U.S. patent application number 16/515074 was filed with the patent office on 2019-11-07 for extraction of lipid from cells and products therefrom.
The applicant listed for this patent is DSM IP Assets B.V.. Invention is credited to Kirk E. APT, Paul Warren BEHRENS, Stephen Robert CHERINKO, Jon Milton HANSEN, Robert Cody KERTIS, Neil Francis LEININGER, Joseph W. PFEIFER, III, Krishna RAMAN, Tracy Lynn STAHL, Nasrin TABAYEHNEJAD, Ross E. Zirkle.
Application Number | 20190338214 16/515074 |
Document ID | / |
Family ID | 45022637 |
Filed Date | 2019-11-07 |
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United States Patent
Application |
20190338214 |
Kind Code |
A1 |
CHERINKO; Stephen Robert ;
et al. |
November 7, 2019 |
EXTRACTION OF LIPID FROM CELLS AND PRODUCTS THEREFROM
Abstract
The present invention relates to processes for obtaining a lipid
from a cell by lysing the cell, contacting the cell with a base
and/or salt, and separating the lipid. The present invention is
also directed to a lipid prepared by the processes of the present
invention. The present invention is also directed to microbial
lipids having a particular anisidine value, peroxide value, and/or
phosphorus content.
Inventors: |
CHERINKO; Stephen Robert;
(Georgetown, SC) ; KERTIS; Robert Cody;
(Timmonsville, SC) ; APT; Kirk E.; (Ellicott City,
MD) ; BEHRENS; Paul Warren; (Ellicott City, MD)
; HANSEN; Jon Milton; (Temple, AZ) ; PFEIFER, III;
Joseph W.; (Westminster, MD) ; STAHL; Tracy Lynn;
(Pasadena, MD) ; Zirkle; Ross E.; (Mt. Airy,
MD) ; TABAYEHNEJAD; Nasrin; (Lexington, KY) ;
RAMAN; Krishna; (Wilmington, DE) ; LEININGER; Neil
Francis; (Winchester, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DSM IP Assets B.V. |
TE Heerlen |
|
NL |
|
|
Family ID: |
45022637 |
Appl. No.: |
16/515074 |
Filed: |
July 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15875733 |
Jan 19, 2018 |
10385289 |
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16515074 |
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13151014 |
Jun 1, 2011 |
10392578 |
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15875733 |
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61452721 |
Mar 15, 2011 |
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61378923 |
Aug 31, 2010 |
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61350363 |
Jun 1, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11B 1/10 20130101; Y02E
50/16 20130101; C11B 3/16 20130101; Y02E 50/10 20130101; C11B 3/02
20130101 |
International
Class: |
C11B 1/10 20060101
C11B001/10; C11B 3/16 20060101 C11B003/16; C11B 3/02 20060101
C11B003/02 |
Claims
1. A process for obtaining a lipid from a microbial cell
composition, comprising: raising the pH of the cell composition to
8 or above; and separating a lipid from the cell composition,
wherein the lipid contains less than 5% by weight of an organic
solvent.
2. The process of claim 1, wherein the raising the pH lyses the
cell composition.
3. The process of claim 1, wherein the raising the pH demulsifies
the cell composition.
4. The process of any of claims 1-3, further comprising adding a
salt to the cell composition to demulsify the cell composition.
5. The process of claim 4, wherein the adding a salt is performed
after the raising the pH.
6. The process of any of claims 1-5, further comprising heating the
cell composition to demulsify the cell composition.
7. The process of claim 6, wherein the heating is performed after
the raising the pH.
8. The process of any of claims 1-7, further comprising raising the
pH of the cell composition a second time to demulsify the cell
composition.
9. The process of claim 8, wherein the raising the pH a second time
is performed after the adding a salt or the heating.
10. A process for obtaining a lipid from a cell, comprising: lysing
a cell to form a lysed cell composition; raising the pH of the
lysed cell composition to 8 or above to demulsify the cell
composition; adding a salt to the lysed cell composition to
demulsify the cell composition; and separating a lipid from the
demulsified cell composition, wherein the lipid contains less than
5% by weight of an organic solvent.
11. A process for obtaining a lipid from a cell composition,
comprising: raising the pH of a cell composition to 8 or above to
lyse the cell composition and demulsify the cell composition;
adding a salt to the cell composition; and separating a lipid from
the demulsified cell composition, wherein the lipid contains less
than 5% by weight of an organic solvent.
12. The process of claim 10 or 11, further comprising heating the
lysed cell composition to demulsify the cell composition.
13. The process of claim 12, wherein the heating is performed after
the adding a salt.
14. The process of any of claims 1-13, further comprising agitating
the lysed cell composition to demulsify the cell composition.
15. The process of any of claims 10-14, further comprising raising
a pH of the lysed cell composition to demulsify the cell
composition.
16. The process of claim 15, wherein the raising the pH of the
lysed cell composition is performed after the adding a salt or the
heating.
17. The process of any of claims 1-16, wherein raising the pH
comprises adding a base.
18. The process of any of claims 1-17, wherein the base has a
pK.sub.b of 1 to 12.
19. The process of any of claims 14-18, wherein the process
comprises agitating the lysed cell composition by stirring, mixing,
blending, shaking, vibrating, or a combination thereof.
20. The process of any of claims 1-19, wherein the lysing comprises
mechanical treatment, physical treatment, chemical treatment,
enzymatic treatment, or a combination thereof.
21. The process of claim 20, wherein the mechanical treatment is
homogenization.
22. The process of any of claims 4-21, wherein the salt is added in
an amount of 0.1% to 20% by weight of the lysed cell
composition.
23. The process of any of claims 4-22, wherein the salt is selected
from the group consisting of: alkali metal salts, alkali earth
metal salts, sulfate salts, and combinations thereof.
24. The process of any of claims 1-23, wherein the separating
comprises centrifuging.
25. The process of any of claims 1-24, wherein the process provides
a lipid comprising at least 50% by weight triglyceride.
26. The process of any of claims 1-25, wherein the process provides
a lipid having an anisidine value of 26 or less.
27. The process of any of claims 10-26, wherein the cell is a
microbial cell.
28. The process of any of claims 1-9 and 27, further comprising
concentrating a fermentation broth comprising the microbial
cell.
29. The process of any of claims 10-26, wherein the cell is an
oilseed.
30. The process of claim 29, wherein the oilseed is selected from
the group consisting of sunflower seeds, canola seeds, rapeseeds,
linseeds, castor oil seeds, coriander seeds, calendula seeds, and
genetically modified variants thereof.
31. The process of any of claims 1-30, further comprising washing
the cell or cell composition.
32. The process of any of claims 1-31, further comprising
pasteurizing the cell or cell composition.
33. The process of any of claims 1-32, further comprising
concentrating the lysed cell composition.
34. The process of any of claims 1-33, further comprising refining
the lipid.
35. The process of claim 34, wherein the refining is selected from
the group consisting of: refining, degumming, acid treatment,
alkali treatment, cooling, heating, bleaching, deodorizing,
deacidification, and combinations thereof.
36. The process of any of claims 1-35, further comprising
harvesting the lipid, wherein the harvesting comprises pumping the
lipid without agitation.
37. A process for obtaining a lipid from a cell, comprising: lysing
a cell to form a lysed cell composition; agitating the cell
composition to demulsify the cell composition; and separating a
lipid from the demulsified cell composition, wherein the lipid
contains less than 5% by weight of an organic solvent.
38. A process of claim 37, wherein the agitating comprises
agitating the cell composition with an impeller having a tip speed
of 350 centimeters per second to 900 centimeters per second.
39. A lipid obtained by the process of any of claims 1-38.
40. The lipid of claim 39, wherein the lipid comprises one or more
polyunsaturated fatty acids.
41. The lipid of any of claims 39-40, wherein the lipid comprises
at least 35% by weight docosahexaenoic acid.
42. The lipid of any of claims 39-41, wherein the lipid has an
overall aroma intensity of 3 or less.
43. The lipid of any of claims 39-41, wherein the lipid has an
overall aromatic intensity of 2 or less.
44. The lipid of any of claims 39-43, wherein the lipid has an
anisidine value of 26 or less.
45. The lipid of any of claims 39-44, wherein the lipid has a
phosphorus content of 100 ppm or less.
46. The lipid of any of claims 39-45, wherein the lipid has a
peroxide value of 5 or less.
47. The lipid of claim 39, comprising at least 20% by weight
eicosapentaenoic acid and less than 5% by weight each of
arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic
acid, linolenic acid, eicosenoic acid, erucic acid, and stearidonic
acid.
48. The lipid of claim 39, comprising a triacylglycerol fraction of
at least 10% by weight, wherein at least 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, and wherein less
than 5% by weight of the fatty acids in the triacylglycerol
fraction is arachidonic acid.
49. The lipid of claim 47 or 48, wherein the lipid has a peroxide
value of 5 or less.
50. The lipid of any of claims 39-49, wherein the lipid is a crude
oil.
51. A crude microbial lipid having an anisidine value of 26 or
less, a peroxide value of 5 or less, a phosphorus content of 100
ppm or less, and less than 5% by weight of an organic solvent.
52. An extracted microbial lipid comprising a triglyceride fraction
of at least 70% by weight, wherein the docosahexaenoic acid content
of the triglyceride fraction is at least 50% by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, and wherein the oil
has an anisidine value of 26 or less.
53. An extracted microbial lipid comprising a triglyceride fraction
of at least 70% by weight, wherein the docosahexaenoic acid content
of the triglyceride fraction is at least 40% by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, wherein the ratio of
docosahexaenoic acid to docosapentaenoic acid n-6 is greater than
6:1, and wherein the oil has an anisidine value of 26 or less.
54. An extracted microbial lipid comprising a triglyceride fraction
of at least about 70% by weight, wherein the docosahexaenoic acid
content of the triglyceride fraction is at least 60% by weight and
wherein the oil has an anisidine value of 26 or less.
55. The extracted microbial lipid of any of claims 52-54, wherein
the oil has a peroxide value of 5 or less.
56. The extracted microbial lipid of any of claims 52-55, wherein
the oil has a phosphorus content of 100 ppm or less.
57. The extracted microbial lipid of any of claims 52-56, wherein
the lipid is a crude oil.
58. A process for obtaining a lipid, the process comprising
refining a crude lipid of any of claims 50, 51, or 57.
59. The process of claim 58, wherein the refining is selected from
the group consisting of: caustic refining, degumming, acid
treatment, alkali treatment, bleaching, deodorizing,
deacidification, and combinations thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Application No.
61/350,363, filed on Jun. 1, 2010, U.S. Application No. 61/378,923,
filed Aug. 31, 2010, and U.S. Application No. 61/452,721, filed
Mar. 15, 2011, each of which is hereby incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to processes for obtaining a
lipid from a cell by lysing the cell, raising a pH of the cell
and/or contacting the cell with a salt, and separating the lipid.
The present invention is also directed to lipids prepared by the
processes of the present invention. The present invention is also
directed to microbial lipids having a particular anisidine value,
peroxide value, and/or phosphorus content.
Background Art
[0003] A typical process for obtaining lipids from a microbial
cell, such as polyunsaturated fatty acids, involves growing
microorganisms that are capable of producing the desired lipid in a
fermentor, pond or bioreactor, separating the fermentation broth
comprising a microbial cell biomass, drying the microbial cell
biomass, and separating the lipids by solvent extraction. Steps in
the separation can include diluting a fermentation broth with
water, centrifuging the diluted broth, lysing the microbial cells,
and extracting an intracellular lipid from the lysed cells by
adding a water-immiscible solvent to the mixture in which the lipid
is soluble, e.g., hexane.
[0004] Another method of extraction to remove a lipid from a
microbial cell is lysing a cell in a fermentation broth using
mechanical force (e.g., homogenization), enzymatic treatment, or
chemical treatment to disrupt the cell walls. Lipid can be
extracted from the resulting composition comprising lipids,
microbial cell biomass, and water using an organic solvent, e.g.,
isopropyl alcohol. The lipid can be separated mechanically from the
composition and the alcohol must be removed from both the lipid and
the aqueous biomass waste stream. See, e.g., International Pub.
Nos. WO 01/76385 and WO 01/76715.
[0005] However, industrial scale production of lipids using either
of the above processes requires a large amount of volatile and
flammable organic solvent, thereby creating hazardous operating
conditions. The use of organic solvents in the extraction process
can also necessitate using an explosion-proof lipid recovery
system, thereby adding to the cost of lipid recovery. Moreover, use
of an organic solvent in extracting lipid from a microbial cell can
generate an organic solvent waste stream that requires a complete
solvent recovery system or a proper method of disposal, which
further increases the overall production cost of lipid extraction.
For example, strict limits on volatile organic compound (VOC)
emissions require greater manpower and added cost to vessels and
other equipment.
[0006] Therefore, there is a need for a process for obtaining
lipids from a cell which does not use an organic solvent. Several
processes have been proposed for separating a lipid from a cell
without the use of an organic solvent. For example, U.S. Pat. No.
6,750,048 discloses an aqueous washing process whereby an emulsion
is washed with aqueous washing solutions until a substantially
non-emulsified lipid is obtained. However, in some embodiments,
this process requires multiple washing steps, which require
substantial cost and time. U.S. Pat. No. 7,431,952 discloses a
process whereby lysed cells are centrifuged to remove cell wall
debris and then oils are extracted and purified. However, this
process provides a crude oil that requires extensive further
purification. Thus, what is needed is a process that does not
utilize a volatile solvent to extract a lipid from a cell, and
which can be performed using readily available equipment and a
minimum number of steps to provide a highly pure lipid.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is directed to a process for obtaining
a lipid from a microbial cell composition, the process comprising
raising the pH of the cell composition to 8 or above, and
separating a lipid from the cell composition, wherein the lipid
optionally contains less than 5% by weight or volume of an organic
solvent.
[0008] In some embodiments, the raising the pH lyses the cell
composition. In some embodiments, the raising the pH demulsifies
the cell composition.
[0009] In some embodiments, the process comprises adding a salt to
the cell composition to demulsify the cell composition. In some
embodiments, the adding a salt is performed after the raising the
pH.
[0010] In some embodiments, the process further comprises heating
the lysed cell composition to demulsify the cell composition. In
some embodiments, the heating is performed after the raising the
pH.
[0011] In some embodiments, the process further comprises raising
the pH of the cell composition a second time to demulsify the cell
composition. In some embodiments, the raising the pH a second time
is performed after the adding a salt or the heating.
[0012] The present invention is also directed to a process for
obtaining a lipid from a cell, the process comprising lysing a cell
to form a lysed cell composition, raising the pH of the lysed cell
composition to 8 or above to demulsify the cell composition, adding
a salt to the lysed cell composition to demulsify the cell
composition, and separating a lipid from the demulsified cell
composition, wherein the lipid optionally contains less than 5% by
weight or volume of an organic solvent.
[0013] The present invention is directed to a process for obtaining
a lipid from a cell composition, the process comprising raising the
pH of the cell composition to 8 or above to lyse the cell
composition and demulsify the cell composition, adding a salt to
the cell composition, and separating a lipid from the demulsified
cell composition, wherein the lipid optionally contains less than
5% by weight or volume of an organic solvent.
[0014] The present invention is also directed to a process for
obtaining a lipid from a cell, the process comprising lysing a cell
to form a lysed cell composition, agitating the cell composition to
demulsify the cell composition, and separating a lipid from the
demulsified cell composition, wherein the lipid optionally contains
less than 5% by weight or volume of an organic solvent.
[0015] In some embodiments, the process further comprises heating
the lysed cell composition to demulsify the cell composition. In
some embodiments, the heating is performed after the adding a
salt.
[0016] In some embodiments, the process further comprises agitating
the lysed cell composition to demulsify the cell composition. In
some embodiments, the agitating is for 5 minutes to 96 hours.
[0017] In some embodiments, the agitating comprises agitating the
cell composition with an impeller having a tip speed of 350
centimeters per second to 900 centimeters per second.
[0018] In some embodiments, the process further comprises raising
the pH of the lysed cell composition to demulsify the cell
composition. In some embodiments, raising the pH of the lysed cell
composition to demulsify the cell composition comprises adding a
base. In some embodiments, a second base is added after the adding
of a salt or the heating.
[0019] In some embodiments, the heating is for 10 minutes to 96
hours.
[0020] In some embodiments, the cell composition is heated to a
temperature of 60.degree. C. to 100.degree. C. In some embodiments,
the cell composition is heated to a temperature of 90.degree. C. to
100.degree. C.
[0021] In some embodiments, raising the pH comprises adding a base.
In some embodiments, the base has a pK.sub.b of 1 to 12.
[0022] In some embodiments, the separating a lipid occurs at a
temperature of 10.degree. C. to 100.degree. C.
[0023] In some embodiments, the process comprises agitating the
lysed cell composition by stirring, mixing, blending, shaking,
vibrating, or a combination thereof. In some embodiments, the
process comprises agitating the lysed cell composition at 0.1
hp/1000 gal to 10 hp/1000 gal of lysed cell composition. In some
embodiments, the process comprises agitating the lysed cell
composition with an agitator having an impeller tip speed of 200
ft/min to 1,000 ft/min.
[0024] In some embodiments, the lysing comprises mechanical
treatment, physical treatment, chemical treatment, enzymatic
treatment, or a combination thereof. In some embodiments, the
mechanical treatment is homogenization.
[0025] In some embodiments, the salt is added in an amount of 0.1%
to 20% by weight of the lysed cell composition. In some
embodiments, the salt is added to the lysed cell composition in an
amount of 0.5% to 15% by weight of the lysed cell composition. In
some embodiments, the salt is added to the lysed cell composition
in an amount of 2% to 10% by weight of the lysed cell
composition.
[0026] In some embodiments, the salt is selected from the group
consisting of: alkali metal salts, alkali earth metal salts,
sulfate salts, and combinations thereof. In some embodiments, the
salt is sodium chloride. In some embodiments, the salt is sodium
sulfate.
[0027] In some embodiments, the separating comprises centrifuging.
In some embodiments, the separating comprises centrifuging at a
temperature of 30.degree. C. to 90.degree. C.
[0028] In some embodiments, the process provides a lipid comprising
at least 50% by weight triglyceride.
[0029] In some embodiments, the process provides a lipid having an
anisidine value of 26 or less, 25 or less, 20 or less, 15 or less,
10 or less, 5 or less, 2 or less, or 1 or less.
[0030] In some embodiments, the process provides a lipid having a
peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3
or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or
less, 0.2 or less, or 0.1 or less.
[0031] In some embodiments, the process provides a lipid having a
phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or
less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or
less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less.
[0032] In some embodiments, the process provides a lipid having at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, or at least 50% by weight
of a desired polyunsaturated fatty acid (PUFA). In some
embodiments, the process provides a lipid having at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, or at least 50% by weight of
docosahexaenoic acid ("DHA"), and/or at least 10%, at least 15%, or
at least 20% by weight of docosapentaenoic acid ("DPA n-6"), and/or
at least 10%, at least 15%, or at least 20% by weight of
eicosapentaenoic acid ("EPA"), and/or at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least
40%, at least 45%, or at least 50% by weight of arachidonic acid
("ARA").
[0033] In some embodiments, the cell is a microbial cell. In some
embodiments, the process comprises concentrating a fermentation
broth comprising the microbial cell.
[0034] In some embodiments, the cell is an oilseed. In some
embodiments, the oilseed is selected from the group consisting of
sunflower seeds, canola seeds, rapeseeds, linseeds, castor oil
seeds, coriander seeds, calendula seeds, and genetically modified
variants thereof.
[0035] In some embodiments, the process comprises washing the cell
or cell composition.
[0036] In some embodiments, the process comprises pasteurizing the
cell or cell composition.
[0037] In some embodiments, the process comprises concentrating the
lysed cell composition.
[0038] In some embodiments, the process comprises refining the
lipid. In some embodiments, the refining is selected from the group
consisting of: caustic refining, degumming, acid treatment, alkali
treatment, cooling, heating, bleaching, deodorizing,
deacidification, and combinations thereof.
[0039] In some embodiments, the process comprises harvesting the
lipid, wherein the harvesting comprises pumping the lipid without
agitation.
[0040] The present invention is also directed to a lipid obtained
by any of the processes of the present invention.
[0041] In some embodiments, the lipid comprises one or more
polyunsaturated fatty acids. In some embodiments, the lipid has at
least 10%, at least 15%, at least 200%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, or at least 50% by weight
of a desired PUFA. In some embodiments, the lipid has at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 450%, or at least 50% by weight of DHA,
and/or at least 10%, at least 15%, or at least 20% by weight of DPA
n-6, and/or at least 10%, at least 15%, or at least 20% by weight
of EPA, and/or at least 100%, at least 15%, at least 20%, at least
25%, at least 30%, at least 35%, at least 40%, at least 45%, or at
least 50% by weight of ARA.
[0042] In some embodiments, the lipid has an overall aroma
intensity of 3 or less. In some embodiments, the lipid has an
overall aromatic intensity of 2 or less.
[0043] In some embodiments, the lipid comprises a triacylglycerol
fraction of at least 10% by weight, wherein at least 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, and
wherein less than 5% by weight of the fatty acids in the
triacylglycerol fraction is arachidonic acid.
[0044] In some embodiments, the lipid comprises at least 20% by
weight eicosapentaenoic acid and less than 5% by weight each of
arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic
acid, linolenic acid, eicosenoic acid, erucic acid, and stearidonic
acid.
[0045] In some embodiments, the lipid has an anisidine value of 26
or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less,
2 or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5
or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less.
[0046] In some embodiments, the lipid is a crude lipid. In some
embodiments, the crude lipid optionally has less than 5% by weight
or volume of an organic solvent.
[0047] The present invention is also directed to a crude microbial
lipid having an anisidine value of 26 or less, a peroxide value of
5 or less, a phosphorus content of 100 ppm or less, and optionally
less than 5% by weight or volume of an organic solvent.
[0048] In some embodiments, the crude microbial lipid has an
anisidine value of 26 or less, 25 or less, 20 or less, 15 or less,
10 or less, 5 or less, 2 or less, or 1 or less, and/or a peroxide
value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less,
2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or
less, or 0.1 or less, and/or a phosphorus content of 100 ppm or
less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or
less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or
less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or
less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or
less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less,
3 ppm or less, 2 ppm or less, or 1 ppm or less.
[0049] In some embodiments, the crude microbial lipid has at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, or at least 50% by weight of
a desired PUFA. In some embodiments, the crude microbial lipid has
at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, or at least 50% by
weight of DHA, and/or at least 10%, at least 15%, or at least 20%
by weight of DPA n-6, and/or at least 10%, at least 15%, or at
least 20% by weight of EPA, and/or at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, or at least 50% by weight of ARA.
[0050] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least 70%
by weight, wherein the docosahexaenoic acid content of the
triglyceride fraction is at least 500 by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, and wherein the oil
has an anisidine value of 26 or less.
[0051] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least 70%
by weight, wherein the docosahexaenoic acid content of the
triglyceride fraction is at least 40% by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, wherein the ratio of
docosahexaenoic acid to docosapentaenoic acid n-6 is greater than
6:1, and wherein the oil has an anisidine value of 26 or less.
[0052] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least
about 70% by weight, wherein the docosahexaenoic acid content of
the triglyceride fraction is at least 60% by weight and wherein the
oil has an anisidine value of 26 or less.
[0053] In some embodiments, the extracted lipid has an anisidine
value of 26 or less, 25 or less, 20 or less, 15 or less, 10 or
less, 5 or less, 2 or less, or 1 or less, and/or a peroxide value
of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5
or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or
less, or 0.1 or less, and/or a phosphorus content of 100 ppm or
less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or
less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or
less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or
less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or
less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less,
3 ppm or less, 2 ppm or less, or 1 ppm or less.
[0054] In some embodiments, the extracted microbial lipid is a
crude lipid or a crude oil. In some embodiments, the crude lipid
optionally has less than 5% by weight or volume of an organic
solvent.
[0055] The present invention is also directed to a process for
obtaining a lipid, the process comprising refining a crude lipid of
the present invention. In some embodiments, the refining is
selected from the group consisting of: caustic refining, degumming,
acid treatment, alkali treatment, cooling, heating, bleaching,
deodorizing, deacidification, and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0056] The accompanying drawings, which are incorporated herein and
form a part of the specification, illustrate the present invention
and, together with the description, further serve to explain the
principles of the invention and to enable a person skilled in the
pertinent art to make and use the invention.
[0057] FIGS. 1-4 provide schematic flow charts describing processes
of the present invention.
[0058] FIG. 5 is a graph providing the electron paramagnetic
resonance (EPR) over time of lysed cells compositions at various
pHs.
[0059] The present invention will now be described with reference
to the accompanying drawings. In the drawings, like reference
numbers indicate identical or functionally similar elements.
Additionally, the left-most digit(s) of a reference number can
identify the drawing in which the reference number first
appears.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention is directed to a process for obtaining
a lipid from a microbial cell composition, the process comprising
raising the pH of the cell composition to 8 or above and separating
a lipid from the cell composition, wherein the lipid optionally
contains less than 5% by weight or volume of an organic solvent. In
some embodiments, the process further comprises one or more of
adding a salt to the cell composition to demulsify the cell
composition, heating the cell to demulsify the cell composition,
agitating the cell composition to demulsify the cell composition,
and raising the pH of the cell composition a second time to
demulsify the cell composition.
[0061] The present invention is also directed to a process for
obtaining a lipid from a cell, the process comprising lysing a cell
to form a lysed cell composition, raising the pH of the lysed cell
composition to 8 or above to demulsify the cell composition, adding
a salt to the lysed cell composition to demulsify the cell
composition, and separating a lipid from the demulsified cell
composition, wherein the lipid optionally contains less than 5% by
weight or volume of an organic solvent. The cell can be a microbial
cell or an oilseed cell. In some embodiments, the process further
comprises one or more of: heating the lysed cell composition to
demulsify the cell composition, agitating the lysed cell
composition to demulsify the cell composition, and raising the pH
of the lysed cell composition a second time to demulsify the cell
composition.
[0062] The present invention is directed to a process for obtaining
a lipid from a cell composition, the process comprising raising the
pH of the cell composition to 8 or above to lyse the cell
composition and demulsify the cell composition, adding a salt to
the cell composition, and separating a lipid from the demulsified
cell composition, wherein the lipid optionally contains less than
5% by weight or volume of an organic solvent. In some embodiments,
the process further comprises one or more of heating the cell
composition to demulsify the cell composition, agitating the cell
composition to demulsify the cell composition, and raising the pH
of the cell composition a second time to demulsify the cell
composition.
[0063] The present invention is directed to a process for obtaining
a lipid from a microbial cell, the process comprising lysing a
microbial cell to form a lysed cell composition, adding a base to
the lysed cell composition to demulsify the cell composition, and
separating a lipid from the demulsified cell composition, wherein
the lipid optionally contains less than 5% by weight or volume of
an organic solvent. In some embodiments, the process further
comprises one or more of: adding a salt to the lysed cell
composition to demulsify the cell composition, heating the lysed
cell composition to demulsify the cell composition, agitating the
lysed cell composition to demulsify the cell composition, and
adding a second base to the lysed cell composition to demulsify the
cell composition.
[0064] The present invention is also directed to a process for
obtaining a lipid from a cell, the process comprising lysing a cell
to form a lysed cell composition, adding a base to the lysed cell
composition to demulsify the cell composition, adding a salt to the
lysed cell composition to demulsify the cell composition, and
separating a lipid from the demulsified cell composition, wherein
the lipid optionally contains less than 5% by weight or volume of
an organic solvent. The cell can be a microbial cell or an oilseed
cell. In some embodiments, the process further comprises one or
more of: heating the lysed cell composition to demulsify the cell
composition, agitating the lysed cell composition to demulsify the
cell composition, and adding a second base to the lysed cell
composition to demulsify the cell composition.
[0065] The present invention is also directed to a process for
obtaining a lipid from a cell, the process comprising lysing a cell
to form a lysed cell composition, agitating the cell composition to
demulsify the cell composition, and separating a lipid from the
demulsified cell composition, wherein the lipid optionally contains
less than 5% by weight or volume of an organic solvent.
[0066] The present invention is also directed to a lipid obtained
by any of the processes of the present invention.
[0067] The present invention is also directed to an extraction
process for obtaining a lipid from a cell, the process comprising
lysing the cell to form a lysed cell composition, contacting the
lysed cell composition with a first base, contacting the lysed cell
composition with a salt, heating the lysed cell composition for 5
minutes to 96 hours, contacting the lysed cell composition with a
second base, and separating a lipid from the lysed cell composition
at a temperature of 10.degree. C. to 100.degree. C.
[0068] The present invention is also directed to an extraction
process for obtaining a lipid from a cell, the process comprising
lysing the cell to form a lysed cell composition, contacting the
lysed cell composition with a salt, and agitating the lysed cell
composition for 5 minutes to 96 hours to provide a treated lysed
cell composition, and separating a lipid from the treated lysed
cell composition at a temperature of 10.degree. C. to 100.degree.
C.
[0069] The present invention is also directed to an extraction
process for obtaining a lipid from a cell, the process comprising
lysing the cell to form a lysed cell composition, contacting the
lysed cell composition with a salt, and separating a lipid from the
lysed cell composition at a temperature of 10.degree. C. to
100.degree. C.
[0070] In some embodiments, the base or second base have a pK.sub.b
of 1 to 12. In some embodiments, the base or second base have a
pK.sub.b of 3 to 5.
[0071] In some embodiments, a process comprises agitating the lysed
cell composition for 5 minutes to 96 hours, 10 minutes to 96 hours,
10 minutes to 4 hours, 12 hours to 84 hours, or 24 hours to 72
hours.
[0072] In some embodiments, the process comprises agitating the
lysed cell composition by stirring, mixing, blending, shaking,
vibrating, or a combination thereof. In some embodiments, the
process comprises agitating the lysed cell composition at 0.1
hp/1000 gal to 10 hp/1000 gal of lysed cell composition. In some
embodiments, the process comprises agitating the lysed cell
composition with an agitator having an impeller tip speed of 200
ft/min to 1000 ft/min.
[0073] In some embodiments, lysing comprises a process selected
from: mechanically treating, physically treating, chemically
treating, enzymatically treating, or a combination thereof.
[0074] In some embodiments, the lysed cell composition is contacted
with a salt in an amount of 0.1% to 20% by weight, 0.5% to 15% by
weight, or 2% to 10% by weight of the lysed cell composition.
[0075] In some embodiments, the salt is selected from the group
consisting of: alkali metal salts, alkali earth metal salts,
sulfate salts and combinations thereof. In some embodiments, the
salt is sodium chloride. In some embodiments, the salt is sodium
sulfate.
[0076] In some embodiments, the process comprises heating the lysed
cell composition for 5 minutes to 96 hours, 10 minutes to 4 hours,
12 hours to 84 hours, or 24 hours to 72 hours.
[0077] In some embodiments, the separating comprises centrifuging.
In some embodiments, the separating comprises centrifuging at a
temperature of 10.degree. C. to 100.degree. C.
[0078] In some embodiments, the process comprises prior to the
lysing: washing, centrifuging, evaporating, or a combination
thereof, a broth that includes the cell.
[0079] In some embodiments, the process provides a lipid having an
anisidine value of 15 or less. In some embodiments, the process
provides a lipid comprising at least 50% by weight
triglycerides.
[0080] In some embodiments, the process does not add an organic
solvent to the lysed cell composition. Organic solvents include
polar solvents, non-polar solvents, water-miscible solvents,
water-immiscible solvents, and combinations thereof.
[0081] In some embodiments, the process comprises concentrating a
broth comprising a cell. In some embodiments, the process comprises
concentrating the lysed cell composition.
