# US 10,100,340 B2 · Method for culturing microalgae of the aurantiochytrium genus in a culture medium without chloride and without sodium for the production of DHA

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,100,340 B2
- Document ID: US10100340B2
- Status: Granted
- Kind: B2
- Application number: 15/301,091
- Filed: 2015-04-03
- Published: 2017-01-19
- Granted: 2018-10-16
- Earliest claimed: 2014-04-03
- Assignees: Fermentalg
- Applicants: Fermentalg
- Inventors: Pierre Calleja; Julien Pagliardini; Olivier Cagnac; Francois Godart
- Web page: https://patentgrep.com/patent/US10100340B2

## CPC

- C12P 7/6434: Docosahexenoic acids [DHA]
- A23D 9/00: Other edible oils or fats, e.g. shortenings or cooking oils (preservation of other edible oils A23B20/30)
- A23K 20/158: Fatty acids; Fats; Products containing oils or fats
- A23K 50/80: for aquatic animals, e.g. fish, crustaceans or molluscs
- A23L 33/115: Fatty acids or derivatives thereof; Fats or oils
- C11B 1/10: by extracting
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- 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)}
- A23D: EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS (preservation thereof A23B20/00; production, refinement or preservation of animal or vegetable fats or oils C11B, C11C)
- A23K: FODDER
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- 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)

## Abstract

A method for culturing a protist of the Aurantiochytrium mangrovei genus. The genus is characterized genetically and by virtue of the lipid profile thereof. The method makes it possible to obtain a high biomass yield and a lipid, and more particularly docosahexaenoic acid (DHA), enrichment of the protists thus cultured. The development of a culture medium allows the production, at high cell density, of a DHA-rich protest of the Aurantiochytrium mangrovei genus. The medium is chemically defined at low sodium ion (Na+) and chloride ion (Cl−) content.

## Claims

### Claim 1

A method for producing DHA in a culture of protists of the genus Aurantiochytrium comprising the following step:
a) culturing, in heterotrophic or mixotrophic conditions, one or more strains of the genus Aurantiochytrium in a chemically defined culture medium,
b) maintaining said culture for several generations, and
c) recovering the biomass thus cultured,
wherein
the one or more strains of the genus Aurantiochytrium has a genetic identity of at least 92% to sequence SEQ ID NO: 1 and
the chemically defined culture medium has less than 3.5 g/L of sodium ions and less than 1 g/L of chloride ions and has 200 mM to 500 mM organic carbon-containing substrate.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the strain of the genus Aurantiochytrium also has a genetic identity of at least 96% to sequence SEQ ID NO: 2, and/or at least 91% to sequence SEQ NO. 3 and/or at least 95% to sequence SEQ ID NO: 4.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the culture medium has less than 3.5 g/L of sodium ions and less than 200 mg/L of chloride ions.

### Claim 4 (depends on 3)

The method of claim 3, wherein the culture medium has less than 1 g/L of sodium ions.

### Claim 5 (depends on 3)

The method of claim 3, wherein the culture medium has less than 6 mg/L of sodium ions.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the medium does not contain an osmotic pressure regulatory agent.

### Claim 7 (depends on 1)

The method according to claim 1, further comprising the steps of:
d) recovering the lipids of the strains, and optionally,
e) extracting the DHA (docosahexaenoic acid).

### Claim 8 (depends on 1)

The method according to claim 1, wherein the culture medium consists of:
Ingredients
Concentration
KCl
0.05-5
g/L
H3BO3
0.01-0.3
g/L
MgSO4, 7H2O
2-10
g/L
CaCl2, 2H2O
0.2-0.9
g/L
KNO3
0.01-0.06
g/L
KH2PO4, 7H2O
0.2-1
g/L
Na2EDTA, 2H2O
0.001-0.005
g/L
ZnSO4•7H2O
0.01-0.1
mg/L
CoCl2•6H2O
0.01-0.1
mg/L
MnCl2•4H2O
0.05-1
mg/L
Na2MoO4, 2H2O
0.0005-0.1
mg/L
Na2SeO3
0.01-0.5
mg/L
NiSO4•6H2O
0.5-5
mg/L
CuSO4•5H2O
0.0025-1
mg/L
EDTA—Fe
10-50
mg/L
Glucose
20-60
g/L
(NH4)2SO4
2-9
g/L
Thiamine
1-50
mg/L
Vitamin B12
0.025-5
mg/L
Pantothenate
0.1-25
mg/L.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the biomass derived from step b) represents at least 100 g/L of dry matter.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the DHA concentration at the conclusion of step b) represents at least 15 g/L.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the DHA contained in the biomass at the conclusion of step b) represents more than 30% of the total lipids.

### Claim 12 (depends on 1)

The method according to claim 1, wherein it has a DHA productivity of at least 0.1 g/L/h.

### Claim 13 (depends on 1)

The method according claim 1, wherein said organism of the genus Aurantiochytrium corresponds to strain FCC 1324, deposited with the CCAP (Culture Collection of Algae and Protozoa), under accession number CCAP 4062/1.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the medium does not contain an osmotic pressure regulatory agent selected among the group consisting of mannitol, sorbitol, polyethylene glycol and sucrose.