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

Abstract

The invention is directed to microbial oils containing omega-3 polyunsaturated fatty acids comprising docosahexaenoic acid, eicosapentaenoic acid, and optionally docosapentaenoic acid and dosage forms containing such oils.

Claims

1. A microbial oil comprising omega-3 polyunsaturated fatty acids comprising docosahexaenoic acid and eicosapentaenoic acid in an amount of >90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids, wherein the amount of eicosapentaenoic acid, by weight, is from 19% to 55% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and the amount of docosahexaenoic acid, by weight, is from 35% to 71% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and wherein the oil comprises from 120 mg to 220 mg eicosapentaenoic acid per one gram of oil and from 240 mg to 450 mg docosahexaenoic acid per one gram of oil, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

2. A microbial oil comprising omega-3 polyunsaturated fatty acids comprising docosahexaenoic acid and eicosapentaenoic acid in an amount of 90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids, wherein the amount of eicosapentaenoic acid, by weight, is from 19% to 55% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and the amount of docosahexaenoic acid, by weight, is from 35% to 71% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and wherein the oil comprises from 130 mg to 195 mg eicosapentaenoic acid per one gram of oil and from 320 mg to 480 mg docosahexaenoic acid per one gram of oil, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

3. A microbial oil comprising omega-3 polyunsaturated fatty acids comprising docosahexaenoic acid and eicosapentaenoic acid in an amount of 90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids, wherein the amount of eicosapentaenoic acid, by weight, is from 10% to 25% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and the amount of docosahexaenoic acid, by weight, is from 75% to 90% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and where the omega-3 polyunsaturated fatty acids further comprise docosapentaenoic acid in an amount of from 0% to 2%, by weight, of the total amount of omega-3 polyunsaturated fatty acids, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

4. The oil of claim 3, wherein the total amount of omega-3 polyunsaturated fatty acids in the microbial oil is at least about 400 mg per gram of oil.

5. A microbial oil comprising omega-3 polyunsaturated fatty acids comprising docosahexaenoic acid and eicosapentaenoic acid in an amount of about >90%, by weight, of the total amount of omega-3 polyunsaturated fatty acids, wherein the amount of eicosapentaenoic acid, by weight, is from about 19% to about 55% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, and the amount of docosahexaenoic acid, by weight, is from about 35% to about 71% of the total amount of docosahexaenoic acid and eicosapentaenoic acid, wherein the total amount of omega-3 polyunsaturated fatty acids is at least about 400 mg per one gram of oil, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

6. The oil of any one of claims 1-5, wherein said oil is produced by Schizochytrium sp.

7. The oil of any one of claims 1-5, wherein said oil comprises less that about 5% by weight of each of arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid and stearidonic acid.

8. A microbial oil comprising a triacylglycerol fraction of at least 10% by weight, wherein at least about 12% by weight of the fatty acids in the triacylglycerol fraction is eicosapentaenoic acid, wherein at least 25% by weight of the fatty acids in the triacylglycerol fraction is docosahexaenoic acid, wherein said oil has a peroxide value of 5 or less, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

9. The oil of any one of claims 1-5, wherein said oil comprises a ratio of EPA:DHA selected from 1:1 to 1:1.5, 1:1 to 1:2, 1:1.5 to 1:2, 1:1 to 1:2.5, 1:2 to 1:2.5, and 1:4 to 1:7 by weight of total omega-3 polyunsaturated fatty acids.

10. A microbial oil comprising 120 mg to 220 mg eicosapentaenoic acid per one gram of oil and from 240 mg to 450 mg docosahexaenoic acid per one gram of oil, and wherein said microbial oil further comprising an effective amount of at least one added antioxidant.

11. The oil of claim 9, wherein said oil is produced by Schizochytrium sp.

Description

EXAMPLE 1

(1) Isolation of Microorganisms

(2) Samples were collected from intertidal habitats during low tide, including bays and estuaries along the West Coast of North America (California, Oregon, and Washington) and Hawaii. Water, sediment, living plant material, and decaying plant/animal debris were placed into sterile 50 ml tubes. Portions of each sample along with the water were spread onto solid agar plates of isolation media. Isolation media consisted of: 500 ml of artificial seawater, 500 ml of distilled water, 1 g of glucose, 1 g of glycerol, 13 g of agar, 1 g of glutamate, 0.5 g of yeast extract, 0.5 g casein hydrolysate, 1 ml of a vitamin solution (100 mg/L thiamine, 0.5 mg/L biotin, 0.5 mg B.sub.12), 1 ml of a trace mineral solution (PII metals, containing per liter: 6.0 g FeCl.sub.36H.sub.2O, 6.84 g H.sub.3BO.sub.3, 0.86 g MnCl.sub.24H.sub.2O, 0.06 g ZnCl.sub.2, 0.026 CoCl.sub.26H.sub.2O, 0.052 g NiSO.sub.4H.sub.2O, 0.002 g CuSO.sub.45H.sub.2O and 0.005 g Na.sub.2MoO.sub.42H.sub.2O), and 500 mg each of penicillin G and streptomycin sulfate. The agar plates were incubated in the dark at 20-25 C. After 2-4 days the agar plates were examined under magnification, and colonies of cells were picked with a sterile toothpick and restreaked onto a fresh plate of media. Cells were repeatedly streaked onto fresh media until contaminated organisms were removed. Two of the isolated microorganisms were deposited under ATCC Accession Nos. PTA-10212 and PTA-10208.

(3) Taxonomic Characteristics of the Isolated Microorganism Deposited Under ATCC Accession No. PTA-10212

(4) Cultures of the isolated microorganism deposited under ATCC Accession No. PTA-10212 (PTA-10212) appeared as white, wet, smeared colonies without visible isolated sori.

(5) PTA-10212 was grown on solid and liquid FFM, solid KMV, KMV slush (1%), KMV broth, and MH broth to further examine growth characteristics. PTA-10212 was observed to grow rapidly on KMV and MH. See, e.g., Porter D., 1989. Phylum Labyrinthulomycota. In Margulis, L., Corliss, J. O., Melkonian, M., Chapman, D. J. (Eds.) Handbook of Protoctista, Jones and Bartlett, Boston, pp. 388-398 (KMV); Honda et al., Mycol. Res. 102:439-448 (1998) (MH); and U.S. Pat. No. 5,130,242 (FFM), each of which is incorporated herein by reference in its entirety.

(6) The following observations were made after growth of PTA-10212 over several days on solid FFM media, after 72 hours growth in KMV medias, and MH broth. Sporangia were not clumped in/on any media and were very small (5-10 m). PTA-10212 did not demonstrate the copious tetrads characteristic of Schizochytrium cleavage patterns. Amoeboid cells appeared about 24 hours after transfer to fresh solid media. These amoeboid cells were gone after a few days and were not evident in liquid or slush media. Unlike Aurantiochytrium, described by Yokoyama, R. et al., Mycoscience 48(6): 329-341 (2007), as having the appearance of small sandgrains on the bottom of the flask when grown in liquid media, PTA-10212 did not settle at the bottom of the flask but was suspended in both KMV and MH liquid media. The sporangia were not as dense as typical of Schizochytrium or Oligochytrium, which also have robust ectoplasmic networks that were absent from PTA-10212. While most species undergo vegetative cleavage of small sporangia or assimilative cells by the division of a larger sporangium over several hours, PTA-10212 formed dumbbell-shaped elongated assimilative cells, which then formed a thin isthmus that pulled apart as the ends of the dumbbell separated. The resulting cells appeared to be small assimilative cells. Direct transformation of an amoeboid cell into the dumbbell shaped assimilative cell was not observed. Typical biflagellate zoospores were observed swimming but were relatively rare. PTA-10212 was non-prolific, dividing by vegetative cleavage. Direct release of zoospores was not observed, although zoospores were observed swimming. Vegetative cells were very small (2 m to 5 m).

(7) PTA-10212 was further examined using the flow-through technique, in which microscopic slides were prepared by suspending a small portion of an agar-grown colony in a drop of half-strength sea water. With this technique, primary sporangia were observed to be globose and approximately 10 m in diameter. Walls were very thin and remnants were not observed when binary division of the protoplast was initiated. Repeated binary division produced 8-16 smaller (4-5 m in diameter) secondary sporangia. The secondary sporangia remained quiescent for several hours before again releasing an amorphous protoplast. The amorphous protoplast divided by pinching and pulling, initially producing typical dumbbell-shaped intermediate stages and finally resulting in 4-8 small globose bodies 2.5-2.8 m in diameter. The latter rested for several minutes up to 1-2 hours, then changed shape (elongated) and turned into biflagellate zoospores, 2.3-2.53.7-3.9 m. Zoospores were abundant and could be precisely measured when they came to rest. Zoospores then rounded off and started a new cycle of development. The zoospores were larger than Sicyoidochytrium minutum and smaller than Ulkenia visurgensis.

(8) PTA-10212 was further characterized based on the similarity of its 18s rRNA gene to that of known species. Genomic DNA was prepared from PTA-10212 by standard procedures. See, for example, Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Briefly: (1) 500 L of cells were centrifuged from mid-log culture. The cells were re-centrifuged, and all traces of liquid were removed from the cell pellet with a small-bore tip; (2) pellets were resuspended with 200 L lysis buffer (20 mM Tris pH 8.0, 125 g/mL Proteinase K, 50 mM NaCl, 10 mM EDTA pH 8.0, 0.5% SDS); (3) cells were lysed at 50 C. for 1 hour; (4) the lysis mixture was pipetted into phase-lock gel (PLG-Eppendorf) 2 mL tubes; (5) equal volume of P:C:I was added and allowed to mix for 1.5 hours; (6) the tubes were centrifuged at 12,000g for 5 minutes; (7) the aqueous phase was removed from above the gel within the PLG tube and an equal volume of chloroform was added to the aqueous phase, and mixed for 30 minutes; (8) the tubes were centrifuged at 14,000g for approximately 5 minutes; (9) the top layer (aqueous phase) was pipetted away from the chloroform, and placed in a new tube; (10) 0.1 volume of 3 M NaOAC was added and mixed (inverted several times); (11) 2 volumes of 100% EtOH were added and mixed (inverted several times) with genomic DNA precipitant forming at this stage; (12) the tubes were centrifuged at 4 C. in a microcentrifuge at 14,000g for approximately 15 minutes; (13) the liquid was gently poured off with genomic DNA remaining at the bottom of the tube; (14) the pellet was washed with 0.5 mL 70% EtOH; (15) the tubes were centrifuged at 4 C. in a microcentrifuge at 14,000g for approximately 5 minutes; (16) the EtOH was gently poured off and the genomic DNA pellet was dried; and (17) a suitable volume of H.sub.2O and RNase was added directly to the genomic DNA pellet. The PCR amplification of the 18s rRNA gene was carried out with primers previously described (Honda et. al., J. Euk. Micro. 46(6): 637-647 (1999). The PCR conditions with chromosomal DNA template were as follows: 0.2 M dNTPs, 0.1 M each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 U Herculase II Fusion DNA Polymerase (Stratagene), and Herculase buffer (Stratagene) in a 50 L total volume. The PCR Protocol included the following steps: (1) 95 C. for 2 minutes; (2) 95 C. for 35 seconds; (3) 55 C. for 35 seconds; (4) 72 C. for 1 minute and 30 seconds; (5) repeat steps 2-4 for 30 cycles; (6) 72 C. for 5 minutes; and (7) hold at 4 C.

(9) PCR amplification yielded a distinct DNA product with the expected size using chromosomal template described above. The PCR product was cloned into the vector pJET1.2/blunt (Fermentas) according to the manufacturer's instructions, and the insert sequence was determined using supplied standard primers.

(10) Phylogenetic analysis places PTA-10212 within the lineage that includes Thraustochytrium pachydermum and Thraustochytrium aggregatum with moderate support. The sporangia of T. pachydermum have very thick cell walls. T. aggregatum forms clearly visible clumps of sori that are opaque. PTA-10212 shows neither of these characteristics. The presence of many amoeboid cells has been described in other taxa, such as Ulkenia, T. gaertnerium, A. liiniacinuin, and S. mangrovei; however, the descriptions associated with those taxa differ from the observed characteristics of the isolate. Moreover, PTA-10212 did not show phylogenetic affinity towards any of these taxa.

(11) Table 3 shows a comparison of the 18s rRNA sequence from the microorganism deposited under ATCC Accession No. PTA-10212 to DNA sequences in the National Center for Biotechnology Information (NCBI) electronic database. The percent identity was determined using two different calculations. Calculation #1 takes into consideration any gaps that occur in the sequences, either from non-homologous regions or partial sequence (AlignX-VectorNTI default settings). Calculation #2 does not include calculated penalties for gaps (AlignX-VectorNTI IDENTITY matrix setting).

(12) TABLE-US-00003 TABLE 3 Comparison of 18s rRNA Sequences % Identity % Identity Thraustochytrids Calculation #1 Calculation #2 Thraustochytrium pachydermum 85% 93% Thraustochytrium aggregatum (p) 83% 92% Thraustochytrium gaertnerium 82% 92% Ulkenia visurgensis 82% 92% Schizochytrium sp. PTA-9695 80% 92% Schizochytrium mangrovei 80% 91% Schizochytrium sp. ATCC 20888 80% 90% Aurantiochytrium limiacinum 79% 90% (p): indicates partial sequence

(13) As shown in Table 3, it was found that, in terms of % identity, the 18s rRNA gene sequence (SEQ ID NO:1) from the microorganism deposited under ATCC Accession No. PTA-10212 is related, though not identical, to 18s rRNA gene sequences available in the NCBI database. It is generally recognized that organisms can have closely related 18s rRNA gene sequences while belonging to a different genus or species.

