NZ712497B2

NZ712497B2 - Eicosapentaenoic acid-producing microorganisms, fatty acid compositions, and methods of making and uses thereof

Info

Publication number: NZ712497B2
Application number: NZ712497A
Authority: NZ
Inventors: Kirk E Apt; Paul Warren Behrens; Xiao Daniel Dong; Jose R Garcia; Jon Milton Hansen; Iii Joseph W Pfeifer
Original assignee: Dsm Ip Assets Bv
Priority date: 2011-07-21
Filing date: 2012-07-20
Publication date: 2017-01-05

Abstract

Disclosed is a method of making a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: (a) fermenting the microorganism in a fermenter vessel to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids; and (b) adjusting the amount of the biomass in the vessel to attain a desired EPA level in the biomass. Also disclosed is an oil extracted from the biomass and its use in a feed product. the total weight of the fatty acids; and (b) adjusting the amount of the biomass in the vessel to attain a desired EPA level in the biomass. Also disclosed is an oil extracted from the biomass and its use in a feed product.

Description

EICOSAPENTAENOIC ACID-PRODUCING RGANISMS, FATTY ACID COMPOSITIONS, AND METHODS OF MAKING AND USES THEREOF BACKGROUND OF THE INVENTION The present application is a divisional application from New d patent application number 710646, which in turn is a divisional application from New Zealand patent application number 620578, the entire disclosures of which are incorporated herein by reference.

Field of the Invention [0001a] The present invention is ed to isolated microorganisms as well as strains and mutants thereof, biomasses, microbial oils, compositions, and cultures; methods of producing the microbial oils, biomasses, and mutants; and methods of using the isolated microorganisms, biomasses, and microbial oils.

Background Art Fatty acids are classified based on the length and saturation characteristics of the carbon chain. Fatty acids are termed short chain, medium chain, or long chain fatty acids based on the number of carbons present in the chain, are termed saturated fatty acids when no double bonds are present between the carbon atoms, and are termed unsaturated fatty acids when double bonds are present. Unsaturated long chain fatty acids are saturated when only one double bond is present and are polyunsaturated when more than one double bond is present.

Polyunsaturated fatty acids (PUFAs) are classified based on the position of the first double bond from the methyl end of the fatty acid: omega-3 (n-3) fatty acids contain a first double bond at the third carbon, while omega-6 (n-6) fatty acids contain a first double bond at the sixth carbon. For example, docosahexaenoic acid ("DHA") is an omega-3 long chain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22 s and 6 double bonds, often ated as "22:6 n-3." Other omega-3 LC-PUFAs e pentaenoic acid ("EPA"), designated as "20:5 n-3," and omega-3 docosapentaenoic acid ("DPA n-3"), designated as "22:5 n-3." DHA and EPA have been termed "essential" fatty acids. Omega-6 LC-PUFAs include donic acid ("ARA"), -1a- designated as "20:4 n-6," and omega-6 docosapentaenoic acid ("DPA n-6"), designated as "22:5 n-6." Omega-3 fatty acids are biologically important molecules that affect cellular physiology due to their presence in cell membranes, te production and gene expression of biologically active compounds, and serve as thetic substrates.

Roche, H. M., Proc. Nutr. Soc. 58: 397-401 . DHA, for example, accounts for approximately 15%-20% of lipids in the human cerebral cortex, % of lipids in the - retina, is concentrated in the testes and sperm, and is an important ent of breast milk. Berge’, J .P., and an, G.. Adv. m. Eng. Biotechnol. 96:49-125 (2005).

DHA accounts for up to 97% of the omega-3 fatty acids in the brain and up to 93% of the omega-3 fatty acids in the retina. Moreover, DHA is essential for both fetal and infant development as well as maintenance of cognitive functions in adults. [(1. Because omega-3 fatty acids are not synthesized de novo in the human body, these fatty acids must be derived from ional sources.

~ Flaxseed oil and ﬁsh oils are considered good dietary sources of omega-3 fatty acids. Flaxseed oil contains no EPA, DHA, DPA, or ARA but rather contains linolenic acid (Cl8z3 n-3), a building block enabling the body to manufacture EPA. There is evidence, however, that the rate of metabolic conversion can be slow and variable, particularly among those with impaired health. Fish oils vary erably in the type and level of fatty acid composition depending on the particular species and their diets.

For example, ﬁsh raiSed by aquaculture tend to have a lower level of omega-3 fatty acids than those in the wild. Furthermore, ﬁsh oils carry the risk of containing environmental contaminants and can be ated with stability ms and a ﬁshy odor or taste.

. Thraustochytrids are microorganisms of the order Thraustochytriales.

Thraustochytrids include members of the genus Schizochytrium and Thraustochytrium and have been recognized as an alternative source of omega-3 fatty acids, including DHA and EPA. See US. Patent No. 5,130,242. Oils produced from these marine heterotrophic microorganisms often have simpler polyunsaturated fatty acid proﬁles than corresponding ﬁsh or microalgal oils. Lewis, T.E., Mar. Biotechnol. 1: 580-587 (1999). Strains of thraustochytrid species have been reported to e omega-3 fatty acids as a high percentage of the total fatty acids produced by the organisms. US. Patent No. 5,130,242; Huang, J. et al., J. Am. 0le Chem. Soc. 78: 605-610 (2001); Huang, J. et al., Mar.

Biotechnol. 5: 450-457 (2003). However, isolated thraustochytrids vary in the identity and amounts of LC-PUFAs produced, such that some previously described strains'can have undesirable levels of 6 fatty acids and/or can demonstrate low productivity in culture. As such, a continuing need exists for the isolation of microorganisms demonstrating high productivity and desirable LC-PUFA profiles. [0006a] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these s formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. [0006b ] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not ding the presence of one or more other features, integers, steps or components, or group thereof.

BRIEF SUMMARY OF THE INVENTION The ants have found that the amount of EPA and DHA, ed by a tochytrid that produces a biomass having at least 3% EPA, can be modulated by varying the amounts of dissolved carbon dioxide (CO2) in an aqueous phase of a fermentation broth during the tation of the microorganism. Provided herein is a method of making a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: fermenting the microorganism in a tor vessel having a dissolved gas in a fermentation broth to produce a biomass, wherein the rganism comprises a Thraustochytrid that es a biomass having at least 3% EPA of the total weight of the fatty acids; and ing the dissolved CO2 levels in the dissolved gas. In one embodiment, the dissolved CO2 levels may be adjusted to attain a desired EPA and/or DHA level in the biomass. In a r ment the amount of dissolved CO2 in the aqueous phase of a fermentation broth ranges from about 38 to about 600 ppm of the total dissolved gas, and particularly from about 38 to about 135 ppm of the total dissolved gas.

Further provided herein is a method of making a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: fermenting the microorganism in a fermentor vessel, comprising a gas, to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total -3a- weight of the fatty acids; and supplementing the gas with CO2 . Supplementing means to add to or charge the vessels with CO2 in an amount additional to the amount produced by the fermentation of the cells or an amount at ambient conditions. In one ment the CO2 is supplemented to the vessel to attain a desired EPA and/or DHA amount in the [0008a] Also ed herein is method of making a biomass of a microorganism having fatty acids and a tration of EPA, comprising: (a) fermenting the microorganism in a fermentor vessel to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids; and (b) adjusting the amount of the biomass in the vessel to attain a desired EPA level in the biomass.

Also provided herein is a method of making a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: fermenting the rganism in a fermentor vessel to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids; and adjusting the amount of the biomass in the vessel. In an embodiment of the invention the biomass is adjusted to attain a desired EPA or DHA level in the biomass.

A method of making a microorganism having fatty acids and a concentration of EPA, sing: fermenting the microorganism in a fermentor vessel to produce a biomass n the microorganism comprises a Thraustochytrid that es a biomass having at least 3% EPA 0f the total weight. of the fatty acids;. and adjusting the pressure on the biomass, for example, but not limited to, controlling the back pressure of the vessel. In one embodiment the pressure is adjusted to attain a desired EPA or DHA level in the biomass.

In another embodiment provided herein is a method of making a microorganism having fatty acids and a concentration of EPA, comprising: fermenting the microorganism in a fermentor vessel to e a fermentation broth and a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids; and adjusting the temperature in the broth. In one embodiment the temperature is adjusted to attain a desired EPA and/or DHA level the biomass.

In various embodiments, the amounts of EPA and DHA can also be modulated by adjusting the amount of dissolved C02 in an aqueous phase or fermentation broth of the vessel by increasing or decreasing the amount of C02 in the vessel. The amount of ved CO2 can be adjusted by additionally adjusting the amount of the biomass fermented. For example, by fermenting the cells in ﬂasks and larger fermentation vessels.

The EPA and DHA can also be varied according to embodiments provided herein by varying the temperature. The amount of dissolved CO2 can be additionally adjusted for example by adjusting the ature in the vessel. For example, lower vessel temperatures will produce higher concentrations of EPA and lower concentrations of DHA. The amount of dissolved CO2 can further be adjusted by adjusting the pressure in the vessel. For example, increasing pressure will likely se dissolved CO2 which will increase the amount of EPA and sethe amount of DHA in the s. Each of the above bed adjustments, e.g., mented CO2, increase or decrease in biomass, se or decrease in temperature, or increase or decrease in pressure, can each be combined with any of the other adjustments to attain a desired EPA and DHA level in 2012/047728 the biomass and any oil extracted from the biomass. Dissolved C02 can also be adjusted by a change in pH.

In some embodiments, the total amount of EPA and DHA remains relatively constant compared to the ainount, by weight, of the total weight of the fatty acids and omega-3 fatty acids. [00141' In further embodiments, the EPA or DHA content of the biomass is measured prior to making an adjustment to the amount of the supplemented CO2, pressure, temperature, or biomass.

While not wishing to be bound to any theory, it is hypothesized that the increase and decrease in the s of the EPA or DHA is directly related to the amount of dissolved CO2 in the aqueous phase of the fermentation broth and that the above described ments of CO2, of pressure, and of temperature, vary the amount of dissolved CO2 in the biomass.

In some embodiments, the invention es a method of making a biomass of a microorganism having increased concentration of EPA, the method comprising growing the microorganism in a culture medium comprising less than 0.1 mg/L vitamin B12 to produce a biomass. In some embodiments, the culture medium comprises less than 0.01 mg/L n BIZ. In someiembodiments, the culture medium comprises less than 0.001 mg/L vitamin BIZ. In further embodiments, the e medium comprises less than 0.0001 mg/L vitamin B12. In some embodiments, the culture medium contains no vitamin BIZ.

In some embodiments, the e medium further comprises less than 1 g of yeast extract per 50 g of lipid-free biomass. In some embodiments, the culture medium further comprises less than 0.5 In further g of yeast extract per 50 g of lipid-free biomass. embodiments, the e medium further comprises less than 0.1 g of yeast extract per 50 g of lipid—free s.

In some embodiments, the EPA concentration is sed by at least 400% compared to the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprising greater than 0.1 mg/L n BIZ. In some embodiments, the EPA, concentration is increased by at least 300% compared to the EPA concentration in a s from a microorganism grown in a culture medium comprising greater than 0.01 mg/L vitamin 312. In further embodiments, the EPA concentration is increased by at least 200% red to the EPA concentration in a biomass from a microorganism grown in a culture medium comprising greater than 0.001 mg/L vitamin B12. In some embodiments, the EPA concentration is sed by at least 100% compared to the EPA tration in a biomass from a microorganism grown in a culture medium comprising greater than 0.0001 mg/L vitamin B12.

In some embodiments, the invention provides a method of making a biomass of a microorganism having increased concentration of EPA, sing growing the microorganism in a culture medium comprising less than 0.1 mg/L cobalt to produce a biomass. In some ments, the culture medium comprises less than 0.01 mg/L cobalt. In some embodiments, the e medium comprises less than 0.00] mg/L cobalt. In further embodiments, the culture medium comprises less than 0.0001 mg/L cobalt. In some embodiments, the culture medium contains no cobalt.

‘ In some embodiments, the microorganism is a Thraustochytrid. In some embodiments, the microorganism produces at least 3% EPA of the total weight of the fatty acids.

In some embodiments, the culture medium has a dissolved C02 level of at least %. In further ments, the culture medium has a dissolved C02 level of at least %. In some embodiments, the» culture medium has a dissolved C02 level of at least %.

The invention also provides an isolated biomass, and a microbial oil extracted from the biomass of any of the methods .

BRIEF DESCRIPTION OF DRAWINGS The various embodiments of the invention can be more fully understood from the following detailed description, the , and the anying ce descriptions, which form a part of this application. shows the performance of PTA-9695 in a thiamine gradient. shows the performance of PTA-9695 in a vitamin B12 gradient. shows the performance of PTA-9695 in a biotin gradient. shows the performance of PTA-9695 in a Ca-pantothenate gradient. shows the performance of PTA-9695 in TSFM standards. — show the performance of PTA-9695 in a‘vitarnin B12 gradient at % C02. — show the performance of PTA-10208 in a n Bl2 gradient at 10% C02.

DETAILED DESCRIPTION OF THE INVENTION The s and compositions provided herein are particularly applicable to a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids it produces. A particular Thraustochytrid that produces a biomass having at least 3% EPA provided herein is an ed rganism of the Species ted under ATCC Accession No. PTA-10212. The isolated microorganism associated with ATCC Accession No. PTA-10212 was deposited under the Budapest Treaty on July 14, 2009 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110—2209..

A particular Thraustochytrid that produces a biomass having at least 3% EPA is selected from an isolated microorganism deposited under‘ATCC Accession No. PTA- 10212, PTA-10213, PTA-10214, PTA-10215, PTA-10208, PTA-10209, PTA-10210, or PTA-10211.

A particular ment provided here is directed. to an isolated microorganism sing an 185 rRNA comprising a polynucleotide sequence of SEQ ID NO:1 or a polynucleotide sequence having at least 94% identity to SEQ ID NO: I.

A particular embodiment provided here is directed to an isolated microorganism comprising an 183 rRNA polynucleotide sequence that has at least 94% identity to an 18s rRNA polynucleotide sequence of the rganism deposited under ATCC Accession No. PTA-10212.

A particular Thraustochytrid that produces a biomass having at least 3% EPA provided herein is ed to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208 A particular ment provided here is directed to an isolated microorganism of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprises more than about 10% by weight eicosapentaenoic acid. 2012/047728 A particular Thraustochytrid that produces a biomass having at least 3% EPA he present invention is directed to an isolated rganism having the characteristics of the species ted under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprises more than about 10% by weight eicosapentaenoic acid. A particular Thraustochytn'd that produces a biomass having at least 3% EPA provided herein is selected from an isolated microorganism selected from the a mutant strain deposited under ATCC Accession No. PTA-10209, PTA—10210, or PTA-10211. The microorganisms associated with ATCC Accession Nos. PTA-10209, PTA-10210, and PTA-1021 l were deposited under the Budapest Treaty on ber 25, 2009 at the American Type Culture Collection, Patent Depository, 10801 sity Boulevard, Manassas, VA 201 10-2209.

Embodiments provided herein are directed to the microorganisms described, above, their mutant strains and the microorganisms identiﬁed in US. Patent Application No. 12/729,013 incorporated by reference herein in its entirety.

An embodiment provided herein is directed to an ed rganism that produces a triacylglycerol fraction, n pentaenoic acid content of the triacylglycerol fraction is at least about 12% by Weight.

An embodiment provided herein is ed to an isolated biomass, wherein at least about 20% by weight of a dry cellweight of the biomass are fatty acids, wherein more than about 10% by weight of fatty acids is eicosapentaenoic acid, and wherein the fatty acids comprise less than about 5% by weight each of arachidonic acid and docosapentaenoic acid n-6. In some embodiments, at least about 25% by weight of the fatty acids is docosahexaenoic acid.

In some embodiments, the present invention is directed to an isolated biomass sing triacylglycerol, wherein at least about 12% by weight of triacylglycerol is eicosapentaenoic acid.

' In some embodiments, the present invention is directed to any of the isolated biomasses of the 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.

The present ion is directed to an isolated thraustochytrid microorganism of the thraustochytrid Species deposited under ATCC Accession No. 95 or a strain derived therefrom, wherein the total fatty acids produced by said microorganism or strain derived therefrom comprise about 10% or less by weight pentaenoic acid. An embodiment provided herein is direct to the microorganism describe above or a strain therefrom and other related rganism describe in US. Patent Application Publication No. US 2010/0239533 incorporated by reference herein in its entirety.

Also provided herein is a method of increasing the concentration of EPA in a s of a microorganism having fatty acids and a concentration of EPA comprising: fermenting the microorganism in a ferrnentor vessel, comprising a gas, to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a s having at least 3% EPA of the total weight of the fatty acids ; and supplementing the gas with C02 in an amount sufficient to increase the concentration of the EPA in the s.

The increase in the concentration of the EPA can be compared for example to the concentration of EPA similarly fermented microorganism not supplemented with CO2 or when ed to a similarly fermented microorganism at ambient conditions.

In another ment, the amount of CO2 sufﬁcient to increase the concentration of the EPA is greater than or equal to 2% of the total gas in the vessel. In another embodiment, the amount of the C02 in the vessel is greater than or equal to about 5% up to about 20% ’of the total gas in the vessel. In another ment, the amount of the C02 in the vessel is greater than or equal to about 5% up to about 15% of the total gas in the vessel.

In a further embodiment, the amount of C02 supplemented is greater than or equal to 2% of the total gas in the vessel to increase the concentration of the EPA in the biomass to greater than about 4%, by weight, of the total weight of the fatty acids, more particularly from about greater than 4% up to about 45%, by weight, of the total weight of the fatty acids, more particularly, from about r than 4% up to about 40% of the total weight of the fatty acids.

In a r embodiment, the amount of CO2 supplemented is sufﬁcient to increase the EPA levels from about 4% to a range of about 6 to 30%, by weight, of the total weight of thefatty acids. In r embodiment, the amount of CO2 provided is sufﬁcient to increase the concentration of the EPA from about 15% up to about 40%, by weight of the total weight of the fatty acids. In another embodiment, the amount of CO2 provided is sufﬁcient to increase the concentration of the EPA to greater than 20%, by weight, of the WO 13208 total weight of the fatty acids. In another embodiment, the C02 is ed in an amount sufﬁcient to se the concentration of the EPA from about 20% up to about.25%.

In another embodiment, ed herein is a method of increasing the ~ tration of EPA in a biomass of a microorganism having fatty acids and a concentration of EPA comprising: fermenting the microorganism in a fermentor vessel to produce a biomass; providing a pressure on the biomass sufﬁcient to increase the. concentration of the EPA in the biomass. The increase in the concentration of the EPA can be compared for e to the concentration of EPA similarly fermented microorganism not provided with the pressure or when compared to a similarly fermented microorganism at ambient conditions. In further embodiment, the re provided on the biomass is about 0.5 psiabove atmospheric pressure. In another embodiment, the vessel has a head pressure (or back pressure) of greater than or equal to about 0.4 psi, from more particularly from about 0.4 psi up to about 30 psi, even more particularly of about 1 up to about 30 psi. In another embodiment, the vessel has a head pressure of from about 1 up to about 20 psi. In another embodiment the pressure provided is provided for a time sufﬁcient to adjust the amount of EPA in the biomass, particularly for a time up to 120 hours.

In another embodiment provided herein is a method of making a biomass of a microorganism producing fatty acids and a concentration of EPA comprising fermenting the microorganism in a fermentor vessel to produce a biomass wherein the microorganism comprises a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids at a temperature sufﬁcient to increase the tration of the EPA in the biomass. In some embodiments, the temperature sufﬁcient to increase the EPA levels is less than about 30 °C, more particularly is less than or equal to about 22 °C, and more particularly the temperature is at less than or equal to about 21°C. The increase in the tration of the EPA can be compared for e to the concentration of EPA in a similarly fermented rganism where the temperature is not adjusted or when compared to a similarly fermented microorganism at ambient conditions.

In a r embodiment, the methods provided herein vary the amounts of EPA: generated during fermentation to produce a biomass, and an oil extracted, wherein the amount of EPA provided is greater than 4%, particularly from about greater than 4% up to about 45%, more particularly from about greater than 4% up to about 40% by weight, of the total weight of the fatty acids. In another embodiment the amount of EPA produced by a method provided herein is in an amount from about 6% up to about 30% by weight of the total weight of the fatty acids. In a further embodiment the amount of EPA ed by a method provided herein is in an amount of from about 15% up to about 40% by weight of the total weight of the fatty acids. In a further embodiment the amount of EPA produced by a method provided herein is in an amount of r than about 20%, more particularly from about 20% up to about 25% by weight of the total weight of the fatty acids'.

In a further ment, the desired EPA level ed is greater than 4%, particularly from about greater than 4% up to about 45%, more particularly from about greater than 4% up to about 40% by weight, of the total weight of the fatty acids. In another embodiment the desired EPA level produced by a method provided herein is in an amount from about 6% up to about 30% by weight of the total weight of the fatty acids.

In a further embodiment the desired EPA level produced by a method provided herein is in an amount of from about 15% up to about 40% by weight of the total weight of the fatty acids. In a further embodiment the desired EPA level produced by a method provided herein is in an amount of greater than about 20%, more particularly from about % up to about 25% by weight of the total weight of the fatty acids.

A method of increasing the concentration of EPA in a biomass of a microorganism having fatty acids and a concentration of EPA comprising: fermenting the microorganism in a fermentor vessel to produce a biomass wherein the microorganism a Thraustochytrid that produces a s having at least 3% EPA of the total weight of the fatty acids; and increasing the s in an amount sufﬁcient to increase the concentration of the EPA in the biomass. In some embodiments the amount of biomass sufficient to increase the concentration of the EPA has a density of greater than or equal to lOg/l. In some embodiments the amount of biomass sufficient to increase the concentration of the EPA has a density of about 10 g/l up to about 250 g/l. The increase in the concentration of the EPA can be compared for example to the concentration of EPA in a similarly fermented microorganism wherein the s is not increased.

In some embodiments, the EPA concentration of the biomass grown in a culture medium sing a higher C02 level (for example, in a vessel comprising C02 in an amount of 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% % of the total gas in the vessel) 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 l300%, at least 1400%, at least 1500%, at least 1600%, at least 1700%, at least 1800%, at least l900%, or at least 2000% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium sing lower CO2 levels (for example, in a vessel sing 'C02 in an amount of less than 2%, less than %, less than 10%, less than 15%, less than 20%, 0% to 4%, or 1% to 3% of the total gas in the vessel, respectively). For example, the EPA concentration of the biomass grown in a culture medium comprising a higher C02 level (for e, in a vessel comprising CO2 in an amount of greater than or equal to 2%, greater than or equal to 5%, greater than to 20%, or equal to 10%, greater than or equal to 15%, greater than or equal to 20%, 5% ‘ or 5% to 15% of the total gas in the vessel) is at least 10%, at least 50%, at least 100%, least 250%, at least 500%, at least 250%, at least 1000%, at least 1100%, at least 1200%, at least l300%, at least 1400%, at least 1500%, at least I600%, at least 1700%, at least 1800%, at least l900%, or at least 2000% higher than the EPA concentration in a biomass obtained from the microorganism grown in a vessel at an ambient C02 level.

Vitamin BIZ in Culture Medium The term "vitamin B12" as used herein refers to a class of ally related compounds in both naturally occurring and synthetic forms, including, but not limited to, vitamin BIZ, cobalamin,‘ cyanocobalamin, and hydroxocobalamin. In some embodiments, the invention provides methods of sing the EPA concentration in the biomass of a microorganism that produces EPA by growing the microorganism in a e medium having low levels of vitamin BIZ or in a culture medium having no vitamin BIZ. In some embodiments, the invention provides methods of making a biomass of a microorganism having increased concentration of EPA, sing growing the microorganism in a culture medium comprising less than 0.1 mg/L n BIZ to produce a 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 0.00] mg/L, less than 0.0005 mg/L, less than 0.0001 mg/L, or no vitamin B12.

In some ments, the culture medium comprises less than 1 g of sources of vitamin B12 (such as yeast extract, corn steep solids, soy ﬂour, and other complex nitrogen sources) per 50 g of lipid-free biomass. In some embodiments, the culture medium comprises 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 sources of vitamin B12 per 50 g of lipid-free biomass. In some embodiments, the culture medium ﬁthher comprises less than 1 g of yeast extract per 50 g of lipid-free biomass, less than 0.8 g of yeast t per 50 g of lipid-free biomass, less than 0.5 g of yeast extract per 50 g of lipid-free biomass, less than 0.3 g of yeast extract per 50 g of lipid-free biomass, less than 0.1' g of yeast extract per 50 50 g of lipid-free s, or g of lipid-free biomass, less than 0.05 g of yeast extract per less than 0.01 g of yeast extract per 50 g of lipid-free s. As used herein, the term "lipid-free biomass" refers to the target fat-free dry cell weight of the microorganism after cultivation.

In some embodiments, the EPA concentration of the biomass grown in a culture medium comprising a lower vitamin BIZ level (for example, in a culture medium comprising 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 0.00] mg/L, less than 0.0005 mg/L, less than 0.0001 mg/L, or no n BIZ) 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 a s obtained from the microorganism grown in a culture medium comprising higher vitamin BIZ levels (for example, in a culture medium sing 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 vitamin BIZ, respectively). For e, the EPA concentration of the biomass grown in a culture medium containing no vitamin B12 is at least 10%, at least 25%, at least 50%, 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 a biomass obtained from the microorganism grown in a culture medium comprising vitamin BIZ (such as at least 0.0001 mg/L vitamin B12). 2012/047728 In some embodiments, the EPA concentration of the biomass grown in a culture medium sing lower vitamin B12 levels (for example, in a culture medium sing 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 vitamin B12) under an ambient C02 level is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in a biomass obtained from the. microorganism grown in a cultUre medium comprising higher vitamin B12 levels (for example, 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, at least 0.0001 mg/L, or at least 0.00005 mg/L vitamin BIZ, respectively) under an ambient CO2 level; , In some ments, the EPA concentration of the biomass grown in a culture medium comprising lower vitamin 812 levels under an ambient CO2 level is 100% to 700%, 150% to 650%, 200% to 600%, 250% to 550%, or 300% to 500% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprising higher vitamin BIZ levels under an ambient CO2 level. In some embodiments, the EPA concentration of the biomass grown in a culture medium comprising lower vitamin 812 levels under high CO2 level (for example, a dissolved C02 level of at least 5%, at least 10%, at least 15%, or at least 20%) is at least 5%, at least %, 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 tration in a biomass obtained from the microorganism grown in a culture medium ' comprising higher vitamin B12 levels under a high CO2 level. In some embodiments, the EPA concentration of the biomass grown in a e medium comprising lower vitamin B12 levels under high CO2 level (for example, a dissolved CO2 level of at least 5%, at least 10%, at least 15%, or at least 20%) is 5% to 200%, 10% to 175%, 15% t01150%, % to 125%, or 25% to 100% higher than the EPA concentration in a biomass obtained from the rganism grown in a culture medium comprising higher vitamin B12 levels under a high CO2 level. For example, the EPA concentration of the biomass grown in a culture medium comprising no vitamin 812 under an ambient C02 level is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium sing vitamin B12 (such as at least 0.0001 mg/L vitamin 812) under an ambient C02 level. As another example, the EPA concentration of the biomass grown in a culture medium comprising no vitamin 812 under a dissolved C02 level of at least 10% is at least %, 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 a biomass obtained from the microorganism grown in a e medium comprising vitamin B12 (such as at least 0.0001 mg/L vitamin B12) under a dissolved C02 level of at least 10%.

In some ments, the EPA concentration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.1 mg/L vitamin BIZ compared to the same microorganism grown in a culture medium having greater than 0.1 mg/L vitamin B12. In some embodiments, the EPA concentration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.01 mg/L vitamin B12 compared to the same microorganism grown in a culture medium having greater than 0.01 mg/L vitamin BIZ. In some embodiments, the EPA concentration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.001 mg/L vitamin B12 compared to the same rganism grown in a culture medium having greater than 0.001 mg/L vitamin B12. In r embodiments, the EPA concentration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the s of a microorganism grown in a culture medium having less than 0.0001 mg/L vitamin B12 ed to the same microorganism grown in a culture medium having greater than 0.0001 mg/L vitamin B12. In some embodiments, the EPA concentration is sed by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having no vitamin 812 compared to the same microorganism grown in a culture medium containing an amount of n 312.

