Eicosapentaenoic acid producing microorganism, fatty acid composition and preparation method and application thereof

Abstract

The invention relates to a microorganism for producing eicosapentaenoic acid, a fatty acid composition as well as a preparation method and application thereof, in particular to an isolated microorganism as well as a strain and a mutant strain thereof, biomass, microbial oil, a composition and a culture. Methods of producing the microbial oils, biomasses, and mutants; and methods of using the isolated microorganisms, biomass, and microbial oils.

Description

[0001] This invention application is a divisional application of an invention patent application with an application date of July 20, 2012, Chinese application number “201510526715.5”, and invention name “Microorganisms producing eicosapentaenoic acid, fatty acid compositions, and production methods and uses thereof”; and 201510526715.5 is a divisional application of an invention patent application with an application date of July 20, 2012, Chinese application number “2012800359352”, international application number “PCT / US2012 / 047728”, and invention name “Microorganisms producing eicosapentaenoic acid, fatty acid compositions, and production methods and uses thereof”. Technical Field

[0002] The present invention relates to isolated microorganisms and strains and mutants thereof, biomass, microbial oils, compositions and cultures; methods for producing microbial oils, biomass and mutants; and methods for using isolated microorganisms, biomass and microbial oils. Background Art

[0003] The classification of fatty acids is based on the length of the carbon chain and the saturation characteristics. Depending on the number of carbons present in the chain, fatty acids are called short-chain, medium-chain or long-chain fatty acids. When there are no double bonds between the carbon atoms, they are called saturated fatty acids, and when double bonds are present, they are called unsaturated fatty acids. Unsaturated long-chain fatty acids are monounsaturated when only one double bond is present, and polyunsaturated when more than one double bond is present.

[0004] The classification of polyunsaturated fatty acids (PUFAs) is based on the position of the first double bond from the methyl end of the fatty acid: ω-3 (n-3) fatty acids have the first double bond on the third carbon, while ω-6 (n-6) fatty acids have the first double bond on the sixth carbon. For example, docosahexaenoic acid ("DHA") is an ω-3 long-chain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22 carbons and 6 double bonds, commonly designated as "22:6n-3". Other ω-3 LC-PUFAs include eicosapentaenoic acid ("EPA"), designated as "20:5n-3", and ω-3 docosapentaenoic acid ("DPA n-3"), designated as "22:5n-3". DHA and EPA have been referred to as "essential" fatty acids. The ω-6 LC-PUFAs include eicosatetraenoic acid ("ARA"), designated "20:4 n-6," and ω-6 docosapentaenoic acid ("DPA n-6"), designated "22:5 n-6."

[0005] Because it is located in the cell membrane, ω-3 fatty acids are important biological molecules that affect the physiological function of cells. They can regulate the production and gene expression of bioactive compounds and play the role of biosynthetic substrates. Roche, HM, Proc.Nutr.Soc.58:397-401 (1999). For example, DHA accounts for about 15% to 20% of lipids in the human cerebral cortex, 30% to 60% of lipids in the retina, is enriched in testicles and sperm, and is an important component in breast milk. Bergé, JP and Barnathan, G..Adv.Biochem.Eng.Biotechnol.96:49-125 (2005). DHA accounts for up to 97% of ω-3 fatty acids in the brain and up to 93% of ω-3 fatty acids in the retina. In addition, DHA is necessary for fetal and infant development and for maintaining adult cognitive function. Same as above. Because ω-3 fatty acids cannot be synthesized de novo in the human body, these fatty acids must be obtained from nutritional sources.

[0006] Flaxseed oil and fish oil are considered to be good dietary sources of omega-3 fatty acids. Flaxseed oil does not contain EPA, DHA, DPA or ARA, but it does contain linolenic acid (C18:3n-3), which is a structural unit that enables the body to make EPA. However, there is evidence that the rate of metabolic conversion is slow and variable, especially in those individuals with poor health. Depending on the specific species and its diet, fish oil has significant differences in the type and level of fatty acid composition. For example, compared with wild fish, fish raised in aquaculture have lower levels of omega-3 fatty acids. In addition, fish oil has the risk of containing environmental pollutants and may be accompanied by stability problems and fishy odor or taste.

[0007] Thraustochytrids are microorganisms of the order Thraustochytriales. Thraustochytrids include members of the genera Schizochytrium and Thraustochytrium and have been identified as an alternative source of ω-3 fatty acids, including DHA and EPA. See U.S. Pat. No. 5,130,242. Oils produced by these marine heterotrophic microorganisms typically have simpler polyunsaturated fatty acid profiles than corresponding fish oils or microalgae oils. Lewis, TE, Mar. Biotechnol. 1:580-587 (1999). It has been reported that strains of Thraustochytrid species produce ω-3 fatty acids that account for a high percentage of the total fatty acids produced by the organism. No. 5,130,242; Huang, J. et al., J. Am. Oil. Chem. Soc. 78:605-610 (2001); Huang, J. et al., Mar. Biotechnol. 5:450-457 (2003). However, isolated thraustochytrids vary in the types and amounts of LC-PUFAs produced, which may result in some previously described strains having undesirable levels of ω-6 fatty acids and / or exhibiting low productivity in culture. Therefore, there is a need for the isolation of microorganisms that exhibit high productivity and a desired LC-PUFA profile. Summary of the invention

[0008] Applicants have discovered that the amount of EPA and DHA produced by thraustochytrids can be regulated by changing the amount of dissolved carbon dioxide (CO2) in the aqueous phase of the fermentation broth during microbial fermentation, wherein the thraustochytrids produce biomass with at least 3% EPA. Provided herein is a method for preparing microbial biomass having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentation tank container having a fermentation broth dissolved with gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and adjusting the level of dissolved CO2 in the dissolved gas. In one embodiment, the level of dissolved CO2 can be adjusted to achieve a desired level of EPA and / or DHA in the biomass. In another embodiment, the amount of dissolved CO2 in the aqueous phase of the fermentation broth is in the range of about 38 to about 600 ppm of the total dissolved gas, and specifically in the range of about 38 to about 135 ppm of the total dissolved gas.

[0009] Also provided herein is a method for preparing microbial biomass having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermenter vessel containing gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and supplementing the gas with CO2. Supplementation refers to adding or charging CO2 to the vessel in an amount that is additional to the amount produced by cell fermentation or in an amount under ambient conditions. In one embodiment, CO2 is supplemented to the vessel to achieve a desired amount of EPA and / or DHA in the biomass.

[0010] Also provided herein is a method for preparing microbial biomass having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentor vessel to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and adjusting the amount of biomass in the vessel. In one embodiment of the invention, the biomass is adjusted to achieve a desired level of EPA or DHA in the biomass.

[0011] A method of preparing a microorganism having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentor vessel to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and regulating the pressure on the biomass, such as, but not limited to, controlling the back pressure of the vessel. In one embodiment, the pressure is adjusted to achieve a desired level of EPA or DHA in the biomass.

[0012] In another embodiment, provided herein is a method of making a microorganism having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentor vessel to produce a fermentation broth and biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and regulating the temperature in the fermentation broth. In one embodiment, the temperature is adjusted to achieve a desired level of EPA or DHA in the biomass.

[0013] In a number of embodiments, the amount of dissolved CO2 in the water phase or fermented liquid of the container can also be adjusted by increasing or decreasing the amount of CO2 in the container, thereby regulating the amount of EPA and DHA. The amount of dissolved CO2 can be adjusted by additionally adjusting the amount of fermented biomass. For example, by fermenting cells in bottles and larger fermentation containers. EPA and DHA can also be changed according to the embodiments provided herein by changing the temperature. For example, the amount of dissolved CO2 can be additionally adjusted by adjusting the temperature in the container. For example, a lower container temperature will produce a higher concentration of EPA and a lower concentration of DHA. The amount of dissolved CO2 can also be adjusted by adjusting the pressure in the container. For example, an increased pressure is likely to increase dissolved CO2, which will increase the amount of EPA in the biomass and reduce the amount of DHA. Each of the above-mentioned adjustments, such as supplementing CO2, increasing or decreasing biomass, increasing or decreasing temperature, or increasing or decreasing pressure, can be combined with any one of the other adjustments, thereby achieving the desired EPA and DHA levels in the biomass and any oil extracted by the biomass. Dissolved CO2 can also be adjusted by a change in pH.

[0014] In some embodiments, the total amount of EPA and DHA (by weight) remains relatively constant compared to the total amount of fatty acids and omega-3 fatty acids by weight.

[0015] In other embodiments, the EPA or DHA content of the biomass is measured before adjustments are made to the amount of supplemental CO2, pressure, temperature, or biomass.

[0016] While not wishing to be bound by any theory, it is speculated that the increase or decrease in the amount of EPA or DHA is directly related to the amount of CO2 dissolved in the aqueous phase of the fermentation broth, and the above adjustments to CO2, pressure and temperature will change the amount of CO2 dissolved in the biomass.

[0017] In some embodiments, the present invention provides a method for preparing a microbial biomass having an elevated concentration of EPA, the method comprising: growing the microorganism in a culture medium containing less than 0.1 mg / L vitamin B12 to produce biomass. In some embodiments, the culture medium comprises less than 0.01 mg / L vitamin B12. In some embodiments, the culture medium comprises less than 0.001 mg / L vitamin B12. In other embodiments, the culture medium comprises less than 0.0001 mg / L vitamin B12. In some embodiments, the culture medium does not comprise vitamin B12.

[0018] In some embodiments, the culture medium further comprises less than 1 g of yeast extract per 50 g of fat-free biomass. In some embodiments, the culture medium further comprises less than 0.5 g of yeast extract per 50 g of fat-free biomass. In other embodiments, the culture medium further comprises less than 0.1 g of yeast extract per 50 g of fat-free biomass.

[0019] In some embodiments, compared with the EPA concentration in the biomass obtained by the microorganism grown in the culture medium containing greater than 0.1mg / L vitamin B12, the EPA concentration has increased by at least 400%. In some embodiments, compared with the EPA concentration in the biomass of the microorganism grown in the culture medium containing greater than 0.01mg / L vitamin B12, the EPA concentration has increased by at least 300%. In other embodiments, compared with the EPA concentration in the biomass of the microorganism grown in the culture medium containing greater than 0.001mg / L vitamin B12, the EPA concentration has increased by at least 200%. In some embodiments, compared with the EPA concentration in the biomass of the microorganism grown in the culture medium containing greater than 0.0001mg / L vitamin B12, the EPA concentration has increased by at least 100%.

[0020] In some embodiments, the present invention provides a method for preparing a microbial biomass having an elevated concentration of EPA, comprising: growing the microorganism in a culture medium containing less than 0.1 mg / L cobalt to produce biomass. In some embodiments, the culture medium comprises less than 0.01 mg / L cobalt. In some embodiments, the culture medium comprises less than 0.001 mg / L cobalt. In other embodiments, the culture medium comprises less than 0.0001 mg / L cobalt. In some embodiments, the culture medium does not comprise cobalt.

[0021] In some embodiments, the microorganism is a thraustochytrid. In some embodiments, the microorganism produces at least 3% EPA by total weight of fatty acids.

[0022] In some embodiments, the culture medium has a level of dissolved CO2 of at least 5%. In some embodiments, the culture medium has a level of dissolved CO2 of at least 10%. In some embodiments, the culture medium has a level of dissolved CO2 of at least 15%.

[0023] The present invention also provides isolated biomass and microbial oil extracted from the biomass by any of the methods herein.

[0024] Specifically, the present invention relates to the following:

[0025] 1. A method for preparing microbial biomass having fatty acids and a certain concentration of EPA, comprising:

[0026] (a) fermenting the microorganism in a fermentor vessel to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and

[0027] (b) Adjusting the amount of biomass in the vessel to obtain the desired EPA level in the biomass.

[0028] 2. A method for preparing microbial biomass having fatty acids and a certain concentration of EPA, comprising:

[0029] (a) fermenting the microorganism in a fermentor vessel to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and

[0030] (b) adjusting the pressure on the biomass to obtain a desired level of EPA in the biomass.

[0031] 3. A method for preparing microbial biomass having fatty acids and a certain concentration of EPA, comprising:

[0032] (a) fermenting the microorganism in a fermentor vessel to produce a fermentation broth and a biomass, wherein the microorganism comprises a thraustochytrid that produces a biomass having at least 3% EPA by total weight of fatty acids; and

[0033] (b) adjusting the temperature in the fermentation broth to obtain a desired level of EPA in the biomass.

[0034] 4. A method for preparing microbial biomass having fatty acids and a certain concentration of EPA, comprising:

[0035] (a) fermenting the microorganism in a fermentor vessel containing gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and

[0036] (b) adjusting the CO2 level in the gas to obtain the desired EPA level in the biomass.

[0037] 5. A method for preparing microbial biomass having fatty acids and a concentration of EPA, comprising:

[0038] (a) fermenting the microorganism in a fermentor vessel to produce a fermentation broth having an aqueous phase and a biomass, wherein the aqueous phase has dissolved gases, wherein the microorganism comprises a thraustochytrid that produces a biomass having at least 3% EPA by total weight of fatty acids; and

[0039] (b) adjusting the dissolved CO2 in the dissolved gas to >2%.

[0040] 6. The method of any one of items 1 and 3-5, further comprising adjusting the pressure on the biomass to obtain a desired EPA level in the biomass.

[0041] 7. The method of any one of items 1, 2, and 4-6, further comprising adjusting the temperature in the fermentation broth to obtain a desired EPA level in the biomass.

[0042] 8. A method for increasing the concentration of EPA in a biomass of a microorganism having fatty acids and a concentration of EPA, comprising:

[0043] (a) fermenting the microorganism in a fermentor vessel containing gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids;

[0044] (b) supplementing the gas with CO2 sufficient to increase the concentration of EPA in the biomass.

[0045] 9. A method as described in claim 8, wherein the amount of CO2 is greater than or equal to 2% (molar) of the gas in the chamber and the EPA concentration in the biomass is increased to greater than about 4% by weight of the total weight of fatty acids.

[0046] 10. The method of claim 9, wherein the amount of CO2 is greater than or equal to about 5% to at most about 20% (molar) of the gas in the chamber.

[0047] 11. A method as described in claim 10, wherein the amount of CO2 is greater than or equal to about 5% to at most about 15% (molar) of the gas in the container.

[0048] 12. A method as described in claim 9, wherein the EPA concentration is increased from greater than about 4 weight % to up to about 45 weight % of the total weight of fatty acids.

[0049] 13. A method as described in claim 11, wherein the EPA concentration is increased from greater than about 4 weight % to up to about 40 weight % of the total weight of fatty acids.

[0050] 14. A method as described in claim 12, wherein the EPA concentration is increased from about 4 weight % of the total weight of fatty acids to a range of about 6 weight % to about 30 weight % of the total weight of fatty acids.

[0051] 15. A method as described in claim 9, wherein the EPA concentration is increased from about 15 weight % of the total weight of fatty acids to up to about 40 weight % of the total weight of fatty acids.

[0052] 16. A method as described in claim 9, wherein the EPA concentration is increased to greater than 20 weight % of the total weight of fatty acids.

[0053] 17. A method as described in claim 9, wherein the EPA concentration is increased from about 20 weight % to up to about 25 weight % of the total weight of fatty acids.

[0054] 18. A method for increasing the concentration of EPA in a biomass of a microorganism having fatty acids and a concentration of EPA, comprising:

[0055] (a) fermenting the microorganism in a fermenter vessel to produce biomass;

[0056] (b) providing a pressure greater than or equal to 0.5 psi above atmospheric pressure on the biomass for a time sufficient to increase the concentration of EPA in the biomass.

[0057] 19. The method of claim 18, wherein the container has a headspace pressure of from about 0.4 psi to at most about 30 psi.

[0058] 20. The method of claim 19, wherein the container has a headspace pressure of from about 1 psi to at most about 30 psi.

[0059] 21. The method of claim 20, wherein the container has a headspace pressure of from about 1 psi to at most about 20 psi.

[0060] 22. The method of claim 18, wherein the pressure is provided for a period of up to about 120 hours.

[0061] 23. A method as described in claim 2, wherein the biomass has a density greater than or equal to 10 g / l.

[0062] 24. A method as described in claim 23, wherein the biomass has a density of about 10 g / l to at most about 250 g / l.

[0063] 25. The method of claim 3, wherein the temperature is adjusted to less than about 30°C.

[0064] 26. The method of claim 25, wherein the temperature is less than about 22°C.

[0065] 27. The method of claim 26, wherein the temperature is about 21°C.

[0066] 28. Biomass obtained by the method according to any one of items 1 to 27.

[0067] 29. Oil extracted from the biomass of claim 28, wherein the biomass is produced by an isolated microorganism deposited under ATCC Accession No. PTA-10208, wherein the oil comprises fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of approximately ≥90 weight % of the total amount of the ω-3 polyunsaturated fatty acids, and the amount of EPA is from about 6 weight % to at most about 65 weight % of the total amount of EPA and DHA.

[0068] 30. The oil of claim 29, wherein the amount of EPA is from about 6 wt % to up to about 28 wt % of the total amount of EPA and DHA.

[0069] 31. The oil of claim 29, wherein the amount of EPA is from about 36 wt % to up to about 65 wt % of the total amount of EPA and DHA.

[0070] 32. The oil of claim 29, wherein the amount of EPA is from about 28 wt % to up to about 36 wt % of the total amount of EPA and DHA.

[0071] 33. Oil extracted from the biomass of claim 28, wherein the biomass is produced by an isolated microorganism deposited under ATCC Accession No. PTA-10212, wherein the oil comprises fatty acids, wherein the fatty acids further comprise DHA and EPA, and the amount of EPA is from about 15 weight % to up to about 70 weight % of the total amount of EPA and DHA.

[0072] 34. Oil extracted from the biomass of claim 28, wherein the biomass is produced by an isolated microorganism deposited under ATCC Accession No. PTA-9695, wherein the oil comprises fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about 50 to 70 weight % of the total amount of the ω-3 polyunsaturated fatty acids, and the amount of EPA is from about 5 weight % to up to about 60 weight % of the total amount of EPA and DHA.

[0073] 35. The oil of claim 34, wherein the amount of DHA and EPA is about 60 weight % of the total amount of ω-3 fatty acids.

[0074] 36. A method for preparing microbial biomass having fatty acids and a concentration of EPA, comprising:

[0075] (a) fermenting the microorganism in a fermentor vessel containing gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids;

[0076] (b) supplementing the gas with CO2.

[0077] 37. A method as described in claim 2, wherein the amount of the biomass, EPA or DHA is determined before adjusting the pressure on the biomass.

[0078] 38. A method as described in claim 3, wherein the amount of the biomass, EPA or DHA is determined before adjusting the temperature in the fermentation broth.

[0079] 39. A method as described in claim 4, wherein the amount of biomass, EPA or DHA is determined before adjusting the CO2 in the container.

[0080] 40. A method as described in claim 5, wherein the amount of biomass, EPA or DHA is determined before adjusting the dissolved CO2.

[0081] 41. A method for producing oil, the method comprising preparing the biomass as described in any one of items 1-27 and 36-40, and obtaining oil from the biomass.

[0082] 42. A method as described in claim 41, wherein the oil is extracted from the biomass.

[0083] 43. A method for preparing biomass of a microorganism having an increased concentration of EPA, the method comprising growing the microorganism in a culture medium containing less than 0.1 mg / L vitamin B12 to produce the biomass.

[0084] 44. A method as described in claim 43, wherein the culture medium contains less than 0.01 mg / L vitamin B12.

[0085] 45. A method as described in claim 44, wherein the culture medium contains less than 0.001 mg / L vitamin B12.

[0086] 46. ​​A method as described in claim 45, wherein the culture medium contains less than 0.0001 mg / L vitamin B12.

[0087] 47. A method as described in claim 46, wherein the culture medium does not contain vitamin B12.

[0088] 48. A method as described in any one of items 43 to 47, wherein the culture medium further comprises less than 1 g of yeast extract per 50 g of fat-free biomass.

[0089] 49. A method as described in any one of items 43 to 48, wherein the culture medium further comprises less than 0.5 g of yeast extract per 50 g of fat-free biomass.

[0090] 50. A method as described in any one of items 43 to 49, wherein the culture medium further comprises less than 0.1 g of yeast extract per 50 g of fat-free biomass.

[0091] 51. A method as described in any one of items 43 to 50, wherein the EPA concentration is increased by at least 400% compared to the EPA concentration in biomass obtained from microorganisms grown in a culture medium containing greater than 0.1 mg / L vitamin B12.

[0092] 52. A method as described in any one of items 44 to 50, wherein the EPA concentration is increased by at least 300% compared to the EPA concentration in biomass obtained from microorganisms grown in a culture medium containing greater than 0.01 mg / L vitamin B12.

[0093] 53. A method as described in any one of items 45 to 50, wherein the EPA concentration is increased by at least 200% compared to the EPA concentration in biomass obtained from microorganisms grown in a culture medium containing greater than 0.001 mg / L vitamin B12.

[0094] 54. A method as described in any one of items 46 to 50, wherein the EPA concentration is increased by at least 100% compared to the EPA concentration in biomass obtained from microorganisms grown in a culture medium containing greater than 0.0001 mg / L vitamin B12.

[0095] 55. A method of preparing microbial biomass having an elevated concentration of EPA, comprising growing the microorganism in a culture medium containing less than 0.1 mg / L cobalt to produce biomass.

[0096] 56. A method as described in claim 55, wherein the culture medium contains less than 0.01 mg / L cobalt.

[0097] 57. A method as described in claim 56, wherein the culture medium contains less than 0.001 mg / L cobalt.

[0098] 58. A method as described in claim 57, wherein the culture medium contains less than 0.0001 mg / L cobalt.

[0099] 59. A method as described in claim 58, wherein the culture medium does not contain cobalt.

[0100] 60. The method of any one of items 43 to 59, wherein the microorganism is a thraustochytrid.

[0101] 61. A method as described in any one of items 43 to 60, wherein the microorganism produces at least 3% EPA based on the total weight of the fatty acids.

[0102] 62. A method as described in any one of items 43 to 61, wherein the culture medium has a dissolved CO2 level of at least 5%.

[0103] 63. A method as described in any one of items 43 to 62, wherein the culture medium has a dissolved CO2 level of at least 10%.

[0104] 64. A method as described in any one of items 43 to 63, wherein the culture medium has a dissolved CO2 level of at least 15%.

[0105] 65. The isolated biomass of any one of items 43 to 64.

[0106] 66. Microbial oil extracted from the isolated biomass as described in any one of items 43 to 65.

[0107] The present invention further relates to the following:

[0108] 1. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0109] (a) fermenting the microorganism in a fermenter vessel to produce biomass, and

[0110] (b) providing a pressure greater than or equal to 0.5 psi above atmospheric pressure on the biomass for a time sufficient to increase the concentration of EPA in the biomass.

[0111] 2. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0112] (a) fermenting the microorganism in a fermentor vessel to produce a fermentation broth having an aqueous phase and a biomass, wherein the aqueous phase has dissolved gases, wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA by total weight of fatty acids; and

[0113] (b) obtaining a desired EPA level in the biomass by (i) adjusting dissolved CO2 to >2% of the dissolved gas, (ii) optionally adjusting the pressure on the biomass, and (iii) optionally adjusting the temperature in the fermentation broth.

[0114] 3. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0115] (a) fermenting the microorganism in a fermentor vessel to produce biomass, wherein the microorganism comprises a Thraustochytrid that produces biomass having at least 3% EPA by weight of total fatty acids; and

[0116] (b) obtaining a desired EPA level in said biomass by (i) adjusting the amount of said biomass in said container and (ii) optionally adjusting the pressure on said biomass.

[0117] 4. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0118] (a) fermenting the microorganism in a fermentor vessel to produce a fermentation broth and a biomass, wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA by weight of total fatty acids; and

[0119] (b) obtaining a desired level of EPA in the biomass by (i) regulating the temperature in the fermentation broth and (ii) optionally regulating the pressure on the biomass.

[0120] 5. The biomass as described in item 4, wherein the temperature is adjusted to less than about 30°C.

[0121] 6. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0122] (a) fermenting the microorganism in a fermentor vessel containing gas to produce biomass, wherein the microorganism comprises a Thraustochytrid that produces biomass having at least 3% EPA by weight of total fatty acids; and

[0123] (b) obtaining a desired EPA level in the biomass by (i) adjusting the CO2 level in the gas and (ii) optionally adjusting the pressure on the biomass.

[0124] 7. A biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising:

[0125] (a) fermenting the microorganism in a fermentor vessel containing gas to produce biomass, wherein the microorganism comprises a Thraustochytrid that produces biomass having at least 3% EPA by weight of total fatty acids; and

[0126] (b) supplementing the gas with CO2 sufficient to increase the concentration of EPA in the biomass.

[0127] 8. The biomass of claim 7, wherein the amount of CO2 is greater than or equal to 2 molar % of the gas in the chamber and the EPA concentration in the biomass is increased to greater than about 4 weight % of the total weight of fatty acids.

[0128] 9. The biomass of claim 8, wherein the EPA concentration increases from greater than about 4% to about 45% by weight of the total weight of fatty acids.