(14) Based on the above characterization, the isolated microorganism deposited under ATCC Accession No. PTA-10212 is believed to represent a new Thraustochytrium species and is therefore also designated as Thraustochytrium sp. ATCC PTA-10212.

(15) Taxonomic Characteristics of the Isolated Microorganism Deposited Under ATCC Accession No. PTA-10208

(16) The microorganism deposited under ATCC Accession No. PTA-10208 (PTA-10208) was identified as a sub-isolate (an individual cell isolated from a culture and maintained as a new separate and distinct culture) of the microorganism deposited under ATCC Accession No. PTA-9695 (PTA-9695), described in U.S. application Ser. No. 12/407,687 and PCT/US2009/001720, each of which is incorporated herein by reference in its entirety.

(17) PTA-10208 shares taxonomic characteristics with PTA-9695. PTA-9695 was found to have biflagellate zoospores at discharge that swim actively away from the mature sporangium, wall remnants of which were clearly visible (in phase contrast) after spore release. PTA-9695 sporangia measured 12.5 m to 25 m in diameter, and zoospores were 2.5 m to 2.8 m4.5 m to 4.8 m in size. There were 8 to 24 spores per individual PTA-9695 sporangium. Settled PTA-9695 zoospores enlarged and rapidly underwent binary divisions leading to tetrads, octads, and finally to clusters of sporangia. Tetrad formation commenced at a very early stage prior to maturity of the sporangia. These characteristics are in agreement with the genus Schizochytrium. In terms of % identity, the PTA-9695 18s rRNA gene sequence (SEQ ID NO:2), which is shared by PTA-10208, was found to be closely related, though not identical, to the 18s rRNA gene sequence of T. aggregatum provided in Honda, D. et al., J. Euk. Micro. 46(6): 637-647 (1999). The 18s rRNA sequence published for Thraustochytrium aggregatum is a partial sequence, with an approximately 71 DNA nucleotide gap in the middle of the sequence. PTA-9695 is believed to represent a new Schizochytrium species. As such, the sub-isolate PTA-10208 is also designated as Schizochytrium sp. ATCC PTA-10208.

EXAMPLE 2

(18) Growth Characteristics of the Isolated Microorganism Deposited Under ATCC Accession No. PTA-10212

(19) The isolated microorganism deposited under ATCC Accession No. PTA-10212 was examined for growth characteristics in individual fermentation runs, as described below. Typical media and cultivation conditions are shown in Table 1.

(20) In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cell weight of 26.2 g/L after 138 hours of culture in a 10 L fermentor volume. The lipid yield was 7.9 g/L; the omega-3 yield was 5.3 g/L; the EPA yield was 3.3 g/L; and the DHA yield was 1.8 g/L. The fatty acid content was 30.3% by weight; the EPA content was 41.4% of fatty acid methyl esters (FAME); and the DHA content was 26.2% of FAME. The lipid productivity was 1.38 g/L/day, and the omega-3 productivity was 0.92 g/L/day under these conditions, with 0.57 g/L/day EPA productivity and 0.31 g/L/day DHA productivity.

(21) In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. with 20% dissolved oxygen at pH 7.3, PTA-10212 produced a dry cell weight of 38.4 g/L after 189 hours of culture in a 10 L fermentor volume. The lipid yield was 18 g/L; the omega-3 yield was 12 g/L; the EPA yield was 5 g/L; and the DHA yield was 6.8 g/L. The fatty acid content was 45% by weight; the EPA content was 27.8% of FAME; and the DHA content was 37.9% of FAME. The lipid productivity was 2.3 g/L/day, and the omega-3 productivity was 1.5 g/L/day under these conditions, with 0.63 g/L/day EPA productivity and 0.86 g/L/day DHA productivity.

(22) In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. with 20% dissolved oxygen at pH 6.8-7.7, PTA-10212 produced a dry cell weight of 13 g/L after 189 hours of culture in a 10 L fermentor volume. The lipid yield was 5.6 g/L; the omega-3 yield was 3.5 g/L; the EPA yield was 1.55 g/L; and the DHA yield was 1.9 g/L. The fatty acid content was 38% by weight; the EPA content was 29.5% of FAME; and the DHA content was 36% of FAME. The lipid productivity was 0.67 g/L/day, and the omega-3 productivity was 0.4 g/L/day under these conditions, with 0.20 g/L/day EPA productivity and 0.24 g/L/day DHA productivity.

(23) In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5-28.5 C. with 20% dissolved oxygen at pH 6.6-7.2, PTA-10212 produced a dry cell weight of 36.7 g/L-48.7 g/L after 191 hours of culture in a 10 L fermentor volume. The lipid yield was 15.2 g/L-25.3 g/L; the omega-3 yield was 9.3 g/L-13.8 g/L; the EPA yield was 2.5 g/L-3.3 g/L; and the DHA yield was 5.8 g/L-11 g/L. The fatty acid content was 42.4%-53% by weight; the EPA content was 9.8%-22% of FAME; and the DHA content was 38.1%-43.6% of FAME. The lipid productivity was 1.9 g/L/day-3.2 g/L/day, and the omega-3 productivity was 1.2 g/L/day-1.7 g/L/day under these conditions, with 0.31 g/L/day-0.41 g/L/day EPA productivity and 0.72 g/L/day-1.4 g/L/day DHA productivity.

(24) Growth Characteristics of the Isolated Microorganism Deposited Under ATCC Accession No. PTA-10208

(25) The isolated microorganism deposited under ATCC Accession No. PTA-10208 was examined for growth characteristics in individual fermentation runs, as described below. Typical media and cultivation conditions are shown in Table 2.

(26) In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10% dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 95 g/L after 200 hours of culture in a 10 L fermentor volume. The lipid yield was 53.7 g/L; the omega-3 yield was 37 g/L; the EPA yield was 14.3 g/L; and the DHA yield was 21 g/L. The fatty acid content was 57% by weight; the EPA content was 27.7% of FAME; and the DHA content was 39.1% of FAME. The lipid productivity was 6.4 g/L/day, and the omega-3 productivity was 4.4 g/L/day under these conditions, with 1.7 g/L/day EPA productivity and 2.5 g/L/day DHA productivity.

(27) In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. at pH 7.5 with 20% dissolved oxygen during the nitrogen feed and 10% dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 56 g/L after 139 hours of culture in a 10 L fermentor volume. The lipid yield was 53 g/L; the omega-3 yield was 34 g/L; the EPA yield was 11.5 g/L; and the DHA yield was 22 g/L. The fatty acid content was 58% by weight; the EPA content was 21.7% of FAME; and the DHA content was 41.7% of FAME. The lipid productivity was 9.2 g/L/day, and the omega-3 productivity was 5.9 g/L/day under these conditions, with 2 g/L/day EPA productivity and 3.8 g/L/day DHA productivity.

(28) In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10% dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 93.8 g/L after 167 hours of culture in a 2000 L fermentor volume. The lipid yield was 47.2 g/L; the omega-3 yield was 33.1 g/L; the EPA yield was 10.5 g/L; and the DHA yield was 20.4 g/L. The fatty acid content was 50.6% by weight; the EPA content was 23% of FAME; and the DHA content was 42.6% of FAME. The lipid productivity was 6.8 g/L/day, and the omega-3 productivity was 4.7 g/L/day under these conditions, with 1.5 g/L/day EPA productivity and 2.9 g/L/day DHA productivity.

(29) In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10% dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 105 g/L after 168 hours of culture in a 2000 L fermentor volume. The lipid yield was 46.4 g/L; the omega-3 yield was 33 g/L; the EPA yield was 10.7 g/L; and the DHA yield was 20.3 g/L. The fatty acid content was 43.9% by weight; the EPA content was 24% of FAME; and the DHA content was 43.7% of FAME. The lipid productivity was 6.6 g/L/day, and the omega-3 productivity was 4.7 g/L/day under these conditions, with 1.5 g/L/day EPA productivity and 2.9 g/L/day DHA productivity.

(30) In carbon (glucose) and nitrogen-fed cultures with 1000 ppm Cl.sup. at 22.5 C. at pH 7.0 with 20% dissolved oxygen during the nitrogen feed and 10% dissolved oxygen thereafter, PTA-10208 produced a dry cell weight of 64.8 g/L after 168 hours of culture in a 2000 L fermentor volume. The lipid yield was 38.7 g/L; the omega-3 yield was 29.9 g/L; the EPA yield was 8.5 g/L; and the DHA yield was 16.7 g/L. The fatty acid content was 59.6% by weight; the EPA content was 23% of FAME; and the DHA content was 42.3% of FAME. The lipid productivity was 5.53 g/L/day, and the omega-3 productivity was 3.8 g/L/day under these conditions, with 1.2 g/L/day EPA productivity and 2.3 g/L/day DHA productivity.

EXAMPLE 3

(31) Fatty Acid Profiles of Microorganism Strains ATCC PTA-10208 and PTA-10212

(32) Four samples of biomass (PTA-10208 Sample #1; PTA-10208 Sample #2; PTA-10212 Sample #1; and PTA-10212 Sample #2) were analyzed for total crude oil content by solvent extraction, lipid classes were determined by high performance liquid chromatography/evaporative light scattering detection (HPLC/ELSD), triacylglycerol (TAG) were analyzed by HPLC/mass spectrometry (HPLC/MS), and fatty acid (FA) profiles were determined by gas chromatography with flame ionization detection (GC-FID). The crude lipid content of each freeze dried biomass was determined using solvent grinding with hexane and compared to the sum of FAME (mg/g) generated by direct transesterification, and the resultant fatty acid methyl esters (FAME) were quantified by GC/FID analysis. Fatty acids in the extracted crude lipid were also quantified by transesterification and quantified using GC/FID analysis of the resultant FAME. The weight percent of all neutral lipids (NL) and free fatty acids (FFA) were determined in the extracted crude lipid using normal phase HPLC with ELSD and atmospheric pressure chemical ionization-MS (APCI-MS) identification. The method separates and quantifies sterol esters (SE), TAG, free fatty acids (FFA), 1,3-diacylglycerols (1,3-DAG), sterols, 1,2-diacylglycerols (1,2-DAG), and monoacylglycerols (MAG). Results are shown in Tables 4 and 5, below.

(33) TAG and phospholipids (PL) were isolated from the crude oils extracted from the four samples of biomass (PTA-10208 Sample #1; PTA-10208 Sample #2; PTA-10212 Sample #1; and PTA-10212 Sample #2). TAG was isolated using low pressure flash chromatography and PL was isolated using solid phase extraction (SPE). The identity of each isolated fraction was confirmed by thin layer chromatography (TLC). The fatty acid profiles of the isolated TAG and PL fractions were determined following direct transesterification using GC-FID as FAME. Results are shown in Tables 6 and 7, below.

(34) The total crude oil content and fatty acid profiles of isolated lipid classes were also determined for two additional samples of biomass from microorganism strain ATCC PTA-10212 (PTA-10212 Sample #3 and PTA-10212 Sample #4). Crude oil was obtained from each sample by hexane extraction, and individual lipid classes were isolated using low pressure flash chromatography. The fatty acid profiles of the biomass, crude oil, and isolated fractions were determined using GC-FID as FAME. Results are shown in Tables 8-11, below.

(35) Individual lipid classes were isolated from a sample of crude oil from microorganism strain ATCC PTA-10212 (PTA-10212 Sample #5) previously extracted using the FRIOLEX process, and the fatty acid profiles of each class were determined using GC-FID as FAME. Results are shown in Tables 12 and 13, below.

(36) Individual lipid classes were isolated from a sample of crude oil from microorganism strain ATCC PTA-10208 (PTA-10208 Sample #3) using normal HPLC with ELSD and APCI-MS identification.

(37) Experimental Procedures

(38) Crude Oil Extraction

(39) Crude oil was extracted from samples of freeze-dried biomass using solvent grinding. For example, approximately 3 grams of biomass was weighed into a Swedish tube. Three ball bearings and 30 mL of hexane were added to the Swedish tube, which was sealed with a neoprene stopper and placed in a shaker for 2 hours. The resultant slurry was filtered using a Buchner funnel and Whatman filter paper. The filtered liquid was collected, the solvent removed under vacuum, and the amount of remaining crude lipid determined gravimetrically.

(40) Fatty Acid Analysis

(41) The samples of biomass, extracted crude lipid, and isolated lipid classes were analyzed for fatty acid composition as FAME. Briefly, freeze-dried biomass and isolated lipid classes were weighed directly into a screw cap test tubes, while samples of the crude oil were dissolved in hexane to give a concentration of approximately 2 mg/mL. Toluene, containing internal standard, and 1.5 N HCl in methanol was added to each tube. The tubes were vortexed, then capped and heated to 100 C. for 2 hours. The tubes were allowed to cool, and saturated NaCl in water was added. The tubes were vortexed again and centrifuged to allow the layers to separate. A portion of the organic layer was then placed in a GC vial and analyzed by GC-FID. FAME was quantified using a 3-point calibration curve generated using Nu-Check-Prep GLC Reference Standard (NuCheck, Elysian, Minn.). Fatty acids present in the extract were expressed as mg/g and as a weight percent. Fat content in the samples was estimated assuming equal response to the internal standard when analyzed by GC-FID.