The determination of the increase in EPA concentration in the biomass can be made by growing a microorganism in a culture medium having higher s of vitamin BIZ, growing the same microorganism in a culture medium having lower amounts of vitamin B 12, and comparing the EPA concentration in the biomass resulting from each culture. In this determination, the content of the culture media having loWer or higher amounts of vitamin BIZ are the same except for their level of vitamin BIZ.

In some embodiments, the EPA concentration of the biomass of the 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 0.00] mg/L, at least 0.0005 mg/L, or at least 0.0001 mg/L vitamin B12 is at least 1%, at least 2%, at least 3%, at least 4%, at least 5% EPA by weight of the total fatty acids. In some embodiments, the EPA concentration of the biomass of the microorganism grown in a culture medium sing at least 0.1 mg/L, at least 0.05 mg/L, at least 0.01 mg/L, at least 0.005 ‘rng/L, at least 0.001 mg/L, at least 0.0005 mg/L, or at least 0.0001 mg/L n 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 % EPA by weight of the total fatty acids.

Cobalt in Culture Medium In some embodiments, the invention provides methods of making a biomass of a microorganism having increased concentration of EPA, comprising growing the microorganism in a culture medium comprising less than 0.1 mg/L cobalt to produce a biomass. In some embodiments, the culture medium ses less than 0.05 mg/L, less than 0.01 mg/L, less than 0.005 mg/L, less than 0.00] mg/L, less than 0.0005 mg/L, less than 0.0001 mg/L, or no cobalt.

In some embodiments, the culture medium r comprises less than 1 g of yeast extract per 50 g of lipid-free biomass, less than 0.8 g of yeast extract per 50g of lipid-free biomass, less than 0.5 g of yeast extract per 50 g of lipid-free biomass, less than 0.3 gof 50 g of yeast extract per 50 g of lipid-free biomass, less than 0.1 g of yeast extract per lipid—free biomass, less than 0.05 g of yeast extract per 50 g of lipid—free biomass, or less than 0.01 g t extract per 50 g of lipid-free biomass.

In some embodiments, the EPA concentration of the s grown in a culture medium comprising a lower cobalt level (for example, in a culture medium comprising 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) is at least %, 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%, least 550%, at least 600%, at least 650%, or at least 700% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprising higher cobalt levels (for example, 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 myL, at least 0.0005 mg/L, at least 0.0001 mg/L, or at least 0.00005 mg/L cobalt, respectively).

For example, the EPA tration of the biomass grown in a culture medium. containing no 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 a biomass ed from the microorganism grown in a culture mediumcomprising cobalt (such as at least 0.0001 mg/L cobalt).

In some embodiments, the EPA tration of the biomass grown in a culture medium comprising lower cobalt levels (for example, in a culture medium comprising 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) under an ambient C02 level is at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprising higher cobalt levels (for example, 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, at least 0.0001 mg/L, or at least 0.00005 mg/L cobalt, respectively) under an ambient CO2 level. In some embodiments, the EPA concentration of the biomass grown in a culture medium comprising lower cobalt levels under an ambient CO2 level is 100% to 700%, 150% to 650%, 200% to 600%, 250% to 550%, or 300% to 500% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprising higher cobalt levels under an ambient CO2 level. In some ments, the EPA concentration of the biomass grown in a e medium sing lower cobalt levels under high C02 level (for example, a dissolved CO2 level of at least 5%, at least 10%, at least 15%, or at least %) 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 a biomass obtained from the microorganism level. In grown in a e medium comprising higher cobalt levels under a high CO2 some embodiments, the EPA tration of the biomass grown in a culture medium comprising lower cobalt levels under high C02 level (for example, a dissolved CO2 level of at least 5%, at least 10%, at least 15%, or at least 20%) is 5% to 200%, 10% to 175%, % to 150%, 20% to 125%, or 25% to 100% higher than the EPA concentration in a s obtained from the microorganism grown in a culture medium comprising higher cobalt levels under a high C02 level. For example, the EPA concentration of the biomass is at least grown in a culture medium comprising no cobalt under an t CO2 level 100%, at least 200%, at least 300%, at least 400%, or at least 500% higher than the EPA concentration in a biomass obtained from the microorganism grown in a culture medium comprisingcobalt (such as at least 0.0001 mg/L cobalt) under an ambient C02 level. As another example, the EPA concentration of. the biomass grown in a culture medium comprising no cobalt under a dissolved C02 level of at least 10% is at least 5%, at least %, 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 tration in a biomassobtained from the microorganism grown. in a culture medium comprising cobalt (such as at least 0.0001 mg/L cobalt) under a dissolved CO2 level of at least 10%.

In some embodiments, the EPA tration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or _at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.1 mg/L cobalt compared to the same microorganism grown in a culture medium having greater than 0.1 mg/L cobalt.

In some embodiments, the EPA concentration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism‘ grown in a culture medium having less than 0.01 mg/L cobalt compared to the same microorganism grown in a culture medium having greater than 0.01 mg/L cobalt. In some embodiments, the EPA tration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.001 mg/L cobalt compared to the same microorganism grown in a culture medium having greater than 0.001 mg/L cobalt. In further embodiments, the EPA tration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having less than 0.0001 mg/L cobalt compared to the same microorganism grown in a culture medium having greater than 0.0001 mg/L cobalt. In some embodiments, the EPA tration is increased by at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% in the biomass of a microorganism grown in a culture medium having no cobalt compared to the same microorganism grown in a culture medium containing an amount of cobalt. The determination of the se in EPA concentration in the biomass can be made by growing a microorganism in a culture medium having higher amounts of cobalt, growing the same microorganism in a culture medium having lower amounts of cobalt, and comparing the EPA concentration in the biomass ing from each culture. In this determination, the content of the culture media having lower or higher amounts of cobalt are the same except for their level of In some ments, the EPA tration of the biomass of the 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 the total fatty acids. In some embodiments, the EPA concentration of the biomass of the rganism grown in a e 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 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 by weight of the total fatty acids.

The culture medium containing low amounts of vitamin B12, yeast extract, and/or cobalt could further comprise a dissolved C02 level of at least 5%, at least 10%, at least %, or at least 20%. The present invention is directed to an isolated biomass of the methods disclosed herein, as well as to a microbial oil extracted from the biomass of the methods.

The present invention is directed to an ed culturecomprising any of the microorganisms of the invention or mixtures thereof.

The present invention is directed to a food product, cosmetic, or pharmaceutical composition for a non-human animal or human, comprising any of the microorganisms or biomasses of the invention or mixtures thereof.

The present invention is ed to a microbial oil comprising at least about 20% by weight eicosapentaenoic acid and less than about 5% by weight each of arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid, and stearidonic acid. In some embodiments, the microbial oil further comprises at least about 25% by weight docosahexaenoic acid.

The present invention is directed to a microbial oil comprising a triacylglycerol ﬁaction of at least about 10% by weight, wherein at least about 12% by weight of the fatty acids in the triacylglycerol fraction is eicosapentaenoic acid, n at least about % by weight of the fatty acids in the triacylglycerol on is docosahexaenoic acid, and wherein less than about 5% by weight of the fatty acids in the triacylglycerol fraction is arachidonic acid.

The present invention is directed to a food t, cosmetic, or pharmaceutical composition for a non—human animal or human, comprising any of the microbial oils of the invention. In some embodiments, the food product is an infant fOrmula. In some embodiments, the infant formula is suitable for premature infants. In some embodiments, the food product is a milk, a beverage, a eutic drink, a nutritional drink, or a combination thereof. In some embodiments, the food product is an additive for the non- human animal or human food. In some embodiments, the food product is a nutritional supplement. In some embodiments, the food product is an animal feed. In some embodiments, the animal feed is an aquaculture feed. In some embodiments, the animal feed is a domestic animal feed, a zoological animal feed, a work animal feed, a livestock feed, or a combination thereof. , The present invention is directed to a method for producing a ial oil comprising omega-3 fatty acids, the method comprising: growing any of the isolated microorganisms of the invention or mixtures f in a culture to produce an oil comprising omega-3 fattyapids. In some embodiments, the methbd further comprises extracting the oil. \ The present invention is directed to a method for producing a microbial oil comprising omega-3 fatty acids, the method sing extracting an oil comprising omega-3 fatty acids from any of the biomasses of the ion. In some embodiments, the microbial oil is extracted using an c solvent extraction process, for example hexane extraction. In some embodiments, the microbial oil is extracted using a tless extraction process.

The present invention is directed to a ial oil produced by a method of the invention.

The present ion is ed to a method for producing a biomass of the invention, comprising: g any of the isolated microorganisms of the invention or mixtures thereof in a culture to produce a biomass.

The present invention is directed to a s ed by a method of the invention.

The present invention is directed to a method for producing a mutant strain of the invention, comprising: mutagenizing any of the microorganisms of the invention, and isolating the mutant strain.

The present ion is directed to use of any of the isolated microorganisms, ses, or microbial oils of the invention, or mixtures thereof, for the manufacture of a medicament for treatment of ation or a condition related thereto.

The present invention is directed to use of any of the isolated microorganisms, biomasses, or microbial oils of the invention, or mixtures thereof, for treatment of inﬂammation or a condition related thereto.

The present invention is directed to any of the isolated microorganisms, biomasses, or microbial oils of the invention, or mixtures thereof, for use in treatment of inﬂammation or a condition related thereto.

The present invention is directed to a method for treating inﬂammation or a condition d thereto in a subject in need f, comprising administering to the subject any of the isolated microorganisms, biomasses, or microbial oils of the invention, or mixtures thereof, and a pharmaceutically able carrier.

The present ion is directed to methods of producing microbial oils, and biomasses, from the microorganisms of the invention, and methods of using the microorganisms, biomasses, and microbial oils.

Microorganisms In some embodiments, a microbial cell for use with the present invention is a microorganism of the phylum Labyrinthulomycota. In some embodiments, a microbial cell of the phylum Labyrinthulomycota is a thraustochytrid, such as a Schizochytrium or Thraustochytrium. According to the t invention, the term "thraustochytrid" refers to any member of the order Thraustochytriales, awhich includes the family Thraustochytriaceae, and the term "labyrinthulid" refers to any member of the order Labyrinthulales, which includes the family Labyrinthulaceae.

Members of the family Labyrinthulaceae were previously considered to be members of the order tochytriales, but in more recent revisions of the taxonomic classiﬁcation of such organisms, the family Labyrinthulaceae is now considered to be a member of the order Labyrinthulales. Both Labyrinthulales and tochytriales are considered to be members of the phylum Labyrinthulomycota: Taxonomic theorists now generally place both of these groups of microorganisms with the algae or algae-like protists of the Stramenopile lineage. The current taxonomic placement of the thraustochytrids and labyrinthulids can be summarized as follows: Realm: Stramenopila (Chromista) Phylum: Labyrinthulomycola (Heterokonta) Class: Labyrinthulomycetes (Labyrinthulae) Order: Labyrinthulales Family: Labyrinthulaceae Order: Thraustochytriales Family: Thraustochytriaceae For purposes of the present invention, strains of microbial cells described as tochytrids include the following organisms: Order: tochytriales; Family: :Thraustochytriaceae; Genera: tochytrium es: sp., arudimentale, , benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, erum, roseum, and striatum), Ulkem‘a (Species: sp., amoeboidea, kerguelensis, minuta, profunda, radiata, sailens, sarkariana, schizochytrops, ensis, yorkensis, and Sp.

BP-5601), Schizochytrium (Species: sp., aggregatum, limnaceum, mangrovei, minutum, and octosporum), Japonochytrium (Species: sp., marinum), Aplanochytrium (Species: Althorm‘a (Species: sp., haliotidis, kerguelensis, profurzda, and stocchinoi), sp., crouchii), or Elina (Species: sp., marisalba, and sinoriﬁca). For the purposes of this ion, species described within Ulkem‘a will be considered to be. members of the genus Thraustochytrium. Aurantiacochytrium and Oblogospora are two additional s encompassed by the phylum Labyrinthulomycota in the present invention. In some embodiments, a microbial cell is of the genus Thraustochystrium, Schizochytrium, and mixtures thereof.

The invention is directed to isolated microorganisms and s d therefrom. A strain that is "derived" from an isolated microorganism of the invention can be a natural or artiﬁcial derivative such as, for example, a mutant, variant, or recombinant strain. The term "isolated" as used herein does not necessarily reﬂect the extent to which form or an isolate has been puriﬁed, but indicates isolation or separation from a native native nment. An isolate can include, but is not limited to, an isolated microorganism, an isolated s, an isolated culture, an isolated ial oil, and an ed sequence (such as an isolated polynucleotide sequence disclosed herein). The term "microorganism" as used herein includes, but is not limited to, the terms algae," "thraustochytrid," and mic classiﬁcations associated with any of the deposited microorganisms described herein. The terms "Thraustochytriales," "thraustochytrid," "Schizochytrium," and "Thraustochytrium" as used in reference to any of the microorganisms of the invention, including the deposited microorganisms described herein, are based on present taxonomic classiﬁcations including available phylogenetic information and are not intended to be limiting in the event that the taxonomic classiﬁcations are revised aﬁer the ﬁling date of the present application.

In some embodiments, the invention is directed to an isolated rganism of the species deposited under ATCC ion No. PTA-10212. The isolated microorganism associated with ATCC ion No. 212 is also known herein as Thraustochytrium Sp. ATCC PTA-10212. The isolated microorganism associated with ATCC Accession No. PTA-10212 was deposited under the Budapest Treaty on July 14, 2009 at the American Type Culture tion, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209. In some embodiments, the invention is directed to an isolated strain deposited under ATCC Accession No. PTA-10212. In some embodiments the invention is directed to an isolated microorganism deposited under ATCC ion No. PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-10208, PTA-10209, PTA-10210, or PTA-10211.

In some embodiments, the invention is directed to an isolated microorganism having the characteristics of the s deposited under ATCC Accession No. PTA- 10212 or a strain derived therefrom. The characteristics of the species deposited under ATCC Accession No. PTA-10212 can include its growth and phenotypic properties (examples of phenotypic properties include morphological and reproductive properties), its physical and chemical properties (such as dry weights and lipid proﬁles), its gene in which the characteristics sequences, and combinations thereof, distinguish the species over previously ﬁed species. In some embodiments, the invention is directed to an isolated microorganism having the characteristics of the Species ted under ATCC Accession No. PTA-10212, wherein the characteristics include an 185 rRNA comprising the polynucleotide ce-of ‘SEQ ID N021 or a polynucleotide ce having at least 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1, the morphological and reproductive properties of the s deposited under ATCC Accession No. PTA- 10212, and the fatty acid proﬁles of the species ted under ATCC Accession No.

PTA-10212. In some embodiments, isolated rganisms of the invention haVe ypic properties substantially identical to those of the microorganism deposited under ATCC ion ,No. PTA-10212. In some embodiments, isolated microorganisms of the invention have growth properties substantially identical to those of the microorganism deposited under ATCC Accession No. PTA-10212. In some embodiments, the invention is directed to an isolated microorganism comprising an 18S rRNA comprising the polynucleotide sequence of SEQ ID NO:1 or a polynucleotide 99% identity to SEQ ID NO:1. sequence having at least 94%, 95%, 96%, 97%, 98%, or In some embodiments, the invention is directed to an isolated microorganism comprising an 185 rRNA polynucleotide sequence that has at least 94% identity to the 185 rRNA polynucleotide sequence of the rganism ted under ATCC Accession No.

PTA-10212.

In some embodiments, the invention is directed to a mutant strain of the microorganism deposited under ATCC Accession No. PTA-10212. In further embodiments, the mutant strain is a strain deposited under ATCC Accession No. PTA- 10213, PTA-10214, or PTA-10215. The microorganisms associated with ATCC Accession Nos. PTA-10213, PTA-10214, and PTA—10215 were ted under the Budapest Treaty on July 14, 2009 at the American Type Culture Collection, Patent tory, 10801 University Boulevard, Manassas, VA 201 10-2209.

In some embodiments, the invention is directed to an isolated rganism of the species deposited under ATCC Accession No. PTA-10208. The isolated microorganism associated with ATCC Accession No. PTA-10208 is also known herein as Schizochytrium sp. ATCC 208. The microorganism associated with ATCC Accession No. PTA-10208 was ted under the Budapest Treaty on July 14, 2009 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, Manassas, VA 20110-2209. In some embodiments, the invention is directed to an isolated strain deposited under ATCC ion No. PTA-10208.

In some embodiments, the ion is directed to an ed microorganism of the species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprises more than about 10%, more than about 1 1%, more than about 12%, more than about 13%, more than about 14%, more than about .1 5%, more than about 16%, more than about 17%, more than about 18%, more than about 19%, or more than about 20% by weight EPA. In some embodiments, the invention is directed to an ed rganism of the Species deposited under ATCC Accession No. PTA-10208, wherein the total fatty acids produced by the microorganism comprises 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 % 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% by weight EPA.

In some embodiments, the ion is directed to an isolated'microorganism having the characteristics of the species deposited under ATCC ion No. PTA- 10208, wherein the total fatty acids produced by the microorganism comprises more than about 10% by weight eicosapentaenoic acid. The characteristics of the microorganism deposited under ATCC Accession No. PTA-10208 include its growth and phenotypic properties (examples of phenotypic ties include morphological and reproductive properties), its physical and chemical properties (such as dry weights and lipid proﬁles), its gene sequences, and combinations thereof, in which the characteristics distinguish the species over previously identiﬁed species. In some embodiments, the invention is directed to an isolated microorganism having the characteristics of the s deposited under ATCC Accession No. PTA-10212, wherein the characteristics include an 185 rRNA comprising the polynucleotide sequence of SEQ ID NO:2, the morphological and ~26- reproductive properties of the species deposited under ATCC ion No. PTA-10208, and the fatty acid proﬁles of the species deposited under ATCC Accession No. PTA- 10208. In some embodiments, isolated microorganisms of the invention have physical and chemical properties ntially identical to those of the microorganism deposited under ATCC ion No. PTA-10208.

In some embodiments, the ion is directed to a mutant strain of the microorganism deposited under ATCC Accession No. PTA-10208. In r embodiments, the mutant strain is a strain deposited under ATCC Accession No. PTA- 10209, PTA-10210, or PTA-10211. The microorganisms associated with ATCC Accession Nos. PTA-10209, PTA-10210, and PTA—10211 were deposited under the Budapest Treaty on ber 25, 2009 at the American Type Culture Collection, Patent Depository, 10801 University Boulevard, as, VA 20110-2209.

In some embodiments, the invention is directed to an isolated microorganism of the ion that produces a triacylglycerol fraction, wherein the EPA content of the triacylglycerol fraction is 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 invention is directed to an isolated microorganism that produces a triacylglycerol fraction, wherein the EPA t of the triacylglycerol fraction is about 12% to about 55%, about 12 % to about 50%, about 12% to about 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, or about 20% to about 30% by .

In some ments, the invention is directed to a mutant, variant, or recombinant of an isolated microorganism of the invention that produces a triacylglycerol fraction, wherein the EPA content of the triacylglycerol 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 %, 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 invention is directed to a mutant, variant, or recombinant of an isolated microorganism of the invention that produces a triacylglycerol fraction, wherein the EPA content of the triacylglycerol fraction is about 12% to about 55%, about 12% to about 50%, about 12% to about 45%, about 12% to about 40%, about 12% to about 35%, about 12% to about 30%, about 15% to about 55%, WO 13208 about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% about to about 35%, about 15% to about 30%, about 20% to about 55%, about 20% to 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, or about 20% to about 30% by weight. Mutant strains can be produced by well-known procedures. Common procedures include irradiation, treatment at high temperatures, and treatment with a mutagen. t strains can be other naturally occurring isolates and/or sub-isolates of the species described herein. Recombinant strains can be produced by any nown methods in molecular biology for the expression of exogenous genes or. alteration of endogenous gene function or expression. In some embodiments, the mutant, variant, or recombinant strain produces a higher amount of omega—3 fatty acids, particularly 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 lower amounts of DHA, ARA, DPA n—6, or combinations thereof. In some embodiments, the of e mutant, variant, or recombinant strain produces a larger dry cell weight per liter than the ype strain. Such mutant, variant, or recombinant strains are eXamples of s derived from an isolated microorganism of the ion.

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

The present invention is also directed to an isolated thraustochytrid microorganism, or a strain derived therefrom, comprising a ceride fraction, wherein the hexaenoic acid content of the triglyceride fraction is at least about 40% by weight, wherein the docosapentaenoic acid n-6 content of the triglyceride fraction is from at least about 0.5% by weight to about 6% by , and wherein the total fatty acids produced by said microorganism or strain derived therefrom comprise about 10% or less by weight pentaenoic acid.

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

In some embodiments, the strain d from the isolated thraustochytrid. microorganism of the invention is a mutant strain.

The present invention is also directed to an isolated microorganism deposited under ATCC Accession No. PTA-9695, PTA-9696, PTA-9697, or PTA-9698.

The present invention is also directed to a thraustochytrid biomass comprising any one of the thraustochytrid microorganisms of the invention or mixtures thereof.

The present invention is also directed to an isolated thraustochytrid biomass, wherein at least about 50% by weight of the dry cell weight of the biomass are 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 is docosahexaenoic acid. The present invention is also directed to an isolated thraustochytrid biomass, n at least about 25% by weight of the dry cell weight of the s is docosahexaenoic acid.

In some embodiments, the present invention is also directed to an isolated thraustochytrid biomass wherein about 10% or less by weight of the fatty acids is eicosapentaenoic acid, and n the weight ratio of docosahexaenoic acid to acid is at least about 5:1. . eicosapentaenoic In some ments, the present invention is also directed to an isolated thraustochytrid s wherein about 1.5% or less by weight of the fatty acids is arachidonic acid, and n the weight ratio of docosahexaenoic acid to arachidonic acid is at least about 20:1.

In some embodiments, the present invention is also directed 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 invention is ed to a thraustochytrid of the species deposited under ATCC Accession No. PTA-9695. isolated tochytrid is also known herein as Schizochytrium sp. ATCC PTA—9695.

The thraustochytrid associated with ATCC Accession No. PTA-9695 was deposited under the Budapest Treaty on January 7, 2009 at the American Type Culture Collection, Patent tory, 10801 University Boulevard, Manassas, VA 20110-2209. -29.

In some embodiments, the invention is directed to an isolated thraustochytrid strain deposited under ATCC Accession No. PTA-9695. In some embodiments, the invention is directed to an isolated tochytrid rganism of the same s as the thraustochytrid deposited 'under ATCC Accession No. PTA-9695. [0.107] In some embodiments, the invention is directed to an isolated thraustochytrid having the characteristics of the species deposited under ATCC Accession No. PTA—9695 or a strain derived therefrom. The characteristics of the thraustochytrid species deposited under ATCC Accession No. PTA-9695 include its growth and phenotypic properties (examples of phenotypic ties include morphological and reproductive properties), its physical and chemical properties (such as dry weights and lipid proﬁles), and its gene sequences. In some embodiments, the isolated thraustochytrids of the invention have substantially identical phenotypic properties of the thraustochytrid deposited under ATCC Accession No. PTA-9695. In some embodiments, the isolated thraustochytrids of the invention have ntially identical growth properties of the tochytrid deposited under ATCC Accession No. 95.

In some embodiments, the invention is directed to a mutant, variant, or recombinant of an isolated thraustochytrid of the ion, wherein the total fatty acids produced by the mutant, variant, or recombinant comprise about 10% or less by weight eicosapentaenoic acid. Mutant strains can be produced by well-known ures.

Common procedures include irradiation; treatment at high temperatures; and treatment with a mutagen. Variant strains can be other naturally occurring isolates and/or subisolates of the species described herein. Recombinant strains can be produced by any well-known s in molecular y for the expression of exogenous genes or the alteration of endogenous gene on or expression. In some embodiments, the mutant, variant, or inant strain produces a higher amount of omega-3 fatty acids, including DHA and/or EPA, than the wild—type . In some embodiments, the mutant, variant, or recombinant strain es a lower amount of one or more fatty acids, such as lower amounts of EPA, ARA, DPA n-6, or combinations thereof. In some embodiments, the mutant, variant, or recombinant strain produces a larger dry cell weight per liter of culture than the wild—type strain. Such mutant, variant, or recombinant s are examples of strains derived from an isolated thraustochytrid of the invention.

' In embodiments, the invention is directed to a mutant strain of the some thraustochytrid deposited under ATCC Accession No. PTA-9695. In further ments, the mutant strain is a strain deposited under ATCC Accession Nos. PTA- 9696, PTA-9697, or PTA-9698. The thraustochytrid strains associated with ATCC ion Nos. PTA-9696, PTA-9697, and PTA-9698 were deposited under the Budapest Treaty on January 7, 2009 at the American Type Culture Collection, Patent Depository, 10801 University ard, Manassas, VA 20110-2209. These deposited mutant strains PTA-9695. are derivatives of the thraustochytrid deposited under ATCC Accession No.

In some embodiments, an isolated thraustochytrid of the invention, including mutants, variants, or inants thereof, comprises a fatty acid proﬁle in one or more fractions isolated from the tochytrid. The one or more fractions isolated from the thraustochytrid includes the total fatty acid fraction, the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof.

The t invention is also ed. to an isolated thraustochytrid culture comprising any one of the thraustochytrid microorganisms of the invention or mixtures thereof. In some embodiments, the culture comprises at least about 5% dissolved oxygen.

The present invention is also directed to a food product, ic, or pharmaceutical composition for animals or humans comprising any one of the thraustochytrid microorganisms or biomasses of the invention or mixtures f.

The present ion is also directed to a microbial oil comprising a triglyceride fraction of at least about 70% by weight, wherein the docosahexaenoic acid t of the triglyceride fraction is at least about 50% by weight, and wherein the docosapentaenoic acid n-6 content of the triglyceride fraction is from about 0.5% by weight to about 6% by weight. In some embodiments, the microbial oil further comprises an arachidonic acid content of the triglyceride fraction of about 1.5% or less by weight.

The present invention is also directed to a ial oil comprising a ceride fraction of at least about 70% by weight, wherein the docosahexaenoic acid content of the triglyceride fraction is at least about 40% by , wherein the docosapentaenoic acid n-6 content of the triglyceride fraction is from 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.

The present invention is also directed to a microbial oil comprising a triglyceride fraction of at least about 70% by weight, wherein the hexaenoic acid content of the triglyceride on is at least about 60% by weight.

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

In some ments, an ed microorganism of the invention, including mutants, variants, and recombinants thereof, comprises a fatty acid proﬁle in one or more fractions isolated from the microorganism. The one or more fractions isolated from the microorganism e the total fatty acid fraction, the sterol esters fraction, the triacylglycerol fraction, the free fatty acid fraction, the sterol fraction, the diacylglycerol fraction, the polar fraction (including the phospholipid fraction), and combinations thereof. The fatty acid proﬁle for a speciﬁc fraction can include any of the fatty acid proﬁles associated with the speciﬁc on as sed herein.

The ion is directed to a method of producing a mutant comprising mutagenizing any of the microorganisms of the invention and isolating the mutant strain.

Cultures and Isolated Biomasses The invention is directed to a culture comprising one or more ed microorganisms of the ion. Various fermentation parameters for inoculating, growing, and recovering‘microﬂora, such as microalgae and thraustochytrids, are known in the art. See, herein in its e.g., U.S. Patent No. 5,130,242, incorporated by reference entirety. Liquid or solid media can contain natural or ial sea water. Carbon sources for heterotrophic growth e, but are not limited to, glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, fucose, 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 acid, corn steep liquor, organic en sources, sodium glutamate, urea, inorganic en sources, ammonium acetate, ammonium sulfate, ammonium chloride, and ammonium nitrate.

