NZ618123B2 - Algal lipid compositions and methods of preparing and utilizing the same - Google Patents

Abstract

process of making an algal biomass comprising at least 67% total fat comprises culturing an algae in two or more types of culture medium sequentially, culturing the algae in a culture medium comprising a carbon source, yeast extract, a magnesium source, and a calcium source; and subsequently, culturing the algae in a culture medium comprising a carbon source, a yeast extract, a nitrogen source, a phosphate source, a magnesium source, a calcium source, and 4 g/L or less of sodium chloride where the nitrogen and phosphate are in a ratio of 50:1 to 4:1. uring the algae in a culture medium comprising a carbon source, a yeast extract, a nitrogen source, a phosphate source, a magnesium source, a calcium source, and 4 g/L or less of sodium chloride where the nitrogen and phosphate are in a ratio of 50:1 to 4:1.

Description

PCT/U82012/046696 ALGAE. LIPID COMPOSITIONS AN!) METHODS OF PREPARENG AND UTILIZING THE SAME This Application claims priority to US Provisional Patent Application Serial No. :3 (ii/507,390 filed 13 July 201 it, hereby incorporated hy reference in its entirety.

FIELD OF THE ENVENTEON This invention relates to compositions comprising high lipid content algae and methods of making and utilizing the same. ln particular, the invention relates to high lipid it) content algae biomass and algal lipid materials d from the same. methods ofnialiing the same, as well as to hiolitels (ea, hiodiesel) and dietary compositions (e.g., animal feeds) sing or made from the same. Compositions and methods of the invention lind use in a variety of anplieations including biofuel, dietary (ego, human and animal nutrition): therapeutic as well as research applications.

BACKGROEIND (FF THE INVENTION Within the last several years, the production ofhiohiel (e. 0’0 from algae has .6 hiodiesel) been an area of interest. in part, this is due to high quality agricum tttral land not being ed to grow algae (algal biomass). However, commercial production of bioluel t e.g, hiodiesel) from algae has remained a nge. in addition over the last fifty years, approaches toward providing animal nutrition have changed. No longer are animals fed whatever forage or other material that may be available. instead, the diets of s are closely red for total nutrition value and cost. Very often. animals on ic diets are red for quality and performance characteristics with the mttritional components of the feed being adiusted to maximize nutrition value or” the feed and optimization of animal performance characteristics.

However, cost is a critical factor. There is a continual search for ffective animal feedsg not only to sustain animalsq hut in many cases to cause enhanced growth and value.

LA! we SUMMARY OF THE lNVENTlQN The it vention relates to compositions sing high lipid content algae and methods ofmalring and utilizing the same. in particular, the invention relates to high lipid PCT/U82012/046696 content algae biomass and algal lipid materials derived from the same, methods ot‘making the same, as well as to biolircls (eg, biodiesei) and dietary compositions (cg, animal feeds) conmrisiug or made from the same, Compositions and methods ot‘tlie invention find use in a variety of ations including biofuel, y leg, human and animal nutrition), U1 therapeutic as well as research applications.

Accordingly, the invention provides a s of ng an algal biomass comprising a desired, high fat content tog, at least 67% total fat) comprising culturing an algae under culture conditions sufficient to provide an algal biomass comprising a desired, high fat content. The ion has identified culture ions under which it is possible to obtain l 0 an algal biomass comprising a desired level of total fat (eg, at least 6796 total fat). The invention is not limited to the total fat content (eta, by weight) of an algal biomass generated according to the invention. in a preferred embodiment, an algal s generated and/or used aceording to the invention comprisesa tat content of at least 679/6er weight. However themyention also des compositions and methods of generating an algal biomass containing greater {ega ,f5reater than 689-6 r than 69%, r than 70%, greater than 7196o, greater than 7296, greater than 739’A), greater than ‘4796, greater than 7596, greater than 76%, greater than 7/9”A), greater than 7896, greater than 7996, greater than 8096, r than 8l96a, greater than 8296.greater than 85%,, or more) or lesser (eg, about 6696, about 659/o, about 6496, about 63%, about 6296, about , about 6096, about 5996, about 5896, about 5796, about 5696about 559.6, about 549/0, or loss) amount ottotal tat lodeed, methods and compositions described herein can be utilized to generate an algal biomass containing any desired level ottotal fat content. in some embodiments, the algae biomass is cultured in two or more types of culture medium in a sequential manner. For example, in some embodiments, one culture medium of the two or more culture medium contains 50 g/L of a L! carbon source, about 75 g/L yeast extract, about 0‘ l5 g/L magnesium sulfate, about Ojlfr g/L calcium chloride and lllfi g/L magnesium chloride. The invention is not limited by the carbon source indeed a variety oi carbon sources may be usedincluding, but not limited to, carbohydrates such as glucose, fructose, , saccharose, maltose or soluble. starch as well as oleic acid, tats such as soybean oil, molasses, glycerol, ‘1nannitol. and sodium acetate, (,2 cotton seed llour, ol, molasses and corn steep liquor. in some embodiments, another e medium ott‘i.to two or more culture medium contains 50g/L of a carbon source about 7.5 sol ye:ast ex tract, about 4.0 g/L magnesium sulfate, about l g/L urea, about 2 g/L m chloride, about 2 g/L magnesium chloride and about (i25 g/L monopotassiun’r phosphate. in some embodiments, one culture medium ofthe two or more culture medium contains a PCT/U82012/046696 carbon source, yeast extract and sea salt. in some embodiments, and as described , algae are cultured a first e medium leg, ccntaining glucose, yeast t and sea salt); erred into and incubated in a second culture medium leg.y containing glucose, yeast extract. magnesium sulfate, calcium chloride and magnesium cl’tloride); and transferred U1 intu and incubated in a third culture medium (ring, cunlainlng glucose, yeast extract, magnesium sulfate” urea, calcium chloride, magnesium chloride and monopotassium phosphate). ln some embodiments, one ofthe e mediums is supplemented with a. fed— batch feed. in a preferred embodimenn the third culture medium is supplemented with a led— batch feed. The invention is not limited by the type, or duration,” oiled-batch feed utilized. l 0 in some embodimentsfi the fed-batch feed comprises urea and monopotassium phosphate.

The invention is not limited by the amounts and/er ratios of media components used in the cultures. Examples that may be utilized as components of each of the various media leg, first culture media, secnnd e media. batch media and ted-batch media) are described in detail herein, ln some embodiments. the algal biomass is harvested from a e leg, from a third cultur medium) between 12—24 hours after ion of the fed-batch process. in some ments, the algal biomass is harvested from the third culture medium after all cf the nutrients have been removed/consumed from the medium. The invention is not limited by the way in wl oh the algal s is harvested. indeed, a variety of ways may be used to t the biomass including, but not limited to, the methods described herein. in some embodiments, the algal blurnass is harvested via ccntritirgation. in some ments, the culture medium comprising the algal biomass is chilled prior to harvesting the algal biomass.

The invention is not limited by the temperature to which the culture n'lcdium sing the algal biomass is chilled prior to ting. indeed, a variety of temperatures may be used including but not limited to, those described . in some embodiments? the culture L! medium comprising the algal biomass is chilled to between about 5. and 35 C. The invention is net limited by the type of algae used in the invention , a variety ofalgae may be used (eg. independently or in combination) including, but not limited to, these described herein. in some embodimentsj the algae is a strain or species from the genus (.hlor'ellu, the genus Schifzadzytr'itmz, or the genus Cryptizecodinimn. in a preferred embodiment, the algae (,2 is Schizochyzrium u.“imacirtum. in some embodiments the first culture medium contains about 50 g/L glucose. about it) g/L yeast extract and about cl— g/L sea salt. in some embodiments the second culture medium contains about 50. g/l. glucose. about 75 gill. yeast extract, about (l.l5 g/L magnesium sulfate, about (1.15 g/l calcium chloride and (llS g/L magnesium chluride. in some embodiments, the third culture medium cunt‘ains about 50 g/L glueoset PCT/U82012/046696 about 7.MgL Least extract, about 4.0 g/L magnesium e, about i g/L urea, about 2 g’L calcium chloride, about 2 g/L magnesium chloride and about 0.25. g/L monopotassium phosphate. in some embodiments, the culture conditions comprise running the algae cu ltur3. at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about U1 lilo/LL. in some embodiments, the third e meriiurn’< e.g, the culture media present at the time olmoL[21tion oi a main tor Leg... 7'13 ,.t)l)l3 L. ill)000 L 256,000 L vessel» contains medium with an initial ratio of nitrogen {N}:phosphoms (Pfipotassium (K) of 46: l3:8.5. In a red embodiment, the N3P1K ratio is the same in the batch and fed~l3atoh cuiiure modes. in some embodiments, the ratio of magnesiumMg):calcium (Cat)is 3: lin 1 0 culture media usedin both batch and LlL nroo3s, at LlLoL gh higher . 4:1 to: l, or more) and lower (eg, 2.5:1, 2:1. 1.521, or lower) ratios may he used. in another embodiment, the ratio of chloride (CTlEjlzsullate ($04)} of l:i is used in culture media used in both batch and tied-hatch modes, although higher (eg, 2:], 3:l, 4: 1, 5: L or more) and lowe (eg, l:?., 1:3. 1:4, L5“, or lower) ratios may be used. In some embodiments, the ratio of e (804:3:phosohate (P04) in media at the time of inoculation ofa rnain l‘er’rnentor (eg. 70,(300 L, ii) L,L256LOOG L vessel)(“\V is: 16:1, although higher (cg, 20:1, 25:1, 30:1, 32:1, or more) and lower {3.0 .L., lt):l, 8:1.5.:l.3:,l or low3r}, ratios new be us3Ll. in some embodiments, the total ratio of sulfate (SO4):phosphate (PO4) that has been batched and fed at the end of a iull culture (e0L iist seed stage, second seed stage and L including inoculun‘LLt main fortnetnor out turn.s) that ganrath an algal hiLLant soofltainiug a d fat content (cg, greater than 67% fat; is5 although higher Log, 5. 5:,1 57: l 6: l 7: 1 8:1 or higher) anr‘lluwoi (cc.2., 51, 4.5.,1 4: i 5: l, or 1cowr) ratiosmay he use(l. in some embodiments, the ratio of chloride ((312)):phosphate (P04) in media at the time ofinooulation of a main fermentor (e.g., 70,000 L, lZO, ll0C3 l- 256,GOGL.VL.essl)is L o laithough , higher Leg, ‘20:}, L! 25:1, 302i, 32:1, or more) and lower Leg, l0: l, 8zl. 5:1, 3th or lower) ratios may he used. in some embodiments, the total ratio of chiioritie (€32) :phL hate (3’04) that has been hatched and fed at the end or a hill culture to.g, including inoculurn, first seed stage. second seed stage and main ferntentor cultures) that generates an algal biomass containing a desired fat contentLog.,greater than ()7A) fat) is 5.3:1, although higher Leg. 5.5:l, 5.7:1. 6:1, 7zl, 8zl or (,2 higher) and lower Leg, 5:1, 45:21, 4:1. 3:i, or lower) ratios may he used.

The invention also provides an algal biomass having a desired, high fat content Leg. total fat cont-Lnt of at least 67% by weight). in some embodiments, the biomass comprises about 17ll~250 nit/"g Liocosahexaenoio acid (DHA) and/or about l5 (1400 trig/g palnntie acid in some embodiments, the ion provides a lipid oornposition. a food product or other PCT/U82012/046696 al comprising the algal biomass (eg, {tried algal biomass) or a component f (eg, a fatty acid component thereof} in some etnbociiments, the aigai biomass tag, a dried aigai biomass (gig, generated according to a method described herein» contains a desired amount of total fat and/or other components tag. gteatei than about 689/1, total fat, greater U1 than about 69% total fat, greater than about 70% totai fat, greater than about 7l9/t'1 total fat, greater than about/2941 total fat greater than about 739/21 total fat greater than about 7.49/13totai fat, greater than about 75% total fit, greater than about 76% total fat, r than about 77% total fat, or greater than about 7894:; total fat). In some enib )tii'ments, an aigal biomass of the inventiont:‘g, containing ”reater 9/E1 total tar3 is tlried such that the s contains l 0 less than 5% moisture (eg less than 4.5% moisture, iess thaii=1% moisture, iess than 315% moisture, less than2% moisture, less than9 5.9/0, moisture, less than 2% moisture or tess than 1.5% moisture). in some embodiments, an algal biomass of the invention (eg, a {tried biomass containing less than 5% moisture) contains about ”ti-250 ing/g or more tineosahexaenoic acid (DHA) (eg, about Wit—180 ing/g DEA, about 180—190 trig/g DHA, about 190200 trig/g DH/‘t, about 200thi trig/g DHA, about 210220 trig/g DHA, about 220- 230 trig/g/-o DEA, about 230-240 trig/g Di—LA about 249-250 nag/g Di—iA, or more than 250 trig/g DHA) insome embodiments, an aigai hiontass of the invention (eg, a dried biomass containing less than 5% moisture) ns about l50—400 or more paintitic acid (lUPAC nanie: hexadettanoio acid (e.g, about ) trig/L9: about 200225 trig/g about 225-250 trig/g, about 250-275 nag/g, ahont 275-3130 nigxp, about 306-325 inc/g about 375- 350 tog/g, about 3503175 nag/g, about 375—400 trig/g, or more than 400 ing.«’g)), l n some embodiments, an aigai biomass of the invention (cg, a dried biomass containing less than '9/13 moisture) contains about 300-600 nig/g or more totai fatty acids (eg, about EGG-350 ing/g? about 0 nag/g, about 400—450 nig/g, about 450600 nag/g, about 500-559 trig/g, L! about 550-691) 1“fig/e or more than 600. mg; 2" fatty acidsi) in some embodiments an algal biomass of the invention tag, a dried biomass containing less than 5943 moisture) contains less than ahonti{5% n (e.a, less than about l)0 protein, toss than about 13'721 protein, less than about 12% protein, less than about t 1% protein, less than about M21941 n, less than about 990 protein, or less than about 8% protein), ln some embodiments, an algal (,2 biomass or component tltereot‘of the invention is used in preparing biofnei (eg. biodiesei). in some embodiments, an algal s or component thereofoi‘the invention is used in preparirtigz-111011 proouetteg an i feeder feedtomponent).

BRIEF DESCREPTEON OF THE DRAWINGS PCT/U82012/046696 Figure 1 depicts data generated during Sarge scale, i‘ieterotrophie algae biomass production according to aspects of the invention.

Figure 2 shows the fatty acid profiie of aigae biomass harvested from l, independent large scale algal cuitures.

Ui Figure 3 shows a eemposite fatty acid proiiie eta harvested biomass utilizing materials and methods described herein.

BEFENI'HQNS As used herein, "phospholipid" refers to err c compound having the i‘oilowing i0 general stmemre: wherein RE is a fatty acid residue, R2 is a fatty neid residue or MOE-L and R3 is a «H or nitrogen containing eompound ehoiine (HOCHg’CHENYCHmOH’), ethanoiamine (HGCHzCHgNiig), innsitoi or serine. RE and R2 cannot anennsiy he OH. When R3 is an ~OH; the compound is a diaoyiglyeerophosphaie, while when R3 in a nitrogen—containing compound, the compound is. a atirie such as ieeithinF oephalin, phosphatidyi serihe or piesmalogen, A11 ”ether olipid” as used herein refers to a phesphoiipid having an ether bond at position i the giyceroi backbone. Examples of ether phosphoiipids inoinde, but are not iimited to” eh yieeyiphosphatidyicholine , ly50--aliiyiaeylohosphatidyiehoiine (LAAPC‘), and nihyiaeyiphosphatidyleti’ianoiamine {AAPE}. A, ”non—ether phosphoiipid” is a phosphoiipid that does not have an ether bond at position i of the nl backbone.

As used herein, the term "omega-3 fatty acid" refers to peiyunsaturaied fatty acids that have the. finai doubie bond in the arbon chitin between the third anti fourth carbon PCT/U82012/046696 atoms from the methyl end of the molecule. iting examples of omega—3 fatty acids e, 5,8,1l,l4,l7«eicosapentaenoic acid (EPA), 4,7,1ll,l3,l(3,19ll-docosahexanoic acid (DHA) and 7,} l),l3,lt),l9~docosapentanoic acid (EPA).

As used herein, the terms "triacylglycerld" "triglyceride” and "triacylglycerol ’ and U1 “TAG” refer to is an ester derived from glycerol and three fatty acids, wherein "fatty acid" refers to a carhosylie acid with a long unbrancliecl aliphatic tail (chain), which is either saturated or unsaturated. l’alniitic acitl is one, non~limiting example of a. triacylglyceride.