[0129] 10. Biomass of a microorganism having fatty acids and a concentration of EPA, wherein the biomass is produced by a method comprising growing the microorganism in a culture medium containing less than 0.1 mg / L vitamin B12 to produce the biomass.

[0130] 11. An oil extracted from the biomass of any one of items 1 to 10, wherein the biomass is produced by an isolated microorganism deposited under ATCC Accession No. PTA-10208, wherein the oil comprises fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of approximately ≥90 weight % of the total amount of the ω-3 polyunsaturated fatty acids, and the amount of EPA is from about 6 weight % to about 65 weight % of the total amount of EPA and DHA.

[0131] 12. The oil of claim 11, wherein the oil is a crude oil extracted from biomass without further processing.

[0132] 13. The oil of claim 11, wherein the oil is a refined oil.

[0133] 14. A feed product comprising the biomass of any one of items 1-10 or comprising the oil of any one of items 11-13.

[0134] 15. The feed product according to claim 14, wherein the feed product is a feed or a feed supplement.

[0135] 16. The feed product of claim 14, wherein the feed product is animal feed.

[0136] 17. The feed product according to claim 14, wherein the feed product is a feed for aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Various embodiments of the present invention can be more fully understood from the following detailed description, drawings and accompanying sequence description, which form a part of this patent application.

[0138] Figure 1 The performance of PTA-9695 in a thiamine gradient is shown.

[0139] Figure 2 The performance of PTA-9695 in a vitamin B12 gradient is shown.

[0140] Figure 3 The performance of PTA-9695 in a biotin gradient is shown.

[0141] Figure 4 The performance of PTA-9695 in a calcium pantothenate gradient is shown.

[0142] Figure 5 The performance of PTA-9695 in TSFM standards is shown.

[0143] Figure 6-Figure 19 The performance of PTA-9695 in a vitamin B12 gradient at 10% CO2 is shown.

[0144] Figure 20-Figure 49 The performance of PTA-10208 in a vitamin B12 gradient at 10% CO2 is shown. DETAILED DESCRIPTION

[0145] The methods and compositions provided herein are particularly useful for thraustochytrids that produce biomass having at least 3% EPA by weight of the total fatty acids produced. Specific thraustochytrids provided herein that produce biomass having at least 3% EPA are isolated microorganisms of species deposited under ATCC Accession No. PTA-10212. The isolated microorganism Thraustochytrium sp. 27797 associated with ATCC Accession No. PTA-10212 was deposited under the Budapest Treaty on July 14, 2009 in the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, in the Patent Depository.

[0146] A specific thraustochytrid that produces biomass having at least 3% EPA is selected from the group consisting of isolated microorganisms deposited under ATCC Accession Nos. PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-10208, PTA-10209, PTA-10210, or PTA-10211.

[0147] Specific embodiments provided herein relate to isolated microorganisms comprising 18s rRNA comprising the polynucleotide sequence of SEQ ID NO:1 or a polynucleotide sequence having at least 94% identity to SEQ ID NO:1.

[0148] Specific embodiments provided herein relate to an isolated microorganism comprising an 18s rRNA polynucleotide sequence that is at least 94% identical to the 18s rRNA polynucleotide sequence of the microorganism deposited under ATCC Accession No. PTA-10212.

[0149] Specific thraustochytrids that produce biomass having at least 3% EPA provided herein relate to isolated microorganisms of the species deposited under ATCC Accession No. PTA-10208.

[0150] Specific embodiments provided herein relate to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprise greater than about 10% by weight eicosapentaenoic acid.

[0151] The specific thraustochytrids of the present invention that produce biomass with at least 3% EPA relate to isolated microorganisms having the characteristics of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprise greater than about 10% by weight of eicosapentaenoic acid. The specific thraustochytrids provided herein that produce biomass with at least 3% EPA are selected from isolated microorganisms selected from mutant strains deposited under ATCC Accession Nos. PTA-10209, PTA-10210, or PTA-10211. The microorganisms associated with ATCC Accession Nos. PTA-10209, PTA-10210, and PTA-10211 were deposited on September 25, 2009, under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.

[0152] Various embodiments provided herein relate to the above-described microorganisms, their mutants, and microorganisms identified in US Patent Application No. 12 / 729,013, which is incorporated herein by reference in its entirety.

[0153] One embodiment provided herein relates to an isolated microorganism that produces a triglyceride fraction, wherein the eicosapentaenoic acid content of the triglyceride fraction is at least about 12% by weight.

[0154] One embodiment provided herein relates to an isolated biomass, wherein at least about 20% by weight of the cell dry weight of the biomass is fatty acids, wherein more than about 10% by weight of the fatty acids are eicosapentaenoic acid, and wherein the fatty acids comprise less than about 5% by weight of eicosatetraenoic acid and docosapentaenoic acid n-6, respectively. In some embodiments, at least about 25% by weight of the fatty acids are docosahexaenoic acid.

[0155] In some embodiments, the invention relates to isolated biomass comprising triacylglycerols, wherein at least about 12% by weight of the triacylglycerols are eicosapentaenoic acid.

[0156] In some embodiments, the present invention relates to any one of the isolated biomass of the present invention, wherein the fatty acids further comprise less than about 5% by weight each of oleic acid, linoleic acid, linolenic acid, eicosenoic acid, and erucic acid.

[0157] The present invention relates to an isolated thraustochytrid microorganism or a strain derived therefrom of a thraustochytrid species deposited under ATCC Accession No. PTA-9695, wherein the total fatty acids produced by the microorganism or the strain derived therefrom comprise about 10% by weight or less of eicosapentaenoic acid. Embodiments provided herein relate to the above-mentioned microorganism or strain derived therefrom and other related microorganisms described in U.S. Patent Application No. US2010 / 0239533, which is incorporated herein by reference in its entirety.

[0158] Also provided herein is a method of increasing the concentration of EPA in a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentor vessel containing a gas to produce biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and supplementing the gas with CO2 in an amount sufficient to increase the concentration of EPA in the biomass. The increase in EPA concentration can be compared to, for example, the concentration of EPA in a similarly fermented microorganism without supplementation of CO2, or to the concentration of EPA in a similarly fermented microorganism under ambient conditions.

[0159] In another embodiment, the amount of CO2 sufficient to increase the EPA concentration is greater than or equal to 2% of the total gas in the container. In another embodiment, the amount of CO2 in the container is greater than or equal to about 5% to at most about 20% of the total gas in the container. In another embodiment, the amount of CO2 in the container is greater than or equal to about 5% to at most about 15% of the total gas in the container.

[0160] In another embodiment, the amount of supplemental CO2 is greater than or equal to 2% of the total gas in the container to increase the EPA concentration in the biomass to greater than about 4 weight % of the total weight of the fatty acids, more specifically greater than about 4 weight % to up to about 45 weight % of the total weight of the fatty acids, and more specifically greater than about 4 weight % to up to about 40 weight % of the total weight of the fatty acids.

[0161] In another embodiment, the amount of supplemented CO2 is enough to increase the EPA level from about 4 weight % of the total weight of the fatty acid to about 6-30 weight %. In another embodiment, the amount of provided CO2 is enough to increase the EPA concentration from about 15 weight % of the total weight of the fatty acid to about 40 weight % at most. In another embodiment, the amount of provided CO2 is enough to increase the EPA concentration to about 20 weight % greater than the total weight of the fatty acid. In another embodiment, CO2 is provided with an amount that is enough to increase the EPA concentration from about 20% to about 25% at most.

[0162] In another embodiment, provided herein is a method for increasing the concentration of EPA in the biomass of a microorganism having fatty acids and a certain concentration of EPA, comprising: fermenting a microorganism in a fermenter container to produce biomass; providing a pressure sufficient to increase the concentration of EPA in the biomass. The increase in EPA concentration can be compared with, for example, the concentration of EPA in a similarly fermented microorganism without providing pressure, or with the concentration of EPA in a similarly fermented microorganism under ambient conditions. In another embodiment, the pressure provided on the biomass is about 0.5 psi higher than atmospheric pressure. In another embodiment, the container has a head pressure (head pressure or back pressure) greater than or equal to about 0.4 psi, more specifically about 0.4 psi to at most about 30 psi, even more specifically about 1 to at most about 30 psi. In another embodiment, the container has a head pressure of about 1 to at most about 20 psi. In another embodiment, the pressure provided is provided for a time sufficient to adjust the amount of EPA in the biomass, specifically up to 120 hours.

[0163] In another embodiment, provided herein is a method for preparing a microbial biomass that produces fatty acids and a concentration of EPA, comprising fermenting a microorganism in a fermentation vessel to produce biomass at a temperature sufficient to increase the concentration of EPA in the biomass, wherein the microorganism comprises a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids. In some embodiments, the temperature sufficient to increase the level of EPA is less than about 30°C, more specifically less than or equal to about 22°C, and more specifically the temperature is less than or equal to about 21°C. The increase in EPA concentration can be compared to, for example, the concentration of EPA in a similarly fermented microorganism that is not temperature-adjusted, or to the concentration of EPA in a similarly fermented microorganism under ambient conditions.

[0164] In another embodiment, the amount of EPA produced during fermentation is changed by the method provided herein to produce biomass and extracted oil, wherein the amount of EPA provided is greater than 4 wt % of the total weight of fatty acids, specifically from greater than about 4 wt % to about 45 wt %, more specifically from greater than about 4 wt % to about 40 wt % at the most. In another embodiment, the amount of EPA produced by the method provided herein is about 6 wt % to about 30 wt % of the total weight of fatty acids. In another embodiment, the amount of EPA produced by the method provided herein is about 15 wt % to about 40 wt % of the total weight of fatty acids. In another embodiment, the amount of EPA produced by the method provided herein is greater than about 20 wt % of the total weight of fatty acids, more specifically from about 20 wt % to about 25 wt % at the most.

[0165] In another embodiment, the EPA level of the expectation provided is greater than 4 wt %, specifically from greater than about 4 wt % to about 45 wt %, more specifically from greater than about 4 wt % to about 40 wt % at the most of the total weight of the fatty acid. In another embodiment, the EPA level of the expectation produced by the method provided herein is about 6 wt % to about 30 wt % of the total weight of the fatty acid. In another embodiment, the EPA level of the expectation produced by the method provided herein is about 15 wt % to about 40 wt % of the total weight of the fatty acid. In another embodiment, the EPA level of the expectation produced by the method provided herein is greater than about 20 wt %, more specifically from about 20 wt % to about 25 wt % at the most of the total weight of the fatty acid.

[0166] A method of increasing microbial biomass having fatty acids and a concentration of EPA, comprising: fermenting a microorganism in a fermentor vessel to produce biomass, wherein the microorganism is a thraustochytrid that produces biomass having at least 3% EPA by total weight of fatty acids; and increasing the biomass in an amount sufficient to increase the concentration of EPA in the biomass. In some embodiments, the amount of biomass sufficient to increase the concentration of EPA has a density greater than or equal to 10 g / l. In some embodiments, the amount of biomass sufficient to increase the concentration of EPA has a density of about 10 g / l to at most 250 g / l. The increase in EPA concentration can be compared to, for example, the concentration of EPA in a similarly fermented microorganism in which no biomass is increased.

[0167] In some embodiments, the EPA concentration of biomass grown in a medium containing higher CO2 levels (e.g., in a container containing CO2 in an amount greater than or equal to 2%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, 5% to 20%, or 5% to 15%, of the total gas in the container) is higher than that of biomass grown in a medium containing lower CO2 levels (e.g., in a container containing CO2 in an amount greater than or equal to 2%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, or 5% to 15%, respectively, of the total gas in the container). In some embodiments, the EPA concentration in the biomass obtained from microorganisms grown in a medium containing CO2 at least 4%, or 1% to 3% (in a container containing CO2 at least 10%, 50%, 100%, 250%, 500%, 750%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900%, or 2000% higher. For example, biomass grown in a culture medium comprising elevated CO2 levels (e.g., in a container containing CO2 in an amount greater than or equal to 2%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, between 5% and 20%, or between 5% and 15% of the total gas in the container) has an EPA concentration that is at least 10%, at least 50%, at least 100%, at least 250%, at least 500%, at least 750%, at least 1000%, at least 1100%, at least 1200%, at least 1300%, at least 1400%, at least 1500%, at least 1600%, at least 1700%, at least 1800%, at least 1900%, or at least 2000% higher than the EPA concentration in biomass resulting from microorganisms grown in the container at ambient CO2 levels.

[0168] Vitamin B12 in culture medium

[0169] As used herein, the term "vitamin B12" refers to a class of chemically related compounds in naturally occurring and synthetic forms, including but not limited to vitamin B12, cobalamin, cyanocobalamin, and hydroxocobalamin. In some embodiments, the present invention provides a method for increasing the concentration of EPA in microbial biomass by growing a microorganism producing EPA in a medium with low levels of vitamin B12 or in a medium without vitamin B12. In some embodiments, the present invention provides a method for preparing a biomass of a microorganism having an increased concentration of EPA, comprising growing a microorganism in a medium containing less than 0.1 mg / L vitamin B12 to produce biomass. In some embodiments, the culture medium contains less than 0.05 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L vitamin B12 or does not contain vitamin B12.

[0170] In some embodiments, the culture medium contains less than 1 g of a vitamin B12 source (e.g., yeast extract, corn steep solids, soy flour, and other complex nitrogen sources) per 50 g of fat-free biomass. In some embodiments, the culture medium contains less than 0.8 g, less than 0.5 g, less than 0.3 g, less than 0.1 g, less than 0.05 g, or less than 0.01 g of such a vitamin B12 source per 50 g of fat-free biomass. In some embodiments, the culture medium further comprises less than 1 g of yeast extract per 50 g of fat-free biomass, 0.8 g of yeast extract per 50 g of fat-free biomass, less than 0.5 g of yeast extract per 50 g of fat-free biomass, less than 0.3 g of yeast extract per 50 g of fat-free biomass, less than 0.1 g of yeast extract per 50 g of fat-free biomass, less than 0.05 g of yeast extract per 50 g of fat-free biomass, or less than 0.01 g of yeast extract per 50 g of fat-free biomass. As used herein, the term "fat-free biomass" refers to the target fat-free cell dry weight of the microorganism after culture.

[0171] In some embodiments, biomass grown in a medium containing lower levels of vitamin B12 (e.g., in a medium containing less than 0.1 mg / L, less than 0.05 mg / L, less than 0.01 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L of vitamin B12, or no vitamin B12) has a higher EPA concentration than biomass grown in a medium containing higher levels of vitamin B12 (e.g., in a medium containing at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, respectively). In some embodiments, the EPA concentration in biomass obtained from microorganisms grown in culture medium containing at least 0.0001 mg / L, at least 0.0001 mg / L, or at least 0.00005 mg / L vitamin B12 is at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, or at least 700% higher. For example, the EPA concentration in biomass grown in a medium that does not contain vitamin B12 is at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, or at least 700% higher than the EPA concentration in biomass obtained from a microorganism grown in a medium that contains vitamin B12 (e.g., at least 0.0001 mg / L vitamin B12).

[0172] In some embodiments, biomass grown in a medium containing lower levels of vitamin B12 at ambient CO2 levels (e.g., in a medium containing less than 0.1 mg / L, less than 0.05 mg / L, less than 0.01 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L of vitamin B12, or no vitamin B12) has a higher EPA concentration than biomass grown in a medium containing higher levels of vitamin B12 at ambient CO2 levels (e.g., containing at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, respectively). In some embodiments, the EPA concentration in the biomass obtained by growing microorganisms in a medium containing at least 0.0001 mg / L, at least 0.00001 mg / L, or at least 0.00005 mg / L vitamin B12 is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in the biomass obtained by growing microorganisms in a medium containing at least 0.0001 mg / L, or at least 0.00005 mg / L vitamin B12 at ambient CO2 levels. In some embodiments, the EPA concentration in the biomass grown in a medium containing lower vitamin B12 levels at ambient CO2 levels is 100% to 700%, 150% to 650%, 200% to 600%, 250% to 550%, or 300% to 500% higher than the EPA concentration in the biomass obtained by growing microorganisms in a medium containing higher vitamin B12 levels at ambient CO2 levels. In some embodiments, the EPA concentration in biomass grown in a medium containing lower vitamin B12 levels under high CO2 levels (e.g., dissolved CO2 levels of at least 5%, at least 10%, at least 15%, or at least 20%) is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than the EPA concentration in biomass obtained from a microorganism grown in a medium containing higher vitamin B12 levels under high CO2 levels. In some embodiments, the EPA concentration in biomass grown in a culture medium containing lower vitamin B12 levels under high CO2 levels (e.g., dissolved CO2 levels of at least 5%, at least 10%, at least 15%, or at least 20%) is 5% to 200%, 10% to 175%, 15% to 150%, 20% to 125%, or 25% to 100% higher than the EPA concentration in biomass obtained from microorganisms grown in a culture medium containing higher vitamin B12 levels under high CO2 levels.For example, the EPA concentration in biomass grown in a medium without vitamin B12 at ambient CO2 levels is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in biomass obtained from microorganisms grown in a medium containing vitamin B12 (e.g., at least 0.0001 mg / L vitamin B12) at ambient CO2 levels. As another example, the EPA concentration in biomass grown in a medium without vitamin B12 at a dissolved CO2 level of at least 10% is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than the EPA concentration in biomass obtained from a microorganism grown in a medium containing vitamin B12 (e.g., at least 0.0001 mg / L vitamin B12) at a dissolved CO2 level of at least 10%.

[0173] In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.1 mg / L vitamin B12 is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the same microorganism grown in a culture medium with greater than 0.1 mg / L vitamin B12. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.01 mg / L vitamin B12 is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% compared to the same microorganism grown in a culture medium with greater than 0.01 mg / L vitamin B12. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.001 mg / L vitamin B12 increases by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with greater than 0.001 mg / L vitamin B12. In other embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.0001 mg / L vitamin B12 increases by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with greater than 0.0001 mg / L vitamin B12. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium without vitamin B12 increases by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with a certain amount of vitamin B12. Increased concentrations of EPA in biomass can be determined by growing a microorganism in a medium with a higher amount of vitamin B 12, growing the same microorganism in a medium with a lower amount of vitamin B 12, and comparing the concentrations of EPA in the biomass obtained in each culture. In this assay, the contents of the medium with the lower or higher amounts of vitamin B 12 are identical except for their vitamin B 12 levels.

[0174] In some embodiments, the EPA concentration of the biomass of a microorganism grown in a culture medium comprising at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, or at least 0.0001 mg / L vitamin B12 is at least 1 weight %, at least 2 weight %, at least 3 weight %, at least 4 weight %, or at least 5 weight % EPA of total fatty acids. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium comprising at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, or at least 0.0001 mg / L vitamin B12 is 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 2% to 50%, 2% to 40%, 2% to 30%, or 2% to 20% EPA of total fatty acids.

[0175] Cobalt in the culture medium

[0176] In some embodiments, the invention provides a method for preparing biomass of a microorganism having an elevated EPA concentration, comprising growing the microorganism in a culture medium containing less than 0.1 mg / L cobalt to produce the biomass. In some embodiments, the culture medium comprises less than 0.05 mg / L, less than 0.01 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L cobalt, or is free of cobalt.

[0177] In some embodiments, the culture medium further comprises less than 1 g of yeast extract per 50 g of fat-free biomass, less than 0.8 g of yeast extract per 50 g of fat-free biomass, less than 0.5 g of yeast extract per 50 g of fat-free biomass, less than 0.3 g of yeast extract per 50 g of fat-free biomass, less than 0.1 g of yeast extract per 50 g of fat-free biomass, less than 0.05 g of yeast extract per 50 g of fat-free biomass, or less than 0.01 g of yeast extract per 50 g of fat-free biomass.

[0178] In some embodiments, biomass grown in a medium containing lower cobalt levels (e.g., in a medium containing less than 0.1 mg / L, less than 0.05 mg / L, less than 0.01 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L of cobalt, or no cobalt) has a higher EPA concentration than a medium containing higher cobalt levels (e.g., in a medium containing at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, respectively). In some embodiments, the present invention relates to a biomass obtained by growing microorganisms in a culture medium containing at least 0.01 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, at least 0.0001 mg / L, or at least 0.00005 mg / L cobalt that has at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, or at least 700% higher EPA concentration than that obtained by growing microorganisms in a culture medium containing at least 0.01 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L cobalt. For example, the EPA concentration in biomass grown in a medium without cobalt is at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, or at least 700% higher than the EPA concentration in biomass obtained from a microorganism grown in a medium comprising cobalt (e.g., at least 0.0001 mg / L cobalt).

[0179] In some embodiments, the EPA concentration in biomass grown in a medium containing lower cobalt levels (e.g., in a medium containing less than 0.1 mg / L, less than 0.05 mg / L, less than 0.01 mg / L, less than 0.005 mg / L, less than 0.001 mg / L, less than 0.0005 mg / L, less than 0.0001 mg / L, or no cobalt) at ambient CO2 levels is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in biomass obtained from microorganisms grown in a medium containing higher cobalt levels (e.g., in a medium containing at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, at least 0.0001 mg / L, or at least 0.00005 mg / L cobalt, respectively). In some embodiments, the EPA concentration of biomass grown in a medium containing lower cobalt levels at ambient CO2 levels is 100% to 700%, 150% to 650%, 200% to 600%, 250% to 550%, or 300% to 500% higher than the EPA concentration in biomass obtained by microorganisms grown in a medium containing higher cobalt levels at ambient CO2 levels. In some embodiments, the EPA concentration of biomass grown in a medium containing lower cobalt levels at high CO2 levels (e.g., at least 5%, at least 10%, at least 15%, or at least 20% dissolved CO2 levels) is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% higher than the EPA concentration in biomass obtained by microorganisms grown in a medium containing higher cobalt levels at high CO2 levels. In some embodiments, the EPA concentration of biomass grown in a medium containing lower cobalt levels under high CO2 levels (e.g., at least 5%, at least 10%, at least 15%, or at least 20% dissolved CO2 levels) is 5% to 200%, 10% to 175%, 15% to 150%, 20% to 125%, or 25% to 100% higher than the EPA concentration in biomass obtained by microorganisms grown in a medium containing higher cobalt levels under high CO2 levels. For example, the EPA concentration of biomass grown in a medium without cobalt under ambient CO2 levels is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than in a medium containing cobalt (e.g., at least 0.0001 mg / L cobalt) under ambient CO2 levels.As another example, as another example, the EPA concentration in biomass grown in a medium without cobalt at a dissolved CO2 level of at least 10% is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% higher than the EPA concentration in biomass obtained from microorganisms grown in a medium containing cobalt (e.g., at least 0.0001 mg / L cobalt) at a dissolved CO2 level of at least 10%.

[0180] In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.1 mg / L cobalt is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with greater than 0.1 mg / L cobalt. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.01 mg / L cobalt is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with greater than 0.01 mg / L cobalt. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium with less than 0.001 mg / L cobalt is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, compared to the same microorganism grown in a culture medium with greater than 0.001 mg / L cobalt. In some embodiments, compared with the same microorganism grown in a culture medium with greater than 0.0001 mg / L cobalt, the EPA concentration in the biomass of the microorganism grown in the culture medium with less than 0.0001 mg / L cobalt increases by at least 100%, at least 200%, at least 300%, at least 400% or at least 500%. In some embodiments, compared with the same microorganism grown in a culture medium with a certain amount of cobalt, the EPA concentration in the biomass of the microorganism grown in the culture medium without cobalt increases by at least 100%, at least 200%, at least 300%, at least 400% or at least 500%. The rise of EPA concentration in the biomass can be determined by growing the microorganism in a culture medium with a higher amount of cobalt, growing the same microorganism in a culture medium with a lower amount of cobalt, and comparing the EPA concentration in the biomass obtained in each culture. In this determination, except for their cobalt levels, the contents of the culture medium with lower or higher amounts of cobalt are the same.

[0181] In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium comprising at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, or at least 0.0001 mg / L cobalt is at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% EPA by weight of total fatty acids. In some embodiments, the EPA concentration in the biomass of a microorganism grown in a culture medium comprising at least 0.1 mg / L, at least 0.05 mg / L, at least 0.01 mg / L, at least 0.005 mg / L, at least 0.001 mg / L, at least 0.0005 mg / L, or at least 0.0001 mg / L cobalt is between 1% and 50%, between 1% and 40%, between 1% and 30%, between 1% and 20%, between 2% and 50%, between 2% and 40%, between 2% and 30%, or between 2% and 20% EPA by weight of total fatty acids.

[0182] The medium containing small amounts of vitamin B12, yeast extract and / or cobalt may also contain dissolved CO2 levels of at least 5%, at least 10%, at least 15%, or at least 20%. The present invention relates to the isolated biomass of the methods disclosed herein, and to microbial oils extracted from the biomass of these methods.

[0183] The present invention relates to an isolated culture comprising any of the microorganisms of the present invention or mixtures thereof.

[0184] The present invention relates to an edible product, cosmetic or pharmaceutical composition for non-human animals or humans comprising any of the microorganisms or biomass of the present invention or a mixture thereof.

[0185] The present invention relates to a microbial oil comprising at least about 20% by weight of eicosapentaenoic acid and less than about 5% by weight of eicosapentaenoic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid and octadecatetraenoic acid, respectively. In some embodiments, the microbial oil further comprises at least about 25% by weight of docosahexaenoic acid.