[0082] The present invention is also directed to the lipid prepared
by a process described herein. In some embodiments, the lipid
comprises one or more polyunsaturated fatty acids. In some
embodiments, the lipid has at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, or at least 50% by weight of a desired PUFA. In some
embodiments, the lipid has at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, or at least 50% by weight of DHA, and/or at least 10%,
at least 15%, or at least 20% by weight of DPA n-6, and/or at least
10%, at least 15%, or at least 20% by weight of EPA, and/or at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, or at least 50% by weight
of ARA. In some embodiments, the lipid has an anisidine value of 26
or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less,
2 or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5
or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less.
[0083] In some embodiments, the lipid comprises a triacylglycerol
fraction of at least 10% by weight, wherein at least 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, and
wherein less than 5% by weight of the fatty acids in the
triacylglycerol fraction is arachidonic acid. In some embodiments,
the lipid has an anisidine value of 26 or less, 25 or less, 20 or
less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less,
and/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5
or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less,
0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphorus
content of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm
or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or
less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or
less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or
less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or
less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or
less. In some embodiments, the lipid is a crude lipid.
[0084] In some embodiments, the lipid comprises at least 20% by
weight eicosapentaenoic acid and less than 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 lipid has an anisidine value of 26
or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less,
2 or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5
or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, the lipid is a crude
oil.
[0085] The present invention is also directed to a crude microbial
lipid having an anisidine value of 26 or less, a peroxide value of
5 or less, a phosphorus content of 100 ppm or less, and optionally
less than 5% by weight or volume of an organic solvent. In some
embodiments, the crude microbial lipid has an anisidine value of 26
or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less,
2 or less or 1 or less, and/or a peroxide value of 5 or less, 4.5
or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, the crude microbial lipid
has at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, or at least
50% by weight of a desired PUFA. In some embodiments, the crude
microbial lipid has at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
or at least 50% by weight of DHA, and/or at least 10%, at least
15%, or at least 20% by weight of DPA n-6, and/or at least 10%, at
least 15%, or at least 20% by weight of EPA, and/or at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, or at least 50% by weight of
ARA.
[0086] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least 70%
by weight, wherein the docosahexaenoic acid content of the
triglyceride fraction is at least 50% by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, and wherein the oil
has an anisidine value of 26 or less. In some embodiments, the
extracted lipid has an anisidine value of 26 or less, 25 or less,
20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or
less, and/or a peroxide value of 5 or less, 4.5 or less, 4 or less,
3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or
less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphorus
content of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm
or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or
less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or
less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or
less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or
less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or
less. In some embodiments, the extracted lipid is a crude
lipid.
[0087] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least 70%
by weight, wherein the docosahexaenoic acid content of the
triglyceride fraction is at least 40% by weight, wherein the
docosapentaenoic acid n-6 content of the triglyceride fraction is
from at least 0.5% by weight to 6% by weight, wherein the ratio of
docosahexaenoic acid to docosapentaenoic acid n-6 is greater than
6:1, and wherein the oil has an anisidine value of 26 or less. In
some embodiments, the extracted lipid has an anisidine value of 26
or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less,
2 or less, or 1 or less, and/or a peroxide value of 5 or less, 4.5
or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, the extracted lipid is a
crude lipid.
[0088] The present invention is also directed to an extracted
microbial lipid comprising a triglyceride fraction of at least
about 70% by weight, wherein the docosahexaenoic acid content of
the triglyceride fraction is at least 60/o by weight and wherein
the oil has an anisidine value of 26 or less. In some embodiments,
the extracted lipid has an anisidine value of 26 or less, 25 or
less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or
1 or less, and/or a peroxide value of 5 or less, 4.5 or less, 4 or
less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less,
1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a
phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or
less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or
less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, the extracted lipid is a
crude lipid.
[0089] The present invention is also directed to a crude lipid
extracted from a microorganism of the species Crypthecodinium
cohnii, having a phosphorus content of 100 ppm or less. In some
embodiments, the crude lipid has an anisidine value of 26 or less,
25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or
less, or 1 or less, and/or a peroxide value of 5 or less, 4.5 or
less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less.
[0090] The present invention is also directed to a process for
obtaining a lipid, the process comprising refining a crude lipid of
the present invention. In some embodiments, the refining is
selected from the group consisting of: caustic refining, degumming,
acid treatment, alkali treatment, cooling, heating, bleaching,
deodorizing, deacidification, and combinations thereof.
Overview
[0091] Generally, the processes of the present invention do not
utilize an organic solvent in order to extract or otherwise
separate a lipid. Thus, in some embodiments, an organic solvent is
not added to a cell broth comprising plant material or fermentation
broth comprising a microbial cell, is not added to a cell
composition, is not added to a lysed cell composition, or is not
added to a lipid during a process of the present invention in an
amount or concentration sufficient to extract a lipid. In some
embodiments, an organic solvent can be added to a cell composition,
a lysed cell composition, or a demulsified cell composition. In
such embodiments, the organic solvent is added in a concentration
less than 5%, less than 4%, less than 3%, less than 2%, less than
1%, less than 0.5%, less than 0.1%, or less than 0.05% by volume.
As used herein, an "organic solvent" refers to a solvent that
includes at least one carbon atom. As used herein, "solvent" refers
to an agent that is hydrophobic or lipophilic, and is not a lipid.
As used herein, "hydrophobic" refers to an agent that is repelled
from a mass of water. As used herein, "lipophilic" refers to an
agent that dissolves lipids. Organic solvents that are not used in
a process of the present invention include, but are not limited to,
polar solvents, non-polar solvents, water-miscible solvents,
water-immiscible solvents, and combinations thereof. Non-limiting
examples of organic solvents include substituted and unsubstituted
C.sub.4-C.sub.8 alkyls (e.g., hexane and the like),
C.sub.5-C.sub.12 cylcolalkyls, C.sub.4-C.sub.12 alkenes,
C.sub.1-C.sub.8 alcohols (e.g., iso-propanol and the like),
C.sub.1-C.sub.8 aldehydes, C.sub.4--C ethers, C.sub.1-C.sub.8
esters, C.sub.6-C.sub.12 aryls, C.sub.1-C.sub.8 amides,
C.sub.5-C.sub.12 heteroaryls, and combinations thereof. An organic
solvent as defined herein can be optionally added to a lysed cell
composition, for example, as a component of a base and/or a salt
for contacting with the lysed cell composition. However, in such
embodiments the organic solvent is present in a concentration such
that the lipid is not substantially extracted from the cell
composition, lysed cell composition, or demulsified cell
composition by the solvent (i.e., in a concentration of less than
5%, less than 4%, less than 3%, less than 2%, less than 1%, less
than 0.5%, less than 0.1%, or less than 0.05% by volume or
weight).
[0092] In some embodiments, a process of the present invention does
not include washing, e.g., with water, or the process reduces the
number of washings of, a lysed cell composition or a demulsified
cell composition. "Washing" refers to a process of diluting a
composition with, e.g., water or buffer and removing the water or
buffer, e.g., by centrifugation. Washing a cell composition can
decrease the overall yield of a lipid obtained from a cell. In the
present invention, the washing can be decreased by 1 time, 2 times,
3 times or more.
Definitions
[0093] As used herein, "lipid" or "oil" refers to one or more fatty
acids (including free fatty acids and esters of fatty acids),
phospholipids, triacylglycerols (i.e. triglycerides),
diacylglycerides, monoacylglycerides, lysophospholipids, soaps,
phosphatides, waxes, sterols and sterol esters, carotenoids,
xanthophylls, hydrocarbons, and other lipids known to one of
ordinary skill in the art. Lipids include polar lipids and neutral
lipids.
[0094] As used herein, "polar lipid" refers to lipids that contain
a polar group and are more readily soluble in polar solvents. Polar
lipids include phospholipids. As used herein, "phospholipid" refers
to lipids having a phosphate group. As used herein, "neutral lipid"
refers to lipids that do not contain areas of polarity and are more
readily soluble in non-polar solvents. Neutral lipids include
triacylglycerols (TAG).
[0095] 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. Fatty acids 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 monounsaturated when only one double bond is present and are
polyunsaturated when more than one double bond is present.
[0096] Fatty acids present in the lipid can have 4 to 28 carbon
atoms. In some embodiments, a lipid comprises one or more
polyunsaturated fatty acids. 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
carbons and 6 double bonds, often designated as "22:6 n-3." For the
purposes of this application, long chain polyunsaturated fatty
acids (LC-PUFAs) are defined as fatty acids of 18 and more carbon
chain length, and are preferably fatty acids of 20 or more carbon
chain length, containing 3 or more double bonds. LC-PUFAs of the
omega-6 series include, but are not limited to,
di-homo-gammalinoleic acid (C20:3n-6), arachidonic acid (C20:4n-6)
("ARA"), docosatetraenoic acid or adrenic acid (C22:4n-6), and
docosapentaenoic acid (C22:5n-6) ("DPA n-6"). The LC-PUFAs of the
omega-3 series include, but are not limited to, eicosatrienoic acid
(C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid
(C20:5n-3) ("EPA"), docosapentaenoic acid (C22:5n-3), and
docosahexaenoic acid (C22:6n-3). The LC-PUFAs also include fatty
acids with greater than 22 carbons and 4 or more double bonds
including, but not limited to, C24:6(n-3) and C28:8(n-3).
[0097] The terms "fatty acid," "polyunsaturated fatty acid," and
"PUFA" include not only the free fatty acid form, but other forms
as well, such as the triacylglycerol (TAG) form, the phospholipid
(PL) form and other esterified forms. As used herein, the terms
"ester" and "esterified" refer to the replacement of the hydrogen
in the carboxylic acid group of a PUFA molecule with another
substituent. Typical esters are known to those in the art, a
discussion of which is provided by Higuchi, T. et al., Pro-drugs as
Novel Delivery Systems, Vol. 14, A.C.S. Symposium Series,
Bioreversible Carriers in Drug Design, Edward B. Roche ed., Amer.
Pharma. Assoc., Pergamon Press (1987), and Protective Groups in
Organic Chemistry, McOmie ed., Plenum Press, New York (1973), each
of which is incorporated herein by reference in its entirety.
Examples of common esters include methyl, ethyl, trichloroethyl,
propyl, butyl, pentyl, tert-butyl, benzyl, nitrobenzyl,
methoxybenzyl and benzhydryl.
[0098] In some embodiments, a lipid comprises at least 10%, at
least 20%, at least 30%, at least 35%, at least 40%, at least 50%,
at least 60%, at least 70% or at least 80% by weight PUFA. In some
embodiments, a lipid comprises at least 10%, at least 20%, at least
30%, at least 35%, at least 40%, at least 50%, at least 60%, at
least 70% or at least 80% by weight DHA. In some embodiments, a
lipid comprises less than 50%, less than 40%, less than 30%, less
than 20%, less than 15%, less than 10%, or less than 5% by weight
EPA. In some embodiments, a lipid comprises less than 10%, less
than 5%, less than 2%, less than 1%, or less than 0.5% by weight
sterols. In some embodiments, one or more PUFAs are present in a
lipid in one or more forms, such as triglycerides, diglycerides,
monoglycerides, phospholipids, free fatty acids, esterified fatty
acids, alkali metal salts of fatty acids, alkali earth metal salts
of fatty acids, and combinations thereof.
[0099] In some embodiments, a lipid separated after centrifuging in
a process of the present invention comprises at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 95%, or 50%
to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 60% to 95%,
60% to 90%, 60% to 85%, 70% to 95%, 70% to 90%, 70% to 85%, 75% to
95%, 75% to 90%, or 75% to 85%, by weight of triglycerides.
[0100] In some embodiments, the triglycerides comprise at least
10%, at least 20%, at least 30%, at least 35%, at least 40%, at
least 50%, at least 60%, at least 70% or at least 80% by weight
DHA. In some embodiments, the triglycerides comprise at least 50%,
at least 40%, at least 30%, at least 20%, at least 15%, at least
10%, or at least 5% by weight EPA.
[0101] As discussed herein, additional refining of a lipid after
the centrifuging can provide a lipid comprising at least 80%, at
least 85%, at least 90%, at least 95%, at least 99%, or 80% to
99.5%, 80% to 99%, 80% to 97%, 80% to 95%, 80% to 90%, 85% to
99.5%, 85% to 99%, 85% to 97%, 85% to 95%, 85% to 90%, 90% to
99.5%, 90% to 99%, 90% to 97%, 90% to 95%, 95% to 99.5%, 95% to
99%, 95% to 97%, 97% to 99.5%, or 98% to 99.5% triglyceride by
weight.
[0102] As used herein, a "cell" refers to a lipid-containing
biomaterial, such as biomaterial derived from plants or
microorganisms. In some embodiments, suitable plant material
includes, but is not limited to, plant parts and oilseeds. Oilseeds
include, but are not limited to, sunflower seeds, canola seeds,
rapeseeds, linseeds, castor oil seeds, coriander seeds, calendula
seeds or the like, and genetically modified variants thereof. Oil
produced from plant material and/or microorganisms, such as
oleaginous microorganisms, according to the processes described
herein, is also referred to as vegetable oil. Oil produced from
algae and/or fungi is also referred to as algal and/or fungal oil,
respectively.
[0103] As used herein, a "microbial cell" or "microorganism" refers
to organisms such as algae, bacteria, fungi, protist, and
combinations thereof, e.g., unicellular organisms. In some
embodiments, a microbial cell is a eukaryotic cell. A microbial
cell suitable for use with the present invention includes, but is
not limited to, golden algae (e.g., microorganisms of the kingdom
Stramenopiles), green algae, diatoms, dinoflagellates (e.g.,
microorganisms of the order Dinophyceae including members of the
genus Crypthecodinium such as, for example, Crypthecodinium cohnii
or C. cohnii), yeast (Ascomycetes or Basidiomycetes), and fungi of
the genera Mucor and Mortierella, including but not limited to
Mortierella alpina and Mortierella sect. schmuckeri. A microbial
cell suitable for use with the present invention can further
include, but is not limited to genera found in the following groups
of organisms: Stramenopiles, Hamatores, Proteromonads, Opalines,
Develpayella, Diplophrys, Labrinthulids, Thraustochytrids,
Biosecids, Oomycetes, Hypochytridiomycetes, Commation,
Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus,
Parmales, Diatoms, Xanthophytes, Phaeophytes, Eustigmatophytes,
Raphidophytes, Synurids, Axodines (including Rhizochromulinaales,
Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales,
Hydrurales, Hibberdiales, and Chromulinales.
[0104] 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 present
invention, the term "thraustochytrid" refers to any member of the
order Thraustochytriales, which includes the family
Thraustochytriaceae, and the term "labyrinthulid" refers to any
member of the order Labyrinthulales, which includes the family
Labyrinthulaceae.
[0105] Members of the family Labyrinthulaceae were previously
considered to be members of the order Thraustochytriales, 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
Thraustochytriales 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:
[0106] Realm: Stramenopila (Chromista) [0107] Phylum:
Labyrinthulomycota (Heterokonta) [0108] Class: Labyrinthulomycetes
(Labyrinthulae) [0109] Order: Labyrinthulales [0110] Family:
Labyrinthulaceae [0111] Order: Thraustochytriales [0112] Family:
Thraustochytriaceae
[0113] For purposes of the present invention, strains of microbial
cells described as thraustochytrids include the following
organisms: Order: Thraustochytriales; Family: Thraustochytriaceae;
Genera: Thraustochytrium (Species: sp., arudimentale, aureum,
benthicola, globosum, kinnei, motivum, multirudimentale,
pachydermum, proliferum, roseum, and striatum), Ulkenia (Species:
sp., amoeboidea, kerguelensis, minuta, profunda, radiata, sailens,
sarkariana, schizochytrops, visurgensis, yorkensis, and sp.
BP-5601), Schizochytrium (Species: sp., aggregatum, limnaceum,
mangrovei, minutum, and octosporum), Japonochytrium (Species: sp.,
marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis,
profunda, and stocchinoi), Althornia (Species: sp., crouchii), or
Elina (Species: sp., marisalba, and sinorifica). For the purposes
of this invention, species described within Ulkenia will be
considered to be members of the genus Thraustochytrium.
Aurantiacochytrium and Oblogospora are two additional genuses
encompassed by the phylum Labyrinthulomycota in the present
invention. In some embodiments, a microbial cell is of the genus
Thraustochystrium, Schizochytrium, and mixtures thereof.
[0114] Microbial cells suitable for use with the present invention
include, but are not limited to, Labyrinthulids selected from:
Order: Labyrinthulales, Family: Labyrinthulaceae, Genera:
Labyrinthula (Species: sp., algeriensis, coenocystis, chattonii,
macrocystis, macrocystis atlantica, macrocystis macrocystis,
marina, minuta, roscoffensis, valkanovii, vitellina, vitellina
pacifica, vitellina vellina, and zopfii), Labyrinthuloides
(Species: sp., haliotidis, and yorkensis), Labyrinthomyxa (Species:
sp., marina), Diplophrys (Species: sp., archeri), Pyrrhosorus
(Species: sp., marinus), Sorodiplophrys (Species: sp., stercorea),
and Chlamydomyxa (Species: sp., labyrinthuloides, and montana)
(although there is currently not a consensus on the exact taxonomic
placement of Pyrrhosorus, Sorodiplophrys, and Chlamydomyxa).
[0115] Host cells of the phylum Labyrinthulomycota include, but are
not limited to, deposited strains PTA-10212, PTA-10213, PTA-10214,
PTA-10215, PTA-9695, PTA-9696, PTA-9697, PTA-9698, PTA-10208,
PTA-10209, PTA-10210, PTA-10211, the microorganism deposited as
SAM2179 (named "Ulkenia SAM2179" by the depositor), any
Thraustochytrium species (including former Ulkenia species such as
U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U.
radiata, U. minuta and Ulkenia sp. BP-5601), and including
Thraustochytrium striatum, Thraustochytrium aureum,
Thraustochytrium roseum; and any Japonochytrium species. Strains of
Thraustochytriales include, but are not limited to Thraustochytrium
sp. (23B) (ATCC 20891); Thraustochytrium striatum (Schneider)(ATCC
24473); Thraustochytrium aureum (Goldstein) (ATCC 34304);
Thraustochytrium roseum (Goldstein) (ATCC 28210); Japonochytrium
sp. (L1) (ATCC 28207); ATCC 20890; ATCC 20892; a mutant strain
derived from any of the aforementioned microorganisms; and mixtures
thereof. Schizochytrium include, but are not limited to
Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium
sp. (S31) (ATCC 20888), Schizochytrium sp. (S8) (ATCC 20889),
Schizochytrium sp. (LC-RM) (ATCC 18915), Schizochytrium sp, (SR
21), deposited strain ATCC 28209, deposited Schizochytrium
limacinum strain IFO 32693, a mutant strain derived from any of the
aforementioned microorganisms, and mixtures thereof. In some
embodiments, the host cell is a Schizochytrium or a
Thraustochytrium. Schizochytrium can replicate both by successive
bipartition and by forming sporangia, which ultimately release
zoospores. Thraustochytrium, however, replicate only by forming
sporangia, which then release zoospores. In some embodiments, the
host cell of the invention is a recombinant host cell.
[0116] Effective culture conditions for a microbial cell for use
with the invention include, but are not limited to, effective
media, bioreactor, temperature, pH, and oxygen conditions that
permit lipid production. An effective medium refers to any medium
in which a microbial cell, e.g., Thraustochytriales microbial cell,
is typically cultured. Such media typically comprises an aqueous
medium having assimilable carbon, nitrogen, and phosphate sources,
as well as appropriate salts, minerals, metals, and other
nutrients, such as vitamins. Microbial cells for use with the
present invention can be cultured in conventional fermentation
bioreactors, shake flasks, test tubes, microtiter dishes, and petri
plates. In some embodiments, culturing is carried out at a
temperature, pH, and oxygen content appropriate for a recombinant
cell.
[0117] In some embodiments, a microbial cell is capable of growth
at a salinity level of 12 g/L or less, 5 g/L or less, or 3 g/L or
less of sodium chloride.
[0118] In some embodiments, a microbial cell produces a lipid
comprising omega-3 and/or omega-6 PUFAs. In some embodiments, a
microbial cell produces a lipid comprising DHA, DPA (n-3), DPA
(n-6), EPA, arachidonic acid (ARA), or the like, and combinations
thereof. Non-limiting examples of microorganisms that produce a
lipid comprising a PUFA are disclosed above and are also found in
U.S. Pat. Nos. 5,340,594, 5,340,742 and 5,583,019, each of which is
incorporated by reference herein in its entirety.
[0119] In some embodiments, a microbial cell comprises at least 30%
by weight lipids, at least 35% by weight lipids, at least 40% by
weight lipids, at least 50% by weight lipids, at least 60% by
weight lipids, at least 70% by weight lipids, or at least 80% by
weight lipids. In some embodiments, a microbial cell for use with
the present invention is capable of producing at least 0.1 grams
per liter per hour (g/L/h) of DHA, at least 0.2 g/L/h of DHA, at
least 0.3 g/L/h of DHA, or at least 0.4 g/L/h of DHA.
Processes
[0120] The processes of the present invention comprises lysing a
cell or cell biomass to form a lysed cell composition. As used
herein, the term "cell biomass" refers to a population of plant or
microbial cells. As used herein, the terms "lyse" and "lysing"
refer to a process of rupturing the cell wall and/or cell membrane
of a cell. In some embodiments, lysing comprises a process such as:
mechanically treating, chemically treating, enzymatically treating,
physically treating, or combinations thereof.
[0121] As used herein, mechanically treating includes, but is not
limited to, homogenizing a cell, applying ultrasound to a cell,
cold-pressing a cell, milling a cell or the like, and combinations
thereof. In some embodiments, a process comprises lysing the cell
by homogenization. In some embodiments, a process comprises lysing
the cell with a homogenizer.
[0122] Homogenizing a cell can include, but is not limited to,
processes utilizing a French pressure cell press, a sonicator, a
homogenizer, a ball mill, a rod mill, a pebble mill, a bead mill, a
high pressure grinding roll, a vertical shaft impactor, an
industrial blender, a high shear mixer, a paddle mixer, a polytron
homogenizer or the like, and combinations thereof. In some
embodiments, a cell is flowed through a homogenizer that is
optionally heated. In some embodiments, suitable homogenization can
include 1 to 3 passes through a homogenizer at either high and/or
low pressures. In some embodiments, a pressure during
homogenization can be 150 bar to 1,400 bar, 150 bar to 1,200 bar,
150 bar to 900 bar, 150 bar to 300 bar, 300 bar to 1,400 bar, 300
bar to 1,200 bar, 300 bar to 900 bar, 400 bar to 800 bar, 500 bar
to 700 bar, or 600 bar.
[0123] As used herein, physically treating can include, but is not
limited to, heating a cell, drying a cell, or the like, and
combinations thereof.
[0124] Heating a cell can include, but is not limited to, resistive
heating, convection heating, steam heating, heating in a fluid
bath, heating with solar energy, heating with focused solar energy,
and the like, any of which can be performed in a tank, pool, tube,
conduit, flask, or other containment device. In some embodiments, a
cell is heated in a tank that includes resistive coils in/on its
walls. In some embodiments, a cell is heated in a liquid bath that
includes a tubing passing there through.
[0125] Drying a cell can include, but is not limited to, exposing
to air flow, exposing to heat (e.g., convection heat, a heated
surface, and the like), exposing to solar energy, freeze drying
(lyophilizing), spray drying, and combinations thereof. In some
embodiments, drying comprises applying a cell to a rotating drum
that is optionally heated.
[0126] As used herein, chemically treating includes, but is not
limited to, raising a pH of a cell, contacting a cell with a
chemical or the like.
[0127] Raising a pH of a cell can include, but is not limited to,
adding a base to a cell composition. In some embodiments, bases
suitable for use with the present invention include, but are not
limited to, hydroxide bases (e.g., LiOH, NaOH, KOH, Ca(OH).sub.2,
and the like, and combinations thereof), carbonate bases (e.g.,
Na.sub.2CO.sub.3, K.sub.2CO.sub.3, MgCO.sub.3, and the like, and
combinations thereof), bicarbonate bases (e.g., LiHCO.sub.3,
NaHCO.sub.3, KHCO.sub.3, and the like, and combinations thereof),
and combinations thereof. A base can be in the form of a solid
(e.g., crystals, a granulate, pellets, and the like) or a liquid
(e.g., an aqueous solution, an alcoholic solution such as a
hydroxide base in methanol, ethanol, propanol, and the like), and
combinations thereof. In some embodiments, the pH of the cell
composition is raised to 8 or above, 9 or above, 10 or above, 11 or
above, 12 or above, or a pH of 7 to 13, 7 to 12, 7 to 11, 7 to 10,
7 to 9, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to
11, 9 to 10, 10 to 12, or 10 to 11.
[0128] In some embodiments, raising a pH of a cell can include, but
is not limited to, performing a chloralkali process. In some
embodiments, a fermentation broth containing sodium chloride and a
cell composition is subjected to electrolysis, which would result
in the formation of sodium hydroxide. The formation of sodium
hydroxide raises the pH of the cell. In some embodiments, a
fermentation broth can include calcium chloride or potassium
chloride in place of or in addition to sodium chloride. Subjecting
such a fermentation broth to electrolysis results in the formation
of calcium hydroxide or potassium hydroxide, respectively, thereby
raising the pH of the cell.
[0129] Enzymatic lysing refers to lysis of a cell wall or cell
membrane of a cell by contacting the cell with one or more enzymes.
Enzymes suitable for use with the present invention include, but
are not limited to, proteases, cellulases, hemicellulases,
chitinases, pectinases, and combinations thereof. Non-limiting
examples of proteases include serine proteases, theronine
proteases, cysteine proteases, aspartate proteases,
metalloproteases, glutamic acid proteases, alacase, and
combinations thereof. Non-limiting examples of cellulases include
sucrase, maltase, lactase, alpha-glucosidase, beta-glucosidase,
amylase, lysozyme, neuraminidase, galactosidase, alpha-mannosidase,
glucuronidase, hyaluronidase, pullulanase, glucocerebrosidase,
galactosylceramidase, acetylgalactosaminidase, facosidase,
hexosaminidase, iduronidase, maltase-glucoamylase, and combinations
thereof. A non-limiting example of a chitinase includes
chitotriosidase. Non-limiting examples of pectinases include
pectolyase, pectozyme, polygalacturonase, and combinations thereof.
In some embodiments, some enzymes are activated by heating.
[0130] As used herein, a "lysed cell composition" refers to a
composition comprising one or more lysed cells, including cell
debris and other contents of the cell, in combination with a lipid
(from the lysed cells), and optionally, broth that contains
microbial cells or plant material. In some embodiments, plant
material is contained in a broth or media comprising the plant
material and water. In some embodiments, a microbial cell is
contained in a fermentation broth or media comprising the microbial
cell and water. In some embodiments, a lysed cell composition
refers to a composition comprising one or more lysed cells, cell
debris, a lipid, the natural contents of the cell, and aqueous
components from a broth. In some embodiments, a lysed cell
composition is in the form of an oil-in-water emulsion comprising a
mixture of a continuous aqueous phase and a dispersed lipid phase.
In some embodiments, a dispersed lipid phase is present in a
concentration of 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to
20%, 5% to 60%, 5% to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to
60%, 10% to 50%, 10% to 40%, 20% to 60%, 20% to 50%, 20% to 40%,
30% to 60%, 30% to 50%, or 40% to 60% by weight of an emulsified
lysed cell composition.
[0131] While not being bound to any particular theory, it is
believed the processes of the present invention break up or
demulsify an emulsified lysed cell composition, allowing a lipid to
be separated from the lysed cell composition. As used herein, the
terms "emulsion" and "emulsified" refers to a mixture of two or
more immiscible phases or layers wherein one phase or layer is
dispersed in another phase or layer. As used herein, the terms
"break," "break up," "demulsify," "demulsification,"
"demulsifying," and "breaking" refer to a process of separating
immiscible phases or layers of an emulsion. For example,
demulsifying or breaking an emulsified lysed cell composition
refers to a process by which an emulsified lysed cell composition
changes from an emulsion having one or more phases or layers to a
composition having two or more phases or layers. For example, in
some embodiments, a process of the present invention breaks an
emulsified lysed cell composition from a single-phase to two or
more phases. In some embodiments, the two or more phases include a
lipid phase and an aqueous phase. In some embodiments, a process of
the present invention breaks an emulsified lysed cell compositions
from one or more phases to at least three phases. In some
embodiments, the three phases include a lipid phase, an aqueous
phase, and a solid phase. In some embodiments, the three phases
include a lipid phase, an emulsion phase, and an aqueous phase.
[0132] In some embodiments, the processes of the present invention
demulsify a lysed cell composition to form a demulsified cell
composition by removing or breaking at least 75% of the emulsion,
at least 80% of the emulsion, at least 85% of the emulsion, at
least 90% of the emulsion, at least 95% of the emulsion, at least
99% of the emulsion. In some embodiments, the process of the
present invention demulsify a lysed cell composition by removing or
breaking 75% of the emulsion to 99% of the emulsion, 75% of the
emulsion to 95% of the emulsion, 75% of the emulsion to 90% of the
emulsion, 75% of the emulsion to 85% of the emulsion, 75% of the
emulsion to 80% of the emulsion, 80% of the emulsion to 99% of the
emulsion, 80% of the emulsion to 95% of the emulsion, 80% of the
emulsion to 90% of the emulsion, 80% of the emulsion to 85% of the
emulsion, 85% of the emulsion to 99% of the emulsion, 85% of the
emulsion to 95% of the emulsion, 85% of the emulsion to 90% of the
emulsion, 90% of the emulsion to 99% of the emulsion, 90% of the
emulsion to 95% of the emulsion, or 95% of the emulsion to 99% of
the emulsion by weight or volume.
[0133] In some embodiments, prior to lysing the cell, the cell can
be washed and/or pasteurized. In some embodiments, washing the cell
includes using an aqueous solution, such as water, to remove any
extracellular water-soluble or water-dispersible compounds. In some
embodiments, the cell can be washed once, twice, thrice, or more.
In some embodiments, pasteurizing the cell includes heating the
cell to inactivate any undesirable enzymes, for example any enzymes
that might degrade lipid or reduce the yield of PUFAs. In some
embodiments, the cell can be washed first and then pasteurized.
[0134] In some embodiments, the cell is plant biomaterial and the
plant biomaterial is formed prior to lysing. In some embodiments,
the plant biomaterial is formed by removing or extracting oilseeds
from a plant. In some embodiments, an interior of an oilseed is
removed from an outer hull of an oilseed by grinding, milling,
extruding, aspirating, crushing, or combinations thereof. In some
embodiments, the dehulled oilseeds can be homogenized or expelled
using processes known in the art, such as by passing the oilseeds
through a press to grind the dehulled oilseeds into a cake. In some
embodiments, water can be added to the cake to form an emulsified
lysed cell composition. In some embodiments, the emulsified lysed
cell composition can be filtered using processes known in the art
to remove any excess hull fragments from the lysed cell
composition.
[0135] In some embodiments, treating a lysed cell composition with
a first base breaks up (i.e., demulsifies) an emulsified lysed cell
composition. In some embodiments, treating a lysed cell composition
with a second base breaks (i.e., demulsifies) an emulsified lysed
cell composition. In some embodiments, treating a lysed cell
composition with a salt breaks (i.e., demulsifies) an emulsified
lysed cell composition. In some embodiments, heating a lysed cell
composition breaks (i.e., demulsifies) an emulsified lysed cell
composition. In some embodiments, agitating a lysed cell
composition breaks (i.e., demulsifies) an emulsified lysed cell
composition. In some embodiments, simultaneous heating and
agitating of a lysed cell composition breaks (i.e., demulsifies) an
emulsified lysed cell composition. In some embodiments, one or more
of the preceding treatments breaks up (i.e., demulsifies) an
emulsified lysed cell composition.