As used herein, the terms "9/6 w/W {weight/weight) ” and "w/w %” and grammatical equivalents re or to the amount (percent) of a given substance in a composition on it) tzweight basis, For example, a composition comprising 50% w/W phospholipitls means that the mass ofthe phospholipids is 50% of the total mass of the composition (ie 50 grams ot‘phospholipids in lOO grams of the ition, such as an oil).

As used herein the term "algae” refers to a unicellular or rnulticellular organism ly classified as plants, occurring in fresh or saltwater, autotrophic or heterotrophic, hut l5 that lack true stems, roots, and leaves. As used herein the term "lietertitronhic” refers to an organism that cannot size its own food and is dependent on organic substances (cg, complex anal/or ‘imple organic nces) for nutrition, Thus, the term otronhic algae" refer to an algae that cannot synthesize its own food and is dependent on organic substances for nutrition. As used herein, the term "atitotrophic" refers to an organism capable ol‘synthcsiz its own food from inorganic nces, using light: or chemical . The use ol‘tlic term "algal” also relates to microalgac and thus encompasses the meaning of Hnncioalgal.‘41 fl"The term H " , "' 17.. v .17 . . ". .. algal ition refers to any composition that comprises algae, such as an aquatic composition, and is not limited to the hotly ofW ater or the culture in which the algae are cultivated. An algal composition can he an algal culture, algal hiomass, a [\3 L!i concentrated algal culture, or a (lewatereil mass of algae, and can he in a liquid, semi—solid, or solid form. A non—liquid algal composition can he bed in terms of moisture level or percentage weight ot‘the solids. An "algal culture” is an algal composition that comprises live algae, The term "algae" includes niacroalgae (commonly known as seaweed) and microalgae.

(A! .

, As used herein, the terms ”algal biomass" or ”biomass" refers to a collection or mass of algal cells grown in a given area or ecosystem at a given time. The area or ecosystem may he a naturally occurring, environment (cg, body of water) or a synthetic environment tag, in a l‘errnentor or hioreactor leg, open or closed».

WO 10090 PCT/U82012/046696 As used herein, the term "total fat” refers to the sum ol‘triglyeerides, phosphoiipids, ‘ present in a material. For example, ”total fat” content oiian algal biomass refers to the sum of triglycerides, phospholipids, wax ester, and sterols present in the biomass. in on, total fat includes both saturated and unsaturated fats, U1 As used herein, the term "preservative" refers to an agent that extends the storage life offend and non-food products by retarding or preventing deterioration of flavor, odor, color, texture, ance nutritive value. or safetyu A preservative need not provide a lethal, irreversible action resulting in partial or complete microbial eell destruction or ineapacit’at‘ion.

Sterilants, sanitizers, disinfectants, sporioides, oes and tttbereuloeidal agents provide l 0 such an irreversible mode of , sometimes referred to as "bactericidal” action. in contrast, a preservative can provide an inhibitory or haeteriostatie action that is reversible, in that the target microbes can resume multiplication it~ the preservative is removed. The principal ences between a preservative and a sanitizer primarily involve mode of action (a preservative ts growth rather than killing microorganisms) and exposure time (a presetyative has days to months to act whereas a sanitizer has at most a few minutes to act}.

As used herein, the term "yeast" and “yeast cells" refers to ettlraiyotie microorganisms classified in the kingdom Fungi, having a. cell wall, eell membrane and intracellular components. Yeasts do not form a specific taxonomic or phylogenetic grouping. tly about 1.500 steeies are known; it is estimated that only 0/6 of all yeast species have been describedrThe term "yeast" is often taken as a, synonym for S. corsair/Image, but the phylogenetic diversity ofyeasts is shown by their placement in both divisions Asoomyeota and Basirliomyeota. The g yeasts ("tine yeasts”): are fied in the order Saeeharomyeetales. Most species of yeast reproduce asexually by budding, although some reproduce by binary n. Yeasts are unicellular, although some species become L! multieeilniar through the formation of a string; of ted budding cells known as prettdoiz)?phae, orfizise ill/‘tvr’piicie. Yeast size can vary greatly depending on the species, typically measuring 3___4 pin in diameter, although some yeast can reach over 40 pm.

As used herein, the terms ”st:lenium—enriehed yeast" and "selenized yeast” refer to any yeast leg, Saeeharomyees eerevisiae) that is cultivated in a medium containing inorganic (,2 selenium salts. The t invention is not limited by the um salt used. indeed, a variety niuni salts are contemplated to be useful in the present invention includinO, but not limited to, sodium selenite, sodium tte, cobalt seienite or eohalt selenate. Free selenomethionine (’eg” not associated with a cell or yeast) can also he used as the um sottree for selenium enriched yeast as yeast does incorporate this form of selenium. During PCT/U82012/046696 cultivation, e of the chemical similarity between selenium and sulfur, yeast incorporate selenimn in place of sulfur in what are normally Stiller-containing organic compounds within the cell, A seleniuinwcontaining compound in such yeast ations is selenomethionine which will be present in a form that is incorporated into polypeptides/proteins. The amount U1 ol‘total cellular selenium present in the form of selenomethiunine in such preparations will vary, but can be between it) and 109%, 20-60%, 50-75% and between 60 and 7594;. The remainder of the organic selenium in selenized yeast ations is predominantly made up of inteimediates in the pathway for selenomethionine hiosynthesis. These e, but are not limited to, selenocystein’e, selenueystalhionine, pelenuhomuey’steine and seleno- l 0 adenosylselen methionine, The amount of residual inorganic selenium salt in the finished product is generally quite low t< 2%). However, the present invention is not limited by this percentage, as preparations that contain more leg between 2 and 70%) or less (e.g., between (ll and 2%) than this percentage are also encompassed by the ion.

As used herein, the term ”SElL—l’LEX” refers to a dried, nonviahle selenium—enriched yeast leg, Sacehoromyces cerevisiae of accession number CNCM 1—3060, tion Nationale De Cultures De Mieroorganismes (CNCM), lnstitut Pasteur, Paris, France) cultivated in a fed-batch fermentation that provides incremental s of cane es and selenium salts in a manner that minimizes the detrimental effects of selenium salts on the growth rate of the yeast and allows for optimal ineorpora ion of inorganic selenium into cellular organic material al nic selenium is eliminated (eg, using a rigorous washing process) and does not exceed 2% of the total selenium content.

As used herein, the term "organic selenium” refers to any organic compound n selenium replaces sulfur Thus, organic selenium can refer to any such compound hiosynthesized by yeast, or it can refer to free organic seleno—compounds that are chemically L! synthesized, An examnle of the latter is free selenomethionine, As used herein, the term "inorganic selenium" generally refers to any selenium salt (eg, sodium selenite, sodium selenate, cobalt selenite and cobalt selenate). There. are also a variety of other inorganic selenium sources ( See e,g., those listed in the Merck index). zed yeast may be generated using a source of inorganic selenium ing, but not (,2 limited to, sodium selenite, sodium selenale, cobalt selenite, cobalt selenate, selenic acid, selenious acid, selenium bromide, selenium de, selenium henal‘luoridec selenium oxide, selenium oxylu'omide, selenium oxychloride, selenium oxylluoride, selenium sulfides, selenium romide, selenium tetrachloride and selenium tetrai’luoride.

PCT/U82012/046696 As used herein the term ” east cell wall" also referred to as "YCW" refers to the cell 7 y wall of a yeast organism that nds the plasma membrane and the intracellular components of the yeast. Yeast cell wall includes both the outer layer (mainly mannan) and the inner layer (mainly glucau and chitin) of the yeast cell wall. A function of the cell wall is Ui to provide structure and protect the metabolically active cytoplasm. Signaling and recognition pathways take place in the yeast cell wall. The composition of yeast cell wall varies from strain to strain and according to (frowth conditions or” yeast.

As used herein, the term "purified” or "to purify” refers to the removal ol‘components from a sam lie. For e, yeast cell walls or yeast cell wall extracts are purified by it) removal of non—yeast cell wall components (egu plasma membrane and/or yeast intracellular components); they are also purified by the removal of contaminants or other agents other than yeast cell wall. The removal of nonyeast cell wall components and/or ast cell wall contaminants s in an increase in the percent of yeast cell wall or components thereof in a sample As used herein, the term ”in vivo” refers to studies and“or experiments conducted within a living sm, occurring Within a ical organism.

As used herein, the term ”in Vitro” refers to an artificial nment outside the living organism and to biological processes or reactions that would normally occur within an organism but are made to occur in an artificial nment ln vitro environments can comprise, but are not: limited to, test tubes and cell culture.

As used herein, the term ”high—pcrformancc liquid chromatography” and the term "l—lPLC" refer to a form ofliquid chromatography to separate compounds. The compounds are dissolved in solution. Compounds are separated by injecting a plug of the sample mixture onto the column. EFLC instruments comprise a reservoir of mobile phase, a. pump, an injector, a separation column, and a detector. The presence of analytes in the column efi‘luent is recorded by quantitatively detecting a change in refractive index, U V~VlS absorption at a set ngth, fluorescence after excitation with a suitable wavelength, or electrochemical response As used herein, the term ”scanning electron microscopy” and the term ”SEEM" refer to '30 use of a type oi“ electron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms that make up the sample producing signals that contain information about the samples surface topography, composition and other properties such as electrical conductivity.

PCT/U82012/046696 As used herein, the term ”fixation agent” refers to a chemical that is capable oftixing one substance to another in order to "fix", stabilize, or otherwise preserve the substance in its current form :0 prevent the substance from degrading or otherwise changing. Often, fixation agents are used in scanning electron copy (SEM) to prepare the sample. y Ui fixation agent: as used herein, the terms "primary fixation agent" refers to the first fixation agent used to ”fix" a substance._Seeondary fixation agent: as used herein, the terms ”secondary fixation agent" refers to the second fixation agent used to "fix” a nce.

Tertiary fixation agent: as used , the terms ”tertiary fixation agent” refers to the third fixation agent used to "fix” a substance. it) As used herein, the term ”analyte" refers to an atom, a molecule, a grouping of atoms and/or molecules, a nce, or chemical constituent. An analyte, in and of itself cannot be measured; ; aspects or properties (physical, chemical, hioiogicai, etc.) of the analyte can he determined using an analytical procedure, such as HPLC. For example, one cannot n ensure a "chair” (analyte-cornpnnent) in and of itself, hut, the height, Width, etc, of a chair l5 can be measured Likewise, one cannot measure a inycotoxin but can e the xin fluorescence that is related to its concentration.

As used herein, the term ”signal" is used generally in reference to any detectable process that tes that a reaction has occurred (for example binding of antibody to antigen). Signals can be assessed qualitatively as weii as quantitatively. Examples of tynes of"signais" include, but are not d to, radioactive signals 'liuorintctr'ie signals or metric product/reagent signals.

As used herein, the term "hioavailahility" re fcrs to the fraction ofa molecule or component that is available to an organism or reaches the systemic Cireuiation. When a molecule or component is stered intravenously, its hioavailahility is lit “/0 However, [\3 L!i when a melee do or component is administered Via other routes (such as orally), its hioavailahilily decreases (due to incomplete absorption and first~pass metabolism). in a nutritional setting, bioavailahility refers to the rates of absorption and utiliaation of a nutrient.

Different fonns of the sante nutrient, for example, may have different bioavailabiiities, As used herein, the term ”ef ective amount” re “ers to the amount of a composition (A! t , sufficient to effect beneficial or desired results. An efiectire amount can he administered and/or combined with another al in one or more administrations, applications or s and is not intended to be limited to a particular tornntlation or administration route, 2012/046696 As used herein, the term ”digest" refers to the sion od, ut‘ts, or other organic compounds into absorbahle form; to soften, decompose, or hreals: down by heat and moisture or chemical action, As used herein, "digestive system" refers to a system {including gastrointestinal Ui system) in which digestion can or does occur.

As used herein, the term tuffs" refers to rna.terial(s) that are consumed by mammals (e,g,, humans and animals) and contribute energy and/or nutrients to a manrmal's diet. Examples of leedstufts include, but are not limited to, Total Mixed Ration (TMR), foragds}, pelletts), eoneentratets), premix(es) eoproduett’s), graints), distiller ), l0 molasses, tibert’ls), fodderts) grass<es), hay, kernelt'sft, leaves, meal, solublets) and supplentent(s).

As used herein, the terms "food ment“ "dietary supplement” "dietary supplement composition“ and the like refer to a food product formulated as a dietary or nutritional supplement to he used as part of a diet. Exam lary dietary supplement compositions are bed herein.

As used , the term l" refers to those ot‘kingdom Animalia. This includes, but is not limited to livestock, farm animals domestic, animals, pet animals, marine and freshwater animals, and wild animals.

As used herein, the terms istration” and the term ”athninistering" refer to the act of giving a substance, ing a drug, g, or other agent, or therapeutic treatment to a subject tag, a subject or in Vivo, in Vitto, or ex ViVO cells, tissues, and organs).

Exemplary routes of administration can be through the eyes (ophthalmic), mouth (oral), shirt (topical or transdermai), nose (nasal), lungs (inhalant), oral mucosa (buoeal), ear, rectal, vaginal, by in'eotion (eg, intravenously, subcutaneously, mtratumorally, intraperitoneally, [\3 L!i etef) anti the like.

As used herein, the term "eo—administration" and the term ”eo-administering" refer to the administration of at least two agentt's) or therapies to a snbj eet and/or material (egg, feedstuii’), Co~adtninistration of two or more agents or therapies can be concurrent, or a first agent/therapy can be administered prior to a seeond agenfltherapy.

(A! e , As used herein, the term "treatment" refers to measures taken that facilitate the improvement and/or reversal of the symptoms ol’disease. The term "treatment” refers to both eutic treatment and propl‘tylaetie or preventative measures. For example, subjects that may 1oenefit from treatment with compositions and methods of the present invention include PCT/U82012/046696 those already with a disease and/or disorder as welt as those in which a e and/or disorder is; to he p evented (cg, using a prophylactic treatment of the present invention).

As used herein, the term "at risk for disease" refers to a subject that is predisposed to experiencing a particular disease, This predisposition may he genetic (cg, a particular U1 genetic tendency to experience the disease, such as heritable disorders), or due to other factors (eg age, weight, environmental conditions, exposures to detrimental compounds present in the SthV: onment, etc).

As used herein, the term "disease”, the term ”infection” and the term logical condition or response" refer to a state, signs, anti/or symptoms that are associated with an l 0 impairment of the normal state of a living animal or of any of its organs or tissues that interrupts or es the performance of normal functions, and may he a response to environmental factors (such as malnutrition, industrial s, or climate, including mycotoxicosis), specific infective agents (such as worms, hacteria, or viruses), to inherent defect of the organism (such as various genetic anomalies), or combinations of these and other factors.

As used , the term ”suffering from disease“ refers to a subject (eg, an animal or human subject}, that is experiencing a ular disease and is not d to any particular signs or symptoms, or disease.

As used herein, the term " refers to any detrimental: deleterious, harmful, or otherwise negative tt{s) on a subject, a cell, or a tissue as compared to the same cell or tissue prior to the contact or administration of the toxin/ toxicant.

As used herein, the term "pirannaccuticai compositionH refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitahic for diagnostic or eutic use in Vitro, in Vivo or ex .

L! As used herein, the term "pharmaceutically acceptahie” and the term ”pharn‘iacoiogicaliy acceptable" refer to compositions that do not substantially e more known adverse reactions than known beneficial reactions.

As used herein, the term ”inoculation” refers to the act of introducing a rganism or suspension of microorganisms (cg, algae, yeast, fungi, bacteria, etc) into 1;.) a culture medium. lnocuiation is the act or process or” introducing something into an environment in which it will grow or reproduce.

As used herein, the term "lt’lOCultml" and the term "pre-inoculun1” refer to cells used in an inoculation, such as cells: added to start a culture. m. (a) PCT/U82012/046696 As used herein, the term ”centrifugation" refers to the ting of molecules by size or y using fugal forces generated by a spinning rotor that puts an obj ect in rotation around a fixed axis, applying a force perpendicular tn the axis. The centrifuge werke using the sedimentation principle, where the centripetal acceleration is used to evenly distribute U1 substances oi“ greater and lesser density into ent 'tayers of density.

As used herein the term "concentration" refers to the amount oi" a substance per defined spacer, Ce-ncentration usually is sed in ternn; of tnase per unit ofvoiutne. To dilute a solution, one must add more solvent, or reduce the amount of solute teg. by selective evaporation, spray drying, freeze drying, egg, coneentra etl yeast cell wall extract or i 0 concentrated ntoditied yeast cell wall extract). By contrast, to trate a solution, one must add more solute, or reduce the amount of t.

