us United States c2) Patent Application Publica oy 76) en @y (60) Peteavich METHOD OF PRODUCING HYDROCARBON BIOFUEI 3 GENETICALLY Robert Petcavich, The Woodlands TX (US) Inventor Comespondenee Address GREER, BURNS & CRAIN 300S WACKER DR, 25TH FLOOR CHICAGO, TL 60606 (US) Appl.No: 124617,767 File: Now: 13, 2009 Related US. Application Data Provisional application No. 61/1996, filed on Nov 13,2008. ion ‘US 20100120111 (io) Pub. No: US 2010/0120111 Al (43) Pub, Date May 13, 2010 ication Classification GI) Inc, CHP sao (52) US.CL 6 ABSTRACT | method of producing hydrocarbon biofuels using ge cally modified seaweed by inserting genes from high hydro- carbon producing micro algae ino high growth seaweed spe- fies, In an embodiment of the present inveation, Botryococcus braunil(BB)a green micro algasthat produces Jarge amounts of aliphatic hydrocarbon molecules is used as ‘source for genetic material. The genes for hydrocarbon production in BB are identified, removed, cloned and subse {quently inserted into high growth brown seasseccs such as Kelp and hydrocarbons subsequently expressed in the plant. The genetically modified kelp is grown in coastal ocean waters, harvested, the hydrocarbons produced extracted, and catalytically converted into useful biofuels, 200601) 438/166,US 2010/0120111 AI METHOD OF PRODUCING HYDROCARBON BIOFUELS USING GENETICALLY MODIFIED SEAWEED 0001) sonal Application Ser. No, 61/199,059, fled Now. L under 38 U.S.C. §119, This application claims the Beneft of US. Provi- 2008, FIELD OF THE INVENTION 10002] |The present invention relates to hydrocarbon pro- ‘duetion by marine aquatic plant life, In particular, genetically modified fast growing seawood species, such as kelp, are used Jor the production of hydrocarbons. More particularly, fast growing seaweed species are genetically modified to produce hydrocarbons that can subsequently be used as biofuels, jnchiding for ansportation vehicles and chemical industry feedstocks, BACKGROUND OF THE INVENTION 10003] Photosynthetic light-driven. biological processes have enormous capacity for sustainable, carbon neutral, solar powered replacement of fossil fuels, Typically such fuels fix ‘more than 100Giga ons (Gtons) of earbon annually, which is ‘equivalent to 100 terawatts (TW) of energy. However, this, ‘kati rates currently in belance with respiration and other facets ofthe globalearbon cyele. Accordingly, adding another 10 TW of fixation would require enormous land arcs at present, Agriculturally productive land is just one problem ‘wih this approach, Land based plant production also requires tremendous amount of nitrogen fertilizer which represents up 10 25% of the east of biomass production, Worldwide, ‘appeoximstely’ 160 million tons of ammonia is produced fnnvally by an energetically expensive fossil fel dependent process which is or may not be sustainable fora Tong period of time. 004} _ Photosynthesis provides greater than 90% ofthe net ‘eneray into the biosphere. I produces the oxygen we breathe tnd drives the biogeochemical eycles. The primary reactions ‘of photosynthesis ean operate at near perfect quantum efi ciency. Its therefore desirable o utilize the photosynthesis, process in the harvesting of useful energy. 10005] One goal ofthe present invention i to Link photo- synthesis and micro alga metabolism, to prodoce liquid hydrocarbons and useful chemical produes. Ths is accom plished by genetically engineering into fst growing seaweed, algae genes for hydrocarbon production, Whilethe beneftsof ‘extrctng fuel hydrocarbons fom algae ae great the process in the past has encountered significant difficulties. See May- field, Genetic Engineering of Alene for Biofuel Production Department of Cell Biology and the Skages Insitute for ‘Chemical Biology, incorporated by reference herein 10006] I te present inwention, a species of algae, Botryo- ‘coceus braun, has been reported to produce liquid hydro= ‘carbons that represent upto 75% of the dey mass cell weight Botryococcus braun, green colonial microalgae is an "unusually rich renewable resource of hydrocarboas and othee ‘chemicals. One of the most interesting facets of the hydo= carbon production of this species is thatthe hydrocarbons produced are in the earban (C25 to C40) chain length that ‘makes them ideal candidates for catalytic conversion into fel for transportation vehicles and chemical feedstocks. By aquatic marine sea plant life in particular alpae, 0 May 13, 2010 zener large amounts of hydrocarbons one solves the prob- Jemof using lange amounts of land area and energy intensive Ierilzer to produce biobels, SUMMARY OF THE INVENTION [0007] The object of this invention isto provide @ renew able resource for hydroearbon prodcion used as biofuels for ‘eansperation and feedstock for chemical production. [0008] Another object of his invention isto create genet cally modified fst growing seawced that produces hydro ’bons that can be used as biofuel and molecular feedstock for chemical predaction, [0009] "Another object ofthis inventions to provide hyo- carbon producing seaweed that can be grown in marine ‘coastal areas for easy harvesting and