OA16793A

OA16793A - Algal lipid compositions and methods of preparing and utilizing the same.

Info

Publication number: OA16793A
Application number: OA1201300507
Authority: OA
Inventors: Kyle A. RANEY; Rebecca A. TIMMONS
Original assignee: Alltech, Inc.
Priority date: 2011-07-13
Filing date: 2012-07-13
Publication date: 2016-01-04

Abstract

This invention relates to compositions comprising high lipid content algae and methods of making and utilizing the same. In particular, the invention relates to high lipid content algae biomass and algal lipid materials derived from the same, methods of making the same, as well as to biofuels (e.g., biodiesel) and dietary compositions (e.g., animal feeds) comprising or made from the same. Compositions and methods of the invention find use in a variety of applications including biofuel, dietary (e.g., human and animal nutrition), therapeutic as well as research applications.

Description

ALGAL LIPID COMPOSITIONS AND METHODS OF PREPARING AND

UTILIZING THE SAME

This Application claims priority to U.S. Provisional Patent Application Serial No.

61/507,390 filed 13 July 2011, hereby incotporated by reference în its entirety.

FIELD OF THE INVENTION

This invention relates to compositions comprising high lipid content algae and methods of making and utilizing the same. In particular, the invention relates to high lipid 10 content algae biomass and algal lipid materials derived from the sanie, methods of making the same, as well us to biofuels (e.g., biodiesel) and dietary compositions (e.g., animal feeds) comprising or made from lhe same. Compositions and methods of the invention find use in a variety of applications including biofuel, dietary (e.g., human and animal nutrition), therapeutic as well as rcsearch applications.

BACKGROUND OFTHE INVENTION

Within the last several years, the production of biofuel (e.g., biodiesel) from algae has been an area of interest. In part, this is due to high quality agricultural land not being required to grow algae (algal biomass). However, commercial production of biofuel (e.g., 20 biodiesel) from algae has remained a challenge.

In addition, over the last fïfty years, approaches toward providing animal nutrition havechangcd. No longer are animais fed whatever forage or other material that may be available. Instead, the dicts of animais arc closely monitored for total nutrition value and cost. Vcry often, animais on spécifie dicts arc monitored for quality and performance 25 charactcristics with the nutritional components of the feed being adjusted to maximize nutrition value of the feed and optimization of animal performance charactcristics.

However, cost is a critical factor. There is a continuai scarch for cost-cffcctivc animal feeds, not only to sustain animais, but in many cases to cause enhaneed growth and value.

SUMMARY OF THE INVENTION

The invention relates to compositions comprising high lipid content algae and methods of making and utilizing the same. In particular, the invention relates to high lipid

Y

content algae biomass and algal lipid materials derived from the same, methods of making lhe same, as well as to biofuels (e.g., biodicscl) and dictary compositions (e.g., animal feeds) comprising or made from the same. Compositions and methods of the invention find use in a variety of applications including biofuel, dietary (e.g., human and animal nutrition), thcrapeutic as well as research applications.

Accordingly, the invention provides a process of making an algal biomass comprising a desired, high fat content (e.g., 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 invention has identified culture conditions under which it is possible to obtain 10 an algal biomass comprising a desired level of total fat (e.g., at least 67% total fat). The invention is not limited to the total fat content (e.g., by weight) of an algal biomuss generated according to the invention. In a preferred embodiment, an algal biomass generated and/or used according to the invention comprises a fat content of at least 67% by weight. However, the invention also provides compositions and methods of generating an algal biomass containing greater (e.g., greater than 68%, greater than 69%, greater lhan 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, greater than 76%, greater than 77%, greater than 78%, greater than 79%, greater than 80%, greater than 81%, greater than 82%, greater than 85%, or more) or fesser (e.g., about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about

57%, about 56%, about 55%, about 54%, or less) amount of total fat. Indccd, methods and compositions described herein can bc utilized to gcncratc an algal biomass containing any desired level of total fat content. In some embodiments, the algae biomass is cultured in two or more types of culture medium in a scquential tnanner. For exemple, in some embodiments, onc culture medium of the two or more culture medium contains 50 g/L of a carbon source, about 7.5 g/L yeast cxtract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and 0.15 g/L magnésium chloride. The invention is not limited by the carbon source. Indccd, a variety of carbon sources may be used including, but not limited to, carbohydrates such as glucose, fructose, xylosc, saccharose, maltosc or soluble starch as well as oleic acid, fats such as soybean oil, molasses, glycérol, mannitol, and sodium acetate, cotton seed tlour, glycérol, molasses and corn steep liquor. In some embodiments, another culture medium ofthe two or more culture medium contains 50 g/L of a carbon source, about

7.5 g/L yeast extract, about 4,0 g/L magnésium sulfate, about t g/L urea, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and about 0.25 g/L monopotassium phosphate. In some embodiments, one culture medium of the two or more culture medium contains a

carbon source, yeast extract and sea sait. In some embodiments, and as described herein. algac arc culturcd in a first culture medium (e.g., containing glucose, yeast cxtract and sea sait); transferred into and incubated in a second culture medium (e.g., containing glucose, yeast extract, magnésium sulfate, calcium chloride and magnésium chloride); and transferred into and incubated in a third culture medium (e.g., containing glucose, yeast extract, magnésium sulfate, urea, calcium chloride, magnésium chloride and monopotassium phosphate). In soinc embodiments, one of the culture médiums is supplemcntcd with a fedbatch feed. In a preferred embodiment, lhe third culture medium is supplemenled with a fedbatch feed. The invention is not limited by the type, or duration, of fcd-batch feed utilized.

In some embodiments, the fcd-batch fccd comprises urea and monopotassium phosphate. The invention is not limited by the amounts and/or ratios of media components used in the cultures. Exemples that may be utilized as components of each of the various media (e.g., first culture media, second culture media, batcli ntedia and fcd-batch ntedia) arc described in detail herein. In some embodiments, the algal biomass is harvested from a culture (e.g., from

I5 a third culture mediutn) between 12-24 hours after cessation of the fed-batch process. In some embodiments, the algal biontass is harvested from tlie third culture medium after all of the nutrients hâve been rcmovcd/consuincd from the mediunt. The invention is not limited by the way in which the algal biomass is harvested. Indced, a variety of ways may be used to harvest the biomass including, but not limited to, the ntethods described herein. In some embodiments, the algal biontass is harvested via centrifugation. In some embodiments, the culture medium comprising the algal biomass is chillcd prior to harvesting the algal biontass. The invention is not limited by the température to which the culture medium comprising the algal biomass is chillcd prior to harvesting. Indced, a variety of températures may bc used including, but not limited to, those described herein. In some embodiments, the culture medium comprising the algal biomass is chillcd to between about 5 and 25 C. The invention is not limited by tlie type of algae used in the invention. Indeed, a variety of algae may be used (e.g., independently or in combination) including, but not linrited to, those described herein. In some embodiments, the algac is a strain or species from the genus Chiorella, the genus Schizochytrium, or the genus Cryptliecodinium. In a preferred embodiment, the algae is Schizochytrium limacinum. In some embodiments, the first culture medium contains about g/L glucose, about 10 g/L yeast cxtract and about 4 g/L sca sait. In some embodiments, the second culture medium contains about 50 g/L glucose, about 7.5 g/L yeast cxtract, about

0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and 0.15 g/L magnésium chloride. In some embodiments, the third culture medium contains about 50 g/L glucose.

aboul 7.5 g/L yeast extract, about 4.0 g/L magnésium sulfate, about l g/L urea, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and about 0.25 g/L nionopotassium phosphate. In some embodiments, thc culture conditions comprise running thc algac culture at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about

10%. In some embodiments, the third culture medium (e.g., the culture media présent at the time of inoculation of a main fermenter (e.g., 70,000 L, 120,000 L, 256,000 L vessel)) contains medium with an initial ratio of nitrogen (N):phosphorus (P):potassium (K) of

46:13:8.5. In a preferred embodiment, the N:P:K ratio is the same in the batch and fed-batch culture modes. In some embodiments, the ratio of magnésium (Mg)icalcium (Ca) is 3:1 in culture media used in both batch and fed-bateh modes, although higher (e.g., 4:1,4,5:1, or more) and lower (e.g., 2.5:1, 2:1. 1.5:1, or lower) ratios may be used. In another embodiment, the ratio of chloride (CI2):sulfate (SO4)) of 1:1 is used in culture media used in both batch and fed-balch modes, although higher (e.g., 2:1, 3:1,4:1, 5:1, or more) and lower (e.g., 1:2, 1:3. 1:4, 1:5, or lower) ratios may be used. In soinc embodiments, thc ratio of sulfate (SO4):phosphate (PO4) in media at the time of inoculation of a main fermenter (e.g., 70,000 L, 120,000 L, 256,000 L vessel) is 16:1, although higher (c.g„ 20:1, 25:1, 30:1, 32:1, or more) and lower (e.g., 10:1, 8:1.5:1, 3:1, or lower) ratios may be used. In some embodiments, the total ratio of sulfate (SO4):phosphate (PO4) that has been batched und fed at the end of a full culture (e.g., including inoculum, first seed stage, second seed stage and main fermenter cultures) that generales an algal biomass containing a desired fat content (e.g., grcater than 67% fat) is 5.3:1, although higher (e.g., 5.5:1, 5.7:1. 6:1, 7:t, 8:1 or higher) and lower (e.g., 5:1,4.5:1,4:1.3:1, or lower) ratios may be used. In some embodiments, the ratio of chloride (C12):phosphatc (PO4) in media at the finie of inoculation of a main fermenter (e.g., 70,000 L, 120,000 L, 256,000 L vessel) is 16:1, although higher (e.g., 20:1,

25:1,30:1,32:1, or more) and lower (e.g., 10:1, 8:1. 5:1,3:1, or lower) ratios may be used.

In some embodiments, the total ratio of chloride (CI2):phosphate (PO4) that has been batched and fed at thc end of a fu 11 culture (e.g., including inoculum, first seed stage, second seed stage and main fermentor cultures) that générâtes an algal biomass containing a desired fat content (e.g., greater than 67% fat) is 5.3:1, although higher (e.g., 5.5:1, 5.7:1. 6:1,7:1,8:1 or higher) and lower (e.g., 5:1,4.5:1, 4:1. 3:1, or lower) ratios may be used.

Thc invention also provides an algal biotnass having a desired, high fat content (e.g., total fat content of at least 67% by weight). ïn some embodiments, the biomass comprises about 170-250 mg/g docosahexaenoic acid (DHA) and/or about 150-400 tng/g palniitic acid.

In some embodiments, thc invention provides a lipid composition, a food product or other

material comprising the algal biomass (e.g., dried algal bioniass) or a component thereof (e.g., a fatty acid component thereof). In sonie embodiments, the algal biomass (e.g., a dried algal biomass (e.g., generated according to a method described herein)) contains a desired amount of total fat and/or other components (e.g.. greater than about 68% total fat, greuter than about 69% total fat, greater than about 70% total fat, greater than about 71% total fat, greater than about 72% total fat, greater than about 73% total fat, greater than about 74% total fat, greater than about 75% total fat, greater than about 76% total fat, greater than about 77% total fat, or greater than about 78% total fat). In some embodiments, an algal biomass ofthe invention (e.g., containing greater than 67% total fat) is dried such that the biomass contains less than 5% moisture (e.g., less than 4.5% moisture, less than 4% moisture, less than 3.5% moisture, less than 3% moisture, less than 2.5% moisture, less than 2% moisture, or less than l.5% moisture). In sonie embodiments, an algal biomass of the invention (e.g.. a dried biomass containing less than 5% moisture) contains about 170-250 mg/g or more docosahcxaenoic acid (DHA) (e.g., about 170-180 mg/g DHA, about 180-190 mg/g DHA, about 190-200 mg/g DHA, about 200-210 mg/g DHA, about 210-220 mg/g DHA, about 220230 mg/g DHA, about 230-240 mg/g DHA, about 240-250 mg/g DHA, or more than 250 mg/g DHA). In soinc embodiments, an algal bioniass ofthe invention (e.g., a dried biomass containing less than 5% moisture) contains about 150-400 mg/g or more palmitic acid (IUPAC name: hexadccanoic acid (e.g., about 150-200 mg/g, about 200-225 mg/g, about

225-250 mg/g. about 250-275 mg/g, about 275-300 mg/g, about 300-325 mg/g, about 325350 mg/g, about 350-375 mg/g, about 375-400 ing/g, or more than 400 mg/g)). In some embodiments, an algal bioniass ofthe invention (e.g., a dried biomass containing less than 5% moisture) contains about 300-600 mg/g or inorc total fatty acids (e.g., about 300-350 mg/g. about 350-400 mg/g, about 400-450 mg/g, about 450-500 mg/g, about 500-550 mg/g.

about 550-600 mg/g, or more than 600 mg/g fatty acids)). In some embodiments, an algal biomass of the invention (e.g., a dried biomass containing less than 5% moisture) contains less than about 15% protein (e.g., less than about 14% protein, less than about 13% protein, less than about 12% protein, less than about 11% protein, less than about 10% protein, less than about 9% protein, or less than about 8% protein). în some embodiments, an algal biomass or component thereof of the invention is used in preparing biofuel (e.g., biodiesel). In some embodiments, an algal biomass or component thereof of the invention is used in preparing a food product (e.g., an animal feed or feed component).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure l depïels data generated during large scale, heterotrophic algae biomass production according to aspects of the invention.

Figure 2 shows the fatty acid profile of algae biomass harvested from several, independent large scale algal cultures.

Figure 3 shows a composite fatty acid profile of a harvested biomass utilizing materials and methods described herein.

DEFINITIONS

As used herein, phospholipid refera to an organic compound having the following general structure:

O

wherein RI îs a fatty acid residue, R2 is a fatty acid residue or -OH. and R3 is a -H or nitrogen containing compound cholinc (HOCf-UCHjN ⁺(CH₃)jOH'), cthanolaminc (HOCFbCHiNFB), inositol or serine. R1 and R2 cannot simultaneously be OH. When R3 is an -OH, the compound is a diacylglycerophosphate, while when R3 is a nitrogen-containing compound, the compound is a phosphatidc such as Iccithin, cephalin, phosphatidyl serine or plasmalogcn.

An ether phospholipid as used herein refers to a phospholipid having an ether bond at position 1 the glycerol backbone. Examples of ether phosphulipids include, but are not limited to, alkylacylphosphatidylcholinc (AAPC), lyso-alkylacylphosphatidylcholine (LAAPC), and alkylacylphosphatidylethanolamine (AAPE). A non-ether phospholipid is a phospholipid that does not hâve an ether bond at position 1 of the glycerol backbone.

As used herein, the term oméga-3 fatty acid refers to polyunsalurated fatty acids that hâve the final double bond in the hydrocarbon chain between the third and fourth carbon

atoms from the methyl end of lhe molécule. Non-hmitmg examples of oméga-3 fatty acids include. 5,8,11,14,17-cicosapcntacnoic acid (EPA), 4,7,10,13,16,19-docosahcxanoic acid (DHA) and 7,10,13,16,19-docosapcntanotc acid (DPA).

As used herein, the terms triacylglycerid triglycéride and triacylglycerol and TAG refer to is an ester derived from glycerol and three fatty acids, wherein fiitty acid refers to a carboxylic acid with a long unbranched aliphatic tail (chain). which is either saturated or unsaturated. Palmitic acid is one, non-limiting example of a triacylglyccridc.

As used herein, the terms % w/w (weight/weight) and w/w % and grammatical équivalents refer to the amount (percent) of a given substance in a composition on wcight:wcight basis. For cxample, a composition comprising 50% w/w phospholipids means that the mass of the phospholipids is 50% of the total mass of the composition (i.e., 50 grams of phospholipids in 100 grams of the composition, such as an oil).