As used herein, the terrn "layer“ refers to a usually horizontal deposit organized in stratum of a material thrrning an oy erlying part or segment obtained after separation hy fugation in relation with the detteity properties of the material.

As used herein, the tern: ”harvest“ refers to the act of collecting or bringing together materials that have been produced (e.g, bringing together materials produced during yeast productionfu As used herein, the term "drying” refers to spray drying, fieeze drying, air drying, vacuum drying or any other kind of process that reduces or eliminates; liquid in a substance.

As used herein, the term ”spray drying” refers to a commonly used method oi‘drying a substance containing liquid using hot gas to evaporate the liquid to reduce or eliminate liquid in the substance, in other words the material is dried by way of ng or atnrni7ing into a draft, of heated dry air.

AS used herein, the term e—drying" and the term "iyophilization" and the term L! "cryodesiccation" refer to the removal of a solvent from matter in a frozen state by sublimation. This is accomplished hy freezing the material to he dried below its eutectic paint and then prnyiding the latent heat of sublimation Precise control 0f heat input permits drying front the frozen state Without product melt—hack in practical application, the proceee is accelerated and eiy controlled under reduced pressure ions. (,2 As used , the ternr "dry free flowing powder” refers to a free flowing dry powder, eg. a powder that can be poured from a, container” bag” vessel etc without hindrance of large clumps.

As used herein the term "grinding” refers to reducing particle size by impact, shearing, or attrition.

PCT/U82012/046696 As used herein, the term ”sample" is used in a broad sense including a specimen or e ed from any source, as well as biological and environmental samples.

Biological samples may he obtained from animals ding humans) and encompass fluids, solids, tissues, and gases ical samples include blood products, such as , serum U1 and the like. Environmental samples include envirom’nental material such as surface matter, soil, water, crystals and industrial samples.

I) IAILED DESCRIPTION OF THE lNVEN't‘lON This invention relaes to compositions comprising high lipid content algae and l0 methods of making and. utilizing the same. in particular, the ion relates to high lipid content algae biomass and algal lipid materials derived from the same, methods ofntahing the same, as well as to biot‘uels (e.g., hiodiesel) and dietary compositions leg, animal feeds} comprising or made from the same. itions and methods of the ion find use in a variety of applications including biofuel, dietary (egg human and animal nutrition), therapeutic as well as research applications.

Accordingly, in one aspect of the invention, there is ed a process for the ation or" an algal biomass containing elevated amounts tag, on a \v/vv basis) of total fat. For examples, as described herein, in some embodiments, the invention provides a method of generating an algal biomass containing a desired, high level of total fat content (cg, greater than 66% total fat, in contrast to conventional methods that generate algal biomass containing a significantly lower level of total fat content (eg 60% or less total fat».

A great challenge of algal~hascd hiolncl (cg, hiodioscl) is to ensure that the 1oiomass is not made at the expense of more energy than is obtained in the final fuel product. Accordingly, in some embodiments, the invention provides a method of generating an algal biomass [\3 Ln containing r than 65% total fat. in some embodiments, the invention provides a method ot‘generating an algal biomass containing greater than 66% total fat. in some embodiments, the invention provides a method of generating an algal biomass containing greater than (37% total fat. in some embodiments, the invention provides a method of generating an algal biomass containing greater than 68% total fat. in some embodiments, the invention provides (,2 r v a method of ting an algal biomass containing greater than 69% total fat. in some embodiments, the invention provides a method of generating an algal biomass containing greater than 76% total fat. in some en’tbodiments, the invention provides a in ethod of generating an algal s ning greater than 70% (e.g., greater than "ll, 72, 73, '74, '75, 76, 77, 78 79, 8'0, ill, 8;, 83, 84, 35, 86, 87, 88, 89, 93% or mo e) total fat on a 'w/w basis, PCT/U82012/046696 in some embodiments, the method utilizes a closed hioreactor system ( 9g, 21 1131111911101), although the 1011 is not so limited19g , in 391119 unhodiments, open hioreaetors may he utilized). ln a1319-9T9r19d 9111bo<li1neut, growth of an algal T omass ofthe invention is ted under aseptic conditions. in another preferred 911'1hodi111911t alga9 are grown (91g, U1 to generate an algal biomass containing a high fat content (9.5.3.1 greater than 679/6 fat» in a fed-hatch process, in some embodiments, the invention provides a method of culturing algae to produce an algal biomass comprising a desired, high total fat 9111119111 (9g 67% or more total fat) as deseiih9d111 Examines i and14 For 9xa11111i9, in 1101119 embodiments, the i11'v9ntion provides a l 0 method of culturing algae comprising culturing the algae in a stepwise manner so as to produce an algal s eomprisingaa d911nd hisIl1 total fat content (9.1167041 111 more total fat). in some embodiments, a stepwise process for culturing algae comprises thawing 21 stored strain of algae and adding (9g aseptically) the thawed algae to a 1L shake flash eontain medium1‘unprising a carbon source (9g sugar (9g, glueose‘3 ‘3, yeast extract and sea salt. in some embodiments, the carbon source is present in a concentration of 50 g/L, the yeast extract is present in a concentration of it) g/‘L and/or the sea salt is present in a concentration oi‘I-l g/L, in some embodiments“ the ill; shake flask ning algae and medium are maintained at 30 EC and shaken (eg, at about 11111-400 RPM) until surh time that the algae have entered exponential growth phase but have not fully ed the carbon source (9g, sugar (9g, e». ments conducted (luring pment of embodiments of the invention have determined that the algae enter exponential growth but do not fully dc lete the carbon source (9g, sugar (9,511., glueose)) at a time period between 72— 144 hours, Thus, in some ments, algae cultivated in a lL culture flask at 30 C for 72- l44 hours at UG—éllli} RPM ,250 RPM)111 medium 9oinprising a carbon source £11 (91g, sugar (9.15,, 111111111593), yeast extract and sea salt is used to inoculate a first seed stage culture (9.31., in a larger vessel (9g, 40, 27 or iii L vessel/i). in some embodiments, the culture inedir 'usedin a first 599d stage composes a caihon 5011199 (9.0 g, sugar (9g, glut;osel) yeast extract magnesium sulfate, 99131111111 rhloride and/or magnesium 1hlorid9.ln a pi‘efened embodiment the 1.11lture medium used111 9 hrst seed stage comprises about fillg/L (,2 of a carbon souree (9.11., sugar (9.5.3., 11111903911}, about 7.5 gfL 921st extract about {1 15 g/ inagn9siuin suliat9,ahou1 0 l5 g/L caldum chloiid9 ‘ndjorit) 15 g/L magnesium chloride. in some embodiments, the first seed stage cultureis 11111 atlll ( under airflow and agitation conditions so as to maintain dissolved oxvgen at about7iii/.1 (9.11,,8, 9, ll), ll, 12, 13, 149.41), gh lower and higher dissolved oxygen conditions may be utilized. in a prel91‘retl PCT/U82012/046696 embodin‘ient, the first seed stage culture is run at 38 {2 outlet airtlow and agitation conditions so as to maintain dissolved oxygen at about 10%. in some embodiments, the first seed stage e containing ‘ lgae and medium are maintained at 30 C and cultivated until such time that the algae have entered exponential growth phase and at least 20 g/l, of carbon source U1 (eg sugar leg, glucosel) has been consumed but the carbon source has not been fully depleted. Experiments conducted during development ol‘emhodiments ol‘the invention determined that the algae enter exponential growth, consume at least 20 g/L of carbon source (eg, sugar (eg, glucose)) but do not luiiy deplete the carbon source leg, sugar (e.g., gitteose)) at a, time period between 24-48 hours after inoculation of ti e l‘irst seed stage l 0 culture, in some embodiments, algae cultivated in first seed stage culture at 30 C for 2-41—48 hours in medium comprising a carbon source (eg, sugar (cg, glucose», yeast t, magnesium sulfate, calcium de and magnesium chioride are used to ate a second seed stage culture in yet a larger vessel (eg, 2000 L vessel), In some embodiments, the culture medium used in a second seed stage culture comprises a carbon source (eg, sugar (eg, giucosell, yeast extract, ium sulfate, calcium de and/or magnesium chloride. In a preferred embodiment, the culture medium used in a second seed stage culture comprises about 50 g/l. of a carbon source {eg sugar (egg glucose/i), about 7.5 g/L yeast extract, about 0. i5 g/is magnesium sulfate, about 0. l5 g/L calcium chloride and/or ll. i5 g/l..r magnesium chloride. in some embodiments, the second seed stage culture is run at 30 C under airtlow and agitation conditions so as to maintain dissolved oxygen at about: 7--l 5% leg, 8., 9, l0, l l, l2 l3, ill-92;), although lower and higher dissolved oxygen conditions may be utilized. in a preferred cmhmlimcnt, the second seed sta0c e is run at '30 C undo airflow and ion conditions so as to in dissolved oxygen at about iOQ/éi. in some embodiments, tl e second seed stage culture containing algae and medium are maintained at L! 30 C and cultivated until such time that the algae have entered exponential growth phase, and at least 20 g/L ofcarhon source leg, sugar {e.g., glucose» has been consun‘ied, but the carbon source has not been litlly depleted. Experiments conducted during pment of embodiments of the invention determined that the algae enter exponential growth, consume at least 20 g/L ofcarhon source (eg, sugar (cg, glucosell but do not fully deplete the carbon (,2 source leg, sugar leg, glucose» at a time period between 2448 hours alter inoculation of the second seed stage culture. in some ments, algae cultivated in second seed stage culture at 30. C for 24—48 hours in medium comprising a carbon source (eg, sugar (eg, glucoseD, yeast extract, ium sulfate, m chloride and magnesium chloride are used to inoculate a large scale vessel leg, ”@300 L, i20,t300 L, 220,990 L or larger vessel PCT/U82012/046696 (e g, a main fermentorl) containing medium used for thither culturing/termentation of the algae. in some embodiments. upon transfer ofthe s-cond s2ed:tage e to the large scale vessel leg. main fermentor). the culture medium (eg, the hatched medium) present in the latge scale vessel (sag, i’nain ferinentor) comprises a carbon source (2.21> sugar leg.

U1 glucosefi. yeast ex. tract, magnesium sulfate, urea, calcium chloride, magnesium chloride and/or monopotassiuni phosphate. In a preferred ment. the culture medium used in a large scale (e.g.. 70.000 L. 120,000 I... 1220.000 l. or larger vessel , main fermentorll culture ses about ’50 g/L ol‘a carbon source (eg, sugar (e.U about 7.5 g g. . glucosel), yeast t. about 4.0 g/L ium sulfate. about l g/L urea, about 2 g/L calcium l 0 chloride. about 2 ml. magnesium chloride and/or about 0.25 g/l. mono-potassium phosphate. in some embodiments, the large scale culture is run at 30 C under airflow and agitation conditions so as to maintain dissolved oxvgen at about7 -l59/:2 (e9 9 . 8, ll) ll l2. l3, 149/21). although lower and higher dissolved oxygen conditions may be utilized. in a preferred embodiment. the large scale culture is tun at 30 C under airflow and ion conditions so as to maintain dissolv ed oxygen at about ltl%. in a preferred embodiment, the carbon source (e.g., sugar (e.g , glucose»is maintained at to 1:1 tor a period oi time (e.g. l or more days (2g... .2. 3. 4, 5. 6. 7, 8, 9. l0. ll. 12. l3, 14 or more days (ea. using a fed-hatehprocessll.

For example, in some embodiments. after a desired amount of e has been consumed by algaein the large scale t(eg, after about 203C- g/‘L l-l glucose has been consumed by the algae in the large scalev' essel (c.g” atter ill gl of 17lu2os2 has been consumed1) nineosc and tch feeds are started. Experiments conducted during development of embodiments of lVLntitun determined that the fed—batoh foods be added for about 34 hours. although shorter leg. about 32, 28. 24, 20 hours or fewer) and longerafe 36 38 42. 46 so 84 96. l2l8, 1120, l3’2. lit-4, 150’. loll. hours or more) time periodsémay he used. in further L! preferred embodu-nnts upon 2empletion ot the fedbat2h process 2ultivaticn of the alrga2 is continued in the large scale vessels until all nutrients are letuntied/consumed from the medium. Experiments conducted during development 21. embodiments of the invention determined that tl1et1utrients are depleted trom the medium b2tween about l2 and 24 hours after cessation of tl1 fed—batchinrceess. in some 211rbo(iii”tents the algal biomassis harvested (,2 from the large scale culture mediunubroth and utilized as described herein in some embodiments. the large .oale culture broth is centrifuged to obtain the algal biomass. in some embodiments, the large 52ale culture brothis cooled prior to centrifugation. Al tlmugh an tanding of a mechanism is not needed to practice the invention and the invention is not d to any particular mechanism ofaction. in some ments. chilling the culture WO 10090 PCT/U82012/046696 broth increases the density of the algal biomass comprising e evated levels of total fat tag, lipids/oil) and aliows a larger recovery ot’the biomass than is achieved in the absence of chilling the culture. broth ( Sec, e.g., Exampic 3)., The invention. is not. limited by the. temperature to which the large scale culture is chilled prior to centrifirgarion. in some Ui embodiments, the large scale culture is chilied to a temperature between 0-50 C, between 5- 40 Ce 5-25 C, 5-15 C or 5-10 C. 'l‘hns. the invention utilizes both batch and tch modes of culturing algae (egg alone and/or subsequent to a first and/or second seed stage) in order to generate an algal s that: contains a desired fat content (tag a, fat content greater than 67%). The invention is not limited by the individual components present in the media used in either the batch or fed—hatch modes. in some embodiments, culture media present at the time of inoculation ot‘a main fermentor (e.g., 70,000 L, 120,000 L, 220,000 L ) contains medium with an initial ratio of nitrogen osnhorns (P):notassinm (K) of46213:8.5. ln a preferred embodiment, the N :l’2K ratio is the same in the hatch and fed—hatch culture modes in some embodiments, the ratio of magnesium (Mg):calcinm (Ca) is 3:1 in culture media used in both hatch and fed—hatch modes. in another embodiment, the ratio of chloride (€12):solfate (SO-4)) is l:‘t in culture ntedia used in both hatch and fed-hatch modes. in some embodiments, the ratio of sulfate (“804):phosphate (1’04) in media at the time of ation ot‘a main fermentor (tag, 70,000 L, 0 L, 220,000 1;. ) is 16:1. in some etnhodintents, the total ratio of sulfate (804)2nhosphate (P04) that has been batched and tied, at the end of a full culture (cg, including inocolumfl first seed stage, second seed stage and main ternicntor cultures) that generates an algal biomass containing a desired fat content ‘3“In 00. than 67% fat) is 5.3:l. in some embodiments, the ratio of chloride ., greater (C,W2):}9hnsphate (PO-4) in media at the time of inoculation of a main fermentor (cg, 70,000 [\3 L!i L, 130,000 1-, 220,000 L vessel) is 16:}, in some embodiments, the total ratio of chloride (C12):phosphate (1304) that has been batched and tied at the end of a lull e (eg including inocuhini, first seed stage, second seed stage and main fermentor cultures) that generates an atgat biomass containing a desired fat content (eg, r than 67% fat) is .311.

(A! i , As described in Example 2 below, the invention also provides a composition comprising an algal biomass reg”, a dried algal biomass (ego generated according to a method described herein» containing a desired amount or" total fat and/or other components For example, in some embodiments, the ion provides an algal biomass (eg, a dried biomass) containing r than a % total fat (out; greater than about 6894;» totai fat, greater PCT/U82012/046696 than about 699/; total fat, (greater than about 7094o tot3i tat, r than about 7l9t> total lat, greater than about 72% total fat, greater than about,-96 total tat, greater than about 74% total fat, greater than about 75% total lat, greater than 094) total tat, greater than about 77% total fat, greater than about, 72"% total Fat or higher amount of total fat) ln some U1 embodiments, an algal biomass (e.g, containing r than (179A) total tat)is dried such that the biomass oontains less than 5"/o moisture egt less than 4.5% moisture, less than 4% moisture loss than 3.5% moisture. loss than 3% rootstore less thanfl.-5% moisture, loss than 29/61rtoisture,or less than l.59o re) in some embodiments an algal biomass of the in ’tlilllO‘ill:I..g, a dried s containing less than 5% moisture) contains about 170-251) l 0 mg’g or more dooosaltexaenoic arid (DHA)teg about 170180 mg/g DHA about 180— lQCI mg/‘ggl)HA, about l90200 mgIg1,)HA, about 2002th mgg DH,A about 2i 0220 mgr/g DHA, about 220230 rug/g DHA, about 230-240 mg/g DHA, about 240250 mg/g DHA, or more than 25% mg/g DHA). in some embodiments, an algal biomass of the invention te.g., a dried biomass containing less than 5% moisture) contains about Mil—400 1ngg or more oahnitio acid (lUPAC name: hexadeoanoie acid (eg, abo it 156-200 rug/g, about 200—225 mgr/g, about 225--520 hog/g about 25.0-275 rug/g, about 0 mg/g,about 300325 mg/g, about 325-SSJII1-thg, about 350‘ 33175 tug/g, about 3.75 403:1" or more than 400 mg/g)‘ in some embodiments, an algal biomass of the invention (eg, a dried biomass containing less than 5% moisture) contains about Still—600 tug/g or more total fatty aoids (e. 3;, about 300—350 tog/g, o490 mg/g,about 40t)456}, trig/g, about 450-5,00 mg’g, about 500--550 rug/g;, about 550—('meng‘gor more than 60.0 mg/g tatty acids/l} in some embodiments an algal s of the invention (og, a dried s containing less than 5% moisture) contains less than about i594; protein (fog, less than about l49/i; protein, toss than about l3°A; n less than about‘t 2% protein, less than about i l°o protein, less than about M393 protein, less L! than about 9vb p1otein,or lessthan about 8% protein).

