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(12) Patent Application Publication (10) Pub. No.: US 2011/0275118A1 De Crecy (43) Pub US 20110275118A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0275118A1 De Crecy (43) Pub. Date: Nov. 10, 2011 (54) METHOD OF PRODUCING FATTY ACIDS (52) U.S. Cl. ........... 435/42:435/257.1: 435/243; 435/41 FOR BIOFUEL, BIODIESEL, AND OTHER (57) ABSTRACT VALUABLE CHEMICALS The present invention relates to a method of producing fatty acids, by inoculating a mixture of at least one of cellulose, (76) Inventor: Fus De Crecy, Gainesville, FL RNicial S. and iA with a microorganism strain and an algaeE. strain, Ario? and growing said inoculatedand either Strains anaerobic-pho under Suc (21) Appl. No.: 13/123,662 SW1OS1SE. Weesplitres Sa1S. UC citiesa SU ae)1C-- Eg SR condition, the microorganism strain produces (22) PCT Filed: Oct. 9, 2009 extracellulases that hydrolyze cellulose, hemicellulose and lignin, to produce Sugars, such as glucose, cellobiose, Xylose, mannose, galactose, rhamnose, arabinose or other hemicel (86). PCT No.: PCT/USO9/60199. lulose i. that are metabolized by the algae strain which S371 (c)(1) also metabolizes acetic acid, glucose and hemicellulose from (2), (4) Date: Aug. 1, 2011 pretreatment. Then, either under a Subsequent anaerobic-het s 9. erotrophic condition, the microorganism uses cellulose and produces fermentation products, and the algae Strain uses part Related U.S. Application Data of the released Sugars and exhibits a slower growth rate, or pp under a further anaerobic-phototrophic condition, the micro (60) Provisional application No. 61/104,046, filed on Oct. organism uses cellulose and produces fermentation products 9, 2008. and CO, and the algae strain uses the CO and part of the released Sugars and the at least one fermentation product. Under a further aerobic-heterotrophic condition, the algae Publication Classification strain uses the fermentation products produced by the micro organism strain in a previous anaerobic step to produce one or (51) Int. Cl. more fatty acids, and the microorganism strain continues to CI2P 39/00 (2006.01) produce extracellulases. The microorganism and algae strains CI2N I/00 (2006.01) are evolved for tolerance to furfural. The fatty acids can CI2P I/00 (2006.01) optionally be recovered and used for production of biodiesel CI2N I/2 (2006.01) fuel. Patent Application Publication Nov. 10, 2011 Sheet 1 of 6 US 2011/0275118A1 Bio-Ethanol current art Biomass Preparation Chipped feedstock Pretreatment Heating 160-220 C Cellulose Hemicellulose Tignin, Furfural, Acetic acid Detoxification O Separation of inhibitors: furfural, acetic acid & Salts Enzymatic Hydrolysis Hydrolysis of cellulose in: glucose, cello biose, Xylose & other Sugars. Presence of inhibitors: lignin, glucose & cel Supplied by lobiose Genencor or Novozymes Solids separation e Separation of lignin & unprocessed biomass Conversion-Fermentation Inoculation of Strain o Fermentation e Strain metabolizes mainly glucose O Inhibition by ethanol (secondary metabolite) o Presence of lignin. o Production of CO Filtration-centrifugation Waste biomass Distillation / dehydratation s 78°C (172F) Evaporator/scrubber Patent Application Publication Nov. 10, 2011 Sheet 2 of 6 US 2011/0275118A1 Producing Fatty Acids Upstream Transesterification Glycerol-1 Culture unit: Using Raw Glycerol as S S W/ Inicroorganism S S y Microorganism processing unit: s Filtering Microorganism Glycerol-1 SS Chemicals Reactor unit: Transesterifying Other Applications Fatty Acids y S Separator unit s^ Fig. B O Patent Application Publication Nov. 10, 2011 Sheet 3 of 6 US 2011/0275118A1 Biotork biodiesel pathway 3-A Chipped Pretreatment feedstock Heating 120-140°C Cellulose Hemicellulose Lignin, Furfural, Acetic acid Fermentation-Respiration-Conversion in heterotrophic conditions oInoculation & growth of Strain1 & Strain2 OBoth strains are aerobic AND anaerobic oSymbiosis: successive of aerobic & anaerobic conditions PAerobic: Strain1 produces extracellular cellulase, oAerobic: Strain2 uses acetic acid from pretreat ment + glucose & hemicellulose hydrolyzed by cellulase produced by Strainl Anaerobic: Strain1 use cellulose & produces sec ondary metabolites as acetic acid, ethanol, Succi nate, butirate, pyruvate & other fermentation prod ucts + CO2 Anaerobic: Strain2 uses part of released Sugars oAerobic: Strain2 uses secondary metabolites pro duced by Strain1 in previous anaerobic conditions Strain1 & 2 evolved for tolerance to furfural oStrain1 evolved for tolerance to acetic acid Cells Recovery Waste oFiltration & drying cells glycerol Direct Transesterification of dry cells with: OUltraSonication OEthanol or methanol oAcid or base Patent Application Publication Nov. 10, 2011 Sheet 4 of 6 US 2011/0275118A1 Biotork biodiesel pathway 3-A Chipped