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

• US 20030180898A1
• Filed: 02/21/2003
• Published: 09/25/2003
• Est. Priority Date: 01/28/2000
• Status: Abandoned Application

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: Martek Biosciences Boulder Corp. (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.
• Application Number:
  US10/371,394
• Publication Number:
  US 20030180898A1
• Time in Patent Office: Days
• 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 eukaryotic microorganisms capable of producing at least about 20% of their biomass 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/L.

2. The process of claim 1, wherein said process comprises a biomass density increasing stage and a production stage, wherein said biomass density increasing stage comprises adding said carbon source and said nitrogen source and said production stage comprises adding said carbon source without adding said nitrogen source to induce nitrogen limiting conditions which induces lipid production.

3. The process of claim 2, wherein the amount of dissolved oxygen present in said fermentation medium during said production stage is lower than the amount of dissolved oxygen present in said fermentation medium during said biomass density increasing stage.

4. The process of claim 3, wherein the amount of dissolved oxygen in said fermentation medium during said biomass density increasing stage is at least about 4%.

5. The process of claim 4, wherein the amount of dissolved oxygen in said fermentation medium during said production stage is less than about 1%.

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

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

8. The process of claim 7, wherein said nitrogen source comprises ammonium hydroxide.

9. The process of claim 8, wherein pH of the fermentation medium is controlled by said nitrogen source.

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

C.

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

12. The process of claim 11, wherein at least about 15% of said lipids are polyunsaturated lipids.

13. The process of claim 11, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said lipids.

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

15. The process of claim 1, wherein said microorganisms are selected from the group consisting of algae, fungi, protists, and mixtures thereof, wherein said microorganisms are capable of producing polyenoic fatty acids or other lipids which requires molecular oxygen for their synthesis.

16. The process of claim 15, wherein said microorganisms are capable of producing lipids at a fermentation medium oxygen level of about less than 3% of saturation.

17. The process of claim 15, wherein said microorganisms are grown in a fed-batch process.

18. The process of claim 17 further comprising maintaining an oxygen level of less than about 3% of saturation in said fermentation medium during second half of said fermentation process.

19. The process of claim 1, wherein said microorganisms are algae.

20. The process of claim 19, wherein said microorganisms are of the order Thraustochytriales.

21. The process of claim 20, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

22. A process for growing eukaryotic microorganisms capable of producing at least about 20% of their biomass 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/L, wherein said process produces at least about 0.5 g/L/hr of lipids, and wherein at least about 15% of the total lipids produced by said microorganisms is polyunsaturated lipids.

23. The process of claim 22, wherein said process further comprises:

(a) a biomass density increasing stage, wherein the dissolved oxygen level in the fermentation medium is at least about 4%; and

(b) a high lipid production stage, wherein the dissolved oxygen level in the fermentation medium is less than about 1%.

24. The process of claim 22, wherein said carbon source is a non-alcohol carbon source.

25. The process of claim 22, wherein said nitrogen source comprises an inorganic ammonium salt.

26. The process of claim 22, wherein said microorganisms are algae.

27. The process of claim 26, wherein said microorganisms are of the order Thraustochytriales.

28. The process of claim 27, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

29. The process of claim 22, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said lipids.

30. The process of claim 22, wherein at least about 25% of said lipids is docosahexaenoic acid.

31. A process for growing eukaryotic microorganisms 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/L, wherein said microorganisms are selected from the group consisting of algae, fungi, protists and mixtures thereof, and wherein said microorganisms are capable of producing at least about 20% of their biomass as lipids comprising polyenoic fatty acids.

32. The process of claim 31, wherein said process produces microbial lipids at a rate of least about 0.5 g/L/hr.

33. The process of claim 32, wherein greater than about 15% of said lipids is polyunsaturated lipids.

34. The process of claim 32, wherein the total amount of omega-3 and omega-6 polyunsaturated fatty acids is at least about 20% of said lipids.

35. The process of claim 32, wherein at least about 25% of said lipids is docosahexaenoic acid.

36. The process of claim 31, wherein said process produces at least about 0.2 g/L/hr of docosahexaenoic acid.

37. The process of claim 31, wherein said carbon source is non-alcoholic.

38. The process of claim 37, wherein said carbon source is a carbohydrate.

39. The process of claim 31, wherein said nitrogen source comprises an inorganic ammonium salt.

40. The process of claim 39, wherein said nitrogen source comprises ammonium hydroxide.

41. A process for producing eukaryotic microbial lipids comprising:

(a) growing eukaryotic microorganisms in a fermentation medium to increase the biomass density of said fermentation medium to at least about 100 g/L;

(b) providing a fermentation condition sufficient to allow said microorganisms to produce said lipids; and

(c) recovering said lipids,

wherein greater than about 15% of said lipids are polyunsaturated lipids.