The invention is not limited by the strain or species otalgae utilized in the methods and compositions described hermein lndeed, a vaiietyooatlgaae find use in theintention including, but not limited to, one or more species of the genus Thruwaowyizum in some embodiments, the algae is a species of the genus Chime?to in some embodiments, the algae (,2 is a species of the genus , chizmhvtItrimn. in some embodiments, the algae is a species of the genus Crivplhecodinizmt. in some embodiments he algae is Tomznlooaymmt wanton T71mattochyirium roseum, Ifirzzuttochviiwm (tweenI,C eoodrmum submit, and/or [lit/‘Lly'lldul’Zl/l}IIIm so in a red embodiment ooizwrmm Zlmnynum is utilizedin the methods and compositions bed herein. The invention is not: limited by the type ot‘lipitls .o;an PCT/U82012/046696 produced by a process to generate an algal biomass with elevated levels ol‘lipids disclosed herein. in some embodiments, the lipids generated by a. process of the invention include, but are not limited to, myristic acid, palmitic acid, oleic acid, linoleic acid, doccsapentaenoic acid (GPA), ahesaeuolc acid (DHA), and stearic acid. "l‘hese lipids have been useful for U1 both animal and human health, for prevention of various es such as cardiovascular" and atory diseases and in infant nutrition for proper brain development and retinal vision in children. in another embodiment, the ion provides a process for production of an algal biomass containing elevated levels (cg, greater than 67%) oi‘tolal fat from an algae species l 0 Cog” Schizochytrium limacinurn), wherein the process comprises culturing algae in a first feed hatch vessel comprising medium (are, comprising about 50 g/ll. of a carbon source (eg, sugar reg, glucosefl, about 75 g/L yeast extract, about 0. l5 g/L magnesium sulfate, about 0.l5 g/L calcium chloride and/or 0. l5 g/L magnesium chloride), tiransieiring (e01, aseptically) the first feed hatch culture to a second seed hatch. culture mediumteg, comprising about 50 g/L of a carbon source leg, sugar (cg, glucoseljt, about 75 g/L yeast extract, about 0. l5 g,L magnesium sulfate, about 0.15 g/L calcium de and/or 0.l5 g/L magnesium chloride}, transferring (cg, aseptically) the second seed hatch culture to a large scale e vessel containing medium (eg, a main term-enter (e.g,, 70,000 L, 120,000 L, 220,000 L vessel, containing, for example, medium sing about 50 g/L ol‘a carbon source (cg, sugar (cg, glucose», about 7.5 sails yeast t, about 40 g/L magnesium sulfate, about l g/L urea, about 2 g/L calcium chloride, about 2 g/ln magnesium chloride and/or about 0.25 g/L monopotassium ate), wherein the glucose level of the large scale culture vesse is maintained at 10 g/L using a fed-hatch process, wherein the algal cells are harvested from the large scale culture between l2~24 hours after ion of the let — L! batch process after all of the nts have been removed"consumed from the medium.

Another ment of the invention provides a process for production ot‘an algal biomass containing ed levels (eg. r than (37"775) of total fat from an algae species (eg, Sclrizochytrium lirnacinum), wherein the culture medium (cg, during each stage of fermentation (cg, first seed stage, second seed stage and/or hatch culture (fed—hatch) (,2 cultivation stagell comprises a carbon source tea, a sugar), yeast extract, a phosphate source (e.g,, monopotassium phosphate, magnesium sulfate and/or zinc sulfate), a nitrogen source (eg, urea), magnesium chloride, and/or calciun‘i chloride. In a preferred en‘ibodiment, the invention es a process for production of an algal biomass containing elevated levels (ewg greater than "'7‘3/13) of total fat from a strain ae whe ‘ein the culture medium leg, 2012/046696 during each stage of fermentation (6.3;, first seed stage, second seed stage and/or batch culture (fed-hatch) cultivation stagej) comprises sugar, yeast extract, nionoootassium phosphate, magnesium sulfate, zinc sultate}, urea, magnesium de, and/or calcium chloride. However, the invention is not limited by the type of nutrient utilized in a culture U1 medium in WI .cli algae are grown. In some ettthodiments, one or more carbon sources are added to the medium. Examples of carbon sources include, but are not d to, carbohydrates such as glucose, fructose, xylose, saccharose, maltose or soluble starch as well as oleic acid, fats such as soybean oil, molasses, glycerol, mannitol, and sodium acetate, cotton see flour, glycerol, molasses and corn steep liquor. in son e embodiments, one or l 0 more nitrogen sources are added to the medium, Examples of en sources include, but are not d to, neutral nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid and corn steep , c nitrogen sources such as sodium glutamate and urea, or inorganic nitrogen sources such as ammonium acetate, ammonium e, ammonium chloride, ammonium nitrate and sodium sulfate. in some embodiments, one or more phosphate sources are added to the medium. Examples of phosphate sources include, but are not limited to, potassium phosphate and potassium dihydrogen ate, inorganic salts, such as ammonium sulfate, sodium sulfate, magnesium sulfate, iron sulfate, zinc sulfate, and copper e. ln some embodiments, magnesium chloride, calcium chloride, and/or vitamins are included in the culture .

The invention is not limited by the amount (cg, concentration) of each ot‘tltcsc components in the culture medium. in some embodiments, an amount is utilized that is not harmful to algal growth. in a tire lorrcd embodiment, the amount (cg, concentration and/"or ratio) ofcach medium ingredient is set at a level (egg during each stage of tation (cg, first seed stage, second seed stage and/or batch culture {fed—hatch) cultivation stage) that L! promotes the formation of higit fat content algae (eg, an algal biomass comprising 67% or greater fat content). in some embodiments, the carbon source (eg, sugar) is present in culture medirm at about 2t) to 120 grams per liter oi‘medium. in other embodiments, the carbon source (cg, sugar) is t in culture medium at about 50—70 grams per liter of medium. in still other embodiments, the carbon source (eg, sugar) is present in culture (,2 medium at about 40 to 60 grams per liter of medium. in a preferred ment, the carbon source (e.g., sugar} is present in culture medium at about 50 grams per liter of medium. in some embodiments, the ratio of urea to n‘ionopotassium pl’iosphate (i,trea:l(ll2l304) is between about Sill leg, about 4.5:O.l; 4:025; 3:025; 420.3; 5:03; 510.5; 420.5; 3:05; 2:05; or l:ll.5); although higher and lower ratios may he used (eg, lzl, l12, l:3 etc.) in a ix.) ix} PCT/U82012/046696 ired embodiment, the ratio of urea to monopotassium phosphate in culture medium is 421‘ in some embodiments, a culture medium does not contain sodium chloride. in other mentse a culture medium contains sodium chloride. in some embodiments, the ratio of magnesium sulfate (:hllgSOAl):ealoium chloride tCaClZ} is l:l. in some en'ihodiinents. the UH ratio gnesium sulfate (MgSO4):calcium chloride {CZECH} is i2. indeed, a y of ratios of magnesium sulfate (MgSOLl):oalcium chloride (CaClE) may he used including, but not limited to. 1:1; lzl.125; 111.5; l:l.75; lift; 1:2.l25; 1:22.25; 112.5; 2511; 2.25zl; 232521; 2:1; l.75:l; l.5:1; 1.25:1 or 1.l25:l. In a preferred embodiment, the ratio utimagnesium suliate {MgSOd-lwalcium de (CaClZ) in a first seed culture medium is l:l. in r it) preferred embodiment the ratio of magnesium sulfate gS(_)/il):ealcirtnt chloride (CaClZ) in a second seed culture medium is 1:1. in yet another preferred embodiment, the ratio of magnesium sulfate (h/IgSlemalcium chloride tCaClE} in a large scale culture medium (cg, main t‘ertnentor reg, 70,0th L, 120,000 L, 230,000 L vessel) also referred to as a third culture medium herein) is l22.

In a turther red ment, after preparing the medium, the pit of the medium need not be adjusted. For example. during a se fermentation process ofthe invention the pll of the culture medium in which algae is grown need not be adjusted. Although an understanding of the mechanism is not necessary to practice the invention and the invention is not limited to any particular ism ot‘action, in some embodiments, sterile and/or aseptic conditions of the stepwise fermentation process of the ion negates the need to adjust the pH of the culture medium during fermentation. in some embodiments the pH of the culture medium is between 4.0 and 65. Cultivation oi‘thc algae during a se tatinn process of the invention may he carried out at a temperature between in and all) C, preferably 17 to 35 C, and most preferably around 3!”) C. Cultivation may he performed [\3 L!i by aeration—agitation culture, shaking culture, stationary culture or the like. in a preferred embodiment, al, 'ae are cultured under conditions such that dissolved oxygen is maintained at{1C or slightly above 10%.

In some embodiments, the invention provides a food, tieedq nutritional or therapeutic supplement ctmprising all or a portion of an algal biomass“ (e.g., a dried algal biomass (A! 1 , described herein and/or generated according to the methods and compositions described herein) comprising ed levels 1:e. g, greater the s 7-1II 0/4)) ol‘total fat. For example in some embodiments, the ion provides a food, feed, initritional or therapeutic supplement comprising a spray dried algal biomass comprising elevated levels (egg, greater the 67%) of total fat. in oti er ments” tl e invention provides a food, feed, nutritional or ti erapeutic a} a} PCT/U82012/046696 supplement con‘iprislng lipirls ted and/or isolated from an algal biomass comprising elevated levels tog, grater the 67%) oftotal fat, The invention is not limited by the type of lipid extracted and/or isolated from an algal biomass comprising elevated levels ( e.g, grater the 67%) of total fat. in some en’rbodiments, the lipids comprise rnyristic acid, palniltic acid, Ui oleic acid, linoleic acid, alpha—linolenic acid (ALA), stearidonic acid (SBA), eicosatrienoic acid, eicosatetraenoic acid, eicosapenlaenoic acid , tlocosapentaenoic acid (DPA) clupanorlonio acitl, docusabexaenoic acid (DHAL tetraocsapentaenoic acid, and/or tetracosahexaenoic acid, in a preferred embodiment, the lipids comprise DHA anti/or palmitic acid, it) in some embodiments, the invention provides a process for the preparation ot‘lipitls (eg, those sed herein (cg, docosahexaenoic article) comprising: culturing an algae strain (eg Schizocizyrrz'um Zimacimtm) in a first culture medium (eg, containing> 50 g/L of a carbon source (eg, sugar (eg, glucosei), ll) g/L yeast extract and 4 g/L sea salt) and incubating the culture at a temperature in the range of 25—35 C for a periorl of about 72~ l 44 hours; transferring the e to a second culture medium (cg, containing 50 g/L oia carbon source leg, sugar leg, glucose», about 7.5 5311. yeast extract, about lilo” g/L magnesium e, about 0. l 5 gi calcium chloride anti/or 0,15 g/L magnesium chloride) and incubating the culture at a temperature in the range of 25—35 C for a period of about 24-48 hours; transferring the culture to a third culture medium (cg, containing 50 g/L of a carbon source (eg, sugar (cg, glueoscfi, about 7.5 g/l. yeast t, about: (HS g/L magnesium sult‘aic, about 0. l5 g/l. calcium chloride and/or (ll 5 g/L magnesium chloride) and incubating the culture at a temperature in the range of 25-35 C for a period of about 24—48 hours; iratisierririg the culture to a fourth cu liure medium (eg containing 50 g/L ofa carbon source (cg, sugar (cg glucose», about 7.5 g/l. yeast extract, about 4.0 g/l; magnesium sulfate, [\3 L!i about l g/l... urea, about 2 g/L m chloride, about 2 g/L magnesium chloride r about 0.25 g/L nronopotassium phosphate) and inc ubating the culture at a temperature in the range ol‘25u35 C (cg, 3C5 C) for a time period ut 24-492 hours (cg, about 36, about 38, about 42, about 45, about bl) about 72., about 84, about 96-, about , lllli, about 120, about 132, about l44, about lilo, about lo8, about ltitl or about l92 hours); separating the cell biomass (A! i . from the culture; and extracting lipids from the s. in some embodiments, algae cultures (eg. grown to produce an algal biomass) are grown in suitable volumes and vessels, ranging from ill-ft ml to hundreds of thousands of liters, in flasks or large fermentors, using various nt media as described herein. in yet r , the separation of the cell biomass containing lipids is obtained PCT/U82012/046696 using centi‘ifiigation, filtration and/01' flocculation or similar ques. in a niefei‘i‘eo ment, an algal biomass is obtained from a culture using eentrii‘ugation. in a thriller profound ment, ifiigation occurs after tlte cell culture is cooled ( e. g., to allow recoveiy of cells containing elevated levels ofllpi-zl). E11 50'13 embodiments an algal biomass Ui obtained is spray—dried and used (eg, directly used in animal feeds or for biofuel production). in one embodiment, the algae is a e of different algae species (e313,, one ot more of the species ol‘algae described herein). in some embodiments, an algal s containing ed levels oftotal fat and/or lipids ted from an algal biomass containing elevated l0 levels of total tatis supplemented Withliipidsto,“ V111tsatuiated fatty acids) 1‘1ont otliei 3, , pol sources including, but not limited to, plant sources. in some embodiments, an algal biomass containing elevated levels of total 1‘91: compiise lipids at a concentiation tw’w)in a tango f1oin about 60-90% (eg about 65-90‘41 about 66—89%, about 67—88%, about 68—87%, about 68—86%, about 69—85%, or about 70 1.5. 0/6} Thus, an lgal biomass containing 6levatedlevels of lipids may comprise lipids ata concentration ol‘6i8/9,6728/63, 6386. 64"/9,65%, 66% 6/ 9,688%o, 69%, 70%, 7l%, 72%, 73%, 74%, 7584, 76%, 77‘678%,79%,80/6, 81%, 8862, 83%,84 ‘3/6 85/9, 86% 1,88%,89‘36, 90/o antltlme like in one embodiment an algal biomass containing elevcttfiid levels of total fat comprise lipids at a concentration of at least 67%. in some embodiments, DHA is included in an algal biomass composition oftlic invention in a range from 1‘36 to 75% oftotal lipitis’fattv acids. Thus, the DHA can be provided in the itit’ln in an amount of total fatty aoi-its of £8413, 2‘36, 38/93, ‘ 86,5 - 413 6‘36, 78/63, 8‘36, 9‘36, W89, ll‘36, 3‘341,l ‘36, l5“/63, l6‘34l.,7%, 18%, l9‘341,20‘36, M86, 22%, 23"6, 24%, 2589, 2’‘36, 2 "3/9, 28%, 29%,30‘41, about 3.50 {Hut 409‘/9, about 45%, about ‘36, about 55‘/41 about 60%, about 65%, about 70%, about 7.5//9 and the like. in other ments, the DllAan be includedin a contposition in an amount ot total latty acidsin a range from 1% to 5‘36, ‘36 to 10%, % to 15%, 1% 020%, l‘36 to 25%,"36 to 30%, 58/616 “7%, 5% to 15%, 5% to 2 ‘36, 58/9 to 25%,5,.6 to 308/9, l0‘36 to 1589,1096to 20%, 10% to 2.5%, l0‘36 to 30%, l5‘36 to 20%,115°6 to 25%, l5‘3’6 613086, 20% to 25%, 21 8/6 to030%, 25% (A! . 1 to 30%, 30841 to 35%, 358.41 to 408/, 40% to 45%, 45% to 50% 50‘36 to 5 old.) 600/o, 60% to 65%, 65‘76 to 70%, 70% to 75%, and the like. in some embodiments, palinitie acid is lnolucled in an algal s composition of the invention in a 1ange iiom l°39to75,/-11 oi total lipids/titty acids Titus the palniitie acid can be provided111 the composition in an amount oitotal latty acids oll‘““1,22%. 3%, 4%, 5%, PCT/U82012/046696 6%, 7’96, 896, 9941, 10‘7'.‘119/11, 12%, 13%, 1491 15%,1113921, 17%, 11196, 19%, 203/11 21%, 22%, 396,249/-. 25941311942102.7941.28%,2991,30922111011135011111111404, about 45/0, about 50%, about 55%, about 00%, about 65%, about out 759/11, and the iike. in other embodiments, the pain‘iitie acid can be ineinded in a composition in an amount of totai fatty acids in a range from 19/6.- to 59/6, 1% 10 10% 1% 11; 15%,19/1- to 209/, 1% to 259/11, 96 to 3096, 596 to 109/11, 5.9610 15%, 5% 10 20%, 59/110 23/11, 596 to 31194.1, 109610 1590,10"610 2196, 1096 109/010 309/11. 15% to 20%. 15/111259015/610 31‘296, 209’m 102 20% to 30/6, % 105096, 309/1110 35%, 359/1110 40%, 40911 to 45%, £15941t0 509/11, 51 9601:5594:, 559610 0096, 609/11 to 05%, 1559/11 to 70%, 70% to 759/21, and the 1i1<1e Additionai ments of the invention in0-.11110 131000sses 01" making aniinai feed additives. Thus, one aspect of the t invention is a process of making an 1 feed additive comprising iinids from an aigae (e.g., an a1ga1 biomass}, the process comprising: coitivating aigae tn produce a aigae biomass ning a desired, elevated ievei l fat (0.};, "reater than 0ta1 flat); and extracting a1gee iipid front. the aigae biomass to produce a 111g;1e (iii; an' 1’0r retrieving 1 'ater from a1gae biomass to produce a ziigae biomass with a soiids centeni from about 5% to 1009/11 weight percent; wherein the aniniai feed additive comprises 1ipids 110111 the :11 gee. 111 some einhndintents, the fatty acids coiieeted front the a1gae are short chain, medium or iong chain omega—3 fatty acids. 111 further embodiments, the a1gae 1ipi<1 extracted from the a1gae biomass is ed with a aigae biomass with a s01ids content "rtrem ahc-tit 59/13 to 100% weight porecnt.

