US 2010.0236137A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0236137 A1 Wu et al. (43) Pub. Date: Sep. 23, 2010 (54) SYSTEMS AND METHODS FOR PRODUCING (60) Provisional application No. 61/099,503, filed on Sep. ECOSAPIENTAENOIC ACID AND 23, 2008. DOCOSAHEXAENOIC ACID FROMALGAE Publication Classification (75) Inventors: Benjamin Chiau-pin Wu, San Ramon, CA (US); David Stephen, (51) Int. Cl. Davis, CA (US); Gaye Elizabeth CI2P 7/64 (2006.01) Morgenthaler, Woodside, CA CIIB I/O (2006.01) (US); David Vancott Jones, CIOL L/88 (2006.01) Woodside, CA (US) AOIK 6L/00 (2006.01) (52) U.S. Cl. ................... 44/385:554/8: 435/134: 554/1; Correspondence Address: 119/215 JONES DAY 222 EAST 41ST ST (57) ABSTRACT NEW YORK, NY 10017 (US) Provided herein are systems and methods for producing (73) Assignee: LiveFuels, Inc., San Carlos, CA eicosapentaenoic acid (EPA) and docosahexaenoic acid (US) (DHA) and/or derivatives and/or mixtures thereof by growing algae that produce the oils containing EPA and/or DHA and/ (21) Appl. No.: 12/731,024 or derivatives and/or mixtures thereof, harvesting the algae with fish in one or more enclosed systems, and then process (22) Filed: Mar. 24, 2010 ing fish to separate and purify the EPA and/or DHA. The multi-trophic systems provided herein comprise at least one Related U.S. Application Data enclosure that contains the algae and the fishes, and means for (63) Continuation-in-part of application No. 12/565,612, controllably feeding the algae to the fishes. Also provided filed on Sep. 23, 2009. herein are the lipid compositions extracted from the fishes. CO2 Source 2OO 250 -- O o Algae 2f1 O o Algae \ O (222 Algae Enclosure Passageway Fish Enclosure 210 240 220 1 - 221 N. Cl3 Fish Gathering Device 270 Nutrient Source 260 Fish Conveyor 280 Fish Processing Plant 300 Lipid Separator 400 Grid Purifier Patent Application Publication Sep. 23, 2010 Sheet 1 of 3 US 2010/0236137 A1 CO2 Source 2OO 250 O o Algae ? 211 O o Algae \ sts to O (222 Algae Enclosure Passageway Fish Enclosure 210 240 220 C 1- St. 9-) 221 N. C Fish Gathering Device 270 Nutrient Source 260 Fish Conveyor 280 Fish Processing Plant 300 Lipid Separator 400 FIG. 1 Ond Purifier Patent Application Publication Sep. 23, 2010 Sheet 2 of 3 US 2010/0236137 A1 FIG 2 Patent Application Publication Sep. 23, 2010 Sheet 3 of 3 US 2010/0236137 A1 S S. C120 methyl laurate C140 methyl myristate C141 methyl myrtistoleate (cis-9) C150 methyl pentadecanoate C15:1 methyl pentadecinoate (cis-10) C160 methyl palmitate C16. 1 methyl palmitoleate (cis-9) C170 methyl heptadecanoate C171 methyl heptadecenoate (cis-10) C180 methyl stearate C18.1 methyl oleate (cis-9) C18.2 methyl linoleate (cis-9,12) C18. J methyl y-linolenate (cis-6,912) C18J methyl ?inolenate (cis-9,12,15) C18.4 methyl Stearidonate (cis-69, 1215) C200 methyl arachidate C20:1 methyl eicosenoate C20:2 methyl eicosadienoate (cis-11, 14) C20 J methyl eicosatrienoote (cis-8,11,14) C21:0 methyl heneicosanoate C20 J methyl eicosatrienoate (cis-11, 14, 17) C20: 4 methyl arachidonate (cis-5,8,11,14) C205 methyl eicosapentanoate (cis-5,8,11,14, 17) C220 methyl behenate C22:1 methyl erucate (cis-13) C222 methyl decoSadienoate (cis-13,16) C230 methyl tricosanoate C240 methyl lignocerate C226 methyl docosahexaenoate (cis 4,7,10,13,16, 19) FIG 3 US 2010/0236137 A1 Sep. 23, 2010 SYSTEMIS AND METHODS FOR PRODUCING While still being studied, many experts believe that EPA and ECOSAPIENTAENOIC ACID AND DHA encourage the production of body chemicals that help DOCOSAHEXAENOIC ACID FROMALGAE control inflammation. 0004. The most widely available source of EPA and DHA is oily fish Such as Salmon, herring, mackerel, anchovies, and 0001. This application is a continuation-in-part applica sardines. Fish oil supplements containing EPA and DHA are tion of U.S. application Ser. No. 12/565,612, filed Sep. 23, often made from pelagic oily fish like menhaden, Sardines, 2009, which claims the benefit of U.S. Provisional Applica and anchovies. Alarmingly, these fish resources are disap tion No. 61/099,503, filed Sep. 23, 2008, each of which is pearing at a rapid pace. Leading researcher, Boris Worm, incorporated by reference in its entirety. predicts that 90% of fish and shellfish used to feed people worldwide may be gone by 2048 with present trends. The 1. INTRODUCTION global production of fish oil and fishmeal has reached a pla teau over the past few years. Present methods to manufacture 0002 Provided herein are systems and methods for pro EPA and DHA through fermentation biotechnological pro ducing lipid compositions containing eicosapentaenoic acid cesses have steadily improved, but remain too costly for most (EPA) and/or docosahexaenoic acid (DHA) and/or deriva applications, except as an additive to high-priced infant for tives and/or mixtures thereof by growing algae that