As used herein the term algae refera to a uniccllular or multicellular organism formcrly elassified as plants, occurring in fresh or sait water, autotropbic or hctcrotrophic, but that lack true stems, roots, and lcaves. As used herein the term heterotrophic refera to an organisai that cannot synthesize its own food and is dépendent on organic substances (e.g., complex and/or simple organic substances) for nutrition. Thus, the term hctcrotrophic algae refer to an algae that cannot synthesize its own food and is dépendent on organic substances for nutrition. As used herein, the tenn autotrophic refera to un organism capable of syntliesizing its own food from inorganic substances, using light or chemical cnergy. The use ofthe tenu algal also relates to microalgae and thus encompasscs the mcaning of microalgal. The term algal composition refera to any composition that comprises algae. such as an aquatic composition, and is not limited to the body of water or the culture in which the algae arc eultivated. An algal composition can be an algal culture, algal biomass, a concentrated algal culture, or a dewatered mass of algae, and can be in a liquid, semi-solid, or solid form. A non-liquid algal composition can be described in terms of moisture level or pcrcentagc weight ofthe solids. An algal culture is an algal compositionthatcomprises livc algae. The term algae includes macroalgac (conimonly known as scawccd) and microalgae.

As used herein, the terms algal biomass or biomass refera to a collection or mass of algal cells grown in a given area or ccosystcm at a given time. The area or ccosystcm may be a naturally occurring environment (e.g., body of wuter) or u synthetîc environment (e.g., in a fermenter or bioreactor (e.g., open or closed)).

As used lièrent, lhe terni total fat refers to the sutn of triglycérides, phospholipids, wax ester, and sterols présent in a material. For exemple, total fat content ofan algal biomass rcfcrs to the sum of triglycérides, phospholipids, wax ester, and sterols présent in the biomass. Tn addition, total fat includes both saturated and unsaturated fats.

As used herein, the term preservutive refera to an agent that extends the storage life of food and non-food products by rctarding or preventing détérioration of flavor, odor, color, texture, appearance, nutritive value, or safety. A prcservatîvc need not providc a léthal, irréversible action resulting in partial or complété inicrobial cell destruction or incapacitation. Stcrilants, sanitizers, disinfectants, sporicides, virucides and tuberculocidal agents provide such an irréversible mode of action, sometimes referred to as bactericidal action. In contrast, a preservative can provide an inhibïtory or buctcriostutic action that is réversible, in that the target microbes can résumé multiplication if the preservative is removed. The principal différences between a preservative and a sanitizer primarily involve mode of action (a preservative prevents growth rather than kill ing microorganisms) and exposure rime (a preservative has days to montlis to act whereas a sanitizer has at most a few minutes io acl).

As used herein, the term ycast and ycast cells rcfcrs to eukaryotic microorganisms elassified in the kingdom Futigi, having a ccll wall. ccll membrane and intraccllular components. Ycasts do not form a spécifie taxonomie or phylogenetic grouping. Currently about 1,500 species are known; it is cstiinated that only 1% of all yeast species hâve been dcscribcd.-Thc terin yeast is often taken as a synonym for £. cerevisiae, but the phylogenetic diversity of ycasts is shown by their placement in both divisions Ascomycota and Basidiomycota. The budding yeasts (true yeasts) are classifïed in the order Saccharomycetales. Most species of yeast rcproduce asexually by budding, although some rcproducc by binary fission. Ycasts arc uniccllular, although some spccies bccomc multicellular through the formation of a string of connected budding cells known as pseudohyphae, oxfalse hyphae. Yeast size can vary greatly depending on the species, typically measuring 3-4 μηι in diameter, although some yeast can reach over 40 pm.

As used herein, the terms sclenium-cnriehcd ycast and sclcnizcd ycast refer to any yeast (e.g., Saccharomyces cerevisiae) that is eultivated in a medium containing inorganic sélénium salts. The présent invention is not limited by the sélénium sait used. Indecd, a variety of sélénium salts arc eontcmplatcd to be useful in the présent invention including, but not limited to, sodium selcnite, sodium selcnate, cobalt selenite or cobult selenate. Free selenomethionine (e.g., not associated with a cell or yeast) can also be used as the sélénium source for sélénium enriched ycast as ycast docs incorporatc this form of sélénium. During

cultivation, because of (he chemical similanty between sélénium and sulfur, yeast ïncorporate sclenium in place of sulfur in what arc normally sulfur-containing organic compounds within the cell. A sclcnium-containing compound în such yeast préparations is sclcnomcthioninc which will bc présent in a form that is incorporated into polypeptides/proteins. The amount of total cellular sélénium présent in the fonn of sclenomcthioninc in such préparations will vary, but can be between 10 and 100%, 20-60%, 50-75% and between 60 and 75%, The remainder of the organic sélénium in sclenized yeast préparations is predominantly made up of intermediates in the pathway for selenometliionine biosynthesis. These include, but are not limited to, sclenocystcine, sclenocyslathionine, selcnohoinocysteine and selenoadcnosylsclenomcthiomnc, The amount of residual inorganic sélénium sait in the finished product is generally quite low (< 2%). However, the présent invention is not limited by this percentage, as préparations lhat contain more (e.g., between 2 and 70%) or less (e.g., between 0.1 and 2%) than this percentage are also cncompasscd by lhe invention.

As used herein, the term SEL-PLEX refers to a dried, nonviablc sclcnïum-cnriched yeast (e.g., Sacchoromyces cerevisiae of accession number CNCM1-3060, Collection Nationale De Cultures De Microorganismes (CNCM), Institut Pasteur, Paris, France) eultivated in a fcd-batch fermentation that provides incrémental amounts of cane molasses and sélénium salts in a manner that minimizes the detriniental effects of sélénium salts on the growth rate of the yeast and allows for optimal incorporation of inorganic sélénium into cellular organic material. Residual inorganic sélénium is eliminated (e.g., using a rigorous washing process) and does not cxcccd 2% of the total sélénium content.

As used herein, the terni organic sélénium refers to any organic compound wherein sélénium replaces sulfur. Thus, organic sélénium can refer to any such compound bïosynthesized by yeast, or it can refer to free organic sclcno-compounds that arc chcinically synthesized. An example of the latter is free selcnomethioninc.

As used herein, the term inorganic sélénium generally refers to any sélénium sait (e.g., sodium sclcnïte, sodium selenate, cobalt selenltc and cobalt sclenatc). There are also a varicty of other inorganic sélénium sources (Sec e.g., those listed in the Merck index). Selenizcd yeast may be generated using a source of inorganic sélénium including, but not limited to, sodium selenite, sodium selenate, cobalt selenite, cobalt selenate, selenic acid, selcnious acid, sélénium biomidc, sélénium chloride, sélénium hcxafluoridc, sélénium oxide, sélénium oxybromide, sélénium oxychloride, sélénium oxyfluoridc, sélénium sulfides, sélénium tetrabromide, sélénium tetrachloride and sélénium tetrafluoride.

As used herein, the terni yeast cell wall also referred to as YCW refers to the cell wall of a yeast organisai that surrounds the plasma membrane and the intraccllular components of the yeast, Yeast cell wall includes both the outer layer (mainly mannan) and the inner layer (mainly glucan and chitin) of the yeast ccll wall. A function of the cell wall is to provide structure and protect the metabolically active cyloplastn. Signaling and récognition pathways take place in the yeast ccll wall. The composition of yeast cell wall varies from strain to strain and according to growth conditions of yeast.

As used herein, the terni purified or to purify refers to the removal of components from a sample. For cxample, yeast ccll walls or yeast cell wall extracts are purified by removal of non-yeast ccll wall components (e.g., plasma membrane and/or yeast intraccllular components); they are also purified by the removul of contaminants or other ugents other than yeast cell wall. The removal of non-yeast cell wall components and/or non-yeast cell wall contaminants results in an increase in the percent of yeast ccll wall or components thereof in a sample.

As used herein, the terni in vivo refers to studies and/or experiments conducted within a living organisai, occurring within a biological organisai.

As used herein, the term in vitro reters to an artificial environment outside the living organisai and to biological processes or reactions that would normally occur within an organism but arc made (o occur in an artificial environment. In vitro environments can comprise, but arc not limited to, test tubes and cell culture.

As used herein, the term high-pcrformancc liquid chromatography and the term HPLC refer to a form of liquid chromatography to separate compounds, The compounds arc dissolved in solution. Compounds are separated by injecting a plug of tlie sample mixture onto the column. HPLC instruments comprise a réservoir of mobile phase, a pump. an injecter, a séparation column, and a detector. The presence of analytes in the column effluent is recorded by quantitatively detecting a change in refractive index, UV-VIS absorption at a set wavelength, fluorescence after excitation with a suitable wavelength, or electrochemical response.

As used herein, the term scanning électron microscopy and the term SEM refer to use of a type of électron microscope that images the sample surface by scanning it with a high-cncrgy bcatn of électrons in a raster scan pattern. The électrons intcract with the atoms that make up the sample producing signais that contain information about the sample's surface topography, composition and other properties such as electrical conduclivity.

As used herein, the terni fixation agent refers to a chemical lhat is capable of fixing one substance to another in order to fix, stabilise, or otherwise préserve the substance in its curTcnt form to prevent the substance from degrading or otherwise changing. Often, fixation agents are used in scanning électron mieroscopy (SEM) to préparé the sample. Primary fixation agent: as used herein, the terms primary fixation agent refers to the first fixation agent used to fix a substancc._Secondary fixation agent: as used herein, the terms secondary fixation agent refers to the second fixation agent used to fix a substance. Tertiary fixation agent: as used herein, the terms tertiary fixation agent refers to the third fixation agent used to fix a substance.

As used herein, the term unalytc refers to an atoin, a molécule, a grouping of atoms and/or molécules, a substance, or chemical constituent. An anulyte, in and of itself cannot be measured; rather, aspects or properties (physical, chemical, biological, etc.) ofthe analyte can bc determined using an analytical procedure, such as HPLC. For cxample, one cannot mcasure a chair (analyte-componcnt) in and of itself, but, the height, width, etc. of a chair can be measured. Likewise, one cannot mcasure a mycotoxin but can measure the mycotoxin fluorescence that is related to its concentration.

As used herein, the term signal is used generally in reference to any détectable process that indicatcs that a réaction lias occurred (for example, binding of antibody to antigen). Signais can be assessed qualitatively as well as quantitatively. Examples of types of signais” include, but arc not limited to, radioactive signais, fl uo rime trie signais or colorimétrie product/rcagent signais.

As used herein, the terni bioavailability refers to the fraction of a molécule or component that is available to an organism or rcachcs the systemie circulation. When a molécule or component is administered intravenously, its bioavailability is 100%. However, when a molécule or component is administered via other routes (such as orally), its bioavailability decreases (due to incomplète absorption and first-pass nietabolism). In a nutritions 1 setting, bioavailability refera to the rates of absoiption and utilization of a nutrient. Different fomis of the same nutrient, for cxample, may hâve different bioavailabilitics.

As used herein, the term effective amount refers to the amount of a composition sufficient to effect bénéficiai or desired results. Ail effective amount can be administered and/or combined with another material in one or tnorc administrations, applications or dosages und is not intended to be limited to a particular formulation or administration route.

As used herein, the terni digest reférs to the conversion of food, feedstuffs, or other organic compounds into absorbable form; to soflen, décompose, or break down by heat and inoishirc or chemical action.

As used herein, digestive system refers to a system (including gastrointestinal system) in which digestion can or does occur.

As used herein, the tenn feedstufls refers to material(s) that are consumcd by mammals (e.g., humans and animais) and contributc energy and/or nutrients to a mammal's diet. Examples of feedstuffs include, but are not limited to. Total Mixed Ration (TMR), forage(s), pellet(s), conccntrate(s), premix(es) coproduct(s), grain(s), distiller grain(s), JO molasses, fibcr(s), fodder(s), grass(cs), hay, kcmel(s), leaves, meal, solublc(s), and supplement(s).

As used herein, the terras food supplément dietary supplément dietary supplément composition and the like refer to a food product formulated as a dietary or nutritional supplément to bc used as part of a diet. Exemplary dietary supplément compositions are described herein.

As used herein, the term animal refers to those of kingdoin Animalia. This includes, but is not limited to livestock, farm animais, domestic animais, pet animais, marine and freshwater animais, and wild animais.

As used herein. the terins administration and the term administering refer to the 20 act of giving a substance, including a drug, prodrug, or other agent, or therapeutic treatment to a subject (e.g., 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), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), car, rectal, vaginal, by injection (e.g,, intravenoiisly, subcutancously, intratumorally, intrapcritoneally, 25 etc.) and the like.

As used herein, the term co-administration and the term co-administering” refer to the administraiion of at least two agcnt(s) or thérapies to a subject and/or material (e.g., fccdstuff). Co-administration of two or more agents or thérapies can bc concuncnt, or a first agent/therapy can bc administered prior to a second agent/therapy.

As used herein, the term treatment refers to measures taken that facilitate the improvement and/or reversai of the symptoms of disease. The term treatment refers to both therapeutic treatment and prophylactic or preventative measures. For example, subjects that may benefit from treatment with compositions and methods of the présent invention include (hose already with a disease and/or disorder as well as those in which a disease and/or disorder is to be prevented (e.g., using a prophylactic treatment of lhe présent invention).

As used herein, the terni at risk for disease refers to a subject that is predisposed to expcriencing a particular disease. This prédisposition may be genetic (e.g., a particular genetic tendency to expérience the disease, such as heritable disorders), or due to other factors (e.g., âge, weight, cnvironmenial conditions, exposures to detrimental compounds présent in the environment, etc.).

As used herein, the term disease, the tenn infection and the term pathological condition or response refer to a state, signs, and/or symptoms that are associatcd with an impairment ofthe normal state ofa living animal or ofany of its organs or tissues that intemipts or modifies the performance of normal fonctions, and muy be a response to environmental factors (such as malnutrition, industrial hazards, or cliniate, including mycotoxicosis), spécifie infcctive agents (such as worms, bacteria, or viruses), to inhérent dcfect of the organisai (such as varions genetic anomalies), or combinations of thèse and other factors.

As used herein, (hc terni suffering from disease refers to a subject (e.g.. an animal or human subject) that is expcriencing a particular disease and is not limited to any particular signs or symptoms, or disease.

As used herein, the term toxic refers to any detrimental, deleterious, hamifol, or otherwise négative cftcct(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 terni pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making tlic composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term pharmaceutically acceptable and the term pharmacologically acceptable refer to compositions that do not substantially produce more known adverse réactions than known bénéficiai reactions.

As used herein, the term inoculation refers to the act of introducing a microorganism or suspension of microorganisms (e.g., algae, yeast, fongi, bacteria, etc.) into a culture medium. Inoculation is the act or process of introducing something into an environment in which it will grow or rcproduce.

As used herein, the term inoculum and the term pre-inoculum refer to cells used in an inoculation, such as cells added to start a culture.

As used herein, the term centrifugation refers to the separating of molécules by size or dcnsity using ccntrifugal forces generated by a spinning rotor thaï puts an object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sédimentation principle, where the centripetal accélération is used to evenly distributc substances of greater and lesser density into different layers of density.

As used herein, the term concentration refers to the amount of a substance per defined spacc. Concentration usually is expressed in terms of mass per unit of volume. To dilute a solution, one must add more solvent, or reduce (be amount of soluté (e.g., by sélective évaporation, spray drying, frceze drying, e.g., concentrated yeast cell wall cxtract or 10 concentrated modified yeast cell wall cxtract). By contrast, to conccntratc a solution, one must add more soluté, or reduce the amount of solvent.

As used herein, the term layer refers to a usually horizontal deposil organized in stratum of a material forming an overlying part or segment obtained after séparation by centrifugation in relation with the dcnsity properties of the material.

As used herein, the terni harvest refers lo the act of collecting or bringing together materials that hâve been produced (e.g. bringing together materials produced during yeast production).