Pretreatment feedstock Heating 120-140°C Cellulose Hemicellulose Lignin. Furfural, Acetic acid Fermentation-Respiration-Conversion in heterotrophic conditions oInoculation & growth of Strain1 & Strain2 Both Strains are aerobic AND anaerobic Symbiosis: successive of aerobic & anaerobic conditions OAerobic: Strain1 produces extracellular cellulase, oAerobic: Strain2 uses acetic acid from pretreat ment + glucose & hemicellulose hydrolyzed by cellulase produced by Strain1 oAnaerobic: Strain1 use cellulose & produces sec ondary metabolites as acetic acid, ethanol, Succi nate, butirate, pyruvate & other fermentation prod ucts + CO2 OAnaerobic: Strain2 uses part of released sugars OAerobic: Strain2 uses secondary metabolites pro duced by Strain1 in previous anaerobic conditions oStrain1 & 2 evolved for tolerance to furfural oStrain1 evolved for tolerance to acetic acid Cells Recovery Waste oFiltration & drying cells glycerol Direct Transesterification of dry cells with: Ultrasonication eEthanol or methanol oAcid or base Patent Application Publication Nov. 10, 2011 Sheet 5 of 6 US 2011/0275118A1 Biotork biodiesel pathway 3-A Chipped Pretreatment Sulfuric switchgrass Heating 120-200°C Acid 0.5-2% Cellulose Iemicellulose Lignin. Furfural, Acetic acid Fermentation-Respiration-Conversion in heterotrophic conditions O EVG41025 a lusarium oxysporum derivative evolved to use pretreated Switchgrass as carbon source in anaerobic environment producing: acetic acid, ethanol, and other fermentation products like Succinate, bu tyrate, pyruvate as byproducts, and evolved for production of more (or more efficient) cellulases in aerobic environment also using pretreated Switchgrass as carbon source. In addition, the strain was evolved for toler ance to furfural & acetic acid and presence of lignin. O EVG17020 a Chlorella protothecoides derivative evolved in heterotrophic environment to use as carbon sources the fermentation products released by EVG41025, acetic acid, ethanol, and other fermentation prod lucts like Succinate, butyrate, pyruvate, any soluble Sugars released by pretreatment of Switchgrass not used by EVG4 102.5, waste glycerol, and any Sugars released by enzymes activity released in aerobic environ ment by EVG4 1025. o Co-inoculation & growth of EVG41025 and EVG17020 in an heterotrophic cnvironment, in the same fer mentOr O Both strains are aerobic AND anaerobic Symbiosis: succession of aerobic & anaerobic conditions, after measurement of acetic acid and for ethanol concentrations in the mcdium O In aerobic conditions: EVG41025 produces extracellular cellulase, Xylanase and EVG 17020 uses acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate, waste glycerol and any Solu ble sugars released by pretreatment of switchgrass by EVG4102.5, and any sugars released by enzymes activ ity released in aerobic environment by EVG4 1025 In anaerobic conditions: EVG41025 use cellulose & hemicelluloses and produces secondary metabolites as acetic acid, ethanol, succinate, butyrate, pyruvate & other fermentation products + CO, EVG17020 uses part of the released Sugars. O EVG17020 produces 40% and more fatty acid (cell dry weight) Cells Recovery oDouble Filtration & cell drying Waste glycerol Direct Transesterification of dry cells with: Ultrasonication, membrane rupture Ethanol or methanol Acid or base Fig.s 4 Gede)Biodiesel Patent Application Publication Nov. 10, 2011 Sheet 6 of 6 US 2011/0275118A1 Biotork biodiesel pathway 3 Example B Chipped Pretreatment Sulfuric Switchgrass Heating 120-200°C Acid O.5-2% Cellulose Hemicellulose Lignin. Furfural, Acetic acid Fermentation-Respiration-Conversion in heterotrophic conditions O EVG42050 a Fusarium oxysporum derivative evolved to use pretreated Switchgrass as carbon source in an aerobic environment producing: acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate as byproducts, and evolved for production of more (or more efficient) cellulases in aerobic environ ment also using pretreated Switchgrass as carbon source. In addition, the strain was evolved for tolerance to furfural & acetic acid and presence of lignin, DEVG17075 a Chlorella protothecoides derivative evolved in heterotrophic environment to use as carbon Sources the fermentation products released by EWG42050, acetic acid, ethanol, and other fermentation products like Succinate, butyrate, pyruvate, any Soluble Sugars released by pretreatment of Switchgrass not used by EVG42050, waste glycerol, and any Sugars released by enzymes activity released in aerobic environment by EWG42O5O. oCo-inoculation & growth of EVG42050and EVG17075 in an heterotrophic environment, in the same fermen tOr Both Strains are aerobic AND anaerobic Symbiosis. Succession of aerobic & anaerobic conditions, after measurement of acetic acid and for ethanol
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