A feed additive according to the invention can be combined with other food components to produce proncss011 food or feed [110(1LL1‘1 (co, 0111111211 and’01 human teed products}. Such other food components ineiude one or mere enzyme ments, vitamin food additives and niinerai food ves. The ing (combined) feed additive may be mixedin an appropriate amount with the other 10011 compo-tents such as cerea1 and p1ant ns to 101111 a sed food product. Piece ingt1‘ these components into a processed food product can be performed using any conventionahy used processing apparatuses‘.

Peed/food additives of the present invention maybe used as a suppientent in a hind/feed by itse1f, in addition with vitamins, minerais, other feed enzymes, agrieuitura1 co—produets (13.51.. '30 wheat middiings or corn gluten meal), or in a combination ith. 111 a further aspect, the invention provides a process 01‘ predncing an aniinai and/01‘ human having an increased tissue content 1211203, fatty acids, the process comprising, feeding to an anirnai/human a feed additive comprising 1ipids/ta't’ty acids co11ected 110111 aigae, the feed additive further comprising: (a) an aigae 1ipid extracted from a cuitivated WO 10090 PCT/U82012/046696 algae biomass and/or (is) a algae biomass from a cultivated algae, wherein water is d from algae biomass to achieve a solids t from about 5 to lOli‘f/o weight percent, wherein the /human displays increased tissue content {if omega} fatty acids, The invention is not limited to any particular mammal (ea, animal or human) that may benefit from a U1 composition of the ion. indeed, animals oldie invention include, but are not limited to, any animal whose eggs, meat, mills or other products are consumed by humans or other animals. Tints, animals of the invention include, but are not limited to, fish, poultry (chickens, s, ducks, eta), pigs, sheep, goats, rabbits, beef and dairy cattle. in some embodiments, tl e invention provides a method for treating a mammalian l0 disease in a t in need thereofby administration to the subject a therapeutically effective amount of a composition of the invention, in some embodiments, a mammalian disease that is treated includes, but is not limited to, a cardiovascular disease, an inflammatory disease, and various cancer diseases. In other embodiments, the cardiovascular diseases to be treated include, lint are not limited to, liypeitriglyeeridemia, coronary heart disease, stroke, acute lS myocardial infarction and atherosclerosis. in further embodiments, the inflammatory diseases to be treated e, but are not limited to, asthma, arthritis, allergic rhinitis, psoriasis, atopic demiatitis, inflammatory bowel diseases, Crohn‘s disease, and allergic rhinoconjonctitis, in still further embodiments, the cancer diseases to he treated include, but are not limited to, te cancer, breast cancer and colon cancer. in additional embodiments, the mammalian diseases to he treated include psychiatric disorders Psychiatric ers include, but are not limited to, depression, hipolar disorder, schixophrenia. in addition, the compositions of the invention can be used to maintain and"or enhance cognitive function. in some embodiments, the invention provides a method of treating a ian [\3 L!i disease in a subject in need the eotby administration to the suhj ect a therapeutically effective amount of a lipid composition provided by and/or obtained from an algal biomass containing an elevated level l fat (eg, greater than 67% total 1‘ Subjects that may find benefit from treatment e but are not limited to, avian and mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, bovines, caprines, (A! , , equines, , porcines, rodents (egg. rats and mice}, lagomornhs, es (including, non- human primates), humans, and the lilie, and mammals in utero. Any mammalian subject in need of being treated according, to the present invention is suitable, Mammals of the present invention include, but are not limited to, s, felines, hovines, caprines, equities, , es, rodents (eg, rats and mice), lagomorphs, primates (including non—human primates), is) ~J PCT/U82012/046696 111111121115; and the like, and s in titero. ing to some embodiments of the present in *ention, the mammalis a non“human mammal. 111some;=11hodiments, the mammal is a human subject. M alia11 subjects of both genders and at anv stage 0i development (so . , neonate, infant, juvenile, cent, adult} can he treated according to the present invention.

U1 illustrative aviansaecordingto the present invention e chickens ducks turkeys Reese. quail, pheasant. raiites leg, tistrich). doinestir;ated birds: eg parrots and eanaiies), and birds in ovo.

Algae l 0 Any algas capahls of producing, using the processes bed . elevated levels oftotal fat or algal biomass containing elevated levels of total fat can he usedin the processes, e in tositions y supplements hiofuei and/or hiotuel precursor and/or feed additi es of the invention. Thus. in some embodiments, the algae of the present invention is selected from litmusoch Wit/1m Difiophvceae {ijvpzopht’ceae zit/6170101011221” ”as, pingaiophvcsee. and combinations thereof in other embodiments the algae of thein 1,!ntion are selected from ThrowL.slochvtrzwn strzalzmz, N'meiwocll’IVI‘i/lm Inseam, ioch‘lliitffli at;cum Cray-1'heeonlz’iziwn coMia Parieloehk?its 1pp Rhodomomts spp Captains: as app Parietochim‘is spa 1161111512511er sop Pwprzwzdmm shop Glossomastzx spp.. and combinations thereof. in further embodiments, the algae of the invention are selected from Pariefochloris impish, onus saline, Hemiselmis bi‘trztrzstrens, P()Ij0lzyridiuln premium and nmstix clpysopi’asa‘cn and combinations thereof. in still finther embodiments, the altea tl’il’lfi,lilVLlll"l’l{[12 is Sehz'orhvfrmmimarimim. in some embodiments of the invention the algae is 3 mixture ol'dilfirent algae species. in other ments. the algae is a single algae species. in some ments of the present invention the algae lipids/lam! acids are provided as an algal oil. in other embodiments, the algae lipids/latty acids are provided as an algal biomass leg, a dried (e.g., powdered) hi00111as:9}.

Further, the algae of the invention include. but are not limited to, Wild~type, mutant ally or induced) or genetically engineered algae. 'n a red embodiment, an algae used in the processes, compositions, dietary supplements, hiol'uel or blofuel precursor and/"or eed additives of the invention is a non-genetically modified organism. As used herein. the terms "genetically modified variant," and ”genetically modified organism'refer to an algae strain that has a genome whichis modified (e from its normal (e.g . mutated Changed) . wild-ty e, naturally ing) form such that a desired result is achieved.

PCT/U82012/046696 Additionally, the algae of the invention includes algae having cells with cell walls of reduced thickness: as: ed to the cells ol‘wild-tyne algae, whereby the cell wall cf reduced thickness improves extractability and/or hioa‘y'ailahility of the algae lipid fraction (egg, ing the ease of (ll Vestihillty of the algae and the ease of extractahility cf the algae(3» U1 lipids/fatty acids from the cells of the algal hlun’lassl. Algae having cells with cell walls cl“ reduced thickness as retl to the cells of wild-type algae can be lly occurring, mutated and/or genetically engineered to have cell walls of reduced thickness as ed to wild~type strains. Thus, in one embodiment of the invention the algae is an algae having a cell wall of reduced thickness as compared to the Wild-type algae, whereby the cell wall of l0 reduced thickness improves tabiliw and/or hioavailahility of the algae lipid fraction, Methods of producing algae with reduced cell walls include those found in W 2006/1 07736 Al, herein incorporated by reference in its entirety. Thus, the algae can he mutagenized with nrutagens knewrt tn those of skill in the art ing, but net limited tn, chemical agents er radiation, In particular embodiments the chemical mutagens e, but are not limited to, l5 ethyl rnethanesulf nate (EMS), tnethylrnethane sult‘onate (HMS), N—ethyl—N—rntrosourea (ENU), triethylrnelarnine (TEM), N-—nretl1yl-—N-—nitrusourea (MNU), prucarhazine, chloramhucil, hosphartridc, l e, actylamide monomer, rnelphalan, nitrogen mustard, Vincristine, (llmethylnitrosamine, N—methyl—Nl—nitro~Nitmsoguanldine (MNN Cs), rtitrosoguanidine, E—aminopurine, 7,l2 dinrethyl~henz(a)anthracene (DMBA), ethylene oxide, {.40 hexamcthylphosphoramidc, hisult‘an, diepnxyallranes (tjlicpnxyoctane (DEG), dicpuxyhutanc (BBB), and the like), 2—mothoxy—6—chlcro—QQB—(ethyl—Z—chlcr~o~ cthfiaantinnpropylaminrfiacridinc rochloridc (lCR— 70:)“ thrntaltlchy dc, and the like.

Methods of radiation mutagenesis include, but are not limited to, Xurays, gamma-radiation, ultra—Violet light, and the like.

Cell wall mutants can he selected for on the basis of increased sensitivity to detergents 01‘ by microscopic observation of tions in cell wall thickness (See, e. 9;, WC 2006/? 07.736 Al) or any other method known in the art tn detect reduced cell wall thickness or reduced cell wall integrity.

The algae of the invention can he cultured according to techniques described in '30 Examples l—3.

Accordingly in some embodiments the algae are cultured at a, temperature in a range from lOGC to 3500 Thus, the algae can he cultured at a temperature uf l 00C, lloC, 12°C, 130C, l40C, lSOC, lt.°C, l70C, lSDC, l9°C, 200C, 210C, 220C. 230C, 240C, 250C, 26°C., 270C, 2804:, 29°C, 300C, 31%; 32°C, SST, 340C, and the like. In other embodiments, the algae can PCT/U82012/046696 be grown in 1213-gesh'ont 20°C to 35°C, although colder (eg, less than 20°C) and warmer eg more that , ) may be used lo a preteried embodiment, the algae are grown at about 300C. in some embodiments, following cultivation, algae ale liatVested in some Ui embodiments, harvesting of algae is performed using, conventional procedures lillt’lwn to those of shill in the art including, but not, limited to, cenlril‘ugaticn, flocculation or filtration. in a preferred embodiment, prior to harvesting, the algae re is cooled, theiehva‘ ig algal cells containing elevated levels oftolal fat to he successliilly harvested. The harvested algal cells or algal biomass can then be used directly as a lipid/fatty acid source or extrac ‘eil l0 to obtain algal oil 130-nprising thehipidS,’fatty acids in some embodimentsin which the algal biomass is to he used directly, water is removed from the algal biomass to achieve a solids content from about 5 to l00 weight percent. In additional embodiments, an algal hiemass that is to he used directly is sed of algal cells further comprising cell walls that are at least partially disrupted to se the extraotabilitj anti/or hioavailahility of the algal oil l5 within the cells. The disruption et‘the algal cells can he carried out aceerdlng te knewn techniques ing, but not limited to, treating the cells with boiling water or by mechanical breaking such 5 s grinding, pulveriziiig, tion, French press, or any other method known to an ordinary artisan, When the algal biomass is used directly. water is ved from the algal biomass to achieve a solids content from about 5 to “30%, Accordingly, in seine embodiments, water is removed from the algal hiontass to achieve a solids content of about 50/95, l09‘6, l5%, "20%, 2595,3594, «95,4995.4594., 51395, 55 5.50.94. 5:595, 595 5794. 5595,5597 7574. 7195, 7295. 73955494575,595 5,77%, 759,5 799555934,;5195, 5295,:53°4.,54955595,55°4:.,.795,88%, 89%, 90%, 9l%, 92%, 93%, 9495,,5%,3595, 97%, 98%, 99%, l00%, and the like. in [\3 L!i onal enthodiments, water is removed from the algal biomass to achieve a solids content in the 1ange l1out about 5% to 509’s, 5'70 it; {3095, 59/3) to 70%59’s to E909/5, 590 to 90%, 5% to 95%, 1096th 5094', l-’9’5 to 40%, 109’40 to 509t), l0':‘4'5 to 60% l004'th {35%, l04’oln '09E), l0'4’5 lo 75. ,109(Mo 80%, l)% to 85%,10% to 90%, l09’6 to 95%, l09'45 to 10“/o,l 95 to 40%,l 5% to 5095,l 5% to 60%,l 5%tto (15%, iii/5 to 709/15, l59’(3 t175%, l5% to 80%, l59€> to 85%, (A! , 9 lilo/i) l0 900/8, isll/h EC 9597), l57/h it) l00%, 207/6 l0 50°75 0 60%- 657/5, 20%) it) 70%), 20°47 10 75°46, 20°43 10 80Al, 20°43 l0 85°13, 200’0 l0 Ell/9’'13, 209(ill) 95°’,2 9?) 10 “90943, 25°46 €03900’, 25//0 it) 60%;, 25/0 it) 70°40, 25//'0 £0 75 9;), 25%) it”; 80%,25°’o 10 85°/'o,A250/0, 10 90°14), °43 10 93°45 3590 it) 100%, )00’'1) l0 50°43. 30961( it) 60943, 3092’) l0 7‘l%300/0 [0 75°40, 309/5 l0 809/1'1,30°’to 35% 304’ to 90%, 3045143 59,2) 45‘44'5 lo l0l96, 5092; to 70%, 50% to 75%, 509/5 51.4 ”“551 PCT/U82012/046696 £0 80%, 50‘36 to 85%, 50941 «190%, 50/‘3, to 95'36 51336 to l00'36 55‘361’0 ”5‘36, 55% to 8ti3‘36, 55‘36m 85%c, 55‘36 to 90%,553610 95%, 553610 30036 600/: to 75‘3‘6 6004:.» to 803o 60% to 8, ‘36, 60% to 9‘36,60”/0 to 95% 60% to 3000'm 70% to 80“Al. 70% to 85% I 3‘36 to 90%, 70% to 95%, 70% to 100%, 75 6to85°.6, 75% lo 90%, 7.596111 95%,” 5% to l00‘36, 80% 115.85 ‘36, 809-6 to 9096., 80% to 95%, 80"6 to 1009/: 8596 to 9i3061,8596 to 95%, 85% to ltll’l‘i/o, 90%. to 95%, 95% to H3096, and the like. in some embodiments the alsralcells ot t11e biomass are disrupted or lysed and the algal lipids extracted. The algal cells can be extracted wet or dry according to tional techniques to produce a composition ning lipids/iatty acids. The disruption or lysis of l0 the alcal cells or-n he carried out atcording, to conventional techniques including, but not limited to, treating the cells with boiling water or by mechanical breaking such as grinding, pulverizing, sonication, French press, or any other known method. Extraction of the lipids/latty acids from the lysed cells follow standard procedures used with algal and other sms that are known including, but not limited to, separating the liquid phase from the l5 solid phase 'olllowingcell lysis,ext1acting the lipids,I’fatty acids in the liquid phase by the addition of a solvent evapmating the solvent, and recovt.ring the lipids/fatty acids obtained from the liquid phase of the lysed cells. ”'lheinvention is not d to any particular SUlVL>1t med for extraction. Solvents include, but are not limited to, hexane, chloroform, ethanol, methanol, isopropanol, diethyl {.0 ether, (lies an, isopropyl ether, tilichloronicthane, ydrotiiran, eum ether and combinations thereof in some embodiments, lipids/"fatty acids derived from an algal biomass oi‘thc invention are provided in the form of free fatty acids, cholesterol esters, salt esters, fatty acid esters, nionoglycerides, erides, triglycerides, diacylglycerols, ycerols, sphin{ophosphcl:pids, splnnaovlvcoipids, 01 any ation thire11t(e,1“11r use in ses, compositions, hiolnels, oducts, dietary supplements, teed additives 151 othe compositions described herein).