As used herein, the term drying refers to spray drying, frceze drying, air drying, vacuum drying or any other kind of process that reduces or éliminâtes liquid in a substance.

As used herein, the term spray drying refers to a commonly used method of drying a substance containing liquid using hot gas to evaporate the liquid to rcducc or climinatc liquid in the substance. In other words the material is dried by way of spraying or atomizing into a draft of heated dry air.

As used herein, the term frceze-drying and the term lyophilization and the term cryodcsiecation refer to the removal of a solvent from matter in a frozen state by sublimation. This is accomplished by freezing the material to be dried below ils eutectic point and then providing the latent heat of sublimation. Précise control of heat input permits drying from the frozen state without product mclt-back. In practical application, the process is accelcratcd and prcciscly controlled under reduced pressure conditions.

As used herein, the term dry free flowing powder refers to a free flowing dry powder, e.g. a powder that can be poured from a container, bag, vessel etc without hindrancc of large clumps.

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

As used herein, the terni sample is used in a broad sense including a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may bc obtained from animais (including humans) and cncompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, sérum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions comprising high lipid content algae and methods of making and utilizing the saine. In particular, the invention relates to high lipid content algae biomass and algal lipid materials derived from the same, methods of making the same, as well as to biofuels (e.g., biodiesel) and dietary compositions (e.g., animal feeds) comprising or made from the same. Compositions and methods of tlie invention fînd use in a variety of applications including biofuel, dietary (e.g., human and animal nutrition), therapeutic as well as research applications.

Accordingly, in one aspect of the invention, there is provided a process for tlie préparation of an algal biomass containing elevated amounts (e.g., on a w/w basïs) of total fat. For exemples, 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 (e.g., greater than 60% total fat. in contrast to conventional methods that générale algal biomass containing a significantly lower level of total fat content (e.g., 60% or less total fat)). A great challenge of algal-based biofuel (e.g., biodiesel) is to ensure that the biomass 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 containing greater than 65% total fat. In some embodiments, the invention provides a method of 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 67% 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 a method of generating 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 70% total fat. In some embodiments, the invention provides a method of generating an algal biomass containing greater than 70% (e.g., greater than 71, 72. 73, 74,75, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87. 88, 89, 90% or more) total fat on a w/w basis.

In sonie embodiments, the method utilises a closed bioreactor system (e.g., a fermentor), allhough the invention is not so limited (e.g., in soinc embodiments, open biorcactors may be utilized). in a preferred embodiment, growth of an algai biomass of the invention is conducted under aseptie conditions. In another preferred embodiment, algae are grown (e.g., to generate an algal biomass containing a high fat content (e.g., greater than 67% fat)) in a fcd-batch process.

In some embodiments, the invention provides a method of culturing algae to produce an algal biomass comprising a desired, high lolal fat content (e.g., 67% or more total fat) as described in Examples 1 and 2. For cxample, in some embodiments, the invention provides a incthod of culturing algae comprising culturing the algae in a stepwisc manner so as to produce an algal biomass comprising a desired, high total fat content (e.g., 67% or more total fat). In some embodiments, a stepwise process for culturing algae comprises thawing a stored strain of algae and adding (e.g., aseptically) lhe thawed algae lo a IL shake flask contain medium comprising a carbon source (c.g„ sugar (e.g., glucose)), yeast cxtract and sea sait, In some embodiments, the carbon source is présent in a concentration of 50 g/L, lhe yeast cxtract is présent in a concentration of 10 g/L and/or the sea sait is présent in a concentration of 4 g/L. in some embodiments, the IL shako flask containing algae and medium are maintuined at 30 C and shaken (e.g., at about 100-400 RPM) until such time that the algae hâve entered exponential growth phase but hâve not fully depleted the carbon source (e.g., sugar (e.g., glucose)). Experiments conducted during development of embodiments of the invention hâve determined that the algae enter exponential growth but do not fully deplete the carbon source (e.g., sugar (e.g., glucose)) at a time period between 72144 hours. Thus, in some embodiments, algae cultivatcd in a IL culture flask at 30 C for 72144 hours at aboutl00-400 RPM (e.g., 250 RPM) in medium comprising a carbon source (e.g., sugar (e.g., glucose)), yeast cxtract and sea sait is used to inoculate a first seed stage culture (e.g., in a larger vessel (e.g., 40,27 or 18 L vessel)). In some embodiments, the culture medium used in a first sced slagc comprises a carbon source (e.g., sugar (e.g., glucose)), yeast cxtract, magnésium sulfate, calcium chloride and/or magnésium chloride. In a preferred embodiment, the culture medium used in a first seed stage comprises about 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast exlract, about 0.15 g/L magnésium sulfate, aboul 0.15 g/L calcium chloride and/or 0.15 g/L magnésium chloride. lu some embodiments, the first seed stage culture is run at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about 7-15% (e.g., 8,9, 10, 11, 12, 13, 14%), allhough lower and higher dissolved oxygen conditions may be utilized. In a preferred

embodiment, the first seed stage culture is run at 30 C under airflow and agitatiou conditions so as to niaintain dissolved oxygen at about 10%. In some embodiments, the first seed stage culture containing algac and medium arc maintained at 30 C and cultivated until such time that the algae hâve entered exponcntial growth phase and at least 20 g/L of carbon source (e.g., sugar (e.g., glucose)) has been consumed but the carbon source has not been fully dcpletcd. Experiments conducted during development of embodiments of the invention determined that the algac enter exponcntial growtb, consume at least 20 g/L of carbon source (e.g., sugar (e.g., glucose)) but do not fully deplete the carbon source (e.g., sugar (e.g., glucose)) at a time period between 24-48 hours after inoculation ofthe first seed stage culture, in some embodiments, algac cultivated in first seed stage culture at 30 C for 24-48 hours in medium comprising a carbon source (e.g., sugar (e.g., glucose)), yeast extract, magnésium sulfate, calcium chloride and magnésium chloride are used to inoculate a second seed stage culture in yet a larger vessel (e.g., 2000 L vessel). In some embodiments, the culture medium used in a second seed stage culture comprises a carbon source (e.g., sugar (e.g., glucose)), yeast extract, magnésium sulfate, calcium chloride and/or magnésium chloride. In a preferred embodiment, the culture medium used in a second seed stage culture comprises about 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and/or 0.15 g/L magnésium chloride. In some embodiments, the second seed stage culture is run at 30 C under airflow and agitation conditions so as to malntain dissolved oxygen at about 7-15% (e.g., 8,9, 10, 11, 12, 13, 14%). although lower and higher dissolved oxygen conditions may be utilized. In a preferred embodiment, lhe second seed stage culture is run at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about 10%. In some embodiments, the second seed stage culture containing algac and medium arc maintained at 30 C and cultivated until such time that the algae hâve entered exponcntial growth phase, and al least 20 g/L of carbon source (e.g., sugar (e.g., glucose)) has been consumed, but the carbon source has not been fully dcpletcd. Experiments conductcd during development of embodiments of the invention determined that the algac enter exponcntial growth, consume at least 20 g/L of carbon source (c.g„ sugar (e.g., glucose)) but do not fully deplete the carbon source (e.g., sugar (e.g., glucose)) at a time period between 24-48 hours after inoculation of the second seed stage culture. In sotne embodiments, algac cultivated in second seed stage culture at 30 C for 24-48 hours in medium comprising a carbon source (e.g., sugar (e.g., glucose)), yeast extract, magnésium sulfate, calcium chloride and magnésium chloride are used to inoculate a large scale vessel (e.g., 70,000 L, 120.000 L, 220,000 L or larger vessel

(e.g., a main fermentor)) containing medium used for further cultunng/fennentation of the algae. In some embodiments, upon transfcr of the second seed stage culture to the large scale vessel (e.g., main fermentor), the culture medium (e.g., the batchcd medium) présent in the large scale vessel (e.g., main fermentor) comprises a carbon source (e.g., sugar (e.g., glucose)), yeast exlract, magnésium sulfate, urea, calcium chloride, magnésium chloride and/or monopotassium phosphate. In a preferred embodiment, the culture medium used in a large scale (e.g., 70,000 L, 120,000 L, 220,000 L or larger vessel (e.g., main fermentor)) culture comprises about 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast extract, about 4.0 g/L magnésium sulfate, about 1 g/L urea, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and/or about 0.25 g/L monopotassium phosphate. In some embodiments, the large scale culture is run at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about 7-15%(e.g., 8, 9, 10, 11, 12, 13, 14%), although lower and higher dissolved oxygen conditions may be utilized. In a preferred embodiment, the large scale culture is run at 30 C under airflow and agitation conditions so as to maintain dissolved oxygen at about 10%. In a preferred embodiment, the carbon source (e.g., sugar (e.g., glucose)) is tnainlained at 10 g/L for a period of time (e.g., 1 or more days (e.g., 2,3,4,5, 6, 7, 8,9, 10, 11, 12, 13, 14 or more days (e.g., using a fcd-batch process)). For example, in some embodiments, after a desired amount of glucose bas been consumed by algae in the large scale vessel (e.g., after about 20-30 g/L of glucose has been consumed by the algae in the large scale vessel (e.g., after 30 g/L of glucose has been consumed)), glucose and fcd-batch feeds are started. Experiments conducted during development of embodiments of the invention determined that the fed-batch feeds be added for about 34 hours, although shorter (e.g., about 32,28, 24,20 hours or fewer) and longer (e.g,, 36, 38,42,46, 60,72, 84, 96, 108, 120, 132, 144, 156, 168 hours or more) time periods may bc used. In further preferred embodiments, upon completion of the fcd-batch process, cultivation of the algae is continued in the large scale vessels until ail nutrients are removed/consuined from the medium. Experiments conducted during development of embodiments of the invention determined that the nutrients arc dcpletcd from the medium between about 12 and 24 hours after cessation of the fed-batch process. fn some embodiments, the algal biomass is harvested from the large scale culture medium/broth and utilized as described herein. In some embodiments, the large scale culture broth is ccntrifuged to obtain the algal biomass. In some embodiments, the large scale culture broth is cooled prior to centrifugation. Although an understanding of a mechanism is not needed to practice the invention, and the invention is not limited to any particular mechanism of action, in somc embodiments, chilling the culture

broth increases the density of the algal bioinass comprising elevated levels of total fat (e.g., lipîds/oil) and allows a larger recovery of thc biomass than is achicved in thc absence of chilling thc culture broth (See, e.g., Examplc 3). Thc invention is not limited by thc température to which the large scale culture is chilied prior to centrifugation. In some embodiments, the large scale culture is chilled to a température between 0-50 C, between 540 C, 5-25 C, 5-15 Cor 5-10 C.

Thus, thc invention utilizcs both batch and fed-batch modes of culturing algac (c.g., alone and/or subséquent to a first and/or second seed stage) in order to generate an algal biomass that contains a desired fat content (e.g., a fat content greater than 67%). The invention is not limited by the individual components présent in the media used in either thc batch or fed-batch modes. In some embodiments, culture media présent at the time of inoculation of a main fermentor (e.g., 70,000 L, 120,000 L, 220,000 L vessel) contains medium with an initial ratio of nitrogen (N):phosphorus (P):potassium (K.) of 46:13:8.5. In a preferred embodiment, the N:P:K ratio is the saine in the batch and fed-batch culture modes. In some embodiments, the ratio of magnésium (Mg):calcium (Ca) is 3:1 inculture media used in both batch and fed-batch modes. In another cmbodiinent, thc ratio of chloride (C12):sulfatc (SO4)) is 1:1 in culture media used in both batch and fcd-batch modes. In some embodiments, thc ratio of sulfate (S04):phosphate (PO4) in media at the time of inoculation of a main fermentor (e.g., 70,000 L, 120,000 L, 220,000 L vessel) is 16:1. In some embodiments, thc total ratio of sulfate (S04):phosphatc (PO4) that has been batched and fed at thc end of a full culture (c.g., including inoculum, first seed stage, second seed stage and main fermentor cultures) that generates an algal bioinass containing a desired fat content (c.g., grcater than 67% fat) is 5.3:1. In some embodiments, thc ratio of chloride (CI2):phosphatc (PO4) in media at thc time of inoculation of a main fermentor (c.g., 70,000 L, 120,000 L, 220,000 L vessel) is 16:1. In some embodiments, thc total ratio of chloride (C12):phosphate (PO4) that has been batched and fed at the end of a full culture (e.g., including inoculum, first seed stage, second seed stage and main fermentor cultures) that generates an algal biomass containing a desired fat content (c.g., grcater than 67% fat) is 5.3:1.

As described in Example 2 below, the invention also provides a composition comprising an algal bioinass (c.g., a dried algal biomass (c.g., generated according to a method described herein)) containing a desired amount of total fat and/or other components.

For example, in soine embodiments, the invention provides an algal biomass (e.g., a dried bioinass) containitig grcater than 67% total fat (e.g.. grcater than about 68% total fat, grcater

than about 69% total fat, greater than about 70% total fat, greater than about 71% total fat, grcatcr than about 72% total fat, greater than about 73% total fat, greater than about 74% total fat, grcatcr than about 75% total fat, greater than about 76% total fat, grcatcr than about 77% total fat, greater than about 78% total fut or higher amount of total fat). In some embodiments, an algal biotnass (e.g., containing greater than 67% total fat) is dried such that the biomass contains less than 5% moisture (e.g., less than 4.5% moisture, less than 4% moisture, less than 3.5% moisture, less than 3% moisture, less than 2.5% moisture, less than 2% moisture, or less than l.5% moisture). In some embodiments, an algal biotnass of the invention (e.g., a dried biontass containing less than 5% moisture) contains about 170-250 mg/g or more docosahcxaenoic acid (DHA) (e.g., about 170-180 mg/g DHA, about 180-190 mg/g DHA, about 190-200 mg/g DHA, about 200-210 mg/g DHA, about 210-220 mg/g DHA, about 220-230 mg/g DHA, about 230-240 mg/g DHA, about 240-250 mg/g DHA, or more than 250 mg/g DHA). In some embodiments, an algal biontass of the invention (e.g., a dried biomass containing less than 5% moisture) contains about 150-400 mg/g or more pahnitic acid (IUPAC name: hexadecanoic acid (e.g.. about 150-200 mg/g, about 200-225 mg/g, about 225-250 mg/g, about 250-275 ntg/g, about 275-300 ntg/g, about 300-325 mg/g, about 325-350 mg/g, about 350-375 mg/g, about 375-400 ing/g, or more than 400 mg/g)). In some embodiments, an algal biontass of the invention (e.g., a dried biontass containing less titan 5% moisture) contains about 300-600 ntg/g or more total fatty acids (e.g., about 300-350 ntg/g, about 350-400 tng/g, about 400-450 mg/g, about 450-500 mg/g, about 500-550 mg/g, about 550-600 mg/g, or more than 600 mg/g fatty acids)). In some emboditnents, an algal biomass ofthe invention (e.g., a dried biontass containing less than 5% moisture) contains less than about 15% protein (e.g., less than about 14% protein, less titan about 13% protein, less than about 12% protein, less than about 11% protein, less than about 10% protein, less than about 9% protein, or less than about 8% protein).

The invention is not limited by the strain or species of algae utilized in the methods and compositions described herein. Indeed, a variety of algac find use in the invention including, but not limited to, onc or more species of the genus Thraustochytrium. In some embodiments, the algae is a species of the genus Chlorella. In some embodiments, the algae 30 is a species of the genus Sehizochytrium. In some embodiments. the algae is a species of the genus Crvpthecodinium. In some embodiments, the algae is Thraustochytrium striatum, Thraustochytrium roseum, Thraustochytrium aureum, Crypthecodinium cohnii, and/or Aurantiochytrium sp. In a preferred embodiment, Sehizochytrium limacinum is utilized in the methods and compositions described herein. The invention is not limited by the type of lipids produced by a process to generate an algal biomass with elevated levels of lipids disclosed herein. In some embodiments, the lipids generated by a process of the invention include, but arc not limited to, myristic acid, palmitic acid, olcic acid, linolcic acid, docosapcntacnoic acid (DPA), docosahexaenoic acid (DHA), and stearic acid. These lipids bave been useful for both animal and human health, for prévention of various diseases such as cardiovascular and inflammatory diseases and in infant nutrition for proper brain development and rctinal vision in childrcn.

