3. Supercritical CO 2 A substance above its critical temperature and critical pressure Property tunability near the critical point Supercritical fluids such as CO 2 are considered “green solvents” and are relatively cheap “Liquid-like” density and “gas-like” viscosity Suitable solvent for extracting volatile oils and fragrances Relatively low critical temperature and pressure 31 ºC 73 bar

4. Lab scale Continuous Batch SolidsLiquids Pilot Scale

5. Supercritical Extractor 300 bar 40 ºC 10 g Supercritical Extractor 300 bar 40 ºC 10 g CO 2 -sc OilOil Neochloris vulgarisExtraction time: 1h 2.1 g of oil extracted (>95% of total amount of oil) Chlorella protothecoidesExtraction time: 1h 2.4 g of oil extracted (>95% of total amount of oil)

6. Fatty acid methyl ester (Biodiesel) Triglyceride Overallmechanism

7. Industrial conventional process Low value glycerol Soap formation Purification process that produces waste FFA concentration control High value glycerol Less energy intensive Direct esterification of FFA Reusable catalyst Enzyme

8. Sample analysis On-column GC Reaction stage Separation stage Enzymatic Packed-bed reactor Enzymatic Packed-bed reactor

9. Materials Oil from biomassCO2MethanolLipozyme TL IM® Reaction conditions Temperature: 30 – 50 ºCPressure: 200 barOil to methanol molar ratio: 1:36 – 1:12

10. Fatty Acid Methyl Esters (Biodiesel) Unreacted mono-, di- and triglycerides Fatty Acid Methyl Esters (Biodiesel) Unreacted mono-, di- and triglycerides 70% Conversion 98.6% Conversion

11. Sub-critical Water Suitable properties for hydrolytic, hydrothermal and oxidative processes At near-critical and supercritical conditions, water and gases, e.g., O 2, N 2, NH 3, CO 2,are completely miscible Becomes less suitable for solvating ionic species Dipole moment decreases from ambient to the critical region

12. BiomassBiomass Gaseous Products Water Soluble Products CharChar

13. Ligno-cellulosic material Glucose / Fructose / Xylose FurfuralsFurfurals PhenolsPhenols Acids / Aldehydes GasesGases Higher molecular weight products

14. WaterWater 65% of initial mass hydrolyzed into water soluble compounds Reactor 150 bar 200 ºC 2 g Reactor 150 bar 200 ºC 2 g

15. BiomassBiomass Sc-CO 2 Anti-oxidantsAnti-oxidants Oil Oil ResidueResidue Sub-critical Water 150 – 350 ºC 100 - 250 bar Water+SugarsWater+Sugars Fermentation EthanolEthanol Enzymatic Transesterification BiodieselBiodiesel GlycerolGlycerol Sc-CO 2

16.  PTDC/CTM100244/2008  PTDC/EQU-EQU/104452/2008  PTDC/ QUI/ 64744/ 2006  PTDC/EBB-EBI/099237/2008  Project “ReThink”, in collaboration with Delta Cafés  PT20.0607/PTA10376507  European Patent application 08398007.8-2115-19/062008 (US, JP, KR)  PT 41867/10 Patents