Method tor Preparing An Algaltl Biomass '30 in some embodiments, the invention provides a method for preparing a algal biomass comprising elevated levels oi total fat (e.g greater than 67'36 lipidsl comprising: culturino algae under a culture condition sufficient to provide an algal biomass comprising elevated levels of total fat (cg, r than 67% lipids), wherein the algal s is harvested at the termination ofa logarithmic growth phase oftl e algae (See,e.gE11aniples l and 2). As v.2 1,.“ PCT/U82012/046696 used herein, the term "logarithmic growth phase," refers to a stage of culturing characterized by exponentially increasing s ofalgal cells. ‘enerally, in a culture system, there is a characteristic growth pattern following ation that includes a lag phase, an exponential or "logarithmic growth phase," a negative growth acceleration phase, and a plateau or U1 "stationary phase.” For example, in the logarithmic growth phases as growth of the algae continues, cells can reach their maximum rate ol‘cell division and numbers of cells increase in log onship to time. Within time after the commencement of the log phase, the rate of cell division may begin to e and some of the cells can begin to die. This is rellected on a growth curve by a gradual flattening out of the line. Eventually the rate ol‘cells dying is l 0 essentially equal to the rate of cells dividing and the total Viable population can remain the same for a period of time, This is known as the stationary or plateau phase and is represented on a growth curve as a ning out ol‘the line where the slope approaches zero. in a preferred embodiment, the algal biomass is cultured under aseptic conditions (eg, to prevent contamination and/or growth of contaminating microorganisms (cg, yeast, bacteria,” VlFUSl etc.) in the culture).

In some embodiments, the culture condition is sutlicient for the algae to produce elevated levels- of total fat (egg, greater than 67% on a w/w basis), The culture conditions comprise a culture medium suitable for g the algae thereby providing the algae biomass comprising elevated levels of total fat tag greater than 67% on a w/‘w basis} Suitable culture mediums are bed herein. The medium may also comprise salts, Vitamins, mincralm metals, and other nutrients. Preferably, the culture condition is sufficient, to provide a suitahlc amount rient and ature for the algae to grow under conditions that: generate an algal biomass comprising elevated levels of total fat. ln some embodiments, culturing comprises limiting a nutrient leg, nitrogen, L! phosphorous) for a suitable time to increase the amount total fat. For example, the culture can be starved of a certain nutrient or transferred to a separate culturing medium lacking a specific nutrient (cg, phosphorus—tree or nitrogen-lice medium, or a culture medium containing lower levels of a nt). in some embodiments, the culture medium contains an initial content of a nutrient such that that nutrient s depleted at a later time during (,2 exponential growth but prior to the ion of other nutrients. in some ments, culturing does not comprise limiting a nutrient ife.g, nitrogen, phosphorous) during culture. ln some embodiments, culturing of a single algal biomass takes place in two or more types of medium in a sequential manner. in some embodiments, culturing of a single algal biomass talres place in three or more types of medium in a sequen manner. in like manne ~14 {x} WO 10090 PCT/U82012/046696 ing of a single algal biomass may take place in two or more vessels, wherein a first vessel is used to ate a subsequent vessel, the subsequent vessel is used to lnoeolare yet another subsequent vessel, and so 011. Although an understanding ol’a ism is not needed to practice the it ventiott, and the invention is not limited to any particular mechanism Ui ol‘action, in some embodiments” sequential culturing of a single algal biomass in multiple vessels containing multiple types ol‘medium allows the algal biomass to grow in such a way that the total fat content of the biomass is elevated eotrtuared to growth of an algal biomass (e.g., of the same algal species) grown in a single vessel ‘ ltd/or growth medium.

Culturing of the algae can be performed in a conventional loioreactor suitable for l0 culturing the algae to provide an algae biomass] For example the algae can be cultured by a process ing, but not limited to, batch, fed~batch, cell recycle, and continuous fermentation. in a preferred embodiment, the algae are cultured in a t”erl»»bateh process.

The invention is not limited to any particular manner or method of ting the algae from the culture medium. A variety of methods can he used to harvest the algal cells l5 hunt the culture . in one embodiment, harvesting comprises recovering the algal biomass front the culture medium by separating, for example by filtration (ego belt filtration, rotary oltnni filtration;t and/or centrifugation. if desired, the harvested algal cells can then be washed, frozen, lyophilizerl, spray dried, and/or stored under a non—oxidizing atmosphere of a gas (e.g., (70;, N2) to reduce or ate the presence of 03. ally, synthetic and/or natural antioxidants including but not limited to, h’otylatcrl hydroxytolnenc (Bill), butylatcrl hydroxyanisole (BRA) tett—hutylhydroquinorte (lBHQ) ethoxyquin, beta~earotcne, vitamin E, and vitamin C also can be added to the hart‘ested cells.

In some enihotilirnents, the ion provides a method for ing an algal biomass comprising elevated levels of total fat, the method comprising: culturing algae under [\3 Ln a culture condition sufficient to e an algal biomass comprising elevated levels of total fat and l‘larvestlng the algal biomass.

Microalgae s The invention provides, in some embodiments, an algal biomass and/or a fraction (A! l , and/or an extract thereof (eg for use in bioluel production and/or as a food or feed t). in some embodiments, the algal hion‘rass comprises: an omega-3 fatty acid content of at least lOO/b (lay weight of the biomass, illustt‘atively, about lilo/h to about 50%, about lllli/o, to about 40%7 about l0% to about 30%. about llli/tt to about 26% dry weight of the biomass. In one enrhodimenh the algal biomass is prepared in accordar ce with the methods oftl e 11.2 (a) PCT/U82012/046696 invention. For example, in some embodiments, the algal biomass is prepared by a method comprising: culturii ' an algae under a culture condition sutiicient to provide a algal biomass comprising elevated total fat levels (cg, greater than 67% w/w‘}, wherein the algal biomass is harvested at a negative growth acceleration phase or a stationary phase, in another U1 embodiment, the algal biomass is harvested from the culture during the exponential, logarithmic growth phase.

Lipid Compositions Prepared From Algal Biomass in some embodiments, tl e invention provides a method for preparing a lipid/fatty acid l 0 t leg” a lipid/fatty acid composition) from an algal biomass grown under conditions to contain elevated levels of total fat, the method sing obtaining lipids from an algal biomass cultured under a culture condition sufficient to e an algal biomass with elevated total fat content (e.g., total fat content greater than 6.7% of the s), wherein the trial biomass is harvested at a ve growth acceleration phase or a stationary phase of the algae. in another embodiment, the algal biomass is harvested during a logarithmic growth phase of the algae.

Methods for obtaining a lipid composition from an algal biomass of the invention include, but are not limited to, extraction, heat, pressure, saponification, tion, freezing, grinding, ion ge, chromatography, membrane separation, electrodialysis, reverse osmosis, distillatirm, chemical dcrivatization, lliaation, etc, For example, algal lipids can be extracted from the algal cells by any suitable method including, but not limited to, extraction with a solvent ing, but not limited to, ethanol, ethyl acetate, isopropyl alcohol: methanol, ethyl acetate, hexane, ene chloride, methanol, petroleum, chloroform, and the like, or by pressurized liquid hydrocarbons such as butane, pentane, L! propane, or others (with our Without (Jo-solvents), or through supercritical fluid extraction (with or without (so—solvents). Optionally, the ted lipid/tatty acid oil are evaporated under reduced pressure to reduce or remove the solvent anchor produce a sample of concentrated lipid material, in. other embodiments, the cells are broken or lysed to obtain the lipid composition, for example into an oil form leg, for use as a hiofitel or a biofnel (,2 sor). in some embodiments, the extracted oils are subjected to g. The invention is not limited by the type ol‘rel’ining. In some embodiments, the extracted oils are chemically d. in some embodiments, the extracted oils are physically refined. in some ments, the extracted oils are both chemically and ally refined. Extracted oils (ego from an algal biomass grown under conditions to ele’r ate the total fat content of the 11.2 .4}.

PCT/U82012/046696 algal cell (6.9;, to above (Wt/bl) may be refined using any conventional refining method. ”hie ‘etining process may remove some or all impurities from hc- extracted lipids/fatty acids/oils. ln some embodiments, the refining process comprises one or more steps to degum, bleach, filter, deodorize and/or polish the extracted lipids/fatty acids/oils.

U1 in some embodiments, the /Fatty oils contained in the extracted lipid composition is concentrated by hydrolyzing the lipids to concentrate the lipid fraction by employing a method such as, for example, urea. ction, fractional distillation, column tography, and/or supercritical fluid fractionation.

Accordingly, in one embodiment, the s ‘ep of ing a lipid composition from an l 0 algal biomass of the invention. comprises extracting the lipid composition from the biomass. ln another embodiment, the step of obtaining a lipid composition from an algal biomass of the invention comprises contacting the biomass with a polar solvent.

For example, in some embodiments, lipid/fatty acid/oil is extracted from the algal biomass to provide a lipid composition using a solvent under an extraction condition sufficient to extract lipids and/or fatty acids but not sufficient to extract compounds that are insoluble in the solvent. in one embodiment, a lipid/fatty acid composition is extracted from an algal biomass of the ion wherein cellular debris and/or precipitated insoluble compounds are separated from the fraction containing lipid/fatty acid and solvent. in another embodiment, the method further comprises separating the cellular debris and precipitated compounds using a separation method such as filtration, ccntrithgation, and/or cotrtbinatiirms thereof. ln some embodiments, the cellular debris and"or precipitated ble nds (cg, that portion 0-9 the algal biomass that are not soluble in a solvent (n.3,, proteins, tibcr, etc) are recovered and utilized tag, in a food or teed product). in some embodiments, the solvent is a. polar t. es ofpolar solvents L! include, but are not limited to, ethanol, ethyl acetate, isopropyl l, methanol, ethyl acetate, and mixtures thereof. in one embodiment, the polar solvent is ethanol. Extraction of the lipid composition with a solvent can be carried out in a variety of ways. For example, the extraction can he a batch process, a continuous s, or a continuous counter-current s. lo a continuous coroner-current process, the solvent contact with the microalgae (,2 leaches the oil into the solvent, providing an increasingly more solvent-oil traction.

Following extraction, the solvent can be d using methods ltnowri in the art. For example, lation, rotary evaporation, or a rising film evaporator and steam stripper or any suitable entizer can be used for removing the solvent. in one embodiment, the extracted /fatty acids are d to an absorption 11.2 Lil PCT/U82012/046696 process (e.g., bleaching) to remove one or more undesirable compomids such as for example, color bodies and/or ohosohaticlcs that may be present, in some embodiments, the absorption process is a bleaching process comprising contacting the spin/em acid extract with a bleaching, material tag neutral earth (eg, natural clay or ‘s , ctivated earth, Ui activated car ion, activated clays, silicates, and or a combination thereof}, The ion is not limited by the amount of bleaching material utilized. in one embodiment, the extracted lipids/Fatty acids are exposed to a degunnning step.

Degumrning methods are ltnown in the art and include, for example, water degumming, acid degumniing, enzynr tic degumming, and membrane degornming. in some ments, ti c it) lipid/fatty acid extract is suhj ected to deguinniing (cg following an. absorption process), wherein the degumnring comprises contacting the lipid/fatty acid extract with a mixture of aqueous acids that are in amounts ei‘iective to precipitate gums and/or chlorophylltype compounds that may he present in the lipid/fatty acid extract ition. The invention is not limited by the type or amount of aqueous acids utilized. in one embodiment, the mixture iii of aqueous acids ses sulfuric acid and/or phosphoric acid. in another embodiment, equal amounts of aqueous acids are mixed with the lipid composition. in a preferred embodiment, when d with the oil, the aqueous acids are in an amount sufficient to provide an acidic pH, Precipitates that form after acid mixing can he removed from the lipid composition, for example using centrifugation and/or filtration tag, membrane filtration). in some embodiments, the dcgnmmcd lipid/fatty acid extract cmnposition is subjected to drying (eg, to reduce moisture content of the composition}. The invention is not d hy the drying condition (cg. tirnc, temperature, and/or a vacuum condition). As described herein, in some embodiments, the moisture content of the dried lipid/fatty acid composition is less than about it)% W/W (cg. less than about 9, S 7, o 5, 4, 3, 2 l, n.9, 0.8, 9.7, (it), 0.5, (ix-l, 0.3, (3.2, til, 0.05, or GOP/23 w/w).

Lipid ition in some embodiments, the invention provides a lipid ition prepared from a. algal biomass of the invention. in some embodiments, the lipid composition is prepared in '30 accordance with a method of the invention. For example, in some embodiments, a lipid composition is an algal s or a portion/fraction thereof from algae of the genus Titan:mtochytrimn. in some embodiments, the algal biomass comprises an algae selected from izyceoe, tophyceaa Trebouxiophyceoe, Pinguz'opizyceue, and/or combinations thereof. in other embodin ents, the algal biomass comprises an algae selected from 11.2 v“, PCT/U82012/046696 Thornton/"ztritium slriuiu‘m. T/zmLts‘toc/zvtrizmz mt, tsSoc/:vzrium azurazmz ~ , J , .) 7 Crvnthewa‘intum comm Parietocleoris 31a Randi-Jammy rm, Cr ultimcmus Sf} J, 1 3 7 . f I l l 7 ,Parietocl’zloris Sim” Hartline/brats 5 J,’ POI‘Ji'Z Widow: Sim, (flossomaslit S and/or , f” a :3. , 11 r combinations thereof in further embodiments, the algal biomass comprises an algae selectet U1 from Parietochiom' incite, Ritmz’omwuw saline, Hazardous firzmescens, Porphw‘idimn tinge/item and Glossomuslix chijto‘twlusla, and ations thereof, In a preferred embodiment. the algal biomass comprises Schizacitvtriwn liraacz’mmi.

Food Products and Animal Feed Additive ‘ l 0 in some embodiments, a Whole—cell algal biomass, fraction, and/or extract thereoiC is used for ption (egg by a mammal (eg, human or animal consumptiunfi or as a food ve leg, to increase the lipid content and/or nutritional components of a food). For example, in some embodiments, when used as animal feed (eg, cattle feed, dairy feed, aquaculture feed, poultry feed, etc), the lipids/fate; acids produced by an algal biomass of the invention is incorporated into a food product (tag, animal feed), in some embodiments, a whole—cell algal biomass, fraction, and/or extract thereof is used for pharmaceutical or nutritional purposes and/or industrial ations, The whole—cell algal biomass, on, and/or t thereof can be provided in any one of variety ot‘fortns/conipositions suitable for a particular application or use. ln some embodiments, the whole-cc l algal s, traction, and/or t thereof is provided. in another embodiment, a whole—cell algal biomass, fraction, and/or extract thereof is provided in a powdered form or as a tree oil in a liquid form (eg lipid composition or a fraction or concentrate thereoi}. A cell algal biomass, fraction, and/or extract thereof may be used for human and/or animal consumption. For example, in some embodiments, a Whole— L! cell algal biomass, fraction, and/or extract thereof is provided as or incorporated into a, feed, a dietary supplement, a food, a pharmaceutical formulation, a dairy product, and/or an infant For example, in one embodiment, a Whole—cell algal biomass, fraction, and/or extract thereof is dried (are, spray drying, tunnel , vacuum drying) and used as a feed or food (,2 supplement for any animal or aquaculture organism (e.g., fish, shrimp, crab, lobster, etc.) whose meat and/or products are consumed by humans or animals (eg. pets, livestock). in another embodiment, a whole—cell algal biomass, fraction, and/or extract thereof is mixed with a dry moisture~reducing agent leg, ground grain such as ground corn).