In another embodiment, the invention provides a process for production of an algal bioniass containing elevated levels (e.g., greater than 67%) of total fat from an algae species (e.g., Schizochytrium limacinum), wherein the process comprises culturing algae in a first feed bateh vessel comprising medium (e.g., comprising about 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast exlract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and/or 0.15 g/L magnésium chloride), transferring (e.g., ascptically) the first feed batch culture to a second seed batch culture medium(c.g., comprising about 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and/or 0.15 g/L magnésium chloride), transferring (c.g., ascptically) the second seed batch culture to a large scale culture vessel containing medium (e.g., a main fermenter (e.g., 70,000 L, 120,000 L, 220,000 L vessel, containing, for example, medium comprising about 50 g/L of a carbon source (c.g., sugar (c.g., glucose)), about 7.5 g/L yeast extract, about 4.0 g/L magnésium sulfate, about 1 g/L urea, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and/or about 0.25 g/L monopotassium phosphate), wherein the glucose level ofthe large scale culture vessel is maintained at 10 g/L using a fcd-batch process, wherein the algal cells arc harvested from the large scale culture between 12-24 hours after cessation of the fedbatch process after ail of the nutrients hâve been rcmovcd/consumed from the medium.

Another embodiment of the invention provides a process for production of an algal biomass containing elevated levels (c.g., greater than 67%) of total fat from an algae species (c.g., Schizochytrium limacinum), wherein the culture medium (c.g., during each stage of fermentation (c.g., first seed stage, second seed stage and/or batch culture (fed-batch) cultivation stage)) comprises a carbon source (e.g., a sugar), yeast extract, a phosphate source (c.g., monopotassium phosphate, magnésium sulfate and/or zinc sulfate), a nitrogen source (e.g., urea), magnésium chloride, and/or calcium chloride. In a preferred embodiment, the invention provides a process for production of an algal biomass containing elevated levels (e.g., greater than 67%) of total fat from a strain of algae wherein the culture medium (e.g.,

during each stage of fermentation (e.g., first seed stage, second seed stage and/or batch culture (fcd-batch) cultivation stage)) comprises sugar, yeast cxtract, monopotassïum phosphate, magnésium sulfate, zinc sulfate), urea, magnésium chloride, and/or calcium chloride. However, the invention is not limited by the type of nutrïent utilized in u culture medium in which algae are grown. In some embodiments, one or more carbon sources are added to tlie medium. Examplcs of carbon sources include, but arc not limited to, carbohydrates such as glucose, fructose, xylosc, saccharose, maltosc or soluble starch as well as oleic acid, fats such as soybean oil, molasses, glycérol, mannitol, and sodium acetate, cotton seed flour, glycérol, molasses and corn steep liquor. In some embodiments, one or more nitrogen sources arc added to tlie medium. Examplcs of nitrogen sources include, but are not limited to, natural nitrogen sources such as peptone, yeast extruct, mult extruct, méat extract, casamino acid and corn steep liquor, organic nitrogen sources such as sodium glutamate and urea. or inorganic nitrogen sources such as ammonium acetate. ammonium sulfate, 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 phosphate, inorganic salts, such as ammonium sulfate, sodium sulfate, magnésium sulfate, iron sulfate, zinc sulfate, and copper sulfate. In some embodiments, magnésium chloride, culcium chloride, and/or vitamîns are included in the cultive medium.

The invention is not limited by the amount (e.g., concentration) of each of these components in tlie culture medium. In some embodiments, an amount is utilized that is not harmful to algal growth. In a preferred embodiment, tlie amount (e.g., concentration and/or ratio) of each medium ingrédient is set at a level (e.g., during each stage of fermentation (e.g., first seed stage, second seed stage and/or balch culture (fcd-batch) cultivation stage) that promûtes the formation of high fat content algae (e.g., an algal biomass comprising 67% or greater fat content). In some embodiments, the carbon source (e.g., sugar) is présent in culture medium at about 20 to 120 grams per liter of medium. In other embodiments, the carbon source (e.g., sugar) is présent in culture medium at about 30-70 grams per liter of medium. In still other embodiments, the carbon source (e.g., sugar) is présent in culture medium at about 40 to 60 grams per liter of medium. In a preferred embodiment, the carbon source (e.g., sugar) is présent in culture medium at about 50 grains per liter of medium. In some embodiments, the ratio of urea to monopotassium phosphate (urea:KH2PO4) is between about 5:0.1 (e.g., about 4.5:0.1; 4:0.25; 3:0.25; 4:0.3; 5:0.3; 5:0.5; 4:0.5; 3:0.5; 2:0.5; or 1:0.5); although higher and lower ratios may bc used (e.g., 1:1, 1:2, 1:3 etc.). In a

preferred embodiment, the ratio of urea to monopotassium phosphate in culture medium is 4: l. In some embodiments, a culture medium does no! contain sodium chloride. In other embodiments, a culture medium contains sodium chloride. In some embodiments, the ratio of magnésium sulfute (MgSO4):calcium chloride (CaCl2) is l : l. In some embodiments, the ratio of magnésium sulfate (MgSO4):calcium chloride (CaCl2) is l :2. Indeed, a variety of ratios of magnésium sulfate (MgSO4):calcium chloride (CaCl2) may be used including, but not limited to, l:l; I:l.l25; 1:1.5; 1:1.75; 1:2; 1:2.125; 1:2.25; 1:2.5; 2.5:1; 2.25:1; 2.125:1; 2:1; 1.75:1; 1.5:1; 1.25:1 or 1.125:1. In a preferred embodiment, the ratio of magnésium sulfate (MgSO4):calcium chloride (CaC12) in a first seed culture medium is 1:1. In another preferred embodiment, the ratio of magnésium sulfate (MgSO4):calcium chloride (CaC12) in a second seed culture medium is 1:1. In yet another preferred embodiment, the ratio of magnésium sulfate (MgSO4):calcium chloride (CaC12) in a large scale culture medium (e.g., main fermentor (e.g., 70,000 L, 120,000 L, 220,000 L vessel) also referred to as a third culture medium herein) is 1:2.

In a further preferred embodiment, after preparing the medium, the pH of tlie medium need not be adjusted. For example, during a stepwisc fermentation process of the invention, the pH of the culture medium in which algac is grown need not be adjusted. AJthougb an understanding of the mechanism is not necessary to practice the invention and the invention is not limited to any particular mechanism of action, in some embodiments, stérile and/or aseptie conditions of the stcpwise fermentation process of lhe invention 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 6.5. Cultivation of the algae during a stepwise fermentation process of the invention may bc carried out at a température between 10 and 40 C„ preferably 17 to 35 C, and most preferably around 30 C. Cultivation may bc performed by aération-agitation culture, shaking culture, stationary culture or the like. In a preferred embodiment, algae are cultured under conditions such that dissolved oxygen is maintained at or slightly above 10%.

In some embodiments, the invention provides a food, feed, nutritional or therapeutic supplément comprising ail or a portion of an algal biomass (e.g., a dried algal biomass described herein and/or generated according to the methods and compositions described herein) comprising clevated levels (e.g., greater the 67%) of total fat. For examplc, in some embodiments, the invention provides a food, feed, nutritional or therapeutic supplément comprising a spray dried algal biomass comprising elevated levels (e.g., greater the 67%) of total fat. In other embodiments, the invention provides a food, feed, nutritional or therapeutic

supplément comprising lipids extracted and/or isolated from an algal biomass comprising elevated levels (e.g., grater the 67%) of total fat. The invention is not limited by the type of lïpid extracted and/or isolated from an algal bioniass comprising elevated levels (e.g., grater the 67%) of totul fat. In some embodiments, the lipids comprise myristic acid, palmitic acid, 5 oleic acid, linoleic acid, alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid, cicosatetracnoic acid, eicosapcntacnoic acid (EPA), docosapcntaenoic acid (DPA), clupanodonic acid, docosahexaenoic acid (DHA), tctracosapcntacnoic acid, and/or tetracosahexaenoic ucid. In a preferred embodiment, the lipids comprise DHA and/or palmitic acid.

In some embodiments, the invention provides a process for the préparation of lipids (e.g., those disclosed herein (e.g., docosahexaenoic acid)) comprising: culturing an algue strain (e.g., Schizochylrium limacinunt) in a first culture medium (e.g., containing 50 g/L ofa carbon source (e.g., sugar (e.g., glucose)), K) g/L yeast extract and 4 g/L sea sait) and incubating the culture at a température in the range of 25-35 C for a period of about 72-144 hours; transferring the culture to a second culture medium (e.g., containing 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0,15 g/L calcium chloride and/or 0.15 g/L magnésium chloride) and incubating the culture at a température in the range of 25-35 C for a period of about 24-48 hours; transferring the culture to a third culture medium (e.g., containing 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and/or 0.15 g/L magnésium chloride) and incubating the culture at a température in the range of 25-35 C for a period of about 24-48 hours; transferring the culture to a fourth culture medium (e.g., containing 50 g/L of a carbon source (e.g., sugar (e.g., glucose)), about 7,5 g/L yeast extract, about 4.0 g/L magnésium sulfate, about I g/L tirca, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and/or about 0.25 g/L monopotassium phosphate) and incubating the culture at a température in the range of 25-35 C (e.g., 30 C) for a time period of about 24-192 hours (e.g., about 36, about 38, about 42, about 46, about 60 , about 72, about 84, about 96, about 108, about 120, about 132, about 144, about 156, about 168, about 180 or about 192 hours); separating the cell biomass fiom tlie culture; and extracting lipids froin the bioniass.

In sonie embodiments, algac cultures (e.g., grown to produce an algal biomass) arc grown in suitable volumes and vessels, ranging from 100 ml to hundreds of thousands of liters, in flasLs or large fertnentors, using various nutrient media as described herein.

In yet another aspect, the séparation of tlie cell biomass containing lipids is obtained r

using centrifugation, filtration and/or flocculation or similar techniques. In a preferred embodiment, an algal biomass is obtained from a culture using centrifugation. In a further preferred embodiment, ccntrifiigatioti occurs after the cell culture is cooled (e.g., to allow recovery of cells containing elevuted levels of lipid). In some embodiments, an algal biomass obtained is spray-dried and used (e.g., directly used in animal feeds or for biofuel production).

In one embodiment, the algae is a mixture of different algae species (e.g., one or more of the species of algae described herein). In some embodiments, an algal biomass containing elevated levels of total fat and/or lipids extracted from an algal biomass containing clevated levels of total fat is supplemented with lipids (e.g., polyunsaturatcd fatty acids) from other sources including, but not limited to, plant sources.

In some embodiments, an algal biomass containing elevated levels of total fat comprise lipids at a concentration (w/w) in a range from about 60-90% (e.g., about 65-90%, about 66-89%, about 67-88%, about 68-87%, about 68-86%, about 69-85%, or about 7080%). Thus, an algal biomass containing elevated levels of lipids may comprise lipids al a concentration of 61%, 62%, 63%, 64%, 65%. 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 8I%, 8%2, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% and the like. In one embodiment, an algal biomass containing elevated levels of total fat comprise lipids at a concentration of at least 67%.

In some embodiments, DHA is includcd in an algal biomass composition ofthe invention in a range from l% to 75% of total lipids/fatty acids. Thus, the DHA can bc provided in the composition in an amount of total fatty acids of l%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%. 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, and the like. In other embodiments, the DHA can be includcd in a composition in an amount of total fatty acids in a range from 1% to 5%. 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1 % to 30%, 5% to 10%. 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 15% to 20%, 15% to 25%, 15% to 30%, 20% to 25%, 20% to 30%, 25% to 30%, 30% to 35%, 35% to 40%. 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, and the like.

In some embodiments, palmitic acid is includcd in an algal biomass composition of the invention in a range from 1% to 75% of total lipids/fatty acids. Thus, the palmitic acid can be provided in the composition in an amount of total fatty acids of 1%, 2%, 3%, 4%, 5%,

6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%. 28%, 29%, 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, and the like. in other embodiments, the palmitic acid cun be included in a composition in an amount of total fatty acids in a range from 1% to 5%, 1% to 10%, 1% to 15%, 1% to 20%, 1% to 25%, 1% to 30%, 5% to 10%, 5% to 15%, 5% to 20%, 5% to 25%, 5% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%. 15% to 20%, 15% to 25%, 15% tu 30%, 20% to 25%, 20% to 30%, 25% to 30%, 30% la 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%. 70% to 75%, and the like.

Additional embodiments of the invention include proccsscs of making animal feed additives. Thus, one aspect of the présent invention is a process of making an animal feed additive comprising lipids from an algae (e.g., an algal biomass), the process comprising: cultivating algae to produce a algae biomass containing a desired, elevalcd level of total fat (e.g., greater than 67% total fat); and cxtracting algae lipid from the algae biomass to produec a algae oil; and/or removîng water from algae biomass lo produce a algae biotnass with a solids content from about 5% to 100% weight percent; wherein the animal feed additive comprises lipids from the algae. In some embodiments, the fatty acids collected from the algae arc short chain, medium or long chain omega-3 fatty acids. In further embodiments, the algae lipid extracted from the algae biomass is combined with a algae biomass with a solids content from about 5% to 100% weight percent.

A feed additive according to the invention can bc combined with other food components to produce processed food or feed products (e.g., animal and/or human feed products). Such other food components include one or inorc enzyme suppléments, vitamin food additives and minerai food additives. The resulting (combined) feed additive may be mixed in an appropriate amount with the other food components such as ccreal and plant proteins to form a processed food product. Processing of these components into a processed food product can be performed using any conventionally used processing apparatuses. Fecd/food additives of the présent invention may bc used as a supplément in a food/fccd by itsclf, in addition with vitamins, minerais, other feed enzymes, agricultural co-products (e.g., wheat mîddlings or corn gluten meal), or in a combination therewith.

In a further aspect, the invention provides a process ofproducing an animal and/or human having an increased tissue content of omega-3 fatty acids, the process comprising feeding to tin animal/human a feed additive comprising lipids/fatty acids collected from algae, the feed additive further comprising: (a) an algae lipid extracted from a cultivated

algae biomass and/or (b) a algae biomass from a cultivated algae, wherein water is removed front algae biomass to achieve a solids content from about 5 to 100% weight percent, wherein the animal/human displays increased tissue content of onicga-3 fatly acids. The invention is not iimîted to any particular mammal (e.g., animal or human) that may benefit from a composition of die invention. Indeed, animais of the invention include. but are not limited to, any animal whose eggs, méat, milk or other products arc consumed by humans or otlicr animais. Thus, animais of the invention include, but arc not limited to, fish, poultry (chickens, turkeys, ducks, etc.), pïgs, sheep, goats, rabbits, beef and dairy cattle.

In some embodiments, the invention provides a method for treating a mammalian disease in a subject in need thereof by 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, (hc cardiovascular diseases lo be treated include, but are not limited to, liypertriglyccridcmia, coronary heart disease, stioke, acute myocardial infarction and atherosclerosis. In further embodiments, the inflammatory discases to be treated include. but are not limited to, asthma, arthritis, allergie rhinitis, psoriasis, atopie dcmiatitis, inflammatory bowcl discases, Crohn's disease, and allergie rhinoconjunctitis. In still further embodiments, the cancer diseuses to be treated include, but are not limited to, prostate cancer, breast cancer and colon cancer. In additional embodiments, the mammalian discases to be treated include psychiatrie disorders. Psychiatrie disorders include, but are not limited to, dépression, bipolar disorder, schizophrénie. In addition, the compositions of the invention can be used to mainlain and/or enhance cognitive fonction.

