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

• US 7,579,174 B2
• Filed: 02/10/2006
• Issued: 08/25/2009
• Est. Priority Date: 01/28/2000
• Status: Expired due to Fees

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Corporation (DSM-Firmenich AG)
• Inventors: Barclay, William R., Bailey, Richard B., Kaneko, Tatsuo, DiMasi, Don, Ruecker, Craig M., Veeder, George T. III, Hansen, Jon M., Mirrasoul, Peter J.
• Primary Examiner(s): Afremova; Vera
• Application Number:
  US11/352,421
• Publication Number:
  US 20060286648A1
• Time in Patent Office: 1,292 Days
• Field of Search: None
• US Class Current: 435/134
• 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 growing microorganisms of the order Thraustochytriales and producing at least about 20% of their biomass as lipids comprising conducting a fermentation of the microorganisms in a medium comprising a carbon source, microorganisms and a limiting nutrient source:

wherein earlier during the fermentation the dissolved oxygen content of the fermentation medium is at least about 4% of saturation and later during the fermentation the dissolved oxygen content of the fermentation medium is less than about 3% of saturation;

wherein the fermentation of the microorganisms in the fermentation medium increases the biomass density of the fermentation medium when the carbon source and limiting nutrient source are present in the fermentation medium and when the dissolved oxygen content of the fermentation medium is at least about 4% of saturation;

wherein the fermentation of the microorganisms in the fermentation medium produces lipids containing polyenoic fatty acids when the dissolved oxygen content of the fermentation medium is less than about 3% of saturation;

wherein the process produces at least an average rate of about 0.5 grams of lipids per liter of fermentation medium per hour of the fermentation; and

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

2. The process of claim 1, wherein the process is a fed-batch process.

3. The process of claim 1, wherein the fermentation of the microorganisms in the fermentation medium produces lipids containing polyenoic fatty acids when the dissolved oxygen content of the fermentation medium is less than about 1% of saturation.

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

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

6. The process of claim 1, wherein the process is a batch or continuous process.

7. The process of claim 1, wherein the dissolved oxygen content of the fermentation medium is less than about 3% of saturation when carbon source is added with no or little additional limiting nutrient source to induce nutrient limiting conditions which induces lipid production.

8. The process of claim 1, wherein the fermentation of the microorganisms in the fermentation medium produces lipids containing polyunsaturated lipids when the dissolved oxygen content of the fermentation medium is less than about 1% of saturation.

9. The process of claim 1, wherein the process is conducted for at least 90 hours, wherein the dissolved oxygen level in the fermentation medium is maintained at about 8% during the first 24 hours, about 4% from the 24th hour to the 40^th hour, and about 0.5% or less from the 40^th hour to the end of the process.

10. The process of claim 1, wherein the dissolved oxygen level in the fermentation medium is maintained at about 0.5% or less after 40 hours of the process until the end of the process.

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

12. The process of claim 1, wherein the limiting nutrient source comprises a nutrient source selected from the group consisting of nitrogen sources, phosphate sources, vitamin sources, trace metal sources, major metal sources, silica sources and mixtures thereof.

13. The process of claim 1, wherein the limiting nutrient source comprises a nutrient source selected from the group consisting of a trace metal source and a major metal source selected from the group consisting of sulfate and chloride salts of these metals and mixtures thereof.

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

15. The process of claim 1, wherein the limiting nutrient source comprises an inorganic ammonium salt.

16. The process of claim 1, wherein the limiting nutrient source comprises ammonium hydroxide.

17. The process of claim 1, wherein pH of the fermentation medium is controlled by a limiting nutrient source.

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

C.

19. The process of claim 1, wherein the process produces at least about 15% of the total lipids produced by the microorganisms as docosahexaenoic acid.

20. The process of claim 1, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of the lipids.

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

22. The process of claim 1, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which can be produced under aerobic conditions.

23. The process of claim 22, wherein the microorganisms produce polyenoic fatty acids or other lipids produced under aerobic conditions.

24. The process of claim 1, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which can be produced under aerobic conditions, and wherein the microorganisms are grown in a fed-batch process.

25. The process of claim 24, wherein the microorganisms produce polyenoic fatty acids or other lipids produced under aerobic conditions.

26. The process of claim 1, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which can be produced under aerobic conditions and wherein the microorganisms are grown in a fed-batch process.

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

28. The process of claim 27, wherein the dissolved oxygen in the fermentation medium is controlled by step selected from the group consisting of controlling the amount of oxygen in the head-space of the fermentor and controlling the speed at which the fermentation medium is agitated.

29. The process of claim 1, further comprising:

removing water from the fermentation medium to provide dry microorganisms; and

isolating the lipids from the dry microorganisms, wherein at least about 15% of the microbial lipids are polyunsaturated lipids.

30. The process of claim 1, wherein at least about 15% of the microbial lipids are docosahexaenoic acid.

31. The process of claim 1, further comprising:

treating the fermentation medium to permeabilize, lyse or rupture the microbial cells; and

recovering the lipids from the fermentation medium by gravity separation, with or without the aid of an agent to aid in breaking the lipid/water emulsion wherein at least about 15% of the microbial lipids are polyunsaturated lipids.

32. The process of claim 1, wherein at least about 15% of the microbial lipids are docosahexaenoic acid.

33. The process of claim 1, further comprising:

evaporating water from the fermentation medium without prior centrifugation to provide dry microorganisms; and

isolating the lipids from the dry microorganisms wherein at least about 15% of the microbial lipids are polyunsaturated lipids.

34. The process of claim 33, wherein at least about 15% of the microbial lipids are docosahexaenoic acid.

35. The process of claim 1, wherein the fermentation of the microorganisms in the fermentation medium produces lipids containing polyenoic fatty acids when the fermentation medium has a biomass density of at least about 100 g/l.

36. The process of claim 1, wherein the fermentation of the microorganisms in the fermentation medium produces lipids containing polyenoic fatty acids when the fermentation medium has a biomass density of at least about 150 g/l.

37. The process of claim 1, further comprising recovering lipids from the microorganisms.

38. The process of claim 37, wherein the lipids comprise polyunsaturated lipids.

39. The process of claim 37, wherein the lipids comprise docosahexaenoic acid.