42. The process of claim 41, wherein a dissolved oxygen level in said fermentation medium during said microorganism growing step is higher than the dissolved oxygen level in said fermentation medium during said lipid producing step.

43. The process of claim 42, wherein the dissolved oxygen level in said fermentation medium during said microorganism growing step is at least about 4%.

44. The process of claim 42, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is less than about 1%.

45. The process of claim 41, wherein said microorganisms are algae.

46. The process of claim 45, wherein said microorganisms are of the order Thraustochytriales.

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 41, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said microbial lipids.

49. The process of claim 41, wherein at least about 25% of said microbial lipids is docosahexaenoic acid.

50. The process of claim 41, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

51. The process of claim 41, 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. The process of claim 41, wherein said microorganism growing step comprises adding a carbon source and a nitrogen source.

54. The process of claim 53, wherein said carbon source is non-alcoholic.

55. The process of claim 54, wherein said carbon source comprises a carbohydrate.

56. The process of claim 53, wherein said nitrogen source comprises an inorganic ammonium salt.

57. The process of claim 53, wherein said nitrogen source comprises ammonium hydroxide.

58. A process for producing eukaryotic microbial lipids comprising:

(a) adding a non-alcoholic carbon source and a nitrogen source to a fermentation medium comprising said microorganisms;

(b) providing conditions sufficient for said microorganisms to produce said microbial lipids; and

(c) recovering said microbial lipids,

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

59. The process of claim 58, wherein the dissolved oxygen level in said fermentation medium during a biomass density increasing step is higher than the dissolved oxygen level in said fermentation medium during said lipid producing step.

60. The process of claim 59, wherein the dissolved oxygen level in said fermentation medium during said biomass density increasing step is at least about 4%.

61. The process of claim 59, wherein the dissolved oxygen level in said fermentation medium during said lipid producing step is about 1% or less.

62. The process of claim 58, wherein said microorganisms are selected from the group consisting of algae, protists, fungi and mixtures thereof.

63. The process of claim 62, wherein said microorganisms are algae.

64. The process of claim 63, wherein said microorganisms are of the order Thraustochytriales.

65. The process of claim 64, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

66. The process of claim 58, wherein said process produces said microbial lipids at a rate of at least about 0.5 g/L/hr.

67. The process of claim 66, wherein the total amount of omega-3 and omega-6 fatty acids is at least about 20% of said microbial lipids.

68. The process of claim 58, wherein at least about 25% of said lipids is docosahexaenoic acid.

69. The process of claim 58, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

70. The process of claim 58, 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.

71. The process of claim 70, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.

72. The process of claim 58, wherein said non-alcoholic carbon source comprises a carbohydrate.

73. The process of claim 58, wherein said nitrogen source comprises an inorganic ammonium salt.

74. The process of claim 58, wherein said nitrogen source comprises ammonium hydroxide.

75. A process for producing eukaryotic microbial lipids comprising:

(a) adding a carbon source and a nitrogen source to a fermentation medium comprising eukaryotic microorganisms and providing conditions sufficient to maintain a dissolved oxygen level of at least about 4% in said fermentation medium to produce a biomass density of at least about 100 g/L;

(b) providing conditions sufficient to maintain the dissolved oxygen level of about 1% 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 said microbial lipids are polyunsaturated lipids.

76. The process of claim 75, wherein said microorganisms are selected from the group consisting of fungi, algae, protists and mixtures thereof.

77. The process of claim 76, wherein said microorganisms are algae.

78. The process of claim 77, wherein said microorganisms are of the order Thraustochytriales.

79. The process of claim 78, wherein said microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

80. The process of claim 75, wherein at least about 20% of said microbial lipids are omega-3 and/or omega-6 fatty acids.

81. The process of claim 75, wherein at least about 25% of said microbial lipids are docosahexaenoic acid.

82. The process of claim 75, wherein said process produces docosahexaenoic acid at a rate of at least about 0.2 g/L/hr.

83. The process of claim 75, 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.

84. The process of claim 83, wherein said water removal step comprises contacting said fermentation medium directly on a drum-dryer without prior centrifugation.