[0136] In some embodiments, the process of the invention comprises
raising the pH of a cell composition to lyse and/or demulsify the
cell composition. In some embodiments, the process of the invention
comprises raising the pH of a lysed cell composition to demulsify
the lysed cell composition. In some embodiments, raising the pH
comprises contacting a cell composition or lysed cell composition
with a base. In some embodiments, the process of the invention
comprises contacting a lysed cell composition with a base to
demulsify the lysed cell composition. As used herein, "contacting"
refers to combining a cell composition or a lysed cell composition
with a second composition (e.g., by adding a composition to a cell
composition or a lysed cell composition, by adding a cell
composition or a lysed cell composition to a composition, and the
like). As used herein, a "composition" can comprise a pure material
or include a combination of two or more materials, substances,
excipients, portions, and the like. Contacting a lysed cell
composition with a first base raises the pH of the lysed cell
composition. In some embodiments, a lysed cell composition is
contacted with a second base. In some embodiments, the pH of a
lysed cell composition or a demulsified cell composition is raised
a second time. In some embodiments, the second raising of the pH
comprises contacting a lysed cell composition or demulsified cell
composition with a second base. In some embodiments, a lysed cell
composition is contacted with a first base, then heated, agitated,
or a combination thereof, and subsequently contacted with a second
base to provide a treated lysed cell emulsion.
[0137] In some embodiments, the first base and/or second base has a
pK.sub.b of 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 2 to 12, 2 to
10, 2 to 8, 2 to 6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to
8, 4 to 6, 5 to 10, or 5 to 8. As used herein, the term "pK.sub.b"
refers to the negative logarithm of the base association constant,
K.sub.b, of the base. K.sub.b refers to the equilibrium constant
for the ionization of the base in water, wherein:
B+H.sub.2O HB.sup.++OH.sup.-; and
the K.sub.b of base, B, is defined as:
K b = [ HB + ] [ OH - ] [ B ] . ##EQU00001##
[0138] Bases suitable for use with the present invention include,
but are not limited to, hydroxide bases (e.g., LiOH, NaOH, KOH,
Ca(OH).sub.2, and the like, and combinations thereof), carbonate
bases (e.g., Na.sub.2CO.sub.3, K.sub.2CO.sub.3, MgCO.sub.3, and the
like, and combinations thereof), bicarbonate bases (e.g.,
LiHCO.sub.3, NaHCO.sub.3, KHCO.sub.3, and the like, and
combinations thereof), and combinations thereof. A base can be in
the form of a solid (e.g., crystals, a granulate, pellets, and the
like) or a liquid (e.g., an aqueous solution, an alcoholic solution
such as a hydroxide base in methanol, ethanol, propanol, and the
like), and combinations thereof. Thus, a solvent can be optionally
present in a base for use with the present invention. As used
herein, "solvent" refers to an agent that is hydrophobic or
lipophilic. As used herein, "hydrophobic" refers to an agent that
is repelled from a mass of water. As used herein, "lipophilic"
refers to an agent that dissolves in lipids.
[0139] In some embodiments, contacting a cell composition or a
lysed cell composition with a base raises the pH of the lysed cell
composition. In some embodiments, contacting a lysed cell
composition with a base raises the pH of the lysed cell composition
to 8 or above, 9 or above, 10 or above, 11 or above, 12 or above,
or a pH of 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 13, 8
to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to 11, 9 to 10, 10 to
12, or 10 to 11. In some embodiments, contacting a lysed cell
composition with a base provides a pH of 8 or below, 7 or below, 6
or below, or 5 or below to the composition.
[0140] In some embodiments, raising the pH of the cell composition
or lysed cell composition with the addition of the base inhibits
lipid oxidation, thereby minimizing the amount of free radicals in
the lysed cell composition so that the crude lipid obtained from
the processes of the invention has a low peroxide value (e.g., 5 or
less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less,
2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1
or less) and/or a low anisidine value (e.g., 26 or less, 25 or
less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or
1 or less). As used herein, the terms "peroxide value" or "PV"
refer to the measure of primary reaction products, such as
peroxides and hydroperoxides, that occur during oxidation of the
lipid. As used herein peroxide value is measured in meq/kg. As used
herein, the terms "anisidine value" or "AV" refer to the measure of
secondary reaction products, such as aldehydes and ketones, that
occur during oxidation of the lipid.
[0141] In some embodiments, free radicals in the lysed cell
composition after adjusting the pH with a base are detected using
an Electron Paramagnetic Resonance spectrometer, e.g., Bruker
BioSpin e-scan EPR (system number SC0274) (Bruker BioSpin,
Billerica, Mass.). In some embodiments, a sample of the lysed cell
composition is diluted in about 1:1 ratio with deionized water
prior to measuring the EPR. In some embodiments, in order to
measure the EPR, a spin trap chemical is added to a sample of the
lysed cell composition. In some embodiments, the spin trap chemical
is any spin trap chemical known in the art, including, but not
limited to, POBN (.alpha.-(4-Pyridyl 1-oxide)-N-tert-butylnitrone)
or DMPO (5,5-dimethyl-1-pyrroline-N-oxide). In some embodiments,
the spin trap chemical is about 1.25 M and about 50 .mu.L is added
to about 0.5 gram sample of the lysed cell composition. In some
embodiments, a sample containing the spin trap chemical is
incubated at room temperature (e.g., about 20.degree. C.). In some
embodiments, the following spectrometer parameters are used:
modulation frequency of about 86 Hz, modulation amplitude of about
2 gauss, microwave power of about 5 mW, time constant of about 20
seconds, sweep time of about 10 seconds, sweep width of about 100
gauss, and a number of scans of about 8. The EPR is measured over
time to determine the concentration of free radicals present in the
lipid. In some embodiments, the EPR is measured hourly over a
course of four hours. In some embodiments, the lysed cell
composition has an EPR signal strength (intensity or amplitude) at
the above listed parameters of less than 0.15.times.10.sup.6, less
than 0.14.times.10.sup.6, less than 0.13.times.10.sup.6, less than
0.12.times.10.sup.6, less than 0.11.times.10.sup.6, less than
0.1.times.10.sup.6, less than 0.09.times.10.sup.6, less than
0.08.times.10.sup.6, less than 0.07.times.10.sup.6, less than
0.06.times.10.sup.6, or less than 0.05.times.10.sup.6 after 4
hours. In some embodiments, the lysed cell composition has an EPR
of 0.05.times.10.sup.6 to 0.15.times.10.sup.6, 0.05.times.10.sup.6
to 0.14.times.10.sup.6, 0.05.times.10.sup.6 to 0.13.times.10.sup.6,
0.05.times.10.sup.6 to 0.12.times.10.sup.6, 0.05.times.10.sup.6 to
0.11.times.10.sup.6, 0.05.times.10.sup.6 to 0.1.times.10.sup.6,
0.05.times.10.sup.6 to 0.09.times.10.sup.6, 0.07.times.10.sup.6 to
0.15.times.10.sup.6, 0.07.times.10.sup.6 to 0.13.times.10.sup.6,
0.07.times.10.sup.6 to 0.11.times.10.sup.6, 0.08.times.10.sup.6 to
0.14.times.10.sup.6, 0.08.times.10.sup.6 to 0.12.times.10.sup.6,
0.08.times.10.sup.6 to 0.1.times.10.sup.6, 0.09.times.10.sup.6 to
0.13.times.10.sup.6, or 0.09.times.10.sup.6 to 0.11.times.10.sup.6.
In some embodiments, the pH of the lysed cell composition resulting
in an EPR specified above is 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9
to 12, 9 to 11, 9 to 10, 10 to 12, or 10 to 11. In some
embodiments, a lysed cell composition having an EPR signal strength
specified above results in a crude lipid having an AV of 26 or
less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2
or less, or 1 or less. In some embodiments, a lysed cell
composition having an EPR specified above results in a crude lipid
having a PV of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or
less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less,
0.2 or less, or 0.1 or less.
[0142] In some embodiments, a process comprises contacting a cell
composition or lysed cell composition with a salt to demulsify the
lysed cell composition. As used herein, a "salt" refers to an ionic
compound formed by replacing a hydrogen ion from an acid with a
metal (e.g., an alkali metal, an alkali earth metal, a transition
metal, and the like) or a positively charged compound (e.g.,
NH.sub.4.sup.+ and the like). Salts suitable for use with the
present invention include, but are not limited to, alkali metal
salts, alkali earth metal salts, or the like, and combinations
thereof. Negatively charged ionic species present in a salt for use
with the present include, but are not limited to, halides, sulfate,
bisulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen
phosphate, carbonate, bicarbonate, or the like, and combinations
thereof. In some embodiments, a salt for use with the present
invention is selected from: sodium chloride, sodium sulfate, sodium
carbonate, calcium chloride, potassium sulfate, magnesium sulfate,
monosodium glutamate, ammonium sulfate, potassium chloride, iron
chloride, iron sulfate, aluminum sulfate, and combinations thereof.
In some embodiments, a salt does not include NaOH. A salt can be
added as a solid (e.g., in crystalline, amorphous, pelletized,
and/or granulated form), and/or as a solution (e.g., a dilute
solution, a saturated solution, or a super-saturated solution)
containing, for example, water, an alcohol, and the like, and
combinations thereof.
[0143] In some embodiments, the salt is added in an amount of 5 g/l
to 25 g/l, 5 g/l to 10 g/l, 10 g/l to 15 g/l, 15 g/l to 20 g/l, 20
g/l to 25 g/l, or 10 g/l to 20 g/l.
[0144] In some embodiments, a temperature of a cell composition or
a lysed cell composition is less than or equal to 60.degree. C.,
less than or equal to 55.degree. C., less than or equal to
45.degree. C., less than or equal to 40.degree. C., less than or
equal to 35.degree. C., less than or equal to 30.degree. C., or
less than or equal to 25.degree. C. when a salt is added to
demulsify the cell composition or the lysed cell composition. In
some embodiments, a temperature of a lysed cell composition is
0.degree. C. to 60.degree. C., 0.degree. C. to 55.degree. C.,
0.degree. C. to 50.degree. C., 0.degree. C. to 45.degree. C.,
0.degree. C. to 4.degree. C., 0.degree. C. to 35.degree. C.,
0.degree. C. to 30.degree. C., 0.degree. C. to 25.degree. C.,
20.degree. C. to 60.degree. C., 20.degree. C. to 55.degree. C.,
20.degree. C. to 50.degree. C., 20.degree. C. to 45.degree. C.,
20.degree. C. to 40.degree. C., 20.degree. C. to 35.degree. C.,
20.degree. C. to 30.degree. C., 30.degree. C. to 60.degree. C.,
30.degree. C. to 55.degree. C., 30.degree. C. to 50.degree. C.,
30.degree. C. to 45.degree. C., 30.degree. C. to 40.degree. C.,
30.degree. C. to 40.degree. C., 40.degree. C. to 60.degree. C.,
40.degree. C. to 55.degree. C., 40.degree. C. to 50.degree. C., or
50.degree. C. to 60.degree. C. when a salt is added to demulsify
the cell composition or the lysed cell composition.
[0145] In some embodiments, the process comprises contacting a cell
composition or a lysed cell composition with 20% or less, 15% or
less, 10% or less, 7.5% or less, 5% or less, or 2% or less salt by
weight, of the lysed cell composition or the cell composition. In
some embodiments, a process comprises contacting a cell composition
or a lysed cell composition with 0.1% to 20%, 0.1% to 15%, 0.1% to
10%, 0.5% to 20%, 0.5% to 15%, 0.5% to 10%, 0.5% to 5%, 0.5% to 4%,
0.5% to 3%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1%
to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 4%, 1% to 3%, 1% to
2.5%, 1% to 2%, 1% to 1.5%, 1.5% to 5%, 1.5% to 4%, 1.5% to 3%,
1.5% to 2.5%, 1.5% to 2%, 2% to 20%, 2% to 15%, 2% to 10%, 2% to
5%, 2% to 4%, 2% to 3%, 2% to 2.5%, 2.5% to 5%, 2.5% to 4%, 2.5% to
3%, 3% to 5%, 3% to 4%, 4% to 5%, 5% to 20%, 5% to 15%, 5% to 10%,
10% to 20%, 10% to 15%, or 15% to 20% salt, by weight, of the cell
composition or lysed cell composition (e.g., a total broth weight).
For example, when a lysed cell composition weighs 1,000 kg,
contacting with 0.5% to 20% salt, by weight, requires combining 5
kg to 200 kg of salt with the lysed cell composition.
[0146] In some embodiments, the process comprises heating a cell
composition or a lysed cell composition to demulsify the lysed cell
composition. In some embodiments the cell composition or the lysed
cell composition is heated for a sufficient period of time for a
base and/or a salt to demulsify a cell composition or a lysed cell
composition. In some embodiments, the process comprises heating a
cell composition or a lysed cell composition for at least 5
minutes, at least 10 minutes, at least 20 minutes, at least 30
minutes, at least 1 hour, at least 2 hours, at least 4 hours, at
least 8 hours, at least 12 hours, at least 18 hours, at least 24
hours, at least 30 hours, at least 36 hours, at least 42 hours, at
least 48 hours, at least 54 hours, at least 60 hours, at least 66
hours, at least 72 hours, at least 78 hours, at least 84 hours, at
least 90 hours or at least 96 hours. In some embodiments, the
process comprises heating a lysed cell composition for 5 minutes to
96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to
1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to
1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours,
1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour
to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84
hours, 4 hours to 72 hours, 4 hours to 60 hours, 4 hours to 48
hours, 4 hours to 36 hours, 4 hours to 24 hours, 8 hours to 96
hours, 8 hours to 84 hours, 8 hours to 72 hours, 8 hours to 60
hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24
hours, 8 hours to 12 hours, 12 hours to 96 hours, 12 hours to 84
hours, 12 hours to 72 hours, 12 hours to 60 hours, 12 hours to 48
hours, 12 hours to 36 hours, 12 hours to 24 hours, 24 hours to 96
hours, 24 hours to 84 hours, 24 hours to 72 hours, 24 hours to 60
hours, 24 hours to 48 hours, or 24 hours to 36 hours.
[0147] In some embodiments, a cell composition or a lysed cell
composition can be heated at a temperature of at least 10.degree.
C., at least 20.degree. C., at least 25.degree. C., at least
30.degree. C., at least 35.degree. C., at least 40.degree. C., at
least 45.degree. C., at least 50.degree. C., at least 55.degree.
C., at least 60.degree. C., at least 65.degree. C., at least
70.degree. C., at least 75.degree. C., at least 80.degree. C., at
least 85.degree. C., at least 90.degree. C., at least 95.degree.
C., or at least 100.degree. C. In some embodiments, a process
comprises heating a cell composition or a lysed cell composition at
a temperature of 10.degree. C. to 100.degree. C., 10.degree. C. to
90.degree. C., 10.degree. C. to 80.degree. C., 10.degree. C. to
70.degree. C., 20.degree. C. to 100.degree. C., 20.degree. C. to
90.degree. C., 20.degree. C. to 80.degree. C., 20.degree. C. to
70.degree. C., 30.degree. C. to 100.degree. C., 30.degree. C. to
90.degree. C., 30.degree. C. to 80.degree. C., 30.degree. C. to
70.degree. C., 40.degree. C. to 100.degree. C., 40.degree. C. to
90.degree. C., 40.degree. C. to 80.degree. C., 50.degree. C. to
100.degree. C., 50.degree. C. to 90.degree. C., 50.degree. C. to
80.degree. C., 50.degree. C. to 70.degree. C., 60.degree. C. to
100.degree. C., 60.degree. C. to 90.degree. C., 60.degree. C. to
80.degree. C., 70.degree. C. to 100.degree. C., 70.degree. C. to
90.degree. C., 80.degree. C. to 100.degree. C., 80.degree. C. to
90.degree. C., or 90.degree. C. to 100.degree. C. In some
embodiments, a salt can be added to the cell composition or the
lysed cell composition during the heating.
[0148] In some embodiments, a cell composition or a lysed cell
composition can be heated in a closed system or in a system with an
evaporator. In some embodiments, a cell composition or lysed cell
composition can be heated in a system with an evaporator such that
a portion of the water present in the cell composition or the lysed
cell composition is removed by evaporation. In some embodiments, a
process comprises heating a cell composition or a lysed cell
composition in a system with an evaporator to remove up to 1%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight of water
present in the cell composition or lysed cell composition. In some
embodiments, a process comprises heating a cell composition or a
lysed cell composition in a system with an evaporator to remove 1%
to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1%
to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 50%, 5% to 45%, 5% to
40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to
10%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%,
10% to 25%, 10% to 20%, 10% to 15%, 15% to 50%, 15% to 45%, 15% to
40%, 15% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%,
20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to
50%, 25% to 45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 50%,
30% to 45%, 30% to 40%, 30% to 35%, 35% to 50%, 35% to 45%, 35% to
40%, 40% to 50%, 40% to 45%, or 45% to 50%.
[0149] In some embodiments, the process comprises holding a cell
composition or a lysed cell composition in a vessel for a
predetermined time to demulsify the lysed cell composition. In some
embodiments, the process comprises holding a cell composition or a
lysed cell composition in a vessel for at least 5 minutes, at least
10 minutes, at least 20 minutes, at least 30 minutes, at least 1
hour, at least 2 hours, at least 4 hours, at least 8 hours, at
least 12 hours, at least 18 hours, at least 24 hours, at least 30
hours, at least 36 hours, at least 42 hours, at least 48 hours, at
least 54 hours, at least 60 hours, at least 66 hours, at least 72
hours, at least 78 hours, at least 84 hours, at least 90 hours or
at least 96 hours. In some embodiments, the process comprises
holding a cell composition or a lysed cell composition for 5
minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5
minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10
minutes to 1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour
to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36
hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours,
4 hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4
hours to 48 hours, 4 hours to 36 hours, 4 hours to 24 hours, 8
hours to 96 hours, 8 hours to 84 hours, 8 hours to 72 hours, 8
hours to 60 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8
hours to 24 hours, 8 hours to 12 hours, 12 hours to 96 hours, 12
hours to 84 hours, 12 hours to 72 hours, 12 hours to 60 hours, 12
hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 24
hours to 96 hours, 24 hours to 84 hours, 24 hours to 72 hours, 24
hours to 60 hours, 24 hours to 48 hours, or 24 hours to 36
hours.
[0150] In some embodiments, the process comprises contacting an
antioxidant with a lysed cell emulsion. Antioxidants suitable for
use with the present invention include, but are not limited to, a
tocopherol, a tocotrienol, a polyphenol, resveratrol, a flavonoid,
a carotenoid, lycopene, a carotene, lutein, ascorbic acid, ascorbyl
palmitate, or the like, and combinations thereof.
[0151] In some embodiments, the process comprises allowing an
emulsified lysed cell composition to stand, wherein the lipid is
separated from the emulsified lysed cell composition using
gravity.
[0152] As used herein, the terms "agitating" and "agitation" refer
to a process of affecting motion in a lysed cell composition
through an application of force. In some embodiments, the process
of the invention comprises agitating a cell composition or a lysed
cell composition by stirring, mixing, blending, shaking, vibrating,
or a combination thereof. In some embodiments, the process of
agitating a cell composition or a lysed cell composition
demulsifies the cell composition or the lysed cell composition.
[0153] In some embodiments, the process of the invention comprises
agitating a lysed cell composition at 0.1 hp/1,000 gal to 10
hp/1,000 gal, 0.5 hp/1,000 gal to 8 hp/1,000 gal, 1 hp/1,000 gal to
6 hp/1,000 gal, or 2 hp/1,000 gal to 5 hp/1,000 gal of lysed cell
composition.
[0154] In some embodiments, the process of the invention comprises
agitating a cell composition or a lysed cell composition using an
agitator. In some embodiments, the agitator is a dispersion style
agitator that disperses a base and/or salt in the cell composition
or the lysed cell composition. In some embodiments, an agitator has
one or more impellers. As used herein, "impeller" refers to a
device arranged to impart motion to a cell composition or a lysed
cell composition when rotated. Impellers suitable for use with the
present invention include straight blade impellers, Rushton blade
impellers, axial flow impellers, radial flow impellers, concave
blade disc impellers, high-efficiency impellers, propellers,
paddles, turbines, or the like, and combinations thereof. In some
embodiments, a process includes agitating a cell composition or a
lysed cell composition using an agitator having an impeller tip
speed of 90 ft/min to 1,200 ft/min, 200 ft/min to 1,000 ft/min, 300
ft/min to 800 ft/min, 400 ft/min to 700 ft/min, or 500 ft/min to
600 ft/min. In some embodiments, a process includes agitating a
cell composition or a lysed cell composition using an agitator
having an impeller tip speed of 350 centimeters/second to 900
centimeters per second, 350 centimeters/second to 850 centimeters
per second, 350 centimeters/second to 800 centimeters/second, 350
centimeters/second to 750 centimeters/second, 350
centimeters/second to 700 centimeters/second, 350
centimeters/second to 650 centimeters/second, 350
centimeters/second to 600 centimeters/second, 350
centimeters/second to 550 centimeters/second, 350
centimeters/second to 500 centimeters/second, 350
centimeters/second to 450 centimeters/second, 350
centimeters/second to 400 centimeters/second, 400
centimeters/second to 900 centimeters per second, 400
centimeters/second to 850 centimeters per second, 400
centimeters/second to 800 centimeters/second, 400
centimeters/second to 750 centimeters/second, 400
centimeters/second to 700 centimeters/second, 400
centimeters/second to 650 centimeters/second, 400
centimeters/second to 600 centimeters/second, 400
centimeters/second to 550 centimeters/second, 400
centimeters/second to 500 centimeters/second, 400
centimeters/second to 450 centimeters/second, 450
centimeters/second to 900 centimeters per second, 450
centimeters/second to 850 centimeters per second, 450
centimeters/second to 800 centimeters/second, 450
centimeters/second to 750 centimeters/second, 450
centimeters/second to 700 centimeters/second, 450
centimeters/second to 650 centimeters/second, 450
centimeters/second to 600 centimeters/second, 450
centimeters/second to 550 centimeters/second, 450
centimeters/second to 500 centimeters/second, 500
centimeters/second to 900 centimeters per second, 500
centimeters/second to 850 centimeters per second, 500
centimeters/second to 800 centimeters/second, 500
centimeters/second to 750 centimeters/second, 500
centimeters/second to 700 centimeters/second, 500
centimeters/second to 650 centimeters/second, 500
centimeters/second to 600 centimeters/second, 500
centimeters/second to 550 centimeters/second, 550
centimeters/second to 900 centimeters per second, 550
centimeters/second to 850 centimeters per second, 550
centimeters/second to 800 centimeters/second, 550
centimeters/second to 750 centimeters/second, 550
centimeters/second to 700 centimeters/second, 550
centimeters/second to 650 centimeters/second, 550
centimeters/second to 600 centimeters/second, 600
centimeters/second to 900 centimeters per second, 600
centimeters/second to 850 centimeters per second, 600
centimeters/second to 800 centimeters/second, 600
centimeters/second to 750 centimeters/second, 600
centimeters/second to 700 centimeters/second, 600
centimeters/second to 650 centimeters/second, 650
centimeters/second to 900 centimeters per second, 650
centimeters/second to 850 centimeters per second, 650
centimeters/second to 800 centimeters/second, 650
centimeters/second to 750 centimeters/second, 650
centimeters/second to 700 centimeters/second, 700
centimeters/second to 900 centimeters per second, 700
centimeters/second to 850 centimeters per second, 700
centimeters/second to 800 centimeters/second, 700
centimeters/second to 750 centimeters/second, 750
centimeters/second to 900 centimeters per second, 750
centimeters/second to 850 centimeters per second, 750
centimeters/second to 800 centimeters/second, 800
centimeters/second to 900 centimeters per second, 800
centimeters/second to 850 centimeters per second, or 850
centimeters/second to 900 centimeters/second. As used herein,
"impeller tip speed" refers to the speed of the outer most portion
of the impeller as it rotates around its central axis.
[0155] In some embodiments, the agitating (and optionally
additional steps as described herein) is performed in a container
comprising an impeller, wherein a ratio of the impeller diameter to
the container volume is 0.1 to 0.5, 0.1 to 0.4, 0.2 to 0.5, 0.2 to
0.4, 0.3 to 0.5, or 0.3 to 0.4.
[0156] In some embodiments, the agitating (and optionally
additional steps as described herein) is performed in a container
comprising an impeller, wherein a ratio of the impeller diameter to
the inner diameter of the container is at least 0.25, at least
0.34, at least 0.65, 0.25 to 0.65, 0.25 to 0.33, 0.3 to 0.6, 0.3 to
0.5, 0.3 to 0.4, 0.34 to 0.65, 0.34 to 0.6, 0.34 to 0.55, 0.37 to
0.55, 0.4 to 0.65, 0.4 to 0.6, 0.4 to 0.5, or 0.42 to 0.55.
[0157] In some embodiments, agitating comprises mixing a cell
composition or a lysed cell composition such that the cell
composition or the lysed cell composition is placed under flow
conditions described by a Reynolds number of 10 to 10,000, 1,000 to
10,000, 1,500 to 10,000, or 2,000 to 10,000. In some embodiments, a
lysed cell emulsion during the agitating has a Reynolds number of
2,000 or more, 3,000 or more, or 5,000 or more, or 2,000 to 10,000,
3,000 to 10,000, or 5,000 to 10,000.
[0158] In some embodiments, a process comprises agitating a cell
composition or a lysed cell composition for at least 5 minutes, at
least 10 minutes, at least 20 minutes, at least 30 minutes, at
least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours,
at least 12 hours, at least 18 hours, at least 24 hours, at least
30 hours, at least 36 hours, at least 42 hours, at least 48 hours,
at least 54 hours, at least 60 hours, at least 66 hours, at least
72 hours, at least 78 hours, at least 84 hours, at least 90 hours
or at least 96 hours. In some embodiments, a process comprises
agitating a cell composition or a lysed cell composition for 5
minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5
minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10
minutes to 1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour
to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36
hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours,
4 hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4
hours to 48 hours, 4 hours to 36 hours, 4 hours to 24 hours, 8
hours to 96 hours, 8 hours to 84 hours, 8 hours to 72 hours, 8
hours to 60 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8
hours to 24 hours, 8 hours to 12 hours, 12 hours to 96 hours, 12
hours to 84 hours, 12 hours to 72 hours, 12 hours to 60 hours, 12
hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 20
hours to 40 hours, 24 hours to 96 hours, 24 hours to 84 hours, 24
hours to 72 hours, 24 hours to 60 hours, 24 hours to 48 hours, or
24 hours to 36 hours.
[0159] In some embodiments, a process comprises simultaneously
agitating and heating a cell composition or a lysed cell
composition to demulsify the cell composition or the lysed cell
composition. In some embodiments, a process comprises agitating a
cell composition or a lysed cell composition at a temperature of at
least 10.degree. C., at least 20.degree. C., at least 25.degree.
C., at least 30.degree. C., at least 35.degree. C., at least
40.degree. C., at least 45.degree. C., at least 50.degree. C., at
least 55.degree. C., at least 60.degree. C., at least 65.degree.
C., at least 70.degree. C., at least 75.degree. C., at least
80.degree. C., at least 85.degree. C., at least 90.degree. C., at
least 95.degree. C., or at least 100.degree. C. In some
embodiments, a process comprises agitating a cell composition or a
lysed cell composition at a temperature of 10.degree. C. to
100.degree. C., 10.degree. C. to 90.degree. C., 10.degree. C. to
80.degree. C., 10.degree. C. to 70.degree. C., 20.degree. C. to
100.degree. C., 20.degree. C. to 90.degree. C., 20.degree. C. to
80.degree. C., 20.degree. C. to 70.degree. C., 30.degree. C. to
100.degree. C., 30.degree. C. to 90.degree. C., 30.degree. C. to
80.degree. C., 30.degree. C. to 70.degree. C., 40.degree. C. to
100.degree. C., 40.degree. C. to 90.degree. C., 40.degree. C. to
80.degree. C., 50.degree. C. to 100.degree. C., 50.degree. C. to
90.degree. C., 50.degree. C. to 80.degree. C., 50.degree. C. to
70.degree. C., 60.degree. C. to 100.degree. C., 60.degree. C. to
90.degree. C., 60.degree. C. to 80.degree. C., 70.degree. C. to
100.degree. C., 70.degree. C. to 90.degree. C., 80.degree. C. to
1000.degree. C., 80.degree. C. to 90.degree. C., or 90.degree. C.
to 100.degree. C.
[0160] In some embodiments, the various combinations of forming a
lysed cell composition, contacting a lysed cell composition with a
base or raising the pH of a lysed cell composition, contacting a
lysed cell composition with a salt, heating the lysed cell
composition, and agitating a lysed cell composition can occur in a
single vessel. In some embodiments, the various combinations of
forming a cell composition, contacting a cell composition with a
base or raising the pH of a cell composition, contacting a cell
composition with a salt, heating the cell composition, and
agitating a cell composition can occur in a single vessel. In some
embodiments, the single vessel includes a fermentation vessel. In
some embodiments, the fermentation vessel can have a volume of at
least 20,000 liters, at least 50,000 liters, at least 100,000
liters, at least 120,000 liters, at least 150,000 liters, at least
200,000 liters, or at least 220,000 liters. In some embodiments,
the fermentation vessel can have a volume of 20,000 liters to
220,000 liters, 20,000 liters to 100,000 liters, 20,000 liters to
50,000 liters, 50,000 liters to 220,000 liters, 50,000 liters to
150,000 liters, 50,000 liters to 100,000 liters, 100,000 liters to
220,000 liters, 100,000 liters to 150,000 liters, 100,000 liters to
120,000 liters, 150,000 liters to 220,000 liters, 150,000 liters to
200,000 liters, or 200,000 liters to 220,000 liters.
[0161] In some embodiments, a quantity of cell composition or lysed
cell composition formed in a vessel can be transferred into one or
more agitation vessels. In some embodiments, the agitation vessels
can have a volume of at least 20,000 liters, at least 30,000
liters, at least 40,000 liters or at least 50,000 liters. In some
embodiments, the agitation vessels can have a volume of 20,000
liters to 50,000 liters, 20,000 liters to 40,000 liters, 20,000
liters to 30,000 liters, 30,000 liters to 50,000 liters, 30,000
liters to 40,000 liters or 40,000 liters to 50,000 liters.
[0162] In some embodiments, the agitation vessels can have any
combination of the following properties. In some embodiments, the
agitation vessels can have two impellers. In some embodiments, the
impellers are Rushton blade impellers. In some embodiments, the
impellers are separated from each other by a distance at least
equal to a diameter of the smallest impeller. In some embodiments,
the impellers are 30 inches to 40 inches, 33 inches to 37 inches,
33 inches, 34 inches, 35 inches, 36 inches or 37 inches from tip to
tip. In some embodiments, the agitation vessels have a volume of at
least 10,000 liters, at least 20,000 liters, at least 30,000
liters, at least 40,000 liters or at least 50,000 liters. In some
embodiments, the agitation vessels have an inner diameter of 90
inches to 110 inches, 95 inches to 105 inches, 98 inches, 99
inches, 100 inches, 101 inches, or 102 inches. In some embodiments,
a first impeller is located 15 inches to 20 inches, 16 inches to 19
inches, or 17 inches to 18 inches from a bottom of the agitation
vessel and a second impeller is located 60 inches to 80 inches, 65
inches to 75 inches, 68 inches, 69 inches, 70 inches, 71 inches, 72
inches, 73 inches, 74 inches, or 75 inches above the first
impeller. In some embodiments, a lysed cell composition is agitated
at least 50 rpm, at least 60 rpm, or at least 70 rpm. In some
embodiments, a lysed cell composition is agitated at 50 rpm to 70
rpm, 50 rpm to 60 rpm, or 60 rpm to 70 rpm.