In some embodiments, the invention provides a method of treating a mammalian disease in a subject in need thereof by administration to the subject a therapeutically effective amount of a lipid composition provided by and/or obtained from an algal biomass containing an elevated level of total fat (e.g., greater than 67% total fat). Subjccts that may frnd benefit from treatment include but arc not limited to, avian and mammalian subjccts. Mammals of the présent invention include, but are not limited to, canines, felines, bovines, caprines, cquines, ovines, porcines, rodent» (e.g. rats and mice), lagomorphs, primates (including nonhunian primates), humans, and the like, and mammals in utero. Any mammalian subject in need of being treated according to the présent invention is suitable. Mammals ofthe présent invention include, but are not limited to, canines, felines, bovines, caprines, cquines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates),

Y humans, and the like, and inainmals ui utero. According to some embodiments of the présent invention, the tnatnmal is a non-huinan mammal. In soinc embodiments, the nianunal is a human subject, Mammalian subjects of both genders and at any stage of development (e.g., neonate, infant, juvénile, adolescent, adult) can be treated according to the présent invention. Illustrative avians according to the présent invention include chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich), domesticated birds (e.g., parrots and Canaries), and bîrds in ovo.

Algae

Any algae capable of producing, using the proccsscs described herein, clevated levels of total fat or algal biomuss containing elevated levels of total fut can be used in the processes, compositions, dietary suppléments, biofuel and/or biofuel precursor and/or feed additives of the invention. Thus. in sonie embodiments, the algae of the présent invention is selected fiom Thraustochytrium, Dinophyceae, Cryptophyceae, Îrebouxiophyceae, Pinguiophyceae, and combinations thereof. In other embodiments, the algae of the invention arc selected from Thraustochytrium striatum, Thraustochytrium roseum, Thraustochytrium aureum, Crypthecodinium cohnii, Parie loch loris spp., Rhodomonas spp., Cryptomonas spp., Parietochloris spp., Hemisebnis spp., Porphyridium spp., Glossomastix spp., and combinations thereof. In further embodiments, the algae of lhe invention are selected from Parietochloris incise, Rhodomonas salifia, Hemiselmis bruneseens, Porphyridium crueiitum and Glossomastix chrysoplasta, and combinations thereof. In still further embodiments, the algae of the invention is Schizochytrium limacinum.

In some embodiments of the invention, the algae is a mixture of different algae species. In other embodiments, the algae is a single algae species. In sonie embodiments of the présent invention, the algae lipids/fatty acids are provided as an algal oil. In other embodiments, the algae lipids/fatty acids are provided as an algal biomass (e.g., a dried (e.g., powdcrcd) biomass).

Further, the algae of the invention include, but arc not limited to, wild-type, mutant (naturally or induced) or genetically engincered algae. In u preferred embodinrontt, an algae used in the processes, compositions, dietary suppléments, biofuel or biofuel precursor and/or fccd additives of the invention is a non-gcnctically modified organism. As used herein, the terms genetically modified variant, and genetically modified organism refer to an algae strain that has a genome which is modified (e.g., mutated, changed) from its normal (e.g., wild-type, naturally occurring) form such that a desired resuit is achieved.

Additionally, the algae of the invention includes algae having cells with ccll walls of rcduccd thickness as compared to tlie cells of wild-type algae, whereby the ccll wall of reduced thickness improves cxtractability and/or bioavailability ofthe algae lipid fraction (e.g., improving the ease of digestibility of the algue and the case of cxtractability of the algae lipids/fatty acids from tlie cells of the algal biomass). Algae having cells with cell walls of rcduccd thickness as compared to the cells of wild-type algae can bc naturally occurring, mutated and/or gcnctically cnginccrcd to have ccll walls of rcduccd thickness as compared to wild-type strains. Thus, in one embodiment of the invention the algae is an algae huving a cell wall of rcduccd thickness as compared to the wild-type algae, whereby the cell wall of rcduccd thickness improves cxtractability and/or bioavailability of the algae lipid fraction. Methods of producing algae with reduced cell walls include those found in WO 2006/107736 Al, herein incorporated by reference in its entirety. Thus, the algae can be mutagenized with mutagens known to those of skill m the art including, but not limited to, chemical agents or radiation. In particular embodiments the chemical mutagens include, but arc not limited to, ethyl methanesulfonate (EMS), methyhnethane sulfonate (MMS), N-ethyl-N-nitrosourea (ENU), tricthylmclaminc (TEM), N-mcthyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, dicthyl sulfate, acrylarnidc tnonomer, mclphalan, nitrogen mustard, vincristine, dimcthylnitrosainine, N-methyl-N'-nitro-Nîtrosoguanidinc (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimelhyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramidc, bisulfan, diepoxyalkanes (dicpoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-mcthoxy-6-chloro-9(3-(cthyl-2-chlor-octhypaminopropylamino)acridine dihydrochloride (ICR-170), formaldéhyde, and the like. Methods of radiation mutagcncsis include, but arc not limited to, x-rays, gamma-radiation, ultra-violet light, and the like.

Cell wall mutants canbc selected for on the basis of increased sensitivîty to détergents or by microscopie observation of alterations in cell wall thickness (See, e.g., WO 2006/107736 Al) or any other method known in the art to dctcct rcduccdccll wall thickness or rcduccd cell wall integrity.

The algae of the invention can bc cultured according to techniques described in

Examples 1-3.

Accordingly, in some embodiments the algae arc cultured at a température in a range from 10C to 35°C. Thus, the algae can bc cultured at a température of 10°C, 11^UC, 12°C,

13°C, 14^l,C, Î5°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24^OC, 25°C, 26°C, 27°C,

28C, 29°C, 30°C, 31ⁿC, 32^ÜC, 33^UC, 34°C, and the like. In other embodiments, the algae can

be grown in ranges from 20°C to 35°C, although colder (e.g., less than 20°C) and warmer (e.g., more than 35C) may bc used. In a preferred embodiment, ihc algae are grown at about

30“C.

In some embodiments, following cultivation, algae are harvested. In some embodiments, harvesling of algae is performed using conventional procedures known to those of skill in the art including, but not limited to, centrifugation, flocculation or filtration. In a preferred embodiment, prior to harvesting, the algac culture is cooled, thereby allowing algal cells containing elevated levels of total fat to be successfully harvested. The harvested algal cells or algal biomass can then be used directly as a lipid/fatty acid source or extracted to obtain algal oil comprising the lipids/fatty acids. In some embodiments in which the algal biomass is to be used directly, water is removed from the algal bioniass to achieve a solids content from about 5 to 100 weight percent. In additional embodiments, an algal biomass that is to be used directly is comprised of algal cells further comprising cell walls that aie at least partially disrupted to increase the cxtractability and/or bioavailability of the algal oil within the cells. The disruption of the algal cells can be carried out according to known techniques including, but not limited to, treating the cells with boiling water or by mechanical breaking such as grinding, pulvcrizing, sonication, Frcnch press, or any other method known to an ordinary artisan.

When the algal biomass is used directly, water îs removed from the algal biomass to achievc a solids content from about 5 to 100%. Accordingly, in some embodiments, water is removed from the algal biomass to achievc a solids content of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%. 74%, 75%, 76%, 77%. 78%, 79%, 80%. 81%, 82%. 83%, 84%, 85%, 86%. 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, 100%, and the like. In additional embodiments, water is removed from the algal biomass to achieve a solids content in the range from about 5% to 50%, 5% tu 60%, 5% to 70%. 5% to 80%, 5% to 90%, 5% to 95%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60% 10% to 65%, 10% to 70%, 10% to 75%, 10% to 80%, 10% to 85%, 10% to 90%, 10% to 95%, 10% to 100%, 15% to 40%, 15% to 50%, 15% to 60%, 15% to 65%, 15% to 70%, 15% to 75%, 15% to 80%, 15% to 85%,

15% to 90%, 15% to 95%, 15% lo 100%, 20% to 50%. 20% lo 60%, 20% to 65%, 20% to

70%, 20% to 75%, 20¾ to 80%, 20?ώ to 85%, 20% to 90%, 20% to 95%, 20% to 100%, 25% to 50%, 25% to 60%, 25% to 70%, 25% to 75%, 25% to 80%, 25% to 85%, 25% to 90%,

25% to 95%, 25% to 100%, 30% to 50%, 301% to 60%, 30% to 70%, 30% to 75%, 30% to

80%, 30% to 85%, 30% to 90%, 30% to 95%, 45% to 100%, 50% to 70%, 50% to 75%, 50%

lo 80%, 50% to 85%, 50% to 90%. 50% to 95%, 50% to 100%, 55% to 75%, 55% to 80%, 55% to 85%, 55% to 90%, 55% to 95%, 55% to 100%, 60% to 75%. 60% to 80%, 60% to 85%, 60% to 90%, 60% to 95%, 60% to 100%, 70% to 80%, 70% to 85%, 70% to 90%, 70% lo 95%, 70% to 100%, 75% to 85%, 75% to 90%, 75% to 95%, 75% to 100%, 80% to 85%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 90%, 85% to 95%, 85% to 100%, 90% lo 95%. 95% to 100%. and the like.

In some embodiments, the algal cells of the biomass are disnipted or lysed and the algal lipids extracted. The algal cells can be extracted wet or dry according to conventional techniques to producc a composition containing lipids/fatty acids. The disruption or lysis of the algal cells can bc carried uut according to conventional techniques including, but not limited to, treating the cells with boiling water or by mechanical breuking such us grinding, pulverizing, sonication, French press, or any other known method. Extraction of the lipids/fatty acids front the lysed cells follow standard procedures used with algal and other organisms that are known including, but not limited to, separating the liquid phase from the solid phase following cell lysis, extracting the lipids/fatty acids in the liquid phase by the addition of a solvent, évapora ting the solvent, and rccovcring the lipids/fatty acids obtained from the liquid phase of the lysed cells.

The invention is not limited to any particular solvent used for extraction. Solvents include, but are not limited to, hexane, chloroform. éthanol, methanol, isopropanol, diethyl ether, dioxan, isopropyl ether, dichloromethane, tetrahydrofuran, petroleum ether and combinations thereof.

In some embodiments, lipids/fatty acids derived from an algal biomass of the invention arc provided in the form of free fatty acids, cholestérol esters, sait esters, fatty acid esters, monoglycerides, diglyccridcs, triglycérides, diacylglyccrols, monoglyccrols, sphingophospholipids, sphingoglycolipids, or any combination thereof (e.g., for use in processes, compositions, biofueLs, food products. dietary suppléments, feed additives or other compositions described herein).

Method for Preparing An Algal Biomass

In sonie embodiments, the invention provides a method for preparing a algal biomass comprising elevated levels of total fat (e.g., greater than 67% lipids), comprising; culturing algae under a culture condition sufficient to provide an algal biomass comprising elevated levels of total fat (e.g., greater than 67% lipids), wherein the algal biomass is harvested at the termination of a logarithinic growth phase of the algae (See, e.g., Examples 1 and 2). As

used herein, the term logarithmic growth phase, refers to a stage of culturing characterized by exponcntially increasing numbers of algal cells. Generally, in a culture system, there is a characteristic growth pattern following inoculation that includes a lag phase, an exponentiel or logarithmic growth phase, a négative growlh accélération phase, and a plateau or statîonary phase. For example, in the logarithmic growth phase, as growth of the algae continues, cells can reach their maximum rate of cell division and numbers of cells increase in log relationship to time. Within time after the commencement of the log phase, the rate of cell division may begin to décliné and some of lhe cells etm begin to die. This is reflected on a growth curve by a graduai flattening out of the line. Evcntually the rate of cells dying is 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 statîonary or plateau phase and is represented on a growth curve as a flattening out of the line where the slope approaches zéro. In a preferred embodiment, the algal biomass is cultured under aseptie conditions (e.g., to prevent contamination and/or growth of contaminating microorganisms (e.g., yeast, bacteria, virus, etc.) in the culture).

In some embodiments, the culture condition is sufficient for the algae to producc elevated levels of total fat (e.g., greater than 67% on a w/w basis). The culture conditions comprise a culture medium suitable for growing the algae thereby providing the algae biomass comprising elevated levels of total fat (e.g., greater than 67% on a w/w basis). Suitable culture médiums are described herein. The medium may also comprise salts, vitamine, minerais, mctals, and other nutrients. Preferably, the culture condition is sufficient to provide a suitable amount of nutrient and température for the algae to grow under conditions that generate an algal biomass comprising elevated levels of total fat.

In some embodiments, culturing comprises limitinga nutrient (e.g., nitrogen, phosphorous) for a suitable time to increase the amount total fat. For exaniple, the culture can be starved of a certain nutrient or (ransferred to a separate culturing medium lacking a spécifie nutrient (e.g,, phosphorus-free or nitrogen-free medium, or a culture medium containing lower levels of a nutrient). In some embodiments. the culture medium contains an initial content of a nutrient such that that nutrient becomes depleted at a later rime during exponential growth but prior to the déplétion of other nutrients. In some embodiments, culturing does not comprise limiting a nutrient (e.g., nitrogen, phosphorous) during culture. In 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 takes place in three or more types of medium in a sequential manner. In like manner,

culturing of a single algal bioniass may take place in two or more vessels, wherein a first vessel is used to inoculatc a subséquent vessel, thc subséquent vessel is used to inoculate yet another subséquent vessel, and so on. Although an understanding of a mechanism is not needed to practice the invention, and the invention is not limited to uny partîcular mechanism of action, in some embodiments, sequentîal culturing of a single algal biomass in multiple vessels containing multiple types of medium allows thc algal biomass to grow in such a way that the total fat content of the biomass is elevated compared to growth of an algal bioinass (e.g., of the same algal species) grown in a single vessel and/or growth medium.

Culturing of the algae can be performed in a conventional bioreactor suitable for culturing thc algae to providc an algae biomass. For example, the algae can be eultured by a process including, but not limited to, batch, fed-batch, cell recycle, and continuous fermentation. In a preferred embodiment, the algae are eultured in a fed-batch process.

Thc invention is not limited to any partîcular manner or method ofharvcsting the algae from thc culture medium. A variety of methods can be used to harvest the algal cells from the culture medium. In one embodiment, harvesting comprises recovering the algal biomass from thc culture medium by separating, for examplc by filtration (e.g., bclt filtration, rotary drum filtration) and/or centrifugation. If desired, the harvested algal cells can then bc washed, frozen, lyophilized, spray dried, and/or stored under a non-oxidiz.ing atmosphère of a gas (e.g., CO2, N2) to reduce or eliminate (he presence of O2. Optionally, synthetic and/or natural antioxidants including. but not limited to, butylated hydroxytoluenc (BHT), butylated hydroxyanisole (BHA), tcrt-butylhydroquinonc (TBHQ), cthoxyquin, bcta-carotcne, vitamin E, and vitamin C also can be added to the harvested cells.

In some embodiments, the invention provides a method for preparing an algal bioniass comprising elevated levels of total fat, thc method comprising: culturing algae under a culture condition suffîcient to provide an algal biomass comprising elevated levels of total fat and harvesting the algal biomass.

Microalgac Biomass

Thc invention provides, in some embodiments, an algal biomass and/or a fraction and/or an exlract thereof (e.g., for use in biofuel production and/or as a food or feed product).

In some embodiments, thc algal biomass comprises an omcga-3 fatty acid content of at least 10% dry weight of the biomass, illustratively, about 10% to about 50%, about I0?4* to about 40%, about 10% to about 30%, about 10% to about 20% dry weight of the biomass. In onc embodiment, thc algal bioinass is prepared in accordance with (he methods of the

invention. For example, in some embodiments. the algal bioinass is prepared by a method comprising: culturing an algac under a culture condition sufficient to providc a algal biomass comprising elevated total fat levels (e.g., grcatcr than 67% w/w), wherein the algal biomass is harvested at a négative growth accélération phase or a stationary phase. In another embodiment, the algal biomass is harvested from the culture during the exponenlial, logarithmic growth phase.

