Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

• US 9,848,623 B2
• Filed: 01/03/2012
• Issued: 12/26/2017
• Est. Priority Date: 01/28/2000
• Status: Expired due to Fees

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: Bailey, Richard B., Dimasi, Don, Hansen, John M., Mirrasoul, Peter J., Ruecker, Craig M., Veeder, III, George T., Kaneko, Tatsuo, Barclay, William R.
• Primary Examiner(s): Afremova, Vera
• Application Number:
  US13/342,623
• Publication Number:
  US 20120178135A1
• Time in Patent Office: 2,184 Days
• Field of Search: None
• CPC Class Codes: A23D 9/00 Other edible oils or fats, e.g. shortenings, cooking oils
• CPC Class Codes: A23K 20/158 Fatty acids; Fats; Products containing oils or fats
• CPC Class Codes: A23K 50/80 for aquatic animals, e.g. fish, crustaceans or molluscs
• CPC Class Codes: A23L 29/065 Microorganisms addition of bacteria for nutritional purposes A23L33/135
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A61P 3/02 Nutrients, e.g. vitamins, minerals
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C12N 1/00 Microorganisms, e.g. protozoa; Compositions thereof medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12P 7/6409 Fatty acids
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: Y10S 435/911 using fungi

Abstract

The present invention provides a process for growing eukaryotic microorganisms which are capable of producing lipids, in particular lipids containing polyenoic fatty acids. The present invention also provides a process for producing eukaryotic microbial lipids.

Claims

1. A process for producing microbial lipids comprising conducting a fermentation of microorganisms of the order Thraustochytriales capable of producing at least 15% of lipids as highly unsaturated fatty acids having at least four unsaturated carbon-carbon bonds in a fermentation medium comprising a carbon source and a limiting nutrient source, the process comprising:

a) providing the fermentation medium comprising the carbon source and limiting nutrient source;

b) adding the microorganisms to the fermentation medium;

c) adding an additional amount of the carbon source and limiting nutrient source to a fermentation medium to increase the biomass density of the fermentation medium to at least 100 g/L on a dry cell weight basis; and

d) adding an additional amount of the carbon source while limiting the amount of the limiting nutrient source added to the formulation medium to induce lipid production by the microorganisms.

2. The process of claim 1, further comprising e) recovering at least some of the lipids from the microorganisms.

3. The process of claim 1, wherein the fermentation of the microorganisms produces lipids containing highly unsaturated fatty acids when the biomass density of the fermentation medium is at least 150 g/L on a dry cell weight basis.

4. The process of claim 1, wherein the process produces lipids at an average rate of at least 0.5 grams lipids per liter of fermentation medium per hour fermentation.

5. The process of claim 1, wherein the microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

6. The process of claim 1, wherein the process produces on average at least 0.2 grams docosahexaenoic acid per liter of fermentation medium per hour fermentation.

7. The process of claim 1, wherein the microorganisms produce lipids containing highly unsaturated fatty acids when the biomass density is at least 100 g/L on a dry cell weight basis.

8. The process of claim 1, wherein the highly unsaturated fatty acids are selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid and mixtures thereof.

9. The process of claim 8, wherein the highly unsaturated fatty acids are docosahexaenoic acid.

10. The process of claim 1, wherein at least 25% of the lipids is docosahexaenoic acid.

11. The process of claim 1, wherein the process produces lipids at an average rate of at least 0.5 g/L/hr and wherein at least 25% of the lipids is docosahexaenoic acid.

12. The process of claim 1, wherein the carbon source comprises a carbohydrate.

13. The process of claim 1, wherein the limiting nutrient source comprises a nitrogen source.

14. The process of claim 1, wherein the pH of the fermentation medium is about pH 5 to about pH 11.

15. The process of claim 1, wherein the fermentation medium is at a temperature of at least 20°

C.

16. The process of claim 1, wherein the microorganisms produce highly unsaturated fatty acids under aerobic conditions.

17. The process of claim 1, wherein dissolved oxygen in the fermentation medium is controlled.

18. The process of claim 1, further comprising:

removing water from the fermentation medium to provide dry microorganisms prior to recovering the lipids from the microorganisms, wherein at least 15% of the lipids are highly unsaturated fatty acids.

19. The process of claim 18, wherein the water is removed from the fermentation medium by evaporation without prior centrifugation to provide dry microorganisms prior to recovering the lipids from the microorganisms, wherein at least 15% of the microbial lipids are highly unsaturated fatty acids.

20. A process for growing microorganisms of the order Thraustochytriales by conducting a fermentation of the microorganisms in a fermentation medium comprising a carbon source, and a limiting nutrient source the process comprising:

a) providing the fermentation medium comprising the carbon source and the limiting nutrient source;

b) adding the microorganisms to the fermentation medium;

c) adding an additional amount of the carbon source and limiting nutrient source to the fermentation medium to increase the biomass density of the fermentation medium to at least 100 g/L on a dry cell weight basis, wherein the additional amount of the carbon source and the limiting nutrient source are added at a rate sufficient to increase the biomass density of the fermentation medium to at least 100 g/L;

d) adding an additional amount of the carbon source while reducing the amount of limiting nutrient source added to induce lipid production by the microorganisms; and

e) recovering the lipids from the fermentation medium,

wherein the primary use of the carbon source by the microorganisms is to produce the lipids after induction of lipid production;

wherein the fermentation of the microorganisms when the biomass density is at least 100 g/L on a dry cell weight basis produces lipids containing polyunsaturated fatty acids;

wherein the lipids are at least 20% of the biomass when the biomass density is at least 100 g/L;

wherein the process produces lipids at least at an average rate of 0.5 g/L/hr; and

wherein at least about 15% of the total lipids produced by the microorganisms are polyunsaturated lipids.