[0163] In some embodiments, the cell composition, the lysed cell
composition, or the lipid are harvested from a vessel by pumping
the cell composition, the lysed cell composition, or the lipid from
the vessel. In some embodiments, the cell composition, the lysed
cell composition, or the lipid are harvested from a vessel without
agitating the vessel. In some embodiments, the cell composition,
the lysed cell composition, or the lipid are harvested from a
vessel by pumping, without agitation, the cell composition, the
lysed cell composition, or the lipid from the vessel. In some
embodiments, the cell composition, the lysed cell composition, or
the lipid are harvested from a vessel without blowing air. In some
embodiments, harvesting the cell composition, the lysed cell
composition, or the lipid by the techniques described above results
in a crude lipid having a low anisidine value (e.g., 26 or less, 25
or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less,
or 1 or less) and/or a low phosphorus content (e.g., 100 ppm or
less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or
less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or
less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or
less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or
less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less,
3 ppm or less, 2 ppm or less, or 1 ppm or less).
[0164] As described herein, the present invention utilizes various
combinations of contacting a lysed cell composition with a first
base or raising the pH of a lysed cell composition, contacting a
lysed cell composition with a salt, heating a lysed cell
composition, and agitating a lysed cell composition to provide a
treated lysed cell composition. As described herein, the present
invention utilizes various combinations of contacting a cell
composition with a first base or raising the pH of a cell
composition, contacting a cell composition with a salt, heating a
cell composition, and agitating a cell composition to provide a
treated cell composition. The treated cell composition or treated
lysed cell composition is at least partially demulsified compared
with an untreated cell composition or treated lysed cell
composition. Thus, a treated cell composition or treated lysed cell
composition can be placed in a centrifuge and a lipid can be
separated therefrom.
[0165] In some embodiments, after raising the pH of a cell
composition or lysed cell composition, e.g., by contacting with a
first base, the heating the cell composition or lysed cell
composition and/or the agitating the cell composition or lysed cell
composition can decrease the pH of the treated cell composition or
treated lysed cell composition. In order for a lipid to be more
effectively separated from a treated cell composition or treated
lysed cell composition by centrifuging, the pH of the treated cell
composition or treated lysed cell composition is raised a second
time, e.g., by contacting the treated cell composition or the
treated lysed cell composition with a second base. In some
embodiments, contacting a treated lysed cell composition with a
second base raises the pH of the treated cell composition or the
treated lysed cell composition. In some embodiments, a treated cell
composition or a treated lysed cell composition is contacted with a
second base to raise the pH of the treated cell composition or the
treated lysed cell composition to 7 or above, 7.5 or above, 8 or
above, 8.5 or above, 9 or above, 9.5 or above, 10 or above, 10.5 or
above, 11 or above, 11.5 or above, or 12 or above. In some
embodiments, a treated cell composition or a treated lysed cell
composition is contacted with a second base to raise the pH of the
treated lysed cell composition to 7 to 13, 7 to 12, 7 to 11, 7 to
10, 7 to 9, 7 to 8, 7 to 7.5, 7.5 to 8, 8 to 13, 8 to 12, 8 to 11,
8 to 10, 8 to 9, 8 to 8.5, 8.5 to 9, 9 to 12, 9 to 11, 9 to 10, 9
to 9.5, 9.5 to 10, 10 to 12, or 10 to 11.
[0166] In some embodiments, the pH of a treated cell composition or
a treated lysed cell emulsion is 7 or less, 6 or less, 5 or less, 4
or less, or 3 or less.
[0167] The processes of the present invention comprise separating a
lipid from a treated cell composition or a treated lysed cell
composition. In some embodiments, a lipid is separated from a lysed
cell emulsion after contacting a lysed cell emulsion with a second
base, after the agitating a lysed cell emulsion, or after
contacting a lysed cell emulsion with a salt by, for example,
permitting the treated lysed cell emulsion to rest for a period of
time sufficient for a lipid to separate from the treated lysed cell
emulsion (e.g., as a separate layer). The lipid can be subsequently
removed, for example, by decanting, skimming, vacuuming, pumping,
sucking off, drawing off, siphoning, or otherwise removing the
lipid from the surface of the treated lysed cell emulsion.
[0168] In some embodiments, the separating comprises centrifuging a
treated cell composition or a treated lysed cell composition (e.g.,
at a temperature of 30.degree. C. to 100.degree. C.), whereby the
centrifuging separates a lipid from the treated cell composition or
the treated lysed cell composition.
[0169] In some embodiments, a process comprises centrifuging a
treated cell composition or a treated lysed cell composition at a
temperature of at least 10.degree. C., at least 20.degree. C., at
least 25.degree. C., at least 30.degree. C., at least 35.degree.
C., at least 40.degree. C., at least 45.degree. C., at least
50.degree. C., at least 55.degree. C., at least 60.degree. C., at
least 65.degree. C., at least 70.degree. C., at least 75.degree.
C., at least 80.degree. C., at least 85.degree. C., at least
90.degree. C., at least 95.degree. C., or at least 100.degree. C.
In some embodiments, a process comprises centrifuging a treated
cell composition or a treated lysed cell composition at a
temperature of 10.degree. C. to 100.degree. C., 10.degree. C. to
90.degree. C., 10.degree. C. to 80.degree. C., 20.degree. C. to
100.degree. C., 20.degree. C. to 90.degree. C., 20.degree. C. to
80.degree. C., 25.degree. C. to 100.degree. C., 25.degree. C. to
90.degree. C., 25.degree. C. to 80.degree. C., 25.degree. C. to
75.degree. C., 30.degree. C. to 100.degree. C., 30.degree. C. to
90.degree. C., 30.degree. C. to 80.degree. C., 40.degree. C. to
100.degree. C., 40.degree. C. to 90.degree. C., 40.degree. C. to
80.degree. C., 50.degree. C. to 100.degree. C., 50.degree. C. to
90.degree. C., 50.degree. C. to 80.degree. C., 50.degree. C. to
70.degree. C., 60.degree. C. to 100.degree. C., 60.degree. C. to
90.degree. C., 60.degree. C. to 80.degree. C., 60.degree. C. to
70.degree. C., 70.degree. C. to 100.degree. C., or 70.degree. C. to
90.degree. C.
[0170] In some embodiments, centrifuging is conducted at a feed
rate (of a treated cell composition or a treated lysed cell
composition into a centrifuge) of 1 kilogram per minute (kg/min) to
500 kg/min, 1 kg/min to 400 kg/min, 1 kg/min to 300 kg/min, 1
kg/min to 200 kg/min, 1 kg/min to 100 kg/min, 1 kg/min to 75
kg/min, 1 kg/min to 50 kg/min, 1 kg/min to 40 kg/min, 1 kg/min to
30 kg/min, 1 kg/min to 25 kg/min, 1 kg/min to 10 kg/min, 10 kg/min
to 500 kg/min, 10 kg/min to 400 kg/min, 10 kg/min to 300 kg/min, 10
kg/min to 200 kg/min, 10 kg/min to 100 kg/min, 10 kg/min to 75
kg/min, 10 kg/min to 50 kg/min, 10 kg/min to 40 kg/min, 10 kg/min
to 30 kg/min, 20 kg/min to 500 kg/min, 20 kg/min to 400 kg/min, 20
kg/min to 300 kg/min, 20 kg/min to 200 kg/min, 20 kg/min to 100
kg/min, 20 kg/min to 75 kg/min, 20 kg/min to 50 kg/min, 20 kg/min
to 40 kg/min, 25 kg/min to 500 kg/min, 25 kg/min to 400 kg/min, 25
kg/min to 300 kg/min, 25 kg/min to 200 kg/min, 25 kg/min to 100
kg/min, 25 kg/min to 75 kg/min, 25 kg/min to 50 kg/min, 30 kg/min
to 60 kg/min, 30 kg/min to 50 kg/min, 30 kg/min to 40 kg/min, 50
kg/min to 500 kg/min, 100 kg/min to 500 kg/min, or 200 kg/min to
500 kg/min.
[0171] The total time required for the separating can vary
depending on the volume of the treated cell composition or the
treated lysed cell composition. Typical total time for separation
(e.g., centrifuge time) is at least 0.1 hour, at least 0.2 hour, at
least 0.5 hour, at least 1 hour, at least 2 hours, at least 4
hours, at least 6 hours, at least 8 hours, at least 10 hours, at
least 12 hours, or 0.1 hour to 24 hours, 0.5 hour to 24 hours, 1
hour to 12 hours, 2 hours to 10 hours, or 4 hours to 8 hours.
[0172] In some embodiments, a process of the invention comprises
centrifuging a treated cell composition or a treated lysed cell
composition at a centrifugal force of 1,000 g to 25,000 g, 1,000 g
to 20,000 g, 1,000 g to 10,000 g, 2,000 g to 25,000 g, 2,000 g to
20,000 g, 2,000 g to 15,000 g, 3,000 g to 25,000 g, 3,000 g to
20,000 g, 5,000 g to 25,000 g, 5,000 g to 20,000 g, 5,000 g to
15,000 g, 5,000 g to 10,000 g, 5,000 g to 8,000 g, 10,000 g to
25,000 g, 15,000 g to 25,000 g, or at least 1,000 g, at least
2,000, g, at least 4,000 g, at least 5,000 g, at least 7,000 g, at
least 8,000 g, at least 10,000 g, at least 15,000 g, at least
20,000 g, or at least 25,000 g. As used herein, "g" refers to
standard gravity or approximately 9.8 m/s.sup.2. In some
embodiments, a process of the invention comprises centrifuging a
treated cell composition or a treated lysed cell composition at
4,000 rpm to 14,000 rpm, 4,000 rpm to 10,000 rpm, 6,000 rpm to
14,000 rpm, 6,000 rpm to 12,000 rpm, 8,000 to 14,000 rpm, 8,000 rpm
to 12,000 rpm, or 8,000 rpm to 10,000 rpm.
[0173] In some embodiments, a process of the invention comprises
drying a lipid after separation of the lipid from a treated cell
composition or a treated lysed cell composition in order to remove
water from the lipid. In some embodiments, drying the lipid can
include, but is not limited to, heating the lipid to evaporate
water. In some embodiments, after drying, the lipid has a water
content by weight percentage of lipid that is less than 3%, less
than 2.5%, less than 2%, less than 1.5%, less than 1%, less than
0.5%, less than 0.1%, or 0%. In some embodiments, after drying, the
lipid has a water content by weight percentage of lipid of 0% to
3%, 0% to 2.5%, 0% to 2%, 0% to 1.5%, 0% to 1%, 0% to 0.5%, 0.1% to
3%, 0.1% to 2.5%, 0.1% to 2%, 0.1% to 1.5%, 0.1% to 1%, 0.1% to
0.5%, 0.5% to 3%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to
1%, 1% to 3%, 1% to 2.5%, 1% to 2%, 1% to 1.5%, 1.5% to 3%, 1.5% to
2.5%, 1.5% to 2%, 2% to 3%, 2% to 2.5%, or 2.5% to 3%.
[0174] In some embodiments, a process further comprises refining a
lipid by one or more processes selected from caustic refining,
degumming, alkali-refining, bleaching, deodorization,
deacidification, or the like, and combinations thereof to remove
one or more phospholipids, free fatty acids, phosphatides, color
bodies, sterols, odors, and other impurities. As used herein, a
"refined oil" is a crude lipid or crude oil that has been refined.
As used herein, "a crude lipid" or "a crude oil" is a lipid or oil
that has not been refined. In some embodiments, the lipid separated
from a demulsified cell composition is a crude lipid.
[0175] Various exemplary processes of the present invention are
described schematically in FIGS. 1-4. Referring to FIG. 1, in some
embodiments, the present invention is directed to a process (100)
for obtaining a lipid (110) from a cell (101), comprising lysing
(102) the cell (101) to form a lysed cell composition (103). The
lysed cell composition is contacted with a first base (104) to
demulsify lysed cell composition (103), contacted with a salt (105)
to demulsify lysed cell composition (103), and heated (106), e.g.,
for 10 minutes to 96 hours, to provide a treated lysed cell
composition (107). The treated lysed cell composition (107) is
contacted with a second base (108) and separated (109), e.g., at a
temperature of 10.degree. C. to 100.degree. C., to provide a lipid
(110).
[0176] Referring to FIG. 2, in some embodiments, the present
invention is directed to a process (200) for obtaining a lipid
(210) from a cell, the process comprising lysing (102) a cell (101)
to form a lysed cell composition (103). The lysed cell composition
is then contacted with a base (204) to demulsify lysed cell
composition (103) and to provide a treated lysed cell composition
(207). The treated lysed cell composition (207) is separated (209),
e.g., at a temperature of 10.degree. C. to 100.degree. C., to
provide a lipid (210).
[0177] Referring to FIG. 3, in some embodiments, the present
invention is directed to a process (300) for obtaining a lipid
(310) from a cell, the process comprising lysing (102) a cell (101)
to form a lysed cell composition (103). The lysed cell composition
is then contacted with a salt (305) to demulsify lysed cell
composition (103) and to provide a treated lysed cell composition
(307), which is separated (309), e.g., at a temperature of
10.degree. C. to 100.degree. C., to provide a lipid (310).
[0178] Referring to FIG. 4, in some embodiments, the present
invention is directed to a process (400) for obtaining a lipid
(410) from a cell, the process comprising lysing (102) a cell (101)
to form a lysed cell composition (103). The lysed cell composition
is then contacted with a salt (405) to demulsify lysed cell
composition (103) and agitated (401), e.g., for 5 minutes to 96
hours, and optionally heated (402), to provide a treated lysed cell
composition (407). The treated lysed cell composition is then
separated (409), e.g., at a temperature of 10.degree. C. to
100.degree. C., to provide a lipid (410).
[0179] In some embodiments, a process of the present invention
comprises concentrating a broth comprising a microbial cell, a
broth comprising plant material and/or concentrating a lysed cell
composition. As used herein, "concentrating" refers to removing
water from a composition. Concentrating can include, but is not
limited to, evaporating, chemical drying, centrifuging, and the
like, and combinations thereof.
[0180] In some embodiments, a broth comprising a microbial cell or
a broth comprising plant material is concentrated to provide a
lipid concentration of at least 4%, at least 5%, at least 10%, at
least 15%, at least 20%, at least 25%, or at least 30% by weight of
the broth. In some embodiments, a broth comprising a microbial cell
or a broth comprising plant material is concentrated to provide a
lipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%,
5% to 40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to
20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or
30% to 40% by weight of the broth.
[0181] In some embodiments, a cell composition or a lysed cell
composition is concentrated to provide a lipid concentration of at
least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at
least 25%, or at least 30% by weight of the lysed cell composition.
In some embodiments, a cell composition or a lysed cell composition
is concentrated to provide a lipid concentration of 4% to 40%, 4%
to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 10%
to 40%, 10% to 30%, 10% to 20%, 15% to 40%, 15% to 30%, 20% to 40%,
20% to 30%, 25% to 40%, or 30% to 40% by weight of the lysed cell
composition.
[0182] In some embodiments, a lipid prepared by a process of the
present invention has an overall aroma intensity of 2 or less. As
used herein, the term "overall aroma intensity" refers to the
olfactory sensory rating given to the lipid by a panel of sensory
analysts. As used herein, the term "sensory analyst" refers to a
trained individual that provides feedback on and/or rates the
sensory characteristics of a substance.
[0183] In some embodiments, a lipid prepared by a process of the
present invention has an overall aromatic intensity of 3 or less.
As used herein, the term "overall aromatic intensity" refers to the
gustatory, or taste, sensory rating given to the lipid by a panel
of sensory analysts. In some embodiments, the Universal Spectrum
descriptive analysis method is used to assess the aroma and
aromatic characteristics of samples. This method uses an intensity
scale of 0-15, where 0=none detected and 15=very high intensity, to
measure the aroma and aromatic attributes of the oils.
[0184] In some embodiments, a lipid prepared by a process of the
present invention does not have an aftertaste characterized as
fishy. As used herein, the term "aftertaste" refers to the
persistence of a sensation of a flavor in the lipid, as
characterized by a panel of sensory analysts.
[0185] In some embodiments, a process of the present invention
provides a crude lipid having a peroxide value (PV) of 5 or less,
4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or
less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or
less. As used herein, the terms "peroxide value" or "PV" refer to
the measure of primary reaction products, such as peroxides and
hydroperoxides, that occur during oxidation of the lipid. In some
embodiments, the PV is an indicator of the quality of the lipid and
the extent of oxidation which has occurred in the lipid having a
low PV (i.e., 5 or less) demonstrates increased stability and
sensory profiles than lipids having a PV greater than 5. In some
embodiments, adding a base to a lysed cell composition, as
discussed above, raises the pH of the lysed cell composition and
inhibits lipid oxidation, thereby minimizing the amount of free
radicals in the lysed cell composition so that the crude lipid
obtained from the processes of the invention has a low PV (i.e., 5
or less).
[0186] In some embodiments, a process of the present invention
provides a crude lipid having an anisidine value (AV) of 26 or
less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2
or less, or 1 or less. As used herein, the terms "anisidine value"
or "AV" refer to the measure of secondary reaction products, such
as aldehydes and ketones, that occur during oxidation of the lipid.
In some embodiments, the AV is an indicator of the quality of the
lipid and the extent of oxidation which has occurred in the lipid.
A lipid having a low AV (i.e., 26 or less) demonstrates increased
stability and sensory profiles than lipids having an AV greater
than 26. In some embodiments, adding a base to a lysed cell
composition, as discussed above, raises the pH of the lysed cell
composition and inhibits lipid oxidation, thereby minimizing the
amount of free radicals in the lysed cell composition so that the
crude lipid obtained from the processes of the invention has a low
AV (i.e., 26 or less).
[0187] In some embodiments, a process of the present invention
provides a crude lipid having a phosphorus content of 100 ppm or
less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or
less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or
less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or
less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or
less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less,
3 ppm or less, 2 ppm or less, or 1 ppm or less.
[0188] In some embodiments, a process of the present invention
provides a crude lipid that has a lower anisidine value, lower
peroxide value, lower phosphorus content and/or a higher extraction
yield than if extraction was performed using a solvent (e.g.,
atypical hexane extraction or a FRIOLEX.RTM. process (Westfalia
Separator AG, Germany)). The FRIOLEX.RTM. process which is a
process of extracting lipids with a water-soluble organic solvent
as described in U.S. Pat. No. 5,928,696 and International Pub. Nos.
WO 01/76385 and WO 01/76715, each of which is incorporated by
reference herein in its entirety.
[0189] In some embodiments, heating the lysed cell composition
causes the secondary reaction products (e.g., aldehydes and
ketones) to participate in a reaction similar to the Maillard
reaction with proteins present in the lysed cell composition. The
reaction is believed to create products that possess antioxidant
activity, which reduces the oxidation of the lipid. In some
embodiments, additional protein, e.g., soy protein, can be added to
the lysed cell composition to increase the antioxidant activity.
The reduction in oxidation of the lipid reduces the AV of the
lipid, reduces any aftertaste of the lipid and/or increases the
stability of the lipid. In some embodiments, the stability is
increased at least 5%, at least 10%, at least 15% or at least
20%.
[0190] In some embodiments, a lipid extracted by a process of the
present invention, the biomass remaining after extraction of the
lipid, or combinations thereof can be used directly as a food or
food ingredient, such as an ingredient in baby food, infant
formula, beverages, sauces, dairy based foods (such as milk,
yogurt, cheese and ice-cream), oils (e.g., cooking oils or salad
dressings), and baked goods; nutritional supplements (e.g., in
capsule or tablet forms); feed or feed supplement for any non-human
animal (e.g., those whose products (e.g., meat, milk, or eggs) are
consumed by humans); food supplements; and pharmaceuticals (in
direct or adjunct therapy application). The term "animal" refers to
any organism belonging to the kingdom Animalia and includes any
human animal, and non-human animal from which products (e.g., milk,
eggs, poultry meat, beef, pork or lamb) are derived. In some
embodiments, the lipid and/or biomass can be used in seafood.
Seafood is derived from, without limitation, fish, shrimp and
shellfish. The term "products" includes any product derived from
such animals, including, without limitation, meat, eggs, milk or
other products. When the lipid and/or biomass are fed to such
animals, polyunsaturated lipids can be incorporated into the flesh,
milk, eggs or other products of such animals to increase their
content of these lipids.
Microbial Lipids
[0191] In some embodiments, the present invention is directed to a
microbial lipid extracted according to the processes of the present
invention. In some embodiments, a crude microbial lipid has an
anisidine value of 26 or less, 25 or less, 20 or less, 15 or less,
10 or less, 5 or less, 2 or less, or 1 or less, and/or a peroxide
value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less,
2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or
less, or 0.1 or less, and/or a phosphorus content of 100 ppm or
less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or
less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or
less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or
less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or
less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less,
3 ppm or less, 2 ppm or less, or 1 ppm or less. In some
embodiments, the crude microbial lipid has less than 5%, less than
4%, less than 3%, less than 2%, or less than 1% by weight or volume
of an organic solvent. In some embodiments, the crude microbial
lipid has at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, or at least
50% by weight of a desired PUFA. In some embodiments, the crude
microbial lipid has at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
or at least 50% by weight of DHA, and/or at least 10%, at least
15%, or at least 20% by weight of DPA n-6, and/or at least 10%, at
least 15%, or at least 20% by weight of EPA, and/or at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least
35%, at least 40%, at least 45%, or at least 50% by weight of ARA.
In some embodiments a crude microbial lipid extracted according to
the processes of the present invention result in a lower anisidine
value, lower peroxide value, lower phosphorus content and/or a
higher extraction yield than if extraction was performed using a
solvent (e.g., a typical hexane extraction or a FRIOLEX.RTM.
process (Westfalia Separator AG, Germany)).
Lipids Extracted From a First Set of Isolated Thraustochytrid
Microorganisms
[0192] In some embodiments, the present invention is further
directed to a microbial lipid extracted from a thraustochytrid as
described in U.S. Pub. No. 2010/0239533 and Int'l. Pub. No. WO
2010/107415, each of which is incorporated by reference herein in
its entirety. In some embodiments, the method comprises growing a
thraustochytrid in a culture to produce a biomass and extracting a
lipid comprising omega-3 fatty acids from the biomass. The lipid
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, and to analyze the fatty acid profile of oils
extracted from the biomass. See, e.g., U.S. Pat. No. 5,130,242,
incorporated by reference herein in its entirety. The lipid can be
extracted according to the processes of the present invention.
[0193] A microbial lipid of the invention can be any lipid derived
from a microorganism, including, for example: a crude oil extracted
from the biomass of the microorganism without further 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 microbial oil; or an enriched oil that is obtained, for
example, by treating a crude or refined microbial oil with further
methods of purification to increase the concentration of a fatty
acid (such as DHA) in the oil.
[0194] In some embodiments, the microbial lipid comprises a sterol
esters fraction of 0%, at least 0.1%, at least 0.2%, at least 0.5%,
at least about 1%, at least 1.5%, at least 2%, or at least 5% by
weight. In some embodiments, the microbial lipid comprises a sterol
esters fraction of from 0% to 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%,
0.2% to 1.5%, 0.2% to 2%, or 0.2% to 5% by weight. In some
embodiments, the microbial lipid comprises a sterol esters fraction
of less than 5%, less than 4%, less than 3%, or less than 2% by
weight.
[0195] In some embodiments, the microbial lipid comprises a
triglyceride fraction of at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, or at least 90% by weight. In some
embodiments, the microbial lipid comprises a triglyceride fraction
of from 65% to 95%, 75% to 95%, or 80% to 95% by weight, or 97% by
weight, or 98% by weight.
[0196] In some embodiments, the microbial lipid comprises a free
fatty acid fraction of at least 0.5%, at least 1%, at least 1.5%,
at least 2%, at least 2.5%, or at least 5% by weight. In some
embodiments, the microbial lipid comprises a free fatty acid
fraction of from 0.5% to 5%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to
1.5%, 0.5% to 1%, 1% to 2.5%, 1% to 5%, 1.5% to 2.5%, 2% to 2.5%,
or 2% to 5% by weight. In some embodiments, the microbial lipid
comprises a free fatty acid fraction of less than 5%, less than 4%,
less than 3%, less than 2%, or less than 1% by weight.
[0197] In some embodiments, the microbial lipid comprises a sterol
fraction of at least 0.5%, at least 1%, at least 1.5%, at least 2%,
or at least 5% by weight. In some embodiments, the microbial lipid
comprises a sterol fraction of from 0.5% to 1.5%, 1% to 1.5%, 0.5%
to 2%, 0.5% to 5%, 1% to 2%, or 1% to 5% by weight. In some
embodiments, the microbial lipid comprises a sterol fraction of
less than 5%, less than 4%, less than 3%, less than 2%, or less
than 1% by weight.
[0198] In some embodiments, the microbial lipid comprises a
diglyceride fraction of at least 1.5%, at least 2%, at least 2.5%,
at least 3%, at least 3.5%, or at least 5% by weight. In some
embodiments, the microbial lipid comprises a diglyceride fraction
of from 1.5% to 3%, 2% to 3%, 1.5% to 3.5%, 1.5% to 5%, 2.5% to 3%,
2.5% to 3.5%, or 2.5% to 5% by weight.
[0199] In some embodiments, the microbial lipid comprises
unsaponifiables of less than 2%, less than 1.5%, less than 1%, or
less than 0.5% by weight of the oil.
[0200] 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
analyzed by other methods know in the art.
[0201] In some embodiments, the microbial lipid 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 at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
at least 75%, or at least 80% by weight DHA. In some embodiments,
the microbial lipid 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 40% to 45%, 40% to 50%, 40% to 60%, 50% to
60%, 55% to 60%, 40% to 65%, 50% to 65%, 55% to 65%, 40% to 70%,
40% to 80%, 50% to 80%, 55% to 80%, 60% to 80%, or 70% to 80% by
weight DHA. In some embodiments, the microbial lipid comprises a
sterol esters fraction comprising 45% or less, 40% or less, 35% or
less, 30% or less, 25% or less, 20% or less, 15% or less, or 13% or
less by weight DHA. In some embodiments, the microbial lipid 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 10% or
less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less,
4% or less, 3% or less, 2% or less, or 1% or less by weight EPA. In
some embodiments, the microbial lipid 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 2% to 3%, 2% to 3.5%, 2.5% to
3.5%, 2% to 6%, 2.5% to 6%, 3.0% to 6%, 3.5% to 6%, 5% to 6%, or 2%
to 10% by weight EPA. In some embodiments, the microbial lipid
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, is substantially free of EPA. In some embodiments, the
microbial lipid 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 EPA of at
least 5:1, at least 7:1, at least 9:1, at least 10:1, at least
15:1, at least 20:1, at least 25:1, at least 30:1, or at least
50:1, wherein the microbial lipid and/or one or more fractions
thereof comprises 10% or less by weight of EPA. In some
embodiments, the microbial lipid 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 EPA of at least 5:1, but less than 20:1. In
some embodiments, the weight ratio of DHA to EPA is from 5:1 to
18:1, from 7:1 to 16:1, or from 10:1 to 15:1. In some embodiments,
the microbial lipid 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 0.1% to 0.25%, 0.2% to
0.25%, 0.1% to 0.5%, or 0.1% to 1.5% by weight ARA. In some
embodiments, the microbial lipid 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 1.5% or
less, 1% or less, 0.5% or less, 0.2% or less, or 0.1% or less by
weight ARA. In some embodiments, the microbial lipid 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, is
substantially free of ARA. In some embodiments, the microbial lipid
and/or one or more fractions thereof selected from the sterol
esters fraction, the triglyceride fraction, the free fatty acid
fraction, the diglyceride fraction, the polar fraction (including
the phospholipid fraction), and combinations thereof, comprises a
weight ratio of DHA to ARA of at least 20:1, at least 30:1, at
least 35:1, at least 40:1, at least 60:1, at least 80:1, at least
100:1, at least 150:1, at least 200:1, at least 250:1, or at least
300:1. In some embodiments, the microbial lipid 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 0.5% to 1%, 0.5% to 2%, 0.5% to 2.5%, 0.5% to 3%, 0.5% to
3.5%, 0.5% to 5%, 0.5% to 6%, 1% to 2%, 2% to 3%, 2% to 3.5%, 1% to
2.5%, 1% to 3%, 1% to 3.5%, 1% to 5%, or 1% to 6% by weight DPA
n-6. In some embodiments, the microbial lipid 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 6%
or less, 5% or less, 3% or less, 2.5% or less, 2% or less, 1% or
less, or 0.5% or less by weight DPA n-6. In some embodiments, the
microbial lipid 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, is substantially free of DPA n-6. In some
embodiments, the microbial lipid 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 6:1, of at least
8:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at
least 50:1, or at least 100:1. In some embodiments, the microbial
lipid 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 5% or less, 4% or less, 3% or less, 2% or less,
1.5% or less, 1% or less, or 0.5% 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 erucic acid (22:1 n-9). In some embodiments,
the microbial lipid 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 5% or less, 4% or less, 3% or
less, 2% or less, 1.5% or less, or 1% or less by weight of
heptadecanoic acid (17:0). In some embodiments, the microbial lipid
and/or one or more fractions thereof comprise 0.01% to 5% by
weight, 0.05% to 3% by weight, or 0.1% to 1% by weight of
heptadecanoic acid.
[0202] In some embodiments, an extracted microbial lipid comprises
a triglyceride fraction of at least 70% by weight, wherein the
docosahexaenoic acid content of the triglyceride fraction is at
least 50% by weight, wherein the docosapentaenoic acid n-6 content
of the triglyceride fraction is from at least 0.5% by weight to 6%
by weight, and wherein the oil has an anisidine value of 26 or
less. In some embodiments, an extracted microbial lipid comprises a
triglyceride fraction of at least 70% by weight, wherein the
docosahexaenoic acid content of the triglyceride fraction is at
least 40% by weight, wherein the docosapentaenoic acid n-6 content
of the triglyceride fraction is from at least 0.5% by weight to 6%
by weight, wherein the ratio of docosahexaenoic acid to
docosapentaenoic acid n-6 is greater than 6:1, and wherein the
lipid has an anisidine value of 26 or less. In some embodiments, an
extracted microbial lipid comprises a triglyceride fraction of at
least 70% by weight, wherein the docosabexaenoic acid content of
the triglyceride fraction is at least 60% by weight and wherein the
lipid has an anisidine value of 26 or less. In some embodiments, an
extracted microbial lipid having any of the above fatty acid
profiles has an anisidine value of 26 or less, 25 or less, 20 or
less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less
and/or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5
or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less,
0.5 or less, 0.2 or less, or 0.1 or less, and/or a phosphorus
content of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm
or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or
less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or
less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or
less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or
less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or
less. In some embodiments, an extracted microbial lipid having any
of the above fatty acid profiles is extracted from an isolated
thraustochytrid microorganism having the characteristics of the
thraustochytrid species deposited under ATCC Accession No.
PTA-9695, PTA-9696, PTA-9697, or PTA-9698. In some embodiments, an
extracted microbial lipid having any of the above fatty acid
profiles is a crude lipid. In some embodiments, the crude lipid has
less than 5% by weight or volume of an organic solvent. In some
embodiments the microbial lipid extracted according to the
processes of the present invention result in a lower anisidine
value, lower peroxide value, lower phosphorus content and/or a
higher extraction yield if extraction was performed using a solvent
(e.g., atypical hexane extraction or a FRIOLEX.RTM. process
(Westfalia Separator AG, Germany)).
Lipids Extracted from a Second Set of Isolated Thraustochytrid
Microorganisms
[0203] In some embodiments, the present invention is further
directed to a microbial lipid extracted from a thraustochytrid as
described in U.S. application Ser. No. 12/729,013 and
PCT/US2010/028175, each of which is incorporated by reference
herein in its entirety. In some embodiments, the method comprises
growing a thraustochytrid in a culture to produce a biomass and
extracting a lipid comprising omega-3 fatty acids from the biomass.
