Extraction and Conversion of Algal Biomass under Supercritical Conditions P.Patila, H. Readya, T. Muppanenia, S.Denga, T.Schuabb, Peter Cookec, N. Khandand , P. Lammerse Chemical Engineering Department a Chemical Analysis and Instrumentation Laboratory b Electron Microscopy Laboratory c Civil and Environmental Engineering Department d Energy Research Laboratory e Las Cruces, NM 88003 ,USA NAABB Upstream-Downstream Final Meeting, Phoenix, AZ, Jan 22- Jan 24, 2013

Outline Supercritical MeOH/EtOH Conversion Process for Wet Algae Microwave-mediated Transesterification of Wet Algal Biomass to Biodiesel under Supercritical Ethanol Conditions Supercritical CO2 Extraction of Algal Lipids Subcritical Water Extraction of Algal Lipids Supercritical Methyl Acetate Process for Algal Biodiesel

Supercritical Conversion Supercritical Fluids Supercritical Conversion Benefits No catalyst is needed, short reaction time (<60 min.) Temp. is the most important process parameter Pressure affects conversion rate High alcohol/oil ratio facilitates conversion Water (60%) don’t affect SCF conversion Small amount of water helps the conversion Conversion rate is faster in SCF than sub-critical High dissolving power (103 – 106 > liquid solvents) Low viscosity High diffusivity Easy separation No harmful solvents Selective extraction/separation Suitable for food/medicine active ingredients CO2, H2O, CH3OH …. Pinnarat and Savage, Ind. Eng. Chem. Res., 47: 6801-6808 (2008) 3

Elemental and Fatty Acid Composition of Nannochloropsis Salina Elements Wt. (%) Carbon (C) 70.43 Oxygen (O) 22.19 Sodium (Na) 0.74 Magnesium (Mg) 1.16 Phosphorous (P) 2.42 Chlorine (Cl) 0.36 Potassium (K) 1.32 Calcium (Ca) 0.94 Sulfur (S) 0.34 Total Lipid Content in algal Biomass : 50-55% on Dry Basis Fatty acids Wt. % Tetradecanoic acid, C14:0 2.72 Hexadecanoic acid, C16:0 37.83 Hexadecenoic acid, C16:1 n7 31.34 Octadecanoic acid, C18:0 5.63 cis-9-Octadecanoic acid, C18:1 n9 11.31 cis-11,14-Eicosandienoic acid, C20:2 n6 1.3 cis-5,8,11,14-Eicosatetraenoic acid, C20:4 n6 2.24 cis-5,8,11,14,17-Eicosapentaenoic acid, C20:5 n3 6.35 Total fatty acids Saturated fatty acids 45.69 Unsaturated fatty acids 54.31

Direct Conversion of Wet Algae to Biodiesel under SCM Optimal conditions:1:9 (wt./vol.) MeOH ratio, around 255 ºC & 25 min. Crude algal biodiesel Pure algal biodiesel Parr Micro-Reactor Patil, P.D., et al. Optimization of direct conversion of wet algae to biodiesel under supercritical methanol conditions. Bioresour. Technol. (2011),102(1),118-122 5

Wet Algae to Biodiesel under Supercritical Ethanol (SCE) Experimental : 3 g of wet algae paste (40% solids); Reaction times:2-30 min; Reaction temperatures-245-270C; and wet algae to ethanol (wt./vol.) ratios : 1:6-1:15. TGA Analysis of Wet Algae: Dehydration: 100-150 C Thermal Degradation : 250-350 C Decomposition (Fast Pyrolysis): 350-500 C Single-step conversion of wet algal biomass to biodiesel was demonstrated. Saves large amount of energy consumed during harvesting & drying. Completely renewable sources of energy such as algae and ethanol used for “Green” biodiesel. Harvind Reddy et al. Direct conversion of wet algae to biodiesel under supercritical ethanol conditions. Applied Energy (Under Review) 6

Microwave-Mediated Supercritical Transesterification R1,R2,R3 are Long chian hydrocarbons may be same or different The microwave effect on the transesterification reaction is twofold: (1) enhancement of reaction by a thermal effect, and (2) evaporation of ethanol due to the strong microwave interaction of the material. The microwave interaction with the reaction compounds (triglycerides and ethanol) results in a large reduction of activation energy due to an increased dipolar polarization Phenomenon and Ionic Conduction yield to FAEE and glycerol. This reaction is Non-catalytic carried out under Supercritical ethanol conditions. Patil, P.D., et al. Microwave-mediated non-catalytci transesterification of algal biomass under supercritical ethanol conditions. Jour of Supercritical Fluids, 2012.

Supercritical CO2 Extraction of Algal Lipid from Dry Alghae TharSFC Reactor GC-MS Analysis Operating range: P : 0-600 bar Max T.: 150 ºc ; 2 Lit 8 8

Subcritical Water Extraction of Lipids from Algal Biomass Experimental : Heating conventional and microwave Biomass loading- 3- 12% solids); Extraction time :10-30min; Extraction temperatures – 170-240oC, Solvent : Hexane Optimum conditions for C-SCW extraction: Extr. temp. 220 oC biomass load. 7.5 % Extr. Time 25 min. Optimum conditions for MW-SCW extraction.: Extr. temp. 205 oC biomass load. 25 % Comparison: Parr Micro-Reactor Anton Parr Microwave Reactor  FAME content in biomass: 24.7% on dry wt. basis (% dry wt. basis) Folch Extraction Conventional -SCW extraction Microwave -SCW extraction Avg. Total lipid or Bio-crude extraction 20.39 ± 1.74 31 ± 1.6 35.7 ± 1.18 FAME content in extracted total lipid or bio-crude extraction (% bio-crude) 40.15 ± 0.59 53.99 ± 2.35 67.10 ± 1.74 Total FAME extracted (% dry wt.) 8.18 ± 0.59 16.75 ± 1.30 24.61 ± 0.33 Subcritical water extracted bio-crude and pure algal oil Harvind, Patil et al. 2012 . Subcritical water extraction of algal oil from algal biomass . Energy and Environmental science (Under Review) 9