processing [0010] Yet another object of this invention i o genetically nodity fast growing seasveeds for production of hydrocar- ‘bons by inserting genes from known high hydrocarbon pro- ‘ducing miero ale into the seaweed eels, [0011] Another object ofthis invention is to directly con- vert solar energy into hydrocarbons for use as biofels and chemical feedstock’ using genetically modified seaweeds. DESCRIPTION OF A PREFERRED EMBODIMENT [0012] Unicel photosynthetic micro alga has the abil- ty to produce lange quaatities of hydroearbous within thei cells. This conversion of solar energy directly into hydrocare bons aad subsequently biofuels is of great eommercil inter cst. One micro alga Botryococeus braun (BB) a member of the ehlorophyta family is regarded asa source of renewable fuel because ofits ability to produce lage amounts of hydeo- carbons. Dependingon he stainand growth conditions, upto 175% of algal dry mass can he hycracarhoas. The chemical nature of the hydrocarbons varies with the stain of BB alga [0013] Three racesof BB have been documented, and these ‘an be differentiated on the bass of hydrocarbons they pro- duce, The rave produces odd numbered carbon C2510 C31, nralkadienes, and trenes. The B race produces triterpenoid hydrocarbons known as botryoeoocenes (Cat 12n-10, n-30- 37), andthe race produces eopadiene, a C40 tetaterpene “Although BB is the prefered hydrocarbon producing micro ‘alga of the present invention miaay others can he wed and & ood summary of the potential candidates ean be found in a ‘report by the United States department of Energy NRELITP- ‘580.2490 entitled “A Look Back at the Department of Ener- y's Aquatic Species Program: Biodiesel fm Algae" incor porated herein by reference, [0014] The genetic manipulation of aluse species to pro- ‘ducea new hybrid stn is wellknown in thea. Forexarmple, the hybridization oftwo species of algse through rowing the species in close proximity ina aulrient solution is shown ia US. Pat, No, 5,365,018 as well as U.S, Pat, No. 5,385,544, both led “Method of Causing Somatic Hybsidization Between Two Species of Algae,” and both of which are incor ported by refereace herein. Similarly, the introduction of orejgn DNA into algae spore protoplasts to form a hybrid fusion is shown in U.S. Pat. No. 5,426,040 “Methods for Producing Improved Sharing of Seawood by Fusion of Spore- Protoplasts, and Resultant Seaweeds and Phycocolloi: [0018] The genetic material responsible for the production fof the various hydrocarbons of BP is isolated, purified, and cloned into the particular organism of interest by techniquesUS 2010/0120111 AI well known in the at of molecular biology, See, for example, Cheney, D. P. 1990, Genetic Improvement of Seaweeds ‘Through Protoplast Pusion, Economically Important Marine Plans of the Atlante: Their Biology and Cultivation, C. Yarish and C. Penniman, (eds) Univ. of Conn, Sea Grant Program, pp. 15.25: Cheney, D., et al, 1998, Genetic Manipulation and Stain Improvement in Commercially Valuable Red Seaweeds, New Developments In Marine Bio technology; Y.1.-Gal and I. Halvorson eds), Plenum Press, NYpp. 101-104; Cheney, DP, 1999, Stain Improvement of Seaweeds Thru Genetic Manipulation: Curent Stats, World Aquaculture, 30: 55-56 & 65; Qi, 8, Tong. S., Zhang. Pet ‘a. 1993, Isolation of Plasmid from se Blue-green Alga, ‘Spirulina platensis, Chinese Journal of Oceanologs and Lim: nology 11(3) 285-288; Qi, S. Jiang, P. Li X. etl. 1988, ‘The Expression of laeZ in Regenerated Sporophytes of Par. thenogenetic laminaria japonica, Proceedings of the 2nd Asia-Pacific Marine Biotechnology Conference and Sr Asa~ Pacific Conference on Algal Biotechnology (in press): Qin, S., ‘Wu, J, Wang, X. etal 1997, Expression of Foreign Gene it Laminaria japonica, The Marine Biology of the Sout China ‘Sea, Morton, B. ed. Hong Kong University Press, Hong Kong, p. 3-11, all of which are incorporated by reference herein, [0016] In the presen invention the organism of interest is seaweed and in particular the pacific giant kelp. Giant kelp is 8 species af marine alga found on the pacific coast of North ‘and Central America, I beuins life as amieroseopic spore but Jnmany species they grow up to 6D meters (270 fect with the "upper fronds forming a dense canopy at the surface. Studies siggestkelp fronds may grow at 1-2 feet perday which makes an ideal species to produce large amounts of hydrocarbons jin a very shor period of time. The giant kelp is perennial plant that isa renewable resource for hydrocarbon production and biofuel generation. See, for example, Copping. etal 2008, “Techno-Economic Feasibility Analysis of Oilshore Seaweed Fanning for Bioenergy and Biobased products, Independent Research and Development Report, Battelle Pacilic Northwest Division, incorporated by relerence herein. “The fronds are harvested and subsequently processed 10 ‘exict the hydrocarbons and other chemicals of interest Although the pacific giant kelp is prefered in the present Jnvention many other species of seatced can be used that are indigenous to various geographic regions throughout the ‘word to maximize hydrocarbon production, Extraction