# US 9,434,898 B2 · Algal lipid compositions and methods of preparing and utilizing the same

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- Number: US 9,434,898 B2
- Document ID: US9434898B2
- Status: Granted
- Kind: B2
- Application number: 13/548,871
- Filed: 2012-07-13
- Published: 2013-01-17
- Granted: 2016-09-06
- Earliest claimed: 2011-07-13
- Assignees: Alltech, Inc.
- Applicants: Kyle A. Raney; Rebecca A. Timmons
- Inventors: Kyle A. Raney; Rebecca A. Timmons
- Web page: https://patentgrep.com/patent/US9434898B2

## CPC

- C10L 1/026: {for compression ignition}
- C11B 1/00: Production of fats or fatty oils from raw materials
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12P 7/64: Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P 7/6463: obtained from glyceride producing microorganisms, e.g. single cell oil
- C10L 2200/0476: Biodiesel, i.e. defined lower alkyl esters of fatty acids first generation biodiesel
- C10L 2200/0484: Vegetable or animal oils
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C10L: FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G OR C10K; LIQUIFIED PETROLEUM GAS; USE OF ADDITIVES TO FUELS OR FIRES; FIRE-LIGHTERS
- C11B: PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES (drying-oils C09F)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## 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.

## Claims

### Claim 1

A process of making an algal biomass comprising at least 67% total fat comprising culturing an algae in two or more types of culture medium sequentially, and wherein said culturing comprises:
culturing the algae in a culture medium comprising a carbon source, yeast extract, a magnesium source, and a calcium source; and subsequently, culturing the algae in a culture medium comprising a carbon source, a yeast extract, a nitrogen source, a phosphate source, a magnesium source, a calcium source, and 4 g/L or less of sodium chloride, wherein the nitrogen and phosphate are in a ratio of 50:1 to 4:1, and wherein the type of algae is selected from the group consisting of Thraustochytrium, Schizochytrium, and Aurantiochytrium.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

The process of claim 7, wherein the carbon source is a sugar.

### Claim 9 (depends on 8)

The process of claim 8, wherein the sugar is glucose.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 11)

The process of claim 11, wherein the fed-batch feed comprises urea and monopotassium phosphate.

### Claim 13 (depends on 11)

The process of claim 11, wherein the algal biomass is harvested from the culture medium between 12-24 hours after cessation of the fed-batch process.

### Claim 14 (depends on 13)

The process of claim 13, wherein the algal biomass is harvested from the culture medium after all of the nutrients have been removed/consumed from the medium.

### Claim 15 (depends on 13)

The process of claim 13, wherein the algal biomass is harvested via centrifugation of the culture medium comprising the algal biomass.

### Claim 16 (depends on 15)

The process of claim 15, wherein the culture medium is chilled prior to harvesting the algal biomass.

### Claim 17 (depends on 16)

The process of claim 16, wherein the culture medium is chilled to between about 5 and 25° C.

### Claim 18 (depends on 1)

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

### Claim 19 (depends on 10)

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

### Claim 20 (depends on 1)

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%.

### Claim 21 (depends on 1)

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

### Claim 22 (depends on 1)

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

### Claim 23 (depends on 1)

The process of claim 1, wherein the nitrogen source is at least one of urea, peptone, malt extract, meat extract, casamino acid, corn steep liquor, sodium glutamate, ammonium acetate, ammonium sulfate, ammonium chloride, or ammonium nitrate.