[0186] The present invention is directed to a microbial oil comprising at least about 10% by weight of a triglyceride fraction, wherein at least about 12% by weight of the fatty acids in the triglyceride fraction are eicosapentaenoic acid, wherein at least about 25% by weight of the fatty acids in the triglyceride fraction are docosahexaenoic acid, and wherein less than about 5% by weight of the fatty acids in the triglyceride fraction are eicosatetraenoic acid.

[0187] The present invention relates to edible products, cosmetics or pharmaceutical compositions for non-human animals or humans, comprising any microbial oil of the present invention. In some embodiments, the edible products are infant formula milk powder. In some embodiments, infant formula milk powder is suitable for premature infants. In some embodiments, the edible products are milk, beverages, therapeutic drinks, nutritional drinks or a combination thereof. In some embodiments, the edible products are additives for non-human animals or human food. In some embodiments, the edible products are nutritional supplements. In some embodiments, the edible products are animal feed. In some embodiments, the animal feed is aquaculture feed. In some embodiments, the animal feed is livestock feed, zoo animal feed, draft animal feed, livestock feed or a combination thereof.

[0188] The present invention relates to a method for producing a microbial oil containing ω-3 fatty acids, the method comprising: cultivating any isolated microorganism of the present invention or a mixture thereof in a culture to produce an oil containing ω-3 fatty acids. In some embodiments, the method further comprises extracting the oil.

[0189] The present invention relates to a method for producing a microbial oil containing ω-3 fatty acids, the method comprising extracting the oil containing ω-3 fatty acids from any biomass of the present invention. In some embodiments, an organic solvent extraction process such as hexane extraction is used to extract the microbial oil. In some embodiments, a solvent-free extraction process is used to extract the microbial oil.

[0190] The present invention relates to microbial oil produced by the method of the present invention.

[0191] The present invention relates to a method for producing the biomass of the present invention, comprising: growing any isolated microorganism of the present invention or a mixture thereof in culture to produce the biomass.

[0192] The present invention relates to biomass produced by the method of the present invention.

[0193] The present invention relates to a method for producing the mutant strain of the present invention, which comprises: mutagenizing any microorganism of the present invention, and isolating the mutant strain.

[0194] The present invention relates to the use of any isolated microorganism, biomass or microbial oil of the present invention or a mixture thereof for the manufacture of a medicament for treating inflammation or related conditions.

[0195] The present invention relates to the use of any isolated microorganism, biomass or microbial oil of the present invention or mixtures thereof for treating inflammation or related conditions.

[0196] The present invention relates to any isolated microorganism, biomass or microbial oil of the present invention or mixtures thereof for use in treating inflammation or related conditions.

[0197] The present invention relates to a method for treating inflammation or related conditions in an individual in need thereof, comprising administering to the individual any isolated microorganism, biomass or microbial oil of the present invention or a mixture thereof and a pharmaceutically acceptable carrier.

[0198] The present invention relates to methods for producing microbial oil and biomass by the microorganisms of the present invention, and methods for using the microorganisms, biomass and microbial oil.

[0199] microorganism

[0200] In some embodiments, the microbial cell for use in the present invention is a microorganism of the Phylum Labyrinthulomycota. In some embodiments, the microbial cell of the Phylum Labyrinthulomycota is a thraustochytrium, such as Schizochytrium or Thraustochytrium. According to the present invention, the term "thraustochytrium" refers to any member of the Thraustochytriales, including the Thraustochytriaceae, and the term "labyrinthulid" refers to any member of the Labyrinthulales, including the Labyrinthulaceae.

[0201] Members of the family Labyrinthaceae were previously considered members of the order Thraustochytriales, but in a recent revision of the taxonomic classification of these organisms, Labyrinthaceae is now considered to be members of the order Labyrinthales. Both Labyrinthales and Thraustochytriales are considered members of the phylum Labyrinthomycota. Taxonomic theory now generally places microorganisms of these two groups with algae or algae-like protists in the Stramenopile lineage. The current taxonomic status of Thraustochytrids and Labyrinths can be summarized as follows:

[0202] Kingdom: Stramenopila (Chromista)

[0203] Phylum: Labyrinthulomycota (Heterokonta)

[0204] Class: Labyrinthulomycetes (Labyrinthulae)

[0205] Order: Labyrinthulales

[0206] Family: Labyrinthulaceae

[0207] Order: Thraustochytriales

[0208] Family: Thraustochytriaceae

[0209] For purposes of the present invention, strains of microbial cells described as thraustochytrids include the following organisms: Order: Thraustochytrides; Family: Thraustochytriaceae; Genus: Thraustochytrium (species: species, arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, and striatum), Ulkenia (species: species, amoeboidea, kerguelensis, minuta, profunda, radiata, sailens, sarkariana, s chizochytrops, visurgensis, yorkensis and species BP-5601), Schizochytrium (species: species, aggregatum, limnaceum, mangrovei, minutum and octosporum), Japonochytrium (species: species, marinum), Aplanochytrium (species: species, haliotidis, kerguelensis, profunda and stocchinoi), Althornia (species: species, crouchii), or Elina (species: species, marisalba and sinorifica). For the purposes of the present invention, species described within the genus Ukenchytropis will be considered members of the genus Thraustochytrium. Aurantiochytrium and Oblongospora are two additional genera included in the phylum Labyrinthomycota in the present invention. In some embodiments, the microbial cell belongs to the genus Thraustochytrium, Schizochytrium and mixtures thereof.

[0210] The present invention relates to isolated microorganisms and strains derived therefrom. Strains "derived" from the isolated microorganisms of the present invention may be naturally derived or artificially derived, such as mutants, variants or recombinant strains. The term "isolated" as used herein does not necessarily reflect the degree to which the isolate has been purified, but refers to separation or isolation from a natural form or natural environment. Isolates may include, but are not limited to, isolated microorganisms, isolated biomass, isolated cultures, isolated microbial oils, and isolated sequences (such as isolated polynucleotide sequences disclosed herein). The term "microorganism" as used herein includes, but is not limited to, the terms "microalgae", "thraustochytrids", and taxonomic classifications associated with any of the deposited microorganisms described herein. The terms "Thraustochytriales", "thraustochytrid", "Schizochytrium", and "Thraustochytrium" as used in connection with any microorganism of the present invention, including the deposited microorganisms described herein, are based on the current taxonomic classification including available phylogenetic information, and are not intended to limit the event of revision of the taxonomic classification after the filing date of the present application.

[0211] In some embodiments, the present invention relates to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10212. The isolated microorganism associated with ATCC Accession No. PTA-10212 is also referred to herein as Thraustochytrium sp. ATCC PTA-10212. The isolated microorganism associated with ATCC Accession No. PTA-10212 was deposited on July 14, 2009, under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, in the Patent Depository. In some embodiments, the present invention relates to an isolated strain deposited under ATCC Accession No. PTA-10212. In some embodiments, the invention relates to an isolated strain deposited under ATCC Accession No. PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-10208, PTA-10209, PTA-10210, or PTA-10211.

[0212] In some embodiments, the present invention relates to isolated microorganisms or strains derived therefrom having characteristics of species deposited under ATCC Accession No. PTA- 10212. Characteristics of species deposited under ATCC Accession No. PTA- 10212 may include their growth properties and phenotypic properties (examples of phenotypic properties include morphological properties and reproductive properties), their physical properties and chemical properties (such as dry weight and lipid profile), their genetic sequences, and combinations thereof, wherein the characteristics distinguish these species from previously identified species. In some embodiments, the present invention relates to an isolated microorganism having characteristics of a species deposited under ATCC Accession No. PTA-10212, wherein the characteristics include: 18s rRNA comprising a polynucleotide sequence of SEQ ID NO: 1 or a polynucleotide sequence at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, morphological and reproductive properties of the species deposited under ATCC Accession No. PTA-10212, and fatty acid profile of the species deposited under ATCC Accession No. PTA-10212. In some embodiments, the isolated microorganism of the present invention has substantially the same phenotypic properties as the microorganism deposited under ATCC Accession No. PTA-10212. In some embodiments, the isolated microorganism of the present invention has substantially the same growth properties as the microorganism deposited under ATCC Accession No. PTA-10212. In some embodiments, the present invention relates to an isolated microorganism comprising an 18s rRNA having a polynucleotide sequence of SEQ ID NO: 1 or a polynucleotide sequence that is at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In some embodiments, the present invention relates to an isolated microorganism comprising an 18s rRNA polynucleotide sequence that is at least 94% identical to the 18s rRNA polynucleotide sequence of the microorganism deposited under ATCC Accession No. PTA-10212.

[0213] In some embodiments, the invention relates to a mutant strain of a microorganism deposited under ATCC Accession No. PTA- 10212. In other embodiments, the mutant strain is a strain deposited under ATCC Accession No. PTA-10213, PTA-10214 or PTA-10215, Thraustochytrium sp. 27797-2, Thraustochytrium sp. 27797-13, and Thraustochytrium sp. 27797-20. Microorganisms associated with ATCC Accession Nos. PTA-10213, PTA-10214, and PTA-10215 were deposited on July 14, 2009, under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.

[0214] In some embodiments, the present invention relates to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208. The isolated microorganism associated with ATCC Accession No. PTA-10208 is also referred to herein as Schizochytrium sp. 27173(9.1)-3-E. The microorganism associated with ATCC Accession No. PTA-10208 is a patent deposit made under the Budapest Treaty on July 14, 2009 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. In some embodiments, the present invention relates to an isolated strain deposited under ATCC Accession No. PTA-10208.

[0215] In some embodiments, the invention relates to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprise greater than about 10%, greater than about 11%, greater than about 12%, greater than about 13%, greater than about 14%, greater than about 15%, greater than about 16%, greater than about 17%, greater than about 18%, greater than about 19%, or greater than about 20% EPA by weight. In some embodiments, the invention relates to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprise about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% EPA.

[0216] In some embodiments, the present invention relates to an isolated microorganism having the characteristics of a species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism contain more than about 10% by weight of eicosapentaenoic acid. The characteristics of the microorganism deposited under ATCC Accession No. PTA-10208 include its growth properties and phenotypic properties (examples of phenotypic properties include morphological properties and reproductive properties), its physical properties and chemical properties (such as dry weight and lipid distribution), its gene sequence and combinations thereof, wherein the characteristics can distinguish the species from previously identified species. In some embodiments, the present invention relates to an isolated microorganism having the characteristics of a species deposited under ATCC Accession No. PTA-10212, wherein the characteristics include 18srRNA containing the polynucleotide sequence of SEQ ID NO: 2, morphological properties and reproductive properties of the species deposited under ATCC Accession No. PTA-10208, and fatty acid profile of the species deposited under ATCC Accession No. PTA-10208. In some embodiments, the isolated microorganisms of the present invention have physical and chemical properties that are substantially the same as the microorganism deposited under ATCC Accession No. PTA-10208.

[0217] In some embodiments, the present invention relates to a mutant strain of a microorganism deposited under ATCC Accession No. PTA- 10208. In other embodiments, the mutant strain is a strain deposited under ATCC Accession No. PTA-10209, PTA-10210 or PTA-10211, Schizochytrium sp. 27173(9.1)-3-E#2, Schizochytrium sp. 27173(9.1)-3-E#4, and Schizochytrium sp. 27173(9.1)-3-E#8. Microorganisms associated with ATCC Accession Nos. PTA-10209, PTA-10210, and PTA-10211 were deposited on September 25, 2009, under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.

[0218] In some embodiments, the present invention relates to an isolated microorganism of the present invention that produces a triglyceride portion, wherein the EPA content of the triglyceride portion is at least about 12% by weight, at least about 13% by weight, at least about 14% by weight, at least about 15% by weight, at least about 16% by weight, at least about 17% by weight, at least about 18% by weight, at least about 19% by weight, or at least about 20% by weight. In some embodiments, the present invention relates to an isolated microorganism that produces a triglyceride portion, wherein the EPA content of the triglyceride portion is about 12% to about 55% by weight, about 12% to about 50% by weight, about 12% to about 45% by weight, about 12% to about 40% by weight, about 12% to about 35% by weight, about 12% to about 30% by weight, about 15% to about 45% by weight, about 15% to about 40% by weight, about 15% to about 35% by weight, about 15% to about 30% by weight, or about 20% to about 30% by weight.

[0219] In some embodiments, the invention relates to a mutant, variant or recombinant strain of an isolated microorganism of the invention that produces a triglyceride fraction, wherein the EPA content of the triglyceride fraction is at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% by weight. In some embodiments, the present invention relates to a mutant, variant or recombinant strain of an isolated microorganism of the present invention that produces a triglyceride portion wherein the EPA content of the triglyceride portion is from about 12% to about 55%, from about 12% to about 50%, from about 12% to about 45%, from about 12% to about 40%, from about 12% to about 35%, from about 12% to about 30%, from about 15% to about 55%, from about 15% to about 50%, from about 15% to about 45%, from about 15% to about 40%, from about 15% to about 35%, from about 15% to about 30%, from about 20% to about 55%, from about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, or from about 20% to about 30%. Mutant strains can be generated by well-known procedures. Commonly used procedures include radiation, high temperature treatment and treatment with mutagen. Variant strains can be other naturally occurring isolates and / or sub-isolates of species described herein. Recombinant strains can be produced by any known method for expressing exogenous genes or changing the function or expression of endogenous genes in molecular biology. In some embodiments, mutant, variant or recombinant strains produce higher amounts of ω-3 fatty acids, especially EPA than wild-type strains. In some embodiments, mutant, variant or recombinant strains produce lower amounts of one or more fatty acids, such as lower amounts of DHA, ARA, DPA n-6 or combinations thereof. In some embodiments, mutant, variant or recombinant strains produce larger dry cell weight per liter of culture than wild-type strains. This mutant, variant or recombinant strain is an example of a strain derived from a microorganism separated by the present invention.

[0220] The present invention also relates to an isolated thraustochytrid microorganism having the characteristics of the thraustochytrid species deposited under ATCC Accession No. PTA-9695, wherein the total fatty acids produced by the microorganism or a strain derived therefrom comprise about 10% by weight or less eicosapentaenoic acid.

[0221] The present invention also relates to an isolated thraustochytrid microorganism or a strain derived therefrom, comprising a triglyceride portion, wherein the docosahexaenoic acid content of the triglyceride portion is at least about 40% by weight, wherein the docosapentaenoic acid n-6 ​​content of the triglyceride portion is at least about 0.5% by weight to about 6% by weight, and wherein the total fatty acids produced by the microorganism or strain derived therefrom comprise about 10% by weight or less of eicosapentaenoic acid.

[0222] The present invention also relates to an isolated thraustochytrid microorganism of the same species as the thraustochytrid or a strain derived therefrom deposited under ATCC Accession No. PTA-9695, wherein the total fatty acids produced by the microorganism or strain derived therefrom comprise about 10% by weight or less eicosapentaenoic acid.

[0223] In some embodiments, the strain derived from the isolated thraustochytrid microorganism of the present invention is a mutant strain.

[0224] The present invention also relates to isolated microorganisms deposited under ATCC Accession Nos. PTA-9695, PTA-9696, PTA-9697 or PTA-9698.

[0225] The present invention also relates to a thraustochytrid biomass comprising any one of the thraustochytrid microorganisms of the present invention or a mixture thereof.

[0226] The present invention also relates to isolated thraustochytrid biomass, wherein at least about 50% by weight of the dry cell weight of the biomass is fatty acids, and wherein at least about 50% by weight of the fatty acids are omega-3 fatty acids. In some embodiments, at least about 50% by weight of the fatty acids are docosahexaenoic acid. The present invention also relates to isolated thraustochytrid biomass, wherein at least about 25% by weight of the dry cell weight of the biomass is docosahexaenoic acid.

[0227] In some embodiments, the present invention also relates to isolated thraustochytrid biomass, wherein about 10% by weight or less of the fatty acids are eicosapentaenoic acid, and wherein the weight ratio of docosahexaenoic acid to eicosapentaenoic acid is at least about 5:1.

[0228] In some embodiments, the present invention also relates to isolated thraustochytrid biomass, wherein about 1.5% by weight or less of the fatty acids are eicosatetraenoic acid, and wherein the weight ratio of docosahexaenoic acid to eicosatetraenoic acid is at least about 20:1.

[0229] In some embodiments, the present invention also relates to an isolated thraustochytrid biomass comprising docosahexaenoic acid and docosapentaenoic acid n-6 ​​in a weight ratio of at least about 10:1. In some embodiments, the present invention relates to a thraustochytrid of the species deposited under ATCC Accession No. PTA-9695. The isolated thraustochytrid herein is also referred to as Schizochytrium sp. 27173. The thraustochytrid associated with ATCC Accession No. PTA-9695 was deposited on January 7, 2009 under the Budapest Treaty at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, in the Patent Depository.

[0230] In some embodiments, the invention relates to an isolated thraustochytrid strain deposited under ATCC Accession No. PTA-9695. In some embodiments, the invention relates to an isolated thraustochytrid microorganism of the same species as the thraustochytrid deposited under ATCC Accession No. PTA-9695.

[0231] In some embodiments, the present invention relates to an isolated thraustochytrid having properties of a species deposited under ATCC Accession No. PTA-9695 or a strain derived therefrom. Properties of the thraustochytrid species deposited under ATCC Accession No. PTA-9695 include its growth properties and phenotypic properties (examples of phenotypic properties include morphological properties and reproductive properties), its physical and chemical properties (such as dry weight and lipid profile), and its gene sequence. In some embodiments, an isolated thraustochytrid of the present invention has substantially the same phenotypic properties as a thraustochytrid deposited under ATCC Accession No. PTA-9695. In some embodiments, an isolated thraustochytrid of the present invention has substantially the same growth properties as a thraustochytrid deposited under ATCC Accession No. PTA-9695.

[0232] In some embodiments, the present invention relates to a mutant, variant or recombinant strain of an isolated thraustochytrid of the present invention, wherein the total fatty acids produced by the mutant, variant or recombinant strain contain about 10% by weight or less of eicosapentaenoic acid. Mutant strains can be produced by known procedures. Common procedures include irradiation, high temperature treatment and treatment with mutagens. Variant strains can be other naturally occurring isolates and / or subisolates of the species described herein. Recombinant strains can be produced by any known method in molecular biology for expressing exogenous genes or altering the function or expression of endogenous genes. In some embodiments, the mutant, variant or recombinant strain produces a higher amount of ω-3 fatty acids (including DHA and / or EPA) than the wild-type strain. In some embodiments, the mutant, variant or recombinant strain produces a lower amount of one or more fatty acids, such as a lower amount of EPA, ARA, DPA n-6 or a combination thereof. In some embodiments, the mutant, variant or recombinant strain produces a greater dry cell weight per liter of culture than the wild-type strain. Such mutants, variants or recombinant strains are examples of strains derived from the isolated thraustochytrid of the present invention.

[0233] In some embodiments, the invention relates to mutant strains of Thraustochytrium deposited under ATCC Accession No. PTA- 9695. In other embodiments, the mutant strains are strains Schizochytrium sp. 27173-C, Schizochytrium sp. 27173-E, and Schizochytrium sp. 27173-G deposited under ATCC Accession No. PTA-9696, PTA-9697, or PTA-9698. Thraustochytrid strains associated with ATCC Accession No. PTA-9696, PTA-9697, or PTA-9698 were deposited under the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA, on January 7, 2009. These deposited mutant strains are derivatives of the Thraustochytrid deposited under ATCC Accession No. PTA-9695.

[0234] In some embodiments, the isolated thraustochytrid of the present invention (including mutants, variants or recombinant strains thereof) comprises fatty acids in one or more fractions isolated from the thraustochytrid. The one or more fractions isolated from the thraustochytrid include a total fatty acid fraction, a sterol ester fraction, a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof.

[0235] The present invention also relates to an isolated thraustochytrid culture comprising any of the thraustochytrid microorganisms of the present invention or mixtures thereof. In some embodiments, the culture comprises at least about 5% dissolved oxygen.

[0236] The present invention also relates to an edible product, cosmetic or pharmaceutical composition for animals or humans comprising any one of the thraustochytrid microorganisms or biomass of the present invention or a mixture thereof.

[0237] The present invention also relates to a microbial oil comprising at least about 70% by weight of a triglyceride portion, wherein the docosahexaenoic acid content of the triglyceride portion is at least about 50% by weight, and wherein the docosapentaenoic acid n-6 ​​content of the triglyceride portion is from about 0.5% by weight to about 6% by weight. In some embodiments, the microbial oil further comprises an eicosatetraenoic acid content of the triglyceride portion of about 1.5% by weight or less.

[0238] The present invention also relates to a microbial oil comprising at least about 70% by weight of a triglyceride fraction, wherein the docosahexaenoic acid content of the triglyceride fraction is at least about 40% by weight, and wherein the docosapentaenoic acid n-6 ​​content of the triglyceride fraction is at least about 0.5% by weight to about 6% by weight, and wherein the ratio of docosahexaenoic acid to docosapentaenoic acid n-6 ​​is greater than about 6:1.

[0239] The present invention also relates to a microbial oil comprising at least about 70% by weight of a triglyceride fraction, wherein the docosahexaenoic acid content of the triglyceride fraction is at least about 60% by weight.

[0240] In some embodiments, at least about 20% of the triglycerides in the triglyceride portion of the microbial oil contain docosahexaenoic acid at two positions selected from any two of the sn-1, sn-2, and sn-3 positions in the triglyceride. In some embodiments, at least about 5% of the triglycerides in the triglyceride portion of the microbial oil contain docosahexaenoic acid at all three positions of the sn-1, sn-2, and sn-3 in the triglyceride.

[0241] In some embodiments, the isolated microorganism of the present invention (including its mutant, variant or recombinant strain) comprises fatty acid distribution in one or more parts separated by this microorganism.One or more parts separated by this microorganism include total fatty acid part, sterol ester part, triglyceride part, free fatty acid part, sterol part, diglyceride part, polar part (including phospholipid part) and combination thereof.The fatty acid distribution of specific part can include any fatty acid distribution associated with this specific part disclosed herein.

[0242] The present invention relates to a method for producing mutant strains, which comprises mutagenizing any one of the microorganisms of the present invention and isolating mutant strains.

[0243] Cultures and isolated biomass

[0244] The present invention relates to a culture comprising one or more isolated microorganisms of the present invention. Various fermentation parameters for inoculating, growing and recovering microbial flora (such as microalgae and thraustochytrid) are known in the art. See U.S. Pat. No. 5,130,242, which is incorporated herein by reference in its entirety. Liquid or solid culture media may contain natural seawater or artificial seawater. Carbon sources for heterotrophic growth include, but are not limited to, glucose, fructose, xylose, sucrose, maltose, soluble starch, molasses, trehalose, glucosamine, dextran, fats, oils, glycerol, sodium acetate and mannitol. Nitrogen sources include, but are not limited to, peptone, yeast extract, polypeptone, malt extract, meat extract, tyrosin amino acids, corn steep liquor, organic nitrogen sources, sodium glutamate, urea, inorganic nitrogen sources, ammonium acetate, ammonium sulfate, ammonium chloride and ammonium nitrate.

[0245] Typical culture media for growing microorganisms deposited under ATCC Accession No. PTA-10212 are shown in Table 1:

[0246] Table 1: PTA-10212 Vessel Culture Medium

[0247]

[0248]

[0249] Typical culture conditions include the following:

[0250]

[0251] In some embodiments, the microorganism deposited under ATCC Accession No. PTA-10212, or a mutant, variant, or recombinant strain thereof, grows heterotrophically on glycerol as a carbon source, but does not grow on glucose as a carbon source.

[0252] A typical culture medium for growing microorganisms deposited under ATCC Accession No. PTA-10208 is shown in Table 2:

[0253] Table 2: PTA-10208 container culture medium

[0254]

[0255]

[0256] Typical culture conditions include the following:

[0257]

[0258] In some embodiments, the fermentation volume (culture volume) is at least about 2 liters, at least about 10 liters, at least about 50 liters, at least about 100 liters, at least about 200 liters, at least about 500 liters, at least about 1000 liters, at least about 10,000 liters, at least about 20,000 liters, at least about 50,000 liters, at least about 100,000 liters, at least about 150,000 liters, at least about 200,000 liters, or at least about 250,000 liters. In some embodiments, the fermentation volume is about 2 liters to about 300,000 liters, about 2 liters, about 10 liters, about 50 liters, about 100 liters, about 200 liters, about 500 liters, about 1,000 liters, about 10,000 liters, about 20,000 liters, about 50,000 liters, about 100,000 liters, about 150,000 liters, about 200,000 liters, about 250,000 liters, or about 300,000 liters.