The lipid 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, and to analyze the fatty acid profile of
oils extracted from the biomass. See, e.g., U.S. Pat. No.
5,130,242, incorporated by reference herein in its entirety. The
lipid can be extracted according to the processes of the present
invention.
[0204] A microbial lipid of the invention can be any lipid derived
from a microorganism, including, for example: a crude oil extracted
from the biomass of the microorganism without further 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 microbial oil; or an enriched oil that is obtained, for
example, by treating a crude or refined microbial oil with further
methods of purification to increase the concentration of a fatty
acid (such as DHA) in the oil.
[0205] In some embodiments, the microbial lipid comprises a sterol
esters fraction of 0%, at least 0.1%, at least 0.2%, at least 0.5%,
at least 1%, at least 1.5%, at least 2%, or at least 5% by weight.
In some embodiments, the microbial lipid comprises a sterol esters
fraction of 0% to 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to
1.5%, 0.2% to 2%, or 0.2% to 5% by weight. In some embodiments, the
microbial lipid comprises a sterol esters fraction of 5% or less,
4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3%
or less, 0.2% or less, 0.5% or less, 0.4% or less, 0.3% or less, or
0.2% or less by weight.
[0206] In some embodiments, the microbial lipid comprises a
triacylglycerol fraction of at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, or at least
90% by weight. In some embodiments, the microbial lipid comprises a
triacylglycerol fraction of 35% to 98%, 35% to 90%, 35% to 80%, 35%
to 70%, 35% to 70%, 35% to 65%, 40% to 70%, 40% to 65%, 40% to 55%,
40% to 50%, 65% to 95%, 75% to 95%, 75% to 98%, 80% to 95%, 80% to
98%, 90% to 96%, 90% to 97%, 90% to 98%, 90%, 95%, 97%, or 98% by
weight.
[0207] In some embodiments, the microbial lipid comprises a
diacylglycerol fraction of at least 10%, at least 11%, at least
12%, at least 13%, at least 14%, at least 15%, at least 16%, at
least 17%, at least 18%, at least 19%, or at least 20% by weight.
In some embodiments, the microbial lipid comprises a diacylglycerol
fraction of 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to
40%, 15% to 35%, or 15% to 30% by weight. In some embodiments, the
microbial lipid comprises a 1,2-diacylglycerol fraction of at least
0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 1%, at
least 5%, at least 10%, at least 11%, at least 12%, at least 13%,
at least 14%, at least 15%, at least 16%, at least 17%, at least
18%, at least 19%, or at least 20% by weight. In some embodiments,
the microbial lipid comprises a diacylglycerol fraction of 0.2% to
45%, 0.2% to 30%, 0.2% to 20%, 0.2% to 10%, 0.2% to 5%, 0.2% to 1%,
0.2% to 0.8%, 0.4% to 45%, 0.4% to 30%, 0.4% to 20%, 0.4% to 10%,
0.4% to 5%, 0.4% to 1%, 0.4% to 0.8%, 0.5% to 1%, 0.5% to 0.8%, 10%
to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 15% to 40%, 15% to 35%,
15% to 30%, or 15% to 25% by weight. In some embodiments, the
microbial lipid comprises a 1,3-diacylglycerol fraction of at least
0.1%, at least 0.2%, at least 0.5%, at least 1%, at least 2%, at
least 2.5%, or at least 3% by weight.
[0208] In some embodiments, the microbial lipid comprises a sterol
fraction of at least 0.3%, at least 0.4%, at least 0.5%, at least
1%, at least 1.5%, at least 2%, or at least 5% by weight. In some
embodiments, the microbial lipid comprises a sterol fraction of
0.3% to 5%, 0.3% to 2%, 0.3% to 1.5%, 0.5% to 1.5%, 1% to 1.5%,
0.5% to 2%, 0.5% to 5%, 1% to 2%, or 1% to 5% by weight. In some
embodiments, the microbial lipid comprises a sterol fraction of 5%
or less, 4% or less, 3% or less, 2% or less, 1.5% or less, or 1% or
less by weight.
[0209] In some embodiments, the microbial lipid comprises a
phospholipid fraction of at least 2%, at least 5%, or at least 8%
by weight. In some embodiments, the microbial lipid comprises a
phospholipid fraction of 2% to 25%, 2% to 20%, 2% to 15%, 2% to
10%, 5% to 25%, 5% to 20%, 5% to 20%, 5% to 10%, or 7% to 9% by
weight. In some embodiments, the microbial lipid comprises a
phospholipid fraction of less than 20%, less than 15%, less than
10%, less than 9%, or less than 8% by weight. In some embodiments,
the microbial lipid is substantially free of phospholipids.
[0210] In some embodiments, the microbial lipid comprises
unsaponifiables of less than 2%, less than 1.5%, less than 1%, or
less than 0.5% by weight of the oil.
[0211] The lipid classes present in the microbial lipid, such as a
triacylglycerol fraction, can be separated by flash chromatography
and analyzed by thin layer chromatography (TLC), or separated and
analyzed by other methods known in the art.
[0212] In some embodiments, the microbial lipid 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, comprises at least 5%, at least
10%, more than 10%, at least 12%, at least 13%, at least 14%, at
least 15%, at least 16%, at least 17%, at least 18%, at least 19%,
at least 20%, at least 25%, at least 30%, least 35%, at least 40%,
or at least 45% by weight EPA. In some embodiments, the microbial
lipid 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,
comprises 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5%
to 30%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%,
10% to 30%, at least 12% to 55%, at least 12% to 50%, at least 12%
to 45%, at least 12% to 40%, at least 12% to 35%, or at least 12%
to 30%, 15% to 55%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%,
15% to 30%, 15% to 25%, 15% to 20%, 20% to 55%, 20% to 50%, 20% to
45%, 20% to 40%, or 20% to 30% by weight EPA. In some embodiments,
the microbial lipid 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 5%, at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 50%,
or at least 60% by weight DHA. In some embodiments, the microbial
lipid 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 5% to 60%, 5% to 55%, 5% to 50%, 5% to 40%, 10% to 60%,
10% to 50%, 10% to 40%, 20% to 60%, 25% to 60%, 25% to 50%, 25% to
45%, 30% to 50%, 35% to 50%, or 30% to 40% by weight DHA. In some
embodiments, the microbial lipid 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 10% or less, 9% or less, 8% or
less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less,
2% or less, or 1% or less by weight DHA. In some embodiments, the
microbial lipid 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 1% to 10%, 1% to 5%, 2% to 5%, 3% to 5%, or 3% to 10% by
weight of the fatty acids as DHA. In some embodiments, the
microbial lipid 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 lipid
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 0.1% to 5%, 0.1% to less than 5%, 0.1% to 4%, 0.1% to 3%,
0.1% to 2%, 0.2% to 5%, 0.2% to less than 5%, 0.2% to 4%, 0.2% to
3%, 0.2% to 2%, 0.3% to 2%, 0.1% to 0.5%, 0.2% to 0.5%, 0.1% to
0.4%, 0.2% to 0.4%, 0.5% to 2%, 1% to 2%, 0.5% to 1.5%, or 1% to
1.5% by weight ARA. In some embodiments, the microbial lipid 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 5% or
less, less than 5%, 4% or less, 3% or less, 2% or less, 1.5% or
less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or
less, or 0.1% or less by weight ARA. In some embodiments, the
microbial lipid 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 ARA. In some embodiments, the microbial lipid
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 0.4% to 2%, 0.4% to 3%, 0.4% to 4%, 0.4% to 5%, 0.4% to
less than 5%, 0.5% to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5%
to 5%, 0.5% to less than 5%, 1% to 2%, 1% to 3%, 1% to 4%, 1% to
5%, or 1% to less than 5% by weight DPA n-6. In some embodiments,
the microbial lipid 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 5%, less than 5%, 4% or less, 3% or less, 2% or less, 1%
or less, 0.75% or less, 0.6% or less, or 0.5% or less by weight DPA
n-6. In some embodiments, the microbial lipid 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 DPA n-6. In some
embodiments, the microbial lipid 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 fatty acids with 5% or less,
less than 5%, 4% or less, 3% or less, or 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.
[0213] In some embodiments, an extracted microbial lipid comprises
at least 20% by weight eicosapentaenoic acid and less than 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, an extracted microbial
lipid comprises a triacylglycerol fraction of at least 10% by
weight, wherein at least 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, and wherein less than 5% by weight of the
fatty acids in the triacylglycerol fraction is arachidonic acid. In
some embodiments, an extracted microbial lipid having any of the
above fatty acid profiles has an anisidine value of 26 or less, 25
or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less,
or 1 or less, and/or a peroxide value of 5 or less, 4.5 or less, 4
or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or
less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and/or a
phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or
less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or
less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, an extracted microbial lipid
having any of the above fatty acid profiles is extracted from an
isolated thraustochytrid microorganism having the characteristics
of the thraustochytrid species deposited under ATCC Accession No.
PTA-10208, PTA-10209, PTA-10210, PTA-10211, PTA-10212, PTA-10213,
PTA-10214, or PTA-10215. In some embodiments, an extracted
microbial lipid having any of the above fatty acid profiles is a
crude lipid. In some embodiments, the crude lipid has less than 5%
by weight or volume of an organic solvent. In some embodiments a
microbial lipid extracted according to the processes of the present
invention result in a lower anisidine value, and/or lower peroxide
value, and/or lower phosphorus content than if extraction was
performed using a typical hexane extraction or a FRIOLEX.RTM.
process (Westfalia Separator AG, Germany).
[0214] In some embodiments, a lipid obtained by any of the
processes of the present invention comprises at least 20% by weight
eicosapentaenoic acid and less than 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, a lipid obtained by any of the processes
of the present invention comprises a triacylglycerol fraction of at
least 10% by weight, wherein at least 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, and wherein less
than 5% by weight of the fatty acids in the triacylglycerol
fraction is arachidonic acid. In some embodiments, a lipid obtained
by any of the processes of the present invention having any of the
above fatty acid profiles has an anisidine value of 26 or less, 25
or less, 20 or less, 15 or less, 10 or less, 5 or less, or 2 or
less, or 1 or less and/or a peroxide value of 5 or less, 4.5 or
less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less,
1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less,
and/or a phosphorus content of 100 ppm or less, 95 ppm or less, 90
ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm
or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, a lipid obtained by any of
the processes of the present invention having any of the above
fatty acid profiles is extracted from an isolated thraustochytrid
microorganism having the characteristics of the thraustochytrid
species deposited under ATCC Accession No. PTA-10208, PTA-10209,
PTA-10210, PTA-10211, PTA-10212, PTA-10213, PTA-10214, or
PTA-10215. In some embodiments, a lipid obtained by any of the
processes of the present invention having any of the above fatty
acid profiles is a crude lipid. In some embodiments, the crude
lipid has less than 5% by weight or volume of an organic solvent.
In some embodiments a lipid extracted according to the processes of
the present invention result in a lower anisidine value, lower
peroxide value, lower phosphorus content and/or a higher extraction
yield if extraction was performed using a solvent (e.g., atypical
hexane extraction or a FRIOLEX.RTM. process (Westfalia Separator
AG, Germany)).
Lipids Extracted from an Isolated Microorganism of the Species
Cryptiecodinium Cohnii
[0215] In some embodiments, the present invention is further
directed to a crude lipid extracted from a microorganism of the
species Crypthecodinium cohnii. In some embodiments, the method
comprises growing a microorganism of the species Crypthecodinium
cohnii in a culture to produce a biomass and extracting a lipid
comprising omega-3 fatty acids from the biomass. The lipid 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 microorganism of the species Crypthecodinium cohnii, and
to isolate a biomass from the culture. See, e.g., U.S. Pat. No.
7,163,811, incorporated by reference herein in its entirety. The
lipid can be extracted according to the processes of the present
invention.
[0216] In some embodiments, the crude lipid extracted from a
microorganism of the species Crypthecodinium cohnii according to
the extraction methods of the present invention can have a lower
phosphorus content compared to using a typical hexane extraction
method. In some embodiments, the crude lipid extracted from a
microorganism of the species Crypthecodinium cohnii comprises a
phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or
less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or
less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or
less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or
less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or
less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less,
or 1 ppm or less. In some embodiments, the crude oil has an
anisidine value of 26 or less, 25 or less, 20 or less, 15 or less,
10 or less, 5 or less, or 2 or less, or 1 or less and/or a peroxide
value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less,
2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or
less, or 0.1 or less. In some embodiments a crude microbial lipid
extracted according to the processes of the present invention
result in a lower anisidine value, lower peroxide value, lower
phosphorus content and/or a higher extraction yield if extraction
was performed using a solvent (e.g., atypical hexane extraction or
a FRIOLEX.RTM. process (Westfalia Separator AG, Germany)).
[0217] Having generally described the invention, a further
understanding can be obtained by reference to the examples provided
herein. These examples are given for purposes of illustration only
and are not intended to be limiting. The following examples are
illustrative, but not limiting, of a process and a lipid prepared
by a process of the present invention. Other suitable modifications
and adaptations of the variety of conditions and parameters
normally encountered in extraction of a lipid from a cell, and
which would become apparent to those skilled in the art, and are
within the spirit and scope of the invention.
EXAMPLES
Example 1
[0218] A cell broth (20,000 kg) containing microbial cells
(Schizochytrium) was heated to 60.degree. C. Enzymes (i.e.,
Alcalase 2.4 L FG 0.5%) were added to the cell biomass to lyse the
cells and form an emulsified lysed cell composition. The emulsified
lysed cell composition was first treated with a first base (NaOH,
250 kg of 50% w/w solution) until the pH of the lysed cell
composition was from 10.4 to 10.6. Next, a salt (solid NaCl, in an
amount of 2%, by weight, of the lysed cell composition) was added
to the lysed cell composition. The lysed cell composition was then
heated to a temperature of 85.degree. C. to 102.degree. C. and held
at that temperature level for 24 hours to 70 hours. A second base
(NaOH, 50% w/w solution, 40 kg) was then added to the lysed cell
composition until the pH was above 8. The lysed cell composition
was then centrifuged to separate the lysed cell composition into
three phases: a top phase containing a lipid layer, a middle phase
containing an emulsion layer, and a bottom phase containing a solid
layer. The lysed cell composition was then centrifuged at
40.degree. C. to 80.degree. C. using a Westfalia RSE110 Centrifuge
(Westfalia Separator Industry GmbH, Germany), operating at 6,000
rpms at a feed rate of 30 kg/min to separate a lipid from the lysed
cell composition. The centrifuging provided three phases: an upper
phase containing a lipid, a middle phase containing an emulsion,
and a bottom phase containing a solid/liquid emulsion. The pH of
the lysed cell composition was maintained at 7.5 to 8.5 during the
centrifuging. The total time to centrifuge the entire 20,000 kg
batch was approximately 10 to 11 hours. The lipid layer was
separated and had a moisture content of approximately 1% by
weight.
Example 2
[0219] A cell broth (500 g) containing microbial cells
(Crypthecodinium cohnii) was concentrated from approximately 7%
biomass to 13.5% biomass, by weight of the broth. The broth was
homogenized at a pressure of 10,000 psi (2 passes) to form a lysed
cell composition. The lysed cell composition was treated with a
base (i.e., NaOH, 10 g of a 50% w/w solution) until the pH of the
lysed cell composition was 10.4 to 10.6. A salt (solid NaCl, in an
amount of 2% by weight of the lysed cell composition) was added to
the lysed cell composition. The lysed cell composition was then
heated to a temperature of 85.degree. C. to 92.degree. C. and held
at that temperature range for 15 minutes to 2 hours. The lysed cell
composition was then centrifuged at a temperature of 70.degree. C.
to 90.degree. C. using a Bench Top Sigma 6K15 Centrifuge (SIGMA
Laborzentrifugen GmbH, Germany), operating at 5,400 rpm to separate
the lysed cell composition into three phases: an upper phase
containing a lipid, a middle phase containing an emulsion, and a
bottom phase containing a solid/liquid emulsion. The pH of the
lysed cell composition during centrifuging was maintained at 6.5 to
8.5. The total time to centrifuge was 5 minutes. The lipid layer
was separated and had a moisture content of approximately 1% by
weight.
Example 3
[0220] A cell broth (20,000 kg) containing microbial cells
(Schizochytrium) was heated to 60.degree. C. Enzymes (i.e.,
Alcalase 2.4 L FG 0.5%) were added to the cell biomass to lyse the
cells and form a lysed cell composition. Next, a salt (solid
Na.sub.2SO.sub.4, 2,000 kg, or 10%, by weight, of the lysed cell
composition) was added to the lysed cell composition. The lysed
cell composition was then agitated for 24 hours to 48 hours at room
temperature. The lysed cell composition was then centrifuged at
40.degree. C. to 75.degree. C. using a Westfalia RSE110 Centrifuge
(Westfalia Separator Industry GmbH, Germany), operating at 6,000
rpm at a feed rate of 40 kg/min to separate a lipid from the lysed
cell composition. The centrifuging provided three phases: an upper
phase containing a lipid, a middle phase containing an emulsion,
and a bottom phase containing a solid/liquid emulsion. The total
time to centrifuge the entire 20,000 kg batch was approximately 8
to 9 hrs. The lipid layer was separated from the centrifuged lysed
cell composition.
Example 4
[0221] A pasteurized cell broth (500 g) containing microbial cells
(ATCC Accession No. PTA-10208) was provided. Enzymes (i.e.,
Alcalase 2.4 L FG 0.5%) were added to the cell biomass to lyse the
cells and form an emulsified lysed cell composition. The emulsified
lysed cell composition was treated with a first base (i.e., a 25%
solution of NaOH) to adjust the pH of the lysed cell composition to
10.5. Next, a salt (solid NaCl, in an amount of 2%, by weight, of
the lysed cell composition) was added to the lysed cell
composition. The lysed cell composition was then heated to a
temperature of 95.degree. C. and held at that temperature level for
2 hours while agitating the lysed cell composition. A second base
(i.e., a 25% solution of NaOH) was then added to the lysed cell
composition until the pH was 8.3. The lysed cell composition was
then centrifuged at 5,100 rpm for 5 minutes to separate the lysed
cell composition and yield a lipid layer and a small emulsion
layer.
Example 5
[0222] A cell broth (500 g) that was concentrated and pasteurized
containing microbial cells (ATCC Accession No. PTA-9695) was
provided. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were added to the
cell biomass to lyse the cells and form an emulsified lysed cell
composition. The emulsified lysed cell composition was treated with
a base (i.e., a 25% solution of NaOH) to adjust the pH of the lysed
cell composition to 10.5. Next, a salt (solid NaCl, in an amount of
2%, by weight, of the lysed cell composition) was added to the
lysed cell composition. The lysed cell composition was then heated
to a temperature of 95.degree. C. and held at that temperature
level for 1 hour while agitating the lysed cell composition and the
pH dropped to 8.5. After an hour in the fermentation broth having a
total of 10 ml, there was an about 1 ml layer of oil (lipid) and an
about 6 ml layer of emulsion. The lysed cell composition was heated
for a total of 220 minutes and the emulsion layer started to
disappear. The lysed cell composition was then centrifuged at 5,100
rpm for 5 minutes to separate the lysed cell composition. The
extraction yield of the lipid was 58.8 by weight %. The anisidine
value (AV) of the crude oil was 11.3. The cell breakage yield was
in a range of 93% to 95% by weight.
Example 6
[0223] A pasteurized cell broth (473 g) containing microbial cells
of the isolated thraustochytrid deposited under ATCC Accession No.
PTA-9695 was provided. Enzymes (i.e., Alcalase 2.4 L FG 0.5%) were
added to the cell biomass to lyse the cells and form an emulsified
lysed cell composition. The emulsified lysed cell composition was
treated with a first base (i.e., a 25% solution of NaOH) to adjust
the pH of the lysed cell composition to 10.62. Next, a salt (solid
NaCl, in an amount of 2%, by weight, of the lysed cell composition)
was added to the lysed cell composition. The lysed cell composition
was then heated to a temperature of 95.degree. C. and held at that
temperature level for 3 hours while agitating the lysed cell
composition. A second base (i.e., a 25% caustic solution of NaOH)
was then added to the lysed cell composition until the pH was 8.13.
The lysed cell composition was then centrifuged at 5,100 rpm for 5
minutes to separate the lysed cell composition and yield a lipid
layer and an emulsion layer in equal amounts. In order to determine
if raising the pH increased the yield of the lipid layer,
additional second base (i.e., a 25% solution of NaOH) was added to
the separated lysed cell composition until the pH was 9.02 and the
lysed cell composition was again centrifuged at 5,100 rpm for 5
minutes. This resulted in a similar yield of lipid layer.
Additional second base was added again to the separated lysed cell
composition until the pH was 10.12 and the lysed cell composition
was again centrifuged at 5,100 rpm for 5 minutes. Again, this
resulted in a similar yield of lipid layer.
Example 7
[0224] A pasteurized cell broth (470 g) containing microbial cells
(ATCC Accession No. PTA-9695) was provided. The cell biomass was
mechanically homogenized to lyse the cells and form an emulsified
lysed cell composition. The emulsified lysed cell composition was
treated with a first base (i.e., a 25% solution of NaOH) to adjust
the pH of the lysed cell composition to 10.5. Next, a salt (solid
NaCl, in an amount of 2%, by weight, of the lysed cell composition)
was added to the lysed cell composition. The lysed cell composition
was then heated to a temperature of 95.degree. C. and held at that
temperature level for 3 hours while agitating the lysed cell
composition. A second base (i.e., a 25% solution of NaOH) was then
added to the lysed cell composition until the pH was 8.07. The
lysed cell composition was then centrifuged at 5,100 rpm for 5
minutes to separate the lysed cell composition and yield a lipid
layer and an emulsion layer wherein the emulsion layer was larger
than the lipid layer. In order to determine if raising the pH
increased the yield of the lipid layer, additional second base was
added to the separated lysed cell composition until the pH was 9.11
and the lysed cell composition was again centrifuged at 5,100 rpm
for 5 minutes. This resulted in a similar yield of lipid layer.
Additional second base was added again to the separated lysed cell
composition until the pH was 10.09 and the lysed cell composition
was again centrifuged at 5,100 rpm for 5 minutes. Again, the
resulted in a similar yield of lipid layer.
Example 8
[0225] A cell broth containing microbial cells (Crypthecodinium
cohnii) was used in a decreased biotin trial fermentor. 20,000 kg
of washed, concentrated, and pasteurized broth was harvested. This
was pulled out at the startup of pasteurization. It was held for
approximately 1 day before being transferred and homogenized. The
material was homogenized at 813 bar/one pass and collected back
into a treatment tank. Through microscopic inspection, it was
estimated that approximately 80% of the cells were lysed.
[0226] The broth was heated to about 40.degree. C. before treatment
began. The pH was adjusted to 10.5 and 2% NaCl was added and heated
to 66.degree. C. At this point a significant oil layer had already
formed and the pH had dropped to 9.5 after 1-2 hours. The broth was
held at 66.degree. C. overnight.
[0227] The next day, the broth was centrifuged on the Westfalia
RSE-110 with a 155 mm ring dam installed. The viscosity was about
180 cP at 40.degree. C. The centrifuge was fed at 48 kg/min, with
5-10 psi backpressure on the light phase and 30 psi backpressure on
the heavy phase. The feed temperature was maintained at 70.degree.
C. No oil was present in the waste phase and only a few drops were
visible after isopropyl alcohol was added.
[0228] Table 1 shows the results of analyses performed on the crude
oil obtained from this procedure.
TABLE-US-00001 TABLE 1 Specifications of crude oil obtained using
process of Example 8. % Oil 87.79 DHA (mg/g) 531.02 % DHA 60.49 PV
1.95 (0.6*) AV 15 % FFA 0.18 Phosphorus (ppm) 8.65 Copper (ppm)
0.22 Iron (ppm) 0.7 Lead (ppm) 0.63 *PV of the centrifuged oil.
[0229] Of the 20,000 kg of broth provided, 10.5% by weight (2,100
kg) was biomass. Of the biomass, 50% by weight was oil (1,050 kg).
Of the oil, 58.9% by weight was DHA (618 kg). After running the
process described above, there was 592.5 kg of material in the
lipid layer, of which about 87.8% by weight (520.2 kg) was oil.
Thus, the extraction yield of oil from the biomass was 49.5%. Of
that oil, 60.5% by weight (314.6 kg) was DHA, thereby resulting in
an extraction yield of 51% by weight DHA from the biomass.
Example 9
[0230] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 25%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 5.8 to 11.2. The addition
of the base and the rise in the pH lysed the cells to form a lysed
cell composition. Next, a salt (solid Na.sub.2SO.sub.4, in an
amount of 5%, by weight, of the lysed cell composition) was added
to the lysed cell composition. The lysed cell composition was then
heated to a temperature in a range of 90.degree. C. to 95.degree.
C. and held at that temperature level for 90 minutes and the pH of
the lysed cell composition dropped to 9.7. After the 90 minutes
there was an about 2.5 ml of oil layer per 45 ml of fermentation
broth and there was no moisture loss. After 3 hours, the pH had
dropped to 9.2. The solution was then centrifuged at about 5,100
rpm for 5 minutes to separate the lysed cell composition and yield
a lipid layer. The extraction yield of the lipid was 81% by weight.
The anisidine value (AV) of the crude oil was 20.1. The cell
breakage yield was in a range of 92% to 98% by weight.
Example 10
[0231] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 25%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 4.8 to 11. The addition of
the base and the rise in the pH lysed the cells to form a lysed
cell composition. Next, a salt (solid NaCl, in an amount of 2%, by
weight, of the lysed cell composition) was added to the lysed cell
composition. The lysed cell composition was then heated to a
temperature in a range of 90.degree. C. to 95.degree. C. and held
at that temperature level for 3.5 hours and the pH of the lysed
cell composition dropped to 8.7 and there was no moisture loss. The
solution was then centrifuged at about 5,100 rpm for 5 minutes to
separate the lysed cell composition and yield a lipid layer. The
extraction yield of the lipid after 3.5 hours was 92% by
weight.
[0232] A portion of the lysed cell composition was held for 6 hours
and the pH of the lysed cell composition dropped to 8.6. The
solution was then centrifuged at about 5,100 rpm for 5 minutes to
separate the lysed cell composition and yield a lipid layer. The
extraction yield of the lipid after 6 hours was 89% by weight. The
anisidine value (AV) of the crude oil was 14.4. The cell breakage
yield was 95% by weight.
Example 11
[0233] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 50%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 5.8 to 11.2. The addition
of the base and the rise in the pH lysed the cells to form a lysed
cell composition. The lysed cell composition was then heated to
70.degree. C. under vacuum to reduce the water content from 88.7%
to 85.5%. During evaporation, the pH dropped to 10.36. The solution
was then centrifuged at about 5,100 rpm for 5 minutes to separate
the lysed cell composition and yield a lipid layer. The extraction
yield of the lipid was 83.9% by weight. The anisidine value (AV) of
the crude oil was 10.5. The cell breakage yield was 93.17% by
weight.
[0234] The process was repeated except the lysed cell composition
was heated to 70.degree. C. under vacuum to reduce the water
content from 88.7% to 79.2%. The extraction yield of the lipid was
87.5% by weight when the water content was reduced to 79.2% and the
cell breakage yield was 92.3% by weight. The process was also
repeated to reduce the water content from 88.7% to 80.8%. The
extraction yield of the lipid was 90% by weight when the water
content was reduced to 80.8% and the cell breakage yield was 95.9%
by weight,
Example 12
[0235] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 50%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 5.6 to 11.1. The addition
of the base and the rise in the pH lysed the cells to form a lysed
cell composition. The lysed cell composition was then heated to
90.degree. C. in a closed system for 40 minutes. After the 40
minutes, there was an about 1 ml layer of oil (lipid) per 40 ml of
fermentation broth. The solution was then centrifuged at about
5,100 rpm for 5 minutes to separate the lysed cell composition and
yield a lipid layer. The extraction yield of the lipid was 85.1% by
weight. The anisidine value (AV) of the crude oil was 16.3. The
cell breakage yield was 97.6% by weight.
Example 13
[0236] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 50%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 4.9 to 11.2. The addition
of the base and the rise in the pH lysed the cells to form a lysed
cell composition. The lysed cell composition was then mixed at room
temperature for 4 hours. The solution was then centrifuged at about
5,100 rpm for 5 minutes to separate the lysed cell composition and
yield a small lipid layer.
[0237] A portion of the lysed cell composition was mixed at room
temperature for about 96 hours. The solution was then centrifuged
at about 5,100 rpm for 5 minutes to separate the lysed cell
composition and yield a larger lipid layer. The extraction yield of
lipid was 61.4% by weight. The anisidine value (AV) of the crude
oil was 22.6.
Example 14
[0238] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (ATCC Accession No.
PTA-9695) was provided. The broth was chemically treated with a
base (i.e., a 50% solution of NaOH) without a prior cell lysis
step. The addition of the base raised the pH of the broth from 5.6
to 11.1. The addition of the base and the rise in the pH lysed the
cells to form a lysed cell composition. The lysed cell composition
was then heated in a range of 70.degree. C. to 75.degree. C. for 3
hours. The solution was then centrifuged at about 5,100 rpm for 5
minutes to separate the lysed cell composition and yield a lipid
layer. The extraction yield of the lipid was 84.4% by weight.
[0239] A portion of the lysed cell composition was heated for a
total of 5 hours. The solution was then centrifuged at about 5,100
rpm for 5 minutes to separate the lysed cell composition and yield
a similar lipid layer. The extraction yield of lipid was 87.3% by
weight. The cell breakage yield was 89.1% by weight.
Example 15
[0240] A cell broth (about 500 g) that was washed, concentrated,
and pasteurized containing microbial cells (Schizochytrium) was
provided. The broth was chemically treated with a base (i.e., a 50%
solution of NaOH) without a prior cell lysis step. The addition of
the base raised the pH of the broth from 7.3 to 11. The addition of
the base and the rise in the pH lysed the cells to form a lysed
cell composition. Next, a salt (solid Na.sub.2SO.sub.4, in an
amount of 5%, by weight, of the lysed cell composition) was added
to the lysed cell composition. The lysed cell composition was then
heated to a temperature of 90.degree. C. and held at that
temperature level for 2 hours. While maintaining a temperature of
90.degree. C. for additional 2 to 4 hours, the vessel containing
the lysed cell composition was opened to allow evaporation of
water. The solution was then centrifuged at about 5,100 rpm for 5
minutes to separate the lysed cell composition and yield a lipid
layer. Extraction yield of the lipid was greater than 70% by
weight. The anisidine value (AV) of the crude oil was 11.6.
Example 16
[0241] A cell broth (9,925 kg) containing microbial cells (ATCC
Accession No. PTA-9695) was provided. The cell broth was diluted
with water in a 1:1 ratio by weight to form a diluted broth of
20,000 kg. The solid content of the broth prior to dilution was
16.13% by weight and after dilution was 8.25% by weight. The
diluted broth was mixed and centrifuged with a desludging
centrifuge at 6,400 rpm to remove extracellular water-soluble or
water-dispersible compounds. The concentrate (10,250 kg) from the
centrifuge was collected and had a solids content of 10.5% by
weight. The collected concentrate was heated to 62.degree. C. to
64.degree. C. to pasteurize the concentrate. Enzymes (i.e.,
Alcalase 2.4 L FG 0.5%) were added to the pasteurized concentrate
to lyse the cells and form an emulsified lysed cell composition.
