1. Development of an integrated algal bio-refinery for polysaccharide and bio-fuel production Cesar Moreira 1, Murali Raghavendran 2, Yatin Behl 2, Spyros Svoronos 2, Edward Philps 3, Pratap Pullammanappallil 1 1 Department of Agricultural and Biological Engineering 2 Department of Chemical Engineering 3 School of Forest Resources and Conservation

2. Current approaches for bio-fuel production from microalgae Algae Anaerobic Digestion Supercritical fluids Pyrolysis Gasification Liquid or vapor fuel Catalytic Upgrading Transportation fuels liquid or gas Biogas Fisher Tropsch synthesis Catalytic Upgrading Liquid hydrogen fuels Hydrogen Higher alcohols synthesis Methanol, Ethanol, etc Ethanol Hydrocarbons Lipids Hydrogen Polysacchari- des. Syngas Modified from: http://www1.eere.energy.gov/biomass/pdfs/algal_biofuels_roadmap.pdf

3. Integrated processes for bio-ethanol and bio-diesel production from microalgae Source: Third generation of bio-fuels from microalgae. Dragone et.al., 2012

4. Algae strain used in this project Synechococcus BG 0011 Courtesy of Dr. Edward Philps

5. Comparison of algae strains currently being studied for byofuels vs Synechococcus Disadvantages of existing microalgae feedstocks Advantages of Synechococcus sp Excellent means of capturing sunlight and atmospheric carbon dioxide Occurs in natural environments (Discovered and isolated by Dr. Edward Phlips from a shallow lake in the Florida Keys) Produces exo-polysaccharide (no need for cell disruption) Nitrogen fixing bacteria (no need for nitrogen addition) Able to grow in high salinity environment (up to 75 psu) The polysaccharide can be used as a feedstock to produce liquid bio-fuels (e.g. butanol) or other bio-products The cyanobacteria culture with the polysaccharide can be fed to the digester to produce methane Mostly fresh water algae Requires harvesting and dewatering of algae Requires addition of nutrients i.e. Nitrogen and Phosphorous Utilize GMO (genetically modified organisms) Possibility of contamination of algal bioreactors

6. harvestpolysaccharides cells Option 1 Options for utilizing Synechococcus BG011 for biofuels and bioproducts Separation Ethanol /Butanol Bio-products Raceway pond/Bioreactor

7. Option 2 Raceway pond/bioreactor Seed and grow out cultures Anaerobic Digestion Harvest Biogas (CH4 + CO 2 ) CH4 Effluent and residue Land application/Nitrogen fertilizer

8. Option 3 Effluent and residue Polysaccharides production Land application/Nitrogen fertilizer Anaerobic Digestion Biogas (CH4 + CO 2 ) CH4 Raceway pond/bioreactor

9. Algae growth -Schematic of the growing chamber

10. Algae growth using air flow 0.5 l/min Semi continuous phase started Sample volume= 5ml/day Water loss by evaporation= 3ml/day

11. Algae growth using air flow 0.5 l/min y = 0.0087 e 0.3314x R 2 = 0.9365 µ = 0.33 days -1

12. Validation of biomass measurement method µ = 0.31 days -1

13. Validation of biomass measurement method

14. Preliminary results on characterization Cyanobacterium suspension (100g) VS 3.82% Ash 1.11% Water 94.41% Supernatant 99.35% >100kDa 4.34% >30kDa<100kDa 3.18% <30 kDa 91.83% >100 kDa 15.84% >30kDa<100kDa 13.67% <30 kDa 9.17% Centrifugation Pellet 0.65% Oven dry Ultrafiltration Freeze dry >100 kDa 0.71g >30kDa<100kDa 0.48g <30 kDa 3.74g Oven dry

15. Preliminary results on characterization Mn av : 4.5648 e 6 g/mol Mw av : 4.6243e 6 g/mol Mz : 4.7065e 6 g/mol P.I.: 1.013

16. Future work Enrichment of air supplied to the photobioreactors with CO 2 Characterization of exopolysaccharide Optimize production of exopolysaccharide with respect to salinity, partial pressure of CO2, light intensity and exposure time, pH and micronutrients. Adapt, optimize and validate techniques commercially developed for saccharification of polysaccharides and ligno-cellulosic biomass for saccharification of exopolysaccharide. Anaerobically digest exopolysaccharide containing cell cultures to quantify methane potential and identify best temperature range for digester operation.

17. Questions