[0259] In some embodiments, the present invention relates to a kind of separated biomass that comprises the distribution of fatty acid of the present invention.In some embodiments, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% or at least about 80% of the dry cell weight of biomass is fatty acid.In some embodiments, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 55% or more than about 60% of the dry cell weight of biomass is fatty acid. In some embodiments, about 20% to about 55%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 30% to about 55%, about 30% to about 70%, about 30% to about 80%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 55% to about 70%, about 55% to about 80%, about 60% to about 70%, or about 60% to about 80% by weight of the dry cell weight of the biomass is fatty acids. In some embodiments, biomass comprises more than about 10 wt %, at least about 12 wt %, at least about 15 wt %, at least about 20 wt %, at least about 25 wt %, at least about 30 wt %, at least about 35 wt %, at least about 40 wt % or at least about 45 wt % fatty acid is EPA.In some embodiments, biomass comprises about 10 wt % to about 55 wt %, about 12 wt % to about 55 wt %, about 15 wt % to about 55 wt %, about 20 wt % to about 55 wt %, about 20 wt % to about 40 wt % or about 20 wt % to about 30 wt % fatty acid is EPA.In some embodiments, biomass comprises triglyceride moiety, wherein at least about 12 wt %, at least about 13 wt %, at least about 14 wt %, at least about 15 wt %, at least about 16 wt %, at least about 17 wt %, at least about 18 wt %, at least about 19 wt % or at least about 20 wt % of triglyceride moiety is EPA.In some embodiments, the biomass comprises a triglyceride portion, wherein the EPA content of the triglyceride portion is at least about 12% to about 55%, about 12% to about 50%, about 12% to about 45%, at least about 12% to about 40%, at least about 12% to about 35%, or at least about 12% to about 30%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, at least about 20% to about 40%, at least about 20% to about 35%, or about 20% to about 30%. In some embodiments, at least about 20% by weight, at least about 25% by weight, at least about 30% by weight, at least about 35% by weight, at least about 40% by weight, at least about 50% by weight or at least about 60% by weight of the cell dry weight of biomass is DHA. In some embodiments, about 20% by weight to about 60% by weight, about 25% by weight to about 60% by weight, about 25% by weight to about 50% by weight, about 25% by weight to about 45% by weight, about 30% by weight to about 50% by weight or about 35% by weight to about 50% by weight in the cell dry weight of biomass is DHA. In some embodiments, about 10% by weight or less, about 9% by weight or less, about 8% by weight or less, about 7% by weight or less, about 6% by weight or less, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less or about 1% by weight or less in the fatty acid weight included in biomass is DHA. In some embodiments, the biomass comprises about 1 wt % to about 10 wt %, about 1 wt % to about 5 wt %, about 2 wt % to about 5 wt %, about 3 wt % to about 5 wt %, or about 3 wt % to about 10 wt % of the fatty acids as DHA. In some embodiments, the biomass is substantially free of DHA. In some embodiments, the biomass comprises between about 0.1% to less than about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.2% to less than about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2% to about 2%, about 0.3% to about 2%, about 0.1% to about 0.5%, about 0.2% to about 0.5%, about 0.1% to about 0.4%, about 0.2% to about 0.4%, about 0.5% to about 2%, about 1% to about 2%, about 0.5% to about 1.5%, or about 1% to about 1.5% of the fatty acids that are ARA.In some embodiments, the biomass comprises less than about 5 wt %, about 4 wt % or less, about 3 wt % or less, about 2 wt % or less, about 1.5 wt % or less, about 1 wt % or less, about 0.5 wt % or less, about 0.4 wt % or less, about 0.3 wt % or less, about 0.2 wt % or less or about 0.1 wt % or less fatty acid as ARA. In some embodiments, the biomass is substantially free of ARA. In some embodiments, the biomass comprises about 0.4% to about 2%, about 0.4% to about 3%, about 0.4% to about 4%, about 0.4% to about 5%, about 0.4% to less than about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 0.5% to less than about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, or about 1% to less than about 5% fatty acid is DPA n-6. In some embodiments, the biomass comprises about 5 wt% or less, less than about 5 wt%, about 4 wt% or less, about 3 wt% or less, about 2 wt% or less, about 1 wt% or less, about 0.75 wt% or less, about 0.6 wt% or less, or about 0.5 wt% or less of fatty acids as DPAn-6. In some embodiments, the biomass is substantially free of DPA n-6. In some embodiments, the biomass comprises fatty acids having about 5 wt% or less, less than about 5 wt%, about 4 wt% or less, about 3 wt% or less, or about 2 wt% or less of oleic acid (18:1n-9), linolenic acid (18:2n-6), linolenic acid (18:3n-3), eicosenoic acid (20:1n-9), sinapic acid (22:1n-9), or a combination thereof.

[0260] The properties of the isolated biomass of the present invention are associated with the endogenous or native properties of the isolated biomass, and not with the properties of externally introduced materials. In some embodiments, the isolated biomass does not contain polyvinylpyrrolidone, or is not isolated from a culture containing polyvinylpyrrolidone.

[0261] The present invention relates to methods of producing biomass. In some embodiments, the method of producing biomass of the present invention comprises growing any isolated microorganism of the present invention or a mixture thereof in culture to produce biomass. The present invention relates to biomass produced by the method.

[0262] In some embodiments, biomass comprises fatty acid, wherein fatty acid also comprises ω-3 polyunsaturated fatty acid, wherein ω-3 polyunsaturated fatty acid comprises DHA and EPA with the amount of about ≥90 weight % of the total amount of ω-3 polyunsaturated fatty acid, and the amount of EPA is about 6 weight % to about 65 weight % of the total amount of EPA and DHA. Especially provided is the biomass of about 6 weight % to about 28 weight % of the total amount of EPA and DHA. Also provided herein is the biomass of about 36 weight % to about 65 weight % of the total amount of EPA and DHA. More specifically provided is the biomass of about 28 weight % to about 36 weight % of the total amount of EPA and DHA.

[0263] Some embodiments provided herein comprise biomass comprising fatty acids, wherein the fatty acids further comprise DHA and EPA, and the amount of EPA is from about 15 wt % up to about 60 wt % of the total amount of EPA and DHA.

[0264] Some embodiments of the present invention also relate to cultures comprising thraustochytrids or mutant strains deposited under ATCC Accession No. PTA-9695. Various fermentation parameters for inoculating, growing and recovering microbial flora are known in the art, for example, see U.S. Pat. No. 5,130,242. Any conventional medium (culture medium) for growing thraustochytrids can be used. The liquid or solid culture medium may contain natural seawater or artificial seawater. Carbon sources include, but are not limited to, glucose, fructose, xylose, sucrose, maltose, soluble starch, molasses, trehalose, glucosamine, dextran, fats, oils, glycerol, sodium acetate and mannitol. Nitrogen sources include, but are not limited to, peptone, yeast extract, polypeptone, malt extract, meat extract, casamino acids, corn steep liquor, organic nitrogen sources, sodium glutamate, urea, inorganic nitrogen sources, ammonium acetate, ammonium sulfate, ammonium chloride, ammonium nitrate, sodium sulfate. Typical culture media are shown in Table 3:

[0265] Table 3: PTA-9695 Vessel Culture Medium

[0266]

[0267]

[0268] Typical culture conditions include the following:

[0269]

[0270] In some embodiments, the culture medium comprises at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% dissolved oxygen as a percentage of the saturation level. In some embodiments, the culture medium comprises about 5% to about 20%, about 5% to about 50%, about 5% to about 100%, about 10% to about 20%, about 10% to about 50%, about 10% to about 100%, about 20% to about 50%, or about 20% to about 100% dissolved oxygen as a percentage of the saturation level.

[0271] The present invention also relates to isolated biomass of the thraustochytrids of the present invention. The isolated thraustochytrid biomass of the present invention is harvested cell biomass obtained by any conventional method for isolating thraustochytrid biomass, such as described in U.S. Pat. No. 5,130,242 and U.S. Application Publication No. 2002 / 0001833.

[0272] In some embodiments, the dry cell weight of biomass isolated from each liter of culture after about 7 days of growth at about 17°C to about 30°C in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about 6.5 to about 8.5, is at least about 50 g, at least about 60 g, at least about 70 g, at least about 80 g, at least about 100 g, at least about 120 g, at least about 140 g, at least about 160 g, at least about 180 g, or at least about 200 g. In some embodiments, the dry cell weight of biomass isolated from liter of culture is at least about 50 g, at least about 60 g, at least about 70 g, at least about 80 g, at least about 100 g, at least about 120 g, at least about 140 g, at least about 160 g, at least about 180 g, or at least about 200 g after about 7 days of growth at about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, or about pH 8.5. In some embodiments, the cell dry weight of biomass isolated from each liter of culture is about 50 g to about 200 g after about 7 days of growth at about 17° C. to about 30° C. in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about 6.5 to about 8.5. In some embodiments, the dry cell weight of biomass isolated from the culture is about 50 g to about 200 g per liter of culture after about 7 days of growth at about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, or about pH 8.5.

[0273] In some embodiments, the isolated thraustochytrid culture has an omega-3 fatty acid productivity of at least about 2 g / L / day, at least about 4 g / L / day, or at least about 8 g / L / day after about 7 days of growth at about 17°C to 30°C in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about pH 6.5 to about pH 8.5. In some embodiments, an isolated thraustochytrid culture has an omega-3 fatty acid productivity of about 1 g / L / day to about 20 g / L / day, about 2 g / L / day to about 15 g / L / day, about 2 g / L / day to about 10 g / L / day, about 3 g / L / day to about 10 g / L / day, or about 4 g / L / day to about 9 g / L / day after about 7 days of growth at about 17°C to 30°C in a culture medium comprising sources of carbon, nitrogen, and nutrients and about 950 ppm to about 8500 ppm chloride ions at a pH of about pH 6.5 to about pH 8.5.

[0274] In some embodiments, the fermentation volume (culture volume) is at least about 2 liters, at least about 10 liters, at least about 50 liters, at least about 100 liters, at least about 200 liters, at least about 500 liters, at least about 1000 liters, at least about 10,000 liters, at least about 20,000 liters, at least about 50,000 liters, at least about 100,000 liters, at least about 150,000 liters, at least about 200,000 liters, or at least about 250,000 liters. In some embodiments, the fermentation volume is about 2 liters to about 300,000 liters, about 2 liters, about 10 liters, about 50 liters, about 100 liters, about 200 liters, about 500 liters, about 1,000 liters, about 10,000 liters, about 20,000 liters, about 50,000 liters, about 100,000 liters, about 150,000 liters, about 200,000 liters, about 250,000 liters, or about 300,000 liters.

[0275] In some embodiments, the present invention relates to a separated thraustochytrid biomass comprising the fatty acid profile of the present invention. In some embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the cell dry weight of the biomass is fatty acid. In some embodiments, more than about 50%, more than about 55%, or more than about 60% of the cell dry weight of the biomass is fatty acid. In some embodiments, about 50% to about 60% by weight, about 50% to about 70% by weight, about 50% to about 80% by weight, about 55% to about 70% by weight, about 55% to about 80% by weight, about 60% to about 70% by weight, or about 60% to about 80% by weight of the cell dry weight of the biomass is fatty acid. In some embodiments, at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, or at least about 80% by weight of the fatty acid contained in the biomass is omega-3 fatty acid. In some embodiments, the fatty acid of about 50% by weight to about 60% by weight, about 50% by weight to about 70% by weight, about 50% by weight to about 80% by weight contained by biomass is omega-3 fatty acid. In some embodiments, the fatty acid of at least about 50% by weight, at least about 55% by weight, at least about 60% by weight, at least about 65% by weight, at least about 70% by weight, at least about 75% by weight or at least about 80% by weight contained by biomass is DHA. In some embodiments, the fatty acid of about 50% by weight to about 60% by weight, about 50% by weight to about 70% by weight or about 50% by weight to about 80% by weight contained by biomass is DHA. In some embodiments, at least about 25% by weight, at least about 30% by weight, at least about 40% by weight, at least about 50% by weight or at least about 60% by weight of biomass dry cell weight is docosahexaenoic acid. In some embodiments, about 25 % by weight to about 65 % by weight, about 25 % by weight to about 50 % by weight, about 30 % by weight to about 40 % by weight, or about 25 % by weight to about 35 % by weight of the biomass cell dry weight is docosahexaenoic acid. In some embodiments, about 10 % by weight or less, about 9 % by weight or less, about 8 % by weight or less, about 7 % by weight or less, about 6 % by weight or less, about 5 % by weight or less, about 4 % by weight or less, about 3 % by weight or less, about 2 % by weight or less, or about 1 % by weight or less fatty acid comprised by biomass is EPA. In some embodiments, about 1 % by weight to about 10 % by weight, about 1 % by weight to about 5 % by weight, about 2 % by weight to about 5 % by weight, about 3 % by weight to about 5 % by weight, or about 3 % by weight to about 10 % by weight fatty acid comprised by biomass is EPA. In some embodiments, biomass is substantially free of EPA.In some embodiments, biomass comprises at least about 5: 1, at least about 7: 1, at least about 10: 1, at least about 11: 1, at least about 14: 1, at least about 15: 1, at least about 17: 1, at least about 20: 1, at least about 25: 1, at least about 50: 1 or at least about 100: 1 weight ratio of DHA to EPA, wherein about 10 wt % or less fatty acid contained by biomass is EPA. In some embodiments, about 0.1 wt % to 0.2 wt %, about 0.1 wt % to about 0.3 wt %, about 0.1 wt % to about 0.4 wt %, about 0.1 wt % to about 0.5 wt % or about 0.1 wt % to about 1.5 wt % fatty acid contained by biomass is ARA. In some embodiments, the fatty acid of about 1.5 % by weight or less, about 1 % by weight or less, about 0.5 % by weight or less, about 0.4 % by weight or less, about 0.3 % by weight or less, about 0.2 % by weight or less or about 0.1 % by weight or less comprised by biomass is ARA.In some embodiments, biomass is substantially free of ARA.In some embodiments, biomass comprises at least about 20:1, at least about 40:1, at least about 60:1, at least about 80:1, at least about 100:1, at least about 150:1, at least about 200:1, at least about 250:1 or at least about 300:1 DHA and ARA weight ratio. In some embodiments, about 0.5% to about 1% by weight, about 0.5% to about 2% by weight, about 0.5% to about 5% by weight, about 0.5% to about 6% by weight, about 1% to about 5% by weight, about 1% to about 6% by weight, about 2% to about 5% by weight, or about 2% to about 6% by weight of the fatty acid contained in the biomass is DPA n-6. In some embodiments, about 6% by weight or less, about 5% by weight or less, about 2% by weight or less, about 1% by weight or less, or about 0.5% by weight or less of the fatty acid contained in the biomass is DPA n-6. In some embodiments, the biomass is substantially free of DPA n-6. In some embodiments, the biomass comprises a weight ratio of DHA to DPA n-6 of more than about 6:1, at least about 8:1, at least about 10:1, at least about 15:1, at least about 20:1, at least about 25:1, at least about 50:1, or at least about 100:1. In some embodiments, the biomass comprises fatty acids each having about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, or about 2% by weight or less of linoleic acid (18:2n-6), linolenic acid (18:3n-3), eicosenoic acid (20:1n-9), and sinapic acid (22:1n-9).

[0276] In another embodiment, provided herein is a biomass comprising fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about greater than or equal to about 58-68 wt %, specifically about 60 wt %, of the total amount of ω-3 polyunsaturated fatty acids, and the amount of EPA is from about 5 wt % to up to about 60 wt % of the total amount of EPA and DHA.

[0277] The characteristics of the isolated biomass of the present invention are associated with the endogenous or original properties of the isolated biomass and not with the properties of externally introduced materials.

[0278] Microbial Oil

[0279] Provided herein are oils, particularly microbial oils, made by the methods described above.

[0280] In some embodiments, the microbial oil comprises fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids account for about 1% of the total amount of the ω-3 polyunsaturated fatty acids. > The amount of 90 % by weight comprises DHA and EPA, and the amount of EPA is about 6 % by weight of EPA and DHA total amount to about 65 % by weight at the most. Specifically provided is that wherein the amount of EPA is about 6 % by weight of EPA and DHA total amount to about 28 % by weight microbial oil at the most. Also provided is that wherein the amount of EPA is about 36 % by weight of EPA and DHA total amount to about 65 % by weight microbial oil at the most. More specifically provided is that wherein the amount of EPA is about 28 % by weight of EPA and DHA total amount to about 36 % by weight microbial oil.

[0281] Some embodiments provided herein comprise a microbial oil comprising fatty acids, wherein the fatty acids further comprise DHA and EPA, and the amount of EPA is from about 15 wt % up to about 60 wt % of the total amount of EPA and DHA.

[0282] In another embodiment, provided herein is a microbial oil comprising fatty acids, wherein the fatty acids further comprise ω-3 polyunsaturated fatty acids, wherein the ω-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about greater than or equal to about 58-68 wt %, specifically about 60 wt %, of the total amount of ω-3 polyunsaturated fatty acids, and the amount of EPA is from about 5 wt % to up to about 60 wt % of the total amount of EPA and DHA.

[0283] The present invention relates to microbial oils comprising the fatty acid distribution of the present invention. The microbial oils of the present invention are "crude oils" or "refined oils" comprising at least about 35% by weight of a triglyceride portion. "Crude oil" is an oil extracted from the biomass of a microorganism and not further processed. "Refined oil" is an oil obtained by treating crude oil with standard procedures for refining, decolorizing and / or deodorizing. See, for example, U.S. Pat. No. 5,130,242, which is incorporated herein by reference in its entirety. Microbial oils also include "final oils" as described herein, which are refined oils diluted with vegetable oils. In some embodiments, the final oil is a refined oil diluted with high oleic sunflower oil. The term "microorganism" as used herein includes, but is not limited to, the terms "microalgae", "thraustochytrids" and taxonomic classifications associated with any one of the deposited microorganisms described herein. The terms "Thraustochytriales", "thraustochytrid", "Schizochytrium" and "Thraustochytrium" as used in connection with any microbial oil of the deposited microorganisms of the present invention are based on the current taxonomic classification including available phylogenetic information, and are not intended to limit the event of revision of the taxonomic classification after the filing date of this application.

[0284] In some embodiments, the fatty acid described herein can be a fatty acid ester. In some embodiments, fatty acid esters include esters of ω-3 fatty acids, esters of ω-6 fatty acids and combinations thereof. In some embodiments, fatty acid esters are DHA esters, EPA esters or combinations thereof. In some embodiments, oil described herein or its portion is esterified to produce an oil or its portion comprising fatty acid esters. The term "ester" refers to replacing the hydrogen in the carboxylic acid group of a fatty acid molecule with another substituent. Typical esters are known to those skilled in the art, and their related discussion is provided in Higuchi, T. and V. Stella in Pro-drugs as Novel Delivery Systems, Vol. 14, ACSSymposium Series, Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association, Pergamon Press, 1987 and Protective Groups in Organic Chemistry, McOmie, Plenum Press, New York, 1973. Examples of esters include methyl, ethyl, propyl, butyl, pentyl, tert-butyl, benzyl, nitrobenzyl, methoxybenzyl, diphenylmethyl and trichloroethyl. In some embodiments, the ester is a carboxylic acid protecting ester group, an ester with an aralkyl group (such as benzyl, phenethyl), an ester with a lower alkenyl group (such as allyl, 2-butenyl), an ester with a lower alkoxy-lower alkyl group (such as methoxymethyl, 2-methoxyethyl, 2-ethoxyethyl), an ester with a lower alkanoyloxy-lower alkyl group (such as acetoxymethyl, trimethylacetoxymethyl, 1-trimethylacetoxyethyl), an ester with a lower alkoxycarbonyl-lower alkyl group (such as methoxycarbonylmethyl, isopropoxycarbonylmethyl), an ester with a carboxyl-lower alkyl group (such as carboxymethyl), an ester with a lower alkoxycarbonyloxy-lower alkyl group (such as 1-(ethoxycarbonyloxy)ethyl, 1-(cyclohexyloxycarbonyloxy)ethyl), an ester with a carbamoyloxy-lower alkyl group (such as a carbamoyloxymethyl), and the like. In some embodiments, the substituent added is a straight chain or cyclic hydrocarbon group, such as C1-C6 alkyl, C1-C6 cycloalkyl, C1-C6 alkenyl or C1-C6 aryl ester. In some embodiments, the ester is an alkyl ester, such as a methyl ester, an ethyl ester or a propyl ester. In some embodiments, when the fatty acid is in a purified or semi-purified state, the ester substituent is added to the free fatty acid molecule. Alternatively, a fatty acid ester is formed when triglycerides are converted to esters.

[0285] The present invention relates to a method for producing microbial oil. In some embodiments, the method comprises growing any separated microorganism of the present invention or its mixture in culture to produce a microbial oil comprising ω-3 fatty acids. In some embodiments, the method also comprises extracting the microbial oil. In some embodiments, the method comprises extracting the microbial oil containing ω-3 fatty acids from any biomass of the present invention or its mixture. In some embodiments, the method comprises the microorganism separated by heterotrophic growth, wherein the culture comprises a carbon source as described herein. The microbial oil can be obtained by extracting from the newly collected biomass, or can be obtained by extracting from the previously collected biomass stored under conditions that prevent deterioration. Known methods can be used to cultivate the microorganism of the present invention, to separate biomass from culture, to extract microbial oil from biomass, and to analyze the fatty acid distribution of the oil extracted from biomass. See, for example, U.S. Patent No. 5,130,242, which is incorporated herein by reference in its entirety. The present invention relates to the microbial oil produced by any method of the present invention.

[0286] In some embodiments, the microbial oil is extracted by an enzymatic extraction method. In some embodiments, the microbial oil is extracted by a mechanical extraction method. In some embodiments, the mechanical extraction method includes one or more of the following steps: (1) treating the pasteurized fermentation broth through a homogenizer to assist in cell lysis and release of oil from the cells; (2) adding isopropanol to the fermentation broth after homogenization to break the emulsion of oil and water; (3) centrifuging the mixture to recover the oil phase; and (4) adding an antioxidant and drying in a vacuum. In some embodiments, the crude oil is purified. In some embodiments, purification of the crude oil includes one or more of the following steps: (1) pumping the crude oil into a refining tank and heating the oil, followed by adding an acid solution under mixing; (2) adding a caustic solution to the oil after the acid treatment;

[0287] (3) reheating the crude oil and then centrifuging to separate the heavy phase from the refined oil; (4) by using, for example, acid, clay and / or filtration to remove remaining polar compounds, trace metals and oxidation products from the refined oil; (5) cold filtering the bleached oil to further remove high melting point components in the oil to achieve the desired clarity level; (6) heating the oil, then cooling and standing the oil for a period of time to cause crystallization of high melting triglycerides and waxes; (7) adding a filter aid to the cooled oil and then removing the crystallized solids by filtration; (8) after cold filtration, using a deodorizer operating at high temperature and vacuum to remove any remaining low molecular weight compounds such as peroxides and off-odor and taste causing compounds; (9) transferring the oil to a deodorizer feed tank, degassing, and deodorizing, for example, in a packed column deodorizer; and (10) cooling, for example, under a nitrogen blanket at the end of the deodorization cycle, and adding a suitable antioxidant to the deodorized oil to provide oxidative stability.

[0288] In some embodiments, the microbial oil comprises about 0 wt %, at least about 0.1 wt %, at least about 0.2 wt %, at least about 0.5 wt %, at least about 1 wt %, at least about 1.5 wt %, at least about 2 wt % or at least about 5 wt % of the sterol ester portion. In some embodiments, the microbial oil comprises about 0 wt % to about 1.5 wt %, about 0 wt % to about 2 wt %, about 0 wt % to about 5 wt %, about 1 wt % to about 1.5 wt %, about 0.2 wt % to about 1.5 wt %, about 0.2 wt % to about 2 wt %, or about 0.2 wt % to about 5 wt % of the sterol ester portion. In some embodiments, the microbial oil comprises about 5 wt % or less, about 4 wt % or less, about 3 wt % or less, about 2 wt % or less, about 1 wt % or less, about 0.5 wt % or less, about 0.3 wt % or less, about 0.2 wt % or less, about 0.5 wt % or less, about 0.4 wt % or less, about 0.3 wt % or less, or about 0.2 wt % or less of sterol ester moieties.

[0289] In some embodiments, the microbial oil comprises at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% by weight of a triglyceride portion. In some embodiments, the microbial oil comprises about 35% to about 98%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about 35% to about 70%, about 35% to about 65%, about 40% to about 70%, about 40% to about 65%, about 40% to about 55%, about 40% to about 50%, about 65% to about 95%, about 75% to about 95%, about 75% to about 98%, about 80% to about 95%, about 80% to about 98%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90%, about 95%, about 97% or about 98% triglyceride portion.