produce mula. A new source of inexpensive EPA and DHA is critically those oils, harvesting the algae with fish, and then processing needed. fish to separate and purify the EPA and/or DHA. 2. BACKGROUND OF THE INVENTION 3. SUMMARY OF THE INVENTION 0003. In the early 1980s, the Inuit were found to have 0005 Provided herein are inexpensive and energy-effi unusually low rates of heart disease despite their high-fat diet cient methods and systems for producing EPA and/or DHA rich in fish. Researchers later showed that it was the omega-3 from algae. As used herein the term “EPA refers to eicosa fatty acids that provided the beneficial effects, specifically the pentaenoate, eicosapentaenoic acid, and/or derivatives marine-derived eicosapentaenoic acid, C20:5n-3 (EPA) and thereof including, but not limited to esters, glycerides, phos docosahexaenoic acid, C22:6n-3 (DHA). In addition to ben pholipids, sterols, and/or mixtures thereof. As used herein the eficial effects on cardiovascular disease, EPA and DHA have term “eicosapentaenoate' refers to all-cis-5,8,11,14, 17 since been linked to beneficial effects on arthritis, sleep dis eicosapentaenoate (Formula 1). Formula 1-eicosapentaenoate orders, high blood pressure, high cholesterol, heart arrhyth 0006. As used herein the term “eicosapentaenoic acid mias, coronary heart disease, insomnia, depression, anorexia, refers to an eicosapentaenoate moiety in acid form, or all-cis Schizophrenia, hypertension, and attention deficit hyperactiv 5,8,11,14, 17-eicosapentaenoic acid (Formula 2). Formula 2-eicosapentaenoic acid HO ity disorder (ADHD). It is estimated that 84,000 deaths annu 0007 EPA in fish oil is commonly found as ethyl eicosa ally are attributable to insufficient dietary omega-3 fatty pentaenoate (Formula 3), but EPA is also found in lipids such acids. Danaei, G. et al., 2009, PLoS Medicine 6(4): 1-23. as acylglycerides, phospholipids, and waxy esters. US 2010/0236137 A1 Sep. 23, 2010 Formula 3-ethyl eicosapentaenoate 0008. As used herein the term “DHA refers to docosa 0012. In certain embodiments, provided herein are meth hexaenoate, docosahexaenoic acid, and/or derivatives thereof ods for producing an EPA and/or DHA-containing oil, said including, but not limited to esters, glycerides, phospholipids, method comprising: sterols, and/or mixtures thereof. As used herein the term 0013 (i) growing one or more algae species that produce "docosahexaenoate' refers to all-cis-4,7,10,13,16,19 EPA and/or DHA in a first enclosed-container system or docosahexaenoate (Formula 4). open-pond system; Formula 4-DHA 0009. As used herein the term “docosahexaenoic acid” 0014 (ii) harvesting said algae, comprising controllably refers to a docosahexaenoate moiety in acid form, or all-cis feeding said algae to one or more Zooplankton and/or fish 4,7,10,13,16,19-docosahexaenoic acid (Formula 5). species that feed on said algae in said first enclosed-container Formula 5-docosahexaenoic acid 0010 DHA in fish oil is commonly found as ethyl docosa system or open-pond system, or in a second enclosed-con hexaenoate (Formula 6), but DHA is also found in lipids such tainer system or open-pond system, wherein said fish species as acylglycerides, phospholipids, and waxy esters. feed on said algae and/or Zooplankton; and Formula 6-ethyl docosahexaenoate 0011. In certain embodiments, provided herein are meth 00.15 (iii) extracting EPA- and/or DHA-containing lipids ods that comprise cultivating autotrophic EPA- and/or DHA from the fish, wherein said EPA- and/or DHA-containing producing algae ("EPA and/or DHA algae'), harvesting them lipids are processed to concentrate and purify EPA and/or with filter-feeding organisms such as fish, and then convert DHA ing the fish to EPA- and/or DHA-containing oils and fishmeal using conventional reduction industry practices. In certain 0016. In one embodiment, the growing step and the har embodiments, methods provided herein avoid standard algae vesting step are carried out simultaneously in said first dewatering and drying steps, which can be expensive interms enclosed-container system or open-pond system. In one of capital and energy. These steps typically require centrifu embodiment, the growing step and the harvesting step are gation and oil extraction with organic solvents. In certain carried out Successively in said first enclosed-container sys embodiments, the methods can further comprise providing a tem or open-pond system and said second enclosed-container multi-trophic system wherein the algae are controllably fed to system or open-pond system, respectively. the fish, while the fish is growing from fry to juvenile, from 0017. In certain embodiments, the harvesting step com juvenile to adult, from fry to adult, or growing adult to larger prises transferring a portion of a population of the fish or the sizes or grow-out.