(42) HPLC/ELSD/MS Method

(43) TABLE-US-00004 Instrument Agilent 1100 HPLC, Alltech 3300 ELSD, Agilent 1100 MSD Column Phenomenex Luna Silica, 250 4.6 mm, 5 m particle size w/ Guard Column Mobile Phase A - 99.5% Hexanes (Omnisolv) 0.4% Isopropyl alcohol (Omnisolv) 0.1% Acetic Acid B - 99.9% Ethanol (Omnisolv, 95:5 Ethanol:IPA) 0.1% Acetic Acid Time, min. % A % B Gradient 0 100 0 5 100 0 15 85 10 20 0 100 25 0 100 26 100 0 35 100 0 Column Temp. 30 C. Flow Rate 1.5 mL/min Injection Volume 5 L ELSD Detection Temperature 35 C., Gas flow 1.2 L/min MSD Mass Range 200-1200, Fragmentor 225 V Drying Gas Temperature 350 C. Vaporizer Temperature 325 C. Capillary Voltage 3500 V Corona Current 10 A

(44) Solid Phase Extraction

(45) PL fractions were separated from the crude lipid by solid phase extraction (SPE) using 2 g aminopropyl cartridges (Biotage, Uppsala, Sweden) placed in a Vac Elut apparatus (Varian Inc, Palo Alto, USA). The cartridge was conditioned with 15 mL of hexane, and 60 mg of each sample was dissolved in 1 mL CHCl.sub.3 and applied to the cartridge. The column was washed with 15 mL of 2:1 CHCl.sub.3:isopropyl alcohol to elute all the neutral lipids, which was discarded. The fatty acids were then eluted with 15 mL of 2% acetic acid (HOAc) in ether, which was discarded. The PL portion was eluted with 15 mL of 6:1 Methanol:Chloroform, which was collected, dried under nitrogen, and weighed.

(46) Flash Chromatography

(47) Flash chromatography was used to separate the lipid classes present in the crude oil. Approximately 200 mg of crude oil dissolved in hexane was injected onto the head of the column. The chromatography system utilized Silica Gel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase composed of Petroleum Ether and Ethyl Acetate at 5 mL/min (Tables 6-7) or 3 mL/min (Tables 8-13). A step gradient was used to selectively elute each lipid class from the column. The mobile phase gradient started from 100% petroleum ether and finished with 50% ethyl acetate. Fractions were collected in 10 mL test tubes using a Gilson FC 204 large-bed fraction collector (Gilson, Inc., Middleton, Wis.). Each tube was analyzed by thin layer chromatography (TLC) and the tubes containing individual lipid classes (as judged by single spots on TLC plate with expected retention factor (Rf)) were pooled, concentrated to dryness, and weighed. The total fraction content was then determined gravimetrically.

(48) TLC Analysis

(49) Thin layer chromatography was conducted on silica gel plates. The plates were eluted using a solvent system consisting of petroleum ether:ethyl ether:acetic acid (80:20:1) and were visualized using iodine vapor. The Rf values of each spot were then compared with reported literature values for each lipid class.

(50) Analysis of TAG and PL Fractions

(51) The isolated TAG and PL fractions were analyzed for fatty acid composition as fatty acid methyl esters (FAME). The TAG fractions were dissolved in hexane to give a concentration of approximately 1-2 mg/mL. 1 mL aliquots of the solutions were concentrated to dryness under nitrogen. Toluene, containing internal standard, and 1.5 N HCl in methanol was added to each tube. The tubes were vortexed, then capped and heated to 100 C. for 2 hours. Internal standard and HCl methanol were added directly to the tubes containing the PL fraction and heated. The tubes were allowed to cool, and saturated NaCl in water was added. The tubes were vortexed again and centrifuged to allow the layers to separate. A portion of the organic layer was then placed in a GC vial and analyzed by GC-FID. FAMEs were quantified using a 3-point calibration curve generated using Nu-Check-Prep GLC 502B Reference Standard (NuCheck, Elysian, Minn.). Fatty acids present in the extract were expressed as mg/g and as a % of FAME.

(52) Results

(53) PTA-10208 Sample #1

(54) The fatty acid profile of the biomass and extracted crude lipid for PTA-10208 Sample #1 was determined using GC/FID. Fatty acids in the biomass were transesterified in situ by weighing 28.6 mg of biomass directly into a FAME tube, while a sample of the extracted crude lipid was prepared by weighing 55.0 mg of crude lipid into a 50 mL volumetric flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 53.2% (as SUM of FAME) using GC with FID detection, while 52.0% (wt/wt) lipid was extracted from the dry biomass, giving a 97.8% recovery of total lipid. The crude lipid was determined to be 91.9% fatty acids (as SUM of FAME) using GC/FID. The major fatty acids contained in the crude lipid were C16:0 (182.5 mg/g), C20:5 n-3 (186.8 mg/g), and C22:6 n-3 (423.1 mg/g).

(55) The lipid class profile of the extracted crude lipid was determined by weighing 55.0 mg of crude lipid into a 50 mL volumetric flask and transferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis. According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2% sterol esters (SE), 95.1% TAG, 0.4% sterols, and 0.5% 1,2-diacylglycerol (DAG). 5% of the TAG fraction included a peak that eluted directly after the TAG peak, but did not give a recognizable mass spectrum.

(56) Isolated TAG from this sample as determined by flash chromatography made up approximately 92.4% of the crude oil. PL was not detected by weight or TLC after SPE isolation. The major fatty acids (>50 mg/g) contained in the TAG were C16:0 (189 mg/g), C20:5 n-3 (197 mg/g), and C22:6 n-3 (441 mg/g).

(57) PTA-10208 Sample #2

(58) The fatty acid profile of the biomass and extracted crude lipid for PTA-10208 Sample #2 was determined using GC/FID. Fatty acids in the biomass were transesterified in situ by weighing 32.0 mg of biomass directly into a FAME tube, while a sample of the extracted crude lipid was prepared by weighing 60.1 mg of crude lipid into a 50 mL volumetric flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 52.4% (as SUM of FAME) using GC with FID detection, while 48.0% (wt/wt) lipid was extracted from the dry biomass, giving a 91.7% recovery of total lipid. The crude lipid was determined to be 95.3% fatty acids (as SUM of FAME) using GC/FID. The major fatty acids contained in the crude lipid were C16:0 (217.5 mg/g), C20:5 n-3 (169.3 mg/g), and C22:6 n-3 (444.1 mg/g).

(59) The lipid class profile of the extracted crude lipid was determined by weighing 60.1 mg of crude lipid into a 50 mL volumetric flask and transferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis. According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2% SE, 95.7% TAG, 0.3% sterols, and 0.7% 1,2-DAG. 5.1% of the TAG fraction included a peak that eluted directly after the TAG peak, but did not give a recognizable mass spectrum.

(60) Isolated TAG from this sample made up approximately 93.9% of the crude oil. PL was not detected by weight or TLC after SPE isolation. The major fatty acids (>50 mg/g) contained in the TAG were C16:0 (218 mg/g), C20:5 n-3 (167 mg/g) and C22:6 n-3 (430 mg/g).

(61) PTA-10208 Sample #3

(62) A sample of crude oil from the microorganism deposited under ATCC Accession No. PTA-10208 (Sample PTA-10208 #3) was analyzed using HPLC/ELSD/MS. A total of 98.38% of lipids were recovered, with the sterol ester (SE) fraction accounting for 0.32%, the TAG fraction accounting for 96.13%, the 1,3-diacylglycerol (DAG) fraction accounting for 0.22%, the 1,2-DAG fraction accounting for 0.78%, and the sterol fraction accounting for 0.93%.

(63) PTA-10212 Sample #1

(64) The fatty acid profile of the biomass and extracted crude lipid for PTA-10212 Sample #1 was determined using GC/FID. Fatty acids in the biomass were transesterified in situ by weighing 27.0 mg of biomass directly into a FAME tube, while a sample of the extracted crude lipid was prepared by weighing 52.5 mg of crude lipid into a 50 mL volumetric flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 38.3% (as SUM of FAME) using GC with FID detection, while 36.3% (wt/wt) lipid was extracted from the dry biomass, giving a 94.6% recovery of total lipid. The crude lipid was determined to be 83.2% fatty acids (as SUM of FAME) using GC/FID. The major fatty acids contained in the crude lipid were C16:0 (328.5 mg/g), C20:5 n-3 (90.08 mg/g), and C22:6 n-3 (289.3 mg/g).

(65) The lipid class profile of the extracted crude lipid was determined by weighing 52.5 mg of crude lipid into a 50 mL volumetric flask and transferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis. According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2% SE, 64.2% TAG, 1.9% FFA, 2.8% 1,3-DAG, 1.4% sterols, 18.8% 1,2-DAG, and 0.5% MAG. 3.4% of the TAG fraction included a peak that eluted directly after the TAG peak, but did not give a recognizable mass spectrum.

(66) Isolated TAG from this sample made up approximately 49.8% of the crude oil. Isolated PL made up approximately 8.1% of the crude oil. The major fatty acids (>50 mg/g) contained in the TAG fraction are C16:0 (400 mg/g), C20:5 n-3 (91 mg/g), and C22:6 n-3 (273 mg/g). The major fatty acids (>50 mg/g) contained in the PL fraction are C16:0 (98 mg/g), C20:5 n-3 (33 mg/g), and C22:6 n-3 (227 mg/g).

(67) PTA-10212 Sample #2

(68) The fatty acid profile of the biomass and extracted crude lipid PTA-10212 Sample #2 was determined using GC/FID. Fatty acids in the biomass were transesterified in situ by weighing 29.5 mg of biomass directly into a FAME tube, while a sample of the extracted crude lipid was prepared by weighing 56.5 mg of crude lipid into a 50 mL volumetric flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 40.0% (as SUM of FAME) using GC with FID detection, while 41.3% (wt/wt) lipid was extracted from the dry biomass, giving a 106.1% recovery of total lipid. The crude lipid was determined to be 82.8% fatty acids (as SUM of FAME) using GC/FID. The major fatty acids contained in the crude lipid were C16:0 (327.3 mg/g), C20:5 n-3 (92.5 mg/g), and C22:6 n-3 (277.6 mg/g).

(69) The lipid class profile of the extracted crude lipid was determined by weighing 56.5 mg of crude lipid into a 50 mL volumetric flask and transferring an aliquot into an HPLC vial for HPLC/ELSD/MS analysis. According to the HPLC/ELSD/MS analysis, the crude lipid contained 0.2% SE, 58.2% TAG, 2.3% FFA, 3.4% 1,3-DAG, 1.7% sterols, 23.4% 1,2-DAG, and 0.6% MAG. 3.3% of the TAG fraction included a peak that eluted directly after the TAG peak, but did not give a recognizable mass spectrum.

(70) Isolated TAG from this sample made up approximately 51.9% of the crude oil. Isolated PL made up approximately 8.8% of the crude oil. The major fatty acids (>50 mg/g) contained in the TAG fraction are C16:0 (402 mg/g), C20:5 n-3 (92 mg/g), and C22:6 n-3 (245 mg/g). The major fatty acids (>50 mg/g) contained in the PL fraction are C16:0 (121 mg/g), C20:5 n-3 (48 mg/g), and C22:6 n-3 (246 mg/g).

(71) TABLE-US-00005 TABLE 4 Fatty Acid Profiles of PTA-10208 and PTA-10212 Biomasses and Extracted Crude Lipids (mg/g) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10212 PTA-10212 PTA-10212 PTA-10212 Sample #1 Sample #1 Sample #2 Sample #2 Sample #1 Sample #1 Sample #2 Sample #2 Biomass Crude Lipid Biomass Crude Lipid Biomass Crude Lipid Biomass Crude Lipid FAME FAME FAME FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) C12:0 1.47 2.43 1.80 3.14 0.99 1.90 0.87 1.91 C14:0 11.62 20.12 16.72 31.03 5.51 12.91 5.97 13.69 C14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 2.43 3.75 3.60 6.22 9.13 20.42 9.39 20.81 C16:0 105.04 182.47 117.72 217.49 145.87 328.45 147.87 327.27 C16:1 0.00 0.00 0.06 0.01 6.26 14.53 7.46 16.89 C18:0 5.37 8.96 4.77 8.37 6.77 15.39 6.77 15.15 C18:1 n-9 0.00 3.26 0.00 3.09 0.03 4.04 0.08 5.87 C18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.48 1.79 1.40 1.85 1.60 3.09 1.67 3.20 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 0.91 1.61 1.10 2.00 2.28 2.56 2.21 2.64 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.10 0.00 0.08 0.00 0.30 0.12 0.35 0.24 C20:4 n-7 0.81 0.45 0.67 0.41 0.00 0.00 0.00 0.00 C20:4 n-6 7.22 12.23 6.84 12.18 1.19 2.26 1.31 2.32 C22:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.63 0.52 0.00 0.46 0.00 0.00 0.00 0.00 C20:4 n-3 3.45 5.45 3.33 5.58 0.00 2.40 0.00 2.34 C20:3 n-3 0.09 0.00 0.11 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 107.31 186.83 92.99 169.32 40.32 90.08 43.15 92.54 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.60 0.00 0.52 0.00 2.81 6.83 2.74 6.53 C24:1 n-9 1.55 3.26 0.85 2.04 0.43 1.34 0.42 1.24 C22:5 n-6 9.66 15.84 10.27 17.98 2.42 4.68 2.32 4.21 C22:5 n-3 20.44 35.13 9.92 17.50 2.41 4.94 2.69 5.23 C22:6 n-3 246.98 423.10 245.96 444.08 139.58 289.34 137.35 277.57 Sum of FAME 527.15 907.18 518.71 942.75 367.89 805.29 372.63 799.68