A typical media for growth of the microorganism deposited under ATCC Accession No. PTA-10212 is shown in Table 1: Table 1: PTA-10212 Vessel Media ient concentration ranges ' 31.0 NaZSO4 g/L 0-50, 15-45, or 25-35 NaCl g/L 0.625 0-25, 01-10, or 0.5-5 KCl g/L 1.0 05, 025-3, or 0.5-2 MgSO4'7H20 g/L 5.0 0-10, 2-8, or 3-6 (NH4)2SO4 g/L 0.44 0-10, 0.25-5, or 0.05-3 MSG'IHZO g/L 6.0 0-10, 4-8, or 5-7 CaClz g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L 6.0 0-20, 01-10, or 1-7 KH2P04 g/L 0.8 01-10, 0.5-5, or 0.6-1.8 Post ave '(‘MetalSl ~ Citric acid mg/L 3.5 01-5000, 10-3000, or 3-2500 FeSO4'7H20 mg/L 10.30 0.1—100, 1-50, or 5-25 4H20 mg/L 3.10 0.1-100, 1—50, or 2-25 ZnSO4'7I-I20 mg/L 3.10 0.01-100, 1-50, or 2-25 CoC12‘6H20 mg/L 0.04 0-1, 0001-01, or 0.01-0.1 NazMoO4'2H20 mg/L 0.04 0001-], 0005-05, or 0.01-0.l CuSO4'5H20 mg/L 2.07 0.1-100, 05-50, or 1-25 NiSO4'6H20 mg/L 2.07 0.1-100, 05-50, or 1-25 Post autoclave {Vitamins} .- Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Vitamin BIZ mg/L 0.16 0.01-100, 0.05-5, or 0.1-1 Ca'A-pantothenate mg/L 2.06 0.1-100, 01-50, or 1-10 Biotin mg/L 3.21 0.1-100, 01-50, or 1—10 Post autoclave (Carbonl ol g/L 30.0 5-150, 10-100, or 20—50 Nitrogen Feed: Ingredient Concentration MSG-IHZO g/L 17 0-150, 10-100, or 15-50 Typical ation conditions would include the following: pH about 6.5 - about 9.5, about 6.5 — about 8.0, or about 6.8 — about 7.8; temperature: about 15 — about 30 degrees Celsius, about 18 — about 28 degrees Celsius, or about 21 to about 23 degrees Celsius; dissolved oxygen: about 0.1 — about 100% saturation, about 5 — about 50% saturation, or about 10 — about 30% saturation; and/or glycerol controlled @: about 5 — about 50 g/L, about 10 — about 40 g/L, or about — about 35 g/L.

In some embodiments, the microorganism deposited under ATCC Accession No. 212, or a , variant, or recombinant thereof, grows heterotrophically on glycerol as the carbon source but does not grow on glucose as the carbon source.

A typical media for growth of the microorganism deposited under ATCC Accession No. PTA-10208 is shown in Table 2: Table 2: PTA-10208 Vessel Media Ingredient concentration ran es NaZSO4 g/L 8.8 0-25, 2-20, or 3-10 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KCl g/L 1.0 0-5, 0.25—3, or 0.5-2 7H20 g/L 5.0 0-10, 2-8, or 3-6 (NH4)2SO4 g/L 0.42 0-10, 0.25-5, or 0.05-3 CaClz g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L ~ 1.0 0—20, 0.1-10, or 0.5-5 KH2P04 g/L 1.765 0.1-10, 05-5, or 1-3 Post autoclave gMetalsl Citric acid ' mg/L‘ - 46.82 0.1-5000, 10-3000, or 40-2500 FeSO4'7H20 mg/L 10.30 0.1-100, 1-50, or 5-25 MnC12'4H20 mg/L 3.10 0, 1-50, or 2-25 ZnSO4'7H20 mg/L 9.3 0.01 -100, 1-50, or 2-25 C0C12'6H20 mg/L 0.04 0-1, 0001-01, or 0.01-0.1 Na2M004'2H20 mg/L 0.04 0001-], 0.005-0.5, or 0.01-0.1 CuSO4'5H20 mg/L 2.07 0.1-100, 05-50, or 1-25 NiSO4'6l-120 mg/L 2.07 0.1-100, 05-50, or 1-25 Post ave (Vitaminsl Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Ca'A-pantothenate mg/L 3.33 0, 0.1—50, or 1-10 Biotin mg/L 3.58 0.1-100, 0.1-50, or 1-10 Post autoclave n) Glucose g/L 30.0 5-150, 10-100, or 20-50 Nitrogen Feed: - Ingredient Concentration NH4OH mL/L 23.6 0-150, 10-100, or 15-50 Typical cultivation ions would include the following: pH about 6.5 _— about 8.5, about 6.5 — about 8.0, or about 7.0 — about 8.0; ature: about 17 — about 30 degrees Celsius, about 20 - about 28 degrees Celsius, or about 22 to about 24 degrees Celsius; dissolved : about 2 — about 100% tion, about 5 — about 50% . saturation, or about 7 — about 20% saturation; and/or glucose controlled @: about 5 — about 50 g/L, about 10 — about 40 g/L, or about 7— about 35 g/L.

In some embodiments, the fermentation volume (volume of culture) 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 ,000 liters, at least about 20,000 , at least abOut 50,000 liters, at least about 100,000 liters, at least about 150,000 , at least about 200,000 liters, or at least about A 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 [000 liters, about 10,000 liters, about 20,000 , 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.

In some ments, the ion is directed to an isolated biomass comprising a fatty acid proﬁle of the 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 leastabout 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 the biomass are fatty acids. In some embodiments, greater than about 20%, greater than about 25%, than greater than about 30%, greater than about 35%, greater than about 40%, greater about 45%, greater than about 50%, greater than about 55%, or greater than about 60% of the dry cell weight of the biomass are fatty acids. 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 are fatty acids. In some embodiments, the biomass comprises more than about 10%, at least about 12%, at least about 15%, at least about %, at least about 25%, at least about 30%, about least about 35%, at least about 40%, or at least about 45% by weight of the fatty acids as EPA. In sOme embodiments, the biomass comprises about 10% to about 55%, about 12% to about 55%, about 15% to about 55%, about 20% to about 55%, about 20% to about 40%, or about 20% to about % by weight of the fatty acids as EPA. In some embodiments, the biomass ses a triacylglycerol fraction, n 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 of the triacylglycerol fraction is EPA.

In some embodiments, the biomass comprises a triacylglycerol fraction, wherein the EPA content of the triacylglycerol fraction is from at least about 12% to about 55%, about 12% about to about 50%, about 12% to about 45%, at least about 12% to about 40%, at least 12% to about 35%, or at least about 12% to about 30%, about 15% to about 55%, about % 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 %, or about 20% to about 30% by weight. 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 50%, or at least about 60% by weight of the dry cell weight of the biomass is DHA.

In some embodiments, about 20% to about 60%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 30% to about 50%, or about 35% to about 50% by weight of the dry cell weight of the biomass is DHA. In some embodiments, the s comprises about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by weight of the fatty acids as DHA. In some embodiments, the biomass comprises about 1% to about 10%, about 1% to about 5%, about 2% to about %, about 3% to about 5%, or about 3% to about 10% by weight of the fatty acids as DHA. In some embodiments, the biomass is substantially free of DHA. In some embodiments, the biomass comprises 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% by weight of the fatty acids as ARA. In some embodiments, the biomass comprises less than about 5%, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less by weight of the fatty acids 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% by weight of the fatty acids as DPA n-6. In some embodiments, the biomass comprises about 5% or less, less than about %, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.75% n-6. or less, about 0.6% or less, or about 0.5% or less by weight of the fatty acids as DPA In some embodiments, the biomass is substantially free of DPA n-6. In some embodiments, the biomass comprises fatty acids with about 5% or less, less than about %, about 4% or less, about 3% or less, or about 2% or less by weight of oleic acid (18:1 n-9), linoleic acid (18:2 n-6), linolenic acid (18:3 n-3), noic acid (20:1 n-9), erucic acid (22:1 n-9), or combinations thereof.

The characteristics of an isolated biomass of the invention are associated with endogenous or native properties of the isolated biomass rather than exogenously introduced als. In some embodiments, the isolated s does not n polyvinylpyrrolidone or is not isolated from a culture containing polyVinylpyrrolidone.

The present invention is directed to a method of producing a biomass. In some embodiments, the method for producing a s of the invention comprises growing invention or mixtures thereof in a culture to any of the isolated microorganisms of the 2012/047728 produce a biomass. The present invention is directed to a biomass produced by'the method.

In some embodiments the biomass ses fatty acids wherein the fatty acids further comprises omega-3 saturated fatty acids wherein the omega-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about _>_90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids and the amount of EPA, by weight, is from about 6% up to about 65% of the total amount of EPA and DHA. Particularly provided is a biomass wherein the amount of EPA, by weight, is from about 6% up to about 28% of the total amount of EPA and DHA. Further provided herein is a biomass wherein the amount of EPA, by weight, is from about 36% up to about 65 of the total amount of the EPA and DHA. More ularly provided is a biomass wherein the amount of EPA, by weight, is from about 28% to about 36% of the total amount of EPA and DHA. [012a] Some embodiments provided herein comprise a biomass comprising fatty acids wherein the fatty acids further comprise DHA and EPA and the amount of EPA, by weight, is from about 15 up to about 60 % of the total weight of EPA and DHA.

Some embodiments of the invention are further directed to a culture comprising a thraustochytrid, or a mutant strain, deposited under ATCC Accession No. PTA-9695.

Various fermentation parameters for inoculating, growing, and recovering microﬂora are known in the art, such as described in US. Patent No. 5,130,242. Any conventional medium for growth of thraustochytrids can be used. Liquid or solid mediums can contain natural or artiﬁcial sea water. Carbon sources include, but are not limited to, e, fructose, xylose, saccharose, maltose, soluble , molasses, fucose, glucosamine, dextran, fats, oils, glycerol, sodium e, and ol. Nitrogen sources include, but are not limited to, e, yeast extract, polypeptone, malt extract, meat extract, casamino acid, corn steep liquor, organic nitrogen sources, sodium glutamate, urea, inorganic en sources, ammonium acetate, ammonium sulfate, ammonium chloride, um nitrate, sodium sulfate. A typical media is shown in Table 3: Table 3: PTA-9695 Vessel Media Ingredient concentration ranges NaCl g/L 12.5 0-25, 5-20, or 10-15 KC] g/L 1.0 0-5, 0.25-3, or 0.5-2 MgSO4'7H20 g/L 5.0 0-10, 2-8, or 3-6 (NH4)2SO4 g/L 0.6 0-10, , or 0.5-3 CaClz g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast t) g/L 6.0 0-20, 1-15, or 5-10 KH2P04 g/L 1.2 01-10, 0.5-5, or 1-3 Post autoclave {Metals} Citric acid mg/L 3.5 01-100, 1—50, or 2-25 7l-l20 mg/L 10.30 0.1-100, 1-50, or 5-25 MnC12'4H20 mg/L 3.10 0.1-100, 1-50, or 2-25 ZnSO4'7H20 mg/L 3.10 0.1-100, l-50, or 2-25 CoC12'6H20 mg/L 0.04 1, 0005-05, or 0.01—0.1 NazMoO4'2I-I20 mg/L 0.04 0001-], 0005-05, or 0.010] CuSO4'5H20 mg/L 2.07 0.1-100, 05-50, or 1-25 NiSO4'6H20 mg/L 2.07 0.1-100, 0.5-50, or 1-25 Post autoclave itamins Thiamine** mg/L 9.75 0.1-100, 1-50, or 5-25 ’VitaminB12** mg/L 0.16 0.1—100, 01-10, or 01—] Cayz-pantothenate" mg/L 3.33 0.1-100, 01-50, or' 1—10 ** Filter ized Post autoclave n} - Glucose g/L 30.0 5-150, 10-100, or 20-50 Nitrogen Feed: Ingredient Concentration NI-I4OH mL/L 21.6 0-150, 10-100, or 15-50 Typical cultivation conditions would include the following: pH about 6.5 — about 8.5, about 6.5 —- about 8.0, or about 7.0 - about 7.5 ' temperature: about 17 — about 30 degrees Celsius, about 20 — about 25 degrees Celsius, or about 22 to about 23 degrees Celsius dissolved oxygen: : about 5 — abOut 100% saturation, about 10 — about 80% saturatione or about 20 —— about 50% saturation glucose controlled @: about 5 - about 50 g/L, about 10 — about 40 g/L, or about 20 — about 35 g/L.

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 saturation level. In some embodiments, the culture medium comprises from 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 tion level. i. [0131] The invention is further directed to an isolated biomass of a thraustochytrid ofthe invention. An isolated thraustochytrid biomass of the invention is a harvested cellular biomass obtained by any conventional method for the isolation of a thraustochytrid biomass, such as described in US. Patent No. 5,130,242 and U.S. Appl. Publ. No. 2002/0001833.

In some embodiments, the dry cell weight of the biomass ed from each 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 aﬁer g for about 7 days at about 17°C to about °C in a culture medium of about pH 6.5 to about 8.5 sing s of carbon, nitrogen, and nutrients, and about 950 ppm to about 8500 ppm chloride ions. In some embodiments, the dry cell weight of the biomass isolated from each 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 aﬂer growing for about 7 days at about 17°C at about 18°C at , , about 19°C, at about 20°C, at about 21°C, at about 22°C, at about 23°C, at about 24°C, at ‘ about 25°C, at about 26°C, at about 27°C, at about 28°C, at about 29°C, or at about 30°C in a culture medium of about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, or about pH 8.5 comprising sources of carbon, en, and nutrients, and about 950 ppm to about 8500. ppm chloride ions. In some embodiments, the dry cell weight of the biomass isolated from each liter of culture is from about 50 g to about 200 g after growing for about 7 days at about 17°C to about 30°C in a culture medium of about pH 6.5 to about about pH 8.5 comprising sources of carbon, nitrogen, and nutrients, and about 950 ppm to 8500 ppm chloride ions. In some embodiments, the dry cell weight of the biomass isolated from each liter of culture is from about 50 g to about 200 g after growing for about 7 days at about 17°C at about 18°C at about 19°C, at about 20°C, at about 21°C, , , at about 22°C, at about 23°C, at about 24°C, at about 25°C, at about 26°C, at about 27°C, at about 28°C, at about 29°C, or at about 30°C in a e medium of about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, or about pH 8.5 comprising sources of carbon, nitrogen, and nutrients, and about 950 ppm to about 8500 ppm chloride ions.

In some embodiments, the isolated thraustochytrid culture has an 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 growing for about 7 days at about 17°C to about 30°C in a culture medium of about pH 6.5 to about pH 8.5 comprising sources of carbon, nitrogen, and nts, and about 950 ppm to about 8500 ppm chloride ions. In some embodiments, the isolated thraustochytrid culture has an omega-3 fatty acid productivity of between 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 y, after growing for about 7 days at about 17°C to about 30°C in a e medium of about pH 6.5 to about pH 8.5 comprising sources of carbon, nitrogen, and nts, and about 950 ppm to about 8500 ppm chloride ions.

In some ments, the fermentation volume (volume of culture) 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 ,000 , 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 , about 200 liters, about 500 liters, about 1000 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 0 liters.

In some embodiments, the invention is directed to an isolated thraustochytrid biomass comprising a fatty acid proﬁle of the ion. In some embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the dry cell -4]- weight of the s are fatty acids. In some embodiments, greater than about 50%, greater than about 55%, or greater than about 60% of the dry cell weight of the biomass are fatty acids, In some embodiments, from 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 are fatty acids. In some ments, the biomass ses at least about 50%, at least about 60%, at least about 70%, or at least about 80% by weight of the fatty acids as omega-3 fatty acids. In some embodiments, the biomass comprises from about 50% to about 60%, about 50% to about 70%, about 50% to about 80% by weight of the fatty acids as omega-3 fatty acids. In some embodiments, the biomass comprises 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% by weight of the fatty acids as DHA. In some ments, the biomass‘comprises from about 50% to about 60%, about 50% to about 70%, or about 50% to about 80% by weight of the fatty acids as DHA. In some embodiments, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% by weight of the dry cell weight of the biomass is ‘ docosahexaenoic acid. In some embodiments, about 25% to about 65%, about 25% to about 50%, about 30% to about 40%, or about 25% to about 35% by weight of the dry cell weight of the biomass is docosahexaenoic acid. In some embodiments, the biomass comprises about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by weight of the fatty acids as EPA. In some embodiments, the biomass comprises from about 1% to about 10%, about 1% to about 5%, about 2% to about 5%, about 3% to about 5%, or about 3% to about 10% by weight of the fatty acids as EPA. In some embodiments, the biomass is substantially free of EPA. In some embodiments, the s comprises a weight ratio of DHA to EPA of at least about 521, 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, wherein the biomass comprises about 10% or less by weight of the fatty acids as EPA. In some embodiments, the biomass comprises from about 0.1% to 0.2%, about 0.1% to about 0.3%, about 0.1% to about 0.4%, about 0.1% to about 0.5%, or about 0.1% to about 1.5% by weight of the fatty acids as ARA. In some embodiments, the biomass comprises about 1.5% or less, about 1% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less by weight of the fatty acids as ARA. In some embodiments, the biomass is substantially free of ARA. In some embodiments, the biomass comprises a weight ratio of DHA to ARA of at least about :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 about 0.5% to some embodiments, the biomass comprises from about 0.5% to about 1%, about 2%, about 0.5% to about 5%, about 0.5% to about 6%, about 1% to about 5%, about 1% to about 6%, about 2% to about 5%, or about 2% to about 6% by weight of the fatty acids as DPA n-6. In some embodiments, the biomass comprises about 6% or less, about 5% or less, about 2% or less, about 1% or less, or about 0.5% or less by weight of the fatty acids as 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 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 biomass comprises fatty acids with about 5% or less, about 4% or less, about 3% or less, or about 2% or less by weight each of linoleic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid (20:1 n-9), and emcic acid (22:1 n-9).

In another ment provided herein is a biomass comprising fatty acids wherein the fatty acids further ses omega-3 saturated fatty acids n the omega-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about of the total greater than or equal to about 58-68%, particularly about 60% , by weight, amount of omega~3 polyunsaturated fatty acids and the amount of EPA, by weight, from about 5% up to about 60% of the total amount of the total weight of the EPA and DHA.

The characteristics of an isolated biomass of the invention are associated with endogenous or native properties of the ed biomass rather than exogenously introduced al.

Microbial Oils Provided herein are oils, particular microbial oils, made by the methods described above. 2012/047728 In some embodiments the microbial oil comprises fatty acids wherein the fatty acids further ses omega-3 polyunsaturated fatty acids wherein the omega-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about _>_90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids and the amount of EPA, by , is from about 6% up to about 65% of the total amount of EPA and, DHA. Particularly provided is a microbial oil wherein the amount of EPA, by weight, is from about 6% up to about 28% of the total amount of EPA and DHA. Further provided herein is a microbial oil n the amount of EPA, by weight, is from about 36% up to about 65 of the total amount of the EPA and DHA. More particularly provided is a microbial oil wherein the amount of EPA, by weight, is from about 28% to about 36% of the total amount of EPA and DHA.

In a further embodiment provided herein comprise a microbial oil comprising fatty acids wherein the fatty acids further comprise DHA'and EPA and the amount of EPA, by weight, is from about 15 up to about 60 % of the total weight of EPA and DHA.

In another embodiment provided herein is a microbial oil comprising fatty acids wherein the fatty acids further comprises omega-3 polyunsaturated fatty acids wherein the omega-3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about of the total greater than or equal to about 58-68%, particularly about 60% , by weight, amount of omega-3 saturated fatty acids and the ar'nount of EPA, by , from about 5% up to about 60% of the total amount of the total weight of the EPA and Dink.

The ion is ed to a microbial oil comprising a fatty acid profile of the invention. A microbial oil of the invention is a "crude oil" or a "reﬁned oil" comprising a triacylglycerol fraction of at least about 35% by weight. A "crude oil" is an oil that is extracted from the biomass of the microorganism without ﬁlrther processing. A "reﬁned oil" is an oil that is obtained by ng a crude oil with standard processing of refining, bleaching, and/or deodorizing. See, e.g., U.S Patent No. 5,130,242, incorporated by reference herein in its ty. A ial oil also includes a "final oil" as described herein, which is a reﬁned oil that has been diluted with a vegetable oil. In some embodiments, a ﬁnal oil is a reﬁned oil that has been diluted with high oleic sunﬂower oil. The term "microbial" as used herein includes, but is not limited to, the terms "microalgal," "thraustochytrid," and taxonomic classiﬁcations associated with any of the deposited microorganisms described herein. The terms "Thraustochytriales," "thraustochytrid," "Schizochytrium," and "Thraustochytrium" as used in nce to any of the microbial oils of the deposited microorganisms described herein are based on present taxonomic classiﬁcations including available phylogenetic information and are not ed to be ng in the event that the taxonomic classiﬁcations are revised after the ﬁling date of the present application.

In some embodiments, a fatty acid as described herein can be a fatty acid ester. In some ments, a fatty acid ester includes an ester of an omega-3 fatty acid, omega-'6 fatty acid, and combinations thereof. In some embodiments, the fatty acid ester is a DHA ester, an EPA ester, or a combination thereof. In; some embodiments, an oil or fraCtion thereof as described herein is esterified to produce an oil or fraction. thereof comprising fatty acid esters. The term " [refers to the replacement of the hydrogen in the carboxylic acid group of the fatty acid molecule with another substituent. Typical esters are known to those in the art, a discussion of which is ed by Higuchi, T. and V.

Stella in Pro-drugs as Novel Delivery Systems, Vol. 14, A.C.S. Symposium Series, Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American ceutical Association, Pergamon Press, 1987, and Protective Groups in Organic Chemistry, McOmie ed., Plenum Press, New York, 1973. es of esters include methyl,'ethyl, , butyl, , t-butyl, benzyl, nitrobenzyl, methoxybenzyl, dryl, and trichloroethyl. In some embodiments, the ester is a carboxylic acid protective ester group, esters with aralkyl (e.g., benzyl, phenethyl), esters with lower alkenyl (e.g., allyl, 2- butenyl), esters with lower-alkoxy-lower-alkyl (e.g., methoxymethyl, 2-methoxyethyl, 2- ethoxyethyl), esters with lower-alkanoyloxy-lower-alkyl (e.g., acetoxymethyl, pivaloyloxyrnethyl, l-pivaloyloxyethyl), esters with lower-alkoxycarbonyl-lower-alkyl (e.g., methoxycarbonylmethyl, isopropoxycarbonylmethyl), esters with carboxy-lower alkyl (e.g., carboxymethyl), esters with lower-alkoxycarbonyloxy-lower-alkyl (e.g., l- (ethoxycarbonyloxy)ethyl, l -(cyclohexyloxycarbonyloxy)ethyl), esters with carbamoyloxy-lower alkyl (e.g., carbamoyloxymethyl), and the like. In some embodiments, the added substituent is a linear or cyclic hydrocarbon group, e.g., a C1—C6 alkyl, C1-C6 cycloalkyl, C13‘C6 alkenyl, or C1-C6 aryl ester. In some embodiments, the ester is an alkyl ester, e.g., a methyl ester, ethyl ester or propyl ester. In some embodiments, theester substituent is added to the free fatty acid molecule when the fatty acid is in a puriﬁed or semi-puriﬁed state. Alternatively, the fatty acid ester is formed upon conversion of a triacylglycerol to an ester.

The present invention is ed to methods of producing microbial oils. In some embodiments, the method comprises growing any of the isolated microorganisms of the invention or mixtures thereof in a culture to produce a microbial oil comprising 3 fatty acids. In some embodiments, the method further comprises extracting the microbial oil. In some embodiments, the method comprises extracting a microbial oil comprising omega-3 fatty acids from any of the biomasses of the invention or mixtures thereof. In some embodiments, the method comprises heterotrophically growing the isolated microorganism, wherein the culture comprises a carbon source as bed . The microbial oil can. be extracted from a freshly ted biomass or can be extracted from a previously harvested biomass that has been stored under ions that t spoilage. Known methods can be used to culture a microorganism of the invention, to isolate a biomass from the culture, to extract a microbial oil from the biomass, and to analyze the fatty acid proﬁle of oils extracted ﬁ'om the s. See, e.g;, US. Patent No. 242, incorporated by reference herein in its entirety. The invention is directed to a microbial oil produced by any of the methods of the invention.

In some embodiments, the microbial oil is extracted by an enzyme extraction method. In some embodiments, the microbial oil is extracted by a ical extraction method. In some embodiments, the mechanical extraction method comprises one or more of: (1) processing a pasteurized fermentation broth h a homogenizer to assist in cell lysis and release of oil from cells; (2) adding isopropyl alcohol to the fermentation broth following homogenization to break the oil and water emulsion; (3) centrifuging the mixture to recover the oil phase; and (4) drying under vacuum with addition of antioxidants. In some embodiments, the crude oil is puriﬁed. In some ments, puriﬁcation of the crude oil comprises one or more of: (l) pumping the crude oil into a reﬁning tank and heating the oil, followed by adding an acid solution with mixing; (2) adding a caustic solution to the oil after acid treatment; (3) reheating the crude oil and then centriﬁiging to te the heavy phase from the reﬁned oil; (4) removing the remaining polar compounds, trace metals, and oxidation products from the reﬁned oil by using, for example, acid, ®, clay, and/or ﬁltration; (5) chill ﬁltering the bleached oil to further remove high melting point components from the oil to achieve the desired level of clarity; (6) heating the oil, after which the oil is then cooled and held for a period of time causing the high melting triglycerides and waxes to crystallize; (7) adding a ﬁlter -aid to the chilled oil and then removing llized solids by ﬁltration; (8) using a deodorizer after chill ion, operated under high temperature and vacuum, to remove, for example, peroxides and any remaining low molecular weight compounds that can cause off-odor and ﬂavors; (9) transferring the oil to the deodorizer feed tank, deaerating, 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 suitable antioxidants to the deodorized oil to e oxidative stability.

In some embodiments, the microbial oil comprises aisterol esters fraction of about 0%, at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 5% by weight. In some embodiments, the microbial oil comprises a sterol esters fraction of about 0% to about %, about 0% to about 2%, about 0% to about 5%, about 1% to about 1.5%, about 0.2% to about 1.5%, about 0.2% to about 2%, or about 0.2% to about 5% by weight. In some embodiments, the ial oil comprises a sterol esters fraction of about 5% or less, about 4% or less, ab0ut 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.3% or less, about 0.2% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, or about 0.2% or less by weight.

In some embodiments, the microbial oil comprises a lglycerol fraction of 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. In some embodiments, the ial oil comprises a triacyl ol fraction of about 35% to about 98%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about % 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% by weight.

In some embodiments, the microbial oil comprises a diacylglycerol fraction of at least about 10%, at least about 11%, at leastoabout 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 microbial oil comprises a diacylglycerol fraction of about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 15% to about 40%, about % to about 35%, or about 15% to about 30% by weight. In some embodiments, the microbial oil comprises a 1,2-diacylglycerol fraction of at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, at least about 5%, 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 microbial oil comprises a diacylglycerol fraction of about 0.2% to about 45%, about 0.2% to about %, about 0.2% to about 20%, about 0.2% to about 10%, about 0.2% to about 5%, about 0.2% to about 1%, about 0.2% to about 0.8%, about 0.4% to about 45%, about 0.4% to about 30%, abOut 0.4% to about 20%, about 0.4% to about 10%, about 0.4% to about 5%, about 0.4% to about 1%, about 0.4% to about 0.8%, about 0.5% to about 1%, about 0.5% to about 0.8%, about 10% to about 45%, about 10% to about 40%, about 10% to about %, about 10% to about 30%, about 15% to about 40%, about 15% to about 35%, about % to about 30%, or about 15% to about 25% by . In some embodiments, the ial oil comprises a 1,,3-diacylglycerol fraction of at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 2%, at least about 2.5 %, or at least about 3% by weight, 'In some embodiments, the microbial oil comprises a sterol fraction of at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 1%, ‘ at least about 1.5%, at least about 2%, or at least about 5% by weight.

In some ments, the microbial oil comprises a sterol fraction of about 0.3% to about 5%, about 0.3% to about 2%, about 0.3% to about 1.5%, about 0.5% to about 1.5%, about 1% to about 1.5%, about 0.5% to about 2%, about 0.5% to about 5%, about 1% to about 2%, or about 1% to about 5% by weight. In some embodiments, the microbial oil comprises a sterol on of about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, or about 1% or less by .

In some embodiments, the microbial oil comprises a phospholipid fraction of at least about 2%, at least about 5%, or at least about 8% by weight. In some embodiments, the microbial oil comprises a phospholipid fraction of about 2% to about 25%, about 2% 2012/047728 to about 20%, about 2% to about 15%, about 2% to about 10%, about 5% to about 25%, about 5% to about 20%, about 5% to about 20%, about 5% to about 10%, or about 7% to about 9% by weight. In some embodiments, the ial oil comprises a phospholipid fraction of less than about 20%, less than about 15%, less than about 10%, less than about ‘9%, or less than about 8% by weight. In some embodiments, the microbial oil is substantially free of phospholipids. In some embodiments, the microbial oil comprises unsaponiflables of less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% by weight of the oil. The lipid classes present in the microbial oil, such as a triacylglycerol fraction, can be separated by ﬂash tography and analyzed by thin layer chromatography (TLC), or separated and analyzed by other methods known in the art.