Supercritical Methyl Acetate Process for Algal Biodiesel All Supercritical process produces large quantity of glycerin -----Over Produced ???? ( Big Problems to Commercial biodiesel processes) Reaction Parameters: Total lipid : 7 ml, Methyl Acetate: 25 ml (1:42 molar ratio) , Time: 120 min, Temperature: 280 C, Pressure: 1178 psi (~ 81 bar) Critical Properties of Methyl acetate : 233.7 C, 45.9 bar, Sp.G- 0.927, Mol wt: 74, Boiling pt at 1 atm- 57 C, Triacetin BP: 258-260 C A mixture of fatty acid methyl esters and triacetin found to be used as biodiesel and can enhance its oxidation stability and fuel properties since the obtained triacetin is miscible with fatty acid methyl esters, both products can readily be used as biodiesel. Crude Biodiesel Fuel (FAME + Triacetin) yield ~ 85-90% Apparent FAME yield ~ 80%

Elemental and Fuel Properties of Algal Extracts Analysis Samples SCCO2 crude lipid SCCO2 pure algal oil SCW crude lipid Pure algal oil SCMA Algal biodiesel SCM Algal Biodiesel   % Carbon (C) 75.09 76.40 76.75 78.20 74.69 78.12 ASTM D5373 % Hydrogen (H) 11.16 11.71 11.11 11.93 11.50 D5373 % Nitrogen (N) 0.46 0.03 0.96 0.18 0.05 D5291 % Oxygen (O) 12.99 11.89 11.20 9.53 13.78 9.91 D5622 % Sulfur (S) 0.09 0.02 0.11 0.04 D1558 Fuel Properties Fuel property 1st generation biodiesel (Corn Biodiesel) Algal SCM biodiesel Diesel ASTM D6751 biodiesel standard HHV (MJ/kg) 40-45 41-43 45.9 --- Kinematic Viscosity (mm2/s) 2.4-4.5 3- 5.2 1.2-3.5 1.9-6.0 Density 0.86-0.89 0.84- 0.86 0.83-0.84 0.86-0.9 Pour point -15 to 10 -12 to -10 -35 to -15

Proposed Scale-up Technologies Integrated SCM to produce algal biodiesel and power cogeneration Integrated algal biodiesel SCM production facility with anaerobic digestion of LEA

Summary of Accomplishments PUBLICATIONS in Peer-Reviewed Journals P.Patil , S.Deng et al. Power Dissipation In Microwave-Enhanced In-situ Transesterification of Algal Biomass Green Chemistry 2012,14, 809-818. P.Patil,, S.Deng et al. Comparison of direct transesterification of algal biomass under supercritical methanol and microwave irradiation conditions. Fuel 2011, 97, 822-831. P.Patil, , S.Deng et al. Direct Conversion of Wet Algal Biomass to Biodiesel under Supercritical Methanol Conditions. Bioresource Technology 2011, 102,118-122. P.Patil, S.Deng et al. Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology. Bioresource Technology 2011, 102, 1399-1405. P. Patil, S.Deng et al. Microwave-Mediated Catalyst-Free Transesterification of Algal Biomass under Supercritical Ethanol Conditions, J of Supercritical Fluids, 2012. P. Patil, S.Deng et al. In-Situ Biodiesel Production from Algal Biomass under Microwave-Mediated Supercritical Ethanol Conditions. Environmental Science & Tech 2013 (Revised) H Reddy, P.Patil, S. Deng et al. Direct conversion of wet algae to biodiesel under supercritical ethanol conditions. Applied Energy (Review). H Reddy, P.Patil, S. Deng et al. Subcritical Water Extraction of Lipids from Wet Algal Biomass to Produce Biofuels. Energy & Environmental Science ( Review) PATENTS PENDING Deng S., Patil Prafulla, Gude Veera. “Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology”. US Patent- 61/426,305 (Filed: December 22, 2010). Deng S., Patil Prafulla, Gude Veera. “Direct Conversion of Wet Algal Biomass to Biodiesel under Supercritical Methanol Conditions”. US Patent- 61/426,333 (Field: Dec’22, 2010).

Concluding Remarks SCM and SCE for wet algal biomass can potentially be an energy efficient and economical route for algal biodiesel production. MW-SCE process improves extractions of algae significantly, with a higher efficiency, reduced extractive-transesterification time and increased yield. An ecofriendly green (subcritical water) extraction process was developed and achieved maximum efficiencies with wet algal biomass SC-CO2 is cleaner production technology (chemically stable, non-toxic, non-flammable) for extraction of algal lipids/oils for algal biodiesel production. Microwave-pretreatment prior to SCCO2 with Azeotropic co-solvents (Hexane+ Ethanol) process gives high quality microalgae oil, rich in fatty acids and increases lipid yield with better recovery of TAG’s. Supercritical methyl acetate process could be the next-generation technology for algal biodiesel production. 14 14

Acknowledgements The (authors) would like to acknowledge funding of this work by the US Department of Energy under contract DE-EE0003046 awarded to the National Alliance for Advanced Biofuels and Bioproducts 15 15