(72) TABLE-US-00006 TABLE 5 Fatty Acid Profiles of PTA-10208 and PTA-10212 Biomasses and Extracted Crude Lipids (%) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10212 PTA-10212 PTA-10212 PTA-10212 Sample #1 Sample #1 Sample #2 Sample #2 Sample #1 Sample #1 Sample #2 Sample #2 Biomass Crude Lipid Biomass Crude Lipid Biomass Crude Lipid Biomass Crude Lipid Fatty Acid % FAME % FAME % FAME % FAME % FAME % FAME % FAME % FAME C12:0 0.28 0.27 0.35 0.33 0.27 0.24 0.23 0.24 C14:0 2.20 2.22 3.22 3.29 1.50 1.60 1.60 1.71 C14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 0.46 0.41 0.69 0.66 2.48 2.54 2.52 2.60 C16:0 19.93 20.11 22.70 23.07 39.65 40.79 39.68 40.93 C16:1 0.00 0.00 0.01 0.00 1.70 1.80 2.00 2.11 C18:0 1.02 0.99 0.92 0.89 1.84 1.91 1.82 1.89 C18:1 n-9 0.00 0.36 0.00 0.33 0.01 0.50 0.02 0.73 C18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 0.28 0.20 0.27 0.20 0.43 0.38 0.45 0.40 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 0.17 0.18 0.21 0.21 0.62 0.32 0.59 0.33 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.02 0.00 0.01 0.00 0.08 0.02 0.09 0.03 C20:4 n-7 0.15 0.05 0.13 0.04 0.00 0.00 0.00 0.00 C20:4 n-6 1.37 1.35 1.32 1.29 0.32 0.28 0.35 0.29 C22:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.12 0.06 0.00 0.05 0.00 0.00 0.00 0.00 C20:4 n-3 0.65 0.60 0.64 0.59 0.00 0.30 0.00 0.29 C20:3 n-3 0.02 0.00 0.02 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 20.36 20.59 17.93 17.96 10.96 11.19 11.58 11.57 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.11 0.00 0.10 0.00 0.76 0.85 0.74 0.82 C24:1 n-9 0.29 0.36 0.16 0.22 0.12 0.17 0.11 0.16 C22:5 n-6 1.83 1.75 1.98 1.91 0.66 0.58 0.62 0.53 C22:5 n-3 3.88 3.87 1.91 1.86 0.65 0.61 0.72 0.65 C22:6 n-3 46.85 46.64 47.42 47.10 37.94 35.93 36.86 34.71 Sum of 100 100 100 100 100 100 100 100 FAME %

(73) TABLE-US-00007 TABLE 6 Fatty Acid Profiles of PTA-10208 and PTA-10212 Isolated TAG PTA-10208 PTA-10208 PTA-10212 PTA-10212 Sample #1 PTA-10208 Sample #2 PTA-10208 Sample #1 PTA-10212 Sample #2 PTA-10212 FAME Sample #1 FAME Sample #2 FAME Sample #1 FAME Sample #2 Fatty Acid (mg/g) % FAME (mg/g) % FAME (mg/g) % FAME (mg/g) % FAME C12:0 2.57 0.27 3.35 0.36 0.00 0.00 0.00 0.00 C14:0 21.07 2.23 31.37 3.41 14.05 1.61 14.45 1.69 C14:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 3.89 0.41 6.17 0.67 23.27 2.66 23.14 2.71 C16:0 189.28 20.07 218.78 23.75 399.51 45.75 402.43 47.07 C16:1 0.00 0.00 0.00 0.00 15.23 1.74 17.62 2.06 C18:0 9.21 0.98 8.07 0.88 22.70 2.60 23.10 2.70 C18:1 n-9 3.35 0.36 3.64 0.40 6.12 0.70 7.48 0.87 C18:1 n-7 0.00 0.00 0.00 0.00 <0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.86 0.20 1.55 0.17 4.76 0.55 5.32 0.62 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 1.64 0.17 2.00 0.22 2.25 0.26 2.24 0.26 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.00 0.00 0.00 0.00 0.55 0.06 0.89 0.10 Un-known 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-7 0.39 0.04 0.05 0.01 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-6 12.79 1.36 11.82 1.28 2.33 0.27 2.25 0.26 C22:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.39 0.04 0.07 0.01 0.00 0.00 0.00 0.00 C20:4 n-3 5.52 0.59 5.09 0.55 2.87 0.33 2.98 0.35 C20:5 n-3 197.14 20.90 166.68 18.10 91.17 10.44 91.78 10.74 C24:0 0.00 0.00 0.00 0.00 6.93 0.79 7.36 0.86 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:1 n-9 1.08 0.11 <0.1 <0.1 0.00 0.00 0.00 0.00 C22:5 n-6 15.88 1.68 16.57 1.80 4.01 0.46 3.39 0.40 C22:5 n-3 36.05 3.82 16.00 1.74 4.53 0.52 5.07 0.59 C22:6 n-3 440.99 46.76 429.83 46.67 273.02 31.26 245.38 28.70 Sum of FAME 943.11 921.03 873.31 854.89 Total % 100.00 100.00 100.00 100.00 FAME

(74) TABLE-US-00008 TABLE 7 Fatty Acid Profiles of PTA-10212 Isolated PL PTA-10212 PTA-10212 Sample #1 PTA-10212 Sample #2 PTA-10212 FAME Sample #1 FAME Sample #2 Fatty Acid (mg/g) % FAME (mg/g) % FAME C12:0 0.00 0.00 0.00 0.00 C14:0 0.93 0.22 1.89 0.39 C14:1 0.00 0.00 0.00 0.00 C15:0 3.44 0.82 5.07 1.05 C16:0 98.29 23.50 120.98 25.00 C16:1 1.15 0.27 3.07 0.63 C18:0 3.25 0.78 3.72 0.77 C18:1 n-9 1.12 0.27 0.95 0.20 C18:1 n-7 <0.1 <0.1 0.02 0.003 C18:2 n-6 0.00 0.00 0.00 0.00 C20:0 <0.1 <0.1 <0.1 <0.1 C18:3 n-3 0.00 0.00 0.00 0.00 C20:1 n-9 0.00 0.00 0.00 0.00 C18:4 n-3 3.71 0.89 3.24 0.67 C20:2 n-6 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 C22:0 0.00 0.00 0.00 0.00 Unknown 42.33 10.12 44.71 9.24 C20:4 n-7 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 C20:4 n-6 0.84 0.20 1.54 0.32 C22:1 n-9 0.00 0.00 0.00 0.00 C20:4 n-5 0.00 0.00 0.00 0.00 C20:4 n-3 <0.1 <0.1 0.27 0.06 C20:5 n-3 33.39 7.98 47.91 9.90 C24:0 <0.1 <0.1 0.01 0.001 C22:4 n-9 0.00 0.00 0.00 0.00 C24:1 n-9 0.00 0.00 0.00 0.00 C22:5 n-6 3.08 0.74 3.82 0.79 C22:5 n-3 <0.1 <0.1 0.66 0.14 C22:6 n-3 226.68 54.20 246.09 50.85 Sum of FAME 418.21 483.94 Total % FAME 100 100
PTA-10212 Sample #3

(75) The lipid content of the biomass for PTA-10212 Sample #3 was estimated to be 34% as the sum of FAME, and the amount of crude oil obtained after solvent extraction was 37% by weight, giving a 109% recovery of fat present in the biomass. After fractionation using flash chromatography, approximately 46% of the crude oil was isolated as TAG, 28% was isolated as DAG, The crude oil contained 309 mg/g DHA and 264 mg/g EPA. The isolated TAG contained 341 mg/g DHA and 274 mg/g EPA. The isolated DAG fraction contained 262 mg/g DHA and 237 mg/g EPA. The total fatty acid profiles of the biomass, extracted crude oil, and isolated fractions are shown below in Table 8 and Table 9 calculated as mg/g and % FAME, respectively.

(76) TABLE-US-00009 TABLE 8 Fatty Acid Profiles of PTA-10212 Sample #3 Biomass and Extracted Crude Lipid (mg/g) Biomass Crude Oil TAG DAG Wt. % NA 37.2% FAME FAME 46.0% 27.9% Fatty Acid (mg/g) (mg/g) FAME (mg/g) FAME (mg/g) C12:0 0.0 0.0 0.0 0.0 C14:0 3.6 10.3 11.5 9.4 C14:1 0.0 0.0 0.0 0.0 C15:0 4.1 10.6 9.8 6.6 C16:0 70.5 181.8 231.7 111.3 C16:1 6.7 19.1 18.7 17.1 C18:0 2.4 10.2 14.2 0.0 C18:1 n-9 0.0 6.7 0.0 0.0 C18:1 n-7 0.0 1.2 0.0 0.0 C18:2 n-6 0.0 1.8 0.0 0.0 C20:0 0.0 2.4 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:1 n-9 0.0 0.3 0.0 1.7 C18:4 n-3 1.9 3.4 3.2 4.4 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 3.3 0.0 0.0 0.0 C20:4 n-7 0.0 2.1 1.5 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 6.8 17.9 21.4 13.8 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 1.3 1.3 0.0 C20:4 n-3 3.0 8.5 10.9 6.4 C20:5 n-3 102.0 263.6 274.2 237.4 C24:0 0.0 1.7 3.9 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 4.2 0.0 C22:5 n-6 3.2 8.3 10.7 6.1 C22:5 n-3 3.8 10.4 15.1 6.6 C22:6 n-3 131.2 309.4 341.1 261.9 Sum of FAME 342.4 871.1 973.2 682.6

(77) TABLE-US-00010 TABLE 9 Fatty Acid Profiles of PTA-10212 Sample #3 Biomass and Extracted Crude Lipid (%) Biomass Crude Oil TAG DAG Wt. % NA 37.2% FAME FAME 46.0% 27.9% Fatty Acid (mg/g) (mg/g) FAME (mg/g) FAME (mg/g) C12:0 0.0 0.0 0.0 0.0 C14:0 1.1 1.2 1.2 1.4 C14:1 0.0 0.0 0.0 0.0 C15:0 1.2 1.2 1.0 1.0 C16:0 20.6 20.9 23.8 16.3 C16:1 2.0 2.2 1.9 2.5 C18:0 0.7 1.2 1.5 0.0 C18:1 n-9 0.0 0.8 0.0 0.0 C18:1 n-7 0.0 0.1 0.0 0.0 C18:2 n-6 0.0 0.2 0.0 0.0 C20:0 0.0 0.3 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:1 n-9 0.0 0.0 0.0 0.2 C18:4 n-3 0.6 0.4 0.3 0.6 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 1.0 0.0 0.0 0.0 C20:4 n-7 0.0 0.2 0.2 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.0 2.1 2.2 2.0 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.1 0.1 0.0 C20:4 n-3 0.9 1.0 1.1 0.9 C20:5 n-3 29.8 30.3 28.2 34.8 C24:0 0.0 0.2 0.4 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 0.4 0.0 C22:5 n-6 0.9 1.0 1.1 0.9 C22:5 n-3 1.1 1.2 1.6 1.0 C22:6 n-3 38.3 35.5 35.1 38.4 Total % FAME 100.0 100.0 100.0 100.0
PTA-10212 Sample #4

(78) PTA-10212 Sample #4 contained approximately 23% lipid determined as the sum of FAME, of which 107% was recovered using hexane extraction. After fractionation using flash chromatography, approximately 42% of the crude oil was isolated as TAG, 18% was isolated as DAG. The crude oil contained 275 mg/g DHA and 209 mg/g EPA. The isolated TAG contained 296 mg/g DHA and 205 mg/g EPA. The isolated DAG fraction contained 245 mg/g DHA and 219 mg/g EPA. The total fatty acid profiles of the biomass, extracted crude oil, and isolated fractions are shown below in Table 10 (mg/g) and Table 11 (% FAME).

(79) TABLE-US-00011 TABLE 10 Fatty Acid Profiles of PTA-10212 Sample #4 Biomass and Extracted Crude Lipid (mg/g) Biomass Crude Oil TAG DAG Wt. % NA 24.7% FAME FAME 42.2% 18.4% Fatty Acid (mg/g) (mg/g) FAME (mg/g) FAME (mg/g) C12:0 0.0 0.0 2.1 2.4 C14:0 2.0 8.3 9.8 9.6 C14:1 0.0 0.0 0.0 0.0 C15:0 4.8 16.8 0.4 0.9 C16:0 63.3 210.6 285.7 138.0 C16:1 1.6 6.7 7.4 7.5 C18:0 2.8 12.2 19.9 4.6 C18:1 n-9 0.0 3.7 0.7 1.1 C18:1 n-7 0.0 0.0 0.3 1.2 C18:2 n-6 0.0 0.0 0.0 0.0 C20:0 0.0 3.3 6.0 1.5 C18:3 n-3 0.0 0.0 0.0 0.0 C20:1 n-9 0.0 0.0 0.7 1.2 C18:4 n-3 1.4 3.8 3.6 5.0 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.4 0.0 C22:0 1.5 0.0 1.9 0.0 C20:4 n-7 0.0 0.0 0.9 0.6 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.5 10.1 13.0 10.3 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.8 0.7 C20:4 n-3 1.4 6.3 8.6 6.0 C20:5 n-3 57.6 209.1 205.4 219.0 C24:0 0.0 2.6 0.8 0.0 C22:4 n-9 0.1 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 1.1 0.5 C22:5 n-6 1.4 6.1 7.9 4.5 C22:5 n-3 4.0 15.8 20.8 12.9 C22:6 n-3 87.7 275.0 296.4 244.8 Sum of FAME 232.2 790.1 894.8 672.4

(80) TABLE-US-00012 TABLE 11 Fatty Acid Profiles of PTA-10212 Sample #4 Biomass and Extracted Crude Lipid (%) Biomass Crude Oil TAG DAG Wt. % NA 24.7% FAME FAME 42.2% 18.4% Fatty Acid (mg/g) (mg/g) FAME (mg/g) FAME (mg/g) C12:0 0.0 0.0 0.2 0.4 C14:0 0.9 1.0 1.1 1.4 C14:1 0.0 0.0 0.0 0.0 C15:0 2.1 2.1 0.0 0.1 C16:0 27.3 26.7 31.9 20.5 C16:1 0.7 0.8 0.8 1.1 C18:0 1.2 1.5 2.2 0.7 C18:1 n-9 0.0 0.5 0.1 0.2 C18:1 n-7 0.0 0.0 0.0 0.2 C18:2 n-6 0.0 0.0 0.0 0.0 C20:0 0.0 0.4 0.7 0.2 C18:3 n-3 0.0 0.0 0.0 0.0 C20:1 n-9 0.0 0.0 0.1 0.2 C18:4 n-3 0.6 0.5 0.4 0.7 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 0.6 0.0 0.2 0.0 C20:4 n-7 0.0 0.0 0.1 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 1.1 1.3 1.5 1.5 C22:1 n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.1 0.1 C20:4 n-3 0.6 0.8 1.0 0.9 C20:5 n-3 24.8 26.5 23.0 32.6 C24:0 0.0 0.3 0.1 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:1 n-9 0.0 0.0 0.1 0.1 C22:5 n-6 0.6 0.8 0.9 0.7 C22:5 n-3 1.7 2.0 2.3 1.9 C22:6 n-3 37.8 34.8 33.1 36.4 Total % FAME 100.0 100.0 100.0 100.0
PTA-10212 Sample #5

(81) A sample of crude oil was extracted from a biomass of PTA-10212 using the FRIOLEX process (GEA Westfalia Separator UK Ltd., Milton Keynes, England) to yield microbial oil PTA-10212 Sample #5. Individual lipid classes were isolated from PTA-10212 Sample #5 using low pressure flash chromatography, and the weight percent of each class was determined. The fatty acid profile of each class was determined using GC-FID.