[0290] In some embodiments, the microbial oil comprises at least about 10 % by weight, at least about 11 % by weight, at least about 12 % by weight, at least about 13 % by weight, at least about 14 % by weight, at least about 15 % by weight, at least about 16 % by weight, at least about 17 % by weight, at least about 18 % by weight, at least about 19 % by weight or at least about 20 % by weight of the diglyceride portion. In some embodiments, the microbial oil comprises the diglyceride portion of about 10 % by weight to about 45 % by weight, about 10 % by weight to about 40 % by weight, about 10 % by weight to about 35 % by weight, about 10 % by weight to about 30 % by weight, about 15 % by weight to about 40 % by weight, about 15 % by weight to about 35 % by weight or about 15 % by weight to about 30 % by weight. In some embodiments, the microbial oil comprises at least about 0.2 weight %, at least about 0.3 weight %, at least about 0.4 weight %, at least about 0.5 weight %, at least about 1 weight %, at least about 5 weight %, at least about 10 weight %, at least about 11 weight %, at least about 12 weight %, at least about 13 weight %, at least about 14 weight %, at least about 15 weight %, at least about 16 weight %, at least about 17 weight %, at least about 18 weight %, at least about 19 weight %, or at least about 20 weight % of the 1,2-diacylglycerol portion. In some embodiments, the microbial oil comprises about 0.2 wt % to about 45 wt %, about 0.2 wt % to about 30 wt %, about 0.2 wt % to about 20 wt %, about 0.2 wt % to about 10 wt %, about 0.2 wt % to about 5 wt %, about 0.2 wt % to about 1 wt %, about 0.2 wt % to about 0.8 wt %, about 0.4 wt % to about 45 wt %, about 0.4 wt % to about 30 wt %, about 0.4 wt % to about 20 wt %, about 0.4 wt % to about 10 wt %, about 0.4 wt % to about 5 wt %. In some embodiments, the microbial oil comprises at least about 0.1 wt %, at least about 0.2 wt %, at least about 0.5 wt %, at least about 1 wt %, at least about 2 wt %, at least about 2.5 wt % or at least about 3 wt % of the 1,3-diaceryl moiety. In some embodiments, the microbial oil comprises at least about 0.3 wt%, at least about 0.4 wt%, at least about 0.5 wt%, at least about 1 wt%, at least about 1.5 wt%, at least about 2 wt%, or at least about 5 wt% of the sterol moiety.

[0291] In some embodiments, microbial oil comprises the sterol moiety of about 0.3 % by weight to about 5 % by weight, about 0.3 % by weight to about 2 % by weight, about 0.3 % by weight to about 1.5 % by weight, about 0.5 % by weight to about 1.5 % by weight, about 1 % by weight to about 1.5 % by weight, about 0.5 % by weight to about 2 % by weight, about 0.5 % by weight to about 5 % by weight, about 1 % by weight to about 2 % by weight, or about 1 % by weight to about 5 % by weight. In some embodiments, microbial oil comprises about 5 % by weight or still less, about 4 % by weight or still less, about 3 % by weight or still less, about 2 % by weight or still less, about 1.5 % by weight or still less or about 1 % by weight or still less.

[0292] In some embodiments, microbial oil comprises at least about 2 % by weight, at least about 5 % by weight or at least about 8 % by weight of the phospholipid part.In some embodiments, microbial oil comprises about 2 % by weight to about 25 % by weight, about 2 % by weight to about 20 % by weight, about 2 % by weight to about 15 % by weight, about 2 % by weight to about 10 % by weight, about 5 % by weight to about 25 % by weight, about 5 % by weight to about 20 % by weight, about 5 % by weight to about 20 % by weight, about 5 % by weight to about 10 % by weight or about 7 % by weight to about 9 % by weight of the phospholipid part.In some embodiments, microbial oil comprises lower than about 20 % by weight, lower than about 15 % by weight, lower than about 10 % by weight, lower than about 9 % by weight or lower than the phospholipid part of about 8 % by weight.In some embodiments, microbial oil does not contain phospholipid substantially.In some embodiments, microbial oil comprises lower than about 2%, lower than about 1.5%, lower than about 1% or lower than the unsaponifiable matter of about 0.5% oil weight. The lipid types present in the microbial oil, such as the triglyceride fraction, can be separated by flash chromatography and analyzed by thin layer chromatography (TLC), or by other methods known in the art.

[0293] In some embodiments, the microbial oil and / or one or more portions thereof selected from the triglyceride portion, the free fatty acid portion, the sterol portion, the diglyceride portion, and combinations thereof, comprises at least about 5 wt%, at least about 10 wt%, greater than about 10 wt%, at least about 12 wt%, at least about 13 wt%, at least about 14 wt%, at least about 15 wt%, at least about 16 wt%, at least about 17 wt%, at least about 18 wt%, at least about 19 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, or at least about 45 wt% EPA. In some embodiments, the microbial oil and / or one or more moieties selected from the triglyceride moiety, the free fatty acid moiety, the sterol moiety, the diglyceride moiety, and combinations thereof, comprises from about 5 wt % to about 55 wt %, from about 5 wt % to about 50 wt %, from about 5 wt % to about 45 wt %, from about 5 wt % to about 40 wt %, from about 5 wt % to about 35 wt %, from about 5 wt % to about 30 wt %, from about 10 wt % to about 55 wt %, from about 10 wt % to about 50 wt %, from about 10 wt % to about 45 wt %, from about 10 wt % to about 40 wt %, from about 10 wt % to about 35 wt %, from about 10 wt % to about 30 wt %, from at least about 12 wt % to about 55 wt %, from at least about 12 wt % to about 50 wt %. %, at least about 12 wt % to about 45 wt %, at least about 12 wt % to about 40 wt %, at least about 12 wt % to about 35 wt %, or at least about 12 wt % to about 30 wt %, about 15 wt % to about 55 wt %, about 15 wt % to about 50 wt %, about 15 wt % to about 45 wt %, about 15 wt % to about 40 wt %, about 15 wt % to about 35 wt %, about 15 wt % to about 30 wt %, about 15 wt % to about 25 wt %, about 15 wt % to about 20 wt %, about 20 wt % to about 55 wt %, about 20 wt % to about 50 wt %, about 20 wt % to about 45 wt %, about 20 wt % to about 40 wt %, or about 20 wt % to about 30 wt % EPA. In some embodiments, the microbial oil and / or one or more portions thereof selected from the triglyceride portion, the diglyceride portion, the sterol portion, the sterol ester portion, the free fatty acid portion, the phospholipid portion, and combinations thereof, comprises at least about 5 wt%, at least about 10 wt%, at least about 15 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, at least about 50 wt%, or at least about 60 wt% DHA.In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a triglyceride portion, a diglyceride portion, a sterol portion, a sterol ester portion, a free fatty acid portion, a phospholipid portion, and combinations thereof, comprises from about 5% to about 60% by weight, from about 5% to about 55% by weight, from about 5% to about 50% by weight, from about 5% to about 40% by weight, from about 10% to about 60% by weight, from about 10% to about 50% by weight, from about 10% to about 40% by weight, from about 20% to about 60% by weight, from about 25% to about 60% by weight, from about 25% to about 50% by weight, from about 25% to about 45% by weight, from about 30% to about 50% by weight, from about 35% to about 50% by weight, or from about 30% to about 40% by weight of DHA. In some embodiments, microbial oil and / or it is selected from one or more parts in triglyceride part, diglyceride part, sterol part, sterol ester part, free fatty acid part, phospholipid part and their combination, comprise about 10 wt % or less, about 9 wt % or less, about 8 wt % or less, about 7 wt % or less, about 6 wt % or less, about 5 wt % or less, about 4 wt % or less, about 3 wt % or less, about 2 wt % or less or about 1 wt % or less DHA. In some embodiments, microbial oil and / or it is selected from one or more parts in triglyceride part, diglyceride part, sterol part, sterol ester part, free fatty acid part, phospholipid part and their combination, and the fatty acid of about 1% to about 10 wt %, about 1 wt % to about 5 wt %, about 2 wt % to about 5 wt %, about 3 wt % to about 5 wt % or about 3 wt % to about 10 wt % contained is DHA. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a triglyceride portion, a diglyceride portion, a sterol portion, a sterol ester portion, a free fatty acid portion, a phospholipid portion, and combinations thereof, are substantially free of DHA.In some embodiments, the microbial oil and / or one or more moieties selected from the group consisting of a triglyceride moiety, a diglyceride moiety, a sterol moiety, a sterol ester moiety, a free fatty acid moiety, a phospholipid moiety, and combinations thereof, comprises about 0.1 wt % to about 5 wt %, about 0.1 wt % to less than about 5 wt %, about 0.1 wt % to about 4 wt %, about 0.1 wt % to about 3 wt %, about 0.1 wt % to about 2 wt %, about 0.2 wt % to about 5 wt %, about 0.2 ... .2 wt % to about 4 wt %, about 0.2 wt % to about 3 wt %, about 0.2 wt % to about 2 wt %, about 0.3 wt % to about 2 wt %, about 0.1 wt % to about 0.5 wt %, about 0.2 wt % to about 0.5 wt %, about 0.1 wt % to about 0.4 wt %, about 0.2 wt % to about 0.4 wt %, about 0.5 wt % to about 2 wt %, about 1 wt % to about 2 wt %, about 0.5 wt % to about 1.5 wt %, or about 1 wt % to about 1.5 wt % ARA. In some embodiments, microbial oil and / or it is selected from one or more parts and their combination in triglyceride moiety, diglyceride moiety, sterol moiety, sterol ester moiety, free fatty acid moiety, the phospholipid part, comprise about 5 weight % or still less, be lower than about 5 weight %, about 4 weight % or still less, about 3 weight % or still less, about 2 weight % or still less, about 1.5 weight % or still less, about 1 weight % or still less, about 0.5 weight % or still less, about 0.4 weight % or still less, about 0.3 weight % or still less, about 0.2 weight % or still less or about 0.1 weight % or ARA still less.In some embodiments, microbial oil and / or it is selected from one or more parts and their combination in triglyceride moiety, diglyceride moiety, sterol moiety, sterol ester moiety, free fatty acid moiety, the phospholipid part, does not contain ARA substantially. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a triglyceride portion, a diglyceride portion, a sterol portion, a sterol ester portion, a free fatty acid portion, a phospholipid portion, and combinations thereof, comprises about 0.4 wt % to about 2 wt %, about 0.4 wt % to about 3 wt %, about 0.4 wt % to about 4 wt %, about 0.4 wt % to about 5 wt %, about 0.4 wt % to less than about 5 wt %, about 0.5 wt % to about 1 wt %, about 0.5 wt % to about 2 wt %, about 0.5 wt % to about 3 wt %, about 0.5 wt % to about 4 wt %, about 0.5 wt % to about 5 wt %, about 0.5 wt % to less than about 5 wt %, about 1 wt % to about 2 wt %, about 1 wt % to about 3 wt %, about 1 wt % to about 4 wt %, about 1 wt % to about 5 wt %, or about 1 wt % to about 5 wt % or less DPA n-6.In some embodiments, the microbial oil and / or one or more moieties selected from the triglyceride moiety, the diglyceride moiety, the sterol moiety, the sterol ester moiety, the free fatty acid moiety, the phospholipid moiety, and combinations thereof, comprises about 5 wt%, less than about 5 wt%, about 4 wt% or less, about 3 wt% or less, about 2 wt% or less, about 1 wt% or less, about 0.75 wt% or less, about 0.6 wt% or less, or about 0.5 wt% or less of DPA n-6. In some embodiments, the microbial oil and / or one or more moieties selected from the triglyceride moiety, the diglyceride moiety, the sterol moiety, the sterol ester moiety, the free fatty acid moiety, the phospholipid moiety, and combinations thereof, are substantially free of DPA n-6. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a triglyceride portion, a diglyceride portion, a sterol portion, a sterol ester portion, a free fatty acid portion, a phospholipid portion, and combinations thereof, comprises fatty acids having about 5 wt % or less, less than about 5 wt %, about 4 wt % or less, about 3 wt % or less, or about 2 wt % or less of oleic acid (18:1n-9), linolenic acid (18:2n-6), linolenic acid (18:3n-3), eicosenoic acid (20:1n-9), sinapic acid (22:1n-9), octadecatetraenoic acid (18:4n-3), or a combination thereof.

[0294] The triglyceride molecule contains 3 central carbon atoms (C(sn-1)H2R1-(sn-2)H2R2-C(sn-3)H2R3) so that different positional isomers can be formed. In some embodiments, the microbial oil comprises a triglyceride portion, wherein based on the relative area percentage of the peak on the HPLC chromatogram, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 35% or at least about 40% of the triglyceride in the triglyceride portion contains DHA (disubstituted DHA) in two positions in any two of the triglycerides selected from sn-1, sn-2 and sn-3 positions. In some embodiments, the microbial oil comprises a triglyceride fraction, wherein the triglyceride fraction is from about 2% to about 55%, from about 2% to about 50%, from about 2% to about 45%, from about 2% to about 40%, from about 2% to about 35%, from about 2% to about 30%, from about 2% to about 25%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 2% to about 30%, from about 2% to about 25%, from about 5% to about 55%, from about 5% to about 50%, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%, from about 5% to about 30%, In some embodiments, the microbial oil comprises a triglyceride fraction wherein at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the triglycerides in the triglyceride fraction contain DHA (trisubstituted DHA) at two positions selected from the sn-1, sn-2, and sn-3 positions, based on the relative area percentages of the peaks on the HPLC chromatogram. In some embodiments, the microbial oil comprises a triglyceride fraction, wherein about 0.5% to about 5%, about 0.5% to about 3%, about 0.5% to about 2.5%, about 0.5% to about 2%, about 1% to about 5%, about 1% to about 3%, or about 1% to about 2% of the triglycerides in the triglyceride fraction contain DHA at all sn-1, sn-2, and sn-3 positions based on the relative area percentages of the peaks on the HPLC chromatogram.In some embodiments, the microbial oil comprises a triglyceride portion, wherein at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% of the triglycerides in the triglyceride portion contain DHA at one position in the triglycerides selected from any one of the sn-1, sn-2, or sn-3 positions, based on the relative area percentage of the peaks on the HPLC chromatogram. In some embodiments, the microbial oil comprises a triglyceride portion wherein about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 35% to about 80%, about 35% to about 75%, about 35% to about 65%, about 35% to about 60%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, or about 40% to about 55% of the triglycerides in the triglyceride portion contain DHA at one position of the triglyceride selected from any one of the sn-1, sn-2, and sn-3 positions, based on the relative area percentage of the peaks on the HPLC chromatogram.

[0295] The present invention also relates to methods for producing microbial oils. In some embodiments, the method comprises growing the thraustochytrids of the present invention in culture to produce biomass, and extracting oil comprising ω-3 fatty acids from the biomass. The oil can be extracted from freshly collected biomass, or can be extracted from previously collected biomass stored under conditions to prevent deterioration. Known methods can be used to cultivate the thraustochytrids of the present invention, separate biomass from culture, extract microbial oil from biomass, and analyze the fatty acid profile of the oil extracted from the biomass. See, e.g., U.S. Pat. No. 5,130,242.

[0296] The invention further relates to microbial oils comprising the fatty acid distribution of the present invention. The microbial oil of the present invention can be any oil derived from a microorganism, for example: a crude oil extracted from the biomass of the microorganism and not further processed; a refined oil obtained by treating a crude microbial oil with further processing steps such as refining, decolorizing and / or deodorizing; a diluted oil obtained by diluting a crude microbial oil or a refined microbial oil; or an enriched oil obtained by, for example, treating a crude microbial oil or a refined microbial oil with an increase in the concentration of fatty acids (e.g., DHA) in the oil by adopting other purification methods.

[0297] In some embodiments, microbial oil comprises about 0 % by weight, at least about 0.1 % by weight, at least about 0.2 % by weight, at least about 0.5 % by weight, at least about 1 % by weight, at least about 1.5 % by weight, at least about 2 % by weight or at least about 5 % by weight of sterol ester moiety.In some embodiments, microbial oil comprises about 0 % by weight to about 1.5 % by weight, about 0 % by weight to about 2 % by weight, about 0 % by weight to about 5 % by weight, about 1 % by weight to about 1.5 % by weight, about 0 % by weight to about 1.5 % by weight, about 0 % by weight to about 2 % by weight, or about 0.2 % by weight to about 5 % by weight of sterol ester moiety.In some embodiments, microbial oil comprises and is less than about 5 % by weight, is less than about 4 % by weight, is less than about 3 % by weight or is less than about 2 % by weight of sterol ester moiety. In some embodiments, microbial oil comprises at least about 65 % by weight, at least about 70 % by weight, at least about 75 % by weight, at least about 80 % by weight, at least about 85 % by weight or at least about the triglyceride moiety of 90 % by weight.In some embodiments, microbial oil comprises about 65 % by weight to about 95 % by weight, about 75 % by weight to about 95 % by weight or about 80 % by weight to about 95 % by weight or about 97 % by weight or about 98 % by weight.In some embodiments, microbial oil comprises at least about 0.5 % by weight, at least about 1 % by weight, at least about 1.5 % by weight, at least about 2 % by weight, at least about 2.5 % by weight or at least about the free fatty acid moiety of 5 % by weight. In some embodiments, microbial oil comprises the free fatty acid moiety of about 0.5 % by weight to about 5 % by weight, about 0.5 % by weight to about 2.5 % by weight, about 0.5 % by weight to about 2 % by weight, about 0.5 % by weight to about 1.5 % by weight, about 0.5 % by weight to about 1 % by weight, about 1 % by weight to about 2.5 % by weight, about 1 % by weight to about 5 % by weight, about 1.5 % by weight to about 2.5 % by weight, about 2 % by weight to about 2.5 % by weight or about 2 % by weight to about 5 % by weight.In some embodiments, microbial oil comprises and is less than about 5 % by weight, is less than about 4 % by weight, is less than about 3 % by weight, is less than about 2 % by weight or is less than about 1 % by weight of free fatty acid moiety.In some embodiments, microbial oil comprises at least about 0.5 % by weight, at least about 1 % by weight, at least about 1.5 % by weight, at least about 2 % by weight or at least about 5 % by weight of sterol moiety. In some embodiments, microbial oil comprises the sterol moiety of about 0.5 % by weight to about 1.5 % by weight, about 1 % by weight to about 1.5 % by weight, about 0.5 % by weight to about 2 % by weight, about 0.5 % by weight to about 5 % by weight, about 1 % by weight to about 2 % by weight or about 1 % by weight to about 5 % by weight. In some embodiments, microbial oil comprises the sterol moiety of being less than about 5 % by weight, being less than about 4 % by weight, being less than about 3 % by weight, being less than about 2 % by weight or being less than about 1 % by weight.In some embodiments, microbial oil comprises at least about 1.5 % by weight, at least about 2 % by weight, at least about 2.5 % by weight, at least about 3 % by weight, at least about 3.5 % by weight or at least about 5 % by weight of diglyceride moiety. In some embodiments, microbial oil comprises about 1.5 % by weight to about 3 % by weight, about 2 % by weight to about 3 % by weight, about 1.5 % by weight to about 3.5 % by weight, about 1.5 % by weight to about 5 % by weight, about 2.5 % by weight to about 3 % by weight, about 2.5 % by weight to about 3.5 % by weight or about 2.5 % by weight to about 5 % by weight of diglyceride moiety. In some embodiments, microbial oil comprises and is less than about 2 % by weight, is less than about 1.5 % by weight, is less than about 1 % by weight or is less than the unsaponifiable matter of about 0.5 % by weight oil weight. Lipid types present in microbial oil, such as triglyceride moiety, can be separated by flash chromatography (flash chromatography) and analyzed by thin layer chromatography (TLC) or separated and analyzed by other methods known in the art.

[0298] In some embodiments, the microbial oil and / or one or more portions thereof selected from the triglyceride portion, the free fatty acid portion, the sterol portion, the diglyceride portion, and combinations thereof, comprises at least about 40 wt%, at least about 45 wt%, at least about 50 wt%, at least about 55 wt%, at least about 60 wt%, at least about 65 wt%, at least about 70 wt%, at least about 75 wt%, or at least about 80 wt% DHA. In some embodiments, the microbial oil and / or one or more portions thereof selected from the triglyceride portion, the free fatty acid portion, the sterol portion, the diglyceride portion, and combinations thereof, comprises about 40% to about 45%, about 40% to about 50%, about 40% to about 60%, about 50% to about 60%, about 55% to about 60%, about 40% to about 65%, about 50% to about 65%, about 55% to about 65%, about 40% to about 70%, about 40% to about 80%, about 50% to about 80%, about 55% to about 80%, about 60% to about 80%, or about 70% to about 80% DHA by weight. In some embodiments, microbial oil comprises the sterol ester portion of DHA that contains about 45 % by weight or less, about 40 % by weight or less, about 35 % by weight or less, about 30 % by weight or less, about 25 % by weight or less, about 20 % by weight or less, about 15 % by weight or less or about 13 % by weight or less.In some embodiments, microbial oil and / or it is selected from one or more parts in triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety and their combination, comprise about 10 % by weight or less, about 9 % by weight or less, about 8 % by weight or less, about 7 % by weight or less, about 6 % by weight or less, about 5 % by weight or less, about 4 % by weight or less, about 3 % by weight or less, about 2 % by weight or less or about 1 % by weight or less EPA. In some embodiments, microbial oil and / or it is selected from one or more parts and their combinations in triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety, comprise about 2 wt % to about 3 wt %, about 2 wt % to about 3.5 wt %, about 2.5 wt % to about 3.5 wt %, about 2 wt % to about 6 wt %, about 2.5 wt % to about 6 wt %, about 3.0 wt % to about 6 wt %, about 3.5 wt % to about 6 wt %, about 5 wt % to about 6 wt %, or about 2 wt % to about 10 wt % EPA. In some embodiments, microbial oil and / or it is selected from one or more parts and their combinations in sterol ester moiety, triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety, polar part (comprising phospholipid moiety), substantially free of EPA.In some embodiments, microbial oil and / or it is selected from one or more parts of sterol ester part, triglyceride part, free fatty acid part, sterol part, diglyceride part, polar part (comprising phospholipid part) and their combination, comprise at least about 5:1, at least about 7:1, at least about 9:1, at least about 10:1, at least about 15:1, at least about 20:1, at least about 25:1, at least about 30:1 or at least about 50:1 DHA and EPA weight ratio, wherein microbial oil and / or it is one or more parts comprise 10 weight % or EPA still less.In some embodiments, microbial oil and / or it is selected from one or more parts of sterol ester part, triglyceride part, free fatty acid part, sterol part, diglyceride part, polar part (comprising phospholipid part) and their combination, comprise at least about 5:1 but less than about 20:1 DHA and EPA weight ratio. In some embodiments, the weight ratio of DHA to EPA is about 5: 1 to about 18: 1, about 7: 1 to about 16: 1, or about 10: 1 to about 15: 1. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of sterol ester portions, triglyceride portions, free fatty acid portions, sterol portions, diglyceride portions, polar portions (including phospholipid portions), and combinations thereof, comprises about 0.1 wt % to about 0.25 wt %, about 0.2 wt % to about 0.25 wt %, about 0.1 wt % to about 0.5 wt %, or about 0.1 wt % to about 1.5 wt % ARA. In some embodiments, microbial oil and / or it is selected from one or more parts and their combination of sterol ester moiety, triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety, polar part (comprising phospholipid moiety), comprises about 1.5 % by weight or still less, about 1 % by weight or still less, about 0.5 % by weight or still less, about 0.2 % by weight or still less or about 0.1 % by weight or ARA still less.In some embodiments, microbial oil and / or it is selected from one or more parts and their combination of sterol ester moiety, triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety, polar part (comprising phospholipid moiety), does not contain ARA substantially. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a sterol ester portion, a triglyceride portion, a free fatty acid portion, a diglyceride portion, a polar portion (including a phospholipid portion), and combinations thereof, comprises a weight ratio of DMA to ARA of at least about 20:1, at least about 30:1, at least about 35:1, at least about 40:1, at least about 60:1, at least about 80:1, at least about 100:1, at least about 150:1, at least about 200:1, at least about 250:1, or at least about 300:1.In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a sterol ester portion, a triglyceride portion, a free fatty acid portion, a sterol portion, a diglyceride portion, a polar portion (including a phospholipid portion), and combinations thereof, comprises about 0.5 wt % to about 1 wt %, about 0.5 wt % to about 2 wt %, about 0.5 wt % to about 2.5 wt %, about 0.5 wt % to about 3 wt %, about 0.5 wt % to about 3.5 wt %, about 0.5 wt % to about 5 wt %, about 0.5 wt % to about 6 wt %, about 1 wt % to about 2 wt %, about 2 wt % to about 3 wt %, about 2 wt % to about 3.5 wt %, about 1 wt % to about 2.5 wt %, about 1 wt % to about 3 wt %, about 1 wt % to about 3.5 wt %, about 1 wt % to about 5 wt %, or about 1 wt % to about 6 wt % DPA n-6. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of sterol ester moieties, triglyceride moieties, free fatty acid moieties, sterol moieties, diglyceride moieties, polar moieties (including phospholipid moieties), and combinations thereof, comprises about 6 wt % or less, about 5 wt % or less, about 3 wt % or less, about 2.5 wt % or less, about 2 wt % or less, about 1 wt % or less, or about 0.5 wt % or less DPA n-6. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of sterol ester moieties, triglyceride moieties, free fatty acid moieties, sterol moieties, diglyceride moieties, polar moieties (including phospholipid moieties), and combinations thereof, is substantially free of DPA n-6. In some embodiments, the microbial oil and / or one or more portions thereof selected from the group consisting of a sterol ester portion, a triglyceride portion, a free fatty acid portion, a sterol portion, a diglyceride portion, a polar portion (including a phospholipid portion), and combinations thereof, comprises a weight ratio of DHA to DPA n-6 of greater than about 6:1, at least about 8:1, at least about 10:1, at least about 15:1, at least about 20:1, at least about 25:1, at least about 50:1, or at least about 100:1. In some embodiments, the microbial oil and / or one or more portions thereof selected from a sterol ester portion, a triglyceride portion, a free fatty acid portion, a sterol portion, a diglyceride portion, a polar portion (including a phospholipid portion), and combinations thereof, contains about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, about 2% by weight or less, about 1.5% by weight or less, about 1% by weight or less, or about 0.5% by weight or less of each of linoleic acid (18:2n-6), linolenic acid (18:3n-3), eicosenoic acid (20:1n-9), and sinapic acid (22:1n-9).In some embodiments, microbial oil and / or one or more parts thereof selected from sterol ester moiety, triglyceride moiety, free fatty acid moiety, sterol moiety, diglyceride moiety, polar moiety (comprising phospholipid moiety) and their combination, comprise about 5 wt % or less, about 4 wt % or less, about 3 wt % or less, about 2 wt % or less, about 1.5 wt % or less or about 1 wt % or less heptadecanoic acid (17:0). In some embodiments, microbial oil and / or one or more parts thereof comprise about 0.01 wt % to about 5 wt %, about 0.05 wt % to about 3 wt % or about 0.1 wt % to about 1 wt % heptadecanoic acid.