The emulsified lysed cell composition was treated with a base
(i.e., a 25% solution of NaOH) to adjust the pH of the lysed cell
composition to 11. Next, a salt (solid Na.sub.2SO.sub.4, in an
amount of 5%, by weight, of the lysed cell composition) was added
to the lysed cell composition. The lysed cell composition was then
heated to a temperature of 95.degree. C. and held at that
temperature level for 10 hours to 12 hours while agitating the
lysed cell composition. After agitation, the pH of the lysed cell
composition was 8.6 and there was a very small emulsion layer. The
agitation tank was allowed to cool to 60.degree. C. and the pH of
the lysed cell composition increased to 9.6 while cooling. The pH
of the lysed cell composition was lowered to 8.2 by adding
phosphoric acid. The addition of the phosphoric acid did not harm
the separation of the lipid layer and the very small emulsion
layer. The lysed cell composition was then centrifuged at 5,100 rpm
at a feed rate of 48 kg/min for 5 minutes at 60.degree. C. to
63.degree. C. to separate the lysed cell composition and yield a
lipid layer having a moisture content of 1.7% to 2.3% by
weight.
Example 17
[0242] A cell broth (500 g) that was washed, concentrated, and
pasteurized containing microbial cells (Crypthecodinium cohnii) was
provided. The broth was homogenized at a pressure of 8,000 to
12,000 psi (2 passes) to form a lysed cell composition. The lysed
cell composition was treated with a base (i.e., a 12.5% solution of
NaOH) until the lysed cell composition reached a pH of 7.8 to 8.2.
A salt (solid Na.sub.2SO.sub.4, in an amount of 5% by weight of the
lysed cell composition) was added to the lysed cell composition.
The lysed cell composition was then heated to a temperature of
60.degree. C. and held at that temperature. The pH of the lysed
cell composition was maintained at the 7.8 to 8.2 level by the
addition of base (i.e., a 12.5% solution of NaOH) for 10 to 15
hours in a closed system with little to no moisture loss. The lysed
cell composition was then centrifuged at about 5,100 rpm for 5
minutes to separate the lysed cell composition and yield an oil
layer. This resulted in an oil layer of about 2 ml in a sample of
40 ml. The extraction yield of the oil was 73% by weight. The
anisidine value (AV) of the crude oil was 13.5. The cell breakage
yield was 82% to 86% by weight.
Example 18
[0243] A pasteurized cell broth (1,000 g) containing microbial
cells (Schizochytrium) was provided. Enzymes (i.e., Alcalase 2.4 L
FG 0.5%) were added to the cell biomass to lyse the cells and form
an emulsified lysed cell composition. The emulsified lysed cell
composition was treated with a base (i.e., a 12.5% solution of
NaOH) to adjust the pH of the lysed cell composition from 7.21 to
10.52. Next, a salt (solid NaCl, in an amount of 2%, by weight, of
the lysed cell composition) was added to the lysed cell
composition. The broth was then separated into 4 portions with each
portion being held at 4 different temperatures and times: 1) Trial
#1 was held at 90.degree. C. for 22 hours; 2) Trial #2 was held at
90.degree. C. for 2 hours and then held at 25.degree. C. for 20
hours; 3) Trial #3 was held at 60.degree. C. for 22 hours; and 4)
Trial #4 was held at 25.degree. C. for 22 hours. The individual
trials were then centrifuged without further pH adjustment. For
Trials #1, #2, and #3, the broth was centrifuged at approximately
6,600 rpm (a g-force of 4,800) for 5 minutes to separate the lysed
cell composition. Because the separation for Trial #4 was not good
(<20%) at a g-force of 4,800, the g-force was increased to
15,000 and the broth was spun at a g-force of 15,000 for 5 minutes.
The extraction yield of the lipid as a weight percent and the
anisidine value (AV) are listed in the table below.
TABLE-US-00002 TABLE 2 Conditions and Results When Varying
Temperature and Heating Time of the Lysed Cell Composition.
Treatment Time Centrifugation Oil Yield Trial # and Temp.
Conditions AV (%) 1 90.degree. C. for 22 hours pH = 6.22 58.7 51.4
g-force = 4,800 2 90.degree. C. for 2 hours, pH = 8.19 109.2 82.2
25.degree. C. for 20 hours g-force = 4,800 3 60.degree. C. for 22
hours pH = 8.38 91.2 27.2 g-force = 4,800 4 25.degree. C. for 22
hours pH = 10.03 105.2 55.7 g-force =15,000
[0244] The anisidine values in Table 2 were higher than expected.
One difference between previous examples and this example was that
the lysed cell composition was allowed to sit for a long period of
time before the lipids were extracted. It is hypothesized that the
long period of time before extraction leads to the oxidation of the
dissolved oxygen present in the lysed cell composition. The
increased oxidation then leads to an increase in the anisidine
value. The fact that the trial heated at the highest temperature
for the longest time (Trial #1) had the lowest anisidine value
supports this hypothesis because the dissolved oxygen content of a
lysed cell composition generally decreases as the temperature is
increased. The increased anisidine values are therefore believed to
be an anomaly that was a result of the delay in extracting the
lipids from the lysed cell composition. In production, there would
be no delay time in extracting the lipids from the lysed cell
composition and the anisidine values would be consistent with
previous results of anisidine values of 26 or less.
Example 19
[0245] A pasteurized cell broth (1,000 g) containing microbial
cells (Schizochytrium) was provided. The broth was then split into
3 portions and diluted as follows: 1) Trial #1 was not diluted at
all and served as the control portion; 2) Trial #2 was diluted 25%
with water; and 3) Trial #3 was diluted 50% with water. Enzymes
(i.e., Alcalase 2.4 L FG 0.5%) were added to the cell biomass to
lyse the cells and form an emulsified lysed cell composition. The
emulsified lysed cell composition was treated with a base (i.e., a
12.5% solution of NaOH) to adjust the pH of the lysed cell
composition from 6.8 to 10.6. Next, a salt (solid NaCl, in an
amount of 2%, by weight, of the lysed cell composition) was added
to the lysed cell composition. The broth was then heated to
90.degree. C. and held for 20 hours. After the hold time, the broth
for each trial was separated into two with one half being
centrifuged as is and the other half having its pH adjusted to
approximately 8.5 before centrifugation. Both portions were then
centrifuged at approximately 8,545 rpm (a g-force of 8,000) for 5
minutes. The extraction yield of the lipid as a weight percent and
the anisidine value (AV) are listed in the table below.
TABLE-US-00003 TABLE 3 Conditions and Results When Varying Dilution
of the Pasteurized Broth. Centrifugation Oil Yield Trial # Diluted?
Conditions AV (%) 1 No dilution pH = 6.0 51.8 81.2 g-force = 8,000
1a No dilution pH = 8.4 44.3 78.1 g-force = 8,000 2 25% dilution pH
= 5.5 76.1 88.9 with water g-force = 8,000 2a 25% dilution pH = 8.4
85.3 82.1 with water g-force = 8,000 3 50% dilution pH = 5.7 68.5
85.0 with water g-force = 8,000 3a 50% dilution pH = 8.5 79.6 84.0
with water g-force = 8,000
[0246] The anisidine values in Table 3 were higher than expected.
One difference between previous examples (excluding Example 18) and
this example was that the lysed cell composition was allowed to sit
for a long period of time before the lipids were extracted. It is
hypothesized that the long period of time before extraction leads
to the oxidation of the dissolved oxygen present in the lysed cell
composition. The increased oxidation then leads to an increase in
the anisidine value. The increased anisidine values are therefore
believed to be an anomaly that was a result of the delay in
extracting the lipids from the lysed cell composition. In
production, there would be no delay time in extracting the lipids
from the lysed cell composition and the anisidine values would be
consistent with previous results of anisidine values of 26 or
less.
Example 20
[0247] Cell broths obtained from various fermentation lots were
treated using the process described in Example 2 except the timing
of adding the salt (e.g., before and after homogenization) and the
amount of salt were varied. The resulting separated lipids were
analyzed and the analyses are provided in Table 4.
TABLE-US-00004 TABLE 4 Specifications of lipids obtained using
processes of the present invention varying the timing of salt
addition and the amount of salt added. Fermentation Lot P2137 P2137
P4167 P4167 P2137 P2137 Addition of Before Before After After After
After NaCl* % NaCl by 2 5 2 5 5 5 weight % Lipid* 27 37 51 72 59 15
% Starting 13 12.9 19.2 19.2 13.3 7.5.sup.+ Solids by weight %
Solids by 19.7 21.7 19.7 20.1 20.2 8.6 weight before Centrifugation
*Addition of NaCl was before or after homogenization; % lipid
refers to the percentage of lipids in triglyceride form .sup.+This
run was diluted to have a low percentage of starting solids.
[0248] The data provided in Table 4 demonstrates that adding the
salt after homogenization results in higher % lipid values than
adding the salt before homogenization. The data provided in Table 4
also demonstrates that diluting the sample resulted in lower a %
lipid value.
Example 21
[0249] A sample of Alcalase enzyme treated lysed cell composition
obtained from microbial cells (Schizochytrium) was used. The sample
had a pH of approximately 5.5. The sample was divided into 4
smaller samples and the pH of three of the samples was adjusted to
approximately 7.4, approximately 10.5, and approximately 12,
respectively, by adding sodium hydroxide. The samples were diluted
in a 1:1 ratio with deionized water. POBN (.alpha.-(4-Pyridyl
1-oxide)-N-tert-butylnitrone, 1.25 M; 50 .mu.L) was added as a spin
trap chemical to 0.5 g of each of the diluted samples. The samples
were measured with a Bruker BioSpin e-scan EPR (Electron
Paramagnetic Resonance) spectrometer (system number SC0274) (Bruker
BioSpin, Billerica, Mass.) to measure the amount of free radicals
present from lipid oxidation. The samples were incubated at room
temperature (20.degree. C.) and 50 .mu.L of each of the POBN
containing samples was tested at hourly intervals for four hours
after adjusting the pHs using the following spectrometer
parameters: modulation frequency of 86 Hz, modulation amplitude of
2.11 gauss, microwave power of 5.19 mW, time constant of 20.48
seconds, sweep time of 10.49 seconds, sweep width of 100 gauss, and
a number of scans of 8. The results of the EPR spectrometer
readings are provided in FIG. 5.
[0250] The data in FIG. 5 demonstrates that initially the level of
free radicals was highest for the sample at pH 5.5 and lowest for
the samples at pH 10.5 and 12. The data also demonstrates that over
the 4 hour period the rate of radical formation was slowest for the
sample at pH 10.5 and highest for the sample at pH 5.5. The data
also demonstrates that the addition of a base to the lysed cell
composition inhibits lipid oxidation, and therefore leads to a low
AV in the crude lipid and refined oil.
Example 22
[0251] Oilseeds are extracted from a rapeseed plant and are then
passed through a grinding mill to crack and break the outer hull of
the oilseeds. The oilseeds are then dehulled through known means,
such as through aspiration, to remove the meat (interior) of the
seeds from the hull of the oilseeds. The dehulled oilseeds are then
homogenized or expelled by passing them through a press to grind
the dehulled oilseeds into a cake in order to lyse the cells of the
oilseeds. Water is added to form an emulsified lysed cell
composition. The emulsified lysed cell composition is filtered to
remove any excess hull fragments from the lysed cell composition.
The emulsified lysed cell composition is treated with a base (i.e.,
a 25% solution of NaOH) to adjust the pH of the lysed cell
composition to 11. Next a salt (solid NaCl, in an amount 2% by
weight of the lysed cell composition) is added to the lysed cell
composition. The lysed cell composition is then heated to a
temperature of 90.degree. C. and held at that level for 6 hours to
48 hours while agitating the lysed cell composition. The lysed cell
composition is then centrifuged at 5,100 rpm for 5 minutes to
separate the lysed cell composition and yield a lipid layer and an
emulsion layer.
Example 23
Comparative Analysis of Crude Lipids Obtained by Hexane
Extraction
[0252] The crude lipids obtained from a lot using the process
described in Example 2 was analyzed to determine various
specifications. Additional crude lipids were obtained using a
typical hexane extraction process on the same microbial cell
utilized in Example 2. The hexane extraction process included spray
drying a fermentation broth, adding hexane to the spray dried
biomass to obtain a solution of 15% to 20% solids by weight. The
solution was then homogenized to lyse the cells to form a lysed
cell composition. The lysed cell composition was centrifuged and a
layer containing lipid and hexane was removed. The hexane was then
removed from the lipid. The results of the analyses are provided in
Table 5.
TABLE-US-00005 TABLE 5 Specifications of lipids obtained using
processes of the present invention or a hexane extraction process.
Fermentation Lot A B C D E F G Extraction Method Ex. 2 Hexane
Hexane Hexane Hexane Hexane Hexane AV* 5.9 ND ND ND 14.7 17.18 6.7
PV* 1.21 0.65 1.56 0.46 ND 0.85 0.3 % Lipid* 89.61 86.94 84.31
86.75 85.53 86.05 86.54 DHA (mg/g) 537.47 508.15 459.32 465.31
510.49 495.82 506.33 % DHA* 59.98 58.39 54.49 53.65 59.71 57.68
58.51 *AV = Anisidine Value; PV = peroxide value; % lipid refers to
the percentage of lipids in triglyceride form; % DHA refers to the
percentage of DHA in the lipid
[0253] The data provided in Table 5 demonstrates that the crude
lipids obtained by the processes of the present invention exhibit
superior anisidine values, percentage of lipid, amount of DHA and
percentage of DHA compared to lipids prepared by typical hexane
extraction processes.
Comparative Analysis of Crude Lipids Obtained by the FRIOLEX.RTM.
Processes
Example 24
[0254] The crude lipids obtained from various fermentation lots
using the processes described in Examples 1 and 3 were analyzed to
determine various specifications. Additional crude lipids were
obtained using a FRIOLEX.RTM. process (Westfalia Separator AG,
Germany), which is a process of extracting lipids with a
water-soluble organic solvent as described in U.S. Pat. No.
5,928,696 and International Pub. Nos. WO 01/76385 and WO 01/76715.
The results of the analyses are provided in Table 6.
TABLE-US-00006 TABLE 6 Specifications of lipids obtained using
processes of the present invention or a FRIOLEX .RTM. process.
Fermentation Lot A B B B B C C C Extraction Method Ex. 1 Ex. 1 Ex.
3 Ex. 1.sup.a Ex. 1.sup.b Ex. 1 Ex. 1 FRIOLEX .RTM. AV* 3.1 1.6 3.9
300 7.1 3.5 4 36 PV* 1.8 0.17 0.14 6.16 0.34 0 0 0.35 % Lipid*
96.27 93.67 92.55 87.20 94.42 95.14 94.31 93.92 DHA (mg/g) 452.4
458.16 455.17 414.66 471.55 416.41 416.05 415.38 Extraction Yield
(%) 93.5 87 ND 96 ND 94 94 94 *AV = Anisidine Value; PV = peroxide
value; % lipid refers to the percentage of lipids in triglyceride
form .sup.aThe lysed cell composition was not heated. .sup.bThe
lysed cell composition was allowed to stand for 3 weeks prior to
extraction.
[0255] The data provided in Table 6 demonstrates that the crude
lipids obtained by the processes of the present invention exhibit
superior anisidine values (with the exception of the lipid obtained
when the lysed cell composition was not heated) compared to lipids
prepared by a FRIOLEX.RTM. process. The lipids prepared by a
process of the present invention exhibit anisidine values that are
from 4.4% to 19.7% of the anisidine values of a lipid prepared
using the FRIOLEX.RTM. process.
[0256] It is believed that a lipid prepared by a process of the
present invention has increased stability. For example, as shown in
Table 6, a process of the present invention was used to extract a
lipid from a lysed cell composition, wherein the lysed cell
composition was allowed to stand for 3 weeks prior to the
extraction process. It is believed that the anisidine value of a
lipid in a lysed cell composition increases with time, and thus, it
would be expected that a lipid extracted from a 3 week old lysed
cell composition have increased anisidine values. However, as shown
in Table 6, the lipid obtained from the 3 week old lysed cell
composition using a process of the present invention had an
anisidine value which was 19.7% of the anisidine value of a lipid
prepared by the FRIOLEX.RTM. process.
Example 25
[0257] The crude lipids obtained from a broth of microbial cells
(ATCC Accession No. PTA-9695) using the process described in
Example 16 were analyzed to determine various specifications.
Additional crude lipids were obtained from a broth of microbial
cells (ATCC Accession No. PTA-9695) using a FRIOLEX.RTM. process
(Westfalia Separator AG, Germany), which is a process of extracting
lipids with a water-soluble organic solvent (e.g., isopropyl
alcohol) as described in U.S. Pat. No. 5,928,696 and International
Pub. Nos. WO 01/76385 and WO 01/76715. The results of the analyses
are provided in Table 7.
TABLE-US-00007 TABLE 7 Crude Lipid Comparison Example 16 FRIOLEX
.RTM. % Oil 9.19 93.73 DHA (mg/g) 570.68 574.33 % DHA 62.1 61.27 %
FFA 1.13 0.22 PV 0 0.74 AV 10 73.9 Iron (ppm) 0.11 0 Copper (ppm)
0.67 0.3 Lead (ppm) 0.21 0 Phosphorus (ppm) 5.22 7.20 Extraction
yield (%) 61.4 45.3
[0258] The data provided in Table 7 demonstrates that the crude
lipids obtained by the processes of the present invention exhibit
superior anisidine values (AV) and peroxide values (PV) compared to
lipids prepared by a FRIOLEX.RTM. process. The data also
demonstrates that the extraction yield of lipid obtained by the
processes of the present invention are superior compared to the
extraction yield of lipid obtained by a FRIOLEX.RTM. process.
Example 26
[0259] Refining of Crude Lipids
[0260] Crude lipids were obtained using the processes outlined in
Example 1 and a FRIOLEX.RTM. process. The crude lipids were further
processed by sequentially: 1) degumming and caustic refining; 2)
bleaching; 3) chilled filtering; and 4) deodorizing with
antioxidants. The data for the crude lipids, caustic refined
lipids, bleached lipids, and deodorized lipids are presented in
Tables 8a and 8b. A comparison of the refined oils is presented in
Table 9.
TABLE-US-00008 TABLE 8a Lipids obtained from the FRIOLEX .RTM.
process (Example 24) FFA DHA Yield % Yield % Processing Step % PV
AV (mg/g) (Lipid) (DHA) Crude Lipid 0.28 0.37 33.6 413.6 N/A N/A
Caustic Refined Lipid <0.1 0.29 -- 416.4 85.7 86.3 Bleached
Lipid <0.1 0.16 13.9 413.1 71.5 70.9 Filtered Lipid <0.1 0.19
13.3 424.86 70.3 76.0 Deodorized Lipid 0.06 <0.1 14.3 401.5 96.6
89.4 w/AOX* *AOX refers to antioxidants
TABLE-US-00009 TABLE 8b Lipids obtained from an extraction process
using NaCl (Example 1) DHA Yield % Yield % Processing Step FFA % PV
AV (mg/g) (Lipid) (DHA) Crude Lipid 1.36 0 3.5 406.7 N/A N/A
Caustic Refined <0.1 0.27 2.3 410.8 85.9 86.7 Lipid Bleached
Lipid <0.1 0.16 0.8 404.3 97.0 95.5 Filtered Lipid <0.1 0.37
1.0 414.1 59.3 60.7 Deodorized Lipid 0.06 <0.1 1.8 379.9* 94.9
87.1 w/AOX *Note: Increased dilution with high oleic sunflower oil
(HOSO) was the reason for the decrease in DHA (mg/g).
TABLE-US-00010 TABLE 9 Refined Oil Comparison FRIOLEX .RTM.
(Example 24) Example 1 DHA (mg/g) 401.5 379.9* % DHA 42.69 40.18
FFA % 0.06 0.06 PV <0.1 <0.1 AV 14.3 1.8 Iron (ppm) 0.05
<0.02 Copper (ppm) <0.02 <0.02 Lead (ppm) <0.1 <0.1
Arsenic (ppm) <0.1 <0.1 Mercury (ppm) <0.01 <0.01 %
Moisture and volatiles <0.01 <0.01 Unsaponifiables (%) 1.17
1.33 Trans-fatty acid by IR (%) <1 <1 *Note: Increased
dilution with HOSO was the reason for the decrease in DHA
(mg/g).
[0261] The data provided in Table 8a, Table 8b, and Table 9
demonstrate that a refined oil prepared by a process of the present
invention exhibits lower anisidine values compared to a refined oil
prepared by the FRIOLEX.RTM. process.
Example 27
Sensory Profile Comparison
[0262] The refined oils obtained in Example 26 were analyzed by a
panel of 8 to 12 sensory analysts. The sensory analysts rated
various lipid specifications based on aroma, aromatics, and
aftertaste to provide an "overall aroma intensity" for each lipid.
The Universal Spectrum descriptive analysis method was used to
assess the aroma and aromatic characteristics of samples. This
method uses an intensity scale of 0-15, where 0=none detected and
15=very high intensity, to measure the aroma and aromatic
attributes of the oils. The results of the sensory data are
provided in Table 10.
TABLE-US-00011 TABLE 10 Sensory specifications of a lipid prepared
by the FRIOLEX .RTM. process (Example 24) and a lipid prepared by a
process of the present invention (Example 1) Specifications Aroma
Overall Fishy Marine Green Nutty Intensity Complex Complex
Herbaceous Roasted Painty Other FRIOLEX .RTM. 3 1 1 1 0 0 0 Ex. 1 2
0 1 1 0 0 0 Aromatics Overall Fishy Marine Green Nutty Intensity
Complex Complex Herbaceous Roasted Painty Other FRIOLEX .RTM. 4 1 1
2 0 0 0 Ex. 1 3 0 1 2 0 0 0 Aftertaste FRIOLEX .RTM.
Herbal/slightly fishy Ex. 1 Herbal
[0263] The data provided in Table 10 demonstrates that a refined
oil prepared by a process of the present invention exhibits
superior sensory data compared to a refined oil prepared by the
FRIOLEX.RTM. process. As shown above, the lipids provided by the
present invention had an overall aroma intensity of 3 and 2,
whereas, the FRIOLEX.RTM. lipids provided an overall aroma
intensity of 4 and 3, respectively.
Example 28
Comparative Example
[0264] An extraction process for obtaining lipids from
microorganisms without the use of an organic solvent is disclosed
in U.S. Pat. No. 6,750,048. A comparison of a refined oil obtained
from a crude lipid prepared by a process of the present invention
and a refined oil obtained from a crude lipid prepared by the
extraction process disclosed in U.S. Pat. No. 6,750,048 is provided
in Table 11.
TABLE-US-00012 TABLE 11 Comparative data for a lipid prepared by a
process of the present invention (Example 1) and a lipid prepared
by a process disclosed in U.S. Pat. No. 6,750,048. Example 1 U.S.
Pat. No. 6,750,048 DHA (mg/g) 379.9 346 % DHA 40.18 37.3 FFA % 0.06
ND PV <0.1 0.46 AV 1.8 ND Iron (ppm) <0.02 0.26 Copper (ppm)
<0.02 <0.05 Lead (ppm) <0.1 <0.20 Arsenic (ppm) <0.1
<0.20 Mercury (ppm) <0.01 <0.20 % Moisture and volatiles
<0.01 0.02 Unsaponifiables (%) 1.33 ND Trans-fatty acid by IR
(%) <1 ND
[0265] The data provided in Table 11 demonstrates that a refined
oil obtained from a crude lipid prepared by a process of the
present invention exhibits superior properties compared to a
refined oil obtained from a crude lipid prepared by the extraction
process disclosed in U.S. Pat. No. 6,750,048.
Example 29
[0266] The isolated thraustochytrid (ATCC Accession No. PTA-9695)
was characterized for taxonomic classification.
[0267] Samples were collected from intertidal habitats during low
tide. 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.degree. 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.
[0268] Colonies from agar plates were transferred to petri dishes
with half-strength seawater and (1 ml) of a suspension of
autoclaved newly hatched brine shrimp larvae. The brine shrimp
larvae became heavily overgrown with clusters of sporangia after
2-3 days. Released zoospores were biflagellate at discharge,
swimming actively away from the mature sporangium, wall remnants of
which are clearly visible (in phase contrast) after spore release.
Sporangia measured 12.5 .mu.m to 25 .mu.m in diameter, and
zoospores were 2.5 .mu.m to 2.8 .mu.m.times.4.5 .mu.m to 4.8 .mu.m
in size. There were 8 to 24 spores per individual sporangium.
Settled 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.
[0269] The isolated thraustochytrid (ATCC Accession No. PTA-9695)
was further characterized based on the similarity of its 18s rRNA
gene to that of known species. Total genomic DNA from the
thraustochytrid (ATCC Accession No. PTA-9695) was prepared by
standard procedures (Sambrook J. and Russell D. 2001. Molecular
cloning: A laboratory manual, 3rd edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.) and used for PCR
amplification of the 18s RNA gene. The PCR amplification of the 18s
rRNA gene was carried out with primers previously described (Honda
et. al., J. Eukaryot. Microbiol. 46(6) 1999). The PCR conditions
with chromosomal DNA template were as follows: 0.2 .mu.M dNTPs, 0.1
uM each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 U
PfuUltra.RTM. II fusion HS DNA polymerase (Stratagene), and
1.times. PfuUltra.RTM. buffer (Stratagene) in a 50 .mu.L total
volume. The PCR Protocol included the following steps: (1)
95.degree. C. for 2 minutes; (2) 95.degree. C. for 45 seconds; (3)
55.degree. C. for 30 seconds; (4) 72.degree. C. for 2 minutes; (5)
repeat steps 2-4 for 40 cycles; (6) 72.degree. C. for 5 minutes;
and (7) hold at 6.degree. C.
[0270] 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.
[0271] Table 12 shows a comparison of the 18s rRNA sequence from
the thraustochytrid (ATCC Accession No. PTA-9695) to DNA sequences
in the National Center for Biotechnology Information (NCBI)
electronic database. Briefly, "% Identity" was determined by the
scoring matrix "swgapdnamt" within the "AlignX" program of the
VectorNTI program (Invitrogen), a standard for DNA alignment. The
"% Coverage" was taken from the results of a Basic Local Alignment
Search Tool (BLAST) calculation from the NCBI electronic database
and is the percent of the query length that is included in the
aligned segments.
TABLE-US-00013 TABLE 12 Comparison of 18s rRNA Sequences % Identity
% Coverage Thraustochytrids Calculation #1 Calculation #2
Thraustochytrium aggregatum (p) 98 90 Thraustochutriidae sp. HU1 84
86 Thraustochutriidae sp. 8-7 84 91 Thraustochytrium
multirudimentale 81 88 Thraustochutriidae sp. PW19 81 85
Schizochytrium sp. ATCC 20888 81 95 (p): indicates partial
sequence
[0272] As shown in Table 12, it was found that, in terms of %
identity, the 18s rRNA gene sequence from the thraustochytrid (ATCC
Accession No. PTA-9695) is 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. In terms of percent coverage, the 18s rRNA gene
sequence of the isolate of the invention is more closely related to
Schizochytrium sp. ATCC 20888 than to T. aggregatum.
[0273] Highly conserved proteins such as actin and beta-tubulin
have been widely used, along with 18s rRNA gene, as markers for
assessing phylogenetic relationships between organisms (Baldauf, S.
M. Am. Nat. 154, S178 (1999)). Total genomic DNA from the
thraustochytrid (ATCC Accession No. PTA-9695) was also used as a
template for PCR amplification of both the actin and beta-tubulin
genes. The PCR amplification was carried out with primers designed
to conserved regions from the actin and beta-tubulin DNA sequences
from T. aggregatum.
[0274] The PCR conditions with chromosomal DNA template were as
follows: 0.2 .mu.M dNTPs, 0.1 uM each primer, 8% DMSO, 200 ng
chromosomal DNA, 2.5 U Herculase.RTM. II fusion DNA polymerase
(Stratagene), and 1.times. Herculase.RTM. buffer (Stratagene) in a
50 .mu.L total volume. The PCR Protocol included the following
steps: (1) 95.degree. C. for 2 minutes; (2) 95.degree. C. for 30
seconds; (3) 55.degree. C. for 30 seconds; (4) 72.degree. C. for 2
minutes; (5) repeat steps 2-4 for 40 cycles; (6) 72.degree. C. for
5 minutes; and (7) hold at 6.degree. C.
[0275] PCR amplification yielded distinct DNA products with the
expected sizes using chromosomal template described above. The
respective PCR products were cloned into the vector pJET1.2/blunt
(Fermentas) according to the manufacturer's instructions, and the
insert sequence of each were determined using supplied standard
primers.
[0276] Table 13 shows identities for the actin amino acid sequence
from the thraustochytrid (ATCC Accession No. PTA-9695) as compared
to actin sequences available in the public database. Identities
were determined through use of the scoring matrix "blosum62mt2"
within the "AlignX" program of the VectorNTI program, a standard
for protein alignment.
TABLE-US-00014 TABLE 13 Comparison of Actin Protein Sequence %
Identities Thraustochytrids % Identity Thraustochytriidae sp. RT49
98 Schizochytrium sp. ATCC 20888 96 Thraustochytrium striatum 96
Thraustochytrium aggregatum 96 Japonochytrium marinum 95
Thraustochytrium aureum 95
[0277] Table 14 shows identities for the beta-tubulin amino acid
sequence from the thraustochytrid (ATCC Accession No. PTA-9695) as
compared to beta-tubulin sequences available in the public
database. Identities were determined through use of the scoring
matrix "blosum62mt2" within the "AlignX" program of the VectorNTI
program, a standard for protein alignment.
TABLE-US-00015 TABLE 14 Comparison of Beta-Tubulin Protein Sequence
% Identities Thraustochytrids % Identity Aplanochytrium kerguelense
100 Aplanochytrium stocchinoi 100 Japonochytrium marinum 100
Labyrinthula sp. N8 100 Thraustochytriidae sp. RT49 100
Thraustochytrium aggregatum 100 Thraustochytriidae sp. HU1 100
Thraustochytrium aureum 100 Thraustochytrium kinnei 100
Thraustochytriidae sp. #32 100 Thraustochytriidae sp. PW19 100
Schizochytrium aggregatum 100 Schizochytrium sp. ATCC 20888 100
[0278] Based on the above characterizations, the isolated
thraustochytrid (ATCC Accession No. PTA-9695) is believed to
represent a new Schizochytrium species and is therefore also
designated as Schizochytrium sp. ATCC PTA-9695.
Example 30
[0279] The isolated thraustochytrid (ATCC Accession No. PTA-9695)
produced high levels of cell growth under varying culture
conditions, as described below. Typical media and cultivation
conditions are shown in Table 15. Also, high levels of fatty acids
and DHA were observed (i.e., greater than 50% by weight of the dry
cell weight were fatty acids and greater than 50% by weight of the
fatty acid methyl esters was DHA).
TABLE-US-00016 TABLE 15 Vessel Media Ingredient concentration
ranges NaCl g/L 12.5 0-25, 5-20, or 10-15 KCl g/L 1.0 0-5, 0.25-3,
or 0.5-2 MgSO.sub.4.cndot.7H.sub.2O g/L 5.0 0-10, 2-8, or 3-6
(NH.sub.4).sub.2SO.sub.4 g/L 0.6 0-10, 0.25-5, or 0.5-3 CaCl.sub.2
g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast g/L 6.0 0-20, 1-15,
or 5-10 extract) KH.sub.2PO.sub.4 g/L 1.2 0.1-10, 0.5-5, or 1-3
Post autoclave (Metals) Citric acid mg/L 3.5 0.1-100, 1-50, or 2-25
FeSO.sub.4.cndot.7H.sub.2O mg/L 10.30 0.1-100, 1-50, or 5-25
MnCl.sub.2.cndot.4H.sub.2O mg/L 3.10 0.1-100, 1-50, or 2-25
ZnSO.sub.4.cndot.7H.sub.2O mg/L 3.10 0.1-100, 1-50, or 2-25
CoCl.sub.2.cndot.6H.sub.2O mg/L 0.04 0.001-1, 0.005-0.5, or
0.01-0.1 Na.sub.2MoO.sub.4.cndot.2H.sub.2O mg/L 0.04 0.001-1,
0.005-0.5, or 0.01-0.1 CuSO.sub.4.cndot.5H.sub.2O mg/L 2.07
0.1-100, 0.5-50, or 1-25 NiSO.sub.4.cndot.6H.sub.2O mg/L 2.07
0.1-100, 0.5-50, or 1-25 Post autoclave (Vitamins) Thiamine mg/L
9.75 0.1-100, 1-50, or 5-25 Vitamin B12 mg/L 0.16 0.1-100, 0.1-10,
or 0.1-1 Ca1/2-panto- mg/L 3.33 0.1-100, 0.1-50, or 1-10 thenate
Post autoclave (Carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50
Nitrogen Feed: NH.sub.4OH 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% saturation, 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.