(82) Briefly, the sample was prepared by dissolving 240 mg of crude oil in 600 L of hexane and applying to the head of the column. After fractionation of the sample using flash chromatography, the combined weights of all the fractions was 240 mg giving a 100% recovery. The sterol ester fraction accounted for 0.9%, the TAG fraction accounted for 42.6%, the free fatty acid (FFA) fraction accounted for 1.3%, the sterol fraction accounted for 2.2%, the DAG fraction accounted for 41.6%. The total fatty acid profiles of the FRIOLEX crude oil and isolated fractions are shown below in Table 12 and Table 13 calculated as mg/g and % FAME, respectively.

(83) TABLE-US-00013 TABLE 12 Fatty Acid Profiles of PTA-10212 Sample #5 Crude Oil (mg/g) Crude Oil TAG DAG Wt. % NA 42.6% 41.6% Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0 0 0.7 1.0 C14:0 7.7 7.7 8.5 C14:1 0 0.0 0.0 C15:0 10.3 11.7 9.3 C16:0 179.3 217.7 134.6 C16:1 18.1 16.3 25.9 C18:0 8.1 13.2 2.3 C18:1 n-9 4.7 8.4 0.7 C18:1 n-7 0 1.8 1.0 C18:2 n-6 1.8 3.3 0.7 C20:0 1.9 3.6 0.2 C18:3 n-3 0 0.0 0.0 C20:1 n-9 0 0.7 1.0 C18:4 n-3 3.1 2.8 3.8 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.6 0.4 C22:0 0 1.5 0.0 C20:4 n-7 0 1.0 0.7 C20:3 n-3 0 0.0 0.0 C20:4 n-6 12.7 16.1 13.6 C22:1 n-9 0 0.0 0.0 C20:4 n-5 0 1.5 0.8 C20:4 n-3 6.5 9.3 6.4 C20:5 n-3 213.3 223.7 252.8 C24:0 2.3 4.4 0.6 C22:4 n-9 0 1.9 0.9 C24:1 n-9 0 0.0 0.0 C22:5 n-6 7.9 9.5 8.3 C22:5 n-3 13 20.6 9.7 C22:6 n-3 305.6 327.4 353.8 Sum of FAME 796.6 905.3 837.4

(84) TABLE-US-00014 TABLE 13 Fatty Acid Profiles of PTA-10212 Sample #5 Crude Oil (%) Crude Oil TAG DAG Fatty Acid % FAME % FAME % FAME C12:0 0 0.1 0.1 C14:0 1 0.9 1.0 C14:1 0 0.0 0.0 C15:0 1.3 1.3 1.1 C16:0 22.5 24.0 16.1 C16:1 2.3 1.8 3.1 C18:0 1 1.5 0.3 C18:1 n-9 0.6 0.9 0.1 C18:1 n-7 0 0.2 0.1 C18:2 n-6 0.2 0.4 0.1 C20:0 0.2 0.4 0.0 C18:3 n-3 0 0.0 0.0 C20:1 n-9 0 0.1 0.1 C18:4 n-3 0.4 0.3 0.5 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.1 0.0 C22:0 0 0.2 0.0 C20:4 n-7 0 0.1 0.1 C20:3 n-3 0 0.0 0.0 C20:4 n-6 1.6 1.8 1.6 C22:1 n-9 0 0.0 0.0 C20:4 n-5 0 0.2 0.1 C20:4 n-3 0.8 1.0 0.8 C20:5 n-3 26.8 24.7 30.2 C24:0 0.3 0.5 0.1 C22:4 n-9 0 0.2 0.1 C24:1 n-9 0 0.0 0.0 C22:5 n-6 1 1.1 1.0 C22:5 n-3 1.6 2.3 1.2 C22:6 n-3 38.4 36.2 42.3 Total % FAME 100 100 100

EXAMPLE 4

(85) The relative amount and fatty acid composition of each TAG isomer present in the extracted crude lipid was determined for each of samples PTA-10208 Sample #1, PTA-10208 Sample #2, PTA-10212 Sample #1, PTA-10212 Sample #2, and PTA-10212 Sample #3, PTA-10212 Sample #4, and PTA-10212 Sample #5 from Example 3 using non-aqueous reversed phase HPLC separation and APCI-MS detection.

(86) TAG Method

(87) TABLE-US-00015 Instrument Agilent 1100 HPLC Agilent 1100 MSD Column(s) Two Phenomenex Luna C18 (2), 150 4.6 mm, 3 m particle size connected in series Mobile Phase A - Acetonitrile B - IPA w/ 0.1% Ammonium Acetate Time, min. % A % B Gradient 0 80 20 120 20 80 125 20 80 126 80 20 140 80 20 Column Temp. 20 C. Flow Rate 0.5 mL/min Injection Volume 5 L MSD Mass Range 350-1150 Fragmentor 225 V Drying Gas Temperature 350 C., Vaporizor Temperature 325 C. Capillary Voltage 3500 V Corona Current 10 A
PTA-10208 Sample #1

(88) The crude lipid isolated from PTA-10208 Sample #1 was prepared for TAG analysis prepared for TAG analysis by weighing 5.5 mg of oil into an HPLC vial and diluting with 1 mL of hexane.

(89) TABLE-US-00016 TABLE 14 Identification of TAG Species in PTA-10208 Sample #1 Retention Area Major (DAG) Time CN Identification Percent [M + H].sup.+ [M + NH4].sup.+ Fragments 41.76 30 EPA/EPA/EPA 1.2 945.8 962.7 643.5 42.97 30 EPA/EPA/DHA 5.4 971.7 988.8 643.4, 669.5 44.17 30 DHA/DHA/EPA 8.8 997.7 1014.7 669.5, 695.5 45.39 30 DHA/DHA/DHA 7.4 1023.7 1040.7 695.5 46.32 32 DHA/EPA/ARA 1.1 973.8 990.8 645.4, 671.5 47.53 32 DHA/DPA/EPA 2.0 999.8 1016.8 671.5, 697.5 48.88 32 DHA/EPA/ARA 2.6 973.8 990.7 645.4, 671.5 DHA/DHA/DPA 1025.7 1042.8 697.5 50.23 32 DHA/DPA/EPA 1.8 999.8 1016.8 671.5, 697.4 51.47 32 DHA/DPA/DHA 1.5 1025.7 1042.8 695.5, 697.5 54.64 34 EPA/14:0/DHA 1.8 897.7 914.7 569.5, 595.5 55.80 34 DHA/DHA/14:0 2.2 923.6 940.8 595.5, 695.5 60.73 36 EPA/EPA/16:0 3.4 899.7 916.8 597.5, 643.3 61.87 36 DHA/16:0/EPA 11.8 925.8 942.7 597.5, 623.5, 669.5 63.0 36 DHA/16:0/DHA 17.7 951.8 968.8 623.5, 695.5 65.47 38 EPA/EPA/18:0 2.3 927.7 944.8 625.5 66.58 38 DHA/DPA/16:0 2.9 953.8 970.8 623.5, 625.5, 697.6 67.31 38 EPA/ARA/16:0 1.0 901.7 918.8 597.6, 599.5, 645.4 68.39 38 DHA/16:0/ARA 2.0 927.7 944.8 625.6 69.52 38 DHA/16:0/DPA 2.3 953.8 970.8 623.5, 625.5 70.16 38 DHA/DHA/18:0 0.9 979.5 996.7 651.5 73.81 40 EPA/16:0/14:0 1.0 825.7 842.7 523.5, 569.5, 597.5 74.73 40 DHA/14:0/16:0 1.5 851.7 868.7 523.4, 623.5 80.96 42 EPA/16:0/16:0 1.8 853.6 870.8 551.5, 597.4 81.93 42 DHA/16:0/16:0 6.5 879.8 896.8 551.5, 623.5 85.50 44 DPA/16:0/16:0 0.9 881.7 898.8 551.4, 625.4 88.92 44 18:0/16:0/DHA 0.9 907.9 924.8 579.4, 651.5
PTA-10208 Sample #2

(90) The crude lipid isolated from PTA-10208 Sample #2 was prepared for TAG analysis prepared for TAG analysis by weighing 5.3 mg of oil into an HPLC vial and diluting with 1 mL of hexane.

(91) TABLE-US-00017 TABLE 15 Identification of TAG Species in PTA-10208 Sample #2 Retention Area Major (DAG) Time CN Identification Percent [M + H].sup.+ [M + NH4].sup.+ Fragments 41.70 30 EPA/EPA/EPA 1.0 945.7 962.8 643.5 42.92 30 EPA/EPA/DHA 4.3 971.7 988.8 643.5, 669.5 44.11 30 DHA/DHA/EPA 6.9 997.8 1014.8 669.5, 695.5 45.33 30 DHA/DHA/DHA 6.2 1023.8 1040.8 695.5 46.26 32 DHA/EPA/ARA 0.5 973.7 990.7 645.4, 671.5 47.47 32 DHA/DPA/EPA 1.1 999.8 1016.8 671.5, 697.5 48.86 32 DHA/EPA/ARA 1.9 973.7 990.8 645.5, 671.5 DHA/DHA/DPA 1025.7 1042.8 697.5 50.16 32 DHA/DPA/EPA 1.5 999.6 1016.9 671.5, 697.4 51.37 32 DHA/DPA/DHA 1.1 1025.7 1042.8 695.5, 697.5 54.57 34 EPA/14:0/DHA 2.0 897.7 914.7 569.5, 595.4 55.78 34 DHA/DHA/14:0 2.9 923.6 940.8 595.5, 695.5 60.74 36 EPA/EPA/16:0 3.1 899.6 916.8 597.5, 643.5 61.94 36 DHA/16:0/EPA 13.00 925.7 942.8 597.5, 623.5, 669.5 63.10 36 DHA/16:0/DHA 20.0 951.8 968.8 623.5, 695.5 65.60 38 EPA/EPA/18:0 1.6 927.7 944.8 625.5 66.71 38 DHA/DPA/16:0 2.0 953.7 970.8 623.6, 625.4, 697.6 67.46 38 DHA/14:0/14:0 1.3 823.5 840.7 495.4, 595.5 EPA/ARA/16:0 901.8 918.8 597.6, 599.5, 645.4 68.60 38 DHA/16:0/ARA 2.0 927.8 944.8 599.5, 623.5, 671.5 69.69 38 DHA/16:0/DPA 2.7 953.7 970.8 623.5, 625.5 70.39 38 DHA/DHA/18:0 0.7 979.8 996.8 651.5 73.88 40 EPA/16:0/14:0 1.1 825.7 842.8 523.5, 569.3, 597.3 74.94 40 DHA/14:0/16:0 2.9 851.8 868.7 523.5, 623.5 81.17 42 EPA/16:0/16:0 1.9 853.8 870.8 551.5, 597.6 82.16 42 DHA/16:0/16:0 9.7 879.7 896.7 551.5, 623.5 85.72 44 DPA/16:0/16:0 0.6 881.7 898.8 551.4, 625.5 89.15 44 18:0/16:0/DHA 1.2 907.7 924.8 579.4, 651.5
PTA-10212 Sample #1

(92) The crude lipid isolated from PTA-10212 Sample #1 was prepared for TAG analysis prepared for TAG analysis by weighing 5.3 mg of oil into an HPLC vial and diluting with 1 mL of hexane.