[0299] The triglyceride molecule contains three central carbon atoms (C sn-1 H2R1-C sn-2 H2R2-C sn-3H2R3), so that different positional isomers can be formed.In some embodiments, microbial oil comprises triglyceride moiety, wherein based on the relative area percentage at the peak on the HPLC chromatogram, at least about 20%, at least about 30%, at least about 35% or at least about 40% triglyceride in the triglyceride moiety contains DHA (disubstituted DHA) in two positions in any two triglycerides selected from sn-1, sn-2 and sn-3 positions.In some embodiments, microbial oil comprises triglyceride moiety, wherein based on the relative area percentage at the peak on the HPLC chromatogram, about 20% to about 40%, about 20% to about 35%, about 30% to about 40%, about 30% to about 35% triglyceride in the triglyceride moiety contains DHA in two positions in any two triglycerides selected from sn-1, sn-2 or sn-3 positions. In some embodiments, the microbial oil comprises a triglyceride portion, wherein at least about 5%, at least about 10%, at least about 15%, or at least about 20% of the triglycerides in the triglyceride portion contain DHA (trisubstituted DHA) at all sn-1, sn-2, and sn-3 positions based on the relative area percentage of the peak on the HPLC chromatogram. In some embodiments, the microbial oil comprises a triglyceride portion, wherein about 5% to about 20%, about 5% to about 15%, about 10% to about 20%, or about 10% to about 15% of the triglycerides in the triglyceride portion contain DHA at all sn-1, sn-2, and sn-3 positions based on the relative area percentage of the peak on the HPLC chromatogram. In contrast, the TAG species reported in U.S. Patent No. 6,582,941 do not contain DHA at all three positions. In some embodiments, the microbial oil comprises a triglyceride portion, wherein at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the triglycerides in the triglyceride portion contain DHA at one position in the triglyceride selected from any one of the sn-1, sn-2, or sn-3 positions, based on the relative area percentage of the peaks on the HPLC chromatogram. In some embodiments, the microbial oil comprises a triglyceride portion, wherein about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 60% to about 75%, about 60% to about 70%, or about 60% to about 65% of the triglycerides in the triglyceride portion contain DHA at one position in the triglyceride selected from any one of the sn-1, sn-2, and sn-3 positions, based on the relative area percentage of the peaks on the HPLC chromatogram.

[0300] Composition

[0301] The present invention relates to a composition comprising the microorganism of the present invention, the isolated biomass of the present invention, the microbial oil of the present invention, or a combination thereof.

[0302] Based on the requirements of the composition, the microorganisms, biomass or microbial oil of the present invention can also be chemically modified or physically modified or processed by any known technology.

[0303] Prior to use in the composition, the microbial cells or biomass can be dried by a variety of methods, including but not limited to freeze drying, air drying, spray drying, tunnel drying, vacuum drying (freeze vacuum drying) and similar methods. Alternatively, the harvested and washed biomass can be used in the composition without drying. See, for example, U.S. Pat. Nos. 5,130,242 and 6,812,009, both of which are incorporated herein by reference in their entirety.

[0304] Can use microbial oil of the present invention as starting raw material, produce the product that is rich in lipid acid such as EPA more efficiently.For example, microbial oil of the present invention can carry out various purification techniques as known in the art, such as distillation or urea inclusion, produces the higher product of the usefulness of EPA or other lipid acid with higher concentration.Microbial oil of the present invention also can be used in the chemical reaction, produces the derived compound from the lipid acid in the oil, such as ester and salt of EPA or other lipid acid.

[0305] The composition of the present invention may include one or more excipients. When used herein, "excipient" refers to a component or a mixture of components used in the composition of the present invention to give the composition desired characteristics, and the composition includes food and pharmaceutical compositions, cosmetic compositions and industrial compositions. When added to a pharmaceutical composition, the excipient of the present invention can be described as a "pharmaceutically acceptable" excipient, which means that the excipient is a compound, material, composition, salt and / or dosage form that is suitable for contacting with the tissues of humans and non-human animals within the scope of correct medical judgment, and does not have excessive toxicity, irritation, allergic reactions or other problematic complications during the expected contact period commensurate with a reasonable benefit / risk ratio. In some embodiments, the term "pharmaceutically acceptable" refers to approved by the competent authority of the federal or state government, or listed in the American Pharmacopoeia or other generally recognized international pharmacopoeias as used for animals and more specifically for people. Various excipients can be used. In some embodiments, excipient can be but not limited to alkali agent, stabilizer, antioxidant, adhesive, separating agent, coating agent, external phase component, controlled release component, solvent, surfactant, wetting agent, buffer, filler, softener or its combination.Except as discussed herein, excipient can also include but not limited to the excipient listed in Remington:The Scienceand Practice of Pharmacy the 21st edition (2005).Excipient is included in the specific category (such as " solvent ") in this article and is intended to illustrate but not limit the effect of excipient.Concrete excipient can belong to multiple categories.

[0306] The compositions of the present invention include, but are not limited to, edible products, pharmaceutical compositions, cosmetics, and industrial compositions.

[0307] In some embodiments, the present composition is an edible product. An edible product is any food for consumption by non-human animals or humans, and includes solid and liquid compositions. An edible product can be an additive in animal or human food. Food includes, but is not limited to, general food; liquid products include milk, beverages, therapeutic drinks and nutritional drinks; functional foods; supplements; nutritional medicines; infant formula, including formula for premature babies; food for pregnant or breastfeeding women; adult food; elderly food; and animal food.

[0308] In some embodiments, the microorganisms, biomass or microbial oils of the present invention can be used directly as one or more of the following substances or as additives included in one or more of the following substances: oils, shortenings, spreads, other fat ingredients, beverages, sauces, milk-based or soybean-based foods (such as milk, yogurt, cheese and ice cream), baked goods, nutritional products such as finished or semi-finished products as nutritional supplements (capsules or tablets), vitamin supplements, dietary supplements, powdered drinks and powdered edible products. In some embodiments, the nutritional supplement is in the form of a vegetarian capsule, which is not formed by any component of animal origin and does not contain any component from animal origin.

[0309] A partial list of food compositions that can include the microbial oil of the present invention includes, but is not limited to, soy-based products (milk, ice cream, yogurt, beverages, fresh cream, spreads, creamers); soups and soup mixes; doughs, batters and baked goods, including, for example, fine pastry products, breakfast cereals, cakes, cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, muffins (biscuits), muffins, pastries, scones (scone), croutons, crackers, desserts, snack cakes, pies, granola / snack bars and shortbread; candy; hard candy desserts; chocolate and other candy desserts; chewing gum; liquid edible products such as milk, energy drinks, infant formula, carbonated drinks, tea, liquid meals, juices, fruit-based drinks, vegetable-based drinks; multivitamin syrups, diet food substitutes, medicinal foods and syrups; powdered drink mix ingredients; pasta; processed fish products; processed meat products; processed poultry products; gravies and sauces; seasonings (ketchup, mayonnaise, etc.); vegetable oil-based spreads; dairy products; yogurt; butter; frozen dairy products; ice cream; frozen desserts; frozen yogurt; semi-solid edible products such as baby food; puddings and gelatin desserts; processed and unprocessed cheeses; pancake ingredients; food bars including energy bars, waffle ingredients; salad dressings; egg substitute ingredients; nuts and nut-based spreads; salted snacks such as potato chips and other crisps or crisps, corn chips, nachos, puffed snacks, popcorn, pretzels, French fries and nuts; and specialty snacks such as dips, dried fruit snacks, meat snacks, pork rinds, health food bars and rice / corn cakes.

[0310] In some embodiments, the microbial oil of the present invention can be used to supplement infant formula. Infant formula can be supplemented with the microbial oil of the present invention or with a physical refined oil combination obtained by a microorganism producing eicosatetraenoic acid (ARA). The microorganism producing ARA is, for example, Mortierella alpina or Mortierella sect. Schmuckeri. Alternatively, the microbial oil of the present invention can be combined with an oil rich in ARA (including (Martek Biosciences, Columbia, MD)) combination to supplement infant formula.

[0311] In some embodiments, the composition is an animal feed. "Animal" includes non-human organisms belonging to the kingdom Animalia, and includes, but is not limited to, aquatic and terrestrial animals. The term "animal feed" or "animal food" means any food intended for non-human animals, whether or not for fish; commercial fish; ornamental fish; fry; shellfish; molluscs; crustaceans; shellfish; shrimp; postlarvae; Artemia; rotifers; brine shrimp; filter feeders; amphibians; reptiles; mammals; livestock; farm animals; zoo animals; sport animals; breeding stock; competition animals; performance animals; heirloom animals; rare or endangered animals; companion animals. Animals; pets such as dogs, cats, guinea pigs, rabbits, rats, mice or horses; primates such as monkeys (e.g., capuchins, rhesus monkeys, African green monkeys, erythrocytes, cynomolgus monkeys and sylvanian monkeys), apes, orangutans, baboons, gibbons and chimpanzees; canines such as dogs and wolves; felines such as cats, lions and tigers; equines such as horses, donkeys and zebras; animals for food such as cows, cattle, pigs and sheep; ungulates such as deer and giraffes; or rodents such as mice, rats, hamsters and guinea pigs, etc. Animal feed includes, but is not limited to, feed for aquaculture, livestock feed including pet feed, zoo animal feed, draft animal feed, livestock feed and combinations thereof.

[0312] In some embodiments, the composition is a feed or feed supplement for any animal whose meat or products are consumed by humans, such as any animal from which meat, eggs or milk are obtained for human consumption. When fed to these animals, nutrients such as LC-PUFAs can be incorporated into the meat, milk, eggs or other products of these animals to increase the content of these nutrients in them.

[0313] In some embodiments, the composition is a spray-dried material that can be broken up to form particles of a suitable size for zooplankton, Artemia, rotifers, and filter feeders. In some embodiments, the zooplankton, Artemia, or rotifers fed with the composition are used to feed larvae, fish, shellfish, shellfish, or crustaceans, respectively.

[0314] In some embodiments, the composition is a pharmaceutical composition. Suitable pharmaceutical compositions include, but are not limited to, anti-inflammatory compositions, drugs for the treatment of coronary heart disease, drugs for the treatment of arteriosclerosis, chemotherapeutic agents, active excipients, osteoporosis drugs, antidepressants, anticonvulsants, anti-Helicobacter pylori drugs, drugs for the treatment of neurodegenerative diseases, drugs for the treatment of degenerative liver diseases, antibiotics, cholesterol-lowering compositions, and triglyceride-lowering compositions. In some embodiments, the composition is a medicinal food. Medicinal food includes foods in a composition taken or applied externally under the supervision of a physician and foods intended for specific dietary management of a condition, the unique nutritional requirements of which are established by medical evaluation based on recognized scientific principles.

[0315] In some embodiments, the microbial oil can be formulated into a dosage form. The dosage form can include, but is not limited to, tablets, capsules, cachets, pellets, pills, powders and granules and parenteral dosage forms containing an effective amount of the microbial oil, including, but not limited to, solutions, suspensions, emulsions and dry powders. It is also known in the art that these formulations can also include pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic carriers, water-soluble carriers, emulsifiers, buffers, humectants, wetting agents, cosolvents, preservatives, etc. The dosage form can include, but is not limited to, tablets, lozenges, capsules, caplet-shaped tablets and pills containing microbial oil and one or more suitable pharmaceutically acceptable carriers.

[0316] For oral administration, microbial oil can be combined with pharmaceutically acceptable carriers known in the art. These carriers can be formulated into tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by individuals to be treated. In some embodiments, the dosage form is a tablet, a pill, or a capsule-shaped tablet. Pharmaceutical preparations for oral use can be obtained by adding solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants if necessary to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers, such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations, such as, but not limited to, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrants may be added, such as, but not limited to, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Pharmaceutical formulations for oral use include, but are not limited to, push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In some embodiments, the dosage form is a vegetarian dosage form that is not formed from, and does not contain, any component of animal origin. In some embodiments, the vegetarian dosage form is a vegetarian capsule.

[0317] In some embodiments, the composition is a cosmetic. Cosmetics include, but are not limited to, emulsions, creams, lotions, facial masks, soaps, shampoos, detergents, creams, conditioners, makeup, bath agents, and dispersions. Cosmetic agents may be medicinal or non-medicinal.

[0318] In some embodiments, the composition is an industrial composition. In some embodiments, the composition is a starting material for one or more articles. Articles include, but are not limited to, polymers, photographic sensitive materials, detergents, industrial oils, or industrial detergents. For example, U.S. Pat. No. 7,259,006 describes the use of fats and oils containing DHA in the production of behenic acid and the use of behenic acid to produce photographic sensitive materials.

[0319] Methods of using the composition

[0320] In some embodiments, the composition can be used to treat a condition in a human or non-human animal. In some embodiments, the composition can be used for nutritional purposes in a human or non-human animal.

[0321] The terms "treatment" and "therapeutic effect" refer to therapeutic treatment and prophylactic or preventative measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disease, or disorder, or to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, mitigating or eliminating symptoms or signs associated with a condition, disease, or disorder; reducing the extent of a condition, disease, or disorder; stabilizing a condition, disease, or disorder (i.e., a condition, disease, or disorder that no longer worsens); delaying the onset or development of a condition, disease, or disorder; ameliorating a condition, disease, or disorder; relieving a condition, disease, or disorder (whether partially or completely, whether detectably or undetectably); or enhancing or ameliorating a condition, disease, or disorder. A therapeutic effect includes inducing a clinically significant response without excessive side effects. A therapeutic effect also includes prolonging survival compared to the expected survival time if not receiving treatment.

[0322] In some embodiments, the composition is used to treat a condition, disease or disorder such as acne, acute inflammation, age-related maculopathy, allergies, Alzheimer's disease, arthritis, asthma, atherosclerosis, autoimmune disease, dyslipidemia, breast cysts, cachexia, cancer, cardiac restenosis, cardiovascular disease, chronic inflammation, coronary heart disease, cystic fibrosis, degenerative disorders of the liver, diabetes, eczema, gastrointestinal disorders, heart disease, high triglyceride levels, hypertension, hyperactivity, immune diseases, inhibiting tumor growth, inflammatory conditions, intestinal disorders, renal dysfunction, leukemia, severe depression, multiple sclerosis, neurodegenerative diseases, osteoarthritis, osteoporosis, peroxisomal disorders, preeclampsia, premature labor, psoriasis, pulmonary disorders, rheumatoid arthritis, heart disease risk or thrombosis.

[0323] In some embodiments, the composition is used to increase the length of gestation of a fetus in the third trimester.

[0324] In some embodiments, the composition is used to control blood pressure.

[0325] In some embodiments, the composition is for improving or maintaining cognitive function.

[0326] In some embodiments, the composition is used to improve or maintain memory.

[0327] The composition or dosage form can be administered to the body of an individual by any route compatible with the composition or dosage form. If a substance is introduced into the body of an individual by an individual, or if a substance is introduced into the body of an individual by another person, a machine or a device, the substance is considered to be "administered". Therefore, "administration" includes self-administration, administration by another person and indirect administration. When used herein, the term "continuous" or "continuous" related to "administration" means that the frequency of administration is at least once a day. However, please note that the frequency of administration can be higher than once a day and still be "continuous" or "continuous", such as twice or even three times a day, as long as the dosage level specified herein is not exceeded. The means and methods for administration are known in the art, and the technician can refer to various pharmacological literature for guidance. For example, "Modern Pharmaceutics," Banker & Rhodes, Informa Healthcare, USA, 4th edition (2002) and "Goodman & Gilman's The Pharmaceutical Basis of Therapeutics," McGraw-Hill Companies, Inc., New York, 10th edition (2001) can be consulted.

[0328] "Subject", "individual" or "patient" refers to any individual, human or non-human, for whom diagnosis, prognosis, treatment or administration of a composition or dosage form is desired. Mammalian subjects include, but are not limited to, humans; livestock; farm animals; zoo animals; sport animals; pets such as dogs, cats, guinea pigs, rabbits, rats, mice or horses; primates such as monkeys (e.g., capuchins, rhesus monkeys, African green monkeys, erythrocebus, cynomolgus monkeys and sylvatic monkeys), apes, orangutans, baboons, gibbons and chimpanzees; canines such as dogs and wolves; felines such as cats, lions and tigers; equines such as horses, donkeys and zebras; animals for food such as cows, cattle, pigs and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; etc. The term subject also encompasses model animals, such as disease model animals. In some embodiments, the term individual includes an economically or otherwise valuable animal, such as an economically important breeding stock, a racing animal, a show animal, a heirloom animal, a rare or endangered animal, or a companion animal. In certain embodiments, the individual is a human individual. In certain embodiments, the individual is a non-human individual.

[0329] The composition can be administered in a "nutritional amount", "therapeutically effective amount", "prophylactically effective amount", "therapeutic dose" or "prophylactic dose". "Nutritional amount" refers to an amount effective to achieve the desired nutritional result under the conditions of the required dosage and time period. The nutritional result can be, for example, an increase in the level of a desired fatty acid component in an individual. "Therapeutically effective amount" or "therapeutic dose" refers to an amount effective to achieve the desired therapeutic result under the conditions of the required dosage and time period. The therapeutic result can be, for example, relief of symptoms, prolonged survival, improved mobility, etc. The therapeutic result is not necessarily a "cure". "Prophylactically effective amount" or "prophylactic dose" refers to an amount effective to achieve the desired preventive result under the conditions of the required dosage and time period. Generally, because the preventive dose is used in individuals before the disease or in an earlier stage, the preventive effective amount will be lower than the therapeutic effective amount used to treat advanced disease.

[0330] Based on the amount of EPA or other fatty acid components of the microorganism, biomass or microbial oil to be administered to an individual, various doses of composition, dosage form or pharmaceutical composition can be administered to an individual. The terms "daily dose", "daily dose level" and "daily dose" herein refer to the total amount of EPA or other fatty acid components administered daily (per 24 hours). Therefore, for example, EPA is administered to an individual with a daily dose of 2 mg, meaning that no matter EPA is administered in a single dosage form comprising 2 mg EPA, or alternatively in four dosage forms (2 mg EPA in total) each containing 0.5 mg EPA, the individual receives a total of 2 mg of EPA on a daily basis. In some embodiments, the daily EPA amount is administered in a single dosage form or in two dosage forms. The dosage form of the present invention can be applied in a single application or in multiple applications. For example, if four tablets are taken daily, each containing 0.5 mg EPA, all four tablets can be taken once a day, or 2 tablets are taken twice a day, or 1 tablet is taken every 6 hours. In some embodiments, the daily dose is about 100 mg to about 15 g of EPA. In some embodiments, the daily dose is about 0.5 mg to about 250 mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about 100 mg to about 1 g, about 1 g to about 2.5 g, about 1 g to about 5 g, about 1 g to about 10 g, about 1 g to about 15 g, about 5 g to about 10 g, about 5 g to about 15 g, about 10 g to about 15 g, about 100 mg to about 10 g, about 100 mg to about 5 g, or about 100 mg to about 2.5 g of EPA, DHA, or a combination thereof. In some embodiments, the composition is a dosage form comprising about 0.5 mg to about 250 mg, 100 mg to about 250 milligrams, about 0.5 mg to about 500 mg, about 100 mg to about 500 mg, about 0.5 mg to about 1 g, or about 100 mg to about 1 g of EPA, DHA, or a combination thereof per dosage form.

[0331] Various methods can be used to achieve the administration of the composition or dosage form of the present invention. For example, in some embodiments, administration is performed daily in succession, or administration is performed every other day (once every two days). Administration can be performed on one day or multiple days.

[0332] The administration of the composition and dosage form can be combined with other methods for treating the condition. For example, the method of the present invention can be combined with diet therapy (such as low carbohydrate diet, high protein diet, high fiber diet, etc.), exercise therapy, weight loss therapy, smoking cessation therapy or a combination thereof. The method of the present invention can also be used in combination with other pharmaceutical products for treating the condition. The composition or dosage form of the present invention can be administered before or after other therapies or pharmaceutical products.

[0333] Kits containing compositions

[0334] The present invention relates to a test kit or packaging containing one or more units of the composition of the present invention. The test kit or packaging can include a food unit, a pharmaceutical composition unit, a cosmetic composition unit or an industrial composition unit containing a microorganism, a biomass or a microbial oil of the present invention or a combination thereof. The test kit or packaging can also include an additive containing a microorganism, a biomass or a microbial oil of the present invention or a combination thereof, which is used to prepare a food, a cosmetic composition, a pharmaceutical composition or an industrial composition.

[0335] In some embodiments, the kit or packaging comprises one or more units of the pharmaceutical composition to be administered according to the method of the present invention. The kit or packaging may comprise one dosage unit or more than one dosage unit (i.e., multiple dosage units). If multiple dosage units are present in the kit or packaging, multiple dosage units may be optionally arranged to be administered in sequence.

[0336] The kit of the present invention may optionally include instructions related to the units or dosage forms of the kit. Such instructions may be in the form specified by a government agency that manages the manufacture, use or sale of pharmaceutical products, which notification reflects the agency's approval for manufacture, use or sale for human administration to treat a condition or illness. The instructions may be in any form that conveys information related to the units or dosage forms in the kit according to the method of the present invention. For example, the instructions may be in the form of a printed matter, or in the form of a pre-recorded media device.

[0337] During the examination of the patient, the medical professional can determine that the use of one of the methods of the present invention is suitable for the patient, or the physician can determine that the patient's condition can be improved by using one of the methods of the present invention. Before specifying any therapy, the physician can provide advice to the patient on various risks and effects associated with the therapy, for example. All known risks and suspected risks associated with the therapy can be fully disclosed to the patient. This advice and counseling can be provided verbally as well as in writing. In some embodiments, the physician can provide the patient with literature data related to the therapy, such as product information, educational materials, etc.

[0338] The present invention relates to a method of educating a consumer about a treatment method, the method comprising dispensing a dosage form at a point of sale along with consumer information. In some embodiments, dispensing is performed at a point of sale with a pharmacist or healthcare provider.

[0339] The term "consumer information" may include, but is not limited to, English text, non-English text, visual images, charts, telephone recordings, websites, and contact with on-site consumer service representatives. In some embodiments, consumer information will provide instructions related to the use of the dosage form according to the method of the present invention, appropriate age of use, indications, contraindications, appropriate dosage, warnings, telephone numbers, or website addresses. In some embodiments, the method also includes providing professional information to relevant personnel, who are responsible for answering questions related to the use of the therapy disclosed by the method of the present invention by the consumer. The term "professional information" includes, but is not limited to, information related to therapy when administered according to the method of the present invention, which is designed to enable medical professionals to answer consumer questions. "Medical professionals" include, for example, physicians, physician assistants, nurses, licensed nurse practitioners, pharmacists, and consumer service representatives.

[0340] Having generally described the present invention, a further understanding may be obtained by reference to the examples provided herein. These examples are for illustrative purposes only and are not intended to be limiting.

[0341] Example 1

[0342] In this example, Schizochytrium sp. was cultured in 250 ml Erlenmeyer shake flasks containing 50 ml of medium. The inoculum was prepared in the same medium consisting of 0.625 g NaCl, 1.0 g KCl, 5 g MgSO4.7H2O, 0.1 g (NH4)2SO4, 0.29 g CaCl 2.2H2O, 1.0 g monosodium glutamate monohydrate, 1.0 g yeast extract and 23.8 g HEPES buffer dissolved in about 900 ml distilled water. The medium was adjusted to pH 7 using NaOH. The final volume of the medium was adjusted to 896 ml and the medium was sterilized by autoclaving. After autoclaving, the following components were aseptically added to the medium: 0.89 ml of 56.5 g / l KH2PO4, 100 ml of 500 g / l glucose, 2 ml of trace metal stock solution and 1 ml of vitamin stock solution. The trace metal stock solution contained the following: 90 g citric acid, 5.15 g FeSO 4. 7H2O, 1.55gMnCl2.4H2O, 0.965g ZnSO4.7H2O, 0.02g CoCl2.6H2O, 0.02gNa2MoO 4. 2H2O, 1.035gCuSO 4. 5H2O, 1.035g NiSO 4. 6H2O is dissolved in one liter of distilled water, and pH is adjusted to 2.5 with HCl. Vitamin stock solution comprises as follows: 0.16g vitamin B12, 9.75g thiamine and 3.33g calcium pantothenate are dissolved in one liter of distilled water. Shake flasks are inoculated with 1ml of inoculum. Bottles in triplicate are placed in a CO2 incubator, which is set to maintain 5, 10 or 15% CO2 in the air. Another group of bottles in triplicate are placed in an incubator under ambient CO2 level. Bottles of all groups are shaken at 200rpm, and all incubators are set at 22.5°C. After growing for 7 days, biomass is collected from shake flasks by centrifugation, and biomass is freeze-dried and the fatty acid profile of biomass is measured using a standard methyl esterification procedure. The rise in CO2 level produces significant changes in biomass, % fat and fatty acid profile. It is noteworthy that the changes in %EPA and %DHA values ​​between CO2 conditions and ambient conditions. In addition, the changes in these fatty acids become more obvious as CO2 levels improve. The results are listed in Table 4.