Lipid Compositions Prepared From Algal Biomass

In some embodiments, the invention provides a method for preparing a lipid/fatty acid cxtract (e.g., a lipid/fatty acid composition) from an algal biomass grown under conditions to contain elevated levels of total fat, the method comprising obtaining lipids from an algal biomass cultured under a culture condition sufficient to provide an algal biomass with elevated total fat content (e.g.. total fat content greater than 67% of the biomass), wherein the algal biomass is harvested at a negative growth accélération phase or a stationary phase of the algae. In another embodiment, the algal biomass is harvested during a logarithmic growth phase of tlie algae.

Methods for obtaining a lipid composition from an algal biomass of the invention include, but are not limited to, extraction, heat, pressure, saponification, sonication, freezing, grinding, ion exchange, chromatography, membrane séparation, electrodialysis, reverse osmosis, distillation, chemical derivatization, crystallization, etc. For example, algal lipids can bc extracted from the algal cells by any suitable method including, but not limited to, extraction with a solvent including, but not limited to, éthanol, ethyl acetate, ïsopropyl alcohol. methanol, ethyl acetate, hexane. méthylène chloride, methanol, petroleum, chloroform, and the like, or by pressurized liquid hydrocarbons such as butane, pcntanc, propane, or others (with our without co-solvents), or through supercritical fluid extraction (with or without co-solvents). Optionally, the extracted lipid/fatty acid oil are evaporated under reduced pressure to rcduce or remove the solvent and/or producc a sample of concentrated lipid material. In other embodiments, the colis arc broken or lysed to obtain the lipid composition, for example into an oil form (e.g., for use as a biofuel or a biofuel precursor). In some embodiments, the extracted oils are subjected to refining. The invention is not limited by the type of refining. In some embodiments, the extracted oils are chemically refined. In some embodiments, the extracted oils are physicully refined. In some embodiments, lhe extracted oils are both chemically and physicully refined. Extracted oils (e.g., from an algal biomass grown under conditions to elevatc the total fat content of the

algal cell (e.g., to above 67%)) may be refined using any conventional refining method. The rcfining process may remove some or ail impuritics from the extracted lipids/fatty acids/oils. In some embodiments, the refïning process comprises onc or more steps to degum, blcach, filter, deodorize and/or polish the extracted lipids/fatty acids/oils.

In some embodiments, the lipids/fatty acids/oils contained in the extracted lipid composition is concentrated by hydrolyzing the lipids to conccntratc the lipid fraction by employing a method such as, for cxample, urca adduction, fractional distillation, column chromatography, and/or supercritical fluid frac donation.

Accordingly, in one embodiment, the step of obtaining a lipid composition from an algal biomass of the invention comprises extracting the lipid composition front the biomass. In another embodiment, the step of obtaining a lipid composition from an algal biomass of the invention comprises contactîng the biomass with a polar solvent.

For example, in some embodiments, lipid/fatty acid/oil is extracted from the algal biomass to providc a lipid composition using a solvent under an extraction condition 15 sufficient to exlract lipids and/or fatty acids but not sufficient to cxtract compounds that are insoluble in the solvent In one embodiment, a lipid/fatty acid composition is extracted from an algal biomass of the invention wherein cellular débris and/or precipitated insoluble compounds arc separated from the fraction containing lipid/fatty acid and solvent. In another embodiment, the method further comprises sépara ting the cellular débris and precipitated compounds using a séparation method such as filtration, centrifugation, and/or combinations thereof. In some embodiments, the cellular débris and/or precipitated insoluble compounds (e.g., that portion of the algal biomass that are not soluble in a solvent (e.g., proteins, fiber, etc.) are rccovcrcd and utilîzed (e.g., in a food or feed product).

In some embodiments, the solvent is a polar solvent. Examples of polar solvents include, but are not limited to, éthanol, ethyl acetate, isopropyl alcohol, methanol, ethyl acetate, and mixtures thereof. In one embodiment, the polar solvent is éthanol. Extraction of the lipid composition with a solvent can bc carricd out in a variety of ways. For cxample, the extraction can be a batch process, a continuons process, or a continuous countcr-currcnt process. In a continuous countcr-currcnt process, the solvent contact with the microalgae leaches the oil into the solvent, providing an incrcasingly more solvent-oil fraction.

Following extraction, tlie solvent can bc removed using methods known in the art. For example, distillation, rotary évaporation, or a rising film evaporutor and steam stripper or any suitable desolventizer can be used for removing lhe solvent.

In one embodiment, the extracted lipids/fàtty acids arc exposed to an absorption

process (e.g., bleaching) to remove one or more undesirable compounds such as, for example, color bodies and/or phosphatidcs that may be présent. In some embodiments, the absorption process is a bleaching process comprising contacting the lipid/fatty acid extract with a bleaching material (e.g,, neutral carth (e.g., natural clay or fulier's earth), acid-activated earth, 5 activated carbon, activated clays, silicates, and or a combination thereof). The invention is not lîmited by the amount of bleaching material utilized.

In onc embodiment, the extracted lipids/fatty acids arc exposed to a degumming step. Degumming methods are known in lhe art and include, for example, water degumming, acid deguinming, cnzymatic degumming, and membrane degumming. In some embodiments, the I0 lipid/fatty acid cxtract is subjectcd to degumming (e.g., following an absorption process), wherein the degumming comprises contacting the lipid/fatty acid extract with a mixture of aqueous acids that are in amounts effective lo precipitate gums and/or chlorophyll-type compounds thaï may bc présent in the lipid/fatty acid extract composition. The invention is not limitcd by the type or amount of aqueous acids utilized. In onc embodiment, the mixture 15 of aqueous acids comprises sulfuric acid and/or phosphoric acid. In another embodiment, equal amounts of aqueous acids are mixed wilh the lipid composition. In a preferred embodiment, when blended with the oil, the aqueous acids arc in an amount sufficient to provide an acidic pH. Précipitâtes that form after acid mixing can be removed from the lipid composition, for example using centrifugation and/or filtration (e.g., membrane filtration). In 20 some embodiments, the degumnted lipid/fatty acid extract composition is subjcctcd to drying (e.g., to rcducc moisturc content of the composition). The invention is not limitcd by the drying condition (e.g., time, température, and/or a vacuum condition). As described herein, in some embodiments, the moisturc content of the dried lipid/fatty acid composition is less than about 10% w/w (e.g., less than about 9,8, 7, 6, 5,4,3, 2, 1, 0.9,0.8,0.7,0.6,0.5,0.4, 25 0.3, 0.2,0.1, 0.05, or 0.01% w/w).

Lipid Composition

In some embodiments, the invention provides a lipid composition prepared from a algal biomass of the invention. Tn some embodiments, the lipid composition is prepared in accordance with a method of the invention. For example, in some embodiments. a lipid composition is an algal biomass or a portion/fraction thereof from algae of the genus

Thraustochytrium. In some embodiments, the algal biomass comprises an algae selected from Dinophyceae, Cryptophyceae, Trebouxiophyceae, Pinguiophyceae, and/or combinations thereof. In other embodiments, the algal biomass comprises an algae selected from °(

Thrausiocliyirium slrialum, Thrauslochylritim roseum, Thrauslochytrium aureiun, Crypthecodinium cohnii, Parietochloris spp., Rhodomonas spp., Cryplomonas spp., Parietochloris spp., Heniisebnis spp.; Porphyridlum spp., Glossomastix spp., and/or combinations thereof. în further embodiments, the algal bioniass comprises an algae selected from Parietochloris incise, Rhodomonas salina, Hemiselmis brunescens, Porphvridium cruentum and Glossomastix chrysoplasta, and combinations thereof. In a preferred embodiment, the algal biomass comprises Schizochytrium lintacinum.

Food Products and Animal Fecd Additives

In some embodiments, a whole-ccll algal biomass, fraction, and/or extract thereof is used for consumption (e.g., by a mammal (e.g., human or animal consumption)) or as a food additive (e.g., to increase the lipid content and/or nutritional components of a food). For cxample, in some embodiments, when used as animal fecd (e.g., cattle feed, dairy feed, aquaculture feed, poultry fecd, etc.), the lipids/fatty acids produced by an algal biomass of the invention is incorporated into a food product (e.g., animal feed). In some embodiments, a wholc-cell algal biomass, fraction, and/or extract thereof is used for pharmaceutical or nutritional purposes and/or industrial applications.

The whole-cell algal bioniass, fraction, and/or extract thereof can be provided in any one of variety of forms/compositions suitable for a particular application or use. In some embodiments, the wholc-eell algal biomass, fraction, and/or cxtract thereof is provided. In another embodiment, a whoJc-ccll algal biomass, fraction, and/or cxtract thereof is provided in a powdered form or as a free oil in a liquid form (e.g., lipid composition or a fraction or concentratc thereof). A whole-cell algal bioinass, fraction, and/or cxtract thereof may bc used for human and/or animal consumption. For cxample, in some embodiments, a wholccell algal biomass, fraction, and/or cxtract thereof is provided as or incorporated into a feed, a dietary supplément, a food, a pharmaceutical formulation, a dairy product, and/or an infant formula.

For cxample, in one embodiment, a wholc-cell algal biomass, fraction, and/or cxtract thereof is dried (e.g., spray drying, tunnel drying, vacuum drying) and used as a feed or food supplément for any animal or aquaculture organism (e.g., fish, shrîmp, crab, lobster, etc.) whosc mcat and/or products arc consumed by humans or animais (e.g., pets, livcstock). In another embodiment, a whole-ccll algal biomuss, fraction, and/or cxtract thereof is mixed with a dry moisture-reducing agent (e.g., ground gTain such as ground corn).

The compositions described herein may be used as a complété food product, as a

coniponent of a food product, as a dietary supplément or as part of a dietary supplément, as a feed additive and may bc either in liquid, semisolid or solid form. The compositions of the invention additionally may bc in the form of a pharmaceutical composition. Tlie compositions, dietary suppléments, food products, buby food products, feed additives, and/or 5 pharmaceutical compositions of the invention may be utilized in methods for pronioting lhe health of an individual. The compositions may be in liquid, semisolid or solid form. For cxamplc, the compositions may bc administered as tablcts, gel packs, capsules, gclatin capsules, flavored drinks, as a powder that can be rcconstituted into such a drink, cooking oil, salad oil or dressing, sauce, syrup, mayonnaise, margarine or the like. Furthermore, the food product, dietary suppléments, and the like, of the présent invention can include, but arc not limited to, dairy products, buby fond, baby formulu, beverages, burs, a powder, a food topping, a drink, a cereal, an ice cream, a candy, a snack mix, a baked food product and a fried food product. Beverages of the invention include but are not limited to energy drinks, nutraceutical drinks, smoothies, sports drinks, orange juicc and other fruit drinks. A bar of the présent invention includes, but is not limited to, a meal replacement, a nutritional bar, a snack bar and an energy bar, an extruded bar, and the like. Dairy products of the invention include, but are not limited to, induding but not limited to yogurt, yogurt drinks, chccsc and milk. Compositions intended for oral administration may be prepared according to any known method for the manufacture of dietary suppléments or pharmaceutical préparations, and such compositions may include at least onc additive selected from the group consisting of tastc improving substances, such as sweetening agents or flavoring agents, stabilizers, cmulsitîcrs, coloring agents and preserving agents in order to provide a dictetically or pharmaceutically palatablc préparation. Vitamins, minerais and trace élément front any physiologically acceptable source may also bc included in the composition of the invention.

In some embodiments, a pharmaceutical composition of the invention comprises the compositions of the invention in a therapeutically effective amount. The compositions of the invention can bc formulated for administration in accordance with known pharmacy techniques. See, e.g., Rcmington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of a pharmaceutical composition according to the invention, the lipid compositions (induding the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier will be compatible with any other ingrédients in the formulation und must not be delctcrious to the subject.

Biofucl

OC

Many of the existing (eclinologies for making biofuel from algae are expensive, inefficient and unsustainable when operated at a scale that is required to dispiacc any mcaningfiil fraction of petrodiescl in the market. The supply and cxpcnditurc of energy to harvest and process algae are often underestimated. To produce biodiesel front algae conventionally, the algae are typically harvested from a culture at a concentration of about 0.2 g/L în water, The harvested algac are then dewatered which incrcases the algal concentration to form an algal pastc of about I5% solids, The pastc 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 conventions!

method for generating biodiescl from algac is prohibitivcly expensive.

For example, when ulgae grows in a natural body of water, the algal biomass is relatively dilute considering the volume of water, Producing a gallon of oil requires processing of about 20,000 to 40,000 gallons of water. The energy cost of (ransporting and processing such a large volume of water is high. As cxamplc, 2,500 gallons of oil/acrc/ycar could be produced if algae with 25% of its mass as lipids could be produced at 25 g/m.sup.2/day. For this exantplc, 50 million gallons of water must be processed to produce the 2,500 gallons of oil, The standard approach of pumping water to a centralized facility for dewatering is simply too energy-intensivc and cost prohibitive. As example, a relatively 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 contained in the oil product, resulting in a net négative energy balance.

Accordingly, in some embodiments, the invention provides a method for preparing an algal biotnass and/or lipid/fatty acid cxtract (e.g., a lipid/fatty acid composition) iront an algal biomass, grown under conditions to contain elevated levels of total fat, the method comprising obtaining lipids from an algal biontass cultured under a culture condition sufficient to provide an algal biomass with elevated total fat content (e.g., total fat content grcatcr than 67% of the biomass), wherein the algal biomass is harvested at a négative growth accélération phase or a stattonary phase of the algac. In another embodiment, the algal biomass is harvested during a logarithmic growth phase of the algae. Methods for obtaining a lipid composition froni an algal biomass of the invention are described herein.

Accordingly, in some embodiments, the invention provides a biofucl feedstock or a biofuel comprising lipids, hydrocarbons, or both, derived from an algal culture and/or algal biomass generated according to the methods of the invention. In some embodiments, lipids or algal compositions comprising the sanie arc subdivided according to poiarity: neutral lipids

and polar lipids. The major neutral lipids are triglycérides and free saturated and unsaturated fatty acids. The major polar lipids are acyl lipids, such as glycolipids and phospholipids. 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 and/or hydrocarbons présent in the composition. In some embodiments, the hydrocarbons présent in algae compositions ofthe invention are mostly straight chain alkanes and alkenes, and may include paraffins and the like having up to 36 carbon atoms.

In some embodiments, the invention provides a method of making a liquid fuel that comprise processing lipids derived from an algal culture and/or algal biomass or lipid fraction 10 thereof described herein. Products of the invention made by the processing algal derived biofuel feedstocks can be incorporated or used in a vuriety of liquid fuels including but not limited to, diesel, biodiesel, kerosene, jet-fuel, gasoline. JP-l, JP-4, JP-5, JP-6, JP-7, JP-8, Jet Propellant Thermally Stable (JPTS), Fischer-Tropsch liquids, alcohol-based fuels including cthanol-containing transportation fuels, other biomass-bascd liquid fiiels including ccllulosic 15 biomass-based transportation fuels.

In some embodiments, triacylglyccridcs in algal oil is converted to fatty acid methyl esters (FAME or bïodicscl), for cxample. by using a basc-catalyzcd transestérification process (for an overview see, e.g., K. Shaine Tyson, Joseph Bozell, Robert Wallace, Eugène Petersen, and Luc Moens, Biomass Oil Analysis: Research Needs and Recommendations, 20 NREL/TP-510-34796, Junc 2004, hereby incorporated by reference in its entirety). In some embodiments, the triacylglycerîdes arc reacted with methanol in the presence of NaOH at 60 C. for 2 hrs to generate a fatty acid methyl ester (biodiesel) and glycérol. In further embodiments, the biodiescl and glycérol co-products arc immisciblc and typieally separated downstream through dccanting or centrifugation, followed by washing and purification. Free 25 fatty acids (FFAs) arc a natural hydrolysis product of triglycéride and formed by reacting triacylglycerîdes and water. In some embodiments, methods of the invention further comprise a step for quickly and substantially drying tlie algal oil by techniques known in the art to limit production of free fatty acids, preferably to less than l%. In another embodiment of the invention, tlie methods can further comprise a step for converting or removing the free 30 fatty acids by techniques known in the art.