of the hydrocarbons produced by the seaweed can be accomplished by several methodologies suchas pressing and solvent extrc- tion, Solvent extractions the preferred method of recovery in the present invention. Under sitable condition 70% ot ydro- ‘carbons can bereleased by 30 minutes of contact with hexane. Hydrocarbons ean be continuously extracted with proper agi tation and recovery methodologies known in the prior at. Supercritical fd (SCF) extraction can also be applied. Flu- Is such as carbon dioxide (CO2) alow rapid extraction is nontoxic, inexpensive, easly removed from extract and reus- able. 10017] _ Hydrocarbons bined in hexane extraction of BB modified seaweed can be drvetly combusted, however for performance in internal combustion engines, the oil must be modified by processes such as pyrolysis and catalytic erack- ing. rade hydrocarbons of BB genetically modified seatvoed ‘can be converted t gasoline, light eyele oil, heavy cycle oil, land coke by subjecting the extract to catalytic cracking as has been taught by Kitazato etal, Sekiy Gakkaishi 32:28, The May 13, 2010 yields of gasoline obtained by catalytic cracking of algal lnydrocarbons are comparable to yields obtained fom peteo- eum, Also the gasoline produced has sufficiently high octane numbers Zor direct use in transportation vehicles [0018]. The invention herein is describod by examples anda particularly desired way’ of practicing the invention bas boca ‘eseribod. However, the invention a claimed herein is not limited to that specific description in any manner. Plements and features. described in conjunction with a particular ‘embodinient are not Limited to use therewith and may be uscd separately or in conjunetion with the other embodiments closed herein, Equivalence to the description as hereinater ‘claimed is considered tobe within the scope of protection of {his patent such as the use micro alga hydrocarbon producing szenes cloned into to land base plant life to generate hyde carbons for biofuels such as fast growing bamboo plants. ‘What is elaimed is 1. A method of producing «liquid hydrocarbon biofuel comprising ‘diving genetic material ia macroalgae Borryococcus ‘raunii which encodes cell to produce hydrocarbons; isolating and purifying ssid genetic material from the ‘mien algae! cloning sid genetic material into designated macroalgne seaweed species to create penetcally modified seaweed! ceximcting Hydrocarbons generated by ssid genetically ‘modified Seaweed through photosyntbesis; and processing said extracted hydrocarbons as liguid biofuel 2. Themethodof claim I wherein said macroalgne seaweed species is Macrocystis pyrifera, '3. The method of claim 1 wherein said hydrocarbons are extracted though supercritical fui extraction, 4. The method of claim 1 ssherein said hydrocarbons are extracted with iguid hydrocarbon solvents '5 The method of claim 4 wherein said liquid hydrocarbon solvents are hexane 6. The method of claim 1 wherein said hydrocarbons are ‘extracted through high pressure pressing. 7. The method of claim 1 Wherein said processing is achieved by catalytic cracking. 8. The method of claim 1 whervin said processing is achieved by pyrolysis 9. The method of elaim 1 wherein said hydrocarbons have carbon chai lengths inthe range of 5 to 60 carbons. 10. The method of claim 9 wherein said carbon chain Jeng is in the range of 20-48 carbons. T1.Themethod of claim further including subjecting said liquid biofuel to catalytic conversion. 12, The method of elaia 11 wherein the catalytically con- verted biofvel is suitable for use in transportation vehicles 13, The method of claim 11 wherein the catalytially con- vert biofuel is witable for use in chemical feed stock. 14, A method of producing a liquid hydrocarbon biofuel comprising identifying genetic material ina stain of the macroaleae Borrvococeus braun, which encodes eels to preduce hydrocarbons: isolating and purifying suid genetic material rom the mac- roaliie; cloning said genetic material into aspocies of Macroeystis _puriferato produce modified seaweed: ‘extracting hydrocarbons generated by s ‘weed through photosynthesis; and processing sad extracted! hydrocarbons aliquid biol ‘modified sea-US 2010/0120111 AI 15, The method of claim 10 whereia ssid stain of Botryo- coceus brawl isan. stain. 16. The method of claim 14 wherein said strain of Borryo- ‘coceus braun 2B sean, 17. The method of claim 14 wherein said strain of Borso- ‘coccus Brawn aL stain, 18, The method of claim 14 wherein said liquid biotel is suitable for use in tansportation vehicles. 19. The method of claim 14 wherein said liquid biofuel is suitable for use in chemical feed stock, 20. \ method of producing liquid hydrocarbon biofuel suitable for use in transportation vehicles comprising: May 13, 2010 isolating and purifying said genetic material from the ‘micro ales cloning std genetic material into «species of Macrocystis prrifera to produce modified seaweed: extracting hydrocarbons generated by stid modified sea- ‘weed through photosynthesis by solvent extraction: rocesig su extracted hydrocarbons as igi bof catalytically converting said liquid biofuel