Transformation of glycerol and cellulosic materials into high energy fuels

• US 9,340,768 B2
• Filed: 07/16/2009
• Issued: 05/17/2016
• Est. Priority Date: 07/16/2008
• Status: Expired due to Fees

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Shaw, Brian D., de Figueiredo, Paul, Li, Lei, Nikolov, Zivko, Dickman, Martin B., Louzada, Eliezer S., Sturino, Joseph M., Chang, Ying-Ying
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/003,931
• Publication Number:
  US 20120115193A1
• Time in Patent Office: 2,497 Days
• Field of Search: None
• US Class Current: 1/1
• 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:

obtaining a sugarcane bagasse biomass and an isolated oleaginous microbe; and

mixing the sugarcane bagasse biomass and the oleaginous microbe in a nitrogen-limiting minimal growth media comprising glycerol, or glucose generated from the sugarcane bagasse biomass, under conditions in which an oleaginous microbe converts the growth media into a triacylglycerol (TAG), wherein the oleaginous microbe overexpress Autophagy Protein ATG8.

2. The method of claim 1, wherein the microbe secretes TAG.

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

4. The method of claim 1, wherein the minimal growth media is defined further as comprising a source of glucose selected from at least one of a cellulose, a cellulosic substrate, cellobiose, carboxymethylcellulose, hemicellulose, a sweet sorghum extract, a sugar cane extract, a sugar cane, or cellulosic substrates derived therefrom.

5. The method of claim 1, wherein the TAG further comprises oleic acid at the Sn-1, Sn-2 or Sn-3 position.

6. The method of claim 1, wherein the oleaginous 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.

7. The method of claim 1, wherein the minimal growth media further comprises 0.1% by weight nitrogen.

8. The method of claim 1, wherein the oleaginous microbes secrete the triacylglycerol, without cell death.

9. The method of claim 1, wherein the oleaginous microbes are induced to overexpress Autophagy Protein ATG8.

10. The method of claim 1, wherein the oleaginous microbes are genetically modified to overexpress Autophagy Protein ATG8.

11. The method of claim 10, wherein the overexpressed Autophagy Protein ATG8 is integrated into the genome of the oleaginous microbes or expressed by an autonomously replicating plasmid.

12. The method of claim 1, wherein the oleaginous microbe is Debaryomyces sp.

13. A method of producing a biofuel comprising:

obtaining a cellulosic biomass and an isolated oleaginous microbe; and

growing the oleaginous microbe in a nitrogen-limiting minimal media to late log phase and/or stationary phase, whereby the oleaginous microbe secretes an oil, wherein the oleaginous microbe overexpress Autophagy Protein ATG8.

14. The method of claim 13, wherein the minimal growth media further comprises 0.1% by weight nitrogen.

15. The method of claim 13, wherein the oleaginous microbe is 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.

16. The method of claim 13, wherein the minimal growth media further comprises 0.5, 1.0, 1.5, or 2.0M salt.

17. The method of claim 13, wherein the minimal growth media further comprises salt that is at least one of NaCl, KCl, or both KCl and NaCl.

18. The method of claim 13, wherein the oleaginous microbe is disrupted by mechanical or chemical treatment to release intracellular oils.

19. A method of reducing bioreactor waste comprising:

obtaining a cellulosic biomass and isolated Debaryomyces hansenii; and

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 TAG comprising oleic acid at the Sn-1, Sn-2 or Sn-3 position; and

recovering the TAG comprising oleic acid at the Sn-1, Sn-2 or Sn-3 position produced thereby, wherein the oleaginous microbe overexpress Autophagy Protein ATG8.

20. 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 TAG comprising oleic acid at the Sn-1, Sn-2 or Sn-3 position; and

recovering the TAG comprising oleic acid at the Sn-1, Sn-2 or Sn-3 position produced thereby, wherein the oleaginous microbe overexpress Autophagy Protein ATG8.