[0343] Table 4

[0344] sample Biomass (g / l) ％16:0 %EPA %DHA %Fat Environment(1) 3.43 33.55 4.24 54.15 54.55 Environment(2) 3.35 33.29 4.30 54.42 52.67 Environment(3) 3.27 33.29 4.29 54.38 50.62 15% CO2(1) 2.96 28.41 26.12 29.52 53.31 15% CO2(2) 2.94 28.13 26.20 29.98 55.46 15% CO2(3) 2.80 28.12 26.61 29.46 53.20 Environment(1) 3.89 35.34 3.95 52.31 60.21 Environment(2) 3.85 34.74 4.00 53.03 58.33 Environment(3) 3.91 34.76 4.04 52.95 58.18 10% CO2(1) 4.91 33.35 12.94 41.13 69.98 10% CO2(2) 5.03 33.28 12.99 41.15 69.58 10% CO2(3) 4.95 33.46 12.84 41.02 69.08 Environment(1) 3.51 29.95 3.00 45.06 58.48 Environment(2) 3.69 30.57 2.97 44.92 54.87 Environment(3) 3.43 29.98 3.13 45.20 53.29 5% CO2(1) 4.18 32.03 5.59 40.55 42.89 5% CO2(2) 4.16 31.87 5.85 40.36 43.47 5% CO2(3) 4.14 31.58 6.13 40.43 44.13

[0345] Example 2

[0346] In this example, Schizochytrium sp. was cultured in 250 ml Erlenmeyer shake flasks containing 50 ml of medium. The inoculum was prepared in the same medium consisting of 0.625 g NaCl, 1.0 g KCl, 5 g MgSO4.7H2O, 0.1 g (NH4)2SO4, 0.29 g CaCl 2.2H2O, 1.0 g monosodium glutamate monohydrate, 1.0 g yeast extract and 23.8 g HEPES buffer dissolved in about 900 ml distilled water. The medium was adjusted to pH 7 using NaOH. The final volume of the medium was adjusted to 896 ml and the medium was sterilized by autoclaving. After autoclaving, the following components were aseptically added to the medium: 0.89 ml of 56.5 g / l KH2PO4, 100 ml of 500 g / l glucose, 2 ml of trace metal stock solution and 1 ml of vitamin stock solution. The trace metal stock solution contained the following: 90 g citric acid, 5.15 g FeSO 4. 7H2O, 1.55gMnCl2.4H2O, 0.965g ZnSO4.7H2O, 0.02g CoCl2.6H2O, 0.02gNa2MoO 4. 2H2O, 1.035gCuSO 4. 5H2O, 1.035g NiSO 4. 6H2O is dissolved in one liter of distilled water, and pH is adjusted to 2.5 with HCl. Vitamin stock solution comprises as follows: 0.16g vitamin B12, 9.75g thiamine and 3.33g calcium pantothenate are dissolved in one liter of distilled water. Shake flasks are inoculated with 1ml of inoculum. Bottles in duplicate are placed in CO2 incubators, which are set to maintain 5, 10 or 15% CO2 in the air. Bottles in duplicate of another group are placed in an incubator under ambient CO2 level. Bottles of all groups are shaken at 200rpm, and all incubators are set at 22.5°C. After growing for 7 days, biomass is collected from shake flasks by centrifugation, and biomass is freeze-dried and the fatty acid profile of biomass is measured using standard methyl esterification procedures. The rise of CO2 level produces significant changes in biomass, % fat and fatty acid profile. It is noteworthy that the changes produced in %EPA and %DHA values ​​between CO2 conditions and ambient conditions. In addition, the changes of these fatty acids become more obvious as CO2 levels improve. The results are provided in Table 5.

[0347] Table 5

[0348]

[0349]

[0350] Example 3

[0351] In this example, Thrasutochrytrium sp. was cultured in 250 ml Erlenmeyer shake flasks containing 50 ml of medium. The inoculum was prepared in the same medium consisting of 42 g Na2SO4, 0.625 g NaCl, 1.0 g KCl, 5 g MgSO4.7H2O, 0.1 g (NH4)2SO4, 0.29 g CaCl 2. 2H2O, 1.0 g monosodium glutamate monohydrate, 1.0 g yeast extract and 23.8 g HEPES buffer dissolved in about 900 ml distilled water. The medium was adjusted to pH 7 using NaOH. The final volume of the medium was adjusted to 961 ml and the medium was sterilized by autoclaving. After autoclaving, the following components were aseptically added to the medium: 0.89 ml of 56.5 g / l KH2PO4, 35 ml of 500 g / l glycerol, 2 ml of trace metal stock solution and 1 ml of vitamin stock solution. The trace metal stock solution contained the following: 9 g citric acid, 5.15 g FeSO 4. 7H2O, 1.55gMnCl2.4H2O, 0.965g ZnSO4.7H2O, 0.02g CoCl2.6H2O, 0.02gNa2MoO 4. 2H2O, 1.035g CuSO 4. 5H2O, 1.035g NiSO 4. 6H2O was dissolved in one liter of distilled water, and pH was adjusted to 2.5 with HCl. Vitamin stock solution contained as follows: 0.16g vitamin B12, 9.75g thiamine and 3.33g calcium pantothenate dissolved in one liter of distilled water. Shake flasks were inoculated with 1ml of inoculum. Bottles in triplicate were placed in a CO2 incubator, which was set to maintain 15% CO2 in the air. Another set of bottles in triplicate were placed in an incubator under ambient CO2 levels. Two groups of bottles were shaken at 200rpm, and two incubators were set at 22.5°C. After 7 days of growth, biomass was collected from shake flasks by centrifugation, freeze-dried and the fatty acid profile of the biomass was determined using a standard methyl esterification procedure. In Thraustochytrium cultivation, an atmosphere of 15% CO2 in the air produced substantial changes. At high CO2, biomass and % fat were lower than under ambient conditions. The %16:0 and %DHA ratios were lower under ambient conditions, and the %EPA ratio was significantly higher under ambient conditions. The results are listed in Table 6.

[0352] Table 6

[0353]

[0354]

[0355] Example 4

[0356] In this example [NBx0614et10], Schizochytrium species (ATCC PTA-10208) were cultured in four 100-liter New Brunswick Scientific BioFlo 6000 fermentors with a target final (formulation) volume of 80 liters under different overpressure conditions using a carbon (glucose) and nitrogen (ammonium hydroxide) batch feed process to evaluate the sensitivity of the culture to elevated dissolved carbon dioxide. Each fermentation was inoculated with 8 liters of culture. For inoculum propagation, 80 liter New Brunswick Scientific BioFlo 5000 fermentors were used. The inoculum medium consisted of 65 liters of culture medium prepared in six separate groups. Group 1 consisted of 585 g MSG*1H2O, 65 g KCl, 325 g MgSO4*7H2O, 24.05 g (NH4)2SO4, 40.625 g NaCl, 390 g T154 (yeast extract) and 13 mL Dow1520US (defoamer). Group 1 was sterilized in batches at 121 degrees in an inoculum fermenter with a volume of approximately 60 liters. Group 2 consisted of 18.85 g CaCl2*2H2O. Group 3 consisted of 33.8 g KH2PO4. Groups 2 and 3 were each autoclaved in separate solutions for approximately 45-60 minutes and aseptically added to Group 1 after sterilization. Group 4 consisted of 201.5 mg MnCl2*4H2O, 201.5 mg ZnSO4*7H2O, 2.6 mg CoCl2*6H2O, 2.6 mg Na2MoO4*2H2O, 134.6 mg CuSO4*5H2O, 134.6 mg NiSO4*6H2O, 669.4 mg FeSO4*7H2O, and 1.522 g citric acid. Group 4 was autoclaved in the same manner as Groups 2 and 3. Group 5 consisted of 633.75 mg thiamine hydrochloride, 10.4 mg vitamin B12, and 216.5 mg pantothenic acid hemi-calcium salt. Group 5 was dissolved in RO water and filter sterilized. Group 6 consisted of 3250 g glucose dissolved in 3000 mL volume of RO water. Groups 2, 3, 4, 5, and 6 were added to the fermentor after the inoculum fermentor was cooled to 22.5 degrees Celsius. Prior to inoculation, the fermentor pH was adjusted to 7 and dissolved oxygen was crossed to 100% using sodium hydroxide and sulfuric acid. The inoculum fermentor was inoculated with 1300 mL of a smaller fermentation culture (prepared and cultured in the same manner as the 65 liter inoculum culture) and cultured at 22.5 degrees Celsius, pH 7, 180 rpm agitation and 32.5 lpm air for a period of 37 hours, at which time 8 liters of inoculum fermentation broth were transferred to each 100 liter fermentor. Each 100 liter fermentor contained 80 liters of fermentation medium.Fermentation medium was prepared in a similar manner into the inoculum fermentor. For each 100 liter fermentor, the fermentation medium consisted of 6 batches of medium. For vessels NB5, NB6, and NB7, Group 1 contained 704 g Na2SO4, 50 g NaCl, 80 g KCl, 400 g MgSO4*7H2O, 33.6 g (NH4)2SO4, 80 g T154 yeast extract, and 8 mL Dow 1520-US defoamer. Group 1 was steam sterilized at 122 degrees Celsius in a 100 liter fermentor for 60 minutes with a volume of approximately 35 liters. Group 2 contained 23.2 g CaCl2*2H2O with a volume of approximately 300 mL. Group 3 contained 141.2 g KH2PO4 dissolved in RO water. Group 4 contained 248 mg MnCl2*4H2O, 744 mg ZnSO4*7H2O, 3.2 mg Na2MoO4*2H2O, 165.6 mg CuSO4*5H2O and 165.6 mg NiSO4*6H2O, 824 mg FeSO4*7H2O and 80 g citric acid, all dissolved in RO water. Group 5 contained 780 mg Thiamine HCl, 266.4 mg Pantothenate hemi-calcium salt and 286.4 ug Biotin, all dissolved and filter sterilized in RO water. Group 6 contained 2400 g glucose in approximately 3 liters of RO water. Groups 2, 3, 4, 5 and 6 were combined and added to the fermentor after the fermentor reached an operating temperature of 22.5 degrees Celsius. For vessel NB8, all groups were identical to the other three conditions except for the citric acid. In NB8, Group 4 contained only ~3.75 g citric acid. Each fermentation tank volume is about 52-53 liters before inoculation.Each fermentation tank is inoculated with 8 liters of fermentation broth from the above-mentioned inoculum fermentation.The pH of fermentation is controlled at pH 7 by dissolving 7.3 liters of 4N ammonium hydroxide until nitrogen is exhausted, at which time 4N sodium hydroxide and 4N sulfuric acid are used to control pH.Dissolved oxygen is controlled to maintain a target of 20% using 180 to 480rpm stirring and 40LPM to 80LPM air flow throughout the fermentation.Each of these containers is controlled at different headspace pressures (NB5=2, NB6=15 and NB7=20PSI) to assess the sensitivity of the organism to the dissolved carbon dioxide raised.In the whole fermentation, 850g / L of 95% glucose solution (corn steep liquor) is fed to maintain a glucose concentration of less than 50g / L. After 8 days, the cell dry weight and ω-3 titer of each 80-L fermentor were similar, NB5 was 110.1 g / L DCW and 44.37 g / L ω-3; NB6 was 117.7 g / L DCW and 45.78 g / L ω-3; NB7 was 114.1 g / L DCW and 48.43 g / L ω-3; NB8 was 119.5 g / L DCW and 43.55 g / L ω-3.As pressure increases, %DHA / FAME decreases, %EPA / FAME increases, and the ratio of DHA to EPA decreases. When comparing the final fatty acid content of 2PSI to 20PSI, %DHA / FAME decreases to 41.26% from 50.48%, and %EPA / FAME increases to 23.28% from 18.95%. The results are listed in Table 7.

[0357] Table 7

[0358]

[0359] Example 5

[0360] In the present embodiment [Nx0719et10], in four 14 liters of New Brunswick Scientific BioFlo310 fermentor tanks, with 10 liters of target final (formula) volumes, carbon (glucose) and nitrogen (ammonium hydroxide) batch feed process were used to cultivate Schizochytrium species (ATCC PTA-10208). At different time points in the fermentation, carbon dioxide was supplemented in three of the four fermentor tanks to assess the sensitivity of the culture to the dissolved carbon dioxide raised. Respectively, at the beginning of the 12th, 24th, 38th hour of log time, NBS1, NBS2 and NBS3 were supplemented with carbon dioxide. Each fermentation was inoculated with 1 liter of culture. For carrying out inoculum propagation (inoculum propagation), 14 liters of Virtis fermentor tanks were adopted. The inoculum culture medium is composed of 10 liters of culture medium prepared in four independent groups. Group 1 consisted of 90 g MSG*1H2O, 10 g KCl, 50 g MgSO4*7H2O, 3.3 g (NH4)2SO4, 6.25 g NaCl, 60 g T154 (yeast extract), 4.97 g KH2PO4, 2.9 g CaCl2*2H2O and 2 mL Dow 1520US (defoaming agent). Group 1 was autoclaved at 121 degrees for 120 minutes in a volume of approximately 9.8 liters. Group 2 consisted of 500 g glucose dissolved in 800 mL RO water. Group 3 consisted of 31 mg MnC12*4H2O, 31 mg ZnSO4*7H2O, 0.4 mg CoC12*6H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O, and 234.1 mg citric acid. Groups 2 and 3 were sterilized by autoclaving for 60 minutes. Group 4 consisted of 97.5 mg thiamine hydrochloride, 1.6 mg vitamin B12, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was dissolved in RO water and filter sterilized. Groups 2, 3, 4, and 5 were added to the fermentor after the fermentor was cooled to 22.5 degrees Celsius. Prior to inoculation, the fermentor pH was adjusted to 7 and the dissolved oxygen was crossed to 100% using sodium hydroxide and sulfuric acid. The inoculum fermentor was inoculated with 150 mL of a smaller fermentation culture (prepared and cultured in the same manner as the 10 liter inoculum culture) and cultured at 22.5 degrees Celsius, pH 7, 433 rpm stirring and 5 lpm air for a period of 44.5 hours, at which time 1 liter of inoculum fermentation broth was transferred to each 14 liter fermentor. Each 14 liter fermentor contained 10 liters of fermentation medium. The fermentation medium was prepared in a similar manner into the inoculum fermentor. For each 14 liter fermentor, the fermentation medium consisted of a culture medium group divided into 6 batches.For all containers, Group 1 contained 60 g Na2SO4, 6.25 g NaCl, 10 g KCl, 50 g MgSO4*7H2O, 0.43 g (NH4)2SO4, 10 g T154 yeast extract, and 1 mL Dow 1520-US antifoam. Group 1 was autoclaved at 121 degrees Celsius for 120 minutes in a 14 liter fermentor in a volume of approximately 6.5 liters. Group 2 contained 2.9 g CaCl2*2H2O in a volume of approximately 20 mL RO water. Group 3 contained 17.61 g KH2PO4 dissolved in 100 mL RO water. Group 4 contained 31 mg MnCl2*4H2O, 93 mg ZnSO4*7H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O and 10 g citric acid, all dissolved in 50 mL RO water. Group 5 contained 97.5 mg thiamine hydrochloride, 33.3 mg pantothenic acid hemi-calcium salt and 36.3 ug biotin, all dissolved and filter sterilized in 10 mL RO water. Group 6 contained 300 g glucose in approximately 0.5 liter RO water. Groups 2, 3, 4, 5 and 6 were combined and added to the fermentor after the fermentor reached an operating temperature of 22.5 degrees Celsius. Each fermentor had a volume of approximately 6.5 liters prior to inoculation. Each fermentor was inoculated with 1 liter of fermentation broth from the above inoculum fermentation. Adopt 0.85 liter of 4N ammonium hydroxide solution that the pH of fermentation is controlled at pH 7, until nitrogen exhaustion, now adopt 4N sodium hydroxide and 3N sulfuric acid to control pH at set point 7.5.Use 357 to 833rpm stirring and 7LPM to 7LPM air stream to control dissolved oxygen to keep 20% target in whole fermentation.Container NBS1, NBS2, NBS3 supplement carbon dioxide in different time periods respectively, to assess the sensitivity of biological to the dissolved carbon dioxide raised.In whole fermentation, 95% glucose solution (corn steep liquor) of feeding 850g / L is less than 50g / L to maintain glucose concentration.After 8 days, the dry cell weight and ω-3 titer of each 10 liters of fermentor tanks are different according to the difference of carbon dioxide supplementation condition. At 183 hours, NBS1 was 109.1 DCW and 45.87 g / Lω-3; NBS2 was 108.9 g / LDCW and 45.41 g / Lω-3; NBS3 was 116.4 g / L DCW and 50.64 g / Lω-3; NBS4 was 95.7 g / L DCW and 40.36 g / Lω-3. Shortly after the start of CO2 supplementation, %DHA / FAME decreased, %EPA / FAME increased, and the ratio of DHA to EPA decreased. When comparing the maximum %EPA / FAME content under CO2 supplementation conditions and ambient conditions, the maximum EPA content increased by 65% ​​under CO2 supplementation conditions.The results are listed in Table 8.

[0361] Table 8

[0362]

[0363] Carbon dioxide is added during the highlighted time period

[0364] Example 6

[0365] In the present embodiment [Nx0106et10], Schizochytrium species (ATCC PTA-10208) was cultivated in four 14 liters of New Brunswick Scientific BioFlo310 fermentors with 10 liters of target final (formulation) volumes using a carbon (glucose) and nitrogen (ammonium hydroxide) fed-batch process. Temperature was controlled throughout the fermentation run to maintain 21.0 ℃, 22.5 ℃, 24.0 ℃ and 25.5 ℃ targets for NBS1, NBS2, NBS3 and NBS4, respectively. Each fermentation was inoculated with 1 liter of culture. For inoculum propagation, 14 liters of Virtis fermentors were used. Inoculum culture medium consisted of 10 liters of culture medium prepared in four separate groups. Group 1 consisted of 90 g MSG*1H2O, 10 g KCl, 50 g MgSO4*7H2O, 3.7 g (NH4)2SO4, 6.25 g NaCl, 60 g T154 (yeast extract), 5.2 g KH2PO4, 2.9 g CaCl2*2H2O and 2 mL Dow 1520US (antifoaming agent). Group 1 was autoclaved at 121 degrees for 120 minutes in a volume of approximately 9.8 liters. Group 2 consisted of 500 g glucose dissolved in 800 mL RO water. Group 3 consisted of 31 mg MnC12*4H2O, 31 mg ZnSO4*7H2O, 0.4 mg CoC12*6H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O, and 234.1 mg citric acid. Groups 2 and 3 were sterilized by autoclaving for 60 minutes. Group 4 consisted of 97.5 mg thiamine hydrochloride, 1.6 mg vitamin B12, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was dissolved in RO water and filtered for sterilization. Groups 2, 3, 4, and 5 were added to the fermentor after the fermentor had cooled to 22.5 degrees Celsius. Prior to inoculation, the fermentor pH was adjusted to 7 and the dissolved oxygen was crossed to 100% using sodium hydroxide and sulfuric acid. The inoculum fermentor was inoculated with 200 mL of a smaller fermentation culture (prepared and cultured in the same manner as the 10 liter inoculum culture) and cultured for a period of 42 hours at 22.5 degrees Celsius, pH 7, 433 rpm agitation and 5 lpm air, at which time 1 liter of inoculum fermentation broth was transferred to each 14 liter fermentor. Each 14 liter fermentor contained 10 liters of fermentation medium. The fermentation medium was prepared in a similar manner into the inoculum fermentor. For each 14 liter fermentor, the fermentation medium consisted of a culture medium group divided into 4 batches.For all containers, Group 1 contained 88 g Na2SO4, 6.25 g NaCl, 10 g KCl, 50 g MgSO4*7H2O, 4.2 g (NH4)2SO4, 2.9 g CaCl2*2H2O, 17.65 g KH2PO4, 10 g T154 yeast extract, and 1 mL Dow 1520-US antifoam. Group 1 was autoclaved at 121 degrees Celsius for 120 minutes in a 14 liter fermentor with a volume of approximately 7.0 liters. Group 2 contained 31 mg MnCl2*4H2O, 93 mg ZnSO4*7H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O, and 468 mg citric acid dissolved in 50 mL RO water. Group 2 was autoclaved for 60 minutes. Group 3 contained 97.5 mg thiamine hydrochloride, 33.3 mg pantothenate hemi-calcium salt, and 35.8 ug biotin, all dissolved and filter sterilized in 10 mL of RO water. Group 4 contained 300 g glucose in approximately 0.5 liters of RO water and autoclaved for 60 minutes. Groups 2, 3, and 4 were combined and added to the fermentor after the fermentor reached an operating temperature of 22.5 degrees Celsius. The volume of each fermentor was approximately 6.5 liters prior to inoculation. Each fermentor was inoculated with 1 liter of fermentation broth from the above inoculum fermentation. The pH of the fermentation was controlled at 7.0 throughout the fermentation using 0.85 liters of 4N ammonium hydroxide solution until the nitrogen was exhausted, at which time 4N sodium hydroxide and 3N sulfuric acid were used to control the pH at the set point. Dissolved oxygen was controlled to maintain a target of 20% until the nitrogen was exhausted. After nitrogen depletion, dissolved oxygen was controlled using 357 to 714 rpm agitation and 8 LPM air flow in the fermentation to maintain a 10% target until the end of the fermentation. Throughout the fermentation, 850 g / L of 95% glucose solution (corn steep liquor) was fed to maintain a glucose concentration of less than 50 g / L. After 8 days, the cell dry weight or ω-3 titer varied slightly with the different evaluation temperatures; however, the lower the fermentation temperature, the higher the % EPA / FAME. At 184 hours, NBS1 was 85.2 DCW and 29.9 g / L ω-3; NBS2 was 92.0 g / L DCW and 35.0 g / L ω-3; NBS3 was 86.8 g / L DCW and 31.7 g / L ω-3; NBS4 was 84.2 g / L DCW and 29.4 g / L ω-3.

[0366] For NBS1, %EPA / FAME from the beginning of fermentation to the end by 12.36% to 19.02%, and a maximum of 21.57%. For NBS2, %EPA / FAME from the beginning of fermentation to the end by 11.72% to 18.11%, and a maximum of 20.21%. For NBS3, %EPA / FAME from the beginning of fermentation to the end by 11.49% to 15.43%, and a maximum of 18.09%. For NBS4, %EPA / FAME from the beginning of fermentation to the end by 11.65% to 13.65%, and a maximum of 15.70%. When comparing the maximum %EPA / FAME, the lowest fermentation temperature makes the maximum EPA content increase by 37% compared with the highest fermentation temperature assessed. The results are listed in the following table 9.