The compositions described herein may be used as a complete food product, as a 11.2 ~J 2012/046696 component of a food product, as a dietary supplement or as part of a dietary supplement, as a ”cred additive and may be either in liquid, semisolid or solid form. The compositions of the ion additionally maybe in the form oi‘a ceutical ition. The compositions, dietary supplements, food products, baby food products, feed additives, and/or U1 pharmaceutical compositions of the invention may he uti. ...ed in methods for promoting the health of an individual. The compositions may be in liquid, semisolid or solid form. For example the compositions may be administered as tablets, gel packs, capsules, gelatin capsules, flavored drinks, as a powder that can he reconstituted into such a drink, coolting oil, salad oil or dressing, sauce, syrup, mayonnaise, ine or the like. Furthermore, the food l 0 product, dietary supplements, and the like of the present invention can include, but. are not limited to, dairy products, baby food, baby formula, beverages, bars, a powder, a food topping, a drink; a cereal, an ice cream, a candy, a snack mix, a haired food product and a fried "cod product. Beverages of the invention include hut are not limited to energy drinks, nutraceutical drinlts, smoothies, sports , orange ju’ce and other fruit drinks, A har of the present invention includes, but is not limited to, a meal replacement, a nutritional bar, a snaclr har and an energy bar, an extruded bar, and the like. Dairy products of the ion include hut are not limited to, including hut not limited to yogurt, yogurt drinks, cheese and millt. Compositions intended for oral administration may he prepared according to any known method for the manutacture of dietary supplements or pharmaceutical preparations, and such compositions may include at least one additive selected from the group consisting of taste improving substances, such as sweetening agents or tlavoring agents, stabilizers, emulsifiers, ng agents and preserving agents in order to provide a dietetieally or phartnaeeutically palatable preparation, Vitamins? minerals and trace element from any physiologically acceptable source may also be included in the ition of the invention.

L! in some embodiments, a pharmaceutical composition ofthe invention comprises the compositions of the invention in a eutically effective amount. The compositions of the invention can be formulated for administration in accordance with ltnown pharmaey techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995‘}. in the cture of a pharmaceutical composition according to the invention, the lipid (,2 compositions {including the physiologically acceptable salts thereof) is lly admixed with, inter alia, an acceptable carrier. The carrier will be ible with any other ients in the fornnilation and must not he deleterious to the subject.

Biohtel PCT/U82012/046696 Many of the existing teclniologies for making hiot‘oel from algae are expensive, inell‘icient and unsustainable when operated at a scale that is required to ce any meaningful fraction ot‘petrodiesel in the market The supply and expenditure of energy to harvest and process algae are often underestimated. To produce biodlesel from algae U1 conventionally, the algae are typically harvested from a culture at a concentration ofahout 9.2 g/L in water. The harvested algae are then dewalered which increases the algal concentration to form an algal paste of about 35% solids, The paste is then fully dried by evaporating the water. Oil is then extracted from the dried algae with an organic solvent. such as hexane, which is removed by distillation from the algal oil. This conventional l 0 method for generating sel from algae is prohibitively expensive.

For example, when algae grows in a natural body of water, the algal biomass is relatively dilute considering the volume of water. ing a gallon of oil requires processing ol‘about 20,000 to 40,000 gallons of water. The energy cost of transporting and processing such a large volume of water is high. As example, 2500 gallons of oil/'acre/year could be produ red it algae with 25% of its mass as lipids could be produced at 25 pQ/day. For this example, 50 million gallons of water must be processed to produce m»he 2,500 gallons of oil. The rd approach of pumping water to a centralized facility for ring is simply too -intensive and cost prohibitive, As example, a vely small algal oil facility that produced 20 million gal/year would expend more energy pumping water from the pond to a central facility than that oontaincrl in the oil product, resulting in a not negative energy balance.

Accordingly, in some embodiments, the invention provides a method for preparing an algal biomass and/or lipid/fatty acid extract (egg a lipid/fatty acid composition) from an algal biomass, grown under conditions to contain elevated levels of total fat, the method L! comprising obtaining lipids from an algal hiomass ed under a culture condition sufficient to provide an algal biomass with elevated total fat t (eg, total fat content greater than (37"% of the biomass), wherein the algal s is harvested at a negative growth acceleration phase or a stationary phase of the algae. in another embodiment, the algal biomass is harvested during a logarithmic growth phase of the algae. Methods for obtaining a (,2 lipid composition from an algal biomass of the invention are described herein.

Accordingly, in some embodiments” the invention provides a biol‘uel feedstock or a hioliiel compliant: lipids, arbons, or both, derived front an algal culture and/or algal biomass generated according to the s of the invention. in some embodiments, lipids or algal compositions comprising the same are subdivided according to polarity: neutral lipids L9~14 PCT/U82012/046696 and polar lipids. The major neutral lipids are triglycerides and free saturated and rated fatty acids. The major polar lipids are acyl lipids, such as glycolipids and phospholinids. is some embodiments, a composition comprising lipids and hydrocarbons of the invention is described and distinguished by the types and relative amounts of fatty acids antifor U1 hydrocarbons present in the ition. in sortie en’ibtniitnents, the hydrocarbons present in algae compositions of the ion are mostly straight chain allianes and alltenesi and may include parafi' ‘ and the like having up to 36 carbon atoms in some ments. the invention provides a method ol‘rnaking a liquid fuel that comprise processing lipids derived from an algal cul ire and/or algal biomass or lipid fraction l 0 thereof described herein. Products of the ion made by the processing algal derived biofuel ocks can be incorporated or used in a variety of liquid fuels including but not limited to, diesel, biodiesel, kerosene, jetluel, gasoline, J?» l, 394,, IFS, lPo, ill-7, 3P8, let l’i‘opellant lly Stable (JPTS), Fischer—Tropsch liquids, alcohol-based fuels including l—containing transportation fuels, other biomassvhased liquid fuels including cellulosic biomass—based transportation fuels. in some embodiments, triacylglycerides in algal oil is ted to fatty acid methyl esters (FAME or biodiesel), for example by using a hasev-eatalyzed transesteritication process (for an oyen'iew see, egg.s K. Shame "l‘yson, Joseph Bozellg Robert e, Eugene Petersen, and Luc Moens, ”Biomass Oil Analysis: Research Needs and Recommendations, NRElJ/TP-S til-347%, lune 200% hereby ratcd hy rcl‘erencc in its entirety) to some embodiments, the triacylglycerides are d with methanol in the ce of NaOll at {it} C. for 2 hrs to generate a fatty acid methyl ester (biodicscl) and glycerol. in thither cmbodimenta the biodiesel and glycerol couproduets are ible and typically separated downstream through decanting or centrifugation, followed by washing and purification. Free L! fatty acids (lets) are a natural hydrolysis product of triglyceride and fanned by reacting triacylglycerides and water. in some embodiments, methods of the invention further comprise a step for quickly and substantially drying the lgal oil by techniques known in the art to limit production ofl‘ree tatty acids, preferably to less than l‘l/i». in another embodiment of the invention, the methods can further comprise a step for converting or removing the free (,2 fatty acids by techniques ltnown in the art. in some embodiments triacylglycerides in algal oil is converted to fatty acid methyl esters (FAR/EVE or biorliesel} by acid—catalyzed transesterilication, enzyme—catalyzed sterilication, or supercritical methanol transesterilication. Supercritical methanol transes erilication does not require a catalyst (See, e.g., Kustiianai l). and Saka, S.., ”El‘lects of PCT/U82012/046696 water on hiodiesel fuel production by supereritieal methane-l treatment," Bieresource Technology 9l (2004), 289-295; Kusdiana, D. and Saka, 8., ”Kinetics oftransesterifieation in rapeeeed nil tn hiediesel fuel as treated in supereritieal methanol,” Fuel 80 (200] ), 6934398; Saha, 5., and Kusdiana, 13., "Biediesel fuel from rapeseed oil as. prepared in supercritical Ui rrretlranel.,H Fuel 8t} ), 225—23 l). The reaction in supereritieal methanel reduces, the reaction time from 2 hrs to 5 nrinuten. In addition, the absence {if the base catalyst NaOi—l greatly fies the downstream purification, reduces raw material cost and eliminates the problem with snaps from free fatty acids. Rather than being a rm the free fatty acids become valuable feedsteeh’e that are cenverted te- hiediesel in the supereritical methanol as it) follows.

In sum: embedimentsa triacylglycerides are reduced with hydrogen to preduce parattins, prnpane, carbon dioxide and water, a prednct lly known as green . The parat‘tins can either be lsonterized to produce diesel or blended directly with diesel. in some ernhedintents, there are ages of hydrogenation over ccnvettticnal hase—eatalgzed lS transesterificalien. For example, the hydrogenatien s (else referred to as racking) is thermochemical and therefore much more robust to teed ties as compared to biechernical processes (eg hytlrecraclx'ing is vely insensitive to free fatty acids and water). Free fatty acids are readily converted to par‘aftirts, and water simply reduces the l thermal efficiency of the preeess but does not significantly alter the chemistry in annthcr hen-limiting example, the paraffin prerluct is a pure hydrocarbon, and therefore indistinguishable from pettnlcum—hascd hydrecarhene. Unlike hiutliesel which has 3 l5% lewer energy content and can freeze in cold r, green diesel has similar energy content and flew characteristics (eg, Viscosity) te petrnleum—hased diesel. In various embodiments? the ds of the inventinn encnmpass the steps of hydrocracking and [\3 L!i isomeriza‘tien, which are we l known in the art to produce liquid fuels, such as jet~fuel, tiieSel, kerosene, gasoline; ill-l, J94, MKS, ill-ti, JFK}, .lP~8., and JPTS.

EXPERlMENTAL The following examples are provided in order to demonstrate and further illustrate (A! t , certain pretErred emhndiments and aspects of the present inventinn and are net tn he censtrued as limiting the scape thereef.

Grewth at" high fat. algal hiemasa PCT/U82012/046696 Experiments were conducted during development ofetrtbodimehts ot‘t’he invention in order to characterize and estabhsh methods for heterotrophie algae production, and in particular, s ofeuiturihg algae in order to generate an algal s containing high fat/1ipid ieveis. A series of conventional trophie atgac production studies was Ui performed and run batch.

A cuiture ofSchizochylrium rttm; was obtained and stored in 15an eryoviais at ~80 C. For each experimentg the process was started by thawing eryoviais and aseptieatiy adding to 1.0L shake flasks of with media. Media in the 1 L flasks contained ’50 g/L sugar; g/L yeast extract, and 4 g/L sea satt. Three liters of 3 to 6 day old shake flash culture was i0 used to inocuiate a 250 L vessei containing media, grown for 2448 hoarse and then transferred to a main vessei (17,000 to 2t 3001:.) and run as a batch process for '36 to 72 hours. The temperature of the hatch runs was kept between 25 and 30 C. The temperature range was large due to lack ofpreeise control of the system. The media used in the seed (25011) and hater: r, 1000 to 283001;)t.» runs was as foliows: Table A. Media used in traditionat hatch and seed euitures.

Total fat t of the algal biomass of the hatch euttures was determined by gas tography (See ADA’C gravimetrie method 92206), acid hydrotysis (See Totai Fat by Aei d i-iydroiysis Atikoni Technology Method 1, 02—10—09), and High Temperature Solvent Extraction (See Atrhom Technology Method 2, 01—30—09 and ADCS Method 5-04). In brief, a t analysis procedure for tation broth was as follows: Broth samples were concentrated by eeotrifiigatiort, Atter decanting the sample was :tireeze dried for 24- hours with resuitant moisture iess than one percent. The samples were weighed prior to acid hydrolysis, washed and dried in an overt. This was followed by an extraction process under PCT/U82012/046696 gradient thermal conditions with petroleum ether. The hydrolysis and extraction process were undertaken utilizing automated instruments. After further drying, results were detennined on the basis ofnrass lose.

As shown in Table lB below, the total late/lipid levels (iv/w} achieved in the hatch productions at a tern Jerature range from 23-30 C was 23-38%. 'l'able l8. Total fat content of algal s grown in batch run between 25—30 C.

Efforts were made to increase the amounts offal/lipid levels as these s were considered too low to he of value and additional experiments were run in an effort to increase the level of lipids produced in cultured algae.

During development of embodiments of the inven'ion, experirtients were conducted in order to determine ifehanges in the tuents and/or amounts or ratios of the same. in the media could provide different algal growth teristiea. in on? experiments were conducted to determine if Scale—up of an algal culture system would alter algal growth characteristics. in particular. the amounts and ratios of; {54304, Urea, CaClg, MgClg, and KHZPO4 were modified in an attempt to increase the level of lipid produced by cultured algae: Results of tiermentations produced in a hatch volume of 10L are shown below: 332E________________________________________________________________________________________________________________________________________________________________________________________________________________ éliiéfiiifiéélllllllllllllllllllllllllllllllllllllllllllllllllllllllllliiiii?”””””””””iiat:””””””””””””””””””””””””link;”””””””””” *3 ..lflll‘l:ll§9§l§.............ll.§§:ll3f§%ll..,,.,,,,,, ..9§2§ll;....... ..ll§?:§l§:.....................................Q3&3???............ (sf Ll Sugar Yeast Extract 2012/046696 Urea 2 2 2 2 1 1 0.1538. E 0.1538 MgCiZ 0.1538 :3 1538 n1538 0.1533 0.5 1 FHFFFF -_ ' i ' Traceé{liguhfi— Fr' CFFFFFF --—" _Zn suteifa --—” Coppe suwate —-'“ strong NH3 strong NHB i ESE???............................sme” .3933]?............................................................................................................................................ ph ISSL s ph Issue; ' foam out foam out TabieZ:10L fermcniatifin condltigns and rewsuits ingredientsiing N83" NBSvflBZSii EN83~0&G?11 Nfié—Gfifl?1l NBfi~BfiG711 {E41911 NWMMlSli NBfiOfil‘f-fll KHZPOA- Trace d) - Ferric Chim‘ide Zn sulfate j‘fln suifate Copper 5L:-ifate Feed : Ureazi<H2P04 2:0.5 2:0.5 220.5 2:013 PCT/U82012/046696 temp set point Table 3: ‘delilOllal 10L fermentation eontlit ms and res i. ingredients/lag 2} {3406141 P48606141 i NBSGSZii NBAQBZ‘li N B603211 iNB§Ofii411 i1 NBSSEZSli NBAOBZBM NBBCSZBM i (gt) ; -n-h : ________ N 30 U rea ZrtSO4 C. 15 38 :15-12............... .........t{-1538 MgCi 2 3.15 38 I<H2Po4 2 Trace (liquid) - mi 10 ' t...................._ .......................

Ferric Chloride Zn sttifate Mn suifate “flatness._____ Copper sulfate Feed UreaziiHZ'C-i temp set point . i E fat "/0 46.5? 65.04 67.51 E 61.28 5?.9‘3 58.53 49.25 :34.41 54.97 i T .

Fomuiar confirmation Natl effects Sait ratio effects .

Tabie 4: onal iOiJ fermentation conditions and t‘esuitst :3 These experiments, ted during development of embodiments of the invention, indicated that n amounts/ratios of substrates present within the media had a direct impact on algal growth teristics (egn, totai biomass achieved as weii as amount off-at and/or other component content Within the biomass its-sit) Parameters that provided a high fat content him-tags in the ML runs were then utilized to determine ifthey would he successfoi for iargc scale production of a high fat content biomass.

EXAMPLE 2 Large scale protiuction of high fat alga} s ihe ini-iai ts to generate a heterotrophic aigai biomass bed in Example 1 above. utiiized procedures based on yeast fermentation processes. The processes were run in PCT/U82012/046696 batch due to limitations in the production facility (Nicln‘dasville, KY) and temperatures that could only be lled between 25 and 38 C. The temperature range was large clue to lacl; ofprecise control cftlte system, As ted in Table l, above, the fat levels achieved at the Nicholasville, KY plant ranged from 853%. However, as indicated above, additional Ui experiments ‘l Vere carried out during development bodimenls of the invention that provided the identification of certain ratios/amounts of substrates that could be utilized during heterotrophic algal biomass production to alter algal growth and biomass generatierr/properties. Modification of the levels and ratios ot‘the media ("cg M53804, Urea, Calf/lg,” MgClz,‘ and Kl-lgi’04) during fermentation was identified and characterized to alter l0 algal growth and to generate a biomass with significantly different properties (cg, a significantly higher fat content biomass). As described below, the process (including media containing the identified ratios/amounts of substrates ett‘ective in generating a high a: t algal biomass (eg greater than 67% fat conterrt)) was harther tested and run in large scale and also as a tch (thereby allowing for modification and l of antounts of l5 nitrogen, orus. potassium, and carbon during the mm).

