Harvesting Algae to Form a Neutraceutical, Specifically Creating a Functional Food

1. Harvesting Algae to Form a Neutraceutical, Specifically Creating a Functional Food Team Alpha Travis Dallas Eric Graves Joaquin Martinez Chris McNinch Ramune Otterson Meskyte Charu Saini Crypthecodinium Cohnii

2. Algae Products • Alternate Energy Sources • Ethanol • Butanol • Transport fuels • Animal Protein Supplement • Docosahexaenoic acid (DHA) • Nutraceuticals • Dietary Supplements • Functional Food • Baby Formulas • Table Spread

3. Algae Acquisition Bio Reactor Fermentation Harvesting Drying Separation Processing Esterification Extraction Refining Food Industry Marketing

4. Algae Acquisition Purchase from suppliers GrowIn-House Less cost upfront Makes the process much simpler Unable to be sure of algae’s consistency and toxicity Will cost more over time Production not self-contained More expensive at first Energy costs of growing algae Will ultimately save money over time Self-contained process, from algae to product

5. Bioreactor Options • Open system • Inconsistency due to climate • Possible contamination • Closed system • Easier to control • Can keep out toxins • Bioreactors • Energy costs • Total control over the fermentation process

6. Fermentation Sugar or sunlight? That's one of the fundamental questions for companies trying to transform algae into transportation fuel or dietary supplements. Key points of fermentation Fermentation offers the most control Temperature, pressure, and other environmental conditions can be minutely controlled Fermentation offers flexibility: A row of vats can be used to make auto fuel, and then scrubbed and prepped for cooking up algae for cooking fuel. Fermentation processes, whether continuous or batch, offer high growth rates from small land areas and provide an ideal opportunity for direct sequestering carbon dioxide. Although fermentation overall does cost more than photosynthesis. As a result, it might make the most sense for higher-value oils and nutraceutical markets. Martek, a leader in this field, in fact ferments algae and sells it as a baby food additive, dietary supplements, energy bars, etc.

7. How fermentation works • From the simplest to complex, biological processes maybe classified as fermentation, elementary physiological processes, and the action of living organisms. • Fermentation is a biological reaction • typical fermentation reaction • Raw material goes into a reactor • Once in reactor mixing with microorganisms • only a small amount of catalytic microorganisms present in the reactor Thus, the conversion of the raw organic material into product is slow, i.e., the rate of reaction is very low • as the concentration, or number, of microorganisms increases, the reaction rate rises, producing more microorganisms and more products • In a typical fermentation reaction, the reaction rate reaches a maximum, there is very little raw organic material and a lot of product, so the rate eventually slows until there is no reaction.

8. AlphaAlgae Fermentation • We will start with Crypthecodinium Cohnii, a classification of algae that will produce our desired product. • The algae will be introduced into our bioreactor which contains a sugar solution of known concentration. • The fermentation process will be initiated via the reactor at certain temps, pressures, and concentrations. • DOCOSAHEXAENOIC ACID – a tryglyceride that is produced from the fermentation process, will be our main product. • 40% of the C-Cohnii algae will be converted to DHA SCO-T oil

9. Crypthecodinium cohnii • Heterotrophic micro algae with red pigments and cellulose cell walls, but no chloroplasts • Higher growth rate and DHA production in dark fermentation • Higher β-carotene production when exposed to sunlight • Produces no significant amount of PUFAs other than DHA • Growth rate is optimized in brackish water and is inhibited by high and low salinity • Aerobic organism that requires mixing with air for oxygen. • Growth is optimized at a pH close to neutral (6.6-7.2).

10. Fermentation Process • A complex medium may be formed to provide a nitrogen source such as yeast extract, meat extract, glutamic acid, waste molasses, and corn steep liquor. KNO3, NH4Cl, and urea may also be used as a nitrogen source. • Carbon sources used for C. cohnii fermentations have been glucose, acetic acid, ethanol, dextrose, sodium acetate, and carob pulp. • The medium must contain salts such as a sea salt at a concentration around 17.8 g/L. Growth inhibition occurs at salt concentrations above 50g/L and below 5g/L. Other inorganic salts may be used. • Growth is optimized for 30°C, although lipid and DHA production is optimized for 15°C. Fermentation will likely take place at ambient temperature. • The fermentation will be either a fed batch or a continuous process has a growth and a stationary phase required to optimize DHA production.

11. Algae Harvesting Methods • Disk Centrifuge • Used to concentrate the algae particles in the broth to facilitate drying • Shear stresses can cause cell damage • Flocculation • Used to precipitate and clump algae together to facilitate harvesting • Can be used concurrently with filtration or centrifugation • Micro-Filtration • C. cohnii diameter ~ 15μm, micro filtration uses pore sizes of .1-1 μm • Driven by pressure • Tangential flow filtration • Dissolved Air Flotation • Air is dissolved into water and causes the algae to float to the top of the broth so that it can be skimmed off.

12. Composition of Fatty Acids • The content of neutral lipids in the lipids is very high (more than 90% of total lipids) • The representative composition of fatty acids in the neutral lipids is as follows: • (n-3) DHA: 40-48%; • Palmitic acid: 30-38%; • (n-6) DPA: 8-13%; • (n-3) EPA: 0-1%; • ARA: 0-0.6%; • other fatty acids: 10-20%.

13. DHA from Algae • Fatty acids are composed of a long hydrocarbon chain and a terminal carboxylate group • Most fatty acids are unbranched and consist of an even number of carbon atoms (10-24) • Schematic representation of docosahexaenoic acid (DHA; ω-3 22:6) is shown below

14. Post-Process Composition

15. Extraction Methods • Expeller Press – mechanical method • Hexane Solvent – Distillation separates cyclo-hexane and oil mixture • Soxhlet Extraction - Repeated washing with an organic solvent such as hexane or petroleum ether, under reflux in a special glassware

16. Extraction Methods • Enzymatic Hydrolysis – a process in digestion in which macromolecules are split from food by the enzymatic addition of water (Carotenes can be separated) • Ultrasonic Cavitations - Waves are used to create cavitation bubbles then these bubbles collapse near the cell walls, it creates shock waves and liquid jets that causes those cells walls to break and release their contents into the solvent. • Cellular Decompression- Pectinase enzyme can be used to break down pectin (polysaccharide substrate) cell wall • Supercritical CO2 - Under pressure and CO2 is heated to the point that it has the properties of both a liquid and a gas, this liquified fluid then acts as the solvent in extracting the oil.

17. Companies Utilizing this Algal Technology • Algenol • Produces ethanol at a rate of 6,000 gal/yr, compared to 370 gal/yr and 890 gal/yr for corn and sugarcane respectively • Continental Airlines • January 7, 2009 flew a plane on a mix of oilgae • Martek • BP contract for $10 million dollars for biofuels with a continuous process • Utilize DHA as a nutraceutical