[0367] Table 9

[0368]

[0369]

[0370] Example 7

[0371] In the present embodiment [Nx0614et10], in two 14 liters of New Brunswick Scientific BioFlo310 fermentors, carbon (glucose) and nitrogen (ammonium hydroxide) batch feed process were used to cultivate Schizochytrium species (ATCC PTA-10208) with 10 liters of target final (formula) volumes. From the beginning to the end of the fermentation run, air supplemented with 15% carbon dioxide was sparged into a fermentor (NBS15), and only air was sparged into another fermentor (NBS17) to assess the sensitivity of the culture to the dissolved carbon dioxide raised. Each fermentation was inoculated with 1 liter of culture. For carrying out inoculum propagation (inoculum propagation), 14 liters of Virtis fermentors were adopted. The inoculum culture medium was composed of 10 liters of culture medium prepared in four separate groups. Group 1 consisted of 90 g MSG*1H2O, 10 g KCl, 50 g MgSO4*7H2O, 3.3 g (NH4)2SO4, 6.25 g NaCl, 60 g T154 (yeast extract), 4.97 g KH2PO4, 2.9 g CaCl2*2H2O and 2 mL Dow1520US (defoaming agent). Group 1 was autoclaved at 121 degrees for 120 minutes in a volume of approximately 9.8 liters. Group 2 consisted of 500 g glucose dissolved in 800 RO water. Group 3 consisted of 31 mg MnC12*4H2O, 31 mg ZnSO4*7H2O, 0.4 mg CoC12*6H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O, and 234.1 mg citric acid. Groups 2 and 3 were sterilized by autoclaving for 60 minutes. Group 4 consisted of 97.5 mg thiamine hydrochloride, 1.6 mg vitamin B12, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was dissolved in RO water and filter sterilized. Groups 2, 3, 4, and 5 were added to the fermentor after the fermentor was cooled to 22.5 degrees Celsius. Prior to inoculation, the fermentor pH was adjusted to 7 and the dissolved oxygen was crossed to 100% using sodium hydroxide and sulfuric acid. The inoculum fermentor was inoculated with 200 mL of a smaller fermentation culture (prepared and cultured in the same manner as the 10 liter inoculum culture) and cultured for a period of 40 hours at 22.5 degrees Celsius, pH 7, 433 rpm agitation and 5 lpm air, at which time 1 liter of inoculum fermentation broth was transferred to each 14 liter fermentor. Each 14 liter fermentor contained 10 liters of fermentation medium. The fermentation medium was prepared in a similar manner into the inoculum fermentor. For each 14 liter fermentor, the fermentation medium consisted of a culture medium group divided into 6 batches.For all containers, Group 1 contained 88 g Na2SO4, 6.25 g NaCl, 10 g KCl, 50 g MgSO4*7H2O, 4.2 g (NH4)2SO4, 10 g T154 yeast extract, and 1 mL Dow 1520-US antifoam. Group 1 was autoclaved at 121 degrees Celsius for 120 minutes in a 14 liter fermentor in a volume of approximately 6.5 liters. Group 2 contained 2.9 g CaCl2*2H2O in a volume of approximately 20 mL of RO water. Group 3 contained 17.65 g KH2PO4 dissolved in 100 mL of RO water. Group 4 contained 31 mg MnCl2*4H2O, 93 mg ZnSO4*7H2O, 0.4 mg Na2MoO4*2H2O, 20.7 mg CuSO4*5H2O, 20.7 mg NiSO4*6H2O, 103 mg FeSO4*7H2O, and 468 mg citric acid dissolved in 50 mL RO water. Groups 2, 3, and 4 were each autoclaved for 60 minutes. Group 5 contained 97.5 mg thiamine hydrochloride, 33.3 mg pantothenate hemi-calcium salt, and 36.3 ug biotin, all dissolved and filter sterilized in 10 mL RO water. Group 6 contained 300 g glucose in approximately 0.5 liters of RO water and autoclaved for 60 minutes. Groups 2, 3, 4, 5, and 6 were combined and added to the fermentor after the fermentor reached an operating temperature of 22.5 degrees Celsius. Each fermentor volume was approximately 6.5 liters before inoculation. Each fermenter was inoculated with 1 liter of fermentation broth from the above inoculum fermentation. The pH of the fermentation was controlled at 7.0 using 0.85 liters of 4N ammonium hydroxide dissolution throughout the fermentation until nitrogen was exhausted, at which time 4N sodium hydroxide and 3N sulfuric acid were used to control the pH at the set point. Dissolved oxygen was controlled to maintain a 20% target until nitrogen was exhausted. After nitrogen was exhausted, dissolved oxygen was controlled to maintain a 10% target using 357 to 833 rpm stirring and 8 LPM air flow in the fermentation until the end of the fermentation. Throughout the fermentation, 850 g / L of 95% glucose solution (corn steep liquor) was fed to maintain a glucose concentration of less than 50 g / L. After 8 days, the dry cell weight and ω-3 titer of each 10-liter fermenter varied depending on the carbon dioxide supplementation conditions. At 188 hours, NBS15 was 54.5 DCW and 13.7 g / L ω-3; NBS17 was 96.1 g / LDCW and 37.5 g / L ω-3. NBS15 (CO2 supplemented conditions throughout the run) had a higher %DHA / FAME than NBS17 (no CO2 supplemented). For NBS15, %EPA / FAME ranged from 25.50% to 35.48% from the start of the fermentation run to the end, with a maximum of 38.34%. For NBS17, %EPA / FAME ranged from 12.31% to 19.80% from the start of the fermentation run to the end, with a maximum of 22.29%.When comparing the maximum % EPA / FAME content under supplemented CO2 conditions and ambient conditions, the maximum EPA content was increased by 73% under supplemented CO2 conditions.

[0372] Throughout the fermentation run, the % DHA / FAME under supplemented CO2 conditions was lower than that under ambient conditions. The results are listed in Table 10 below.

[0373] Table 10

[0374]

[0375]

[0376] Example 8

[0377] In this example [K019], Schizochytrium species (ATCC PTA-10208) was cultured in a 157,000 liter stirred fermenter at a final (formulation) weight of 100,000 kg using a carbon (glucose) and nitrogen (anhydrous ammonia) fed-batch process. The fermentation was inoculated with 4,500 kg of culture. For inoculum propagation, a 7,500 liter stirred seed fermenter was used. The inoculum medium consisted of 4,500 kg of culture medium prepared in four separate groups. Group 1 consisted of 40.5 kg MSG*1H2O, 4.5 kg KCl, 22.5 kg MgSO4*7H2O, 1.7 kg (NH4)2SO4, 2.81 kg NaCl, 27 kg T154 (yeast extract), 2 kg KH2PO4, 985 g CaCl2 and 0.9 kg Dow1520US (defoamer) dissolved in a total weight of 2,300 kg of treated water. Group 2 consisted of 247.5 kg glucose.1H2O dissolved in a total weight of 1500 kg of treated water. Group 1 was sterilized in a seed fermenter and Group 2 was sterilized in a separate container with steam-in-place at 122-123 degrees Celsius for 30 minutes. Group 3 consisted of 14 g MnC12*4H2O, 14 g ZnSO4*7H2O, 180 g CoC12*6H2O, 180 g Na2MoO4*2H2O, 9.3 g CuSO4*5H2O, 9.3 g NiSO4*6H2O, 46.4 g FeSO4*7H2O and 105.3 g citric acid dissolved in 5 L of distilled water. Group 3 was autoclaved at 121 degrees Celsius for 60 minutes. The 4th group is made up of 43.9g thiamine hydrochloride, 720g vitamin B12 and 15g pantothenic acid hemi-calcium salt dissolved in 5L of distilled water and filtered sterilized. After the seed fermenter is cooled to 22.5 degrees Celsius, the 2nd, 3rd and 4th groups are added to the fermenter. Before inoculation, the fermenter pH is adjusted to 7 and the dissolved oxygen is crossed to 100% using sodium hydroxide and sulfuric acid. The seed fermenter is inoculated with a smaller fermentation medium of 12L (a smaller fermentation culture prepared and cultivated in the same manner as the seed culture) and cultivated for 4-5 days at 22.5 degrees Celsius, pH 7, 90rpm stirring and 130-170Nm2 / hr air to obtain a cell dry weight of about 15g / L. The fermentation medium is prepared in a similar manner into the inoculum fermenter. The fermentation medium consists of 5 groups. Group 1 contained 177 kg KH2PO4, 880 kg Na2SO4, 500 kg MgSO4*7H2O, 4.2 kg (NH4)2SO4, 100 kg T154 yeast extract and 10 kg Dow 1520-US antifoam in 9000 kg solution.Group 2 contained 21.9 kg CaCl2, 62.5 kg NaCl, 100 kg KCl in 9000 kg solution. Groups 1 and 2 were both pumped through a heat exchanger into a fermenter and then water was pumped in to obtain a weight of 67,000 kg in the fermenter. Group 3 contained 310 g MnCl2*4H2O, 930 g ZnSO4*7H2O, 4 g Na2MoO4*2H2O, 207 g CuSO4*5H2O, 207 g NiSO4*6H2O, 1.03 kg FeSO4*7H2O and 4.68 kg citric acid dissolved in 1500 kg of process water. Group 4 contained 4300 kg corn steep liquor (DE-95, 70.5%). Groups 3 and 4 were sterilized in separate containers with steam at 122-123 degrees Celsius for 30 minutes. The 5th group contained 975g thiamine hydrochloride, 333g pantothenic acid hemi-calcium salt and 358mg biotin, dissolved and filter sterilized in 5L distilled water. Groups 3, 4 and 5 were added to the fermenter after the fermenter was cooled to 22.5 degrees Celsius. The weight in the fermenter volume was about 73500kg before inoculation. After setting the initial fermentation conditions (temperature: 22.5°C, pressure: 0.34bar, air flow: 3000Nm3 / hr, agitation: 40rpm), the fermentation pH was adjusted to 7, and the dissolved oxygen was crossed to 100%. The weight after inoculation was about 78000kg. At the beginning, anhydrous ammonia was used to control the pH at 7 until 550kg ammonia was added, and then 30% sodium hydroxide solution was used to control the pH at the set point 7.5. Using 40 to 100 rpm stirring and 2000 to 8000 Nm3 / hr air flow, dissolved oxygen was controlled to maintain a target of 20% during ammonia feed, and 10% after ammonia feed. Throughout the fermentation, a 65% DE-95 corn steep liquor solution was fed to maintain a glucose concentration of about 35 g / L. In another embodiment [K020], three changes were made relative to embodiment K019: 1) the pressure was reduced to 0.15 bar, 2) the weight was reduced to 68000 kg after inoculation, and 3) the air flow was increased to more than 5000 Nm3 / hr at 60 hours after inoculation and maintained at a high level throughout the run regardless of the dissolved oxygen concentration. The above three changes reduced the dissolved carbon dioxide concentration in the fermentation broth. The results showed that as CO2 decreased, %DHA / FAME increased from 38.38% to 43.8%, and %EPA / FAME decreased from 24.42% to 20.68%. The DHA:EPA ratio increased from 1.57 to 2.12. The results are listed in Table 11 below.

[0378] Table 11

[0379]

[0380]

[0381] Table 12

[0382]

[0383] Example 9

[0384] In the following table, the maximum dissolved CO2 of various examples was calculated using Henry's constant. The first condition "10L (NBS4 0719et10) at 1 PSI backpressure and 45.5mmol / L / hour CER" is the calculated dissolved CO2 of NBS4 at 45.5mmol / L / hour carbon dioxide release rate, 10 liters fermentation volume, 0.8vvm aeration rate, 0 PAI backpressure in Table 8. The second condition "10L (NBS2 0719et10) at 1 PSI backpressure and 6% CO2 in the inlet gas and 50mmol / L / hour CER" is the calculated dissolved CO2 of NBS2 at 50mmol / L / hour carbon dioxide release rate, 10 liters fermentation volume, 0.8vvm aeration rate, 0 PSI backpressure, and 6% CO2 supplementation of the total gas in the inlet gas flow (measured by mass spectrometry using a Thermo Prima dB mass spectrometer) in Table 8. The third condition "80 liters at 2PSI back pressure and 55mmol / L / hour CER (NB5 0614et10)" is the NB5 dissolved CO2 calculated at a carbon dioxide release rate of 55mmol / L / hour, a fermentation volume of 80 liters, a ventilation rate of 1.0vvm, and a 2PSI back pressure in Table 7. The fourth condition "80 liters at 15PSI back pressure and 50mmol / L / hour CER (NB6 0614et10)" is the NB6 dissolved CO2 calculated at a carbon dioxide release rate of 50mmol / L / hour, a fermentation volume of 80 liters, a ventilation rate of 1.0vvm, and a 15PSI back pressure in Table 7. The fifth condition "80 liters at 20 PSI back pressure and 50 mmol / L / hour CER (NB7 and NB80614et10)" is the dissolved CO2 of NB7 and NB8 calculated at a carbon dioxide evolution rate of 50 mmol / L / hour, a fermentation volume of 80 liters, aeration rate of 1.0 vvm, and 20 PSI back pressure in Table 7. All CER values ​​are calculated using outgassing CO2 data collected using a Thermo Prima dB mass spectrometer. The calculated results are provided in Tables 13 and 14 below.

[0385] Table 13

[0386]

[0387]

[0388]

[0389] Table 14

[0390]

[0391] Example 10

[0392] Experiments were conducted to determine the effect of vitamin composition on performance (cell dry weight (DW), % DHA, % fat, and % EPA of biomass) in Thraustochytrium Shake Flask Medium (TSFM) under ambient CO2 levels using ATCC Accession No. PTA-9695.

[0393] Materials and methods: Four vitamins (thiamine.HCl, B12, biotin and pantothenate Ca) were used in TSFM medium with 0.25 g / L tastone and 0.625 g / L NaCl (see Table 16). Additional MSG and H2PO4 were added to the medium to maintain their total nitrogen and total phosphorus content. Depending on the vitamin studied, the overall vitamin concentration in the medium was 0, 0.5x, 1x, 5x, 10x, 20x or 30x the standard amount (see Table 17). Gradient studies were performed separately for each vitamin. However, in the case of biotin and pantothenate Ca, standard amounts of thiamine hydrochloride and B12 were also added to the culture because the concentrations of the latter two were very low in conventional TSFM. Three TSFM controls were also included in the experiment for comparison. These controls are standard TSFM with 2g / L tastone (see Table 15), 1x thiamine hydrochloride and 1x B12 (C1); TSFM without tastone without any vitamin (A); and TSFM without tastone with 1x thiamine hydrochloride and 1x B12 (B). All controls include 0.625g / L NaCl. Under 0.1g DW / L, three-day cultures of PTA-9695 are used to inoculate duplicate 250-ml shake flasks. All shake flasks (flat bottom, 50ml culture medium in total) are inoculated on a rotary shaker (200RPM) under an aerobic environment at 22.5+ / -1C. All cultures are harvested after 7 days, and final freeze-dried biomass samples are subjected to FAME analysis.

[0394] Table 15 Thraustochytrid shake flask medium (TSFM) with 2 g / L tastone

[0395]

[0396]

[0397] Table 16 Thraustochytrid shake flask medium (TSFM) with 0.25 g / L Tastone

[0398]

[0399]

[0400] Table 17 Vitamin concentrations used in this study (mg / L):

[0401]

[0402] Results: When the amount of thiamine added to the culture medium was 5 times the standard level ( Figure 1 ) the highest DW and % fat for the thiamine gradient were obtained (6.7 g / L and 38.5%, respectively). At this level of thiamine the % DHA was 44.1%. Both DW and % fat began to decline below or above 5x thiamine. % DHA also declined in media with less than 5x thiamine and fluctuated slightly above 5x thiamine with no significant improvement. % EPA in the thiamine gradient cultures was between 8.6 and 11.5. When no tastone or vitamins ( Figure 5 , Medium A), all parameters except %DHA decreased significantly. However, the increase in %DHA seems to be spurious, as both DW and %Fat are extremely low under these conditions. It appears that the 1x concentration of B12 is optimal for DW, %DHA, %Fat, and %EPA ( Figure 2 ). At this vitamin B12 level, the following results were obtained: 7.1 g / L DW, 50.6% DHA, 42.7% fat and 2.1% EPA. The highest %EPA (11.5) was obtained when no B12 was added to the medium. Tastone-free medium without vitamin addition did not further improve the performance of the organism, and the higher %DHA appears to be an artifact for the reasons discussed above ( Figure 5 , culture medium A).

[0403] Likewise, a 1x concentration of biotin was also optimal for DW, %DHA, and %Fat ( Figure 3 ). The corresponding values ​​of these parameters are: 6.8 g / L, 47.7% and 37.9%. The EPA percentage is 1.9. Tastone-free medium with 1x Thiamine and 1x B12 ( Figure 5 , Medium B) significantly reduced the overall performance of PTA-9695.

[0404] When pantothenate Ca was added to the culture medium at 10x the standard amount, this vitamin produced the highest DW, %DHA and %Fat ( Figure 4). The optimal concentrations for these parameters were: 7.0 g / L, 47.3% and 39.1%, respectively. EPA content was less than 2% throughout the pantothenate Ca gradient experiment. A significant decrease in the overall performance of PTA-9695 was noted when the organism was grown in a tastone-free medium with only 1x thiamine and 1x B12 ( Figure 5 , culture medium B).

[0405] The results are also shown in the data table below.

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413] Embodiment 11

[0414] Experiments were conducted to determine the minimum vitamin B12 requirements of ATCC Accession No. PTA-9695 for maximum performance at 10% CO2, including the minimum amount of vitamin B12 required for PTA-9695 cultures to produce maximum dry weight and DHA production at 10% CO2.

[0415] Temperature: 22.5 + / - 1°C (at 10% CO2)

[0416] Shaking speed: 200rpm

[0417] Basal medium: Defined scaled-down fermenter medium (DSDFM-B) (Table 20).

[0418] Inoculum: 3-day culture of PTA-9695 grown in DSDFM-B with various concentrations of vitamin B12.

[0419] First, thaw PTA-9695 in a new cryovial in SDFM-B (Table 19), then culture and transfer several times in a defined SDFM-B at 10% CO2. Use a 3-day culture (4%) of PTA-9695 in DSDFM-B to prepare a starting inoculum bottle, which contains DSDFM-B with various concentrations of vitamin B12 (i.e., 7.68 ml / L to 0 mg / L B12 for treatments A to I, respectively, see Table 18). Maintain an inoculum bottle of each vitamin B12 concentration by transferring the culture to its corresponding fresh medium at 10% CO2 and every 7 days. Weekly transfer of the culture is necessary to wash out any excess vitamin B12 that may have been stored inside the cells, so that the minimum requirement for this vitamin can be determined more accurately. Use optical density at 600nm to inoculate shake flasks in duplicate with their 3-day inoculum at a theoretical DW of 0.1 g / L. The cultures were grown under 10% CO2 for 9 days before being harvested and further analyzed by FAME assay.

[0420] result:

[0421] Note: The 3-week data point was not considered in this study because the supply of CO2 to the incubator was interrupted during laboratory renovations and the shake flasks had to be removed from the incubator for 6 h.

[0422] Long-term exposure (up to 4 weeks) of PTA-9695 to high concentrations of vitamin B12 adversely affected dry weight, % fat, and DHA production, while vitamin B12 concentrations from 1.5 to 768 ug / L had no significant effect on DW, % EPA, and DHA production of PTA-9695.

[0423] Note that there was an increase in the number of single cells (up to 15 compared to 3-4) in cultures grown in the presence of 7 68 mg / L vitamin B12 (treatment A).

[0424] Eliminating vitamin B12 from the media formulation significantly increased %EPA by approximately 10%, while slightly decreasing %DHA and %16:0. Figure 6-19 The experimental results are shown in Table 21 below.

[0425] in conclusion:

[0426] A minimum of about 1.5 ug / L vitamin B12 (1 / 500 of the standard concentration in SDF-B) is required to obtain maximum DW, %DHA, %Fat, and DHA yields. Very high concentrations of vitamin B12 (greater than 3.84 mg / L) can significantly reduce DW, %Fat, and DHA yields, while complete removal of this vitamin from PTA-9695 significantly (about 10%) increases its %EPA. Very high levels of vitamin B12 also play a role in promoting the conversion of "clumpy" cultures to "single cells."

[0427] Table 18 Vitamin B12 treatment

[0428]

[0429] Table 19 Scaled down fermenter medium (SDFM-B) for PTA-9695, pH 7

[0430]

[0431]

[0432] Table 20 Determined SDFM-B (DSDFM-B), pH 7

[0433]

[0434]

[0435] Table 21 Experimental data

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448] Example 12

[0449] ATCC Accession No. PTA-10208 Vitamin B12 Gradient at 10% CO2

[0450] Experiments were performed to determine the vitamin B12 concentration that provided optimal PTA-10208 growth and EPA production.

[0451] Temperature: 23℃

[0452] Shaking speed: 200rpm

[0453] Basal medium: Defined SDFM-O (DSDFM-O)

[0454] Inoculum: Thaw one vial of PTA-10208 into SDFM-O at ambient conditions. Transfer 2 mL of culture into 48 mL of DSDFM-O at 10% CO2. Transfer culture to fresh DSDFM-O (10% CO2) (see Table 22). Transfer culture to fresh DSDFM-O (10% CO2). Use culture to inoculate vitamin 12 gradient experiment (2 mL / bottle) (10% CO2) in Week #1.

[0455] Experimental setup:

[0456] All cultures were cultivated in 50mL shake flasks, and duplicate shake flasks were cultivated under each condition. Every 7 days in DSDFM-O (without tastone), PTA-10208 was inoculated into the 9-day vitamin B12 gradient. The inoculum for the vitamin B12 gradient of each concentration was maintained throughout the experiment. By continuing to transfer PTA-10208 in the vitamin B12 that reduces concentration, excessive vitamin B12 was effectively washed out to the cell outside. Every seven days, the inoculum for the vitamin B12 of each concentration was transferred. Four days inoculum was used to start each 9 days vitamin B12 gradient. Each 9 days gradient was continuously marked as experimental settings A, B, C, D, E, F, G, H, I, J and listed in the following table 23. In order to transfer the cells of roughly the same amount, the optical density of the vitamin B12 of each concentration was measured before each gradient was inoculated. After growing for 9 days, all cultures were collected to measure pH, dry weight and fatty acid profile. The experiment was terminated once the dry weight of each vitamin B12 concentration was found to have stabilized for at least 3 consecutive 9-day gradients.

[0457]

[0458]

[0459] Table 23

[0460] [] Vitamin B12 mg / L Vitamin B12 5x 3.84 1x 0.77 1 / 2x 0.38 1 / 5x 0.15 1 / 10x 0.077 1 / 20x 0.038 1 / 50x 0.015 1 / 100x 0.0077 1 / 500x 0.0015 0x 0

[0461] Seven continuous 5x to 1 / 500x vitamin B12 gradients were performed. At the end of the last experiment, a new experiment was started to compare PTA-10208 growth and EPA production under 1 / 500x (0.0015 mg / L) and 0x (0 mg / L vitamin B12). Four continuous 1 / 500x vs. (vs) 0x vitamin B12 experiments were performed.

[0462] result:

[0463] (For 5x-->1 / 500x Vitamin B12): For seven consecutive nine-day experiments, PAT-10208 dry weight, %EPA, %DHA, %Fat and EPA production did not change in concentrations ranging from 5x (3.84 mg / L) to 1 / 500x (0.0015 mg / L) Vitamin B12.

[0464] (For 1 / 500x and 0x Vitamin B12): Results over the course of four consecutive nine-day experiments showed that PTA-10208 cultured in DSDFM-O containing 0 mg / L Vitamin B12 had much higher %EPA than when cultured at only 1 / 500x (0.0015 mg / L) Vitamin B12. %EPA increased from about 18% at 1 / 500x (0.0015 mg / L) Vitamin B12 to about 27% without any Vitamin B12, and %DHA decreased from about 45% at 1 / 500x (0.0015 mg / L) Vitamin B12 to about 36% without any Vitamin B12. When Vitamin B12 was completely removed from DSDFM-O, PTA-10208 dry weight and %fat increased slightly. PTA-10208 EPA production increased by approximately 60% when vitamin B12 was removed from DSDFM-O due to the increase in %EPA and slight increases in dry weight and %fat.

[0465] The results are also shown in Figure 20-49 and in the table below.

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481] Conclusion: Growth and EPA production of PTA-10208 were not altered when vitamin B12 concentrations ranged between 5x (3.84 mg / L) and 1 / 500x (0.0015 mg / L) in DSDFM-O at 10% CO2. However, when vitamin B12 was completely removed from DSDFM-O, %EPA increased by approximately 50%, while dry weight and %fat also increased slightly, resulting in a 60% increase in EPA production.

[0482] The various aspects, embodiments, and options described herein may be combined in any and all variations.

[0483] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A microbial biomass comprising A microbial oil comprising fatty acids and a triglyceride fraction of at least 65% by weight based on the total weight of the microbial oil, wherein the biomass comprises at least 35% fatty acids based on the total dry cell weight of the biomass and at least 20% by weight eicosapentaenoic acid (EPA) based on the total weight of the fatty acids, and wherein the biomass is produced by a method comprising: (a) fermenting the microorganism in a fermenter vessel comprising a fermentation broth and the microorganism, the microorganism comprising a thraustochytrid microorganism; and (b) obtaining said EPA level concentration by one or more steps comprising: (i) fermenting said microorganism in said fermentation broth at a temperature below 30° C. and (ii) optionally applying a headspace pressure of greater than 0.4 psi to said fermentor vessel during at least a portion of step (a).

2. The biomass according to claim 1, comprising EPA in an amount of 20 to 55 wt% of the total weight of the fatty acids.

3. The biomass according to claim 1, comprising EPA in an amount of at least 25 wt% of the total weight of said fatty acids.

4. The biomass according to claim 1, comprising EPA in an amount of at least 30 wt% of the total weight of said fatty acids.

5. The biomass according to claim 1, comprising EPA in an amount of at least 35 wt% of the total weight of said fatty acids. The biomass of claim 1 , wherein the temperature is 20° C. to 23° C.

7. The biomass of claim 1, further comprising docosahexaenoic acid (DHA) in an amount of at least 20 wt% based on the total weight of the fatty acids. 8 . The biomass according to claim 1 , further comprising arachidonic acid in an amount of 5 wt % or less based on the total weight of the fatty acids.

9. The biomass according to claim 1, comprising EPA in an amount of 20 to 50 wt% based on the total weight of the fatty acids.

10. The biomass according to claim 1, comprising DHA in an amount of 20 to 60 wt% based on the total weight of the fatty acids.

11. The biomass of claim 1 comprising at least 40 wt% fatty acids based on the total dry cell weight of the biomass.

12. The biomass of claim 1, comprising at least 45 wt% fatty acids based on the total dry cell weight of the biomass.

13. The biomass of claim 1, comprising at least 50 wt% fatty acids based on the total dry cell weight of the biomass.

14. The biomass of claim 1, comprising at least 70 wt% triglyceride fraction based on the total weight of the microbial oil.

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