TRANSFORMATION OF GLYCEROL AND CELLULOSIC MATERIALS INTO HIGH ENERGY FUELS

• US 20120115193A1
• Filed: 07/16/2009
• Published: 05/10/2012
• Est. Priority Date: 07/16/2008
• Status: Active Grant

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Nikolov, Zivko, Dickman, Martin B., de Figueiredo, Paul, Li, Lei, Shaw, Brian D., Louzada, Eliezer S., Sturino, Joseph M., Chang, Ying-Ying
• Granted Patent: US 9,340,768 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 1/22 Processes using, or culture media containing, cellulose or hydrolysates thereof
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12P 5/00 Preparation of hydrocarbons or halogenated hydrocarbons
• CPC Class Codes: C12P 5/02 acyclic C12P5/007 takes precedence
• CPC Class Codes: C12P 7/6409 Fatty acids
• 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: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention includes a system and method for making a biofuel comprising: providing a nitrogen-limiting, minimal growth media comprising glycerol, sugars generated from cellulosic biomass or both, under conditions in which an oleaginous microbe converts the growth media into at least one of triacylglycerol, neutral lipids, fatty acids, long-chain fatty acids, and hydrocarbons that is secreted by the microbe.

Claims

1. A method of making a biofuel comprising:

providing a nitrogen-limiting, minimal growth media comprising glycerol, sugars generated from cellulosic biomass or both, under conditions in which an oleaginous microbe converts the growth media into at least one of triacylglycerol, neutral lipids, fatty acids, long-chain fatty acids, and hydrocarbons.

2. The method of claim 1, wherein the oils are secreted by the microbe.

3. The method of claim 1, wherein the media comprises 0.5, 1.0, 1.5, or 2.0 M salt.

4. The method of claim 1, wherein the media comprises at least one of a cellulose, a cellulosic substrate, cellobiose, carboxymethylcellulose, hemicellulose, a sweet sorghum extract, a sugar cane extract, a sugar cane baggasse, or cellulosic substrates derived therefrom.

5. The method of claim 1, wherein the neutral lipid is at least one of a triacylglycerol (TAG) comprising saturated esterified fatty acids;

a TAG comprising unsaturated esterified fatty acids;

a TAG comprising oleic acid;

or a TAG comprising oleic acid at the Sn-1, Sn-2 or Sn-3 position.

6. The method of claim 1, wherein the hydrocarbons comprise C16, C18, C20, C22, C24, C26, C28, C30, and/or C40 and the hydrocarbons are further processed into a lubricant, biodiesel, gasoline, jet fuel, or a liquid transportation fuel.

7. The method of claim 1, wherein the hydrocarbons contain C15, C17, C19, C21, C23, C25, C27, C29, and/or C31.

8. The method of claim 1, wherein the mixture of hydrocarbons is optimized for a specific application, selected from a precursor for liquid transportation fuel biosynthesis, a precursor for lubricant biosynthesis or derivatives thereof.

9. The method of claim 1, wherein the microbes are grown for 48, 72, 96, or 120 hours at a temperature of 25°

C., 30°

C., or 37°

C. and at a pH of 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5.

10. The method of claim 1, wherein the media comprises 0.1% nitrogen.

11. The method of claim 1, wherein the microbes secrete the triacylglycerol, neutral lipids, fatty acids, long-chain fatty acids, and hydrocarbons without cell death.

12. The method of claim 1, wherein the microbes are induced to overexpress one or more autophagy-associated genes.

13. The method of claim 1, wherein the microbes are induced to overexpress one or more autophagy-associated genes selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

14. The method of claim 1, wherein the microbes are genetically modified to overexpress one or more autophagy-associated S. cerevisiae genes selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

15. The method of claim 1, wherein the microbes are genetically modified to overexpress one or more autophagy-associated genes that is orthologous or paralogous to a gene selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

16. The method of claim 1, wherein the microbes are genetically modified to overexpress one or more autophagy-associated genes integrated into the genome or on an autonomously replicating plasmid.

17. The method of claim 1, wherein the microbes are selected from Debaryomyces sp., Saccharomyces sp., Rhodococcus sp., Nocardia sp., Mycobacterium sp., Rhodosporidium sp., Cryptococcus sp., Rhodotorula sp., Yarrowia lipolytica or Lipomyces sp. or combinations of these microbes in a single reaction vessel.

18. A method of producing a biofuel comprising:

growing an oleaginous microbe in a nitrogen-limiting, minimal media to late log phase and/or stationary phase, whereby the oleaginous microbe secretes an oil.