(93) TABLE-US-00018 TABLE 16 Identification of TAG Species in PTA-10212 Sample #1 Retention Area Time CN Identification Percent [M + H].sup.+ [M + NH4].sup.+ (DAG) Fragments 43.01 30 EPA/EPA/DHA 1.2 971.7 988.7 643.5, 669.5 44.24 30 DHA/DHA/EPA 2.6 997.7 1014.8 669.5, 695.4 45.41 30 DHA/DHA/DHA 2.4 1023.8 1040.8 695.5 54.78 34 DHA/EPA/14:0 0.7 897.7 914.8 569.3, 595.4 DHA/DHA/16:1 949.7 966.7 621.3, 695.3 59.53 35 DHA/15:0/DHA 0.9 937.8 954.8 609.6, 695.5 60.88 36 EPA/16:0/EPA 1.5 899.7 916.7 597.5, 643.2 62.03 36 EPA/16:0/DHA 8.3 925.8 942.7 597.6, 623.4 63.17 36 DHA/16:0/DHA 13.1 951.7 968.8 623.5, 695.5 66.82 38 DHA/18:0/EPA 1.5 953.7 970.8 625.6 68.63 38 DHA/ARA/16:0 0.7 927.7 944.7 599.5, 623.3, 671.5 69.79 38 DHA/DPA/16:0 0.7 953.8 970.8 623.5, 625.5 70.41 38 DHA/DHA/18:0 0.7 979.7 996.7 651.5, 695.3 72.79 40 EPA/16:0/16:1 0.5 851.7 868.6 549.4 73.82 40 DHA/18:1/14:0 1.8 877.7 894.7 549.5, 621.5, 623.3 75.00 40 DHA/16:0/14:0 1.7 851.8 868.8 523.5, 623.5 77.55 41 EPA/15:0/16:0 0.8 839.7 856.7 537.5, 583.4 78.59 41 DHA/16:0/15:0 2.5 865.6 882.8 537.3, 623.5 81.32 42 EPA/16:0/16:0 5.5 853.7 870.7 551.5, 597.5 82.19 42 DHA/16:0/16:0 21.7 879.8 896.7 551.5, 623.5 85.74 43 DHA/17:0/16:0 2.0 ND 910.7 565.5, 623.5, 637.7 88.23 44 DPA/16:0/16:0 0.8 881.7 898.8 551.7, 625.5 89.19 44 DHA/16:0/18:0 3.2 907.9 924.8 579.5, 623.3, 651.5 93.18 46 16:0/16:0/16:1 1.2 ND 822.8 549.5, 551.6 94.57 46 16:0/16:0/14:0 1.1 779.7 796.7 523.4, 551.3 95.91 46 DHA/12:0/24:0 0.7 935.9 952.8 567.5, 607.6 97.88 47 16:0/16:0/15:0 1.4 ND 810.7 537.5, 551.4 101.14 48 16:0/16:0/16:0 7.5 807.6 824.7 551.5 104.26 49 16:0/16:0/17:0 0.7 ND 838.7 551.4, 565.5 107.35 50 16:0/16:0/18:0 1.7 ND 852.8 551.5, 579.5 108.58 50 DHA/16:0/24:0 0.7 991.8 1008.9 623.5, 663.7, 735.5 113.28 52 16:0/16:0/20:0 0.6 ND 880.9 551.5, 607.4 124.24 56 16:0/16:0/24:0 0.6 ND 936.8 551.5, 663.5 ND = Not Detected
PTA-10212 Sample #2

(94) The crude lipid isolated from PTA-10212 Sample #2 was prepared for TAG analysis prepared for TAG analysis by weighing 3.6 mg of oil into an HPLC vial and diluting with 1 mL of hexane.

(95) TABLE-US-00019 TABLE 17 Identification of TAG Species in PTA-10212 Sample #2 Retention Area Time CN Identification Percent [M + H].sup.+ [M + NH4].sup.+ (DAG) Fragments 42.99 30 EPA/EPA/DHA 1.4 971.7 988.7 643.4, 669.5 44.22 30 DHA/DHA/EPA 3.3 997.8 1014.7 669.5, 695.5 45.4 30 DHA/DHA/DHA 2.4 1023.7 1040.8 695.5 54.80 34 DHA/EPA/14:0 0.6 897.7 914.7 569.5 DHA/DHA/16:1 ND 966.7 621.3 59.49 35 DHA/15:0/DHA 0.5 ND 954.8 609.6 60.83 36 EPA/16:0/EPA 1.6 899.7 916.6 597.5, 643.3 62.02 36 EPA/16:0/DHA 9.9 925.8 942.7 597.5, 623.5 63.16 36 DHA/16:0/DHA 13.0 951.8 968.8 623.6, 695.5 66.8 38 DHA/18:0/EPA 0.7 953.7 970.6 625.5 68.65 38 DHA/ARA/16:0 0.4 ND 944.8 599.6, 623.4 69.76 38 DHA/DPA/16:0 0.4 953.6 970.8 623.5 70.44 38 DHA/DHA/18:0 0.3 979.8 996.7 ND 72.74 40 EPA/16:0/16:1 0.4 ND 868.6 549.5 73.77 40 DHA/18:1/14:0 1.6 877.6 894.8 549.5, 621.9 74.97 40 DHA/16:0/14:0 1.4 851.8 868.7 523.5, 623.7 77.73 41 EPA/15:0/16:0 0.3 ND 856.7 537.5 78.53 41 DHA/16:0/15:0 2.2 865.8 882.7 537.5 81.32 42 EPA/16:0/16:0 6.3 853.8 870.8 551.4, 597.5 82.15 42 DHA/16:0/16:0 22.8 879.7 896.7 551.5, 623.5 85.67 43 DHA/17:0/16:0 1.6 ND 910.8 565.5 89.08 44 DPA/16:0/16:0 2.6 ND 898.8 551.5 DHA/16:0/18:0 907.8 924.8 579.5 93.09 46 16:0/16:0/16:1 0.9 ND 822.8 549.6, 551.3 94.47 46 16:0/16:0/14:0 1.1 ND 796.7 523.5, 551.5 95.78 46 DHA/12:0/24:0 0.5 ND 952.8 607.5 97.8 47 16:0/16:0/15:0 1.6 ND 810.7 537.3, 551.5 100.99 48 16:0/16:0/16:0 9.5 ND 824.7 551.4 104.11 49 16:0/16:0/17:0 0.6 ND 838.7 551.7, 565.5 107.23 50 16:0/16:0/18:0 2.1 ND 852.8 551.3, 579.5 108.45 50 DHA/16:0/24:0 0.7 ND 1009.0 663.5 113.11 52 16:0/16:0/20:0 0.5 ND 880.9 551.3, 607.7 124.07 56 16:0/16:0/24:0 0.8 ND 937.0 551.5, 663.5 ND = Not Detected
PTA-10212 Sample #3

(96) A sample of the TAG fraction of PTA-10212 Sample #3 was prepared in hexane and analyzed by HPLC/APCI/MS to determine the identities of individual TAG isomers.

(97) TABLE-US-00020 TABLE 18 Identification of TAG Species in PTA-10212 Sample #3 Retention Area Time Peak # Identification Percent [M + H].sup.+ [M + NH4].sup.+ (DAG) Fragments 20.016 1 EPA/EPA/EPA 3.7 945.5 962.7 643.5 20.471 2 EPA/EPA/DHA 8.6 971.6 988.7 643.5, 669.5 20.970 3 DHA/DHA/EPA 6.5 997.7 1014.7 695.5, 669.5 21.441 4 DHA/DHA/DHA 3.7 1023.7 1040.7 695.5 21.855 5 EPA/EPA/DPA 0.7 973.7 990.7 645.3, 671.3 22.107 6 DHA/EPA/DPA 1.9 999.5 1016.7 697.5, 671.4 EPA/EPA/ARA 947.5 964.6 643.3, 645.3 22.573 7 EPA/ARA/DHA 2.2 973.7 990.7 671.5, 645.5 23.057 8 DHA/EPA/DPA 1.4 999.6 1016.7 696.5, 671.4 23.548 9 DHA/DHA/DPA 0.8 1025.7 1042.7 695.5, 697.5 DHA/14:0/EPA 897.7 914.7 569.4, 595.3 24.034 10 DHA/16:1/EPA 1.2 923.5 940.7 595.5, 621.3 24.306 11 EPA/16:1/EPA 0.7 897.6 914.7 595.3 24.509 12 DHA/16:1/DHA 0.8 949.6 966.7 621.3 24.783 13 DHA/14:0/DHA 0.5 923.7 940.8 595.5 25.571 14 EPA/15:0/DHA 1.0 911.7 928.7 583.4, 609.4 26.026 15 DHA/15:0/DHA 0.7 937.7 954.7 609.3 26.376 16 EPA/16:0/EPA 7.2 899.7 916.7 597.5 26.832 17 EPA/16:0/DHA 14.3 925.7 942.7 597.3, 623.4 27.272 18 DHA/16:0/DHA 13.2 951.7 968.7 623.5 27.842 19 DPA/14:0/ARA 0.5 901.6 918.7 599.4, 623.4 28.048 20 EPA/18:0/EPA 1.0 927.6 944.7 623.4 28.271 21 DHA/16:0/ARA 1.0 927.7 944.7 599.5, 623.5 28.564 22 DPA/16:1/DPA 2.3 953.7 970.6 623.5 ARA/16:1/ARA 901.8 918.6 597.4 29.060 23 DHA/16:0/ARA 2.6 927.7 944.7 599.3, 625.5 29.381 24 DHA/18:0/EPA 0.8 953.8 970.3 625.4, 651.5 29.512 25 DHA/16:0//DPA 1.0 953.8 970.8 623.4, 625.4 30.654 26 DHA/16:0/16:1 1.4 877.8 894.7 549.5, 623.5 31.015 27 DHA/16:0/14:0 0.6 851.7 868.7 523.3, 623.7 32.216 28 DHA/16:0/15:0 0.8 865.8 882.7 537.5, 623.3 33.063 29 EPA/16:0/16:0 4.1 853.5 870.7 551.5, 597.5 33.438 30 DHA/16:0/16:0 9.7 879.7 896.7 551.5, 623.5 35.518 31 DPA/16:0/16:0 0.9 881.7 898.7 551.5, ND 35.798 32 DHA/18:0/16:0 1.3 907.8 924.7 579.4, 651.3 39.578 33 16:0/16:016:0 1.3 ND 824.8 551.5 ND = Not Detected
PTA-10212 Sample #4

(98) A sample of the TAG fraction of PTA-10212 Sample #4 was prepared in hexane and analyzed by HPLC/APCI/MS to determine the identities of individual TAG isomers.

(99) TABLE-US-00021 TABLE 19 Identification of TAG Species in PTA-10212 Sample #4 Retention Area Time Peak # Identification Percent [M + H].sup.+ [M + NH4].sup.+ (DAG) Fragments 20.1 1 EPA/EPA/EPA 2.3 945.5 962.7 643.5 20.6 2 EPA/EPA/DHA 4.4 971.6 988.7 643.5, 669.5 21.1 3 DHA/DHA/EPA 4.7 997.7 1014.7 695.5, 669.5 21.6 4 DHA/DHA/DHA 4.1 1023.7 1040.7 695.5 EPA/EPA/DPA 973.7 990.7 645.3, 671.3 22.0 5 EPA/EPA/DPA 0.2 973.7 990.7 645.3, 671.3 22.3 6 DHA/EPA/DPA 1.4 999.5 1016.7 697.5, 671.4 EPA/EPA/ARA 947.5 964.6 645.3 22.7 7 EPA/ARA/DHA 1.1 973.7 990.7 671.5, 645.5 23.2 8 DHA/EPA/DPA 0.5 999.6 1016.7 696.5, 671.4 23.7 9 DHA/DPA/DHA 0.2 1025.8 1043.8 697.7 24.6 10 DHA/16:1/DHA 0.3 949.6 966.7 621.3 24.9 11 DHA/14:0/EPA 0.4 923.5 940.7 595.4 25.3 12 EPA/15:0/EPA 0.4 885.5 902.5 583.5 25.7 13 EPA/15:0/DHA 1.0 911.7 928.7 583.4, 609.4 26.2 14 DHA/15:0/DHA 0.6 937.7 954.7 609.3 26.6 15 EPA/16:0/EPA 4.9 899.7 916.7 597.5 27.0 16 EPA/16:0/DHA 12.8 925.7 942.7 597.3, 623.4 27.5 17 DHA/16:0/DHA 15.2 951.7 968.7 623.5 28.3 18 EPA/18:0/EPA 2.0 927.6 944.7 623.4 28.7 19 DPA/16:1/DPA 2.3 953.7 970.6 623.5 ARA/16:1/ARA 901.8 918.6 597.4 29.3 20 DHA/16:0/ARA 1.4 927.7 944.7 599.3, 623.4 29.6 21 DHA/16:0/DPA 1.5 953.8 970.3 625.4, 651.5 30.0 22 DHA/18:0/DHA 0.7 979.7 996.7 651.5 30.9 23 DHA/16:0/16:1 0.7 877.8 894.7 549.5, ND EPA/16:0/16:1 825.6 842.6 549.5, ND 31.3 24 DHA/14:0/16:0 0.6 851.7 868.7 523.3, 595.3 32.5 25 DHA/16:0/15:0 1.5 865.8 882.7 537.5, 623.4 33.4 26 EPA/16:0/16:0 4.4 853.5 870.7 551.5, 597.5 33.8 27 DHA/16:0/16:0 14.5 879.7 896.7 551.5, 623.5 35.0 28 DPA/16:0/16:0 2.3 881.6 898.7 551.5, 625.5 DHA/15:0/18:0 893.8 910.7 565.5, ND 35.8 29 DPA/16:0/16:0 1.3 881.7 898.7 551.5, ND 36.2 30 DPA/16:0/16:0 2.6 881.7 898.7 551.5, 625.5 DHA/16:0/18:0 907.8 924.7 579.4, 623.5 40.0 31 16:0/16:016:0 3.4 ND 824.8 551.5 42.2 32 16:0/16:0/18:0 0.7 ND 852.8 551.3, 579.5 43.0 33 DHA/16:0/24:0 0.9 991.7 1008.8 623.3, 663.5 ND = Not Detected
PTA-10212 Sample #5

(100) A sample of the TAG fraction of PTA-10212 Sample #5 was prepared in hexane and analyzed by HPLC/APCI/MS to determine the identities of individual TAG isomers.

