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

• US 6,607,900 B2
• Filed: 01/26/2001
• Issued: 08/19/2003
• Est. Priority Date: 01/28/2000
• Status: Expired due to Term

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Boulder Corp. (DSM-Firmenich AG)
• Inventors: Bailey, Richard B., Kaneko, Tatsuo, DiMasi, Don, Hansen, Jon M., Mirrasoul, Peter J., Barclay, William R., Ruecker, Craig M., Veeder, George T. III
• Primary Examiner(s): Marx, Irene
• Assistant Examiner(s): Afremova, Vera
• Application Number:
  US09/771,352
• Publication Number:
  US 20010046691A1
• Time in Patent Office: 935 Days
• Field of Search: 435/134, 435/254.1, 435/911
• 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 producing lipids containing polyenoic fatty acids from microorganisms of the order Thraustochytriales that produce at least about 20% of their dry cell weight as lipids, comprising adding to a fermentation medium comprising said microorganisms a non-alcoholic carbon source and a nitrogen source at a rate sufficient to increase the biomass density of said fermentation medium to at least about 100 g dry cell weight per liter, wherein said process Comprises a biomass density increasing stage and a production stage and wherein the level of dissolved oxygen present in said fermentation medium during said biomass density increasing stage is at least about 4% of saturation in said fermentation medium and wherein the level of dissolved oxygen present in said fermentation medium during said production stage is less than about 3% of saturation in said fermentation medium.

2. The process of claim 1, wherein the amount of dissolved oxygen in said fermentation medium during said production stage is less than about 1% of saturation.

3. The process of claim 1, wherein said non-alcoholic carbon source comprises a carbohydrate.

4. The process of claim 1, wherein said nitrogen source comprises an inorganic ammonium salt.

5. The process of claim 4, wherein said nitrogen source comprises ammonium hydroxide.

6. The process of claim 5, wherein pH of the fermentation medium is controlled by said nitrogen source.

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

C.

8. The process of claim 1, wherein said process produces lipids at a rate of at least about 0.5 g/L/hr.

9. The process of claim 8, wherein at least about 15% by weight of said lipids are polyunsaturated lipids.

10. The process of claim 8, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of the weight of said lipids.

11. The process of claim 8, wherein at least about 25% of the weight of said lipids is docosahexaenoic acid.

12. The process of claim 1, wherein said microorganisms are grown in a fed-batch process.

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

14. A process for growing microorganisms of the order Thraustochytriales that produce at least about 20% of their dry cell weight as lipids, said process comprising adding to a fermentation medium comprising said microorganisms a carbon source and a nitrogen source at a rate sufficient to increase the biomass density of said fermentation medium to at least about 100 g dry cell weight per liter, wherein said process produces at least about 0.5 g/L/hr of lipids, and wherein at least about 15% of the weight of the total lipids produced by said microorganisms are polyunsaturated lipids, wherein said process comprises a biomass density increasing stage and a production stage and wherein the level of dissolved oxygen present in said fermentation medium during said biomass density increasing stage is at least about 4% of saturation in said fermentation medium and wherein the level of dissolved oxygen present in said fermentation medium during said production stage is less than about 3% of saturation in said fermentation medium.

15. The process of claim 14, wherein the dissolved oxygen level in the production stage is less than about 1% of saturation.

16. The process of claim 14, wherein said carbon source is a non-alcohol carbon source.

17. The process of claim 14, wherein said nitrogen source comprises an inorganic ammonium salt.

18. The process of claim 17, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

19. The process of claim 14, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of the weight of said lipids.

20. The process of claim 14, wherein at least about 25% of the weight of said lipids is docosahexaenoic acid.

21. A process for producing eukaryotic microbial lipids comprising:

(a) growing eukaryotic microorganisms of the order Thraustochytriales in a fermentation medium to increase the biomass density of said fermentation medium to at least about 100 g dry cell weight per liter;

wherein the level of dissolved oxygen present in said fermentation medium is at least about 4% of saturation in said fermentation medium;

(b) providing a fermentation condition sufficient to allow said microorganisms to produce said lipids, wherein said condition comprises reducing the amount of dissolved oxygen during lipid production to about 3% of saturation or less; and

(c) recovering said lipids, wherein greater than about 15% of the weight of said lipids are polyunsaturated lipids.

22. The process of claim 21, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is less than about 1% of saturation.

23. The process of claim 21, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

24. The process of claim 21, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of weight of said microbial lipids.

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

26. The process of claim 21, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

27. The process of claim 21, wherein said lipid recovery step comprises:

(d) removing water from said fermentation medium to provide dry microorganisms; and

(e) isolating said lipids from said dry microorganisms.