A culture ofSeltizocitytritmt [imacinwn was obtained and stored in 1.5inL cryovials at —80 C. For each culture, a crycvial was thawed and aseptically added to ltlL shalce llaslr of media. Media in the l L flasks contained the components as shown in Table 5: ingredient Batched Manufacturer , . l—Hantmond, lN, USA Yeast Extract ' . n' lndiananolls, 1N, USA .' Sigma-Aldrich St. Louis, MO USA Table 5. Media used for toll. culture.

The temperature of the shake flasks containing Schizochytrz’wn it'macr‘rrtmt in media was kept at 30 C and shaken at .250 RPM until such time that the algae had entered logarithntic/exponential growth phase hut prior to ion of glucose in the media {usually 72— M4 hours).

The contents of lL e flasks were then aseptically transferred into 2.0L aspirator bottles with sterile connectors that were used to connect to larger vessels (40L or ".27 l. or l8 L vessels). Thus the lL culture flask cultures were used as inoculum and aseptically added to a seed vessel (either 40L or 27L or 18L) containing media described in Table 6 below: PCT/U82012/046696 Manufaemrer Cargill — Hammond IN. USA, Sensiem ~ liidianapnlls, lN, USA Norkmn limited Kutahya, Turkey Occidental al Company — Dallas, TX Norm American SalE y Overland Park, Table 6 sed for E811 or 27L, first seed cultures.

The firsE seed stage /27L) was run at '36.} C, under airflow and agitation L11 conditions so as En mainlain dissolved oxygen aE 01‘ above 10%, and uiiEil at least 20 g/L {if glucose was consumed. When grown under sterile conditions, no pH COITE‘El‘Ol was required.

Kathe} the pH slayeLl Within a healthy lanye> throughout the fermentation ss. Elie first seed stage (40/ lx’l 7L) was consideiecl completed wlien Ell'gal gromli W218 within log,exponential giomh stage glucese had 110E been depleteLl from the EnL.die. but at leasE2 l0 gal Ol glucose had-been Ltor‘isumed (inaeneial this oeLLined bemeen about 24-48 hows). A laiger vessel E400El/2000L} was made readv fer the firs{seed stage culture (e.g., it was filled with media and bmugln EL) 30 C undel stLrile enndltio113‘: Upon completion. Liftlie first seed culture The contents oflhe first seeLl stage (40f! 8/2VllL:uli‘ure vessel was transferred to a vessel with at least 25000}; media described in Table 7 below: _‘ 3 Cargill — Hammond, EN, USA Sensient Indianapolis, 1N, USA MgSOAl Noi‘lLeni Limited l<LLiEaliya~ Turkey ental Chemical Company — , EX North American Salt Company Overland Park: MgClZ KS Eablc7Media used foil4 0.00/2000-le seeond seed cul Euros.

PCT/U82012/046696 This second seed stage (4(300/2000L} e was run at 30 C, under airflow and agitation conditions so as to maintain dissented oxygeia at er above 10%, and until at ieast 2C! gI’L {If e was consumed. When grown under sterile conditions, no pH comm} was required. Rather, the pH stayed within at healthy range ihi‘eughout the tatien process.

U1 The second seed stage (4000/20001.) was considered emepleted when algal growth was within log/experiential growth stage: glucose had not, been depleted free: the media, but at, least 20 gl of glucose had been consumed (in general. this eeeurred between about 24—48 hours).

Upon completion ufthe sewed seed (40(llil/ZGQOL) culture! the cements of the seee-nd I0 seed e weie caliy transferred into a third culture vessel with a volume ranging between 70,090 L, :0 220,000 L of sterile media at 30. C as described in Table 8 below: Batehed Manufacturer 50 g/L Czargill Hammond, IN USA I5 vessei (70,000220fi99 L vessel), glucose and fedbateh feeds were started Glucose was ined at ill g/L {luring large scale culture ofalgae in the third culture vessel (70,000" ZZUAEQD l. vessel). As described in Table 9 belew, the feed used for the fed~b3tel1preeess eentained: Table 9, Feed used fer fed~beteli process, PCT/U82012/046696 The fed batch feed was added over a. 34 hour period. Although an understanding ol‘ the mechanism is not needed to practice the presem‘ haveu’lieu, and while the present ihvehlhm is not limlled to any parlicular mechanism 0? aeliuh, in sum > cllmehte, this Ui time period was fied based upon the valiun lhal ll leek ~ 20 hours fur the feed to Start (fur 30 g,” of glucose to he consumerl by {he algae present in the third culture vessel).

The feed was their stepped (e. go. at aruuml lug huur 54) in urder m allow all of the nutrients m be remeved (cerisumecl) frum the media. Harvesl of the algal hlemass took place upuh the leririiiiatiuri ol‘expuuemial growth, occurring generally between the lug hours 66-76. l0 'l'he culture hrmlr was ale—“sludge centrifuged under (renditions to e 15—30% solids, with the concentrate spray dried to remove water to a final moisture of less than 5%. s of several independent, large scale cultures are shown in Figure l and Tables ML- 12 below: Run Fat % Vol 0/6 recovery Far 9/0 Protein 9/6; lVlOlSlll‘IQ % number st adjusted {rem ll (spray dried (spray spray dried sample) Biomass centrifuge 'l product) dried. product Table l0. Large scale production culture results, l’had harvest sample preeess control problems will: this batch o E l ~2—ll *1 Had a batch v olume 0f7l'l,llll(l L and a harvest volume of 93,700 L a li'l~3—ll I! Had a batch ‘v‘ulume of 70,000 L, and a harvest velume 0? 84,000 L, o Fl—A’M l I! Had a batch volume of 70,000 L and a harvest volume of 92,300 L 0 E l--ll Had a batch volume $70,000 L and a harvest volume of (A! r v 82,300 L 0 Fl -6—l l I“ Had a batch volume uf 80,090 I. and. a harvest volume of 83,600 E. o FZ-le-ll PCT/U82012/046696 .. Had a batch volume of 1 10,000 L and a harvest volume of 113,000L o FZ—Z-H '4 Had a batch velume 0f 1 “£000 L and a harvest vehrme of U1 IESVGOOL The biemass generated from each large scaie, fed-batch culture was characterized; including unaiysis 0f the total fat (saturated and rated fat) t; moisture, doeesehexaenoic acid (DI-iA'menten’r, palmitie acid cmrterrt, crude protein content and ash cement (See; eg Fer cement end/Tviuisrtrre AOCS Am 5-04 ‘Rnpid Determinatien of Oil/Fat Utilizing Big1} Temperature Solvent Extraction” V: 3/31/10; Palmitie — AOCSS Mefimd Ce 113-89 and AOAC Method ef Anaiysis 9'9} 3'9; Protein — AOAC 990.03; Ash - AOAC 942.05 V01 adjusted Biomass (gr/L) Stene, er. a}. Dry Weight Measurement of Micrebiai Biomass and Measurement Variablrty Analysis. Bioreehneiogy Techniques. VGE 6: 207- 211.2 ”Neil ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, ' i T i E E Crude i r = FAT ’60 E a A : TQTAF re DHA i Paimitic Preterm {‘56) ‘5 E i 1.

Run #fCemments (Fmai g ; max g (9;) Max {0/‘3 t J”) (mglg1.} (m'/g g ) Re 0W0“”p t) E " % i : i reiease H11117201391918379143639 ______ germ-11 é ~ - . sens—11 15.92 s 11 SL—Fl—S—ll SL--F2-Z-11 247.2 197.36 SL—FZ-d—llw gr—rz—g-ri sews-11 SL-—F2-~11-11 grams—r1 202.51: 250.01 10.02 3.4g sen—134‘ 70.95 1.56; 326.94 12.39 4.59 sen-1441 PCT/U82012/046696 SL—F1-1E3-11 SL-Fl-lS-l SisF 1 — 16— 11 SL~F3—8—11 SL-Fl-—25-—ll S§.--F3-11 5L~F311 SL~F1~1>12 Crude TOTAL FAT 96 . : Moisture. Protein {‘56} Run #[Ccammems E {Final (‘36) Max 6% Report on Harvest) e E ESL—1514142 159.54 SL-FIi—S‘ZEZ 171.73 . 5- .4.

ELM—3&2 176.68 u. m 6'- Eu.) m ,..x O m {7‘ ,..E 172.91 SL- 2 Si.~F3—9~1 2 E 69.18 1.74 Tabie 12. Characterizatinn of large scale cultures 2012/046696 Additionally, the fatty acid profile oftiie biomass was characterized. As shown in Figure 2, the fatty acid profile ofeaeh algal biomass generated is highly similat/consistent, independent of the total fat content of the s. A eonigmsite fatty acid profile, taking into Ui consideratien the collective profiles ol’all samples analyzed, is provided in Figure 3.

The glyceride profile was also determined for each algal biomass” Oftlie total glyceritle eohteht e-‘f the big-mass? about 4~8% were diglyceritles. less than 1% glycerol, about 3-7043 ycerides and about 84~88% triglycerides.

EXAEWFLE 3 Biomass harvesting Experiments certducted during development of embodiments oi‘the invention identified that the increased total fat levels in the biomass caused significant problems with regard it} eeii‘trilugatioh ofthe algal biomass. Recevery of liemass content nest~ fugatieii ranged from only about 45-85% total biomass weight. This is shown, fer example, in Table 13 helew: ........................................"""""""'"""',“" ......

; Run Fat 0/5 Vol Protein % Fat "m Protein e, l number (harvest adjusted (harvest iron} itspraydriod (spray spreydricd sample) Biomass sample) centrifuge product) dried product Product‘s t, *ljacl harvest sample” process control problems with this hatch E l .,3__1 l N Had a hatch volume $70,000 L and a t velome of 93,700 L la.) Li! F l ~3—ll E Had a hatch volume of 70,000 L and a harvest vohime of 849000 L El 4—11 I“ Had a batch volume $70,001:) L and a harvest volume of U.) :3; 92,390 L 1315—11 1/: {N} 2012/046696 .. Had a hatch volume of '?O,{.){30 L. and a harvest voinme of 82,300 L F 1—6—1 1 '4 Had a hatch veiume of'hflflflh L and a harvest voiume of 83.600 L EZ--i--1 1 I Had a batch volume of i 10,990 L and a harvest volume of 113,()(}()L E'Z-Z—i i I“ Ha i a bateh voiume hi i 10,089 L and. a harvest volume of 1935,6001, The reeoveiy problems were fied to be attributable to the increase in the amount of low density lipid/nil in the biomass. Thus, experiments were centiueted during; embodiments of the invention in an effort to address this prehiem.

One approach that tiispiayed the abiiity to enhance recovery of the biomass was to ehih the euiture eoniprising the aigai biomass prior to e n rifngatien. Aithough an understanding ei’a nieehanism is not needed to practice the ion and the invention is not iirnited to any particular mechanism of action, in some embodiments, ehiiiing the enitnre increased the density of the iipizi/oii and aiiowed a iarger recovery of the biomass Experiments were conducted in order to determine the effects of ehiiiing the biomass before centrifiigation.

Lab triai one: ‘2 gaiinns ofhroth eoiieeted and stored at 7—8 C. for 16 pins hours“ Eight X 50 mi centrifuge tubes were eoiieeteti and pine-ed in a water hath to reach target [\J Li] atures described in table E4 heinw. Aii s were centrifuged at 5000 rpm for 5 minutes.

Temperature iC} Visual Observation ent separation with no floating eeiis.

Ciear supernatant _2_O________"5351i"""séfiéiéiihii"""{t7iiiifiiiimii‘é‘aiiii;wee-ii: Cioudier than 10C ar to 20 C; eionriier Good. separation with no floating Good separation with ne floating eeiis; very e i on riy 1/: wJ PCT/U82012/046696 510 Still separating; floating cells; milky supernatant Poor separation Almost no se iteration with numerous tloatin-‘té: Lab trial 2:: Fresh broth samples were collected and tested over a ature range of lO—Sth. They were not refrigerated overnight as in trial 1, All samples were allowed to sit in an ice water bath to target ature. Samples were fuged at 5009 1pm for 5 minutes.

Temperature Visual Observation Density (fig/[111D EExcellent separation with no floating cells, Clear atant.

E Good separation with no floating cells. 2 Very cloudy supernatant ESample still separating; Visible ; flocculation E‘v’eiy similar to 20C; increasing cloudiness EStill Good Separation; increasing cloudiness Table l3" Cultiire tempo "attire and centrifugation results of trail 2. ll} As described in Example '2 and Figure l, during large scale production chilling oi‘the biomass prior to ry (centrifugation) lend to significant increase in total recovery of the biomass" Multiple large scale rims have been completed with total ry of approximately 95%.

All publications and patents mentioned in the above specification are herein lS incorporated by reference Various modifications and variations ol‘the described compositions and methods oftne invention will be apparent to those skilled in the art Without PCT/U52012/046696 departing {Tom the scope. and spirit ofthe invemion. Aitizougi‘i ihe invention hat: been described in connection with specific preferred embodiments, ii shouid be understand that the invention as claimed shonid 1103‘, be nnduiy d to such specific embodimenfi indeedg various modifications of the described modes for carrying out the ion that are Obi/inns U] to these skilled in the relevant fields are iniended to be within Ehe scope of ihe present inv eniinn.

Claims (24)

1. A s of making an algal biomass comprising at least 67% total fat comprising culturing an algae in two or more types of culture medium sequentially, culturing the algae in a culture medium sing a carbon source, yeast extract, a magnesium source, and a calcium source; and subsequently, culturing the algae in a culture medium comprising a carbon source, a yeast extract, a nitrogen source, a phosphate source, a magnesium source, a calcium , and 4 g/L or less of sodium chloride, n the nitrogen and phosphate are in a ratio of 50:1 to 4:1.

2. The process of claim 1, wherein one culture medium of the two or more e medium contains 50 g/L of a carbon source, 7.5 g/L yeast extract, 0.15 g/L magnesium sulfate, 0.15 g/L calcium de and 0.15 g/L magnesium de.

3. The process of claim 2, wherein the carbon source is a sugar.

4. The process of claim 3, wherein the sugar is glucose.

5. The process of claim 1, wherein one e medium of the two or more culture medium contains 50 g/L of a carbon source, 7.5 g/L yeast extract, 4.0 g/L magnesium sulfate, 1 g/L urea, 2 g/L calcium chloride, 2 g/L magnesium chloride and 0.25 g/L monopotassium phosphate.

6. The process of claim 5, wherein the carbon source is a sugar.

7. The process of claim 6, wherein the sugar is glucose.

8. The process of claim 1, wherein one culture medium of the two or more culture medium contains a carbon source, yeast extract and sea salt.

9. The process of claim 8, wherein the carbon source is a sugar.

10. The process of claim 9, wherein the sugar is glucose.

11. The process of claim 1, further comprising culturing the algae in a culture medium containing e, yeast extract and sea salt prior to the culturing in the culture medium comprising a carbon source, yeast extract, a magnesium source, and a calcium source.

12. The process of claim 1, wherein the culture medium sing a carbon source, a yeast extract, a nitrogen source, a phosphate , a magnesium source, a m source, and 4 g/L or less of sodium chloride, n the nitrogen and phosphate are in a ratio of 50:1 to 4:1, is supplemented with a fed-batch feed.

13. The process of claim 12, wherein the fed-batch feed comprises urea and tassium phosphate.

14. The s of claim 12, wherein the algal biomass is harvested from the culture medium between 12-24 hours after cessation of the fed-batch process.

15. The process of claim 14, n the algal biomass is harvested from the culture medium after all of the nutrients have been removed/consumed from the medium.

16. The process of claim 14, wherein the algal biomass is harvested via centrifugation of the culture medium comprising the algal biomass.

17. The process of claim 16, wherein the culture medium is chilled prior to harvesting the algal biomass.

18. The process of claim 17, wherein the culture medium is chilled to between about 5 and 25º C.

19. The process of claim 1, wherein the algae is Schizochytrium limacinum.

20. The process of claim 11, wherein the culture medium contains 50 g/L e, about 10 g/L yeast extract and about 4 g/L sea salt.

21. The process of claim 1, wherein the culture conditions comprise running the algae culture at 30º C under airflow and ion conditions so as to maintain dissolved oxygen at 10%.

22. The process of claim 1, wherein algae are cultured under sterile conditions.

23. An algal s prepared according to the process of claim 1.

24. The process of claim 1, wherein the culture medium comprising a carbon source, a yeast extract, a nitrogen source, a phosphate source, a magnesium source, a m source, and 4 g/L or less of sodium chloride, wherein nitrogen and phosphate are in a ratio of 50:1 to 4:1, has magnesium and calcium in a ratio of 4.5:1 to 1:1.