[0280] In carbon and nitrogen-fed cultures with 8200 ppm Cl.sup.-
at 22.5.degree. C. with 20% dissolved oxygen at pH 7.0, the isolate
produced a dry cell weight of 140 g/L after 7 days of culture, with
a fatty acid content of 70% by weight. Closed loop ammonia feed was
used and the pH was maintained at 7.0. The omega-3 productivity was
8.92 g/(L*day) under these conditions, with 4.7 g/L EPA (5% by
weight of fatty acids) and 56.3 g/L DHA (57% by weight of fatty
acids) in 7 days.
[0281] In carbon and nitrogen-fed cultures with 3640 ppm Cl.sup.-
at 22.5.degree. C. with 20% dissolved oxygen at pH 7.0, the isolate
produced a dry cell weight of 82 g/L after 7 days of culture, with
a fatty acid content of 58% by weight. The omega-3 productivity was
4.5 g/(L*day) under these conditions, with 2.1 g/L EPA (4.3% by
weight of fatty acids) and 28.5 g/L DHA (58.7% by weight of fatty
acids) in 7 days.
[0282] In carbon and nitrogen-fed cultures with 980 ppm Cl.sup.- at
22.5.degree. C. with 20% dissolved oxygen at pH 7.0, the isolate
produced a dry cell weight of 60 g/L after 7 days of culture, with
a fatty acid content of 53% by weight. The omega-3 productivity was
2.8 g/(L*day) under these conditions, with 1.1 g/L EPA (3.4% by
weight of fatty acids) and 18.4 g/L DHA (56.8% by weight of fatty
acids) in 7 days.
Example 31
[0283] Oils were extracted from a biomass sample (Sample A) of the
isolated thraustochytrid (ATCC Accession No. PTA-9695). The biomass
sample was produced in a carbon and nitrogen-fed culture with 980
ppm CY at 22.5.degree. C. with 20% dissolved oxygen at pH 7.0. Oils
were extracted from biomass Sample A by the hexane extraction
process to yield microbial oil Sample A1. Briefly, dried biomass
was ground with hexane using stainless steel tubes and stainless
steel ball bearings for approximately 2 hours. The slurry was
vacuum filtered and the filtrate was collected. The hexane was
removed using a rotary evaporator. Oils were also extracted from
biomass Sample A using the FRIOLEX.RTM. process (GEA Westfalia
Separator UK Ltd., Milton Keynes, England) to yield microbial oil
Sample A2. Individual lipid classes were isolated from microbial
oil Samples A1 and A2 using low pressure flash chromatography, and
the weight percent of each class was determined. The fatty acid
profile of each class was determined using gas chromatography with
flame ionization detection (GC-FID) as fatty acid methyl esters
(FAME).
[0284] Flash Chromatography--
[0285] Flash chromatography was used to separate the lipid classes
present in the crude oils, and to determine the weight percent of
each class present in the oils. The chromatography system utilized
Silica Gel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase
composed of Petroleum Ether and Ethyl Acetate at 3 mL/min. 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 (followed by a 100% methanol
wash). 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.
[0286] TLC Analysis--
[0287] 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.
[0288] Fatty Acid Analysis--
[0289] The samples of biomass and isolated lipid classes were
analyzed for fatty acid composition as FAMEs. Samples were weighed
directly into screw cap test tubes, and 1 mL of C19:0 internal
standard (NuCheck, Elysian, Minn.) in toluene and 2 mL of 1.5 N HCl
in methanol was added to each tube. The tubes were vortexed briefly
and placed in a heating block for 2 hours at 100.degree. C. The
tubes were removed from the heating block, allowed to cool, and 1
mL of saturated NaCl in water was added. The tubes were vortexed
again, centrifuged, and a portion of the top (organic) layer was
placed in a GC vial and analyzed by GC-FID. FAME's were quantified
using a 3-point internal standard calibration curve generated using
Nu-Chek-Prep GLC reference standard (Nu-Chek Prep, Inc., Elysian,
Minn.) and tentatively identified based on retention time. Fatty
acids present were expressed as mg/g and % of total FAME.
[0290] Sample A1 was prepared by dissolving the crude oil in hexane
and applying to the head of the column. After fractionation of the
sample using flash chromatography, the sterol ester fraction
accounted for 1.2% by weight, the triacylglycerol (TAG) fraction
accounted for 82.7% by weight, the free fatty acid (FFA) fraction
accounted for 0.9% by weight, and the diacylglycerol (DAG) fraction
accounted for 2.9% by weight of the crude oil. The total fatty acid
profiles of the Sample A1 crude oil and isolated fractions are
shown below in Table 16 and Table 17 calculated as mg/g and % FAME,
respectively.
TABLE-US-00017 TABLE 16 Sample A1 Fatty Acid Profiles Calculated as
Milligrams per Gram FAME Biomass Crude Oil Sterol Esters TAG FFA
DAG Wt. % NA 38% 1.2% 82.7% 0.9 2.9% Fatty Acid FAME (mg/g) FAME
(mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0* 0.6
0.0 1.9 3.2 1.7 0.0 C14:0* 5.7 13.6 12.8 20.2 13.0 17.6 C14:1* 0.0
0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.4 3.1 3.1 2.1 2.6 C16:0* 105.5
239.5 222.2 274.3 183.3 225.1 C16:1* 0.0 0.0 0.8 0.0 0.8 0.0 C18:0*
6.4 16.4 43.1 16.8 9.8 14.0 C18:1 N9* 0.0 3.8 1.9 3.3 1.0 3.5 C18:1
N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0*
1.8 5.5 13.0 4.7 2.0 2.9 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1
N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.6 0.0 C20:2
N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0*
0.0 0.8 7.3 0.8 0.0 1.2 C20:4 N7 0.0 0.0 0.8 0.0 0.0 0.0 C20:3 N3
0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.0 3.4 0.0 2.6 2.0 1.9 C22:1 N9*
0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3
1.5 4.1 1.5 3.5 2.1 2.1 C20:5 N3* 18.2 39.5 3.5 38.4 30.6 42.8
C24:0* 0.0 0.0 6.3 0.0 0.0 0.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0
C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 29.5 8.9 26.9 14.8
18.7 C22:5 N3* 1.1 4.7 0.9 3.6 3.4 2.7 C22:6 N3* 253.5 569.7 107.3
556.5 352.8 451.4 Sum of all FAME's 408.6 934.0 435.4 958.0 620.1
786.4
TABLE-US-00018 TABLE 17 Sample A1 Fatty Acid Profiles as a Percent
of Total FAME Biomass Crude Oil Sterol Esters TAG FFA DAG Fatty
Acid % FAME % FAME % FAME % FAME % FAME % FAME C12:0* 0.1 0.0 0.4
0.3 0.3 0.0 C14:0* 1.4 1.5 2.9 2.1 2.1 2.2 C14:1* 0.0 0.0 0.0 0.0
0.0 0.0 C15:0 0.3 0.4 0.7 0.3 0.3 0.3 C16:0* 25.8 25.6 51.0 28.6
29.6 28.6 C16:1* 0.0 0.0 0.2 0.0 0.1 0.0 C18:0* 1.6 1.8 9.9 1.8 1.6
1.8 C18:1 N9* 0.0 0.4 0.4 0.3 0.2 0.4 C18:1 N7 0.0 0.0 0.0 0.0 0.0
0.0 C18:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0* 0.4 0.6 3.0 0.5 0.3
0.4 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 0.0
0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.1 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0
0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.0 0.1
C20:4 N7 0.0 0.0 0.2 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0
C20:4N6* 0.3 0.4 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0
C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.3 0.3
C20:5 N3* 4.5 4.2 0.8 4.0 4.9 5.4 C24:0* 0.0 0.0 1.4 0.0 0.0 0.0
C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0
C22:5 N6* 2.9 3.2 2.1 2.8 2.4 2.4 C22:5 N3* 0.3 0.5 0.2 0.4 0.5 0.3
C22:6 N3* 62.0 61.0 24.6 58.1 56.9 57.4 Sum of FAME % 100.0 100.0
100.0 100.0 100.0 100.0
[0291] Sample A2 was prepared by dissolving the crude oil in hexane
and applying to the head of the column. After fractionation of the
sample using flash chromatography, the sterol ester fraction
accounted for 0.8% by weight, the triacylglycerol (TAG) fraction
accounted for 83.4% by weight, the free fatty acid (FFA) fraction
accounted for 1.8% by weight, and the diacylglycerol (DAG) fraction
accounted for 5.6% by weight of the crude oil. The total fatty acid
profiles of the Sample A2 crude oil and isolated fractions are
shown below in Table 18 and Table 19 calculated as mg/g and % FAME,
respectively.
TABLE-US-00019 TABLE 18 Sample A2 Fatty Acid Profiles Calculated as
Milligrams per Gram FAME Biomass Crude Oil Sterol Esters TAG FFA
DAG Wt. % NA FAME NA 0.8% 83.4% 1.8% 5.6% Fatty Acid (mg/g) FAME
(mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0* 0.6
0.0 0.0 1.5 0.0 1.0 C14:0* 5.7 13.2 8.9 14.1 9.5 5.4 C14:1* 0.0 0.0
0.0 0.0 0.0 0.0 C15:0 1.3 3.3 2.8 3.4 2.1 2.2 C16:0* 105.5 233.7
183.8 246.1 159.7 137.3 C16:1* 0.0 0.0 0.0 0.8 0.0 0.0 C18:0* 6.4
16.6 23.6 16.9 11.3 5.6 C18:1 N9* 0.0 7.6 5.0 4.3 2.4 2.6 C18:1 N7
0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 2.2 0.7 1.6 0.8 5.1 C20:0*
1.8 5.2 12.1 5.5 2.6 1.1 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1
N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.8 1.0 0.0 C20:2
N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.3 0.0 0.0 C22:0*
0.0 0.7 6.0 1.3 0.8 0.0 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3
0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N6* 1.0 3.0 0.0 3.1 2.3 1.2 C22:1 N9*
0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3
1.5 4.1 1.4 4.3 2.7 1.0 C20:5 N3* 18.2 38.6 2.7 38.6 39.5 45.5
C24:0* 0.0 0.0 4.7 0.6 0.0 0.3 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0
C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 28.2 8.6 29.6 18.0
14.7 C22:5 N3* 1.1 3.4 0.0 3.5 2.5 2.2 C22:6 N3* 253.5 566.7 102.2
575.0 475.3 447.2 Sum of all FAME's 408.6 926.5 362.3 951.3 730.4
672.5
TABLE-US-00020 TABLE 19 Sample A2 Fatty Acid Profiles as a Percent
of Total FAME Biomass Crude Oil Sterol Esters TAG FFA DAG Fatty
Acid % FAME % FAME % FAME % FAME % FAME % FAME C12:0* 0.1 0.0 0.0
0.2 0.0 0.2 C14:0* 1.4 1.4 2.4 1.5 1.3 0.8 C14:1* 0.0 0.0 0.0 0.0
0.0 0.0 C15:0 0.3 0.4 0.8 0.4 0.3 0.3 C16:0* 25.8 25.2 50.7 25.9
21.9 20.4 C16:1* 0.0 0.0 0.0 0.1 0.0 0.0 C18:0* 1.6 1.8 6.5 1.8 1.5
0.8 C18:1 N9* 0.0 0.8 1.4 0.5 0.3 0.4 C18:1 N7 0.0 0.0 0.0 0.0 0.0
0.0 C18:2 N6* 0.0 0.2 0.2 0.2 0.1 0.8 C20:0* 0.4 0.6 3.3 0.6 0.4
0.2 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.0 0.0 0.0
0.0 C18:4 N3 0.0 0.0 0.0 0.1 0.1 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0
0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.1 0.0
C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0
C20:4 N6* 0.3 0.3 0.0 0.3 0.3 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0
C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.4 0.4 0.4 0.4 0.4 0.2
C20:5 N3* 4.5 4.2 0.7 4.1 5.4 6.8 C24:0* 0.0 0.0 1.3 0.1 0.0 0.0
C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0
C22:5 N6* 2.9 3.0 2.4 3.1 2.5 2.2 C22:5 N3* 0.3 0.4 0.0 0.4 0.3 0.3
C22:6 N3* 62.0 61.2 28.2 60.4 65.1 66.5 Sum of FAME % 100.0 100.0
100.0 100.0 100.0 100.0
[0292] It is noted that Samples A1 and A2 were extracted using a
typical hexane extraction and a FRIOLEX.RTM. process, respectively.
The fatty acid profiles of Tables 16-19 are expected to be the
substantially the same if the samples were extracted using the
processes of the present invention.
Example 32
[0293] After oil was extracted from the fermentation broth using
the Friolex process, as described in Example 31, the crude oil was
further processed via refining, bleaching, and deodorizing steps to
obtain a final oil. The final oil was diluted with high oleic
sunflower oil to obtain finished commercial oil 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 gas
chromatography with flame ionization detection (GC-FID) as fatty
acid methyl esters (FAME).
[0294] Flash Chromatography--
[0295] Flash chromatography was used to separate the lipid classes
present in the final oil, and to determine the weight percent of
each class present in the oil. The chromatography system utilized
Silica Gel 60 (EMD Chemical, Gibbstown, N.J.) with mobile phase
composed of Petroleum Ether and Ethyl Acetate at 3 mL/min. 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 (followed by a 100% methanol
wash). 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.
[0296] TLC Analysis--
[0297] 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.
[0298] Fatty Acid Analysis--
[0299] The final oil sample and isolated lipid classes were
analyzed for fatty acid composition as FAMEs. Samples were weighed
directly into screw cap test tubes, and 1 mL of C19:0 internal
standard (NuCheck, Elysian, Minn.) in toluene and 2 mL of 1.5 N HCl
in methanol was added to each tube. The tubes were vortexed briefly
and placed in a heating block for 2 hours at 100.degree. C. The
tubes were removed from the heating block, allowed to cool, and 1
mL of saturated NaCl in water was added. The tubes were vortexed
again, centrifuged, and a portion of the top (organic) layer was
placed in a GC vial and analyzed by GC-FID. FAME's were quantified
using a 3-point internal standard calibration curve generated using
Nu-Chek-Prep GLC reference standard (Nu-Chek Prep, Inc., Elysian,
Minn.) and tentatively identified based on retention time. Fatty
acids present were expressed as mg/g and % of total FAME.
[0300] The sample was prepared by dissolving 250 mg of final oil in
600 .mu.L of hexane and applying to the head of the column. After
fractionation of the sample using flash chromatography, the sterol
ester fraction accounted for 1.2% by weight, the triacylglyceride
(TAG) fraction accounted for 92.1% by weight, the free fatty acid
(FFA) fraction accounted for 2.1% by weight, the sterol fraction
accounted for 1.1%, the diacylglyceride (DAG) fraction accounted
for 2.8% by weight of the final oil.
[0301] The TLC analysis of the pooled fractions showed that the FFA
and sterol fractions were mixed with TAG and DAG respectively. The
total fatty acid profiles of the FRIOLEX.RTM. final oil and
isolated fractions are shown below in Table 20 and Table 21
calculated as mg/g and % FAME, respectively.
TABLE-US-00021 TABLE 20 Fatty Acid Profile Calculated as Milligrams
per Gram of FAME Final Sterol Oil Esters TAG FFA Sterol DAG Wt. %
NA 1.2 92.1 2.1 1.1 2.8 FAME FAME FAME FAME FAME FAME Fatty Acid
(mg/g) (mg/g) (mg/g) (mg/g) (mg/g) (mg/g) C12:0* 0.0 0.0 1.0 0.0
1.2 0.6 C14:0* 11.5 5.1 11.3 6.0 9.6 5.7 C14:1* 0.0 0.0 0.0 0.0 0.0
0.0 C15:0 2.3 0.0 2.3 1.2 2.0 1.9 C16:0* 183.3 80.0 180.8 99.9
149.3 132.2 C16:1* 0.0 0.0 0.9 0.0 0.8 0.6 C18:0* 19.6 17.5 19.6
7.5 16.2 6.7 C18:1 N9* 243.3 242.8 249.6 62.9 190.5 84.0 C18:1 N7
1.9 1.7 2.0 0.8 1.9 0.9 C18:2 N6* 13.8 5.6 13.8 6.2 14.3 9.1 C20:0*
4.3 6.6 4.5 1.5 3.6 1.4 C18:3 N3* 0.0 0.0 0.3 0.0 0.0 0.0 C20:1 N9*
0.0 0.0 0.8 0.0 0.8 0.0 C18:4 N3 0.0 0.0 0.7 1.3 0.9 0.4 C20:2 N6*
0.0 0.0 0.6 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.3 0.0 0.0 0.0 C22:0* 3.3
61.0 3.2 1.1 3.0 1.2 C20:4 N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0
0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.7 0.0 2.3 1.4 1.9 1.3 C22:1 N9* 0.0
0.0 0.0 0.0 0.0 0.0 C20:4 N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 2.4
4.5 3.0 2.2 2.6 1.3 C20:5 N3* 28.1 3.0 27.7 38.6 25.6 43.2 C24:0*
1.4 64.3 1.4 0.0 2.0 1.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9*
0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 20.0 7.6 21.0 10.1 17.2 14.4
C22:5 N3* 2.8 0.0 3.1 3.7 3.4 2.9 C22:6 N3* 407.1 72.5 417.4 443.6
350.5 428.5 Sum of all 936.1 572.1 967.6 688.0 797.3 737.3
FAME's
TABLE-US-00022 TABLE 21 Fatty Acid Profiles as a Percent of Total
FAME Final Sterol Oil Esters TAG FFA Sterol DAG % % % % % % Fatty
Acid FAME FAME FAME FAME FAME FAME C12:0* 0.0 0.0 0.1 0.0 0.2 0.1
C14:0* 1.2 0.9 1.2 0.9 1.2 0.8 C14:1* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0
0.2 0.0 0.2 0.2 0.2 0.3 C16:0* 19.6 14.0 18.7 14.5 18.7 17.9 C16:1*
0.0 0.0 0.1 0.0 0.1 0.1 C18:0* 2.1 3.1 2.0 1.1 2.0 0.9 C18:1 N9*
26.0 42.4 25.8 9.1 23.9 11.4 C18:1 N7 0.2 0.3 0.2 0.1 0.2 0.1 C18:2
N6* 1.5 1.0 1.4 0.9 1.8 1.2 C20:0* 0.5 1.1 0.5 0.2 0.5 0.2 C18:3
N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:1 N9* 0.0 0.0 0.1 0.0 0.1 0.0 C18:4
N3 0.0 0.0 0.1 0.2 0.1 0.1 C20:2 N6* 0.0 0.0 0.1 0.0 0.0 0.0 C20:3
N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.4 10.7 0.3 0.2 0.4 0.2 C20:4 N7
0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6*
0.2 0.0 0.2 0.2 0.2 0.2 C22:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N5
0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 0.3 0.8 0.3 0.3 0.3 0.2 C20:5 N3*
3.0 0.5 2.9 5.6 3.2 5.9 C24:0* 0.2 11.2 0.1 0.0 0.2 0.1 C22:4 N9
0.0 0.0 0.0 0.0 0.0 0.0 C24:1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6*
2.1 1.3 2.2 1.5 2.2 1.9 C22:5 N3* 0.3 0.0 0.3 0.5 0.4 0.4 C22:6 N3*
43.6 12.7 43.1 64.5 44.0 58.1 Sum of 100 100 100 100 100 100 FAME
%
[0302] It is noted that fatty acid profiles of Tables 20 and 21
were obtained from samples extracted using a FRIOLEX.RTM. process.
The fatty acid profiles of Tables 20 and 21 are expected to be the
substantially the same if the samples were extracted using the
processes of the present invention.
Example 33
[0303] A two-day old inoculum flask of the isolated thraustochytrid
(ATCC Accession No. PTA-9695) was prepared in a carbon and
nitrogen-fed culture with 980 ppm Cl.sup.- (thraustochytrid
media).
[0304] Mutagenesis was carried out according to following
procedure:
[0305] 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 in tubes containing thraustochytrid media. The
diluted macerate samples were vortexed prior to adding 200 .mu.l of
inoculum to a thraustochytrid media agar petri dish, 100.times.15
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.
[0306] 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.
[0307] Plates were placed in a 22.5.degree. 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 thraustochytrid 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 thraustochytrid media. This
flask was placed on a shaker at 200 rpm in a 22.5.degree. 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 spun down 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-spun. The collected pellet was freeze dried, weighed,
and a FAME analysis was performed. The data in Tables 22-28
represents mutants produced with the above process.
TABLE-US-00023 TABLE 22 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control Fatty Acids ATCC PTA-9695 Mutant 1
Mutant 2 Mutant 3 Mutant 4 Mutant 5 Mutant 8 Mutant 9 Mutant 10 %
08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 12:0
0.10 0.10 0.08 0.08 0.13 0.07 0.11 0.08 0.08 % 12:1 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.17 0.13 0.12 0.18
0.11 0.15 0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
% 14:0 1.79 1.85 1.49 1.37 2.36 1.29 1.85 1.72 1.57 % 14:1 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 16:0 30.98 28.75 29.96 29.97 30.33 29.86
30.97 30.11 29.20 % 16:1 0.27 0.20 0.31 0.14 0.25 0.27 0.16 0.27
0.24 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 16:3
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15 0.13
0.17 0.27 0.12 0.16 0.13 0.13 % 18:0 1.29 1.22 1.38 1.47 1.22 1.57
1.25 1.34 1.34 % 18:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:2
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00 0.03
0.00 0.00 0.07 0.00 0.03 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 20:0 0.39 0.36 0.42 0.45 0.34 0.46 0.37 0.40 0.40
% 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:2 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.32 0.42 0.44 0.32 0.41
0.33 0.36 % 20:4 ARA 0.55 0.55 0.94 0.57 0.80 0.89 0.60 0.73 0.75 %
20:5 n-3 EPA 2.62 2.94 3.01 2.40 3.64 2.83 2.54 2.81 2.81 % 22:0
0.08 0.08 0.09 0.09 0.07 0.10 0.07 0.09 0.09 % 22:1 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
% 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:5 n-6
3.19 3.19 2.94 3.43 3.35 2.87 3.34 3.01 3.15 % 22:5 n-3 0.18 0.18
0.21 0.23 0.20 0.18 0.20 0.17 0.18 % 22:6 n-3 DHA 56.88 58.63 57.56
57.85 54.87 57.98 56.62 57.53 58.52 % 24:0 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.00 0.00 0.00 0.00 0.00 0.09
0.00 % Fat 46.83 46.10 31.23 47.39 49.78 30.62 54.71 37.72 37.87 %
Unknown 0.85 0.46 0.35 0.51 0.51 0.36 0.50 0.38 0.39
TABLE-US-00024 TABLE 23 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control ATCC Fatty Acids PTA-9695 Mutant 11
Mutant 13 Mutant 14 Mutant 15 Mutant 16 Mutant 20 Mutant 21 Mutant
22 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
12:0 0.10 0.10 0.08 0.09 0.11 0.11 0.09 0.09 0.10 % 12:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.15 0.16 0.14 0.13
0.12 0.17 0.16 0.13 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 14:0 1.79 1.89 1.43 1.75 1.83 1.98 1.76 1.77 1.81 % 14:1
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 16:0 30.98 31.08 30.27 29.92 31.79
30.18 28.84 30.05 30.81 % 16:1 0.27 0.32 0.26 0.28 0.21 0.24 0.23
0.23 0.33 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.24
0.15 0.13 0.15 0.12 0.14 0.16 0.14 % 18:0 1.29 1.36 1.44 1.31 1.36
1.21 1.28 1.34 1.33 % 18:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00
0.05 0.00 0.00 0.00 0.03 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.42 0.39 0.40 0.37 0.37 0.38
0.38 % 20:1 n-9 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 % 20:2
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.43 0.36 0.33 0.36 0.37
0.33 0.35 0.34 % 20:4 ARA 0.55 0.79 0.72 0.80 0.64 0.62 0.83 0.73
0.69 % 20:5 n-3 EPA 2.62 3.17 2.72 2.97 2.52 2.66 3.03 2.90 2.87 %
22:0 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:5
n-6 3.19 3.25 3.06 2.97 3.07 3.16 2.98 3.01 3.02 % 22:5 n-3 0.18
0.20 0.19 0.17 0.19 0.16 0.17 0.18 0.18 % 22:6 n-3 DHA 56.88 55.17
57.52 57.63 56.02 57.38 58.58 57.45 56.65 % 24:0 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 0.00 0.07
0.00 0.00 0.08 % Fat 46.83 46.19 37.00 38.41 48.46 47.32 37.71
40.23 43.55 % Unknown 0.85 0.47 0.39 0.36 0.47 0.44 0.37 0.39
0.38
TABLE-US-00025 TABLE 24 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control ATCC Fatty Acids PTA-9695 Mutant 24
Mutant 26 Mutant 27 Mutant 29 Mutant 30 Mutant 33 Mutant 34 Mutant
35 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
12:0 0.10 0.11 0.09 0.09 0.08 0.08 0.10 0.11 0.09 % 12:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.12 0.13 0.14 0.16
0.14 0.12 0.12 0.10 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 14:0 1.79 1.98 1.71 1.69 1.63 1.66 1.93 2.01 1.59 % 14:1
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00
0.00 0.00 0.00 0.70 0.54 0.39 % 16:0 30.98 30.61 30.32 30.21 29.70
29.50 30.26 32.28 30.78 % 16:1 0.27 0.19 0.22 0.22 0.26 0.26 0.29
0.26 0.16 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.15
0.18 0.16 0.13 0.13 0.26 0.16 0.12 % 18:0 1.29 1.24 1.31 1.31 1.32
1.30 1.32 1.37 1.34 % 18:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.10
0.11 0.09 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:0 0.39 0.37 0.39 0.40 0.40 0.39 0.37 0.40
0.40 % 20:1 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.14 % 20:2
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.38 0.37 0.35 0.35 0.35
0.00 0.00 0.00 % 20:4 ARA 0.55 0.61 0.59 0.69 0.68 0.32 0.34 0.24
0.28 % 20:5 n-3 EPA 2.62 2.62 2.70 2.85 2.90 2.91 3.28 2.51 2.59 %
22:0 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 % 22:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:5
n-6 3.19 3.10 3.11 3.05 3.10 3.11 3.43 3.26 3.56 % 22:5 n-3 0.18
0.16 0.18 0.19 0.18 0.18 0.18 0.15 0.24 % 22:6 n-3 DHA 56.88 57.03
57.46 57.46 57.96 58.52 55.92 54.96 56.73 % 24:0 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.08 0.00 0.00 0.00 0.00
0.07 0.07 0.07 % Fat 46.83 47.80 43.50 38.86 38.60 38.16 46.95
46.43 51.55 % Unknown 0.85 0.45 0.42 0.39 0.37 0.82 1.25 1.23
1.25
TABLE-US-00026 TABLE 25 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control ATCC Fatty Acids PTA-9695 Mutant 36
Mutant 37 Mutant 38 Mutant 39 Mutant 40 Mutant 42 Mutant 43 Mutant
44 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
12:0 0.10 0.00 0.11 0.00 0.11 0.09 0.08 0.12 0.09 % 12:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.44 0.09 0.24 0.12
0.11 0.12 0.08 0.15 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 14:0 1.79 1.25 1.99 1.48 1.96 1.76 1.43 2.17 1.75 % 14:1
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 2.12 0.48
0.71 0.54 0.55 0.36 0.62 0.50 % 16:0 30.98 26.95 28.04 32.28 30.84
30.25 25.77 43.37 30.18 % 16:1 0.27 0.00 0.26 0.23 0.22 0.21 0.10
1.05 0.22 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.95
0.13 0.28 0.16 0.16 0.10 0.26 0.13 % 18:0 1.29 1.58 1.11 1.79 1.30
1.29 1.25 2.21 1.34 % 18:1 n-9 0.00 0.37 0.08 0.25 0.09 0.09 0.12
0.09 0.10 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.00 %
18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00
0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:0 0.39 0.34 0.31 0.43 0.38 0.39 0.36 0.61
0.40 % 20:1 n-9 0.00 0.00 0.00 0.43 0.00 0.14 0.15 0.15 0.49 % 20:2
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 20:4 ARA 0.55 0.41 0.31 0.24 0.27 0.24 0.30 0.35
0.23 % 20:5 n-3 EPA 2.62 5.36 2.77 4.00 2.72 2.80 3.21 3.47 2.80 %
22:0 0.08 0.00 0.07 0.14 0.07 0.08 0.07 0.14 0.08 % 22:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 22:4 n-6 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.00 0.00 % 22:5
n-6 3.19 2.40 3.94 2.57 3.48 3.29 3.89 2.37 3.33 % 22:5 n-3 0.18
0.00 0.19 0.00 0.17 0.17 0.30 0.33 0.17 % 22:6 n-3 DHA 56.88 57.52
58.57 54.20 56.24 57.09 60.99 41.61 56.76 % 24:0 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.08 0.00 0.08 0.09
0.08 0.06 0.09 % Fat 46.83 12.73 54.86 18.08 45.74 42.59 42.48
56.44 41.20 % Unknown 0.85 0.29 1.36 0.73 1.28 1.20 1.31 0.90
1.20
TABLE-US-00027 TABLE 26 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control ATCC PTA- Fatty Acids 9695 Mutant 45
Mutant 46 Mutant 47 Mutant 48 Mutant 49 Mutant 50 Mutant 51 Mutant
52 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
12:0 0.10 0.10 0.13 0.11 0.07 0.09 0.09 0.09 0.11 % 12:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0 0.11 0.11 0.10 0.09 0.13
0.09 0.13 0.10 0.09 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 14:0 1.79 1.79 2.07 1.86 1.52 1.62 1.78 1.78 1.85 % 14:1
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.41 0.76
0.57 0.46 0.48 0.55 0.53 0.53 % 16:0 30.98 28.79 24.90 30.07 29.07
31.21 30.46 30.79 32.53 % 16:1 0.27 0.19 0.24 0.18 0.17 0.17 0.18
0.21 0.22 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.12 0.11
0.24 0.16 0.12 0.14 0.17 0.18 0.15 % 18:0 1.29 1.24 1.07 1.28 1.41
1.43 1.36 1.48 1.35 % 18:1 n-9 0.00 0.08 0.07 0.09 0.09 0.08 0.10
0.09 0.06 % 18:1 n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00
0.00 0.12 0.05 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:0 0.39 0.36 0.29 0.37 0.42 0.42 0.39 0.40
0.41 % 20:1 n-9 0.00 0.15 0.13 0.11 0.24 0.13 0.19 0.16 0.19 % 20:2
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-9 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-6 0.00 0.00 0.05 0.00
0.00 0.00 0.00 0.00 0.00 % 20:3 n-3 0.37 0.00 0.12 0.00 0.00 0.00
0.00 0.00 0.00 % 20:4 ARA 0.55 0.29 0.65 0.26 0.18 0.21 0.22 0.24
0.24 % 20:5 n-3 2.62 3.05 4.28 2.66 2.93 2.46 2.71 2.94 2.44 EPA %
22:0 0.08 0.07 0.06 0.07 0.09 0.09 0.08 0.08 0.08 % 22:1 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 22:4 n-6 0.00 0.06 0.07 0.05 0.00 0.00 0.00 0.00 0.00 % 22:5
n-6 3.19 3.59 4.28 3.46 3.07 3.32 3.17 3.18 3.24 % 22:5 n-3 0.18
0.25 0.27 0.18 0.17 0.17 0.16 0.17 0.17 % 22:6 n-3 DHA 56.88 57.74
58.32 56.70 58.65 56.45 56.83 56.19 55.06 % 24:0 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.07 0.15 0.10 0.10 0.11
0.10 0.10 0.07 % Fat 46.83 48.91 58.95 54.80 35.41 48.60 44.93
43.01 51.93 % Unknown 0.85 1.55 1.63 1.57 1.09 1.35 1.31 1.28
1.19
TABLE-US-00028 TABLE 27 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control ATCC Fatty Acids PTA-9695 Mutant 53
Mutant 54 Mutant 55 Mutant 56 Mutant 57 Mutant 58 Mutant 60 Mutant
61 Mutant 65 % 08:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 09:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
10:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 12:0 0.10 0.09 0.08 0.12 0.08
0.08 0.08 0.08 0.10 0.08 % 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 13:0 0.11 0.11 0.12 0.08 0.09 0.13 0.16 0.14 0.09
0.14 % 13:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
14:0 1.79 1.74 1.63 2.13 1.67 1.59 1.59 1.59 1.85 1.58 % 14:1 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 15:1 0.00 0.53 0.52
0.48 0.51 0.52 0.45 0.50 0.51 0.48 % 16:0 30.98 30.13 29.54 33.01
31.08 29.37 30.65 29.39 31.15 30.03 % 16:1 0.27 0.21 0.23 0.26 0.26
0.14 0.25 0.22 0.26 0.25 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 17:0 0.12 0.15 0.14 0.14 0.14 0.16 0.12 0.13 0.14 0.13 %
18:0 1.29 1.30 1.30 1.37 1.38 1.37 1.46 1.30 1.30 1.35 % 18:1 n-9
0.00 0.08 0.08 0.00 0.06 0.11 0.09 0.10 0.07 0.07 % 18:1 n-7 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-6 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 18:3 n-3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 18:4 n-3 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 20:0 0.39 0.38 0.39 0.40 0.42 0.38 0.43 0.39
0.39 0.41 % 20:1 n-9 0.00 0.19 0.16 0.13 0.19 0.20 0.17 0.14 0.13
0.21 % 20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
20:3 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3
n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 n-3
0.37 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:4 ARA 0.55
0.25 0.21 0.26 0.22 0.25 0.51 0.20 0.24 0.19 % 20:5 n-3 EPA 2.62
2.75 2.78 2.81 2.67 2.78 5.76 2.72 2.59 2.82 % 22:0 0.08 0.08 0.08
0.08 0.09 0.08 0.09 0.08 0.08 0.09 % 22:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 % 22:4 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.00 %
22:5 n-6 3.19 3.47 3.20 3.25 3.19 3.43 2.62 3.30 3.42 3.18 % 22:5
n-3 0.18 0.18 0.18 0.17 0.17 0.20 0.59 0.17 0.17 0.17 % 22:6 n-3
DHA 56.88 56.99 58.07 54.04 56.38 57.76 54.09 58.21 55.91 57.56 %
24:0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00
0.09 0.09 0.07 0.10 0.09 0.11 0.10 0.07 0.08 % Fat 46.83 45.83
39.59 48.81 41.92 43.97 33.96 36.97 50.40 36.21 % Unknown 0.85 1.28
1.19 1.19 1.29 1.35 0.77 1.24 1.48 1.17
TABLE-US-00029 TABLE 28 Mutants of Thraustochytrid Strain ATCC
Accession No. PTA-9695 control Mutant 68 Mutant 70 Mutant 72 Fatty
ATCC PTA- ATCC PTA- ATCC PTA- ATCC PTA- Mutant Acids 9695 Mutant 66
Mutant 67 9696 Mutant 69 9697 Mutant 71 9698 Mutant 73 74 % 08:0
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 % 12:0 0.15 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00
% 12:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:0
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 13:1 0.00 0.00
0.00 0.00 0.00 0.00 0.22 0.00 0.00 0.00 % 14:0 2.42 2.29 2.07 2.09
2.11 2.21 2.27 2.29 1.97 2.05 % 14:1 0.00 0.00 0.00 0.00 0.00 0.00
0.19 0.00 0.00 0.00 % 15:1 0.55 0.47 0.48 0.47 0.47 0.44 0.46 0.40
0.50 0.47 % 16:0 39.19 31.02 26.20 25.84 27.79 28.14 28.89 33.49
24.50 23.95 % 16:1 0.43 0.19 0.00 0.00 0.00 0.00 0.19 0.21 0.00
0.00 % 16:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 %
16:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 17:0 0.16
0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00 % 18:0 1.67 1.68 1.22
1.22 1.44 1.49 1.51 2.24 1.11 1.02 % 18:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 n-9 % 18:1 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 n-7 % 18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 % 18:3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 n-6 % 18:3 0.00 0.18 0.20 0.21 0.19 0.17 0.22 0.16 0.22 0.22
n-3 % 18:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-3 %
20:0 0.49 0.41 0.32 0.31 0.35 0.37 0.44 0.52 0.29 0.27 % 20:1 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 20:2 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 20:3 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 n-9 % 20:3 0.00 0.00 0.00 0.00 0.15
0.00 0.00 0.00 0.00 0.00 n-6 % 20:3 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 n-3 % 20:4 0.18 0.16 0.33 0.27 0.24 0.37 0.30
0.27 0.38 0.39 ARA % 20:5 1.76 2.30 3.86 3.97 3.32 4.12 3.09 2.74
4.43 4.53 n-3 EPA % 22:0 0.33 0.46 0.35 0.44 0.48 0.38 0.43 0.12
0.35 0.34 % 22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
% 22:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:3
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 22:4 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 % 22:5 2.62 2.83 3.17
2.66 2.72 2.95 3.46 2.79 3.17 3.19 n-6 % 22:5 0.18 0.18 0.46 0.42
0.34 0.61 0.25 0.27 0.48 0.57 n-3 % 22:6 49.52 57.01 60.60 61.42
59.74 58.03 55.62 53.06 61.83 62.23 n-3 DHA % 24:0 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 % Fat 52.70 49.32 48.51 49.49 48.80 53.65
40.38 63.40 48.27 46.63 % 0.35 0.82 0.73 0.66 0.67 0.73 2.46 1.18
0.78 0.76 Unknown
Example 34
[0308] Isolation of Microorganisms
[0309] 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.degree. 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.