In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the free fatty acid fraction, the sterol fraction, the diacylglycerol fraction, and combinations f, comprises at least about 5%, at least about 10%, more than about 10%, 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%, at least about 20%, at least about 25%, at least about 30%, about least about 35%, at least about 40%, or at least about 45% by weight EPA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the free fatty acid fraction, the sterol fraction, the diacylglycerol fraction, and combinations thereof, ses about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, 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%, at least about 12% to about 55%, at least about 12% to about 50%, at least 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 %, about 15% to about 25%, about 15% to about 20%, about 20% to about 55%, about % to about 50%, about 20% to about 45%, about 20% to about 40%, or about 20% to about 30% by weight EPA-.valn some ments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, comprises at least about 5%, at least about 10%, at leastabout1596,atleastabout2096,atleastabout2596,atleastabout3096,atleastabout %, at least about 40%, at least about 50%, or at least about 60% by weight DHA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the lglycerol fraction, the diacylglycerol fraction,lthe sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, comprises about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about596toabout4096,about1096toabout6096,about1096toabout5096,about1096to about4096,about2096to about6096,about2596to about6096,about2596to about509ﬁ 596to about4596,about3096to 096,about3596to about5096,orabout % to about 40% by weight DHA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the diacylglycerol fraction, the sterol ﬁ‘action, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, comprises about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or leSS, or about 1% or less by weight DHA. In some ments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol on, the diacylglycerol fraction, the sterol on, the sterol esters fraction,‘the free fatty acids fraction, the olipid fraction, and combinations thereof, comprises about 1% to about 10%, about 1% to about 5%, about 2% to about 5%, about 3% to about 5%, or about 3% to about 10% by weight of the fatty acids as DHA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, is substantially free of DHA.. In some embodiments, the microbial oil and/or one or more fractions thereof ed from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, comprises about 0.1% to about 5%, 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%JWm0ﬂMowww%JMMQWMM%Manwﬁ%ﬁmm0ﬁMowwM%, 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% by weight ARA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol on, the diacylglycerol on, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, comprises about 5% or less, less than about 5%, about 4% or less, about 3% or less, about 2% or 1e35, about 1.5% or less, about 1% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less by weight ARA. In some embodiments, the microbial oil and/or one or more fractions thereof ed from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, is substantially free of ARA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol on, the glycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, 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% by weight DPA n-6. In some embodiments, the microbial oil and/or one or more fractions thereof ed from the triacylglycerol fraction, the glycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and ations thereof, comprises about 5%, less than about 5%, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.75% or less, about 0.6% or less, or about 0.5% or less by weight DPA n-6. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and combinations thereof, is ntially free of DPA n-6. In some embodiments, the ial oil and/or one or more fractions thereof selected from the triacylglycerol fraction, the diacylglycerol fraction, the sterol fraction, the sterol esters fraction, the free fatty acids fraction, the phospholipid fraction, and 2012/047728 ations thereof, comprises fatty acids with about 5% or less, less than about 5%, about 4% or less, about 3% or less, or about 2% or less by weight of oleic acid (18:1 n—9), linoleic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid (20:1 n-9), erucic acid (22:1 n-9), stearidonic acid (18:4 n-3), or combinations thereof} The triacylglycerol molecule contains 3 central carbon atoms (C(sn'-1)H2R1-(sn- 2)H2R2-C(sn-3)H2R3), allowing for formation of different positional isomers. In some embodiments, the ial oil comprises a lglycerol fraction in which 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 triacylglycerols in the triacylglycerol fraction n DHA at two positions in the triacylglycerol (di—substituted DHA) selected from any two of the sn-1, sn-2, and sn—3 positions, based on the relative area percent of peaks on an HPLC chromatograph. In which about some embodiments, the microbial oil comprises a triacylglycerol fraction in 2% to about 55%, about 2% to about 50%, about 2% to about 45%, about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, 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 10% to about 25%, about 10% to about 20%, about 20% to about 40%, about 20% to about 35%, or about 20% to about % of the triacylglycerols in the triacylglycerol fraction contain EPA at we positions in the triacylglycerol selected, from any two of the sn-1, sn-2, or sn-3 positions, based on the relative area percent of peaks on an HPLC chromatograph. In some embodiments, the microbial oil comprises a lglycerol fraction in which at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the triacylglycerols in the triacylglycerol fraction contain DHA at all of the sn-1, sn-2, and sn-3 positions (tri- Isubstituted DHA), based on the relative area percent of peaks on an HPLC chromatograph. In some embodiments, the microbial oil comprises a lglycerol fraction in which 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 triacylglycerols in the lglycerol fraction contain DHA at' all of the sn-1, sn-2, and sn-3 positions, based on the relative area percent of peaks on an 2012/047728 HPLC tograph. In some embodiments, the microbial oil comprises a triacylglycerol fraction in which at least about 10%, at least about 15%, at least about %, 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 triacylglycerols in the triacylglycerol fraction contain DHA at one position in the triacylglycerol selected from any one of the sn-1, sn-2, or sn-3 positions, based on the relative area percent of peaks on an HPLC tograph. In some embodiments, the microbial oil comprises a triacylglycerol fraction in which 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 30%, about 35% to about 75%, about 35% to about 65%, about % 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 lglycerols in the triacylglycerol fraction contain DHA at one position in the triacylglycerol selected from any one of the sn-1, sn-2, and sn-3 positions, based on the relative area percent of peaks on an HPLC chromatograph.

The present invention is further ed to methods of producing ial oils.

In some embodiments, the method comprises growing a thraustochytrid of the invention in a culture to produce a biomass and extracting an oil comprising omega-3 fatty acids from the biomass. The oil can be extracted from a freshly harvested biomass or can be extracted from a previously harvested biomass that has been stored under conditions that prevent spoilage. Known methods can be used to culture a thraustochytrid of the invention, to isolate a biomass from the culture, to extract a microbial oil from the biomass, and to analyze the fatty acid proﬁle of oils extracted from the biomass. See, e.g., US. Patent No. 242.

The invention is further directed to a microbial oil comprising a fatty acid proﬁle of the invention. A ial oil. of the invention can be any oil derived from a microorganism, including, for example: a crude oil extracted from the biomass of the microorganism without r processing; a reﬁned oil that is obtained by treating a crude microbial oil with further processing steps such as reﬁning, bleaching, and/or deodorizing; a diluted microbial oil obtained by diluting a crude or reﬁned ial oil; or an enriched oil that is ed, for example, by treating a crude or reﬁned microbial WO 13208 oil with further methods of puriﬁcation to increase the tration of a fatty acid (such as DHA) in the oil.

In some embodiments, the microbial oil comprises a sterol esters fraction of about 0%, at least about 0.1%, at least about 0.2%, at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 5% by weight. In some embodiments, the microbial oil comprises a sterol esters fraction of from about 0% to about 1.5%, about 0% to about 2%, about 0% to about 5%, about 1% to about 1.5%, about 0.2% to about 1.5%, about 0.2% to about 2%, or about 0.2% to about 5% by weight. In some embodiments, the microbial oil comprises a sterol esters fraction of less than about 5%, less than about 4%, less than about 3%, or less than about 2% by weight.

In some embodiments, the microbial oil comprises a triglyceride fraction of 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. In some embodiments, the microbial -oi1 comprises a triglyceride fraction of from about 65% to about 95%, about 75% to about 95%, or about 80% to about 95% by weight, or about 97% by weight, or about 98% by . In some ments, the ial oil comprises a free fatty acid fraction of at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, or at least about 5% by weight. In some embodiments, the microbial oil comprises a free fatty acid fraction of from about 0.5% to about 5%, about 0.5% to about 2.5%, about 0.5% to about 2%, about 0.5% to about 1.5%, about 0.5% to about 1%, about 1% to about 2.5%, about 1% to about 5%, about 1.5% to about 2.5%, about 2% to about 2.5%, or about 2% to about 5% by weight. In some ments, the microbial oil comprises a free fatty acid fraction of less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% by weight. In some embodiments, the microbial oil comprises a sterol fraction of at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, or at least about 5% by weight. In some embodiments, the microbial oil comprises a sterol fraction of from about 0.15% to about 1.5%, about 1% to about 1.5%, about 0.5% to about 2%, about 0.5% to about 5%, about 1% to about 2%, or about 1% to about 5% by weight. In some embodiments, the microbial oil comprises a sterol fraction of less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% by weight. In some embodiments, the microbial oil comprises a eride fraction of at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, or at least about 5% by weight. In some embodiments, the microbial oil comprises a eride fraction of from about 1.5% to about 3%, about 2% to ab0ut 3%, about 1.5% to about 3.5%, about 1.5% to about 5%, about 2.5% to about 3%, about 2.5% to about 3.5%, or about 2.5% to about 5% by weight. In some ments, the microbial oil comprises unsaponiﬂables of less than about 2%, less than about 1.5%, less than about 1%, or less than about 0.5% by weight of the oil. The lipid classes present in the microbial oil, such as the triglyceride fraction, can be separated by ﬂash chromatography and analyzed by thin layer chromatography (TLC), 'or separated and ed by other methods know in the an.

In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol on, the diglyceride on, and combinations thereof, comprises 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% by weight DHA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, and combinations thereof, comprises from 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% by weight DHA. In about some embodiments, the microbial oil comprises a sterol esters fraction comprising 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, or about 13% or less by weight DHA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid on, the sterol fraction, the diglyceride fraction, and combinations f, comprises about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less by weight EPA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the eride fraction, and combinations thereof, comprises from about 2% to about 3%, about 2% to about 3.5%, about 2.5% to about 3.5%, about 2% to about 6%, about 2.5% to about 6%, about 3.0% to about 6%, about 3.5% to about 6%, about 5% to about 6%, or about 2% to about 10% by weight EPA. In some ments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the olipid fraction), and combinations f, is substantially free of EPA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride on, the free fatty acid fraction, the sterol fraction, the eride on, the polar fraction (including the phospholipid fraction), and combinations thereof, comprises a weight ratio ofDHA to EPA of at least about 5:1, at least about 7:1, at least about 9:1, at least about 10:], at least about 15:1, at least about :], at least about 25:1, at least about 30:1, or at least about 50:1, wherein the microbial oil and/or one or more fractions thereof comprises 10% or less by weight of EPA. In from some embodiments, the microbial oil and/or one or more ons thereof selected the sterol esters fraction, the triglyceride on, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, comprises a weight ratio of DHA to EPA of at least about 5:1, - but less than about 20: 1. In some embodiments, the weight ratio of DHA to EPA is from about 5:1 to about 18:1, from about 7:1 to about 16:1, or from about 10:1 to about 15:1.

In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof comprises from about 0.1% to about 0.25%, about 0.2% to about 0.25%, about 0.1% to about 0.5%, or about 0.1% to about 1.5% by weight ARA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid on, the sterol fraction, the diglyceride fraction, the polar fraction ding the phospholipid fraction), and combinations thereof, comprises about 1.5% or less, about 1% or less, about 0.5% or less, about 0.2% or less, or about 0.1% or less by weight ARA. some embodiments, the microbial oil and/or one or more fractions f selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, is substantially free of ARA. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the ceride on, the free fatty acid fraction, the diglyceride fraction, the polar fraction ding the phospholipid fraction), and combinations thereof, comprises a weight ratio of DHA to ARA of at least about 20:1, at least about 30:1, at least about :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 ons thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, comprises from about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 2.5%, about 0.5% to about 3%, about 0.5% to about 3.5%, about 0.5% to about 5%, about 0.5% to about 6%, about 1% to about 2%, about 2% to about 3%, about 2% to about 3.5%, about 1% to about 2.5%, about 1% to about 3%, about 1% to about 3.5%, about 1% to about 5%, or about 1% to about 6% by weight DPA n-6. In some embodiments, the microbial oil and/or one or more ons thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction ding the olipid fraction), and combinations thereof, comprises about 6% or less, about 5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1% or less, or about 0.5% or less by weight DPA n-6. In some ments, the microbial oil and/or one or more fractions thereof selected from the sterol esters on, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and ations thereof, is substantially free of DPA n-6. In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, comprises a weight ratio of DHA to DPA n-6 of greater than about 6:1, of 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 fractions f selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, comprises about 5%:or less, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, about 1% or less, or about 0.5% or less by weight each of ic acid (18:2 n-6), linolenic acid (18:3 n-3), eicosenoic acid (20:1 n-9), and erucic acid (22:1 n- 9). In some embodiments, the microbial oil and/or one or more fractions thereof selected from the sterol esters fraction, the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, the polar fraction (including the phospholipid fraction), and combinations thereof, ses about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, or about 1% or less by weight of’ ecanoic acid (17:0). In some embodiments, the microbial oil and/or one or more fractions thereof comprise about 0.01% to about 5% by weight, about 0.05% to about 3% by , or about 0.1% to about 1% by weight of heptadecanoic acid.

The triglyceride molecule contains 3 central carbon atoms (CsmleRl—szHzRZ- C5,,_3H2R3), ng for formation of different onal isomers. In some embodiments, the ial oil comprises a triglyceride fraction in which at least about %, at least about 30%, at least about 35%, or at least about 40% of the triglycerides in the ceride fraction contain DHA at two positions in the triglyceride (di-substituted DHA) selected from any two of the sn-1, sn-2, and sn-3 positions, based on the relative area percent of peaks on an HPLC chromatograph. In some embodiments, the microbial oil comprises a triglyceride fraction in which from about 20% to about 40%, about 20% to about 35%, about 30% to about 40%, or about 30% to about 35% of the triglycerides in the triglyceride fraction contain DHA at two positions in the triglyceride selected from of peaks on any two of the sn-1, sn-2, or sn-3 positions, based on the relative area percent an HPLC chromatograph. In some embodiments, the microbial oil comprises a triglyceride fraction in which at least about 5%, at least about 10%, at least about 15%, or at least about 20% of the triglycerides in the triglyceride fraction contain DHA at all of the sn-1 and sn-3 ons (tri-substituted DHA), based on the relative area percent , sn-2, of peaks on an HPLC chromatograph. In some embodiments, the microbial oil comprises a ceride fraction in which from about 5% to about 20%, about 5% to about 15%, about 10% to about 20%, ;or about 10% to about 15% of the cerides in the triglyceride ﬁ‘action contain DI-IA at all of the sn-1, sn-2, and sn-3 positions, based on the relative area percent of peaks on an, HPLC chromatograph. In contrast, the TAG species .53_ reported in US. Patent No. 6,582,941 does not contain DHA at all three positions. In some embodiments, the microbial oil ses a triglyceride fraction in which 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 fraction 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 percent of peaks on an- HPLC chromatograph. In some embodiments, the microbial oil comprises a triglyceride fraction in which from 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 on contain DHA at one position in the ceride ed from any one of the sn-1, sn-2, and sn-3 ons, based on the relative area percent of peaks on an HPLC chromatograph.

Compositions The invention is directed to compositions comprising a microorganism of the invention, an isolated s of the invention, a ial oil of the- invention, or combinations thereof.

A microorganism, biomass, or microbial oil of the invention can be further chemically or physically modiﬁed or processed based on the requirements of the ition by any known technique.

Microorganism cells or biomasses can be dried prior to use in a composition by s including, but not,,limited to, freeze drying, air drying, spray drying, tunnel drying, vacuum drying_(lyophilization), and a similar s. Alternatively, a harvested and washed biomass can be used directly in a composition without drying. See, e.g., US.

Patent Nos. 5,130,242 and 6,812,009, each of which is incorporated by reference herein in its entirety. - Microbial oils of the ion can be used as starting material to more efﬁciently produce a product enriched in a fatty acid such as EPA. For example, the microbial oils of the invention can be subjected to various puriﬁcation techniques known in the art, such as distillation or urea adduction, to produce a higher potency product with higher concentrations of EPA or another fatty acid. The microbial oils of the invention can also be used in chemical reactions to produce compounds derived from fatty acids in the oils, such as esters and salts of EPA or another fatty acid.

A composition of the invention can include one or more excipients.‘ As used herein, "excipient" refers to a component, or e of components, that is used in a composition of the t invention to give desirable characteristics to the composition, ing foods as well as pharmaceutical, cosmetic, and industrial itions. An excipient of the present invention can be described as a "pharmaceutically acceptable" excipient when added to a pharmaceutical composition, meaning that the excipient is a compound, material, composition, salt, and/or dosage form which is, within the scope of sound medical judgment, suitable for contact with tissues of human beings and non- human animals without excessive toxicity, irritation, allergic response, or other problematic complications over 'the desired duration of contact commensurate with a reasonable beneﬁt/risk ratio. In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized intemational phannacopeia for use in animals, and more ularly in humans. [Various excipients can be used. In some embodiments, the excipient can be, but is not limited to, an alkaline agent, a stabilizer, an antioxidant, an adhesion agent, a separating agent, a coating agent, an exterior phase component, a lled—release component, a solvent, a surfactant, a humectant, a buffering agent, a ﬁller, an emollient, or combinations thereof. Excipients in addition to those discussed herein can include excipients listed in, though not limited to, Remington: The Science and Practice ofPharmacy, 2151 ed. (2005). Inclusion of an ent in a ular classiﬁcation herein (e.g., "‘solvent") is intended to illustrate rather than limit the role of the excipient. A particular excipient can fall within multiple classifications.

Compositions of the ion include, but are not limited to, food ts, pharmaceutical compositions, cosmetics, and industrial compositions.

In some embodiments, the composition is a food product. A food t is any food for non-human animal or human consumption, and includes both solid and liquid compositions. A food product can be an additive to animal or human foods. Foods include, but are not limited to, common foods; liquid products, including milks, beverages, therapeutic drinks, and nutritional drinks; onal foods; supplements; euticals; infant formulas, including formulas for pre-mature s; foods for pregnant or nursing women; foods for adults; geriatric foods; and animal foods.

In some embodiments, a microorganism, biomass, or microbial oil of the invention can be used directly as or included as an additive within one or more of: an oil, shortening, , other fatty ingredient, beverage, sauce, dairy-based or soy-based food (such as milk, yogurt, cheese and ice-cream), a baked good, a nutritional product, e.g., as a nutritional supplement (in capsule or tablet form), a vitamin supplement, a diet supplement, a powdered drink, and a ﬁnished or semi-ﬁnished powdered food product.

In some embodiments, the nutritional supplement is in the form of a vegetarian capsule that is not formed from and does not contain any ents from an animal source.

A partial list. of food compositions that can include a microbial oil of the invention includes, but is not limited to, soya based products (milks, ice creams, yogurts, drinks; baked food items creams, spreads, whiteners); soups and soup mixes; doughs, batters, and including, for example, fine bakery wares, breakfast cereals, cakes, Cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, biscuits, mufﬁns, pastries, , croutons, crackers, sweet goods, snack cakes, pies, granola/snack bars, and toaster pastries; candy; hard confectionery; chocolate and other confectionery; g gum; liquid food products, for example milks, energy drinks, infant formula, carbonated drinks, teas, liquid meals, fruit juices, fruit—based drinks, vegetable-based ; multivitamin , meal replacers, medicinal foods, and syrups; ed beverage mixes; pasta; sed ﬁsh products; processed meat products; processed y products; gravies and ; condiments (ketchup, mayonnaise, etc.); vegetable oil—based spreads; dairy products; yogurt; butters; frozen dairy ts; ice creams; frozen desserts; frozen s; semi- solid food products such as baby food; puddings and gelatin desserts; sed and unprocessed cheese; pancake mixes; food bars including energy bars; wafﬂe mixes; salad dressings; replacement egg mixes; nut and nut-based spreads; salted snacks such as potato chips and other chips or crisps, corn chips, tortilla chips, extruded snacks, popcorn, pretzels, potato crisps, and nuts; and specialty snacks such as dips, dried fruit snacks, meat snacks, pork rinds, health food bars and rice/com cakes.

In some embodiments, a microbial oil of the invention can be used to supplement infant formula. Infant formula can be supplemented witha microbial oil of the invention alone or in combination with a physically reﬁned oil derived from an arachidonic acid (ARA)-producing rganism. An ARA-producing rganism, for e, is Morlierella alpina or Mortierella sect. schmuckeri. Alternatively, infant formulas can be .61. supplemented with a microbial oil of the invention in combination with an oil rich in ARA, ing ARASCO® (Martek Biosciences, Columbia, MD).

In some embodiments, the composition is an animal feed. An "animal" includes non-human organisms belonging to the kingdom Animalia, and includes, without limitation, aquatic animals and terrestrial animals. The term "animal feed" or "animal food" refers to any food intended for non-human s, whether for ﬁsh; commercial ﬁsh; ornamental ﬁsh; ﬁsh larvae; bivalves; ks; crustaceans; shellﬁsh; shrimp; larval shrimp; artemia; rotifers; brine shrimp; ﬁlter feeders; amphibians; reptiles; mammals; domestic animals; farm animals; zoo animals; sport animals; ng stock; racing animals; show animals; heirloom animals; rare or endangered animals; companion animals; pet s such as dogs, cats, guinea pigs, rabbits, rats, mice, or horses; primates such as monkeys (e.g., cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), apes, orangutans, baboons, gibbons, and chimpanzees; canids such as dogs and wolves; felids such as'cats, lions, and ; equids such as horses, donkeys, and zebras; food animals such as cows, cattle, pigs, and sheep; ungulates such as deer and giraﬁ'es; or rodents such as mice, rats, hamsters and guinea pigs; and so on. An animal feed includes, but is not limited to, an aquaculture feed, a domestic animal feed including combinations pet feed, a zoological animal feed, a work animal feed, a livestock feed, and thereof.

In some ments, the composition is a feed or feed ment for any animal whose meat or products are consumed by humans, such as any animal from which meat, or milk is deriVed for human consumption. When fed to such animals, eggs, nutrients such as As can be incorporated into the ﬂesh, milk, eggs or other ts of such animals to increase their content of these nutrients.

In some ments, the composition is a spray-dried material that can be crumbled to form particles of an appropriate size for consumption by zooplankton, artemia, rotifers, and ﬁlter feeders. In some embodiments, the zooplankton, a, or rotifers fed by the composition are in turn fed to ﬁsh larvae, ﬁsh, shellﬁsh, bivalves, or crustaceans.

In some embodiments, the composition is a pharmaceutical composition. le pharmaceutical compositions include, but are not limited to, an anti-inﬂammatory composition, a drug for treatment of coronary heart disease, a drug for treatment of 2012/047728 arteriosclerosis, a chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an elicobacter pylori drug, a drug for treatment of neurodegenerative disease, a drug for ent of degenerative liver disease, an antibiotic, a cholesterol ng composition, and a triacylglycerol lowering composition. In some embodiments, the composition is a medical food. A medical food includes a food that is in a composition to be consumed or administered externally under the supervision of a physician and that is intended for the c dietary management of scientiﬁc a condition, for which distinctive nutritional requirements, based on recognized ples, are established by medical evaluation.

In some embodiments, the ial oil can be formulated in a dosage form.

Dosage forms can include, but are not limited to, tablets, capsules, cachets, pellets, pills, powders and granules, and parenteral dosage forms, which include, but are not limited to, ons, suspensions, emulsions, and dry powders comprising an effective amount of the microbial oil. It is also known in the art that such formulations can also contain pharmaceutically acceptable diluents, ﬁllers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsiﬁers, buffers, ants, moisturizers, solubilizers, preservatives and the like. Administration forms can include, but are not limited to, tablets, dragees, capsules, caplets, and pills, which contain the ial oil and one or more suitable pharmaceutically acceptable carriers.

For oral administration, the microbial oil can be combined with pharmaceutically able carriers well known in the art. Such. carriers‘ enable the microbial oils of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a- t to be treated. In some embodiments, the dosage form is a tablet, pill or caplet. Pharmaceutical preparations for oral use can be obtained by adding a solid excipient, optionally grinding the ing mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, 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 d to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. ceutical ations that can be used orally include, but are not limited to, push- ﬁt capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as ol or sorbitol. In some embodiments, the dosage form is a vegetarian dosage form, in which the dosage form is not formed from and does not n any components from an animal source. In some ments, the rian dosage form is a vegetarian capsule.

In some embodiments, the composition is a cosmetic. Cosmetics include, but are not limited to, ons, creams, lotions, masks, soaps, shampoos, washes, facial Cosmetic agents can creams, conditioners, make-ups, bath agents, and dispersion s. be medicinal or non-medicinal.

In some embodiments, the composition is an industrial composition. In some embodiments, the composition. is a starting material for one or more manufactures. A manufacture includes, but is not limited to, a polymer; a photographic photosensitive material; a detergent; an industrial oil; or an industrial detergent. For example, US.

Patent No. 7,259,006 describes use of DHA-containing d oil for production of behenic acid and production of photographic sensitive materials using behenic acid.

Methods of Using the Compositions In some embodiments, the compositions can be used in the treatment of a condition in humans or non—human animals. In some embodiments, the compositions can be used for nutrition in humans or non-human animals.

The terms "treat"- and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, e, or disorder, or to obtain beneﬁcial or desired clinical results. For purposes of this invention, beneﬁcial or desired clinical results include, but are not limited to, alleviation or elimination of the symptoms or signs associated with a condition, disease, or er; diminishment of the extent of a condition, disease, or disorder; stabilization of a condition, disease, or disorder, (i.e., where the condition, e, or disorder is not worsening); delay in onset or progression of the condition, disease, or disorder; amelioration of the condition, disease, or disorder; remission (whether l or total and whether detectable or undetectable) of the condition, e, or disorder; or enhancement or improvement of a condition, disease, or -64— disorder. Treatment includes eliciting a clinically signiﬁcant response without excessive side effects. Treatment also includes prolonging survival as compared to ed survival if not receiving treatment.

In some embodiments, the composition is used to treat a condition, disease, or disorder such as acne, acute inﬂammation, age related maculopathy, allergy, Alzheimer's, arthritis, asthma, atherosclerosis, autoimmune disease, blood lipid disorder, breast cysts, cachexia, cancer, cardiac restenosis,_ vascular diseases, chronic inﬂammation, coronary heart e, cystic ﬁbrosis, degenerative disorder of the liver, es, eczema, intestinal disorder, heart disease, high triacylglycerol levels, hypertension, ctivity, immunological diseases, inhibiting tumor growth, inﬂammatory conditions, intestinal disorders, kidney dysfunction, ia, major depression, multiple sclerosis, neurodegenerative disorder, osteoarthritis, osteoporosis, peroxisomal discrder, preeclampsia, preterm birth, psoriasis, pulmonary disorder rheumatoid tis, risk of heart e, or thrombosis.

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

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

In some embodiments, the composition is used to improve or maintain cognitive function.

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

The ition or dosage form can be administered into the body of a subject by A substance is considered to any route compatible with the composition or dosage form. be istered" if the substance is uced into the body of the subject by the subject, or if another person, a e, or a device introduces the nce into the. body of the subject. "Administering," therefore, includes, e.g., self-administration, administration by others, and indirect administration. The term "continuous" or "consecutive," as used herein in reference to "administration," means that the ncy of administration is at least once daily. Note, however, that the frequency of administration can be greater than once daily and still be "continuous" or "consecutive," levels as speciﬁed herein are e.g., twice or even three times daily, as long as the dosage not exceeded. The means and methods for administration are known in the art and an artisan can refer to various pharmacologic references for guidance. For example, n Pharmaceutics," Banker & Rhodes, Informa Healthcare, USA, 4th ed. (2002); and "Goodman & Gilman’s The Pharmaceutical Basis of Therapeutics," McGraw-Hill Companies, Inc., New York, 10th ed. (2001) can be consulted.

By "subject," "individual," or "patient" is meant any subject, whether human or non-human, for whom diagnosis, prognosis, therapy, or administration of the composition or dosage form is desired. ian subjects e, but are not limited to, humans; domestic animals; farm s; 200 animals; sport animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, or horses; primates such as monkeys (e.g., cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), apes, tans, s, gibbons, and nzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and ; food s such as cows, cattle, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. The term subject also encompasses model animals, e.g., disease model animals. In some embodiments, the term subject includes valuable animals, either economically or otherwise, e.g., economically important breeding stock, racing animals, show animals, heirloom animals, rare or endangered animals, or ion animals. In certain embodiments, the subject is a human subject. In n embodiments, the subject is a non-human subject.