In some embodiments. triacylglyccridcs in algal oil is converted to fatty acid methyl esters (FAME or biodiesel) by acid-catalyzed transestérification, enzyme-catalyzed transestérification, or supercritical methanol transestérification. Supercritical methanol transestérification docs not rcquirc a catalyst (See, e.g., Kusdiana, D. and Saka, S., Effects of

Y

water on biodiesel fuel production by supercritical methanol treatment, Bioresource Technology 91 (2004), 289-295; Kusdiana, D. atid Saka, S., Kinetics of transestérification in rapeseed oil to biodîcscl fuel as treated in supercritical methanol, Fuel 80 (2001), 693-698; Saka, S., and Kusdiana, D., Biodiesel fuel from rapeseed oil as prepared in supercritical methanol, Fuel 80 (2001), 225-231). The reaction in supercritical methanol reduces the réaction time front 2 hrs to 5 minutes. In addition, the absence of the base catalyst NaOH grcatly simplifies the downstream purification, rcduccs raw material cost, and éliminâtes the problem with soaps froin free fatty acids. Rather than being a problem, the free fatty acids become valuable feedstocks that are converted to biodicsel in the supercritical methanol as

I0 follows.

In some embodiments, triacylglycerides are reduced with hydrogen to produce paraffins, propane, carbon dioxide and water, a product generally known as green diesel. The paraffins can either be isomerized to produce diesel or blendcd directly with diesel. In some embodiments, there arc advantages of hydrogénation over conventional base-catalyzcd transestérification. For example, the hydrogénation process (also referred to as hydrocracking) is thermochcinical and therefore much more robust to feed inipuriti.es as compared to biochcmical proccsscs (e.g., hydrocracking is relatively insensitive to free fatty acids and water). Free fatty acids are readily converted to paraffins, and water siniply reduces the overall thermal efficiency of the process but does not significantiy aller the chemistry In another non-limiting example, the paraffin product is a pure hydrocarbon, and therefore indistinguishablc from pctrolcum-bascd hydrocarbons. Unlike biodiescl which has a 15% lower energy content and can freeze in cold weather, green diesel has similar energy content and flow characteristics (e.g., viscosity) to pctroleum-bascd diesel. In various cmbodimctits, the methods ofthe invention cncompass the steps of hydrocracking and îsomerization, which are well known in the art to producc liquid fuels, such as jet-fuel, diesel, kerosene, gasoline, JP-l, JP-4, JP-5, JP-6, JP-7, JP-8, and JPTS.

EXPERIMENTAL

The following examples are provided in order to demonstrate and fiirther illustrate certain preferred embodiments and aspects of the présent invention and are not to be construcd as limitîng the scope thereof.

EXAMPLE 1

Growth of high fat algal biomass

ίο

Expérimente were conducted during development of embodiments of the invention in order to charactcrize and cstablish methods for hetcrotrophic algae production, and in particular, methods of culturing algae in order to generate an algal bîomass containing high fat/lipid levels. A sériés of conventional hetcrotrophic algae production studies was performed and run in batch.

A culture of Schizochytrium limaciitum was obtained and stored in 1.5mL cryovials at -80 C. For each experiment, the process was started by thawing eiyovials and aseptically adding to l.OL shake flasks of with media. Media in the l L flasks contained 50 g/L sugar, 10 g/L yeast extract, and 4 g/L sea sait. Three liters of 3 to 6 day old shake flask culture was used to ÎTtoculatc a 250 L vessel containing media, grown for 24-48 hours, and then transferred to a main vessel (17,000 to 28.000L) and run us a butch process for 36 to 72 hours. The température of the batch runs was kept between 25 and 30 C. The température range was large due to lack of précisé control of the system. The media used in the seed (250L) and batch (17,000 to 28,0001,) runs was as follows:

Raw material	Batched
Sugar	50	g/L
Yeust Extract	7.5	g/L
MgSO4	0.1538	g/L
Urea	2	g/L
CaC12	0.1538	g/L
MgC12	0.1538	g/L
Antifoam	0.3	ml

Tab c 1 A. Media used in traditional batch and seed cultures.

Total fat content of the algal bîomass of the batch cultures was determined by gas chromatography (Sec AOAC gravimétrie method 922,06), acid hydrolysis (Sec Total Fat by 20 Acid Hydrolysis Ankom Technology Method 1, 02-10-09), und High Température Solvent Extraction (See Ankom Technology Method 2, 01-30-09 and AOCS Method 5-04). In brief, a typical analysis procédure for fermentation broth was as follows: Broth samples were concentrated by centrifugation. After dccanting, the sample was treeze dried for 24 hours with résultant moisture less than one percent. The samples were weighed prior to acid hydrolysis, washed and dried in an oven. This was followed by an extraction process under

gradient thermal conditions with petroleum ether. The hydrolysis and extraction process were undcrtakcn utilizing automated instruments. Aficr further drying, rcsults were determined on the basis of mass loss.

As shown in Table l B below, the total fut/lipid levels (w/w) achîeved in the batch productions at a température range from 25-30 C was 8-38%,

Log run #	Total fat (%)
A-1-10	33.75
A-2-10	38.59
A-3-10	27.30
A-4-10	38.51
A-5-10	7.82
A-7-10	33.33
A-8-10	34.85
A-9-10	27.21

Table l B. Total fat content of algal biomass grown in batcb ion between 25-30 C.

Efforts were made to increase the amounts of fat/lipid levels as these amounts were

IO considcred too low to bc of value and additional experiments were nm in an effort to increase the level of lipids produced in eultured algae.

During development of embodiments of the invention, experiments were conducted in order to détermine if changes in the constitucnts and/or amounts or ratios of the same in the media could providc different algal growth charactcristics. (n addition, experiments wcrc conducted to détermine if scale-up of an algal culture system would aller algal growth characteristics. In particular, the amounts and ratios of MgSCL, Urea, CaCh, MgCh, and KH2PO4 were modified in an attempt to inctcasc the level of lipid produced by eultured algae.

ResulLs of ferme nia lions produced in a batch volume of 10L are shown below:

lab trial runs 10L

Ingredients/log tt	NB4-030311	NB6030311	NB4032311	NB6032311	NB3032811	NB4032811
(g/L)
Sugar	50	50	50	50	50	50
Yeast Extract	7.5	7,5	7.5	7.5	7.5	7.5
MgSO4	0.1538	0,1538	0.1538	0.1538	0.5	1
NaCI	0	0	0	0	0	0

Urea	2	2	2	2	1	1
ZnSO4	0.1538	0.1538	0.1538	0.1538	0.1538	0.1538
CaCI2	0.1538	0.1538	0.1538	0.1538	1	2
MgCI2	0.1538	0.1538	0.1538	0.1538	O.S	1
KH2PO4	-	-	1.5	l.S	0.5	1
Trace (liquid) ml	10	10	10	10	20	20
Ferrie Chloride
Zn sulfate
Mn sulfate
Borlc acid
Copper sulfate
Feed
Urea:KH2PO4	urea 200g/L	urea 200g/L	2:1	2:1.5	2:0.5	2:0.5

tempset point	30	30	30	30	30	30
fat%	8.13	6.89	88.98	84.28	56.1	64.3
Notes	strong NH3 smell	strong NH3 smell
	ph Issues	ph issues
	foarn out	foam out

Table 2: ÎOL fermentation conditions and results.

Ingredlents/log tt	NB6-032811	N 03-040711	NB4-040711	NB6-O40711	NB3- 041911	NB4041911	NB6041911
(g/L)
Sugar	50	50	50	50	SO	50	50
Yeast Extract	7.5	7.5	7.5	5	7.5	7.5	6.25
MgSO4	2	2	2	2	2	2	2
NaCI	0	0	0	0	0	0	0
Urea	1	1	1	1	1	1	1
ZnSO4	0.1538	0.1538	0.1538	0.1538	0	0	0
CaCI2	4	4	4	4	4	4	4
MgCI2	2	2	2	2	2	2	2
KH2PO4	2	0.25	0.25	0.25	0.25	0.25	0.25
Trace (liquid) ml	20	20	20	20	20	20	20
Ferrie Chloride
Zn sulfate
Mn sulfate
Boric acid
Copper sulfate
Feed
Urea:KH2P04	2:0.5	2:0.5	2:0.5	2:0.5

temp set point	30	30	30	30	30	30	30
fat %	73.7	71.16	73.49	73.88	68.69	76.56	72.56
Tab	e 3: Additions	10L fermentation conditions and results.

Ingredients/log »	NB3061411	ND406141 1	NB606141 1	NB3062U 1	NB406211 1	NB603211 1	NB3062811	NB4062B11	NB6032811
(g/D
Sugar	5Û	50	50	50	50	50	50	50	50
Yeast Extract	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5	7.5
MgSO4	0.1538	2	2	2	2	2	0.1538	2	2
NaCI	0	0	0	4	8	0	0	0	0
Urea	2	1	1	1	1	1	1	1	1
ZnSO4	0.1538	0.1538	0	0	0	0	0	0	0
CaCI2	0.1538	4	4	4	4	4	4	0.1538	4
MgCI2	0.1538	2	2	2	2	2	2	2	0.1538
KH2PO4	2	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Trace (liquid) ml	10	10	0	0	0	0	0	0	0
Ferrie Chloride
Zn sulfate
Mo sulfate
Boricadd
Copper sulfate
Feed
Urea:KH2PO4	2:0.5	2:0.5	2:0.5	2:0.5	2:0.5	2:0.5	2:0.5	2:0.5	2:0.5

temp set point	30	30
fat%	46.52	65.04	67.61	61.28	62.95	68.53	49.25	54.41	64.97
	Fomular confirmation	NaCI effects	Sait ratio effects

Table 4: Additional 10L fermentation conditions and results.

These experiments, conducted during development of embodiments of the invention, indicated that certain amounts/ratios of substrates présent within the media had a direct impact on algal growth characteristics (e.g., total biomass achieved as well as amount of fat and/or other component content within the biomass itself). Parameters that provided a high fat content biomass in the l OL runs were then utilized to déterminé if they would be successfiil for large scale production of a high fat content biomass.

EX AMPLE 2

Large scale production of high fat algal biomass

The initial attempts to gcncrate a hctcrotiophic algal biomass described in Example l above utilized procedures based on yeast fermentation processes. The processcs were run in

batch due to limitations in lhe production facility (Nicholasville, K.Y) and températures that could only bc controlled between 25 and 30 C. The température range was large duc to lack of précise control of the system. As indicated in Table l, above, the fat levels achieved at the Nicholusville, K.Y plant ranged from 8-38%. However, as indicated above, additional experiments were carried out during development of embodiments of the invention that provided the identification of certain ratios/amounts of substrates that could be utilized during hcfcrotrophic algal biomass production to altcr algaJ growth and biomass generation/properties. Modification ofthe levels and ratios ofthe media (e.g.. MgSO₄, Urea, CaCl₂, MgC'h, and KH2PO4) during fermentation was identified and characterized to aller algal growth, and to generate a biomass with significantly different properties (e.g., a significantly higher fat content biomass). As described below, the process (including media containing the identified ratios/amounts of substrates effective in generating a high fat content algal biomass (e.g., greater than 67% fat content)) was further tested and run in large scale and also as a fed-batch (thereby allowing for modification and control of amounts of nîtrogen, phosphores, potassium, und carbon during the run).

A culture of Schizochytrium limacinum was obtained and stored in l.5mL cryovials at -80 C. For cach culture, a cryovial was thawcd and ascptically added to l .0L shake flask of media. Media in the I L flasks contained the components us shown in Table 5:

Ingrédient	Batched	Manufacturer
Sugar	50 g/L	Cargill - Hammond, IN, USA
Yeast Extract	10 g/L	Sensient - Indianapolis, IN, USA
Sea Sait	4 g/L	Sigma-Aldrich - St. Louis, MO USA

Table 5. Media used for l ,0L culture.

The température of the shake flasks containing Schizochytrium limacinum in media was kept at 30 C and shaken at 250 RPM until such time that the algac had entered logarithmic/exponentiul growth phase but prior to déplétion of glucose in the media (usually 25 72-144 hours).

The contents of IL culture flasks were then ascptically transferred into 2.0L aspirator bottles with stérile connectors that were used to connect to larger vessels (40L or 27 L or 18

L vessels). Thus, the IL culture flask cultures were used as inoculuin and aseptically added to a seed vessel (either 40L or27L or 18L) containing media described in Table 6 below:

Ingrédient	g/L	Manufacturer
Sugar	50	Cargill - Hammond, IN, USA
Yeust Extract	7.5	Sensient - Indianapolis, IN, USA
MgSO4	0.1538	Norkem Limited - Kutahya, Turkcy
CaC12	0.1538	Occidental Chemical Company - Dallas, TX
MgC12	0.1538	North Ameriean Sait Company - Overland Park, KS

Table 6. Media used for 181. or 27L, first seed cultures.

The first seed stage (40/18/27L) was ruu at 30 C, under airflow and agitation conditions so as to inaintain dissolved oxygen at or above 10%, and until at least 20 g/L of glucose was consumed. When grown under stérile conditions, no pH control was required. Rather, the pH slayed within a healthy range throughout the fermentation process. The first seed stage (40/18/27L) was considered conipleted when algal growth was within log/cxponcntial growth stage, glucose had not been depleted from the inedia, but at least 20 g/L of glucose had been consumed (in general, this occurrcd between about 24-48 hours). A larger vessel (4000/2000L) was made ready for the first seed stage culture (e.g., it was H lied with media and brought to 30 C under stérile conditions).

Upon completion of the first seed culture, the contents of the first seed stage (40/18/27L) culture vessel was transferred to a vessel with at least 2.000L media described in

Table 7 below:

Ingrédient	g/L	Manufacturer
Sugar	50	Curgill - Hammond, IN, USA
Yeast Extract	7.5	Sensient - Indianapolis, IN, USA
MgSO4	0.1538	Norkem Limited - Kutahya, Turkey
CaC12	0.1538	Occidental Chemical Company - Dallas, TX
MgC12	0.1538	North Ameriean Sait Company - Overland Park, KS

Table 7. Media used for 4,000/20001.., second seed cultures.

This second seed stage (4000/2000L) culture was ntn at 30 C, under airflow and agitation conditions so as to maintaîn dissolved oxygen at or above 10%, and until at least 20 g/L of glucose was consumcd. When grown under stérile conditions, no pH control was required. Rather, the pH stayed within a healthy range throughout the fermentation process.

The second seed stage (4000/2000L) was considered completed when algal growth was within log/cxponcntial growth stage, glucose had not been dcpleted from the media, but at least 20 g/L of glucose had been consumcd (in general, this occurrcd between about 24-48 hours).

Upon completion of the second seed (4000/2000L) culture, the contents of the second 10 seed culture were uscptically transferred into a third culture vessel with a volume ranging between 70,000 L to 220,000 L of stérile media at 30 C as described in Table 8 below:

Batcb
Raw material	Batched	Manufacturer
Sugar	50 g/L	Cargiil - Hammond, IN, USA
Yeast Extract	7.5 g/L	Scnsient- Indianapolis, IN, USA
MgSO4	4 g/L	Norkem Limited - Kutahya, Turkey
Urea	1 g/L	PCS Sales - Northbrook, IL
CaC12	2 g/L	Occidental Chemical Company - Dallas, TX
MgC12	2 g/L	North American Sait Company - Overland Park, KS
KH2PO4	0.25 g/L	Lîdochem - Hazlet, NJ

When 30 g/L of glucose had been consumcd by algae présent in the third culture vessel (70,000-220,000 L vessel), glucose and fcd-batch feeds were started. Glucose was maintained at 10 g/L during large scale culture of algae in the third culture vessel (70,000220,000 L vessel). As described in 'fable 9 below, lhe feed used for the fcd-batch process conta ined:

Feed
Ingrédient	g/L	Manufacturer
Urea	2 g/L	PCS Sales - Northbrook, IL
KH2PO4	0.5 g/L	Lidochem - Hazlet, NJ

Table 9. Feed used for fcd-batch process.