(101) TABLE-US-00022 TABLE 20 Identification of TAG Species in PTA-10212 Sample #5 Retention Area Time Peak # Identification Percent [M + H].sup.+ [M + NH4].sup.+ (DAG) Fragments 21.0 1 EPA/EPA/EPA 1.7 945.7 962.8 643.5 21.5 2 EPA/EPA/DHA 5.5 971.6 988.7 643.5, 669.5 22.0 3 DHA/DHA/EPA 7.6 997.7 1014.7 695.5, 669.5 22.5 4 DHA/DHA/DHA 4.5 1023.8 1040.7 695.5 23.0 5 EPA/EPA/DPA 0.5 973.8 990.8 645.5, 671.5 23.3 6 DHA/EPA/DPA 1.5 999.7 1016.7 697.5, 671.5 EPA/EPA/ARA 947.6 964.8 645.5 23.7 7 EPA/ARA/DHA 1.9 973.6 990.7 671.5, 645.4 DHA/DPA/DHA 1025.7 1042.7 695.5, 697.5 24.2 8 DHA/EPA/DPA 1.1 999.7 1016.7 669.5, 671.4, 696.5 24.8 9 DHA/DHA/DPA 0.6 1025.7 1042.9 695.5, 697.5 DHA/14:0/EPA 897.7 914.7 595.5 25.3 10 DHA/16:1/EPA 0.7 923.8 940.8 595.5, 621.3 25.6 11 DHA/14:0/EPA 0.6 897.7 914.7 569.3, 595.4 25.8 12 DHA/16:1/DHA 0.5 949.7 966.7 621.3, 695.5 26.0 13 DHA/16:1/EPA 0.4 923.7 940.8 595.5, 621.3 26.9 14 EPA/15:0/DHA 0.7 911.7 928.7 583.5, 609.3 27.4 15 DHA/15:0/DHA 0.7 937.6 954.8 609.3 27.8 16 EPA/16:0/EPA 4.9 899.7 916.7 597.5 28.2 17 EPA/16:0/DHA 14.3 925.7 942.8 597.5, 623.5 28.7 18 DHA/16:0/DHA 12.2 951.7 968.8 623.5 29.3 19 DPA/14:0/ARA 0.6 901.7 918.8 597.3 29.5 20 EPA/18:0/EPA 1.5 927.7 944.8 625.5 30.0 21 DHA/16:0/ARA 3.4 953.7 970.8 623.4 30.6 22 EPA/EPA/18:0 2.1 927.7 944.7 599.5, 625.5, 669.3 31.0 23 DHA/18:0/EPA 1.7 953.8 970.8 625.4, 651.5 31.3 24 DHA/18:0/DHA 0.9 979.7 996.8 651.5, 695.3 31.9 26 16:0/DHA/14:0 0.8 851.7 868.7 595.5, 623.5 32.3 27 18:1/14:0/DHA 1.7 877.7 894.7 549.5, 595.5 32.6 28 DHA/16:0/14:0 0.9 851.8 868.7 523.4, 623.5 33.5 29 EPA/15:0/16:0 0.7 839.7 856.7 537.5, 583.3 DHA/20:0/EPA 981.7 998.8 653.5, 679.6 33.9 30 DHA/16:0/15:0 1.2 865.7 882.7 537.5, 623.5 34.8 31 EPA/16:0/16:0 3.9 853.8 870.7 551.5, 597.5 35.2 32 DHA/16:0/16:0 10.6 879.7 896.7 551.5, 623.5 36.4 33 DPA/16:0/16:0 1.5 881.7 898.7 551.5, 625.5 37.4 33 DPA/16:0/16:0 1.2 881.7 898.7 551.5, 625.5 37.7 34 DHA/16:0/18:0 1.9 907.7 924.7 579.4 38.4 35 EPA/24:0/DHA 0.5 1037.8 1054.8 709.5, 735.6 38.8 36 DHA/24:0/DHA 1.0 1064.8 1081.8 735.7

EXAMPLE 5

(102) Crude oils were further processed via refining, bleaching, and deodorizing to obtain refined oils. The refined oils were diluted with high oleic sunflower oil to obtain final oils with a DHA content of approximately 400 mg/g. Individual lipid classes were isolated and the fatty acid profiles of each class was determined using GC-FID as FAME.

(103) PTA-10208 Final Oils

(104) The fatty acid profiles for PTA-10208 Final Oils #1-5 are summarized in Tables 21-22, including profiles associated within the isolated TAG fraction (Tables 23-24) and the isolated sterols/DAG fraction (Tables 24-26).

(105) Individual lipid classes in the final oils were also determined using flash chromatography (Table 27) and normal HPLC with ELSD and APCI-MS confirmation (Table 28).

(106) TABLE-US-00023 TABLE 21 Fatty Acid Profiles of PTA-10208 Final Oils (mg/g) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #4 Final Oil #5 Final Oil #1 Final Oil #2 Final Oil #3 FAME FAME Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) (mg/g) (mg/g) C12:0 2.5 2.4 2.8 2.7 2.7 C14:0 16.1 14.9 21.0 18.4 17.5 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 3.8 3.6 4.4 3.9 3.9 C16:0 192.1 179.1 193.1 184.3 194.6 C16:1 0.4 0.5 0.5 0.5 0.5 C17:0 0.6 0.5 0.9 0.8 2.1 C18:0 12.8 13.9 11.5 12.3 12.9 C18:1 n-9 23.5 82.0 25.7 26.0 29.5 C18:1 n-7 0.2 0.7 0.1 0.1 0.1 C18:2 n-6 3.7 8.1 4.0 4.1 4.3 C20:0 4.3 4.1 3.7 4.0 4.0 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 0.1 <0.1 <0.1 <0.1 C18:4 n-3 2.4 2.5 2.8 2.7 2.8 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.1 0.1 0.1 0.1 C22:0 1.2 1.8 1.0 1.1 1.1 C20:4 n-7 1.7 1.6 1.7 1.8 1.6 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 12.9 12.1 13.5 13.5 13.3 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.4 1.5 1.7 1.5 C20:4 n-3 6.0 5.7 6.0 6.0 6.1 C20:5 n-3 173.8 163.3 196.4 209.6 197.9 C24:0 1.4 1.6 1.3 1.3 1.0 C22:4\n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 3.4 3.2 2.3 2.6 2.3 C22:5 n-6 14.9 14.0 14.4 13.0 12.9 C22:5 n-3 43.9 41.3 32.8 40.3 36.9 C22:6 n-3 394.8 373.7 373.2 374.3 364.2 Sum of FAME 918.1 932.2 914.7 925.1 914.1

(107) TABLE-US-00024 TABLE 22 Fatty Acid Profiles of PTA-10208 Final Oils (%) PTA- PTA- PTA- PTA- 10208 10208 10208 10208 Final Final Final Final PTA-10208 Oil #1 Oil #2 Oil #3 Oil #4 Final Oil #5 Fatty Acid % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.3 0.3 0.3 C14:0 1.8 1.6 2.3 2.0 1.9 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.1 19.9 21.3 C16:1 <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.2 C18:0 1.4 1.5 1.3 1.3 1.4 C18:1 n-9 2.6 8.8 2.8 2.8 3.2 C18:1 n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.4 0.9 0.4 0.4 0.5 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4\n-5 0.2 0.2 0.2 0.2 0.2 C20:4\n-3 0.7 0.6 0.7 0.7 0.7 C20:5 n-3 18.9 17.5 21.5 22.7 21.6 C24:0 0.1 0.2 0.1 0.1 0.1 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.4 0.3 0.2 0.3 0.2 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.0 C22:6 n-3 43.0 40.1 40.8 40.5 39.9

(108) TABLE-US-00025 TABLE 23 Isolated TAG Fatty Acid Profiles: PTA-10208 Final Oils (mg/g) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0 2.5 2.3 2.7 2.5 2.6 C14:0 16.3 15.1 21.3 18.6 18.1 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 3.9 3.6 4.4 4.0 4.0 C16:0 194.2 181.9 196.1 186.1 199.8 C16:1 0.4 0.4 0.6 0.5 0.7 C17:0 0.6 0.5 0.9 0.8 0.8 C18:0 12.9 14.2 11.7 12.5 13.2 C18:1 n-9 24.3 84.0 26.8 26.1 34.0 C18:1 n-7 0.1 0.7 0.1 0.1 0.3 C18:2 n-6 3.2 7.7 3.4 3.5 4.0 C20:0 4.4 4.2 3.8 4.0 4.2 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 0.2 <0.1 <0.1 0.1 C18:4 n-3 2.5 2.4 2.8 2.6 2.7 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.2 0.1 0.1 0.1 C22:0 1.2 1.9 1.0 1.1 1.1 C20:4 n-7 1.7 1.6 1.8 1.8 1.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 13.2 12.3 13.8 13.7 13.8 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.5 1.6 1.7 1.5 C20:4 n-3 6.1 5.7 6.1 5.9 6.2 C20:5 n-3 176.0 166.1 199.0 211.2 204.2 C24:0 1.2 1.3 1.0 1.1 1.2 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 3.3 3.2 2.2 2.5 2.4 C22:5 n-6 15.0 14.2 14.7 13.2 13.5 C22:5 n-3 44.4 42.0 33.3 40.5 38.3 C22:6 n-3 397.9 378.4 376.4 375.5 375.5 Sum of FAME 926.9 945.7 925.5 929.6 944.1

(109) TABLE-US-00026 TABLE 24 Isolated TAG Fatty Acid Profiles: PTA-10208 Final Oils (%) PTA- PTA- PTA- PTA- 10208 10208 10208 10208 Final Final Final Final PTA-10208 Oil #1 Oil #2 Oil #3 Oil #4 Final Oil #5 Fatty Acid % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.2 0.3 0.3 0.3 C14:0 1.8 1.6 0.3 0.3 0.3 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.2 20.0 21.2 C16:1 <0.1 <0.1 0.1 0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.1 C18:0 1.4 1.5 1.3 1.3 1.4 C18:1 n-9 2.6 8.9 2.9 2.8 3.6 C18:1 n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.3 0.8 0.4 0.4 0.4 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 0.2 0.2 0.2 0.2 0.2 C20:4 n-3 0.7 0.6 0.7 0.6 0.7 C20:5 n-3 19.0 17.6 21.5 22.7 21.6 C24:0 0.1 0.1 0.1 0.1 0.1 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.4 0.3 0.2 0.3 0.3 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.1 C22:6 n-3 42.9 40.0 40.7 40.4 39.8

(110) TABLE-US-00027 TABLE 25 Isolated Sterols/DAG Fatty Acid Profiles: PTA-10208 Final Oils (mg/g) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0 1.9 2.1 2.9 2.1 1.9 C14:0 9.9 9.5 9.7 10.3 8.0 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 2.4 2.3 2.2 2.3 2.0 C16:0 132.6 128.6 110.1 116.8 106.4 C16:1 0.2 0.3 <0.1 0.3 0.4 C17:0 0.3 0.2 0.3 0.3 0.3 C18:0 7.3 8.1 6.4 6.8 6.1 C18:1 n-9 15.0 55.1 47.4 19.0 30.1 C18:1 n-7 0.4 0.7 0.1 <0.1 0.2 C18:2 n-6 13.1 16.7 21.6 13.5 18.4 C20:0 2.0 2.1 1.2 1.8 1.4 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 2.3 2.4 2.4 2.4 2.0 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.6 1.0 0.5 0.6 0.5 C20:4 n-7 0.8 0.9 2.1 0.9 0.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 5.7 5.8 4.8 6.1 4.5 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.6 <0.1 C20:4 n-3 2.7 2.7 2.1 2.7 2.0 C20:5 n-3 92.9 94.5 91.9 111.6 84.8 C24:0 1.2 1.3 1.1 1.1 1.3 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 1.9 2.0 1.2 1.5 1.2 C22:5 n-6 7.8 8.0 6.7 7.0 5.5 C22:5 n-3 22.2 22.9 13.9 20.7 14.2 C22:6 n-3 246.3 252.7 223.5 240.3 196.3 Sum of FAME 569.3 619.8 552.1 568.7 488.2

(111) TABLE-US-00028 TABLE 26 Isolated Sterols/DAG Fatty Acid Profiles: PTA-10208 Final Oils (%) PTA- PTA- PTA- PTA- 10208 10208 10208 10208 Final Final Final Final PTA-10208 Oil #1 Oil #2 Oil #3 Oil #4 Final Oil #5 Fatty Acid % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.5 0.4 0.4 C14:0 1.7 1.5 1.8 1.8 1.6 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.4 0.4 0.4 C16:0 23.3 20.8 19.9 20.5 21.8 C16:1 <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.0 0.0 0.1 0.1 0.1 C18:0 1.3 1.3 1.2 1.2 1.2 C18:1 n-9 2.6 8.9 8.6 3.3 6.2 C18:1 n-7 0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 2.3 2.7 3.9 2.4 3.8 C20:0 0.4 0.3 0.2 0.3 0.3 C18:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:1 n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.4 0.4 0.4 0.4 0.4 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.1 0.1 0.4 0.2 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.0 0.9 0.9 1.1 0.9 C22:1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.1 <0.1 C20:4 n-3 0.5 0.4 0.4 0.5 0.4 C20:5 n-3 16.3 15.2 16.6 19.6 17.4 C24:0 0.2 0.2 0.2 0.2 0.3 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:1 n-9 0.3 0.3 0.2 0.3 0.2 C22:5 n-6 1.4 1.3 1.2 1.2 1.1 C22:5 n-3 3.9 3.7 2.5 3.6 2.9 C22:6 n-3 43.3 40.8 40.5 42.3 40.2

(112) TABLE-US-00029 TABLE 27 Lipid class separation by flash chromatography (wt %) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Lipid Class Separation Final Oil #1 Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 TAG 93.4 95.4 94.0 95.7 95.1 Sterols/DAG 3.1 2.9 2.6 3.0 2.9 Recovery (%) 96.5 98.3 96.6 98.7 98.0

(113) TABLE-US-00030 TABLE 28 Lipid class separation by HPLC-ELSD (wt %) Sterol Esters TAG FFA Sterols 1,3-DAG 1,2-DAG MAG Total PTA-10208 0.4 90.8 ND 0.8 0.5 0.5 N.D. 93.0 Final Oil #1 PTA-10208 0.4 88.5 ND 0.6 0.6 0.6 N.D. 90.7 Final Oil #2 PTA-10208 0.3 89.4 ND 0.8 0.6 0.5 N.D. 91.6 Final Oil #3 PTA-10208 0.3 88.0 ND 0.8 0.5 0.5 N.D. 90.1 Final Oil #4 PTA-10208 0.3 86.3 ND 0.7 0.8 0.5 N.D. 88.6 Final Oil #5 PTA-10208 0.36 100.76 ND 0.84 0.54 0.61 N.D. 103.11 Final Oil #6 ND = Not Detected
PTA-10212 Final Oil

(114) DHA was present in a PTA-10212 Final Oil at 41.63% and 366.9 mg/g, while EPA was present at 16.52%. Individual fatty acid profiles were determined and are summarized in Table 29.