28. The process of claim 27, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.

29. The process of claim 21, wherein said microorganism growing step comprises adding a carbon source and a nitrogen source.

30. The process of claim 29, wherein said carbon source is non-alcoholic.

31. The process of claim 30, wherein said carbon source comprises a carbohydrate.

32. The process of claim 29, wherein said nitrogen source comprises an inorganic ammonium salt.

33. The process of claim 29, wherein said nitrogen source comprises ammonium hydroxide.

34. A process for producing lipids with eukaryotic microorganisms of the order Thraustochytriales comprising:

(a) adding a non-alcoholic carbon source and a nitrogen source to a fermentation medium comprising said microorganisms to increase the density of said microorganisms wherein the level of dissolved oxygen present in said fermentation medium is at least about 4% of saturation in said fermentation medium;

(b) providing conditions sufficient for said microorganisms to produce said microbial lipids, wherein said conditions comprise reducing the amount of dissolved oxygen during lipid production to about 3% of saturation or less; and

(c) recovering said microbial lipids,

wherein at least about 15% of the weight of said microbial lipids are polyunsaturated lipids.

35. The process of claim 34, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is about 1% of saturation or less.

36. The process of claim 34, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

37. The process of claim 34, wherein said process produces said microbial lipids at a rate of at least about 0.5 g/L/hr.

38. The process of claim 37, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of the weight of said microbial lipids.

39. The process of claim 34, wherein at least about 25% of the weight of said lipids is docosahexaenoic acid.

40. The process of claim 34, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

41. The process of claim 34, wherein said lipid recovery step comprises:

(d) removing water from said fermentation medium to provide dry microorganisms; and

(e) isolating said lipids from said dry microorganisms.

42. The process of claim 41, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.

43. The process of claim 34, wherein said non-alcoholic carbon source comprises a carbohydrate.

44. The process of claim 34, wherein said nitrogen source comprises an inorganic ammonium salt.

45. The process of claim 34, wherein said nitrogen source comprises ammonium hydroxide.

46. A process for producing eukaryotic microbial lipids comprising:

(a) adding a carbon source and a nitrogen source to a fermentation medium comprising eukaryotic microorganisms of the order Thraustochytriales and providing conditions sufficient to maintain a dissolved oxygen level of at least about 4% of saturation in said fermentation medium to produce a biomass density of at least about 100 g dry cell weight per liter;

(b) providing conditions sufficient to maintain the dissolved oxygen level of about 1% of saturation or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids; and

(c) recovering said microbial lipids,

wherein at least about 15% of the weight of said microbial lipids are polyunsaturated lipids.

47. The process of claim 46, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

48. The process of claim 46, wherein at least about 20% of the weight of said microbial lipids are omega-3 and/or omega-6 fatty acids.

49. The process of claim 46, wherein at least about 25% of the weight of said microbial lipids are docosahexaenoic acid.

50. The process of claim 46, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

51. The process of claim 46, wherein said lipid recovery step comprises:

(d) removing water from said fermentation medium to provide dry microorganisms; and

(e) isolating said lipids from said dry microorganisms.

52. The process of claim 51, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.

53. A process for producing eukaryotic microbial lipids comprising:

(a) adding a carbon source and a nitrogen source to a fermentation medium comprising eukaryotic microorganisms of the order Thraustochytriales and providing conditions sufficient to maintain a dissolved oxygen level of at least about 4% of saturation in said fermentation medium sufficient to increase biomass density of said fermentation medium;

(b) reducing the dissolved oxygen level to about 3% of saturation or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids; and

(c) recovering said microbial lipids;

wherein at least about 15% by weight of said microbial lipids are polyunsaturated lipids.

54. The process of claim 53, wherein step (b) comprises providing conditions sufficient to maintain a dissolved oxygen level of about 1% of saturation or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids.

55. A process for producing eukaryotic microbial lipids comprising:

(a) adding a carbon source and a nitrogen source to a fermentation medium comprising eukaryotic microorganisms of the order Thraustochytriales and providing conditions sufficient to maintain a dissolved oxygen level of at least about 4% of saturation in said fermentation medium sufficient to increase biomass density of said fermentation medium to at least 100 g dry cell weight per liter;

(b) reducing the dissolved oxygen level to about 3% of saturation or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids; and

(c) recovering said microbial lipids.

56. The process of claim 55, wherein step (b) comprises providing conditions sufficient to maintain a dissolved oxygen level of about 1% of saturation or less in said fermentation medium and providing conditions sufficient to allow said microorganisms to produce said lipids.