The composition can be administered as a tional amount," "therapeutically ive amount," a a "prophylactically effective amount," a "therapeutic dose," or "prophylactic dose." A "nutritional amount" refers to an amount effective, at dosages and for periods of time necessary, to e a desired nutritional result. A nutritional result can be, e.g., increased levels of a desirable fatty acid component in a subject. A "therapeutically effective amount" or "therapeutic dose" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired eutic result. A therapeutic result can be, e.g., lessening of symptoms, prolonged survival, improved mobility, and the like. A therapeutic result need not be a "cure." A "prophylactically effective amount" or "prophylactic dose" refers to an amount effective, at s and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, a prophylactically effective amount will be less than a therapeutically effective amount for treatment of an advanced stage of disease.

Various dosage amounts of the composition, dosage form, or pharmaceutical composition can be administered to a subject, based on the amount of EPA or other fatty acid ent of the microorganism, biomass, or microbial oil to be administered to the subject. The terms "daily dosage," "daily dosage level," and "daily dosage amount" refer herein to the total amount of EPA or other fatty acid component administered per day (per 24 hour period). Thus, for example, administration of EPA to a subject at a daily dosage of 2 mg means that the subject receives a total of 2 mg of EPA on a daily basis, whether the EPA is administered as a single dosage form comprising 2 mg EPA, or alternatively, four dosage forms comprising 0.5 mg EPA each (for a total of 2 mg EPA). In some embodiments, the daily amount of EPA is administered in a single dosage form, or in two dosage forms. The dosage forms of the present invention can be taken in a single application or multiple ations. For example, if four tablets are taken daily, each tablet comprising 0.5 mg EPA, then all four tablets can be taken once daily, or 2 tablets can be taken twice daily, or 1 tablet can be taken every 6 hours. In some ments, the daily dosage is about 100 mg to about 15 g of EPA. In some embodiments, the daily dosage 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 gm about 15 g, about 10 g to about 15 g, about 100 mg to about 10 g, about 100 mg to about thereof. In some g, or about 100 mg to about 2.5 g of EPA, DHA, or a combination embodiments, the composition is a dosage form that comprises about 0.5 mg to about 250 about 100 mg to about 500 mg, 100 mg to about 250 mg, about 0.5 mg to about 500 mg, 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. stration of the compositions or dosage forms of the present invention can be achieved using various regimens. For example, in some embodiments, stration occurs daily on consecutive days, or alternatively, occurs every other day (bi—daily).

Administration can occur on one or more days.

Administration of the itions and dosage forms can be combined with other regimens used for treatment of the condition. For example, the method of the t invention can be combined with diet regimens (e.g., low carbohydrate diets, high protein diets, high ﬁber diets, etc), exercise ns, weight loss regimens, smoking cessation regimens, or combinations thereof. The method of the present invention can also be used in combination with other pharmaceutical products in the treatment of the condition. The itions or dosage forms of the present invention can be administered before or after other regimens or pharmaceutical products.

Kits Comprising the Compositions The invention is directed’to kits or packages containing one or more units of a composition of the invention. Kits or packages can include units of a food product, pharmaceutical ition, cosmetic, or industrial composition comprising the microorganism, biomass, or microbial oil of the ion, or combinations thereof. Kits or packages can also include an additive comprising the microorganism, biomass, or microbial oil of the invention, or combinations f for preparation of a food, cosmetic, pharmaceutical composition, or industrial composition.

In some embodiments, the kit or package contains one or more units of a. pharmaceutical composition to be administered according to the methods of the present ion. The kit or package can n one dosage unit, or more than one dosage unit (i.e., multiple dosage . If multiple dosage units are present in the kit or package, the multiple dosage units can be ‘optionally arranged for sequential administration.

The kits of the present invention can optionally contain instructions associated with the units or dosage forms of the kits. Such instructions can be in a form prescribed by a governmental agency regllating the manufacture, use or sale of pharmaceutical ts, which notice reﬂects approval by the agency of the manufacture, use or sale for human administration to treat a condition or disorder. The instructions can be in any form which conveys information on the use of the units or dosage forms in the kit according to the methods of the invention. For example, the instructions can be in the form of d matter, or in the form of a pre-recorded media .

In the course of examination of a patient, a medical sional can determine that administration of one of the methods of the present invention is appropriate for the patient, or the physician can ine that the patient's condition can be improved by the administration of one of the methods of the present invention. Prior to prescribing any regimen, the physician can counsel the patient, for example, on the various risks and beneﬁts associated with the-regimen. The patient can be provided full disclosure of all known and suspected risks associated with the regimen. Such counseling can be provided verbally, as well as in written form. In some embodiments, the physician can provide the patient with literature materials on the regimen, such as product information, educational materials, and the like.

The present ion is directed to methods of educating consumers about the methods of treatment, the method comprising distributing the dosage forms with consumer information at a point of sale. In some ments, the distribution will occur at a point of sale having a pharmacist or healthcare provider.

The term "consumer ation" can include, but is not limited to, an English language text, non-English language text, visual image, chart, telephone recording, website, and access to a live er service representative. In some embodiments, consumer information will provide directions for use of the dosage forms according to the methods .of the present invention, appropriate age use, indication, contraindications, appropriate dosing, warnings, telephone number or website address. In some embodiments, the method further comprises providing sional information to relevant persons in a position to answer consumer questions regarding use of the disclosed regimens according to the methods of the present invention. The term "professional ation" includes, but is not limited to, information concerning the n when administered according to the methods of the t invention that is designed to enable a medical professional to answer costumer questions. A "medical professional," includes, for example, a physician, physician assistant, nurse, nurse practitioner, pharmacist and customer service representative.

Having generally described this invention, a further understanding can be obtained by reference to the examples provided herein. These examples are for purposes of illustration only and are not intended to be ng.

Example 1 In this example, Schizochytrium sp. was ated in 250 ml Erlenmeyer shake ﬂasks containing 50 mls of culture . An um was prepared in the same medium which consisted of 0.625 g NaCl, 1.0 g KCl, 5 g 7I-I20, 0.1 g (NH4)2SO4, 0.29 g CaClz'ZHZO, 1.0 g monosodium glutamate monohydrate, 1.0 g yeast extract, and 23.8 g HEPES buﬁ'er dissolved in approximately 900mls of distilled water.

" The medium was brought to pH 7 using NaOH. The ﬁnal volume of the medium was brought to 896 mls, and the medium was sterilized by aving. Aﬁer autoclaving the following components were sterilely added to the medium: 0.89 ml of 56.5 g/l KH2P04, 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 FeSO4'7H2O, 1.55 g MnCl2'4H2O, 0.965 g ZnSO4'7H2O, 0.02 g CoClz'6H2O, 0.02 g Na2MoO4'21-I2O, 1.035 g CuSO4‘5H2O, 1.035 g NiSO4'6H2O dissolved in one liter of distilled water and pH’ed to 2.5 with HCl. The n stock solution contained the ing: 0.16 g vitamin 'B12, 9.75 g thiamine, and 3.33 g calcium pantothenate dissolved in one liter of distilled water. The shake ﬂasks were inoculated with 1 ml of inoculum. Triplicate ﬂasks were placed in a C02 incubator set to maintain an atmosphere of either 5, 10 or 15% CO2 in air. r set of cate ﬂasks were placed in an incubator at ambient CO2 . All sets of ﬂasks were shaken at 200 rpm and all incubators were set at 22.5C. After seven days of growth the biomass was collected from the shake ﬂasks by centrifugation, the biomass was freeze dried and the fatty acid proﬁle of the biomass was determined using standard methyl esteriﬁcation procedures. Elevated CO2 levels produced cant changes in the biomass, %Fat and the fatty acid proﬁles.

Notable was the change produced in the %EPA and %DHA values between CO2 conditions and ambient conditions. Furthermore, the changes in thcse fatty acids became . more pronounced as the CO2 level increased. The results are presented in Table 4.

SAMPLE Biomass (g/l) : Example 2 In this example, Schizochytrium sp. was cultivated in 250 ml Erlenmeyer shake ﬂasks containing 50 mls of culture medium. An inoeulum was prepared in the same medium which consisted of 0.625 g NaCl, 1.0 g KC], 5 g MgSO4.7H20, 0.1 g (NH4)ZSO4, 0.29 g CaC12.2H20, 1.0 g monosodium glutamate monohydrate, 1.0 g yeast extract, and 23.8 g HEPES buffer ved in approximately 900mls of distilled water.

The medium was t to pH 7 using NaOH. The ﬁnal volume of the medium was brought to 896 mls, and the medium was sterilized by autoclaving. After autoclaving the following components were sterilely 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 on, and 1 ml of vitamin stock solution. The trace metal stock solution contained the following: 90 g citric acid, 5.15 g FeSO4.7H20, 1.55 g MnC12.4H20, 0.965 g ZnSO4.7H20, 0.02 g CoC12.6HZO, 0.02 g Na2MoO4.2H20, 1.035 g CuSO4.5H20, 1.035 g NiSO4.6l-120 dissolved in one liter of distilled water and pH’ed to 2.5 with HCl. The vitamin‘stoek solution contained the following: 0.16 3.33 m g vitamin B12, 9.75 g ne, and g pantothenate dissolved in one liter of distilled water. The shake (ﬂasks were inoculated with 1 m1 of um. Duplicate ﬂasks were placed in a C02 incubator set to maintain an atmosphere 2012/047728 of either 5, 10 or 15% C02 in air. Another set of duplicate ﬂasks were placed in an incubator at ambient CO2 levels. All sets of ﬂasks were shaken at 200 rpm and all biomass was collected from . tors were set at 22.5C. Aﬁer seven days of growth the the shake ﬂasks by centrifugation, the s was freeze dried and the fatty acid proﬁle of the biomass was determined using rd methyl esteriﬁcation procedures. Elevated CO2 levels ed signiﬁcant s in the biomass, %Fat and the fatty acid proﬁles.

Notable was the change produced in the %EPA and %DHA values between C02 conditions and ambient conditions. Furthermore, the changes in these fatty acids became more pronounced as the CO2 level increased. The results are e in Table 5 below.

Table 5 Biomass 3 SAMPLE (; 1) 0 Um11> '11 2°.

Ambient 1 Ambient 2 NM Oxoo VINN4:. wwmoooo"U\coo>° axm C00 coax \1\o axox oxo \106Am % co: 1) _—- A Ox \1 a ;__. N w UI . 00 J> oxN 4>A %c02 2 1431 3609 36.59 63 00 % CO2 1 21.44 19.88 46.40 64.94 % c022 2139 19.88 46.74 65.82 % 002 1 2471 11.56 54.04 77.16 % c022 24.62 1174 54.36 67.94 Example 3 In this example, Thrasutochytrium sp. was cultivated in 250 ml Erlenmeyer shake ﬂasks containing 50 mls of culture medium. An inoculum was prepared in the same medium which consisted of 42g Na2SO4, 0.625 g NaCl, 1.0 g KCl, 5 g MgSO4'7H2O, 0.1 1.0 g yeast g (NH4)2SO4, 0.29 g CaCl2‘2H2O, 1.0 g monosodium "glutamate monohydrate, extract, and 23.8 g HEPES buffer dissolved in approximately 900mls of distilled water.

The medium was brought to pH 7 using NaOH. The ﬁnal volume of the medium was brought to 961 mls, and the medium was sterilized by autoclaving. After autoclaving the following components were sterilely added to the medium: 0.89 ml of 56.5 g/l KH2PO4, 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 FeSO4‘7H2O, 1.55 g MnCl2'4H20, 0.965 g ZnSO4'7H2O, 0.02 g CoCl2'6H20, 0.02 g Na2MoO4'2H20, 1.035 g CuSO4‘5H2O, 1.035 g NiSO4‘6H2O dissolved in one liter of distilled water and pH’ed‘ to 2.5 with HCl. The vitamin stock solution contained the ing: 0.16 g vitamin B12, 9.75 g thiamine, and 3.33 g calcium pantothenate dissolved in one liter of distilled water. The shake ﬂasks were ated with 1 ml of inoculum. Triplicate ﬂasks were placed in a C02 incubator set to maintain an atmosphere of 15% CO2 in air. Another set of triplicate ﬂasks were placed in an incubator at ambient C02 levels. Both sets of ﬂasks were shaken at 200 rpm and both incubators were set at 22.50 After seven days of growth the biomass was collected from the shake ﬂasks by centrifugation, the biomass was freeze dried and the fatty acid proﬁle of the biomass was ined using standard methyl esteriﬁcation procedures. An atmosphere of 15% C02 in air produced substantial changes in the tochytrium culture. At high CO2 the biomass and the %fat were lower than under ambient conditions. The %16:0 and %DHA I conditions. The were lower and the %EPA was signiﬁcantly higher than under ambient results are presented in Table 6 below.

’ Table 6 — °/ Atmoshere ; l % 16:0 % EPA DHA % Fat .m... - - 1 3.39 31.74 11.61 44.65 53.32 Ambient 2 - 3.42 30.88 11.95 44.99 53.30 _---- -3 3.39 32.13 11.46 44.19 53.93 (1 ---- -2.23 22.12 36.94 19.04 38.73 _--- -2 2.05 21.40 37.14 18.57 39.34 _---- -3 2.11 21.68 36.83 18.52 40.86 Example 4 In this example [NBx0614et10], Schizochytrium s (ATCC PTA-10208) was cultivated in four 100-1iter New ick iﬁc BioFlo 6000 ferrnentors at a target ﬁnal e) volume of {SO-liters with a carbon (glucose) and nitrogen (ammonium hydroxide) fed-batch process at various over pressure conditions to evaluate the sensitivity of the culture to increased dissolved carbon dioxide. The fermentations were each ated with 8 liters of culture. For inoculum propagation, an 80-liter New Brunswick iﬁc BioFlo 5000 ferrnentor was utilized. The inoculum medium consisted of 65-liters of medium prepared in six separate groups. Group 1 consisted of ' 585 g MSG*1H20, 65 g KCl, 325 g MgSO4*7H20, 24.05 g (NH4)2SO4, 40.625 g of NaCl, 390 g of T154 (yeast extract), and 13 mL Dow 1520US (antifoam). Group 1 was batch sterilized at 121 degrees in the inoculum ferrnentor at a volume of approximately 60 liters. Group 2 consisted of 18.85 of 33.8» g g CaC12*2H20. Group 3 consisted KH2PO4. Groups 2 and 3 were each autoclaved in separate solutions for approximately 45-60 s and added to group 1 aseptically post-sterilization. Group 4 consisted of 201.5 mg MnC12*4H20, 201.5 mg ZnSO4*7H20, 2.6 mg 6H20, 2.6 mg Na2MoO4*2H20, 134.6 mg CuSO4*5H20, 134.6 mg NiSO4*6H20, 669.4 mg FeSO4*7I—120 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—HCl: 10.4 mg vitamm B12, and 216.5 mg pantothenic acid hemi-calcium salt. Group 5 was dissolved in R0 water and then ﬁlter-sterilized. Group 6 consisted of 3250 ‘g glucose dissolved in a volume of 3000 mL RO water. After the inoculum ntor was cooled to 22.5 degrees Celsius, ferrnentor. groups 2, 3, 4, 5, and 6 were added to the Using sodium hydroxide and ic acid, the ferrnentor was pH adjusted to 7 and the dissolved oxygen was spanned mL of a to 100% prior to inoculation. The um ntor was inoculated with 1300 smaller fermentation e (The smaller tation culture was prepared and cultivated in the same manner as the 65-liter inoculum culture.) and cultivated at 22.5 degrees Celsius, pH 7, 180 rpm agitation, and 32.5 1pm of air for a period of 37 hours, at which point 8 liters of inoculum broth was transferred to each lOO-liter ferrnentor. Each lOO-liter ferrnentor contained 80—liters of fermentation media. The fermentation media was prepared in a similar fashion to the inoculum ferrnentor. For each 100-liter fermentor, the fermentation media consisted of 6 d media groups. For vessels NBS, NB6, and NB7, group 1 contained 704 g Na2SO4, 50 g NaCl, 80 g KCl, 400 g MgSO4*7H20, 33.6 g (NH4)2SO4, 80 g T154 yeast extract, and 8 mL Dow 1520-US antifoam. Group 1 was steam sterilized at 122 s Celsius for 60 minutes in the 100- liter ferrnentors at a volume of approximately 35 liters. Group 2 contained 23.2 g CaC12*2H20 in a volume of approximately 300 mL. Group 3 contained 141.2 g KH2PO4 dissolved in R0 water. Group 4 contained 248 mg MnC12*4H20, 744 mg ZnSO4*7H20, 3.2 mg Na2MoO4*2H20, 165.6 mg CuSO4*5H20, and 165.6 mg NiSO4*6H20, 824 mg FeSO4*7H20 and 80 g citric acid all dissolved in R0 water.

Group 5 ned 780 mg Thiamine-HCI, 266.4 mg pantothenic acid herni-calcium salt, and 286.4 ug of biotin, all dissolved and ﬁlter sterilized in R0 water. Group 6 contained 2400 g of glucose in approximately 3 liters of R0 water. Groups 2, 3, 4, 5, and 6 were ed and added to the fennentor after the ferrnentor d an operating temperature of 22.5 degrees Celsius. For vessel NB8, all of the groups were the same as the other three conditions with the exception of the citric acid. In NB8, group 4 only contained ~3.75 g of citric acid. Each fermentor volume prior to inoculation was approximately 52-53 liters. Each fermentor was inoculated with 8 liters of broth from the inoculum fermentation described above. The fermentation was pH lled utilizing a 7.3 liter solution of 4N ammonium hydroxide at a pH of 7 until nitrogen tion at which point 4N sodium hydroxide and 4N sulfuric acid were utilized for pH control. The dissolved oxygen was controlled to maintain a target of 20% throughout the fermentation using agitation from 180 to 480 rpm and airﬂow from 40 LPM to '80 LPM. Each of these vessels was centrolled at a different head pressUre , NB6=15, and NB7=20 PS1) to evaluate the sensitivity of the organism to increased dissolved carbon dioxide.

Throughout the fermentation, an 850 g/L solution of 95% dextrose (corn syrup) was fed - to maintain a glucose concentration less than 50 g/L. After 8 days, the dry cell weight and omega—3 titer of each 80-liter fermentor was similar with NB5 at 110.1 g/L DCW and 44.37 g/L omega-3; NB6 at 117.7 g/L DCW and 45.78 g/L omega-3; NB7 at 114.1 g/L DCW and 48.43 g/L omega—3; NB8 at 119.5 g/L DCW and 43.55 g/L omega-3. As the the %EPA/FAME increased, and re was increased, the %DHA/FAME decreased, the ratio of DHA to EPA sed. When comparing the ﬁnal fatty acid centent of 2 PS1 to 20 PSI, the %DHA/FAME decreased from 50.48% to 41.26% and the %EPA/FAME increased from 18.95% to 23.28%. The results are presented in Table 7.

WO 13208 Table7 %ﬂ’A/FANIE mNBS(2PSI) N86(ISPSI) NB7(20PS[) N88(20PS[) _—-E_ 13.15 1432 "mm-ailm— .11- 14.03‘ .m- 1534 m-20.04 20.86 23.08 139.0 m- 21.57 .113:- 25.25 Iris-mm man-am 18.89 23.28 23.90 E E/ WWI-m! "mm-mm "mm-m.— m———_ m—__— __mmanna- m—_——- iHAzﬂ’ARatio nut-""- "mm- "mm—-‘ mail—mm -_-—-E- m—M-ﬂ- m‘ 2.66 ---E- Example 5 In this example 9et10], chytrium species (ATCC PTA-10208) was cultivated in four 14-liter New Brunswick Scientiﬁc BioFlo 310 fermentors at a target ﬁnal (recipe) volume of lO-liters with a carbon (glucose) and nitrogen (ammonium hydroxide) fed-batch process. Three of the four fermentors were supplemented with carbon dioxide at different time points during the fermentation to evaluate the sensitivity of the culture to increased dissolved carbon dioxide. NBSl, NBSZ, and N883 were supplemented with carbon dioxide beginning at log hour 12, 24, and 48 respectively. The fermentations were each; inoculated with 1 liters of e each. For inoculum propagation, a 14-liter Virtis fermentor was utilized. The inoculum medium consisted of ers of medium prepared in four te groups. Group 1 consisted of 90 g MSG*1H20, 10 g KC], 50 g MgSO4*7H20, 3.3 g (NH4)ZSO4, 6.25 g ofNaCl, 60 g of T154 (yeast extract), 4.97 g KH2P04, 2.9 g CaC12*2H20, and 2 mL Dow 1520US (antifoam). Group 1 was autoclaved at 121 degrees for 120 minutes at a volume of approximately 9.8 . Group 2 consisted 0500 g glucose dissolved in a volume of 800 mL RO water. . Group 3 ted of31 mg MnCl2*4H20, 31 mg ZnSO4*7H20, 0.4 mg CoC12*6H20, 0.4 20.7 mg Na2MoO4*2H20, 20.7 mg CuSO4*5H20, mg NiSO4*6H20, 103 mg FeSO4*7H20 and 234.1 mg citric acid. Groups 2 and 3 were 1.6 mg each autoclaved 60 minutes. Group 4 consisted of 97.5 mg Thiamine-PIC], vitamin B12, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was dissolved in R0 water and, then ﬁlter—sterilized. Aﬁer the fermentor was cooled to 22.5 degrees Celsius, groups 2, 3, 4, and 5 were added to the fermentor. Using sodium hydroxide and sulfuric acid, the fermentor was pH adjusted to 7 and the dissolved oxygen was spanned to 100% prior to inoculation. The inoculum fermentor was inoculated with 150 mL of a fermentation culture (The smaller fermentation e was prepared and smaller cultivated in. the same manner as the er inoculum culture.) and cultivated at 22.5 s Celsius, pH 7, 433 rpm agitation, and 5 1pm of air for a period’of 44.5 hours, at which point 1 liters of inoculum broth was transferred to each 14-1iter fermentor. Each er fermentor contained 10-liters of fermentation media. The fermentation media For each 14-1iter fermentor, was prepared in a similar fashion to the inoculum fermentor. the fermentation media consisted of 6 batched media groups. For all vessels, group 1 contained 60 g , 6.25 g NaCl, 10 g KCl, 50 g MgSO4*7H20, 0.43. g (NH4)ZSO4, g T154 yeast t, and 1 mL Dow 1520-US antifoam. Group 1 was autoclaved at s for 120 minutes in the l4-liter ferrnentors at a volume of 121 degrees approximately 6.5 liters. Group 2 contained 2.9 g CaC12*2H20 in a volume of approximately 20 mL RO Water. Group 3 contained 17.61 g KH2P04 ved in 100 mL of RO water. Group 4 contained 31 mg MnC12*4H20, 93 mg ZnSO4*7H20, 0.4 .7 mg NiSO4*6H20, 103 mg mg 4*2H20, 20.7 mg CuSO4*5H20, FeSO4*7H2O and 10 g citric acid all dissolved in 50 mL of R0 water. Group 5 contained 97.5 mg Thiamine-HCI, 33.3 mg pantothenic acid hemi-calcium salt, and 36.3 sterilized in 10 mL RO water. Group 6 contained 300 ug of biotin, all dissolved and ﬁlter g of glucose in approximately 0.5 liters of R0 water. Groups 2, 3, 4, 5, and 6 were combined and added to the ntor after the fermentor reached an operating ature of 22.5 degrees Celsius. Each tor volume prior to inoculation was approximately 6.5 liters. Each fennentor was inoculated with 1 liter of broth from the inoculum fermentation described above. The fermentation was pH controlled utilizing a 0.85 liter solution of 4N ammonium hydroxide at a pH of 7 until nitrogen tion at which point 4N sodium ide and 3N sulfuric acid were utilized for pH control at a set-point of 7.5. The dissolved oxygen was controlled to maintain a target of 20% throughout the fermentation using agitation from 357 to 833 rpm and airﬂow from 7 LPM to 7 LPM. Vessels NBSl, NBSZ, NBS3 were each supplemented with carbon dioxide for different timeframes to evaluate the sensitivity of the organism to increased dissolved carbon dioxide. Throughoutthe tation, an 850 g/L solution ‘of 95% dextrose (corn syrup) was fed to maintain a glucose concentration less than 50 g/L. After 8 days, the dry cell weight and omega-3 titer of each 10-liter fermentor varied depending on the carbon dioxide supplementation conditions. At 183 hours, NBS] was at 109.1 g/L DCW and 45.87 g/L omega-3; NBSl was at 108.9 g/L DCW and 45.41 g/L omega-3; NBS3 was at 116.4 g/L DCW and 50.6 g/L omega-3; NBS4 was at 95.7 g/L DCW and 40.36 g/L omega-3. Shortly after the carbon dioxide supplementation was initiated, the AME decreased, the %EPA/FAME increased, and the ratio of DHA to EPA decreased. When comparing the maximum %EPA/FAME content of the carbon dioxide mented conditions and the ambient condition, there is 65% increase in the maximum EPA t under C02 supplemented conditions. The results are presented in Table 8.

Table 8 %EPA/FAME m----@12hours)@ 24 hours) @ 48 hours) added) 3 _-1_ %DHA/FAME HEE- NBsz NBss 4&34 , 4317‘rr _4¢69 "*ﬁ" : ‘14?" ' 3954 3881 - 1 31 4731 "" 45J9 44J3 46A7 4859 4.53 l carbonﬁdioxiduewsupﬂwghientewd duringjijghﬁghted timejrame ..,_M .M__...__,,._,‘.._,.

Example 6 In this example [Nx0106et10], Schizochytrium s (ATCC PTA-10208) was cultivated in four l4-liter New Brunswick Scientiﬁc BioFlo 310 fermentors at a target ﬁnal (recipe) volume of 10—liters with a carbon (glucose) and nitrogen (ammonium hydroxide) fed-batch process. The ature was controlled throughout the fermentation run to in a target of2l.0 °C, 22.5 °C, 24.0 °C, and 25.5 °C for NBSl, NBSZ, NBS3, and NBS4 respectively. The fermentations were each inoculated with 1 WO 13208 liters of culture each. For inoculum propagation, a l4-liter Virtis fermentor was utilized.

The inoculum medium consisted of 10-liters of medium prepared in four separate groups.

Group 1 consisted of 90 3.7 g MSG*1H20, 10 g KCl, 50 g MgSO4*7H20, g SO4, 6.25 g of NaCl, 60 g of T154 (yeast extract), 5.2 g KH2P04, 2.9 g CaC12*2H20, and 2 mL Dow 1520US (antifoam). Group 1 was aved at 121 degrees for 120 minutes at a volume of approximately 9.8 liters. Group 2 consisted of 500 g e ved in a volume of 800 mL R0 water. Group 3 consisted of 31 mg MnCl2*4H20, 31 mg ZnSO4*7H20, 0.4 mg COC12*6H20, 0.4 mg Na2MoO4*2H20, .7 mg CuSO4*51-120, 20.7 mg NiSO4*6H20, 103 mg FeSO4*7H20 and 234.1 mg citric acid. Groups 2‘ and 3 were each autoclaved for 60.minutes. Group 4 consisted of 97.5 mg Thiamine-HCI, 1.6 mg vitamin B12, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was dissolved in R0 water and then ﬁlter-sterilized. Aﬁer the fermentor was cooled to 22.5 degrees Celsius, groups 2, 3, 4, and 5 were added to the fermentor.

Using sodium hydroxide and sulfuric acid, the fermentor was pH adjusted to 7 and the dissolved oxygen was spanned to 100% prior to inoculation. The inoculum fermentor was inoculated with 200 mL of a smaller fermentation culture (The smaller fermentation culture was prepared and cultivated in the same manner as the lO-liter inoculum culture.) and cultivated at 22.5 degreesCelsius, pH 7, 433 rpm agitation, and 5 1pm of air for a period of 42 hours, at which point 1 liters of inoculum broth was transferred to each 14- liter fermentor. Each l4-liter fermentor contained 10-liters of fermentation media. The fermentation media was prepared in a similar n to theinoculum tor. For each 14-liter fermentor, the fermentation media consisted of 4 batched media groups. For all vessels, group 1 ned 88 g Na2SO4, 6.25 g NaCl, 10 g KC], 50 g MgSO4*7I—I20, 4.2 g SO4, 2.9 g CaC12*2H20, 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 the l4-1iter fermentors at a volume of approximately 7.0 liters. Group 2 contained 31 mg MnC12*4H20, 93 mg ZnSO4*7I-120, 0.4 mg Na2MoO4*2H20, 20.7 FeSO4*7H20 and 468 mg of citric mg CuSO4*5H20, 20.7 mg NiSO4*6H20, 103 mg acid dissolved in 50 mL of R0 water. Group 2 was autoclaved for 60 minutes. Group 3 contained 97.5 mg Thiamine-HCI, 33.3 ’mg pantothenic acid hemi-calcium salt, and 35.8 mL RO water. Group 4 contained 300 ug of biotin, all ved and ﬁlter ized in 10 g Of e in approximately 0.5 liters of R0 water and autoclaved for 60 minutes.

