1. 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

2. -3 Polyunsaturated Fatty Acids ---- Chemical Structure
EPA (20:5 -3)
DHA (22:6 -3)

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

4. Example Products with -3 Fatty Acids

5. 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

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

7. -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

8. Development of Biodiesel
Cited from:

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

10. 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

11. 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

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

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

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

15. 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

16. 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

17. 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

18. High Density Process Development ---- Feeding Strategy

19. Oxygen Limitation at Lipid Accumulation Stage in the High Density Culture

20. Effect of C/N Ratio to Cell Density Growth

21. Effect of Temperature to Cell Density Growth

22. Effect of Feeding to Cell Density Growth

23. 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

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

25. 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

26. 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

27. 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

28. Heterotrophic Algae Seed Production and its Trophism Shifting

29. Integrated Heterotrophic and Phototrophic Algae Culture System
Winter Time
Summer Time

30. acknowledgements