19. The method of claim 18, wherein the media comprises 0.1% nitrogen.

20. The method of claim 18, wherein the microbes are grown for 48, 72, 96, or 120 hours at a temperature of 25°

C., 30°

C., or 37°

C. and at a pH of 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5.

21. The method of claim 18, wherein the media comprises 0.5, 1.0, 1.5, or 2.0M salt.

22. The method of claim 18, wherein the media comprises salt that is at least one of NaCl, KCl, or both KCl and NaCl.

23. The method of claim 18, wherein the microbes are disrupted with an agent that increases PI-3 kinase activity.

24. The method of claim 18, wherein the microbes are disrupted by mechanical or chemical treatment to release intracellular oils.

25. The method of claim 18, wherein the microbes have been genetically modified to overexpress PI-3 kinase.

26. The method of claim 18, wherein the microbes have been genetically modified to comprise a PI-3 kinase overexpression cassette into the cell, wherein the PI3-kinase overexpression cassette is integrated into the genome or the PI-3 kinase is in an overexpression cassette on an autonomously replicating plasmid.

27. An oleaginous microbe comprising that has been engineered to secrete enhanced amounts of oil by upregulating PI-3 kinase activity.

28. The microbe of claim 27, wherein the microbes have been genetically modified to overexpress PI-3 kinase.

29. The microbe of claim 27, wherein the microbes have been genetically modified to comprise a PI-3 kinase overexpression cassette into the cell, wherein the PI3-kinase overexpression cassette is integrated into the genome or the PI-3 kinase is in an overexpression cassette on an autonomously replicating plasmid.

30. The microbe of claim 27, wherein the microbes are selected from wherein the organism used for biofuel formation are selected from Debaryomyces sp., Saccharomyces sp. Rhodococcus sp., Nocardia sp., Mycobacterium sp., Rhodosporidium sp., Cryptococcus sp., Rhodotorula sp., and Lipomyces sp. or combinations of these organisms in a single reaction vessel.

31. An oleaginous microbe comprising that has been engineered to secrete enhanced amounts of oil by modulating the expression of autophagy-associated genes.

32. The microbe of claim 31, wherein the microbes are induced to modify the expression of one or more autophagy-associated S. cerevisiae genes selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

33. The microbe of claim 31, wherein the microbes are induced to modify the expression of one or more autophagy-associated genes that are orthologous to the S. cerevisiae genes selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

34. The microbe of claim 31, wherein the microbes are genetically modified to vary the expression of one or more autophagy-associated genes selected from ATG1, ATG2, ATG3, ATG4, ATG5, ATG6, ATG7, ATG8, ATG9, ATG10, ATG11, ATG12, ATG13, ATG14, ATG15, ATG16, ATG17, ATG18, ATG19, ATG20, ATG21, ATG22, ATG23, ATG24, ATG25, ATG26, ATG27, ATG28, ATG29, ATG30, ATG31.

35. The microbe of claim 31, wherein the microbes are genetically modified to vary the expression of one or more autophagy-associated genes by integrated the genes into the genome, by expression of the genes on an autonomously replicating plasmid or by modifying the expression of the genes post-translationally.

36. The microbe of claim 31, wherein the microbes are engineered to under-express the autophagy-associated genes to decrease oil secretion.

37. The microbe of claim 31, wherein the microbes are engineered to over-express the autophagy-associated genes to increase oil secretion.

38. The method of claim 31, wherein microbes are further genetically modified to overexpress PI-3 kinase.

39. A method of reducing bioreactor waste comprising:

mixing a reaction waste product comprising glycerol with a growth media and an inoculum of Debaryomyces hansenii under conditions in which Debaryomyces hansenii converts the glycerol into long-chain fatty acids and hydrocarbons; and

recovering the long-chain fatty acids and hydrocarbons produced thereby.

40. A method of reducing bioreactor waste comprising

generating a biofuel by fermentation;

collecting a glycerol waste stream from the fermentation;

mixing a biofuel reactor waste product comprising glycerol with a growth media and an inoculum of Debaryomyces hansenii under conditions in which Debaryomyces hansenii converts the glycerol into long-chain fatty acids and hydrocarbons; and

recovering the long-chain fatty acids and hydrocarbons produced thereby.

41. A biofuel reactor comprising:

a vessel comprising an internal volume capable of holding a growth medium;

a microbe capable of converting glycerol into a long-chain fatty acids and hydrocarbons in the growth medium; and

a source of glycerol.