WO 13208 Groups 2, 3, and 4 were combined and added to the fermentor after the fermentor reached an operating temperature of 22.5 degrees Celsius. Each fermentor volume prior to inoculation was imately 6.5 liters. Each fermentor was inoculated with 1 liter of broth from the inoculum fermentation described above. The fermentation was pH controlled at 7.0 throughout the fermentation utilizing a 0.85 liter solution of 4N ammonium hydroxide until nitrogen exhaustion at which point 4N sodium hydroxide and 3N sulfuric acid were utilized for pH control at a set-point. The dissolved oxygen was controlled to in a target of 20% until nitrogen exhaustion. Aﬁer en exhaustion, the dissolved oxygen was lled to in a target of 10% until the end of the fermentation using agitation from 357 to 714 rpm and 8 LPM of airﬂow.

Throughout the fermentation, an 850 g/L solution of 95% dextrose (corn syrup) was fed to maintain a glucose concentration less than 50 g/L. Aﬁer 8 days, the dry cell weight or omega-3 titer varied slightly for the different temperatures evaluated; r, the lower- fermentation temperatures resulted in higher %EPA/FAME. At 184 hours, NBSl was at 85.2 g/L DCW and 29.9 g/L 3; NBS2 was at 92.0 g/L DCW and 35.0 g/L omega- 3; NBS3 was at 86.8 g/L DCW and 31.7 g/L omega-3; NBS4 was at 84.2 g/L DCW and . 29.4 g/L omega-3.

For NBS], %EPA/FAME ranged from 12.36% to 19.02% from start to end of the tation run with a maximum of 21.57%. For NBS2, %EPA/FAME ranged from 11.72% to 18.11% from start to end of the fermentation run with a maximum of 20.21%.

NBS3, %EPA/FAME ranged from 11.49% to 15.43% from start to end of the fermentation run with a maximum of 18.09%. NBS4, %EPA/FAME ranged from 11.65% to 13.65% from start to end of the fermentation run with a maximum of 15.70%. When comparing the maximum %EPA/FAME, the lowest fermentation temperature resulted in a 37% increase in the maximum EPA content over the highest fermentation temperature evaluated. The results are provided in Table 9 below.

Table 9 |%EPA / FAME mNBSl (21 °C) NBS2 (22.5 °C) NBS3 (24 °C) NBS4 (25.5 °C) "-36 11-72 11-49 m 14.17 13.61 12.21 13.04 14.48 m 14.23 13.62 NBS4 (25.5 DHA:EPA Ratio Hours NBSl (21 °C) mm (22.5 °C) NBS3 (24 °C) "50325-5 y—s M O\ _-z_ .

Example 7 In this e [Nx0614et10], Schizochytrium species (ATCC PTA-10208) was cultivated in two 14-liter New Brunswick Scientiﬁc BioFlo 310 fermentors at a target final (recipe) volume of 10—liters with a carbon (glucose) and nitrogen (ammonium hydroxide) tch process. One fermentor (NBSlS) was sparged with air WO 13208 supplemented with 15% carbon dioxide from start to end of the fermentation run and the other tor (NBSl7) was sparged with air only to evaluate the sensitivity of the culture to increased dissolved carbon dioxide. The fermentations were each ated with 1 liters of culture each. For inoculum propagation, a l4-liter Virtis ntor was four utilized. The inoculum medium consisted of lO-liters of medium prepared in separate groups. Group 1 consisted of 90 g MSG*1H20, 10 g KCl, 50 g MgSO4*7H20, 2.9 g 3.3 g )ZSO4, 6.25 g of NaCl, 60 g of T154 (yeast extract), 4.97 g KH2PO4, CaC12*2H20, and 2 mL Dow 1520US (antifoam). Group 1 was autoclaved at 121 degrees for 120 minutes at a volume of approximately 9.8 liters. Group 2 consisted of 500 g glucose dissolved in a volume of 800 mL R0 water. Group 3 Consisted of 31 mg MnCl2*4H20, 31 mg ZnSO4*7I—I20, 0.4 mg 6H20, 0.4 mg Na2MoO4*2H20, .7 mg 5H20, 20.7 mg NiSO4*6H20, 103 mg FeSO4*7H2O and 234.1 mg citric acid. Groups 2 and 3 were each autoclaved for 60 minutes. Group 4 consisted of 97.5 mg Thiamine—PIC], 1.6 mg vitamin BIZ, and 33.3 mg pantothenic acid hemi-calcium salt. Group 4 was ved in R0 water and then ﬁlter-sterilized. Aﬁer the fermentor added to the fermentor. was cooled to 22.5 degrees Celsius, groups 2, 3, 4, and 5 were Using sodium hydroxide and sulfuric acid, the tor was pH adjusted to 7 and the dissolved oxygen was spanned to 100% prior to inoculation. The inoculum fermentor smaller fermentation was inoculated with 200 mLof a smaller fermentation culture (The culture was prepared and ated in the same manner as the lO-liter inoculum culture.) and cultivated at 22.5 degrees Celsius, pH 7, 433 rpm ion, and 5 1pm of air for a period of 40 hours, at which point 1 liters of inoculum broth was transferred to each 14; liter tor. Each 14-liter fermentor contained lO-liters of fermentation media. The fermentation media was prepared in a similar fashion to the inoculum fermentor. For each 14-liter fermentor, the fermentation media consisted of 6 batched media groups. For all vessels, group 1 contained 88 g Na2SO4, 6.25 g NaCl, 10 g KCl, 50 g MgSO4*7HZO, 4.2 g (NH4)2SO4, 10 g T154 yeast extract, and 1 mL Dow 1520-US antifoam. Group 1 was aved at 121 degrees Celsius for 120 minutes in the l4-liter fermentors at a volume of approximately 6.5 liters. Group 2 contained 2.9 g CaCl2*2H20 in a volume approximately 20 mL R0 Water. Group 3 contained 17.65 g KH2PO4 dissolved in 100 mL of RO water. Group 4 contained 31 mg MnC12*4I-IZO, 93 mg ZnSO4*7H20, 0.4 Na2MoO4*2H20, 20.7 mg 103 mg CuSO4*5H20, 20.7 NiSO4*6H20, mg FeSO4*7H20 and 468 mg of citric acid dissolved in 50 mL of R0 water. Groups 2, 3, and 4 were each autoclaved for 60 s. Group 5 contained 97.5 mg Thiamine-HCI, 33.3 mg pantothenic acidhemi-calcium salt, and 36.3 ug of biotin, all dissolved and ﬁlter sterilized in 10 mL R0 water. Group 6 contained 300 g of glucose in approximately 0.5 liters ofR0 water and autoclaved for 60 minutes. Groups 2, 3, 4, 5, and 6 were combined and added to the fermentor aﬁer the tor reached an Operating temperature of 22.5 degrees Celsius. Each fermentor volume prior to inoculation was approximately 6.5 liters. Each fermentor was inoculated with 1 liter of broth from the inoculum tation described above. The fermentation was pH controlled at 7.0 throughout the fermentation utilizing a 0.85 liter on of 4N ammonium ide until en exhaustion at which point 4N sodium hydroxide and 3N sulfuric acid were utilized for pH control at a int. The dissolved . was controlled to maintain a target of 20% until nitrogen exhaustion. After nitrogen exhaustion, the dissolved oxygen was controlled to maintain a target of 10% until the end of the fermentation using agitation from 357 to 833 rpm and 8 LPM of airﬂow. Throughout the fermentation, an 850 g/L solution of 95% dextrose (corn syrup) was fed to maintain a glucose concentration less than 50 g/L. After 8 days, the dry cell weight and omega-3 titer of each 10-liter fermentor varied depending on the carbon e supplementation conditions. At 188 hours, NBS15 was at 54.5 g/L DCW and 13.7 g/L omega-3; NBS17 was at 96.1 g/L DCW and 37.5 g/L omega-3.

%EPA/FAME was higher in NBS15 (C02 supplemented condition throughout the run) than NBS17 (no C02 srlpplementation). For NBS15, %EPA/FAME ranged from 25.50% to 35.48% from start to end. of the fermentation run with a maximum of 38.34%. For NBS l7, %EPA/FAME ranged from 12.31% to 19.80% from start to end of the fermentation run with a maximum of 22.29%. When comparing the maximum %EPA/FAME content of the carbon dioxide supplemented conditions and the ambient condition, there is 73% increase in the maximum EPA content under C02 supplemented conditions.

%DHA/FAME was lower for the C02 supplemented conditions than for the ambient conditionlthroughout the tation run. The results are provided in Table 10 belwo. 2012/047728 Table 10 %EPA / FAME mam—_ 184 19.02 » 18 11 15.43 13.65 o/oDHA / FAME NBSZ (22.5 °C) NBS3 (24 °C) NBS4 (25.5 °C) ‘ NBSl (21 °C) m 48.04 48 99 49.39 . 49.38 DHAzEPA Ratio NBS] (21°C) NBSZ (22.5 °C) NBS4(25.5 °C) Ip—l U "— 184 2.53 2.71 3.20 3.62 In this example [K019], Schizochytrimn species (ATCC PTA-10208) was cultivated in 157,000-1iter agitated fennentor at a target ﬁnal (recipe) weight of 100,000 kg with a carbon (glucose) and nitrogen (anhydrous ammonia gas) fed-batch process.

The tation was inoculated with 4500 kg of culture. For inoculum propagation, a 7500—liter agitated seed tor was utilized. The inoculum medium consisted of 4500 kg of medium prepared in four separate groups. Group 1 consisted of 40.5 kg , 4.5 kg KCl, 22.5 kg 7H20, 1.7 kg (NH4)ZSO4, 2.81 kg of NaCI, 27 kg of T154 (yeast extract), 2 kg KH2PO4, 985 g CaC12, and 0.9 kg Dow 1520US (antifoam) dissolved in process water with a total weight of 2300 kg. Group 2 consisted of 247.5 kg glucoseJHZO dissolved in process water with a total weight of 1500 kg.

Group 1 was sterilized in the seed fermentor, and Group 2 was sterilized in a te vessel, with steam—in-place at 122-123 degrees Celsius for 30 minutes. Group 3 consisted of 14g MnC12*4H20, 14 g ZnSO4*7H20, 180 mg 6H20, 180 mg NaZMoO4*2H20, 9.3 g CuSO4*5H20, 9.3 g NiSO4*6H20, 46.4 g FeSO4*7H20 and 105.3 g citric acid dissolved in 5 L of distilled water. Group 3 were autoclaved at 121 s Celsius for 60 s. Group 4 consisted of 43.9 g Thiamine-HCI, 720 mg vitamin B12, and 15 g pantothenic acid hemi—calcium salts dissolved in 5 L distilled water and then ﬁlter-sterilized. After the seed fermentor was cooled to 22.5 degrees Celsius, groups 2, 3, 4 were added to the fermentor. Using sodium hydroxide and sulfuric acid, the ferrnentor was pH adjusted to 7 and the dissolved oxygen was spanned to 100% prior to inoculation. The seed fermentor was inoculated with 12 L of a smaller fermentation culture (The smaller fermentation e was prepared and cultivated in the 90 rpm same manner as the seed e) and cultivated at 22.5 degrees Celsius, pH 7, agitation, and 130-170 Nm3/hr of air for a period of 4-5 days to get a dry cell weight about 15 g/L. The fermentation media was prepared in a similar fashion to the inoculum fermentor. the tation media consisted of 5 groups. Group 1 contained 177 kg KH2PO4, 880 kg NaZSO4, 500 kg MgSO4*7H20, 42 kg (NH4)ZSO4, 100 kg T154 yeast extract, and 10 kg of Dow 1520-US antifoam in a 9,000 kg of solution. Group 2 contained 21.9 kg CaC12, 62.5 kg NaCl, 100 kg KC], in a 9,000 kg solution. Group 1 and 2 were pumped through a heat exchanger into the fermentor, followed by water to get 67,000 kg of weight in ferrnentor. Group 3 contained 310 g MnCl2*4H20, 930 g ZnSO4*7H20, 4 g Na2MoO4*2H20, 207 g CuSO4*5H20, 207 g NiSO4*6H20, 1.03 kg FeSO4*7H20 and 4.68 kg citric acid dissolved in 1500 kg of process water. Group 4 contained 4300 kg of corn syrup (DE-95, 70.5%). Group 3 and Group 4 were sterilized in different vessels, with stearn-in-place at 122—123 degrees Celsius for 30 minutes. Group 5 contained 975 g Thiamine-HG], 333 g pantothenic acid hemi-calcium salt, and 358 mg of , dissolved and ﬁlter sterilized in 5 L distilled water. Groups 3, 4, and 5 were added to the fermentor aﬁer the fermentor was cooled down to 22.5 degrees Celsius. The weight in the ferrnentor volume prior to inoculation was approximately 73,500 kg. After the starting fermentation condition was set (temperature: 22.5 °C, pressure: 0.34 bar, airﬂow: 3000 Nm3/hr, agitation, 40 rpm), the fermentation pH was adjusted to 7 and the dissolved At the oxygen was spanned to 100%. The weight aﬁer inoculation was about 78,000 kg. beginning, pH was controlled at 7 ing anhydrous ammonia until 550 kg of ammonia set- was added, and then 30% sodium hydroxide solution was utilized for pH control at a point of 7.5. The dissolved oxygen was controlled to maintain a target of 20% during ammonia feed and 10% aﬁerward using agitation from 40 to 100 rpm and airﬂow from 2000 to 8000 Nm3/hr. Throughout the fermentation, a 65% DE-95 corn syrup on was fed to maintain a e concentration around 35 g/L. In another e [K020], three changes were made from the e K019: 1) pressure was reduced to 0.15 bar, 2) weight after inoculation was reduced to 68,000 kg, 3) airﬂow was increased. to above 5000 Nm3/hr at 60 hrs after inoculation and was maintained high throughout the run less of the ved oxygen tration. The above three changes d the dissolved carbon dioxide concentration in the broth. The results showed that with the reduced C02, AME was increased from 38.38% to 43.8%, and %EPA/FAME was reduced from 24.42% to 20.68%. The DHA2EPA ratio was increased from 1.57 to 2.12. The results are presented in Table 11 below.

Table 1 1 DHA/FAME ME Hr % % Ratio 11.47 13.2 62 43.99 16.97 74 34.65 19.94 86 32.14 22.56 .3! 30.05 25.2 13 31.45 28.36 158 35.48 . .47 Table 12 K020 Reduced Dissolved C02 hr % % Ratio 4.32 .05 180 I 43.8 Example 9 In the table below, the maximum dissolved C02 is calculated for several of the es using Henry’s constant. The ﬁrst c0ndition, "10 L (NBS4 07l9et10) at 0 PSI back pressure and 45.5 mmol/L/hour CER" is the calculated dissolved C02 for NBS4 in table 8 at a carbon dioxide evolution rate of 45.5 mmol/L/hour, a fermentation volume of -liters, an on rate of 0.8 vvm, and 0 PSI backpressure. The second condition, "10 L (NBSZ 07l9et10) at 0~ PSI back pressure with 6% C02 in the inlet gas and 50 2012/047728 -884 mmol/L/hour CER" is the calculateddissolved CO2 for. NBSZ in Table 8 at a carbon dioxide evolution rate of 50 /hour, a fermentation volume of lO-liters, an aeration rate of 0.8 vvm, at 0 PSI backpressure, and with CO2 supplemented in the inlet stream 6% of the total gas as measured by mass spectrometry using a Thermo Prima dB mass spectrometer. The third condition, "SO-liter (NBS 06l4et10) at 2 PSI backpressure and 55 mmOl/L/hour CER" is the calculated dissolved CO2 for NBS in Table 7 at a carbon dioxide evolution rate of 55 mmol/L/hour, a fermentation volume of 80-liters, an aeration rate of 1.0 vvm, and 2 PSI backpressure. The fourth condition, "80-1iter (NB6 O614et10) at 15 PSI backpressure and 50 mmol/L/hour CER" is the calculated dissolved CO2 for N36 in Table 7 at a carbon e evolution rate of 50 mrriol/L/hour, a tation volume of 80-liters, an aeration rate of 1.0 vvm, and 15 PSI backpressure. The ﬁfth condition, "80-1iter (NB7 & NB8 0614et10) at 20 PSI backpressure and 50 mmol/L/hour CER" is the calculated dissolved CO2 for NB7 and NB8 in Table 7 at a carbon dioxide evolution rate of 50 mmol/L/hour, a fermentation volume of 80-liters, an aeration rate of 1.0 vvm, and 20 PSI backpressure. All CER values were calculated using off-gas C02 data collected with a Thermo Prima dB mass ometer. The results of the calculations are provided in Tables 13 and 14 below.

Table 13 L (NBS4 0719et10) at 0 PSI back pressure and 45.5 mmol/L/hour CER RV 10 L te re input 1.01 bar Airﬂow input 8 LPM va 0.8 Required CO2 in the air inlet output 0.14 % Delta CO2 in the offgas 2.309125 % CO2 in the offgas 2.446301 % co2 Partial Pressure 1 0.024708 1] bar dissolved C02 0.000785 mol/L ved C02 34.53 L (NBSZ 07l9et10) at 0 PSI back pressure with 6% C02in the inlet gas and 50 mmol/L/hour CER RV 10 L Absolute Pressure input 1.01 bar Airflow input 8 LPM va 0.8 Required CO2 in the air inlet output 7.00 % Delta C02 in the offgas 2.5375 % C02 in the offgas 53 % C02 Partial Pressure dissolved C02 0.003059 moi/L dissolved C02 134.58 80-liter (NBS 0614et10) at 2 PSI essure and 55 mmol/L/hour CER Back Pressure Total Pressure Airﬂow Delta C02 C02 in the outlet C02 Partial Pressure dissolved C02 dissolved C02 80—liter (NB6 0614et10) at 15 PSI backpressure and 50 mmoI/L/hour CER RV Back Pressure - 1 input Total Pressure ' Airﬂow input va . -4 Delta C02 from CPR C02 in the outlet C02 Partial Pressure dissolved C02 dissolved C02 59.71 80-liter (NB7 & NBS 0614et10) at 20 PSI backpressure and 50 mmol/L/hour Back Pressure Total Pressure Airﬂow Delta C02 C02 in the outlet C02 l Pressure dissolved C02 dissolved C02 Table 14 N36 (15 N37 (20 NB8 (20 %EPA/FAME m 21.57 m 25.25 27.94 17.81 ‘VoDHA/FAME 40.35 39.22 34.14 32.42 24.36 38.81 maxdissolvedcoz 37.70 —mmm 34-53 Example 10 Experiments were performed to determine the effect of vitamin gradients on performance (dry cell weight of biomass (DW), % DHA, % fat, and % EPA) using ATCC Accession No. PTA-9695 in Thraustochytrium Shake Flask Medium (TSFM) under ambient C02 level.

Materials and Methods: Four vitamins ine.HCl, 312, Biotin, and Ca— pantothenate) were used in TSFM medium with 0.25 g/L tastone and 0.625 g/L NaCl (see Table 16). Additional MSG and KH2PO4 were added to the media to maintain their total nitrogen and phosphorous contents. The overall n concentrations in the media were, 0, 0.5x, 1x, 5x, 10x, 20x, or 30x the standard amount, depending on the ns studied (see Table 17). The gradient study was performed on each vitamin separately. However in the case of biotin and Ca—pantothenate, standard amounts of Thiamine.HCl and B12 were very were also orated in the media, since the trations of the latter two low in the regular TSFM. Three TSFM controls were also ed in the experiment for comparison. These controls were standard TSFM with 2g/L tastone (see Table 15) and 1x Thiamine.HCl and 1x B12 (Cl); tastone-free TSFM without any vitamins (A); and tastone-free TSFM with I); ine.HCl and lx BIZ (B). All controls contained 0.625 g/L NaCl. Three-day old culture of PTA-9695 was used to inoculate duplicate 250-ml shake ﬂasks at 0.1 g DW/L. All ﬂasks (ﬂat-bottom, with total of 50 1111 media) were incubated aerobically at 22.5+/- 1C on a rotary shaker (200 RPM). All es were harvested after 7 days, and FAME analysis was performed on the ﬁnal freeze—dried biomass samples Table 15 Thraustochytrium Shake Flask Medium (TSFM) with 2 g/L tastone Amount per mL of stock to ent liter (g) '[Stock] (g/l) use per liter NaCl 0.625 dry KCl 1 50 20 ml MgSO4.7H20 5 - 227 22 ml CNH4)2$O4 0.2 190 1.05 ml CaCl2 21-120 0.29 dry drate 2 dry Tastone 154 2 dry HEPES (l00 mMO pH 7 23.8 dry KH2P04 0.1 56.5 1.77 ml add aﬁer autoclaving e 50 500 100 ml add aﬁervautoclaving Trace Metals see below 1 ml add aﬁer autoclaving Vitamins see below '1 ml add aﬁer autoclaving Trace Metal Solution FeCl3.6H20 2.9 mg 2.9 CuSO4.5H20 0.02 mg 0.02 MnC12.4H20 8.6 mg 8.6 CoCl2.6H2O 0.26 mg 0.26 ZnCl2 0.6 mg 0.6 Citric Acid 12 mg 12 g (dry) Vitamin Solution .

Thiamine 10 ug l0 mgl Vitamin 812 1 ug 1 mg/L Table 16 Thraustochytrium Shake Flask Medium (TSFM) with 0.25 g/L Tastone mL of stock Amount per [Stock] to use per Component - liter (g) (g/l) liter NaCl 0.625 dry KCl 1 50 20 ml MgSO4.7H20 5 227 22 ml (NH4)ZSO4 0.2 190 1.05 ml CaClZ 2H20 0.29 dry MSG monohydrate 4.554 dry Tastone GC 7189-1 0.25 dry HEPES (100 mM0 pH 7 23.8 dry - KH2P04 0.1 56.5 4.28 m] add aﬂer autoclaving Glucose 50 500 100 ml add atter autoclaving Trace Metals see below 1 ml add aﬂer autoclaving Vitamins see below 1 ml add after autoclaving TSFM Trace Metal FeCl3.6H20 2.9 mg 2.9 CuSO4.5H20 0.02 mg 0.02 MnC12.4H20 8.6 mg 8.6 CoC12.6H20 0.26 mg 0.26 ZnC12 0.6 mg 0.6 Citric Acid 12 mg 12 g (dry) Vitamin Solution ~ Thiamine 10 ug 10 mgl Vitamin BIZ 1 ug 1 mg/L Table 17 Vitamin concentrations used in this study (mg/L): vitamin cone. [XI Thiamine.HCl 312 Biotin Ca-Pantothenate 0 0.0 ' 0.0 0.0 0.0 0.5 0.005 0.00234 . l 0.010 0.001 8 3.33 0.050 0.005 16.65 ' 0.100 0.010 0.0468 33.30 0.200 . 0.020 0.0936 66.60 0.1404 Results: The highest DW and % fat (6.7 g/L and 38.5%, respectively) for the thiamine gradient were ed when the added amount of this vitamin to the medium was 5 times the standard level (Figure 1). The % DHA at this level of thiamine was 44.1%. Below and above 5x thiamine, both DW and % fat started declining. % DHA also ed in the media with less than 5x thiamine, and it slightly ﬂuctuated 'at above 5x thiamine with no cant improvement. % EPA in the thiamine nt cultures ranged between 8.6 and 11.5. When no tastone or vitamins were added to the medium WO 13208 (Figure 5, medium A), everything except %DHA declined cantly. The increase in % DHA, however, appeared to be artiﬁcial since both the DW and % fat were extremely low for this condition. The 1x tration of B12 seemed to be optimal for DW, % DHA, % fat, and % EPA (Figure 2). At this level of n B12, the following were achieved: 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 with and the higher % no added vitamins did not further improve mance of the organism, DHA seemed to be artiﬁcial, as it was described previously e 5, medium A).

Similarly, the 1x concentration of biotin was l for DW, % DHA, and'% fat (Figure 3). The corresponding values for these parameters were: 6.8 g/L, 47.7%, and 37.9%, respectively. Percent EPA at this point was 1.9. Tastone-free medium with 1x thiamine and 1x B12 (Figure 5,‘ medium B) signiﬁcantly compromised the overall performance of PTA-9695.

Ca—pantothenate produced the highest DW, % DHA, and % fat when this vitamin. was added to the medium at 10x the standard amount (Figure 4). The optimal concentrations of these parameters were 7.0 g/L, 47.3%, and 39.1%, accordingly. The EPA content in the entire Cafpantothenate gradient experiment was less than 2%.

Signiﬁcant reduction in the. overall performance of PTA-9695 was noticed when the organism was grown in tastone-free medium with only 1x thiamine and 1x BIZ (Figure 5, medium B).

Results are also shown in the data tables below.

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WO 13208 «as? Example 11 Experiments were med to determine the minimum requirement of ATCC Accession No. 95 for vitamin B12 to e maximal performance at 10% C02, including the minimum amount of vitamin 812 that is required for PTA-9695 culture to produce maximum dry weight and DHA yield under 10% C02 conditions.

Temperature: 22.5 +/- 1C (@ 10% C02) Shaker speed: 200 rpm [0216} Base medium: Deﬁned Scaled-Down Fermentor Medium (DSDFM-B) (Table ' 20).

Inoculum: 3-day old culture of PTA-9695 grown in DSDFM-B with different concentrations of vitamin B12.

A new cryovial of PTA-9695 was lly thawed in SDFM-B (Table 19), and was subsequently grown and transferred several times in Deﬁned SDFM-B under 10% CO2. A 3-day old culture of PTA-9695 in DSDFM-B was used (at 4%) to prepare the initial inoculum ﬂasks containing DSDFM-B with various concentrations of vitamin B12 (i.e., from 7.68 mg/L to zero mg/L of B12 for treatments A through I, accordingly; see Table 18): The inoculum ﬂasks for each concentration of vitamin B12 were maintained under 10% CO2 and by erring the cultures into their respective fresh media every 7 days. The weekly transfer of the cultures was essential for g out any excess vitamin 812 that might have been stored inside the cells, so that the minimum requirement for this vitamin could be more accurately determined. Duplicate shake ﬂasks were inoculated with their respective 3-day old inocula at a theoretical DW of 0.1 g/L, using the Optical Density at 600 nm. Cultures were grown under 10% CO2 for 9 days, prior to being harvested and further ed by FAME assay.

Results : Note: Data points for week 3 are being disregarded in this study, as the supply of C02 to the incubator had been interrupted, and ﬂasks had to be removed from the incubator for 6 hours during lab tion.

Prolonged exposure (up to 4 weeks) of 95 to high concentrations of vitamin B12 (greater than 3.84 mg/L) can adversely impact the dry weight, % fat, and DHA yield, while a concentration of between 1.5 to 768 ug/L n B12 has no significant effect on PTA-969SS' DW, % fat, % EPA, and DHA yield.

An increase in the number of single cells (up to 15, .as opposed to 3-4) were noted in cultures that had been grown in the presence of 7 68 mg/L vitamin BIZ (treatment A).

Eliminating vitamin 812 from the media recipe, signiﬁcantly increases % EPA by about 10%, while it slightly decreases both % DHA and % 16:0. s 6-19 and Table 21 below show the s of the ments.

Conclusion: ’A minimum of about 1.5 ug/L vitamin B12 (l/SOOth the standard concentration in SDFM—B) is essential for obtaining maximal DW, % DHA, % fat, and DHA yield. While very high concentrations of vitamin BIZ (greater than 3.84 mg/L) can substantially reduce DW, % fat, and DHA yield, a complete deprivation of PTA-9695 from this vitamin can significantly (approximately 10%) increase its % EPA. Very high levels of vitamin B12 may also _play a role in promoting transformation of the "clumpy" culture into "single cells".