The icd batch feed was added over a 34 hour period. Although an understanding of thc mechanism is not needed to practice thc présent invention, and while thc présent invention is not limited to uny partîcular mechanism of action, in some embodiments, this time period was identifîed based upon the observation that it look ~ 20 hours for the feed to start (for 30 g/L of glucose to be consumed by the algae présent in thc third culture vessel). The feed was then stopped (e.g,, at around log hour 54) in order to allow ail of the nutrients to be removed (consumed) from thc media. Harvest of the algal biomass took place upon the termination of exponential growth, occurring generally between thc log hours 66-76.

Thc culture broth was dc-sludgc centrifuged under conditions to aehieve 15-30% solids, with the concentrate spray dried to remove water to a final moisture of less than 5%.

Results of several independent, large scale cultures are shown in Figure l and Tables 10-12 below:

Run number	Fat % (harvest sample)	Vol adjusted Biomass (g/L)	% recovery from centrifuge	Fal% (spray dried product)	Protein % (spray dried Product)	Moisture % spray dried product
Fl-2-11	60.7*	86.2		75.7	11,66	1.47
Fl-3-11	69.64	86.4	55	70.25	16.47	1.37
Fl-4-11	74.76	66.5	67	71.56	15.92	2.11
Fl-5-11	73.12	70.8	68	65.65	17,14	2.43
Fl-6-11	62.77**	45.7	89	54.8	13.35	2.14
F2-1-11	72.59	50.9	87	65.89	17.64	2.72
F2-2-11	70.81	59.5	52	66.49	15.29	2.15
Table 10.	Large sca	c production culture resu	ts.

*bad harvest sample *· process control problems with this batch o Fl-2-ll

Had a batch volume of 70,000 L and a harvest volume of 93,700 L o Fl-3-ll

Had a batch volume of 70,000 L and a harvest volume of 84,000 L o Fl-4-ll

Had a batch volume of 70,000 L and a harvest volume of

92.300 L o Fl-5-11

Had a batch volume of 70,000 L and a harvest volume of

82.300 L o Fl-6-11

Had a batch volume of 80,000 L and a harvest volume of 83,600 L o F2-1-11

Had a batch volume of 110,000 L and a harvest volume of H3.000L o F2-2-H

Had a batch volume of 110,000 L and a harvest volume of

125,60ÜL

The biomass gcncratcd from cach large scalc, fed-batch culture was characterized, including analysis of the total fat (saturated and unsaturated fat) content; moisture, docosahexaenoic acid (DHA)content, palmitic acid content, crude protein content and ash content (See, e.g., Fat content and/Moisture - AOCS Am 5-04 ‘Rapid Détermination of Oil/Fat lltilizing High Température Solvent Extraction’ v. 3/3 l/l0; DHA-'Palmitic - AOCS Method Ce lb-89 and AOAC Method of Analysis 991.39; Protein - AO AC 990.03; Ash AOAC 942.05 Vol adjusted Biomass (g/L) - Stone, et. al. Dry Weight Measurement of

Microbial Biomass and Measurement Variablity Analysis. Bîotechnology Techniques. Vol 6:207-212.

Table 11
Run «/Comments	TOTAL FAT % (Final Harvest)	Moisture (%) Max 6%	DHA (mg/g)	Palmitic (mg/g)	Crude Protein (%) Report on release	Ash max 10%
SL-F1-1-11	72.70	1.39	191.8	379	14.96	3.5
SL-F1-3-11	69.64	1.37	181.1	366.2	16.47	3.08
SL-F1-4-11	73.71	2.11	185.8	373.8	15.92	3.11
SL-F1-5-11	73.12	2.43	176.8	351.3	17.14	0.0373
SL-F2-1-11	72.59	2.72	252.4	360.9	17.64	0.0363
SL-F2-2-11	70.81	2.15	247.2	365	15.29	0.041
SL-F2-3-11	72.86	2.50	197.36	269.4	11.58	3.29
SL-F2-4-11	69.03	3.07	177,9	133.65	18.23	3.9
SL-F2-5-11	70.10	2.12%	203.24	193.99	10.93	0.0342
SL-F2-6-11	73.5S	2.61	203.99	206.67	12.01	3.34
5L-F2-8-11	70.86	1.91	190.31	287.79	12.71	3.3
SL-F2-9-11	76.89	1.68	191.69	227.52	9.62	3
SL-F1-8-11	76.31	1.55	191.41	318	10.71	3.8
SL-F2-10-11	72.15	1.75	186.24	256.13	13.37	4.81
SL-F2-11-11	73.64	2.49	184.34	299.77	11.45	4.42
SL-F1-9-11	75.57	1.28	202.51	250.01	10.02	3.48
SL-F2-13-U	70.95	1,66	182.82	326.94	12.39	4.59
SL-F2-14-11	69.13	1.42	196	253.11	14,79	3.65

SL-F1-13-11	67.56	1.73	184.66	212.92	15.1	3.97
SL-F1-14-11	68.57	1.23	170.16	89,35	11.86	4.22
SL-F3-4-11	68.8	1.56	203.25	183.65	15.31	3.84
SL-F1-15-11	70,58%	1.14	175.58	147.37	9.8	3.56
5L-F1-16-11	72.72	1.31	175.28	91.4	10.43	4.06
SL-F3-8-11	71.75	1.8	207.95	138.37	14.32	3.65
SL-F5-5-11	68.7	1.23	189,07	119.72	11.68	3.42
SL-F1-25-11	74.80	1.54			9.16	3.68
SL-F3-14-11	76.24	1.90			8.41	3.08
SL-F3-15-11	75.80	1.08			7.3	3.26
SL-F1-1-12	70.24				8.3	3.51
1. Characterization of large scale ci	iltures
Table 12
Run H/Comments	TOTAL FAT % (Final Harvest)	Moisture (%) Max 6%	DHA (mg/g)	Palmltlc (mg/g)	Crude Protein (%) Report on release	Ash max 10%
SL-F1-25-11	74,80	1.54	170.63	355.6	9.16	3.68
SL-F3-14-11	76.24	1.90	179.85	346.41	8.41	3.08
SL-F3-15-11	75.80	1.08	182.81	367.01	7.3	3.26
SL-F1-1-12	70.24	1.81	160.84	336.77	8.3	Released
SL-F4-112	68.53	1.71	189.06	332.29	10.26	3.56
SL-F6-2-12	69.13	1.73	169,44	351.39	7.75	3.11
SL-F5-2-12	75.00	1.84	175.05	371.57	6.89	3.5
SL-F4-2-12	69.10	1.84	198,24	341.94	8.85	3.88
SL-F6-3-12	69.34	1.80	175.42	340.86	10.44	3.62
SL-F5-3-12	67.28	3.61	176,61	360.01	10.19	3.56
SL-F1-2-12	71.09	1.72	154.22	371.31	12.56	3.78
5L-F1-4-12	68.42	1.66	159.54	375.98	12.88	3.81
SL-F4-5-12	70.89	1.88	171.73	397.13	12.38	3.1
SL-F6-6-12	70.38	1.8	155.05	377.11	12,56	3.71
SL-F5-6-12	68.17	1.75	155.73	389.31	10.56	3.8
SL-F3-3-12	73.64	1.94%	156.25	393.08	12.13	3.52
SL-F1-7-12	71.97	1.58	164.05	362.4	3.75	3.98
SL-F3-4-12	70.93	2.37	183.88	366.83	10.13	2.23
SL-F6-7-12	70.95	2.66	176.68	366,29	10.56	4.31
SL-F1-8-12	72.08	1.94	172.91	407.2	9	3.62
SL-F3-6-12	72.15	1.85			10.S6	7.56
SL-F3-7-12	69.63	2.42			10.57	3.67
SL-F3-812	71.77	1.78
SI.-F3-9-12	69.18	1.74

Table 12. Characterization of large scale cultures

SI

Additionally, the fatty acid profile of the biomass was characterized. As shown in Figure 2, the fatty acid profile of each algal biomass generated is highly similar/consistcnt, independent of the total fat content of tlie biomass. A composite fatty acid profile, taking into 5 considération the collective profiles of ail samples analyzed, is provided in Figure 3.

The glyccride profile was also determined for cach algal biomass. Of the total glyccridc content of the biomass, about 4-8% were diglyccrides, less than l% glyccrol, about 3-7% monoglyccrides and about 84-88% triglycérides.

I0

EXAMPLE 3

Biomass harvesting

Experiments conducted during development of embodiments of the invention identified that the increased total fat levels in the biomass caused significant problcms with 15 regard to centrifugation of the algal biomass. Recovery of biomass content postcentrifugation ranged from only about 45-85% total biomass weight, This is shown, for cxample, in Table 13 below:

Run number	Fat % (harvest sample)	Vol adjusted Biomass (iÿL)	Protein % (harvest sample)	% recovery from centrifuge	Fat % (spray dried product)	Protein % (spray dried Product)	Moisture % spray dried product
Fl-2-ll	60.7*	«6.2	ΝΛ	ΝΛ	75.7	11.66	1.47
Fl-3-l l	69.64	86.4	NA	55	70.25	16.47	1.37
F1-4-H	74.76	66.5	ΝΛ	67	71.56	15.92	2.11
Fl-5-ll	73.12	70.8	16.14	68	65.65	17.14	2.43
El -6-1J	62.77*·	45.7	NA	89	54.8	13.35	2.14
F2-1-11	72.59	50.9	16.29	87	65.89	17.64	2J2
F2-2-11	70.81	59.5	12.58	52	66.49	15.29	2.15

Tuble 13. Comparison of Fat and Protein yield fforn direct harvest sample versus spray dried product.

*bad harvest sample** process control problcms with this batch Fl-2-ll

Had a batch volume of 70,000 L and a harvest volume of 93,700 L

Fl-3-ll

Had a batch volume of 70,000 L and a harvest volume of 84,000 L

Fl-4-ll

Had a batch volume of 70,000 L and a harvest volume of

92,300 L

Fl-5-ll

Had a batch volume of 70,000 L and a harvest volume of

82,300 L

Fl-6-l l

Had a batch volume of 80,000 L and a harvest volume of 83,600 L

F2-1-H

Had a batch volume of 110,000 L and a harvest volume of

113,OOOL

F2-2-11

Had a batch volume of 110,000 L und a harvest volume of l25,600L

The recovery problems wcrc identified to bc attributablc to the incrcasc in the amount of low density ltpid/oil in the biomass. Thus, experiments were conducted during embodiments ofthe invention in an effort to address this problcm.

Onc approach that displayed the ability to enhance recovery of the biomass was to chill the culture comprising the algal biomass prior to centrifugution. Although an understanding of a mechanism is not nceded to practice the invention, and the invention is not limitcd to any particular mechanism of action, in some embodiments, chilling the culture increased the density of the lipid/oil and allowcd a larger recovery of the biomass.

Experiments were conducted in order to détermine the effects of chilling the biomass before centrifugation.

l.ab trial onc: 2 gallons of broth collected and stored at 7-8 C for 16 plus hours. Eight X 50 ml centrifuge tubes wcrc collected and placed in a wuter buth to reach target températures described in table I4 below. Ail samples were centrifuged at 5000 rpm for 5 minutes.

Température (C)	Visual Observation
10	Excellent séparation with no floating cells. Clear supernatant.
20	Good séparation with no floating cells. Cloudicr than 10C
25	Similar to 20 C; cloudicr
30	Good séparation with no floating cells; cloudicr
35	Good séparation with no floating cells; very cloudy

40	Still separating; floating cells; mîlky supematant
45	Poor séparation
50	Almost no séparation with numerous floating cells

Table 14. Culture température and centrifugation results of trail l.

Lab trial 2: Fresh broth samples were collected and tested over a température range of

10-30C. They were not refrigerated overnight as in trial l. All samples were allowed to sit in an icc water bath to target température. Samples were ccntrifuged at 5000 rpm for 5 minutes.

Température (C)	Visual Observation	Density (g/inl)
II)	Excellent séparation with no floating cells. Clear supematant.	i.01967
15	Good séparation with no floating cells. Very cloudy supematant
20	Sample still separating; visible flocculation.
25	Very similar to 20C; increasing cioudincss
30	Still Good séparation; increasing cloudiness	1.02915

Table 13. Culture température and centrifugation results of trail 2.

l() As described in Example 2 and Figure i, during large scale production, chilling ofthe biontass prior to recovery (centrifugation) lead to significant incrcasc in total recovery of the biomass. Multiple large seule nins hâve been completcd with total recovery of approximately 95%.

All publications and patents mentioned in the above spécification are herein incorporatcd by référence. Various modifications andvariations ofthe described compositions and methods of the invention will be apparent to those skilled in the art without

departing from the scope and spirit of tlie invention. Although the invention has been described in connection with spécifie preferred embodiments, it should bc understood that tlie invention as claimed should not bc unduly limited to such spécifie embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious 5 to those skilled in the relevant fields are intended to be within the scope of lhe présent invention.

Claims

What Is Claimed Is:

5 l.A process of making an algal biomass comprising at least 67% total fat comprising culturing an algae under culture conditions sufficient to provide an algal biomass comprising

67% or more total fat, with the algae biomass being cultured in two or more types of culture medium in a sequential manner.

10
2. The process of claim 1, wherein one culture medium of the two or more culture medium contains 50 g/L of a carbon source, about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and 0.15 g/L magnésium chloride.
3. The process of claim 1, wherein one culture medium of the two or more culture

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

20 medium contains a carbon source, yeast extract and sea sait.
5. The process of claim 1, wherein the algae are cultured in a first culture medium containing glucose, yeast extract and sea sait; transferred into and incubated in a second culture medium containing glucose, yeast extract, magnésium sulfate, calcium chloride and

25 magnésium chloride, and transfened into and incubated in a third culture medium containing glucose, yeast extract, magnésium sulfate, urea, calcium chloride, magnésium chloride and monopotassium phosphate.
6. The process of claim 1, wherein the algae is Schizochytrium limacinum.
7. The process of claim 5, wherein the first culture medium contains about 50 g/L glucose, about 10 g/L yeast extract and about 4 g/L sea sait.
8. The process of claim 5, wherein the second culture medium contains about 50 g/L

35 glucose, about 7.5 g/L yeast extract, about 0.15 g/L magnésium sulfate, about 0.15 g/L calcium chloride and 0.15 g/L magnésium chloride.
9. The process of claim 5, wherein the third culture medium contains about 50 g/L glucose, about 7.5 g/L yeast extract, about 4.0 g/L magnésium sulfate, about l g/L urea, about 2 g/L calcium chloride, about 2 g/L magnésium chloride and about 0.25 g/L monopotassium phosphate.
10. The process of claim 1, wherein the culture conditions comprise running the algae culture at 30° C under airflow and agitation conditions so as to maintain dissolved oxygen at 10%.
11. An algal biomass having a total fat content of at least 67% by weight comprising about 170-250 mg/g docosahexaenoic acid (DHA) and about 150-400 mg/g palmitic acid.
12. A lipid composition prepared from the algal biomass of claim 11,
13. A food product comprising the lipid composition of claim 12.
14. A food product comprising the algal biomass of claim 11.
15. Use of an algal biomass having a total fat content of at least 67% by weight in the préparation of a biofuel.