Taxonomic Characteristics of the Isolated Microorganism Deposited
Under ATCC Accession No. PTA-10212
[0310] Cultures of the isolated microorganism deposited under ATCC
Accession No. PTA-10212 ("PTA-10212") appeared as white, wet,
smeared colonies without visible isolated sori.
[0311] 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).
[0312] 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 min). 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 .mu.m to 5 .mu.m).
[0313] 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 .mu.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 .mu.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 .mu.m in diameter. The latter rested
for several minutes up to 1-2 hours, then changed shape (elongated)
and turned into biflagellate zoospores, 2.3-2.5.times.3.7-3.9
.mu.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.
[0314] 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 .mu.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 .mu.L lysis
buffer (20 mM Tris pH 8.0, 125 .mu.g/mL Proteinase K, 50 mM NaCl,
10 mM EDTA pH 8.0, 0.5% SDS); (3) cells were lysed at 50.degree. 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
12,000.times.g 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 14,000.times.g 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.degree. C. in a
microcentrifuge at 14,000.times.g 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.degree. C. in a
microcentrifuge at 14,000.times.g 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 .mu.M
dNTPs, 0.1 .mu.M each primer, 8% DMSO, 200 ng chromosomal DNA, 2.5
U Herculase.RTM. II Fusion DNA Polymerase (Stratagene), and
Herculase.RTM. buffer (Stratagene) in a 50 .mu.L total volume. The
PCR Protocol included the following steps: (1) 95.degree. C. for 2
minutes; (2) 95.degree. C. for 35 seconds; (3) 55.degree. C. for 35
seconds; (4) 72.degree. C. for 1 minute and 30 seconds; (5) repeat
steps 2-4 for 30 cycles; (6) 72.degree. C. for 5 minutes; and (7)
hold at 4.degree. C.
[0315] 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.
[0316] 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.
limiacinum, 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.
[0317] Table 29 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 occurs 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).
TABLE-US-00030 TABLE 29 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
[0318] As shown in Table 29, it was found that, in terms of %
identity, the 18s rRNA gene sequence 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.
[0319] Based on the above characterization, the isolated
microorganism (ATCC Accession No. PTA-10212) is believed to
represent a new Thraustochytrium species and is therefore also
designated as Thraustochytrium sp. ATCC PTA-10212.
[0320] Taxonomic Characteristics of the Isolated Microorganism
Deposited Under ATCC Accession No. PTA-10208
[0321] 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. Pub.
No. 2010/0239533 and Int'l. Pub. No. WO 2010/107415.
[0322] 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 .mu.m in diameter, and
zoospores were 2.5 .mu.m to 2.8 .mu.m.times.4.5 .mu.m to 4.8 .mu.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, 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 35
[0323] Growth Characteristics of the Isolated Microorganism
Deposited Under ATCC Accession No. PTA-10212
[0324] The isolated microorganism (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 30.
TABLE-US-00031 TABLE 30 PTA-10212 Vessel Media Ingredient
concentration ranges Na.sub.2SO.sub.4 g/L 31.0 0-50, 15-45, or
25-35 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 MgSO.sub.4.cndot.7H.sub.2O g/L 5.0 0-10, 2-8, or
3-6 (NH.sub.4).sub.2SO.sub.4 g/L 0.44 0-10, 0.25-5, or 0.05-3
MSG.cndot.1H.sub.2O g/L 6.0 0-10, 4-8, or 5-7 CaCl.sub.2 g/L 0.29
0.1-5, 0.15-3, or 0.2-1 T 154 (yeast g/L 6.0 0-20, 0.1-10, or 1-7
extract) KH.sub.2PO.sub.4 g/L 0.8 0.1-10, 0.5-5, or 0.6-1.8 Post
autoclave (Metals) Citric acid mg/L 3.5 0.1-5000, 10-3000, or
3-2500 FeSO.sub.4.cndot.7H.sub.2O mg/L 10.30 0.1-100, 1-50, or 5-25
MnCl.sub.2.cndot.4H.sub.2O mg/L 3.10 0.1-100, 1-50, or 2-25
ZnSO.sub.4.cndot.7H.sub.2O mg/L 3.10 0.01-100, 1-50, or 2-25
CoCl.sub.2.cndot.6H.sub.2O mg/L 0.04 0-1, 0.001-0.1, or 0.01-0.1
Na.sub.2MoO.sub.4.cndot.2H.sub.2O mg/L 0.04 0.001-1, 0.005-0.5, or
0.01-0.1 CuSO.sub.4.cndot.5H.sub.2O mg/L 2.07 0.1-100, 0.5-50, or
1-25 NiSO.sub.4.cndot.6H.sub.2O mg/L 2.07 0.1-100, 0.5-50, or 1-25
Post autoclave (Vitamins) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25
Vitamin B12 mg/L 0.16 0.01-100, 0.05-5, or 0.1-1 Ca1/2-panto- mg/L
2.06 0.1-100, 0.1-50, or 1-10 thenate Biotin mg/L 3.21 0.1-100,
0.1-50, or 1-10 Post autoclave (Carbon) Glycerol g/L 30.0 5-150,
10-100, or 20-50 Nitrogen Feed: MSG.cndot.1H.sub.2O g/L 17 0-150,
10-100, or 15-50 Typical cultivation 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 glucose controlled @: about 5 - about 50 g/L,
about 10 - about 40 g/L, or about 15 - about 35 g/L.
[0325] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0326] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm
CV at 22.5.degree. 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.
[0327] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0328] In carbon (glycerol) and nitrogen-fed cultures with 1000 ppm
C1 at 22.5-28.5.degree. 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.
[0329] Growth Characteristics of the Isolated Microorganism
Deposited Under ATCC Accession No. PTA-10208
[0330] The isolated microorganism (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 31.
TABLE-US-00032 TABLE 31 PTA-10208 Vessel Media Ingredient
concentration ranges Na.sub.2SO.sub.4 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 MgSO.sub.4.cndot.7H.sub.2O g/L 5.0 0-10, 2-8, or 3-6
(NH.sub.4).sub.2SO.sub.4 g/L 0.42 0-10, 0.25-5, or 0.05-3
CaCl.sub.2 g/L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast g/L 1.0
0-20, 0.1-10, or 0.5-5 extract) KH.sub.2PO.sub.4 g/L 1.765 0.1-10,
0.5-5, or 1-3 Post autoclave (Metals) Citric acid mg/L 46.82
0.1-5000, 10-3000, or 40-2500 FeSO.sub.4.cndot.7H.sub.2O mg/L 10.30
0.1-100, 1-50, or 5-25 MnCl.sub.2.cndot.4H.sub.2O mg/L 3.10
0.1-100, 1-50, or 2-25 ZnSO.sub.4.cndot.7H.sub.2O mg/L 9.3
0.01-100, 1-50, or 2-25 CoCl.sub.2.cndot.6H.sub.2O mg/L 0.04 0-1,
0.001-0.1, or 0.01-0.1 Na.sub.2MoO.sub.4.cndot.2H.sub.2O mg/L 0.04
0.001-1, 0.005-0.5, or 0.01-0.1 CuSO.sub.4.cndot.5H.sub.2O mg/L
2.07 0.1-100, 0.5-50, or 1-25 NiSO.sub.4.cndot.6H.sub.2O mg/L 2.07
0.1-100, 0.5-50, or 1-25 Post autoclave (Vitamins) Thiamine mg/L
9.75 0.1-100, 1-50, or 5-25 Ca1/2-panto- mg/L 3.33 0.1-100, 0.1-50,
or 1-10 thenate Biotin mg/L 3.58 0.1-100, 0.1-50, or 1-10 Post
autoclave (Carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50
Nitrogen Feed: NH.sub.4OH mL/L 23.6 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 8.0;
temperature: about 17 - about 30 degrees Celsius, about 20 - about
28 degrees Celsius, or about 22 to about 24 degrees Celsius;
dissolved oxygen: about 2 - about 100% saturation, 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 20 - about 35 g/L.
[0331] In carbon (glucose) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0332] In carbon (glucose) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0333] In carbon (glucose) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0334] In carbon (glucose) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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.
[0335] In carbon (glucose) and nitrogen-fed cultures with 1000 ppm
Cl.sup.- at 22.5.degree. 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 36
[0336] Fatty Acid Profiles of Microorganism Strains ATCC PTA-10208
and PTA-10212
[0337] 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
32 and 33, below. It is noted that fatty acid profiles of Tables 32
and 33 were obtained from samples extracted using a solvent. The
fatty acid profiles of Tables 32 and 33 are expected to be the
substantially the same if the samples were extracted using the
processes of the present invention.
[0338] 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 34 and 35, below.
[0339] 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 36-39, below. It is noted that fatty
acid profiles of Tables 36-39 were obtained from samples extracted
using a typical hexane extraction. The fatty acid profiles of
Tables 36-39 are expected to be the substantially the same if the
samples were extracted using the processes of the present
invention.
[0340] 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.RTM. process, and
the fatty acid profiles of each class were determined using GC-FID
as FAME. Results are shown in Tables 40 and 41, below. It is noted
that fatty acid profiles of Tables 40 and 41 were obtained from
samples extracted using a FRIOLEX.RTM. process. The fatty acid
profiles of Tables 40 and 41 are expected to be the substantially
the same if the samples were extracted using the processes of the
present invention.
[0341] 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.
[0342] Experimental Procedures
[0343] Crude Oil Extraction--
[0344] 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.
[0345] Fatty Acid Analysis--
[0346] 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.degree. 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.
[0347] HPLC/ELSD/MS Method--
TABLE-US-00033 Instrument Agilent 1100 HPLC, Alltech 3300 ELSD,
Agilent 1100 MSD Column Phenomenex Luna Silica, 250 .times. 4.6 mm,
5 .mu.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
TABLE-US-00034 Gradient Time, min. % A % B 0 100 0 5 100 0 15 85 10
20 0 100 25 0 100 26 100 0 35 100 0
TABLE-US-00035 Column Temp. 30.degree. C. Flow Rate 1.5 mL/min
Injection Volume 5 .mu.L ELSD Detection Temperature 35.degree. C.,
Gas flow 1.2 L/min MSD Mass Range 200-1200, Fragmentor 225 V Drying
Gas Temperature 350.degree. C. Vaporizer Temperature 325.degree. C.
Capillary Voltage 3500 V Corona Current 10 .mu.A
[0348] Solid Phase Extraction--
[0349] 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
.about.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.
[0350] Flash Chromatography--
[0351] 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.
[0352] TLC Analysis--
[0353] 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.
[0354] Analysis of TAG and PL Fractions--
[0355] 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.degree. 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.
[0356] Results
[0357] PTA-10208 Sample #1
[0358] 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).
[0359] 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.
[0360] 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).
[0361] PTA-10208 Sample #2
[0362] 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).
[0363] 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.
[0364] 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).
[0365] PTA-10208 Sample #3
[0366] 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%.
[0367] PTA-10212 Sample #1
[0368] 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).
[0369] 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.
[0370] 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).
[0371] PTA-10212 Sample #2
[0372] 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).
[0373] 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.
[0374] 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).
TABLE-US-00036 TABLE 32 Fatty Acid Profiles of PTA-10208 and
PTA-10212 Biomasses and Extracted Crude Lipids (mg/g) PTA- PTA-
PTA- PTA- PTA- PTA- PTA- 10208 10208 10208 10208 10212 10212 10212
PTA- Sample Sample #1 Sample Sample #2 Sample Sample #1 Sample
10212 #1 Crude #2 Crude #1 Crude #2 Sample #2 Biomass Lipid Biomass
Lipid Biomass Lipid Biomass Crude Lipid Fatty FAME FAME FAME FAME
FAME FAME FAME FAME 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 0.00 3.26 0.00 3.09 0.03 4.04
0.08 5.87 n-9 C18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7
C18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:0 1.48 1.79
1.40 1.85 1.60 3.09 1.67 3.20 C18:3 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 n-3 C20:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9
C18:4 0.91 1.61 1.10 2.00 2.28 2.56 2.21 2.64 n-3 C20:2 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-6 C22:0 0.10 0.00 0.08 0.00 0.30 0.12 0.35 0.24
C20:4 0.81 0.45 0.67 0.41 0.00 0.00 0.00 0.00 n-7 C20:4 7.22 12.23
6.84 12.18 1.19 2.26 1.31 2.32 n-6 C22:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-9 C20:4 0.63 0.52 0.00 0.46 0.00 0.00 0.00 0.00
n-5 C20:4 3.45 5.45 3.33 5.58 0.00 2.40 0.00 2.34 n-3 C20:3 0.09
0.00 0.11 0.00 0.00 0.00 0.00 0.00 n-3 C20:5 107.31 186.83 92.99
169.32 40.32 90.08 43.15 92.54 n-3 C22:4 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-9 C24:0 0.60 0.00 0.52 0.00 2.81 6.83 2.74 6.53
C24:1 1.55 3.26 0.85 2.04 0.43 1.34 0.42 1.24 n-9 C22:5 9.66 15.84
10.27 17.98 2.42 4.68 2.32 4.21 n-6 C22:5 20.44 35.13 9.92 17.50
2.41 4.94 2.69 5.23 n-3 C22:6 246.98 423.10 245.96 444.08 139.58
289.34 137.35 277.57 n-3 Sum 527.15 907.18 518.71 942.75 367.89
805.29 372.63 799.68 of FAME
TABLE-US-00037 TABLE 33 Fatty Acid Profiles of PTA-10208 and
PTA-10212 Biomasses and Extracted Crude Lipids (%) PTA- PTA- PTA-
PTA- PTA- 10208 PTA- 10208 PTA- 10212 PTA- 10212 10208 Sample 10208
Sample 10212 Sample 10212 Sample Sample #1 Sample #2 Sample #1
Sample #2 #1 Crude #2 Crude #1 Crude #2 Crude Biomass Lipid Biomass
Lipid Biomass Lipid Biomass 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 0.00 0.36 0.00 0.33 0.01 0.50
0.02 0.73 n-9 C18:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-7
C18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:0 0.28 0.20
0.27 0.20 0.43 0.38 0.45 0.40 C18:3 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 n-3 C20:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9
C18:4 0.17 0.18 0.21 0.21 0.62 0.32 0.59 0.33 n-3 C20:2 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:3 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-6 C22:0 0.02 0.00 0.01 0.00 0.08 0.02 0.09 0.03
C20:4 0.15 0.05 0.13 0.04 0.00 0.00 0.00 0.00 n-7 C20:4 1.37 1.35
1.32 1.29 0.32 0.28 0.35 0.29 n-6 C22:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-9 C20:4 0.12 0.06 0.00 0.05 0.00 0.00 0.00 0.00
n-5 C20:4 0.65 0.60 0.64 0.59 0.00 0.30 0.00 0.29 n-3 C20:3 0.02
0.00 0.02 0.00 0.00 0.00 0.00 0.00 n-3 C20:5 20.36 20.59 17.93
17.96 10.96 11.19 11.58 11.57 n-3 C22:4 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-9 C24:0 0.11 0.00 0.10 0.00 0.76 0.85 0.74 0.82
C24:1 0.29 0.36 0.16 0.22 0.12 0.17 0.11 0.16 n-9 C22:5 1.83 1.75
1.98 1.91 0.66 0.58 0.62 0.53 n-6 C22:5 3.88 3.87 1.91 1.86 0.65
0.61 0.72 0.65 n-3 C22:6 46.85 46.64 47.42 47.10 37.94 35.93 36.86
34.71 n-3 Sum 100 100 100 100 100 100 100 100 of FAME %
TABLE-US-00038 TABLE 34 Fatty Acid Profiles of PTA-10208 and
PTA-10212 Isolated TAG PTA- PTA- PTA- PTA- PTA- PTA- PTA- PTA-
10208 10208 10208 10208 10212 10212 10212 10212 Sample Sample
Sample Sample Sample Sample Sample Sample #1 #1 #2 #2 #1 #1 #2 #2
Fatty FAME % FAME % FAME % FAME % 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 3.35 0.36 3.64 0.40 6.12
0.70 7.48 0.87 n-9 C18:1 0.00 0.00 0.00 0.00 <0.1 <0.1
<0.1 <0.1 n-7 C18:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
n-6 C20:0 1.86 0.20 1.55 0.17 4.76 0.55 5.32 0.62 C18:3 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 n-3 C20:1 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 n-9 C18:4 1.64 0.17 2.00 0.22 2.25 0.26 2.24 0.26
n-3 C20:2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C20:3 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-6 C22:0 0.00 0.00 0.00 0.00
0.55 0.06 0.89 0.10 Unknown 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
C20:4 0.39 0.04 0.05 0.01 0.00 0.00 0.00 0.00 n-7 C20:3 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 n-3 C20:4 12.79 1.36 11.82 1.28 2.33
0.27 2.25 0.26 n-6 C22:1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
n-9 C20:4 0.39 0.04 0.07 0.01 0.00 0.00 0.00 0.00 n-5 C20:4 5.52
0.59 5.09 0.55 2.87 0.33 2.98 0.35 n-3 C20:5 197.14 20.90 166.68
18.10 91.17 10.44 91.78 10.74 n-3 C24:0 0.00 0.00 0.00 0.00 6.93
0.79 7.36 0.86 C22:4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n-9
C24:1 1.08 0.11 <0.1 <0.1 0.00 0.00 0.00 0.00 n-9 C22:5 15.88
1.68 16.57 1.80 4.01 0.46 3.39 0.40 n-6 C22:5 36.05 3.82 16.00 1.74
4.53 0.52 5.07 0.59 n-3 C22:6 440.99 46.76 429.83 46.67 273.02
31.26 245.38 28.70 n-3 Sum 943.11 -- 921.03 -- 873.31 -- 854.89 --
of FAME Total % -- 100.00 -- 100.00 -- 100.00 -- 100.00 FAME
TABLE-US-00039 TABLE 35 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
[0375] PTA-10212 Sample #3
[0376] 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 36 and Table 37 calculated as mg/g and % FAME,
respectively.
TABLE-US-00040 TABLE 36 Fatty Acid Profiles of PTA-10212 Sample #3
Biomass and Extracted Crude Lipid (mg/g) Crude Biomass Oil TAG DAG
Wt. % NA 37.2% 46.0% 27.9% FAME FAME FAME FAME Fatty Acid (mg/g)
(mg/g) (mg/g) (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
TABLE-US-00041 TABLE 37 Fatty Acid Profiles of PTA-10212 Sample #3
Biomass and Extracted Crude Lipid (%) Crude Biomass Oil TAG DAG Wt.
% NA 37.2% 46.0% 27.9% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g)
(mg/g) (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
[0377] PTA-10212 Sample #4
[0378] 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 38 (mg/g) and Table 39
(% FAME).
TABLE-US-00042 TABLE 38 Fatty Acid Profiles of PTA-10212 Sample #4
Biomass and Extracted Crude Lipid (mg/g) Crude Biomass Oil TAG DAG
Wt. % NA 24.7% 42.2% 18.4% FAME FAME FAME FAME Fatty Acid (mg/g)
(mg/g) (mg/g) (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
TABLE-US-00043 TABLE 39 Fatty Acid Profiles of PTA-10212 Sample #4
Biomass and Extracted Crude Lipid (%) Crude Biomass Oil TAG DAG Wt.
% NA 24.7% 42.2% 18.4% FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g)
(mg/g) (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
[0379] PTA-10212 Sample #5
[0380] A sample of crude oil was extracted from a biomass of
PTA-10212 using the FRIOLEX.RTM. 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.
[0381] Briefly, the sample was prepared by dissolving 240 mg of
crude oil in 600 .mu.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%, and the DAG fraction accounted for 41.6%. The total fatty
acid profiles of the FRIOLEX.RTM. crude oil and isolated fractions
are shown below in Table 40 and Table 41 calculated as mg/g and %
FAME, respectively.
TABLE-US-00044 TABLE 40 Fatty Acid Profiles of PTA-10212 Sample #5
Crude Oil (mg/g) Crude Oil TAG DAG Wt. % NA 42.6% 41.6% FAME FAME
FAME Fatty Acid (mg/g) (mg/g) (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
TABLE-US-00045 TABLE 41 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 37
[0382] 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 were
determined using GC-FID as FAME.
[0383] PTA-10208 Final Oils
[0384] The fatty acid profiles for PTA-10208 Final Oils #1-5 are
summarized in Tables 42-43, including profiles associated within
the isolated TAG fraction (Tables 44-45) and the isolated
sterols/DAG fraction (Tables 46-47).
[0385] Individual lipid classes in the final oils were also
determined using flash chromatography (Table 48) and normal HPLC
with ELSD and APCI-MS confirmation (Table 49).
TABLE-US-00046 TABLE 42 Fatty Acid Profiles of 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 FAME FAME
FAME FAME FAME Fatty Acid (mg/g) (mg/g) (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
TABLE-US-00047 TABLE 43 Fatty Acid Profiles of PTA-10208 Final Oils
(%) 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 % 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
TABLE-US-00048 TABLE 44 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
FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g) (mg/g)
(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
TABLE-US-00049 TABLE 45 Isolated TAG Fatty Acid Profiles: PTA-10208
Final Oils (%) 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 % 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
TABLE-US-00050 TABLE 46 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 FAME FAME FAME FAME FAME Fatty Acid (mg/g) (mg/g) (mg/g)
(mg/g) (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
TABLE-US-00051 TABLE 47 Isolated Sterols/DAG Fatty Acid Profiles:
PTA-10208 Final Oils (%) 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 % 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
TABLE-US-00052 TABLE 48 Lipid class separation by flash
chromatography (wt %) Lipid Class PTA-10208 PTA-10208 PTA-10208
PTA-10208 PTA-10208 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
TABLE-US-00053 TABLE 49 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
[0386] PTA-10212 Final Oil
[0387] 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 50,
TABLE-US-00054 TABLE 50 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 38
[0388] 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 30 and 31.
[0389] Mutagenesis was carried out according to following
procedure:
[0390] 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 .mu.l of inoculum to a media agar plate, 100.times.15
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.
[0391] 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.
[0392] Plates were placed in a 22.5.degree. 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.degree. 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 51 and 52 represents mutants produced
with the above process from strains PTA-10208 and PTA-10212,
respectively.
TABLE-US-00055 TABLE 51 PTA-10208 Mutants control Mutant 1 Mutant 2
Mutant 3 Fatty Acids PTA-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 6.56 6.47 11.99 9.87 EPA % 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 51.58
52.27 48.17 49.35 DHA % 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
TABLE-US-00056 TABLE 52 PTA-10212 Mutants Control Mutant 1 Mutant 2
Mutant 3 Fatty Acids PTA-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 13.33 14.93 14.14 8.54 EPA % 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 59.18 54.31 59.89 56.39 DHA % 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 39
[0393] Two cell broths (approximately 13.3 kg) containing microbial
cells (Schizochytrium) were heated to 60.degree. C. in a 20 liter
fermentor. The fermentor had two 6-blade Rushton impellers having a
diameter of 15 cm. The top impeller was positioned at the 12 liter
mark and the bottom impeller was positioned 10 cm below the top
impeller. The first cell broth was continuously agitated at 307
centimeters/second. The second cell broth was continuously agitated
at 464 centimeters/second. Enzymes (i.e., Alcalase 2.4 L FG 0.5%)
were added to the cell biomass to lyse the cells and form an
emulsified lysed cell composition. The emulsified lysed cell
composition was first treated with a base (NaOH, 250 kg of 50% w/w
solution) until the pH of the lysed cell composition was from 10.4
to 10.6. Next, a salt (solid NaCl, in an amount of 2%, by weight,
of the lysed cell composition) was added to the lysed cell
composition. The lysed cell composition was then heated to a
temperature of 90.degree. C. and held at that temperature level for
20 hours. A sample of each cell broth was taken and the pH was
adjusted to 8.0 and placed in 50 ml test tubes. The test tubes were
centrifuged and the oil extraction data was measured. The oil
extraction data is provided in Table 53.
TABLE-US-00057 TABLE 53 Results from extraction testing in 50 mL
tubes at pH 8.0. Wet broth tested for Mass of Oil % yield % yield
Extraction (g) Recovered (g) (oil/broth) (oil/solids)* 307
centimeters/second 49.990 3.881 7.76 27.81 50.814 2.747 5.41 19.36
50.772 2.418 4.76 17.05 464 centimeters/second 51.154 7.067 13.81
49.13 51.092 7.055 13.81 49.11 50.132 6.606 13.18 46.86 *based on
solids content of untreated pasteurized broth
[0394] The data provided in Table 53 demonstrates that the higher
agitation speed resulted in a greater mass of oil recovered, a
greater % yield of oil from the broth, and a greater % yield of oil
from the solids content of the untreated pasteurized broth.
CONCLUSION
[0395] All of the various embodiments or options described herein
can be combined in any and all variations. While the invention has
been particularly shown and described with reference to some
embodiments thereof, it will be understood by those skilled in the
art that they have been presented by way of example only, and not
limitation, and various changes in form and details can be made
therein without departing from the spirit and scope of the
invention. Thus, the breadth and scope of the present invention
should not be limited by any of the above described exemplary
embodiments, but should be defined only in accordance with the
following claims and their equivalents.
[0396] All documents cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued or foreign patents, or any other documents,
are each entirely incorporated by reference herein, including all
data, tables, figures, and text presented in the cited
documents.
* * * * *