 cells /ml Fed batch culture improved the dry cell weight from 25 g/L to 145 g/L, contains with 16.2 g/L DHA. Crude glycerol from biodiesel industry was used as the major carbon source for the fed batch culture Patent filed

Integrated Biodiesel and -3 Production Crude Glycerol Fatty Acids Enriched Algae Biomass Oil Feedstock Trans-esterification Extracted Oil FAME or FAEE Biodiesel -3 Fatty Acids

Extended Application of Two-stage Strategy — Heterotrophic Culture for Algae Seed Cell Production Phototrophic culture use light as energy source and CO2 as carbon source Heterotrophic culture use organic carbon as both energy source and carbon source

Heterotrophic Algae Seed Production and its Trophism Shifting

Integrated Heterotrophic and Phototrophic Algae Culture System Winter Time Summer Time

acknowledgements