Production of Oil in Microorganisms

• US 20110014665A1
• Filed: 04/30/2010
• Published: 01/20/2011
• Est. Priority Date: 06/01/2007
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Franklin, Scott, Day, Anthony G., Dillon, Harrison F., Trimbur, Donald E., Im, Chung-Soon, Coragliotti, Anna
• Granted Patent: US 8,889,401 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C10L 1/06 for spark ignition
• CPC Class Codes: C10L 2200/0484 Vegetable or animal oils
• CPC Class Codes: C10L 2270/026 for diesel engines, e.g. automobiles, stationary, marine
• CPC Class Codes: C10L 2270/04 for turbines, planes, power generation
• CPC Class Codes: C11C 3/00 Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom sulfonated fats or oils C07C309/62; factice C08H; drying oils C09F
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• CPC Class Codes: C12N 9/2408 acting on alpha -1,4-glucosidic bonds
• CPC Class Codes: C12N 9/2431 Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 5/02 : acyclic C12P5/007 takes precedence
• CPC Class Codes: C12P 7/6409 : Fatty acids
• CPC Class Codes: C12P 7/6458 : by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
• CPC Class Codes: C12P 7/6463 : obtained from glyceride producing microorganisms, e.g. single cell oil
• CPC Class Codes: C12P 7/649 : Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: C12Y 302/01026 : Beta-fructofuranosidase (3.2.1.26), i.e. invertase
• CPC Class Codes: Y02E 50/10 : Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 : Fuel from waste, e.g. synthetic alcohol or diesel
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts
• CPC Class Codes: Y02T 50/678 : Aviation using fuels of non-fossil origin

Abstract

The invention provides methods of cultivating oil-bearing microbes using cellulosic material. Also provided are microorganisms containing one or more exogenous genes that facilitate the use of cellulosic materials as a feedstock. Also provided are microorganisms and methods for manufacturing non-alcohol-based fuels and fuel feedstocks through a process of converting cellulosic materials into oils.

Claims

1. A microalgae of the genus Prototheca containing an exogenous gene.

2. The microalgae of claim 1, wherein the exogenous gene encodes a polysaccharide degradation enzyme and/or a sucrose utilization gene.

3. The microalgae of claim 1, wherein the microalgae is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca wickerhamii, Prototheca moriformis, and Prototheca zopfii.

4. The microalgae of claim 1 that contains a second exogenous gene that encodes a protein selected from the group consisting of a lipid modification enzyme and a hydrocarbon modification enzyme.

5. The microalgae of claim 1 that is capable of growth on a cellulosic feedstock selected from the group consisting of sugarcane bagasse, rice hulls, corn fiber, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, urban paper waste, grass clippings, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane and fiber sorghum.

6. A microalgal culture containing:

(a) a population of obligate heterotrophic microalgae; and

(b) a culture medium comprising depolymerized cellulosic material.

7. The microalgal culture of claim 6, wherein the microalgae are of the genus Prototheca.

8. The microalgal culture of claim 6, wherein the depolymerized cellulosic material is selected from depolymerized sugarcane bagasse, rice hulls, corn fiber, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, urban paper waste, grass clippings, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

9. The microalgal culture of claim 7, wherein the microalgae is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, and Prototheca zopfii.

10. The microalgal culture of claim 6, wherein the culture medium is essentially free of carbon sources other than glucose and xylose.

11. The microalgal culture of claim 6, wherein the culture is axenic.

12. The microalgal culture of claim 6, wherein the culture medium contains a furfural.

13. The microalgal culture of claim 6, wherein the culture medium contains lignin.

14. The microalgal culture of claim 6, wherein the culture medium contains a polysaccharide degradation enzyme.

15. The microalgal culture of claim 6, wherein the microalgae are at least 50% lipid by dry weight.

16. A method of cultivating obligate heterotrophic microalgae, comprising:

(a) providing an obligate heterotrophic microalgae cell;

(b) placing the microalgae cell in culture medium comprising depolymerized cellulosic material as a carbon source; and

(c) culturing the microalgae cell under heterotrophic growth conditions until the microalgae cell accumulates at least 10% of its dry cell weight as lipid.

17. The method of claim 16, wherein the microalgae cell is of the genus Prototheca.

18. The method of claim 17, wherein the microalgae cell is selected from the group consisting of Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca wickerhamii, and Prototheca zopfii.

19. The method of claim 16, wherein the culture medium is axenic.

20. The method of claim 16, wherein the depolymerized cellulosic material is selected from depolymerized sugarcane bagasse, rice hulls, corn fiber, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, paper fractions of municipal solid waste, grass clippings, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

21. The method of claim 16, wherein the culture medium is essentially free of carbon sources other than sucrose, glucose and fructose.

22. The method of claim 16, wherein the culture medium further comprises at least one additional carbon source selected from glucose, glycerol, xylose, fructose, arabinose, mannose, galactose or acetate.

23. The method of claim 22, wherein the culture medium further comprises lignin.

24. The method of claim 23, wherein the culture medium further comprises a furfural.

25. The method of claim 16, wherein the lipid comprises at least 50% C18:

1.

26. The method of claim 16, wherein the microalgae cell is at least 50% lipid by dry weight.

27. The method of claim 16, where the culture medium further comprises at least 1% sucrose.

28. The method of claim 16, wherein the culturing step occurs primarily in an absence of light.

29. The method of claim 16, wherein the microalgae cell has a lipid profile comprising at least 3% C:

18;

0 and at least 8% C;

16;

0.

30. The method of claim 16, further comprising isolating the lipid from the microalgae cell.

31. The method of claim 30, further comprising subjecting the lipid to a chemical reaction that changes the chemical structure of the lipid.

32. The method of claim 31, wherein the chemical reaction is selected from cracking, transesterification, hydrogenation, deoxygenation, and isomerization.

33. The method of claim 32, wherein the lipid is subjected to hydrogenation, deoxygenation and isomerization to produce a renewable diesel fuel that meets ASTM D975 specifications.

34. The method of claim 32, wherein the lipid is subjected to transesterification to produce a biodiesel fuel that meets ASTM D6751 specifications.

35. The method of claim 16, wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid.

36. A method of cultivating microalgae, comprising culturing a recombinant microalgae cell of the genus Prototheca (a) in a culture medium containing depolymerized cellulosic material as a primary carbon source, (b) under heterotrophic growth and limited nitrogen conditions, and (c) until the microalgae cell accumulates at least 10% of its dry cell weight as lipid. wherein the depolymerized cellulosic material is provided to the culture medium in a feedstock selected from the group consisting of depolymerized sugarcane bagasse, rice hulls, corn fiber, corn stover, wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels, hardwood thinnings, softwood thinnings, wood chips, sawdust, pulp mill waste, paper fractions of municipal solid waste, grass clippings, wood construction waste, switchgrass, hybrid poplar wood, miscanthus, fiber cane or fiber sorghum.

37. The method of claim 36, wherein the culturing is continued until the microalgae cell accumulates at least 60% of its dry cell weight as lipid.