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Downstream processing of algal oil Emilio Molina Grima Dpt. Chemical Engineering, University of Almería, SPAIN [email_address] Seminar on Microalgae IMDEA Energy-EOI, April 8 2010, Madrid

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<ul><li>No well-defined demonstration industrial plants </li></ul><ul><li>Microalgae differ from other bioenergy feedstocks in: </li></ul><ul><ul><li>Cell wall chemistry </li></ul></ul><ul><ul><li>Large amount of water </li></ul></ul><ul><ul><li>Small cell size </li></ul></ul><ul><ul><li>Much more polar lipids than traditional crops </li></ul></ul><ul><li>Extraction of lipids from algae requires attention to their polarity </li></ul><ul><li>The strain and the harvest time will determine the solvent election </li></ul>Questions to take into consideration

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Pretreatment of microalgae cells J.Y. Lee et al. (2009 ) Bioresour Technol. DOI10.1016/j.biotech2009.03.058 Comparison of several methods for effective lipid extraction from microalgae Lipid content (wt %) Non-disruption Autoclaving Bead-beating Microwaves Sonication Osmotic shock 30 20 10 0 30 20 10 0 30 20 10 0 Botryococcus sp Chlorella vulgaris Scenedemus sp

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Extraction of lipids Solvent Mixture (dry Isochrysis galbana ) Cl 3 CH/MeOH/H 2 O (1:2:0.8 v/v/v) Hexane/EtOH 96% (1:2.5 v/v) Hexane/EtOH 96% (1:0.9 v/v) n-Butanol EtOH 96% EtOH 96%/H 2 O (1:1 v/v) Hexane/Isopropanol (1:1.5 v/v) Cl 3 CH/MeOH/H 2 O (1:1:0.9 v/v/v) Hexane/EtOH/ H2O (6:1:1 v/v/v) Hexane/EtOH/ H2O (29:4.7:1 v/v/v) Cl 3 CH Cl 3 CH Cl 3 CH Cl 3 CH Extraction (monophasic) Purification (two phases) E. Molina Grima et al. (1994) JAOCS, 71(9), 955-959

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Extraction of lipids Information on saturation curves may be found at: Walter D. Bonner (1910). Experimental Determination of Binodal Curves, Plait Points, and tie lines, in Fifty systems, each consisting of water and two organic liquids. J. Phys. Chem. 14, 738-789 E. Molina Grima et al. (1994) Comparison between extraction of lipids and fatty acids from microalgal biomass. JAOCS, 71(9), 955-959 Optimal tie line

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Extraction of lipids Yields (%) of Extracts and Raffinates ( in parentheses ) of lipids extraction from dry Isochrysis galbana Cl 3 CH/MeOH/H 2 O (1:2:0.8 v/v/v) Hexane/EtOH 96% (1:2.5 v/v) Hexane/EtOH 96% (1:0.9 v/v) Butanol EtOH 96% EtOH 96%/H2O (1:1 v/v) Hexane/Isopropanol (1:1.5 v/v) 92.9 (0.9) 52.2 (27.4) 49.5 (8.3) 70.4 84.4 63.3 66.0 Solvent Mixture

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Extraction of fatty acids Yields (%) of Extracts obtained by direct saponification from dry Isochrysis galbana Cl 3 CH/MeOH/H 2 O (1:2:0.8 v/v/v) Hexane/EtOH 96% (1:2.5 v/v) Hexane/EtoH 96% (1:0.9 v/v) Butanol EtOH 96% EtOH 96%/H 2 O (1:1 v/v) Hexane/Isopropanol (1:1.5 v/v) Solvent system + KOH (47.5 mL solvent /g KOH) - 81.0 48.0 9.0 79.8 46.5 62.0 60ºC-1 h - 75.0 47.1 - 79.2 45.5 61.1 Room-8 h