Table 18. n BIZ Treatments A [----I5-E-I Vit. 1312 u_/L 7680 3840 768 384 153.6 76.8 15.36 7.68 1.536 n Table 19. -Down Fermentor Medium for PTA-9695 (SDFM—B), pH 7 mls of stock Final' - [Stock] (g/L) into grams of dry concentration . 1L shake ingredient in flask 1L shake flask M5047H20 246.5 22 — CaClZ 21120 3 -——— Tastone GC9156-1 _- HEPES 100mM) 117.0 195.2 — KHZP04 Glucose 180 500 100 _____ __—_— _—_-EI-_ _-—-—_ __M— —__—-— 262.84 —_—_.- FeSO4.7H20) 117.5 1.175 —--__— _-___ ' —__-— 0.000768 __M— 0002 2.17 _— Table 20. Deﬁned SDFM-B (DSDFM-B), pH 7 HI. mls of Final MW [Stock] (g/L) stock into grams» of dry concentration 1L shake ingredient in flask 1L shake flask \lA . U10‘ 56 17.9 _ MSO4.7H20 246.5 227 22 — (NH4)ZSO4 132.14 190 1.1483 m CaCl2.2H20 147 60 4.833 — MSG w/ 1 mole H20) 187.1— 2.188 2.188 Na2504 142.04_m-m _17427E1.1240Tastone (GC9156-1)HEPES (100 mM) H 7.0 195 2 . 56 .5 ' -_ Fe504.7H20 278 02. 1 . =_Zn504.7H20 1 NaZMoO4.ZHZO 0.00004 —-—-— 000207 -_ 0-00207 Citric Acid(in Fe504.7H20) Vitamin Solution -——— Vitamin B12 1355. 4 0.768 -— 0.000768 Thiamine HQ 337 3 476 54 3996 244 3 0002 Table 21. Data from Experiments Shake Flask series SF lVit. BIZ concL 7:8 —E15363840 15-36 3. 885361 3. 584331 2. 885361 2.584331 2.186391 1.885361 1 186391 0.885361 0.186391 ml—_ ———_—- 3.. 418. 3.. 3412 3.7. 33.9 ms Oahu new cocau—tAoe —'—'———'——'—'—— s2s‘2' --------- m—m—m nmm_mmmm—m- ————_- _——————-—-- "mm—mum- —mmm——mm——m. -_—————__—- m---__--—--mm- "mmmm—m—m—2m —————_—_——- ".2"? =-_-—----- .7. maximum—m 6—1_———m—-m 6M 6M 67.0 2 0. ﬂask 2 22 7..1 22 SJ 22 4.3 22 36. 22 6.1. avera-c DHA ylcld l 3 11 1.1. L2 5 ﬂask 2 7. 1 1. 1 3 avera-_e % EPA 1 n. 22M6. 11 mm 11... 6&7. 735 20.4 I"M2 1. 7 5. 1555 ﬂask 1 —-E- .1w 1677. averaue lM ﬂask 1 m9.2 .1 2 2 102.5 ﬂask 2 2 HQ0.1.34 1H1 3.7. 63 avera w 3 www 587 84.1. 11] 211 407 767 99mm [063.

EPA yield ﬂask 1 04. ﬂask 2 3. 00 4.4. 00 5.4 00 54" 00 54. 04. 00 .. 54 0.5 averu c l—FDW 21l 1 5. 1 5. l 4.5 1 5 1 5. 1] .4 5 ﬂask 2 avcrane %l6:0 ﬂask I ﬂask 2 22.mum 222 mmu 22233M11 222 331m 687 22 2.3.6.1 222 mum MM.4.5 222333 40.2. 23.6 23 7. avera-c % DPA n 1 2. 1 2 ﬂask 2 1 1. 1 2 1 2 ever e 22 1.2. 22 1.6 22 3.3 222 52.3 222 1 6. .22 222 2.22. 222 4.55 222 423 5. nm—"mmm—m. . -——_———_—_ n__—__———_ Shake - series(SF :- -—._N 7680 3840 ::—21536Eé01536 - 1536 3885361 3.584331 2.885361 2.584331 2.186391 1885361 1. [86391 0.885361 0.186391 __=—7——__17ll — —3916 2572 .ww:0253x) 2.222mum- _'————_——— .?mm -————————— mmmmmmmum qm— Lab’s: —————.——_—m—8——— ———_ m— maxim—"m——_uww 011mg»blah: ———_' ——-—— -——- 207.2mm mum-1m:N""’ 38:; 090;. ——-——-—-_— 1.ny--------- mm——m Mum-ml-MOgoobut —mmnm __——_—- _———___ 26 24 22 29 2 2w . 27 . 22 2 .6 2J 2 .4. 2 .6. 2 .6. ﬂask 2 av: c DHA yield ﬂask l 02 1.2 1.3 1.3 1.4 1.4 1 2. 11 1.2 1.2 1 3. 14 ﬂask 2 . avera_6 22 9 188 174 17 1 18 8 21 6 164. . . . . . . .8 24.5 18.8 m 18.8 17.0 20.0 16.8 21.8 188 17 9 17.0 . 17 9. ﬂask l 117.4 102.4 1083 107. 122.1 115. 8 100.5 79.1 132.4 118.6 114.4 102.8 127.0 108.3 ﬂask 2 71.5 1114 e .

EPA yield "Mk I 0.5 0.5 o4. 0.5 0.5 0.5 0.5 0. ﬂask 2 o5. oA 05 04. 0 5 average L-F DW .L 6 15 1 5. 15 1 7 . .

.L 5 1.5 1.7 1.5 1.5 1.1. 5.7. l 7. 26.0 22.5 21.7 24.3 22.5 23.1 23.0 22.0 23.2 ﬂask l ﬂask 2 295 22.3 24.3 23.2 22.7 23.2 avcmgc "/o DPA n- ﬂask 1 l. 5 1.1 100. ﬂask 2 1.2 1. 1. uveru;c % DPA n- 23 2 3 2 2 1 7. 23 21 2.3 253. 2 l 26.4.. 25 . . a w... __ — — -108— ome8-3 ml_m—"lﬁ-_m—682 mm———W.m —m——mm—mmmm —-_—__—--—- "———-——_—_-m m671 Table 21 continued Shake Flask series SF Treatment lVit. 812 conc. m L 7680 3840 -6 1536O-31.536 3885361 3.584331 2.885361 1 2.186391 1. 885361 1.186391 0.885361 0.186391 — - \l on 0" -_3258 N 2.92 —4108 3524 2534. 3974 3.794 — 3.90374 3106 —439 48 —34 3.013 '_'-'————'——4324 NI4, .1.121; - -__--—- 11.9 - 991 minim—m—mm __ ——————_- —— ———__—_- __ 99.9 _——__m— _m999 38.3 —m 39.9 mmmmm—mm —— _-————— m— --_----161.7 - 1799 10 2199 mmmmm.——_____———_ m-----_-——-_————-———m- mmmmm —-———-——_———' —109— —-______—_-—————_-——m- _-—-——-l-_——- —mW-_2.3 m----------DHA'ld lam-mum"!!- mun—Immun- nmmm ————_—__—-- _—__- ——lmlm_mmmm—m ————--_—__- M--__-_--_- m—1226 _mmmmﬂ" 1197 _m- ——-———__—-- —---------- manna—"_- ——_-_-——-_- 1-1 --------_- mm—mmm—nuﬂ _Iﬂ-I—W —mm———m‘m ---__—-—-_- —____—_———- m_——__——__- _ﬂask2_235=_217_228_214_210_215_220_182m23.6 m—mnmmm _——-—————-- mnmumnmmm "HI!- --!_-l_-I-—lll —-_- —__—_- on n-- ------ mnm "——mm- ave'" —-m "IE-- —__ — —_- oma-J - 1 10 - WEI-M Emu—mm -——______- -_-—_—-__- 6.87 Table 21 continued Flask series SF 4 4 4 4 4 4 . 4 "mm—-, Vit. 312 Cone. mJL 7680 3840 768 334 153.6 76.8 15.36 7.68 1.536 Vit. BIZ conc- (LoglO- mL 3.885361 3.584331 2.885361 2.584331 91_ 1.885361 1.186391 0.885361 0.186391 m_-—_-_—_-- MEIR" ——-_-__-—_- :rii‘é'iy ---------- m__—_--_—_m mmnmm ———_—____-- ———-_———-—- mun-Emm- ____—_--_-- n----------"A m172.7 memm —-_-_-__-_- "mff‘" ---- MEI-"mm _—_-——--- m_--I-———_- ——-m— --m "mum—m —_——— —_———- m--- ------ mun—m- "um-mum -—m-_ "mum-III:- nmmm mum-1.1mm ———— ————————_— —_—_—- mum-mm ———'——__———- m---------- minim-Ian mmmmmm ---- m—m— mum-mum" —'———————_-- ----- m—_-m—m-mm-mmmmum-mumm- n—m—_um--m———1—————-—- ——————— ———- mum-mm —_—'——_———_- m———__——-l--I-m _——_——-l--I--l--l- 11—m-m-lm-m-I-n-mm-m —"—_———————- -----—----- _m———-I--——-m- _-E--E-Ill ﬁmmmu-m-m-m- ———————--—- 3.1:? -----——_-- ———— mo 6.84 Example 12 ATCC Accession No. PTA-10208 Vitamin BIZ Gradients at 10% C02 Experiments performed to determine the tration of Vitamin B12 that provides optimum PTA—10208 growth and EPA tivity.

Temperature: 23 °C Shaker Speed: 200 rpm Base : Deﬁned SDFM-O -O) Inoculum: Thawed a vial of PTA-10208 into SDFM-O at ambient conditions.

Transferred 2 mL' of culture into 48 mL of DSDFM-O at 10% C02. [Transferred culture into fresh DSDFM-O (10% 002) (see Table 22). Transferred culture into fresh DSDFM- O (10% CO2). Used culture to inoculate Vitamin B12 gradient experiment Week #1 (2mL/ﬂask) (10% C02).

Experimental Set-Up: All cultures were grown in 50 mL shake ﬂasks and ate ﬂasks were grown for each condition. PTA-10208 was inoculated into nine day Vitamin B12 gradients in DSDFM-O (no tastone) every seven days. Inocula for each concentration of Vitamin 812 were maintained throughout the course of the experiment. By continuing to er PTA-10208 in reduced concentrations of n B12, excess Vitamin 812 was effectively washed out iof the cells. Inocula for each concentration of Vitamin B12 were transferred every seven days. Four day old inocula were used to start each nine day Vitamin B12 gradient. Each nine day gradient is consecutively labeled as Experiment Set A, B, C, D, E, F, G, H, I, J, and K below in Table 23. Before each gradient ation, optical density was measured for each concentration of Vitamin 812 in order to er approximately the same amount of cells. After nine days of growth, all cultures were harvested to measure pH, dry weight, and fatty acid proﬁle. The experiment was ended once dry weights at each concentration of Vitamin B12 were found to have stabilized for at least three consecutive nine day gradients.

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MO‘ 207 "— Amount liter mg mg per (g) 9.3 2.07 2.07 Jonent FeSO4.7H20 Metal with Solution BIZ Com Trace Solution MnCl2.4HZO ZnSO4.7H20 CuSO4.5H20 NiSO4.6H2O to Totals pH Vitamin n Thiamine CaPantothenate [on Table 23 0.15 0.077 0.038 0.0077 Seven consecutive 5x to l/SOOx n B12 gradients were carried out. At. the end of the last experiment, a new experiment was started comparing PTA-10208 growth and EPA production at l/500x (0.0015 mg/L) and 0x (0 mg/L Vitamin B12). Four consecutive 1/500x vs 0x Vitamin B12 experiments were carried out.

Results: (For 5x --> 1/500x Vitamin BIZ): For seven consecutive nine day experiments, PAT-10208 dry weight, %EPA, %DHA, % fat, and EPA yield did not change in n BIZ trations ranging from 5x (3.84 mg/L) to 1/500x (0.0015 mg/L).

(For 1/500x and Ox Vitamin B12): Results over the course of four consecutive nine day experiments show that PTA-10208 grown in DSDFM-O containing 0 mg/L Vitamin BIZ has much higher % EPA than when grown with just l/SOOX (0.0015 mg/L) Vitamin B12. While % EPA increases from about 18% with 1/500x 5 mg/L) Vitamin B12 to about 27% without any Vitamin B12, % DHA decreasesvfrom about 45% with l/500x (0.0015 mg/L) n BIZ to about 36% without any Vitamin B12. PTA- 10208 dry weight and % fat increase slightly when Vitamin B12 is Completely removed from DSDFM-O. As a result of the increase in % EPA and the slight increases in dry weight and % fat, PTA-10208 EPA yield increases by about 60% when Vitamin BIZ is removed from DSDFM-O.

Results are also shown in Figures 20 — 49 and in the tables below.

Experiment 600nm Q/L lnoc. mglL Vit Tare Out wt Set SAMPLE 0.0. 812 pH (9) (g) dry pellet wt (9) Biomass A 5x (1) 3.84 6.80 10.4757 10.723 0.2473 4.95 5x (2) 3.84 6.80 10.4534 10.689 0.2356 4.71 1x (1) 0.77 6.80 10.5142 10.7449 0.2307 4.61 - I 16 - Exparlment 600nm g/L lnoc. mg/L Vit Tare Out wt Set SAMPLE 0.0. B12 pH (9) (g) dry pellet wt (9) Blomass 1x (2) 0.77 6.81 10.431 10.6512 0.2202 4.40 1/2x (1) 0.38 6.82 10.5684 2 0.2198 4.40 1/2x (2) 0.38 6.82 10.6373 10.8691 0.2318 4.64 1/5x (-1) 0.15 6.81 10.5044 10.7348 0.2304 4.61 1/5x (2) 0.15 6.82 9 10.747 0.2251 4.50 1/10x (1) 0.077 6.83 10.5221 10.7481 0226 4.52 1/10x (2) 0.077 6.83 10.4705 10.6979 0.2274 4.55 1/20x (1) 0.038 6.83 10.4743 10.6988 0.2245 4:49 1/20x (2) 0.038 6.83 10.7322 10.9646 0.2324 4.65 1l50x (1) 0.015 6.84 10.4777 10.6945 0.2168 4.34 1/50x (2) 0.015 6.85 10.4766 10.7018 0.2252 4.50. 1I100x (1) 0.0077 6.84 10.4543 10.6863 0.232 4.64 1I100x (2) 0.0077 6.85 10.5041 10.7452 0.2411 4.82 1/500x (1) 0.0015 6.86 2 10.7563 0.2411 4.82 1/500x 12) 0.0015 6.85 10.574 1 0.2341 4.68 3 5x (1) 4.0445 3.84 6.63 10.5021 10.7338 0.2317 4.63 5x (2) 4.0445 3.84 6.65 10.4735 10.7152 0.2417 4.83 1x (1) 4.3024 0.77 6.64 10.5011 10.7394 0.2383 4.77 1x (2) 4.3024 0.77 6.65 10.4004 10.6362 0.2358 4.72 1/2x (1) 4.1648 0.38 6.64 10.4599 10.71 0.2501 5.00‘ 1/2x (2) 4.1648 0.38 6.64 10.5630 10.804 0.241 4.82 1/5x (1) 4.1600 0.15 6.63 10.6521 10.889 0.2369 . 4.74 1/5x (2) 4.1600 0.15 6.63 10.5032 10.7341 0.2309 4.62 1/10x (1) 4.4997 0.077 6.65 10.4726 10.6998 0.2272 4.54 1/10x (2) 4.4997 0.077 6.64 10.4841 10.7279 0.2438 4.88 1/20x (1) 4.9716 0.038 6.64 10.3785 10.6112 0.2327 4.65 1/20x (2) 4.9716 0.038 6.64 2 10.6154 0.2322 4.64 1l50x (1) 4.3049 0.015 6.65 10.3782 10.612 0.2338 4.68 1l50x (2) 4.3049 0.015 6.65 10.5546 10.7877 0.2331 4.66 1/100x (1) 4.6035 0.0077 6.65 10.4858 10.7172 0.2314 4.63 1/100x (2) 4.6035 0.0077 6.66 10.4771 10.7122 0.2351 4.70 1/500x ' (1) 4.4494 0.0015 6.65 10.6083 10.8163 0.208 4.16 1/500x ' {22 4.4494 0.0015 6.65 10.5722 10.7896 0.2174 4.35 C 5x (1) 4.1784 3.84 6.77 10.4842 10.7237 0.2395 4.79 5x (2) 4.1784 3.84 6.80 10.5261 10.7553 0.2292 4.58 1x (1) 4.2842 0.77 6.80 10.3785 10.6194 0.2409 4.82 1x (2) 4.2842 0.77 6.77 3 10.8459 0.2376 4.75 1/2x (1) 4.8865 0.38 6.81 4 10.7183 0.1909 3.82 1/2x (2) 4.8865 0.38 6.81 10.4713 10.6853 0.214 4.28 1/5x (1) 4.5411 0.15 6.81 10.3831 10.5997 0.2166 4.33 1/5x (2) 4.5411 0.15 6.84 10.5444 10.7666 0.2222 4.44 1/10x (1) 5.2960 0.077 6.80 10.501 107.181 0.2171 4.34 1/10x (2) 5.2960 0.077 6.82 10.3783 10.5978 0.2195 4.39 1/20x (1) 5.2480 0.038 6.82 10.4521 10.6539 0.2018 4.04 1/20x (2) 5.2480 0.038 6.83 10.4018 10.5881 0.1863 3.73 1l50x (1) 5.6680 0.015 6.82 10.5049 10.7248 0.2199 4.40 1/50x (2) 5.6680 0.015 6.83 10.4197 10.6441 0.2244 4.49 1/100x (1) 4.9024 0.0077 6.83 10.5531 10.7847 0.2316 4.63 1/100X (2) 4.9024 0.0077 6.83 10.7098 10.9412 0.2314 4.63 . 1/500x (1) 4.6011 0.0015 6.83 10.5543 10.7942 0.2399 4.80 1/500X ' (2! 4.6011 0.0015 6.83 2 10.5746 0.2364 4.73 2012/047728 - 1 l7 Experiment 600nm g/L lnoc. mg/L Vi! Tare Out wt Set SAMPLE 0.0. B12 pH (9) (g) dry pellet wt (9) Biomass D 5x (1) 4.3297 3.84 6.85 ‘10.3415 10.5976 0.2561 5.12 5x (2) 4.3297 3.84 6.85 10.4549 10.698 0.2431 4.86 1x (1) 5.3731 0.77 6.85 10.3785 10.6143 0.2358 4.72 1x (2) 5.3731 0.77 6.85 10.6117 10.8401 0.2284 4.57 112x (1) 4.9040 0.38 6.85 10.4318 10.6573 0.2255 4.51 1/2x (2) 4.9040 0.38 6.85 2 10.8742 0,217 4.34 1/5x (1) 4.7064 0.15 6.85 10.552 10.7791 0.2271 4.54 1/5x (2) 4.7084 0.15 6.85 10.7118 10.9623 0.2505 5.01 1/10x (1) 4.9843 0.077 6.85 10.6174 10.8589 0.2415 4.83 1/10x (2) 4.9843 0.077 6.86 10.5229 10.7614 0.2385 4.77 1/20x (1) 4.7533 0.038 6.84 10.5045 10.7448 0.2401 4.80 1/20x (2) 4.7533 0.038 6.84 10.5049 10.7281 0.2232 4.46 1/50x (1) 4.3732 0.015 6.86 10.5161 10.7566 0.2405 4.81 1/50x (2) 4.3732 0.015 6.85 10.5234 107574 0234 4.68 1/100x (1) 4.0902 0.0077 6.86 10.4858 10.7193 0.2335 4.67 1/100x (2) 4.0902 0.0077 6.86 10.5523 10.7856 0.2333 4.67 1/500X (1) 4.2472 0.0015 0.2502 5.00 . 6.86 10.553 10.8032 11500x (22 4.2472 0.0015 6.86 10.4488 10.6976 0.2488 4.98 E 5x (1) 4.5635 3.84 6.79 10.3613 10.6103 0.249 4.98 5x (2) 4.5635 3.84 6.80 10.5311 10.7546 0.2235 4.47 1x (1) 4.2673 0.77 6.80 10.3311 1 0.234 4.68 1x (2) 4.2673 0.77 6.80 10.466 10.7162 0.2502 5.00 1/2x (1) 5.1989 0.38 6.79 6 10.9054 0.2258 4.52 1/2x (2) 5.1989 0.38 6.80 10.5552 10.7836 0.2284 4.57 1l5x (1) 5.0275 0.15 6.80 10.4877 10.721 0.2333 4.67 1/5x (2) 5.0275 0.15 6.80 10.5061 10.7331 0.227 4.54 1/10x (1) 5.6680 0.077 6.81 10.3585 10.5946 0.2361 4.72 1/10x (2) 5.6680 0.077 6.80 10.6807 10.9116 0.2309 4.62 1/20x (1) 5.2891 0.038 6.81 10.3664 10.6143 0.2479 4.96 1/20x (2) 5.2891 0.038 6.81 10.3745 10.6253 0.2508 5.02 1/50x (1) 5.0356 0.015 6.81 10.4285 10.682 0.2535 5.07 1/50x (2) 5.0356 0.015 6.80 10.4022 10.6524 0.2502 5.00 1/100X (1) 5.7271 0.0077 6.81 10.3332 7 0.2605 5.21 1/100x : (2) 5.7271 0.0077 6.81 10.525 10.7782 0.2532 5.06 1/500x ’ (1) 5.1000 0.0015 6.81 10.4192 5 0.2593 . . 5.19 1/500x [2! 5.1000 0.0015 6.81 10.3991 10.6668 0.2677 5.70 F 5x (1) 4.5635 3.84 6.75 10.524 10.7696 0.2456 5.23 5x (2) 4.5635 3.84 6.75 10.3291 10.5729 0.2438 4.88 1x (1) 4.2673 0.77 6.75 10.4927 10.7424 0.2497 4.99 1x (2) 4.2673 0.77 6.76 10.5257 10.7652 0.2395 4.79 1/2x (1) 5.1989 0.38 6.75 10.463 10.6908 0.2278 4.56 1/2x (2) 5.1989 0.38 6.76 10.3756 10.6003 0.2247 4.49 1/5x (1) 5.0275 0.15 6.76 10.4212 10.6704 0.2492 4.98 1/5x (2) 5.0275 0.15 6.76 10.5061 10.7509 0.2448 4.90 1/10x (1) 5.6680 0.077 6.76 10.3829 10.6082 0.2233 4.47 1/10x (2) 5.6680 0.077 6.76 1 8 0.2417 4.83 1/20x (1) 5.2891 0.038 6.75 10.5096 10.7442 0.2346 4.69 1/20x (2) 5.2891 0.038 6.77 5 10.6226 0.2351 4.70 1/50x (1) 5.0356 0.015 6.78 10.3584 10.6063 0.2479 4.96 1/50x (2) 5.0356 0.015 6.76 10.5235 10.7671 0.2436 4.87 1/100x (1) 5.7271 0.0077 6.77 10.3664 10.6148 0.2484 4.97 1/100X (2) 5.7271 0.0077 6.78 10.5055 10.7441 0.2386 4.77 Experiment 600nm g/L lnoc. mglL Vit Tare Out wt Set SAMPLE 0.0. 812 pH (9) (g) dry pellet wt (9) Biomass 1/500x - (1) 5.1000 0.0015 6.79 10.3322 10.5868 0.2546 5.09 1/500x (2) 5.1000 0.0015 6.80 10.3279 10.591 0.2631 5.26 G 5x (1) 3.7017 3.84 6.83 10.362 10.6077 0.2457 4.91 5x (2) 3.7017 3.84 6.82 5 10.6145 0.255 5.10 1x (1) 4.2905 0.77 6.84 10.3648 10.6151 0.2503 5.01 1x (2) 4.2905 0.77 6.85 10.3356 10.5972 0.2616 5.23 1/2x (1) 4.4548 0.38 6.84 10.5096 3 0.2587 5.17 1/2x (2) 4.4508 0.38 6.84 10.435 10.6889 0.2539 5.08 1/5x (1) 4.6844 0.15 6.84 10.3305 10.5868 0.2563 5.13 1/5x (2) 4.6844 0.15 6.85 10.4964 10.7476 0.2512 5.02 1l10x (1) 5.8389 0.077 6.85 10.3637 10.6173 0.2536 5.07 1/10x (2) 5.8389 0.077 6.85 10.3351 10.5881 0.253 5.06 1/20x (1) 4.4483 0.038 6.85 10.4393 10.6958 0.2565 5.13 1120x (2) 4.4483 0.038 6.86 10.3798 10.6416 0.2618 5.24 1/50x (1) 3.9821 0.015 6.86 10.3619 10.619 0.2571 5.14 1/50x (2) 3.9821 0.015 6.87 9 10.7721 0.2542 5.08 1l100x (1) 5.0043 0.0077 6.87 10.4756 10.7288 0.2532 5.06 1/100x (2) 5.0043 0.0077 6.87 10.4993 10.7561 0.2568 5.14 1/500x (1) 5.0108 0.0015 6.88 10.434 10.6941 0.2601 5.20 1/500x ‘ ' (2) 5.0108 0.0015 6.87 10.3896 10.6446 0.255 5.10 1/500x H (1) 4.6921 0.0015 6.85 9 10.5954 0.2355 4.71 1/500x ' (2) 4.6921 0.0015 6.85 10.3720 10.6254 0.2534 5.07 0x (1) 4.8144 0.0000 6.85 10.3624 10.6052 0.2428 4.86 0x 32! 4.8144 0.0000 6.86 10.3585 3 0.2508 5.02 1/500x I (1) 4.7906 0.0015 6.89 2 10.7920 0.2428 4.86 1/500x (2) 4.7906 0.0015 6.90 10.5958 10.8508 0.255 5.10 Ox (1) 4.7063 0.0000 6.89 10.5505 10.8037 0.2532 5.06 0x (22 4.7063 0.0000 6.89 10.5120 10.7693 0.2573 5.15 . 1l500x J (1) 5.8805 0.0015 6.99 10.5433 10.8089 0.2656 5.31 1/500x (2) 5.8805 0.0015 7.00 10.5974 10.8714 0.274 5.48 0x (1) 4.6385 0.0000 7.00 10.5509 10.8145 0.2636 5.27 0x 322 4.6385 0.0000 6.98 10.5462 10.8252 0.279 5.58 1/500x K (1) 4.7133 0.0015 6.87 10.5424 5 0.2661 5.32 1l500x (2) 4.7133 0.0015 6.88 10.3444 10.6111 0.2667 5.33 0x (1) 4.2141 0.0000 6.86 10.3873 10.6587 0.2714 5.43 0x 322 4.2141 0.0000 6.86 10.4188 10.6866 0.2678 5.36 Experiment % % % (n-6) (n-3) % % Set SAMPLE 16:0 ARA EPA DPA DPA DHA Fat A 5x (1) 27.53 1.67 16.01 2.22 2.33 41.72 67.22 5x (2) 27.65 1.71 16.02 2.16 2.41 41.17 66.07 1x (1) 27.73 1.72 16.00 2.15 2.44 41.06 66.25 1x (2) 27.72 1.75 16.32 2.10 2.48 40.46 65.42 1/2x (1) 27.56 1.74 16.36 2.09 2.49 40.54 65.45 1/2x (2) 27.73 1.77 16.53 2.14 2.32 40.93 66.57 1/5x (1) 27.60 1.73 16.33 2.13 2.38 41.03 66.59 1/5x (2) 27.88 1.74 15.94 2.17 2.43 41.04 66.28 1/10x (1) 27.87 1.75 16.28 2.09 2.56 40.66 65.14 1l10x (2) 27.81 1.74 16.07 2.13 2.48 40.96 65.39 WO 13208 - 1 19 - Experiment % "/0 % (n-6) (n~3) % % Set SAMPLE 16:0 ARA EPA DPA DPA DHA F31 1/20x (1) 27.70 1.75 16.36 2.08 2.52 40.53 65.64 1/20x (2) 27.63 1.75 16.42 2.13 2.36 40.93 66.30 1/50x (1) 27.83 1.76 16.61 2.07 2.43 40.60 66.02 1/50x (2) 27.82 1.69 16.35 2.11 2.49 40.84 64.97 1/100x (1) 27.50 1.72 16.13 2.16 2.50 41.19 66.21 1/100x (2) 27.60 1.69 15.64 2.24 2.34 41.72 66.56 1/500x (1) 27.58 1.70 15.63 2.24 2.28 41.74 66.99 1/500x - 12! 27.57 1.75 16.86 2.06 2.41 40.59 64.91 B 5x (1) 28.30 1.75 16.44 2.32 2.35 42.35 64.09 5X (2) 28.45 1.74 15.96 2.36 2.36 42.62 62.46 1x (1) 28.55 1.74 16.10 2.32 2.43 42.35 62.96 1x (2) 28.47 1.78 16.33 2.33 2.44 42.26 63.13 1/2x (1) 28.21 1.71 15.90 2.39 2.34 42.95 64.75 1/2)( (2) 28.15 1.75 16.65 2.29 2.33 42.32 63.50 1/5x (1) 28.51 1.74 16.34 2.29 2.37 42.23 62.02 1l5x (2) 28.43 1.75 16.35 2.29 2.48 42.24 63.88 1/10x (1) 28.66 1.77 15.85 2.37 2.45 42.45 62.47 1/10)( (2) 28.52 1.74 15.74 2.40 2.39 42.70 63.14 1/20x (1) 28.67 1.75 15.86 2.33 2.37 42.55 63.10 1/20X (2) 28.36 1.75 16.13 2.35 2.26 42.62 63.90 1/50x (1) 28.36 1.79 16.64 2.29 2.42 42.04 62.31 1/50x (2) 28.43 1.77 15.92 2.40 2.42 42.57 62.77 1/100X (1) 28.81 1.75 16.01 2.30 2.49 42.16 61.70 1/100x (2) 28.61 1.73 15.93 2.35 2.40 42.51 63.60 1/500X (1) 28.62 1.85 17.15 2.17 2.59 41.19 60.91 1/500)( 322 28.30 1.84 17.39 2.20 2.43 41.40 61.56 C ' 5x (1) 27.97 1.95 16.08. 2.60 2.43 42.37 67.30 5x (2) 28.24 1.96 15.99 2.55 2.54 42.02 66.72 1x (1) 28.20 1.94 - 15.96 2.60 2.61 42.06 66.98 1x (2) 28.10 1.98 16.39 2.54 2.64 41.73 66.30 1/2x (1) 27.77 2.14 17.67 2.40 2.85 40.46 64.53 1/2x (2) 27.84 2.10 17.27 2.50 2.62 40.85 65.79 1/5x (1) 27.57 2.06 17.37 2.46 2.64 41.17 66.69 1/5x (2) 27.71 2.05 17.05 2.50 2.70 41.31 65.86 1/10x (1) 28.02 2.06 16.89 2.48 2.70 41.27 65.53 1/10)( (2) 27.66 2.05 16.76 2.55 2.64 41.66 65.85 1/20x (1) 27.64 2.13 17.27 2.52 2.74 41.03 65.00 1/20x (2) 27.56 2.19 18.62 2.27 2.88 39.82 64.23 1/50x (1) 27.78 2.02 16.98 2.51 2.52 41.61 66.22 1/50x (2) 27.82 2.01 16.92 2.53 2.65 41.47 65.85 1/100x .; (1) 27.85 2.01 16.77 2.54 2.67 41.50 65.23 (2) 27.91 2.01 16.61 2.58 2.59 41.72 66.87 1/500x (1) 27.73 2.02 17.42 2.41 2.58 41.10 65.60 1/500x (2) 27.48 2.05 17.93 2.39 2.54 40.85 65.85 D 5x (1) 27.80 1.85 15.97 2.59 2.32 42.99 67.08 5x (2) 27.89 1.86 16.27 2.52 2.42 42.60 66.60 1x (1) 27.60 1.92 16.59 2.54 2.53 42.42 66.32 1x (2) 27.75 1.89 16.40 2.53 2.48 42.60 66.35 1/2x (1) 27.35 1.98 17.19 2.52 2.53 41.97 65.19 1l2x (2) 27.26 2.01 17.65 2.45 2.45 41.58 66.67 1/5x (1) 27.42 1.97 17.53 2.42 2.55 41.72 66.69 1/5x (2) 27.46 1.91 16.60 2.60 2.45 42.44 66.58 2012/047728 Experiment % % % (n-6) (n-3) % % Set SAMPLE 16:0 ARA EPA DPA DPA DHA Fat mm: (1) 27.45 1.97 17.03 2.50 2.61 42.04 66.06 1/10x (2) 27.45 1.90 16.66 2.52 2.46 42.46 66.91 1/20x (1) 27.45 1.96 16.95 2.58 2.41 42.11 66.10 1/20x (2) 27.30 2.00 17.94 2.42 2.45 41.50 65.30 1/50x (1) 27.58 1.87 16.65 2.55 2.33 42.61 66.84 1l50x (2) 27.69 1.89 16.85 2.50 2.44 42.27 65.40 1/100x (1) 27.44 1.92 16.88 2.51 2.51 42.38 65.88 1/100x (2) 27.47 1.92 16.67 2.58 2.43 42.56 65.83 1/500x (1) 26.91 1.98 18.05 2.40 2.47 41.66 66.18 1/500x 2' 27.03 1.99 18.08 2.40 2.36 41.69 65.80 E 5x (1) 27.46 1.89 16.02 2.60 2.30 43.33 66.12 5x (2) 27.86 1.93 16.05 2.50 2.38 43.02 63.64 1x (1) 27.75 1.95 15.91 2.58 2.39 43.09 65.41 1x (2) 27.51 1.94 15.59 2.72 2.29 43.52 67.35 1/2x (1) 27.07 2.04 16.99 2.52 2.42 42.64 65.01 1/2x (2) 26.86 2.05 17.23 2.48 2.35 42.59 65.00 1/5x (1) 27.05 1.96 16.65 2.55 2.29 43.21 65.66 1/5x (2) 27.12 1.98 16.69 2.52 2.41 43.08 63.58 1/10x (1) 27.35 1.95 16.45 2.55 2.48 42.94 64.34 1/10x (2) 27.31 1.92 16.22 2.60 2.41 43.24 64.49 1/20x (1) 26.72 1.93 16.73 2.64 2.31 43.21 65.83 1/20x (2) 26.47 1.94 17.11 2.65 2.21 43.14 66.13 1/50x (1) 26.63 1.86 16.30 2.72 2.19 43.84 65.30 1l50x (2) 26.68 1.86 16.33 2.69 2.26 43.76 65.67 1/100x (1) 26.47 1.87 16.39 2.69 2.28 43.89 63.98 1/100x (2) 26.45 1.89 16.78 2.62 2.38 43.49 65.21 1/500x (1) 26.06 1.88 17.13 2.60 2.23 43.73 66.13 1/500x 32} 25.95 1.88 17.58 2.57 2.10 43.40 65.46 F 5x (1) 25.71 1.96 17.50 2.46 2.34 43.56 65.46 5x (2) 25.91 1.93 17.26 2.47 2.38 43.73 65.71 1x (1) 25.93 1.90 16.70 2.54 2.43 43.93 66.05 1x (2) 26.03 1.94 17.17 2.48 2.39 43.41 65.34 1/2x (1) 25.76 2.03 18.11 2.43 2.38 42.76 65.27 1/2x (2) 25.74 2.05 18.52 2.37 2.31 42.52 65.75 1/5x (1) 25.44 1.95 17.62 2.49 2.27 43.73 65.80 1/5x (2) 25.41 1.94 17.35 2.52 2.32 43.99 65.88 1/10x (1) 26.13 2.04 18.06 2.36 2.55 42.43 64.10 1/10x (2) 25.62 1.98 17.53 2.48 2:44 43.52 65.51 1/20x (1) 25.77 2.01 17.97 2.45 2.34 42.94 65.21 1/20x (2) 25.55 2.03 18.45 2.42 2.24 42.78 66.05 1/50x (1) 25.26 1.96 17.54 2.55 2.18 44.07 66.41 1l50x (2) 25.48 1.95 17.58 2.50 2.30 43.72 65.93 1/100x (1) 25.50 1.93 17.00 2.62 2.23 44.36 65.37 1/100x (2) 25.59 1.96 17.37 2.56 2.26 43.80 65.18 1/500x (1) 25.54 1.99 18.11 2.45 2.20 43.08 65.05 1/500x (2) 25.55 2.00 18.59 2.36 2.18 42.66 64.57 G 5x (1) 25.74 1.95 17.58 2.37 2.08 43.04 63.68 5x (2) 25.79 1.89 16.83 2.51 2.04 43.69 63.67 1x (1) 26.01 1.90 16.69 2.52 2.08 43.42 64.95 1x (2) 25.71 1.91 16.74 2.57 1.98 43.85 65.37 1/2x (1) 25.47 1.93 17.14 2.52 1.97 43.69 64.28 2012/047728 Experiment % % % (n-6) (n-3) % % Set SAMPLE 16:0 ARA EPA DPA DPA DHA Fat 1/2x (2) 25.68 1.95 17.80 2.42 1.94 42.76 64.15 1I5x (1) 25.63 1.94 17.43 2.46 2.02 43.21 63.70 1I5x (2) 25.86 1.91 17.03 2.47 2.04 43.16 64.14 1/10x (1) 26.34 1.95 17.09 2.48 2.14 42.35 63.27 1/10x (2) 26.24 1.93 16.79 2.52 2.04 42.93 64.71 1l20x (1) 25.47 1.97 18.06 2.37 2.12 42.62 63.38 1/20x (2) 25.38 1.98 18.16 2.40 1.95 42.73 62.81 1/50x (1) 25.46 1.94 18.06 2.37 2.02 42.72 63.38 1/50)( (2) 25.50 1.94 17.46 2.46 2.04 43.15 63.66 1/100x (1) 25.52 1.92 16.69 2.60 1.81 44.16 65.74 1/100x (2) 25.96 1.92 17.11 2.48 1.96 43.00 63.89 1/500x (1) 25.71 1.95 17.78 2.41 1.97 42.83 64.61 1/500x 12} 25.56 2.02 18.25 2.37 1.90 42.63 64.80 1/500x .