(115) TABLE-US-00031 TABLE 29 Fatty Acid Profiles of PTA-10212 Final Oil (% FAME) Fatty Acid % FAME C6:0 ND C7:0 ND C8:0 ND C9:0 ND C10:0 ND C11:0 ND C12:0 ND C13:0 ND C14:0 0.84 C14:1 ND C15:0 1.33 C16:0 27.09 C16:1 1.03 C17:0 0.34 C17:1 ND C18:0 1.26 C18:1 n-9 2.14 C18:1 n-7 0.18 C19:0 ND C18:2 n-6 0.58 C20:0 0.32 C18:3 n-3 ND C20:1 n-9 ND C18:3 n-6 ND C20:2 n-6 0.26 C20:3 n-6 ND C22:0 0.14 C20:3 n-3 ND C20:4 n-6 1.34 C22:1 n-9 ND C23:0 ND C20:5 n-3 16.53 C24:0 0.53 C24:1 n-9 ND C22:5 n-6 1.50 C22:5 n-3 1.30 C22:6 n-3 41.63 Unknown 0.87 ND = Not Detected

EXAMPLE 6

(116) An analysis of the triacylglycerides (TAGs) of the PTA-10208 final oils described in Example 5 was performed using techniques described in Example 4. The identification of each fatty acid moiety was made, as summarized in Table 30 below.

(117) TABLE-US-00032 TABLE 30 Identification of TAG Species in PTA-10208 Final Oil PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final Oil #1 Final Oil #2 Final Oil #3 Final Oil #4 Final Oil #5 Identification Area % Area % Area % Area % Area % EPA/EPA/EPA 1.3 1.0 1.9 1.7 1.4 EPA/EPA/DHA 8.2 6.0 7.3 6.8 6.4 DHA/DHA/EPA 14.2 11.1 10.6 9.5 8.7 DHA/DHA/DHA 10.2 8.3 7.6 6.1 5.7 DPA/EPA/EPA 1.2 0.9 1.0 1.1 1.1 DHA/DPA/EPA 3.0 2.4 2.5 2.3 2.9 DHA/EPA/ARA 3.8 3.0 3.0 3.0 2.3 DHA/DPA/DHA DHA/DPA/EPA 2.3 1.9 1.5 1.6 1.7 DHA/DPA/DHA 1.7 1.2 1.1 1.2 1.2 EPA/14:0/DHA 1.1 1.0 1.8 1.8 1.5 DHA/DHA/14:0 1.1 1.0 1.4 1.3 1.3 EPA/EPA/16:0 2.3 2.3 3.4 3.9 3.3 DHA/16:0/EPA 12.1 12.5 12.9 14.0 13.4 DHA/16:0/DHA 16.1 16.8 17.5 14.8 17.2 EPA/EPA/18:0 2.1 2.0 1.7 2.1 2.7 DHA/DPA/16:0 3.0 3.3 2.3 2.9 2.7 DHA/16:0/ARA 1.6 1.6 1.8 2.0 2.2 DHA/16:0/DPA 1.3 2.1 1.4 1.5 2.5 DHA/18:0/DHA 0.8 0.8 0.7 0.8 1.0 DHA/14:0/16:0 0.6 1.0 1.3 1.4 1.3 EPA/16:0/16:0 0.9 1.1 1.5 1.7 2.0 DHA/16:0/16:0 3.6 4.8 5.5 5.7 6.5 18:0/16:0/DHA 0.4 0.8 0.7 0.8 1.2 18:1/18:1/18:1 0.6 4.0 1.0 1.4 1.6

EXAMPLE 7

(118) A two-day old inoculum flask of the isolated microorganisms deposited under ATCC Accession Nos. PTA-10208 and 10212 was prepared as a carbon and nitrogen-fed culture in media according to Tables 1 and 2.

(119) Mutagenesis was carried out according to following procedure:

(120) A sterile T=2 day old flask, approximately 50 ml, was poured into a sterile 40 ml glass homogenizer. The culture received 50 plunges in the homogenizer. The culture was pipeted out and filtered through a sterile 50 micron mesh filter, which was placed in a 50 ml sterile tube (the mesh was used as a means of retaining the larger clumps of colonies while letting the smaller clusters and single cells pass through the 50 micron mesh.). The entire concentrated macerate was collected in a sterile 50 ml tube. The macerated culture was vortexed and dilutions at levels up to 1:100 fold were made. The diluted macerate samples were vortexed prior to adding 200 l of inoculum to a media agar plate, 10015 mm, containing 4-5 glass beads (3 mm glass beads). Each plate was gently agitated in an effort to have the beads spread the inoculum evenly around the plate. Beads were dumped off of plates and plates were left to sit with covers on for approximately 5 minutes to dry. Lights in both the sterile hood and adjoining areas were turned off as the procedure was performed in dim light. There was minimal light available to be able to run the procedure but only indirect and dim.

(121) Five replicate plates were placed on the floor of the XL crosslinker (Spectronics Corporation, New York) with the lids off while the samples were irradiated. The crosslinker delivered power in terms of microjoules and a level was sought that achieved a 90%-95% Kill. Five replicate control plates were inoculated with un-mutagenized cells using the same protocol. These cell counts were used to calculate the % Kill. Once the irradiation was finished the plates were taken out, the lids were replaced, and the plates were wrapped in parafilm followed by a wrap in aluminum foil. It was imperative that the plates grew for the first week in the dark so that they were not able to repair the damaged genes.

(122) Plates were placed in a 22.5 C. room for about 10 days prior to counting the colonies. When final counts were made, individual colonies were picked with a sterile inoculating loop and re-streaked on new media plates. Each colony was plated on an individual plate. As plates grew dense a sample was taken, using a inoculating loop, and inoculated into a sterile 250 ml shake flask containing 50 ml of media. This flask was placed on a shaker at 200 rpm in a 22.5 C. room. On T=7 days the shake flask culture was harvested into a 50 ml sterile tube. The pH was taken and the sample was centrifuged to collect the biomass pellet. Each sample was rinsed and re-suspended in a 50:50 mixture of isopropyl alcohol and distilled water prior to being re-centrifuged. The collected pellet was freeze dried, weighed, and a FAME analysis was performed. The data in Tables 31 and 32 represents mutants produced with the above process from strains PTA-10208 and PTA-10212, respectively.

(123) TABLE-US-00033 TABLE 31 PTA-10208 Mutants control PTA- Mutant 1 Mutant 2 Mutant 3 Fatty Acids 10208 PTA-10209 PTA-10210 PTA-10211 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.11 0.10 0.22 0.19 % 12:1 0.00 0.00 0.00 0.00 % 13:0 0.19 0.19 0.15 0.16 % 13:1 0.00 0.00 0.00 0.00 % 14:0 1.94 1.82 2.98 2.59 % 14:1 0.00 0.00 0.00 0.00 % 15:1 2.66 2.22 1.76 1.66 % 16:0 24.87 24.97 23.71 25.01 % 16:1 0.20 0.25 0.07 0.07 % 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 1.49 1.21 0.62 0.66 % 18:0 1.13 1.14 0.91 1.01 % 18:1 n-9 0.07 0.07 0.06 0.06 % 18:1 n-7 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00 0.00 0.05 0.04 % 18:3 n-3 0.09 0.08 0.17 0.14 % 18:4 n-3 0.00 0.00 0.00 0.00 % 20:0 0.31 0.33 0.24 0.30 % 20:1 n-9 0.00 0.04 0.00 0.00 % 20:2 0.00 0.00 0.05 0.00 % 20:3 n-9 0.00 0.00 0.00 0.00 % 20:3 n-6 0.12 0.13 0.08 0.04 % 20:3 n-3 0.42 0.42 0.08 0.06 % 20:4 ARA 0.68 0.67 1.44 1.11 % 20:5 n-3 EPA 6.56 6.47 11.99 9.87 % 22:0 0.07 0.07 0.06 0.07 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.11 0.09 0.10 0.08 % 22:3 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 % 22:5 n-6 2.32 2.36 2.36 2.36 % 22:5 n-3 0.48 0.66 0.66 0.52 % 22:6 n-3 DHA 51.58 52.27 48.17 49.35 % 24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 % Fat 47.87 49.41 66.00 63.12 % Unknown 4.61 4.45 4.07 4.64

(124) TABLE-US-00034 TABLE 32 PTA-10212 Mutants Control PTA- Mutant 1 Mutant 2 Mutant 3 Fatty Acids 10212 PTA-10213 PTA-10214 PTA-10215 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.00 0.00 0.00 0.00 % 12:1 0.00 0.00 0.00 0.00 % 13:0 0.00 0.00 0.21 0.20 % 13:1 0.00 0.00 0.00 0.00 % 14:0 0.68 0.77 0.62 0.97 % 14:1 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.00 % 16:0 17.36 19.94 15.27 23.61 % 16:1 1.45 2.33 1.40 2.57 % 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 0.20 0.21 0.18 0.27 % 18:0 0.78 0.82 0.79 0.81 % 18:1 n-9 0.00 0.00 0.00 0.00 % 18:1 n-7 0.18 0.27 0.20 0.19 % 18:2 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 % 20:0 0.00 0.00 0.00 0.00 % 20:1 n-9 0.00 0.00 0.00 0.00 % 20:2 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 % 20:3 n-3 0.90 0.77 0.99 0.66 % 20:4 ARA 1.43 1.32 1.65 0.72 % 20:5 n-3 EPA 13.33 14.93 14.14 8.54 % 22:0 0.00 0.00 0.00 0.00 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 % 22:5 n-6 2.39 1.95 2.59 2.18 % 22:5 n-3 0.73 0.79 0.80 0.68 % 22:6 n-3 DHA 59.18 54.31 59.89 56.39 % 24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 % Fat 45.69 38.08 42.88 48.48 % Unknown 1.38 1.58 1.27 2.19

EXAMPLE 8

(125) An oil was prepared according to the method described in Example 5 wherein the oil was diluted with high oleic sunflower oil to achieve a combined DHA+EPA content of at least about 500 mg/g oil. A typical analysis and product specification of an oil made according to this Example is set forth in Table 33.

(126) TABLE-US-00035 TABLE 33 Specification Result Chemical Characteristics DHA Content mg/g oil Min. 320 382 EPA Content mg/g oil Min. 130 179 DHA + EPA Content mg/g oil Min. 500 561 Peroxide Value meq/kg Max. 5.0 0.1 Ansidine Value- Max. 20 4.8 Free Fatty Acid % Max. 0.25 0.1 Moisture and Volatiles % Max. 0.02 <0.01 Unsaponifiable Matter % Max. 4.5 1.1 Trans-fatty Acids % Max. 1 <1 ELEMENTAL COMPOSITION Arsenic ppm MAX 0.1 <0.1 Cadmium ppm MAX 0.1 <0.1 Copper ppm MAX 0.05 <0.02 Iron ppm MAX 0.2 <0.02 Lead ppm MAX 0.1 <0.1 Mercury ppm MAX 0.04 <0.01

(127) Other ingredients contained in the oil include less than 2% of Sunflower Lecithin; Rosemary Extract; Tocopherols and Ascorbyl Palmitate (as antioxidants).

EXAMPLE 9

(128) An oil was prepared according to the method described in Example 5 wherein the oil was diluted with high oleic sunflower oil to achieve a combined DHA+EPA content of at least about 400 mg/g oil. A typical analysis and product specification of an oil made according to this Example is set forth in Table 34.

(129) TABLE-US-00036 TABLE 34 Specification Result Chemical Characterstics DHA Content mg/g oil Min. 240 255 EPA Content mg/g oil Min. 120 155 DHA + EPA Content mg/g oil Min. 400 411 Peroxide Value meq/kg Max. 5.0 0.4 Ansidine Value- Max. 20 <1 Free Fatty Acid % Max. 0.25 0.1 Moisture and Volatiles % Max. 0.02 <0.01 Unsaponifiable Matter % Max. 4.5 0.9 Trans-fatty Acids % Max. 1 <1 ELEMENTAL COMPOSITION Arsenic ppm MAX 0.1 <0.1 Cadmium ppm MAX 0.1 <0.1 Copper ppm MAX 0.05 <0.02 Iron ppm MAX 0.2 0.0 Lead ppm MAX 0.1 <0.1 Mercury ppm MAX 0.04 <0.01

(130) Other ingredients contained in the oil include less than 2% of Sunflower Lecithin; Rosemary Extract; Tocopherols and Ascorbyl Palmitate (as antioxidants).

(131) In some embodiments, Examples 8 or 9 above are provided in a size 20 vegetarian gel capsule in a fill weight of about 999 mg to about 1105 mg oil with the gross weight of the capsule being about 1463 mg to about 1789 mg wherein the capsule has a rupture time of not more than 15 minutes and has a shelf life of about 24 months.

(132) All of the various aspects, embodiments, and options described herein can be combined in any and all variations.