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Comparison between the extraction of Lipids and Fatty acids I. galbana <ul><li>Alcohol content seems to be the main factor in lipid extraction. The higher the polarity of the co-solvent, the higher the yield </li></ul><ul><li>For each solvent mixture the yield of lipid extraction is slightly higher than that of fatty acid extraction </li></ul><ul><li>Direct saponification of the biomass could be induced in two steps, being the lipid extraction the bottleneck </li></ul><ul><li>A LARGE AMOUNT OF SOLVENT WAS USED </li></ul>144 mL/g of dry biomass

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Optimization of fatty acids extraction 810 g 405 g Extract (overflow) 480 g Wet biomass (100 g d.b) ethanol (1 L) KOH ethanol (0,5 L) Residue Raffinate (underflow) Alcoholic solution Fatty acids soaps (25% water) S-L (W) S-L Fatty acids hexane hexane hexane hexane L-L L-L L-L L-L Alcoholic phase pH6 Alcoholic solution Fatty acids soaps (25% water) water Alcoholic solution Fatty acids (40% water) HCl Ramírez Fajardo et al., 2007

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Optimization of fatty acids extraction Influence of Ethanol (96%)/wet biomass M.J. Ibañez González et al. (1998) JAOCS 75, 1735 – 1740. Lyophilized

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Optimization of fatty acids extraction S-L stage V 01 = 810 g (1 L ethanol) +21 g KOH Residue Alcoholic solution Fatty acid soaps (25% water) 2 1 V 1 +V 2 = 1336 g y A =0.0072 V 02 = 405 g (0.5 L ethanol) L 1 X A1 X B1 X S1 Lo =480 g X A0 = 0.023 X B0 = 0.977 L 2 X A2 X B2 X S2 Wet Biomass (100 g d.b) Recovery Yield 87%

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Optimization of fatty acids extraction V 0 Scale-up calculations needed (repeated contact system) L A0 + V 01 = L 1 + V 1 = M 1 X A0 ·L A0 = Y A1 ·V 1 + X A1 ·L 1 S-L stage underflow overflow x A2 L 0 X B0 = L 1 X B1 V 01 = 810 g (1 L ethanol) Residue Alcoholic solution Fatty acid soaps (25% water) 2 1 V 1 +V 2 = 1336 g y A = 0.0072 V 02 = 405 g (0.5 L ethanol) L 1 X A1 X B1 X S1 Lo = 480 g X A0 = 0.023 X B0 = 0.977 L 2 X A2 X B2 X S1 Wet Biomass (100 g d.b) Recovery Yield 91% B L 0 A S x A0 V 1 M 1 V 2 L 1 L 2 M 2 Kg FA soaps solution Kg inert solid P= = 3 X A1 1- X B1 Y A1 = (Ideal stage)

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Optimization of fatty acids extraction Δ Scale-up calculations needed (counter current systems) S-L stage A S B V 2 V 1 x A2 x A0 L n L 2 L 1 M L 0 V n+1 2 1 V 1 y A1 L 1 X A1 X B1 X S1 Lo =480 g X A0 = 0.023 X B0 =0.977 L 2 X A2 X B2 X S2 V 2 y A2 V 0 =1215 g Extract (overflow) Raffinate (underflow)

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Optimization of fatty acids extraction L-L Extraction of FA V 1 y 1 V 1 y 2 V 1 y 3 V 1 y 4 Fatty acids Extract (Hexane phase) V 0 = 0.026 L Hexane V 0 = 0.026 L Hexane V 0 = 0.026 L Hexane Alcoholic phase Alcoholic solution Fatty acids (40% water) L-L L-L L-L L-L V 0 = 0.026 L Hexane Lo = 1,53 kg X A0 = 903 mg/L L 1 X A1 L 2 X A2 L 3 X A3 L 4 X A4