H (1) 25.19 2.03 19.36 2.14 2.02 41.86 65.21 1/500x (2) 25.41 1.94 17.85 2.37 1.83 43.31 . 65.75 0x (1) 21.45 2.62 27.54 1.31 2.50 35.78 67.83 0x122 21.79 2.63 27.33 1.30 2.51 35.66 67.07 1/500x I (1) 25.21 2.02 19.89 2.15 2.10 41.48 64.17 1/500x (2) 25.00 2.02 20.00 2.11 2.09 41.63 64.04 Ox (1) 21.82 2.62 28.55 1.20 2.61 34.16 67.22 0x 122 21.66 2.64 28.83 1.18 2.55 34.20 . 66.93 1I500x _ J (1) 24.44 1.81 16.75 2.49 2.22 45.20 63.43 1I500x (2) 24.34 1.82 17.73 2.39 1.95 44.71 63.88 0x (1) 21.56 2.58 26.96 1.30 2.60 36.04 66.74 0x (2} 21.53 2.61 27.30 1.31 2.43 - 35:96 65.71 1I500x K (1) 23.87 1.82 17.33 2.46 2.00 45.45 64.67 1I500x (2) 24.07 1.82 17.67 2.42 1.93 45.06 64.30 0x (1) 21.77 2.63 26.13 1.39 2.81 36.49 66.14 0x122 21.91 2.63 27.19 1.29 2.63 35.60 66.81 Experiment (g/L) (g/L) (g/L) 9 B12! EPA DHA mglg Set SAMPLE Yield mg/g EPA Yield DHA LFDW g LFDW gIL Fat A 5x (1) 0.53 107.64 1.39 291.68 1.62 0.0024 3.32 5x (2) 0.50 105:83 1.28 282.95 1.60 0.0024 3.11 1x (1) 0.49 105.99 1.26 282.93 1.56 0.00049 3.06 1x (2) 0.47 106.77 1.17 275.34 1.52 0.00051 2.88 1/2x (1) 0.47 107.06 1.17 275.98 1.52 0.00025 2.88 112x (2) 0.51 110.04 1.26 283.39 1.55 0.00025 3.09 1I5x (1) 0.50 108.74 0.00010 3.07 . 1.26 284.18 1.54 1/5x (2) 0.48 105.63 1.22 282.95 1.52 0.00010 2.98 1/10x (1) 0.48 106.01 1.20 275.47 1.58 0.000049 2.94 1/10x (2) 0.48 105.07 1.22 278.58 1.57 0.000049 2.97 1/20x (1) 0.48 107.36 1.19 276.71 1.54 0.000025 2.95 1/20x (2) 0.51 108.87 1.26 282.24 1.57 0000024 3.08 1/50x (1) 0.48 109.67 1.16 278.77 1.47 0000010 2.86 1/50x (2) 0.48 106.19 1.19 275.96 1.58 0.000010 2.93 1/100x (1) 0.50 106.81 1.27 283.69 1.57 0.0000049 3.07 1/100x (2) 0.50 104.11 1.34 288.86 1.61 0.0000048 3.21 1I500x (1) 0.50 104.68 1.35 290.83 1.59 000000094 3.23 - 122 ment (glL) (glL) (glL) g 812/ EPA DHA m9/9 Set SAMPLE Yield mglg EPA Yleld DHA LFDW g LFDW g/L Fat 1/500x (2) 0.51 109.46 1.23 274.01 1.64 0.00000091 3.04 B 5x (1 ) 0.49 105.34 1.26 279.16 1.66 0.0023 2.97 5x (2) 0.48 99.71 1.29 273.81 1.81 0.0021 3.02 1x (1) 0.48 101.34 1.27 274.22 1.77 0.00044 3.00 1x (2) 0.49 103.10 1.26 274.39 1.74 0.00044 2.98 1/2x (1) 0.52 102.99 1.39 286.04 1.76 0.00022 3.24 1/2x (2) 0.51 105.75 1.30 276.38 1.76 0.00022 3.06 1/5x (1) 0.48 101.36 1.24 269.37 1.80 0.000083 2.94 1/5x (2) 0.48 104.41 1.25 277.49 1.67 0.000090 2.95 1/10x (1 ) 0.45 98.98 1.20 272.75 1.71 0.000045 2.84 1/10x (2) 0.48 99.38 1.31 277.29 1.80 0.000043 3.08 1/20x (1 ) 0.47 100.10 1.25 276.16 1.72 0.000022 2.94 1/20x (2) 0.48 103.06 1.26 280.15 1.68 0000023 2.97 1/50x (1) 0.48 103.67 1.22 269.42 1.76 0.0000085 2.91 1/50x (2) 0.47 99.90 1.25 274.84 1.74 0.0000086 2.93 1/100x (1) 0.46 98.77 1.20 267.53 1.77 00000043 2.86 1/100x (2) 0.48 101.29 1.27 278.11 1.71 045 2.99 1/500x 0.43 104.49 1.04 258.03 1.63 0.00000092 2.53 . (1) 1/500x $22 0:47 107.03 1.11 262.14 1.67 0.00000090 2.68 0 5x (1) 0.52 108.25 1.37 292.82 1.57 0.0025 3.22 5x (2) 0.49 106.70 1.29 287.91 1.53 0.0025 3.06 1x (1) 0.51 106.88 1.36 289.31 1.59 0.00048 3.23 1x (2) 0.52 108.87 1.31 284.09 1.60 0.00048 3.15 1/2x(1) 0.44 114.02 1.00 288.13 1:35 8 2.46 1/2x (2) 0.49 113.61 1.15 275.95 1.46 0.00026 2.82 1/5x(1) 0.50 115.85 1.19 281.94 1.44 0.00010 2.89 115x (2) 0.50 112.29 1.21 279.43 1.52 0.00010 2.93 1/10x (1) 0.48 110.70 1.17 277.71 1.50 0.000051 2.85 1/10x (2) 0.48 110.39 1.20 281.73 1.50 0.000051 2.89 1/20x (1) 0.45 112.23 1.08 273.88 1.41 0.000027 2.62 ' 2) 0.45 119.57 0.95 262.65 1.33 0.000029 2.39 1/50x (1) 0.49 112.42 1.21 282.97 1.49 0.000010 2.91 1/50x (2) 0.50 111.40 1.23 280.41 1.53 0.000010 2.96 1/100x (1) 0.51 109.40 1.25 278.02 1.61 00000048 3.02 1/100x (2) 0.51 111.08 1.29 286.49 1.53 0.0000050 3.09 1/500x (1) 0.55 114.30 1.29 276.88 1.65 0091 3.15 1/500x 122 0.56 118.05 1.27 1.61 0.00000093 3.11 . 276.25 D 5x (1) 0.55 107.16 1.48 296.16 1.69 0.0023 3.44 5x (2) 0.53 108.39 1.38 291.37 1.62 0.0024 3.24 1x (1) 0.52 110.03 1.33 288.89 1.59 0.00048 3.13 1x (2) 0.50 108.83 1.29 290.26 1.54 0.00050 3.03 1/2x(1) 0.51 112.05 1.23 281.00 1.57 0.00024 2.94 1/2x (2) 0.51 117.67 1.20 284.69 1.45 0.00026 2.89 1le (1) 0.53 116.92 1.26 285.74 1.51 0.00010 3.03 1/5x (2) 0.55 110.50 1.42 290.20 1.67 0.000090 3.34 1/10x (1) 0.54 112.52 1.34 285.20 1.64 0.000047 3.19 111071 (2) 0.53 111.48 1.36 291.77 1.58 0.000049 3.19 1/20x (1) 0.54 112.02 1.34 285.86 1.63 0.000023 3.17 1/20x (2) 0.52 117.18 1.21 278.32 1.55 0.000025 2.91 1/50x (1) 0.54 111.32 1.37 292.53 1.59 0.0000094 3.22 1/50x (2) 0.52 110.19 1.29 283.91 1.62 0.0000093 3.06 1/100x (1) 0.52 111.21 1.30 286.73 1.59 0.0000048 3.08 1/100x 0.51 109.75 1.31 287.74 1.59 0.0000048 3.07 Hz"? WO 13208 —123- Experiment (g/L) (Q/L) (glL) g 812/ EPA DHA "19/9 Set SAMPLE Yield mglg EPA Yield DHA LFDW g LFDW g/L Fat 1/500x (1) 0.60 119.41 1.38 283.11 1.69. 0.00000089 3.31 1/500x 122 0.59 118.98 1.37 281.74 1.70 0088 3.27 E 5x(1) 0.53 105.94 1.43 294.23 1 .69 0.0023 3.29 5x(2) 0.46 102.13 1.22 281.16 1.63 0.0024 2.84 1x (1) 0.49 104.07 1.32 289.48 1.62 0.00048 3.06 1x (2) 0.53 104.97 1.47 301.01 1.63 0.00047 3.37 1/2x (1) 0.50 110.46 1.25 284.65 1.58 0.00024 2.94 1/2x(2) 0.51 112.01 1.26 284.34 1.60 0.00024 2.97 1/5x(1) 0.51 109.31 1.32 291.38 1.60 0.000094 3.06 1/5x(2) 0.48 105.13 1.24 281.31 1.65 0.000091 2.89 1/10x (1) 0.50 105.82 1.30 283.74 1.68 46 3.04 1/10x(2) 0.48 104.57 1.29 286.39 1.64 0.000047 2.98 1/20x (1) 0.55 110.12 1.41 292.12 1 .69 0.000022 3.26 1/20x (2)' 0.57 113.13 1.43 293.02 1.70 0.000022 3.32 1/50x (1) 0.54 106.44 1.45 294.03 1.76 00000085 3.31 1/50X (2) 0.54 107.21 1.44 295.14 1.72 0.0000087 3.29 1/100x (1) 0.55 104.88 1.46 288.40 1.88 0.0000041 3.33 1/100x (2) 0.55 109.40 1.44 291.26 1.76 0.0000044 3.30 1/500x (1) 0.59 113.27 1.50 296.97 1.76 0.00000085 3.43 1/500x (2) 0.66 115.09 1.62 291.79 1.97 0.00000076 3.73 F 5x (1) 0.60 114.57 1.49 292.02 1 .80 0.00212779 3.42 5x (2) 0.55 113.41 1.40 294.25 1.57 000229687 3.20 1x (1) 0.55 110.28 1.45 297.11 1.70 000045415 3.30 1x (2) 0.54 112.21 1.36 290.41 1.66 0.00046376 3.13 1/2x (1) 0.54 118.20 1.27 285.84 1.58 0.00024017 2.97 1/2x (2) 0.55 121.79 1.26 286.25 1.54 000024686 2.95 1/5x (1 ) 0.58 115.91 1.43 294.66 - 1.70 000008801 3.28 1/5x (2) 0.56 114.29 1.42 296.75 1.67 000006979 3.23 1/10x (1) 0.52 115.79 1.21 278.51 1.60 000004803 2.86 1/10x (2) 0.56 114.86 1.38 291.97 1.67 0.00004619 3.17 1/20x (1) 0.55 117.21 1.31 286.75 1.63 0.00002328 3.06 1/20x (2) 0.57 121.84 1.33 289.39 1.60 000002381 3.11 1/50x (1) 0.58 116.45 1.45 299.69 1.67 0.00000901 3.29 1/50x (2) 0.56 115.90 1.40 295.17 1.66 0.00000904 3.21 1/100x (1) 0.55 111.13 1.44 296.95 1.72 0.00000448 3.25 1/100x (2) 0.54 113.25 1.36 292.35 1.66 000000463 3.1 1 1/500x (1) 0.60 117.81 1.43 286.97 1.78 000000084 3.31 1/500x 122 0.63 120.03 1.45 282.05 1.86 0080 3.40 G 5x(1) 0.55 111.96 1.35 279.27 1.78 0.00215168 3.13 5x (2) 0.55 107.14 1.42 283.43 1.85 0.00207251 3.25 1x (1) 0.54 108.42 1.41 287.31 1.75 000043883 3.25 1x (2) 0.57 109.44 1.50 292.04 1.81 000042495 3.42 1/2x (1) 0.57 110.17 1.45 286.10 1.85 0558 3.33 1/2x (2) 0.58 114.19 1.39 279.49 1.82 0.00020874 3.26 1/5x (1) 0.57 111.03 1.41 280.46 1.86 000008062 3.27 115x (2) 0.55 109.25 1.39 282.05 1.80 0.00008326 3.22 1/10x (1) 0.55 108.14 1.36 273.02 1.86 0.00004134 3.21 1/10x (2) 0.55 108.66 1.41 283.05 1.79 000004313 3.27 1/20x (1) 0.59 114.48 1.39 275.22 1.88 0.00002023 3.25 1/20x (2) 0.60 114.04 1.41 273.40 1.95 0.00001951 3.29 1/50x (1) 0.59 114.46 1.39 275.82 1.88 0.00000797 3.26 Experiment (glL) (glL) (glL) 9 B12! EPA DHA mglg Set SAMPLE Yleld mglg EPA Yield DHA LFDW g LFDW glL Fat 1I50x (2) 0.57 111.16 1.40 279.89 1.85 0.00000812 3.24 1/100x (1) 0.56 109.75 1.47 295.79 1.73 0.00000444 3.33 1/100X (2) 0.56 109.31 1.41 279.92 1.85 000000415 3.28 1/500x (1) 0.60 114.86 1.44 281.92 1.84 0.00000081 3.36 ' 1/500x (22 0.60 118.27 1.41 281.47 1.79 0.00000084 3.31 1I500x H (1) 0.59 126.25 1.29 279.17 1.64 000000092 3.07 1/500x (2) 0.59 117.35 1.44 291.28 1.74 0086 3.33 0x (1) 0.91 186.81 1.18 248.20 1.56 0.00000000 3.29 0x (2) 0.92 183.32 1.20 244.63 1.65 0.00000000 3.36 1/500x l (1) 0.62 127.64 1.29 272.24 1.74 0.00000086 3.12 1/500x (2) 0.65 128.06 1.36 272.66 1.83 0.00000082 3.27 0x (1) 0.97 191.93 1.16 234.87 1.66 0.00000000 3.40 0x (22 0.99 192.97 1.18 234.10 1.70 0.00000000 3.44 1/500x J (1) 0.56 106.22 1.52 292.66 1.94 0.00000077 3.37 1/500x (2) 0.62 113.25 1.57 291.57 ., 1.98 0.00000076 3.50 0x (1) 0.95 179.92 1.27 245.50 1.75 0.00000000 3.52 0x g2} 1.00 179.38 1.32 241.17 1.91 0.00000000 3.67 1/500x K (1) 0.60 112.09 1.56 298.08 1.88 0.00000080 3.44 1I500x (2) 0.61 113.60 1.55 293.84 1.90 000000079 3.43 0x (1) 0.94 172.82 1.31 244.79 1.84 0.00000000 3.59 OX (2! 0.97 181.66 1.27 241.23 1.78 0.00000000 3.58 sion: PTA-10208 growth and EPA productivity does not change at Vitamin BIZ concentrations ranging between 5x (3.84 mg/L) and 1/500x (0.0015 mgL) in DSDFM-O at 10% C02. r, when Vitamin BIZ is completely removed from DSDFMi—O, % EPA increases by about 50%, while dry weight and % fat also increase slightly, resulting in a 60% inerease in EPA productivity.

All of the various aspects, ments, and options described herein can be combined in any and all variations.

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

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of making a biomass of a microorganism having fatty acids and a concentration of EPA, comprising: (a) fermenting the microorganism in a fermentor vessel to produce a biomass wherein the microorganism ses a Thraustochytrid that produces a biomass having at least 3% EPA of the total weight of the fatty acids; and (b) adjusting the amount of the biomass in the vessel to attain a desired EPA level in the biomass.

2. The method of Claim 1, further comprising adjusting the pressure on the s to attain a desired EPA level in the biomass.

3. The method of Claim 1, r comprising adjusting the temperature in the broth to attain a desired EPA level in the biomass.

4. A biomass made ing to the method of Claim 1.

5. An oil extracted from a biomass as claimed in Claim 4 wherein the biomass is ed from an isolated microorganism deposited under ATCC Accession No. PTA-10208 wherein the oil comprises fatty acids wherein the fatty acids further comprise omega-3 polyunsaturated fatty acids wherein the 3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about >90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids and the amount of EPA, by weight, is from about 6% up to about 65% of the total amount of EPA and DHA.

6. The oil as claimed in Claim 5 wherein the amount of EPA, by weight, is from about 6% up to about 28% of the total amount of EPA and DHA.

7. The oil as d in Claim 5 wherein the amount of EPA, by weight, is from about 36% up to about 65 of the total amount of the EPA and DHA.

8. The oil as claimed in Claim 5 where the amount of EPA, by weight, is from about 28% to about 36% of the total amount of EPA and DHA.

9. An oil extracted from a biomass as claimed in Claim 4 wherein the biomass is produced from an ed rganism ted 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, by weight, is from about 15 up to about 70 % of the total weight of EPA and DHA.

10. An oil extracted from a s as d in Claim 4 wherein the biomass is produced from an isolated microorganism deposited under ATCC Accession No. PTA-9695 wherein the oil comprises fatty acids wherein the fatty acids further comprises omega-3 polyunsaturated fatty acids wherein the 3 polyunsaturated fatty acids comprise DHA and EPA in an amount of about 50 to about 70% by weight, of the total amount of omega-3 polyunsaturated fatty acids and the amount of EPA, by weight, is from about 5% up to about 60% of the total amount of the total weight of the EPA and DHA.

11. The oil as claimed in Claim 10 wherein the DHA and EPA is in an amount of about 60% of the total amount of the omega-3 fatty acids.

12. A method of producing an oil, the method comprising making a biomass according to claim 1, and obtaining the oil from the biomass.

13. The method of claim 12, wherein the oil is ted from the biomass.

14. An oil extracted from a biomass as claimed in Claim 4.

15. The oil as claimed in Claim 14 or in any one of Claims 5-11, wherein said oil is a crude oil extracted from the biomass without further processing.

16. The oil as claimed in Claim 14 or in any one of Claims 5-11, wherein said oil is a refined oil.

17. A feed product that contains a biomass of Claim 4 or an oil of any one of Claims 14-16.

18. The feed product of Claim 17, wherein said feed product is a feed or feed supplement.

19. The feed of Claim 18, n said feed is an animal feed.

20. The feed of Claim 18, wherein said feed is an aquaculture feed.

21. The method of claim 1, substantially as hereinbefore described, with reference to the Examples and/or