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Optimization of fatty acids extraction Fatty acids concentration Alcoholic phase C FA (mg·L -1 ) The distribution equilibria of FA between the alcoholic phase and hexane facilitate the calculation of design, scale-up and assessment purposes L/V 930 mg L -1 0 1000 2000 3000 4000 5000 6000 0 50 100 150 200 Fatty acids concentration Hexane phase, C AH (mg·L -1 ) Step1 Step 2 Equililbrium x 1 x 2

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Optimization of fatty acids extraction Conclusions  FA recovery yield (wet biomass)  FA recovery yield (dry biomass)  Important reduction of hexane  Important reduction of alcohol (96% v/v)  Solvent/biomass 87% 96,2% 90% 84% 24 mL/g biomass

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Optimization of fatty acids extraction E. Molina Grima et al. (1996) Spanish patent 9602090

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Direct transesterification of paste Biomass Biodiesel BIOMASS HEXANE ACETONE EPA METHANOL RESIDUE ACETYL CHLORIDE WARTER CONDENSER MIXER EVAPORATOR CROMATOGRAPHY CENTRI- FUGE FILTER SOLIDS EXTRACTOR REACTOR ULTRASONIC MIXER OPCIONAL OPTIONAL E Belarbi et al. (2000 ). Enzyme and Microbial Technology 26 516-529 . EVAPORATOR COOLER EVAPORATOR

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Paste biomass Hexane Methanol Acetyl chloride/ H 2 SO 4 (10%) Extractor Cooler FAMEs Spent biomass Centrifugation Filtration 1000 mL *500 g 50 mL 1000 mL Temperature = 100 ºC Time: 120 min 9.1 g *(500 g of paste biomass of Phaeodactylum tricornutum ≈ 100 g of lyofilized biomass) (9.9% of TFAs) 500 mL Extractor Condenser Direct transesterification of paste Biomass Phaeodactylum tricornutum - Monodus subterraneus – Scenedesmus almeriensis EVAPORATOR

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Direct transesterification of paste Biomass CASE STUDY Slurry reaction products (Spent biomass, water, methanol, FAMES, catalyst) Biodiesel produced per year: 13.550 kg Calculations needed for processing the paste biomass produced per day in one Ha of tubular photobioreactors of Scenedesmus almeriensis. Slurry reaction products (Spent biomass, water, methanol, FAMES, catalyst) V 01 = 605 kg/day Hexane Residue S-L 2 S-L 1 V 1 +V 2 = 773 kg/day y A =0.0483 V 02 = 303 kg/day Hexane L 1 X A1 X B1 Lo =3190 Kg/day X A0 = 0.013 X B0 =0.987 L 2 X A2 X B2 Reaction L= 1375 Kg/day X A0 = 0.03 X B0 =0.997 Wet biomass 1815 kg/day methanol + acetyl chloride or 10% H 2 SO 4 Hexane solution of methyl esters S-L 2 S-L 1 V 1 = 395,15 kg/day y A1 =0.095 L 1 X A1 X B1 L 2 X A2 X B2 V 2 y A2 V 0 =530 kg/day Lo =3190 kg/day X A0 = 0.013 X B0 =0.987 Residue Hexane solution of methyl esters Hexane

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Paste Biomass Hexane Methanol Acetyl chloride / H 2 SO 4 REACTOR Centrifugation Filtration Spent Biomass ** Biodiesel *** EPA EXTRACTOR * Biodiesel Argentated silica gel column chromatography Hexane: Acetone (99.5: 0.5 v/v) EPA (99%): 2154 $/ Kg EPA (70%): 185 $/ Kg (Abayoumi et al., 2009) 1500 mL 500 g 50 mL 1000 mL (9.9 % of TFAs) (9.1 g) (6.9 g) (1.6 g) (76%) (18%) EXTRACTOR Cooler Direct transesterification of paste Biomass * Crude biodiesel with high content of EPA (27.7%) and a 92% recovery yield with respect to the total FA contained in the biomass ** Biodiesel with low EPA content (8.2%) and a 76% recovery yield with respect to the total FA contained in the biomass *** High grade EPA methylester (96.4%). Recovery yield 18% with respect to the total FA contained in the biomass. Cooler EVAPORATOR

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Comparison of experimental conditions and yields obtained by direct transesterification of microalgae biomass and other feedstocks <ul><li>Methylating mixture: mtehanol:heptane:benzene: 2,2 diemthoxypropane: SO 4 H 2 (37:36:20:5:2) </li></ul><ul><li>Methylating mixture: methanol:benzene (3:2) </li></ul>Feedstock Wet biomass 500 g Jatropha curcas seeds 20 g Solvent to extract FAMES Catalyst Methanol Biomass/tissue Working conditions Yield Fresh tissues of olive oil fruit 50-200 mg E. Belarbi et al. (2000) Siew Hoong et al. (2010) R. Garces and M. Mancha (1993) a G. Lapage and C.C.Roy (1984) b Maternal milk or adipose tissue 10 mg Hexane Hexane Heptane Benzene or Toluene Benzene SO 4 H 2 5% wt (CH 3 COCl 5%) SO 4 H 2 15% wt SO 4 H 2 2% wt SO 4 H 2 2 v/w 7,5 v/w 25 v/w 100 v/w 2 h, 100ºC 2,5 atm 24 h, 60ºC 1-2 h, 80ºC 10 min, 80ºC >90% >99% >95% >98%

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Extraction and Fractionation of microalgal Lipids Conclusions <ul><li>Technology seems to be developed at lab scale so far </li></ul><ul><li>Nowadays any pilot or demonstration plant will not be efficient or cost-effective, but it surely evolve to a greater efficiency and less operating cost process </li></ul><ul><li> Other components such as carbohydrates and proteins may need multi-step processes </li></ul>

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Multi-step process 4. Protein and carbohydrate separation from non lipidic residue Carbohydrates Proteins Precipitation 1. Protein and carbohydrate extraction from wet biomass Biomass slurry Wet Biomass Solid-Liquid Extraction Precipitation Proteins Carbohydrates 3. Chlorophyll separation from non lipidic residue Enriched in ethanol VACUUM BATCH DISTILLATION Filtration Chlorophylls 2. Fatty acid extraction by direct saponification and recovery of unsaponifiable products (carotenoids) Carotenoids Spent biomass Liquid-Liquid Extraction Fatty Acids Saponification Filtration Liquid-Liquid Extraction 59,0% 91% 6,6% Chlorophyll a 10,8% Chlorophyll c 41,0 % 16,3% 15,5%

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Multi-step process Carotenoids in hexane 0.33 g L-L L-L L-L 0.9 L hexane L-L L-L 0.9 L hexane 0.9 L hexane 0.9 L hexane Alcoholic phase 0.9 L hexane S-L, solid-liquid extraction L-L , Liquid-Liquid extraction P, Precipitation S-L 1.5 L buffer S-L 1.5 L buffer 476 g Wet biomass (100 g d.b) Proteins in the pellet 17.7 g P (NH 4 ) 2 SO 4 Carbohydrates in water 1.7 g 0.3 L water S-L residue 40 g KOH 1 L ethanol S-L 0.5 L ethanol

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Multi-step process pH 6 HCl Alcoholic Phase Fatty acids in hexane 8.0 g L-L L-L L-L 0.45 L hexane L-L 0.45 L hexane 0.45 L hexane 0.45 L hexane D, Distillation F, Filtration P, Precipitation L-L , Liquid-liquid extraction D Carbohydrates in water enriched in ethanol F P (NH4) 2 SO4 Chlorophylls in torte Chlorophylls a 0.0753 g Chlorophylls c 0.0378 g Proteins in pellet 6.7 g

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Downstream processing of algal oil Acknowledgements Antonio Giménez Giménez Alfonso Robles Medina Maria Jose Ibáñez González Jose M. Fernandez-Sevilla Francisco Gabriel Acién Fernández Emilio Molina Grima El Hassan Belarbi