Fractionation of oil-bearing microbial biomass

• US 9,115,332 B2
• Filed: 10/16/2013
• Issued: 08/25/2015
• Est. Priority Date: 05/26/2009
• Status: Active Grant

• Current Assignee: Corbion Biotech, Inc. (Corbion NV)
• Original Assignee: Solazyme Inc.
• Inventors: Dillon, Harrison F., Elefant, Dan, Day, Anthony G., Franklin, Scott, Wittenberg, Jon
• Primary Examiner(s): Kim, Alexander
• Application Number:
  US14/055,763
• Publication Number:
  US 20140093945A1
• Time in Patent Office: 678 Days
• US Class Current: 1/1
• CPC Class Codes: C10L 1/026 for compression ignition
• CPC Class Codes: C11B 1/10 by extracting
• CPC Class Codes: C11B 1/106 using ultra-sounds
• CPC Class Codes: C11C 3/003 by esterification of fatty acids with alcohols C11C3/02 takes precedence
• CPC Class Codes: C11C 3/04 by esterification of fats or fatty oils
• 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: Y02T 50/678 Aviation using fuels of non-fossil origin

Abstract

The invention generally relates to the production of hydrocarbon compositions, such as a lipid, in microorganisms. In particular, the invention provides methods for extracting, recovering, isolating and obtaining a lipid from a microorganism and compositions comprising the lipid. The invention also discloses methods for producing hydrocarbon compositions for use as biodiesel, renewable diesel, jet fuel, and other materials.

Claims

1. A method of extracting a lipid from recombinant Prototheca cells, the method comprising the steps of:

(a) lysing recombinant Prototheca cells to produce a lysate, wherein the recombinant Prototheca cells;

(i) have not been subjected to a drying step between culturing and lysing; and

(ii) comprise at least 10% lipid by dry weight, and wherein the fatty acid profile of the lipid is at least 10% C8-C14 ;

(b) treating the lysate with an organic solvent for a period of time, wherein the organic solvent is an alcohol selected from ethanol, isopropanol or butanol;

(c) separating the treated lysate into layers comprising a lipid layer and an aqueous layer and, optionally, a lipid;

aqueous emulsion layer, and/or a cell pellet; and

(d) removing the lipid from the other layer(s).

2. The method of claim 1, wherein step (d) further comprises reducing the temperature of the mixture to below 25°

C.

3. The method of claim 1, wherein the recombinant Prototheca cells comprise lipid that is at least 10% C14 triacylglycerols, at least 10% C12 triacylglycerols, or at least 10% C10 triacylglycerols.

4. The method of claim 1, wherein the lysing is accomplished by subjecting the recombinant Prototheca cells to heating, sonication, mechanical lysis, osmotic shock, expression of an autolysis gene, exposure to pH above 8, exposure to an acidic pH, heating and exposure to an acidic pH, or digestion with an enzyme.

5. The method of claim 1, wherein the lysing is accomplished by subjecting the recombinant Prototheca cells to sonication.

6. The method of claim 1, wherein the recombinant Prototheca cells comprise a polynucleotide encoding a sucrose invertase or a lipid pathway enzyme.

7. The method of claim 6, wherein the polynucleotide encodes a lipid pathway enzyme.

8. The method of claim 6, wherein the recombinant Prototheca cells comprise one or more polynucleotides encoding a sucrose invertase and a lipid pathway enzyme.

9. The method of claim 8, wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase.

10. The method of claim 7, wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase.

11. The method of claim 1, wherein the Prototheca cells are Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, or Prototheca zopfii.

12. A method of extracting a lipid from recombinant Prototheca cells, the method comprising the steps of:

(a) lysing recombinant Prototheca cells to produce a lysate, wherein the recombinant Prototheca cells;

(i) have not been subjected to a drying step between culturing and lysing; and

(ii) comprise at least 10% lipid by dry weight, and wherein the fatty acid profile of the lipid is at least 4% C8-C14;

(b) treating the lysate with an organic solvent for a period of time, wherein the organic solvent is an alcohol selected from ethanol, isopropanol or butanol;

(c) separating the treated lysate into layers comprising a lipid layer and an aqueous layer and, optionally, a lipid;

aqueous emulsion layer, and/or a cell pellet; and

(d) removing the lipid from the other layer(s).

13. The method of claim 12, wherein the recombinant Prototheca cells comprise one or more polynucleotides encoding a sucrose invertase and/or a lipid pathway enzyme.

14. The method of claim 13, wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase.

15. The method of claim 12, wherein the Prototheca cells are Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, or Prototheca zopfii.

16. A method of extracting a lipid from recombinant Prototheca cells, the method comprising the steps of:

(a) lysing recombinant Prototheca cells to produce a lysate, wherein the recombinant Prototheca cells ;

(i) have not been subjected to a drying step between culturing and lysing; and

(ii) comprise at least 10% lipid by dry weight, and wherein the fatty acid profile of the lipid is at least 60% C18;

1;

(b) treating the lysate with an organic solvent for a period of time, wherein the organic solvent is an alcohol selected from ethanol, isopropanol or butanol;

(c) separating the treated lysate into layers comprising a lipid layer and an aqueous layer and, optionally, a lipid;

aqueous emulsion layer, and/or a cell pellet; and

(d) removing the lipid from the other layer(s).

17. The method of claim 16, wherein the fatty acid profile of the lipid is at least 80% C18:

1.

18. The method of claim 16, wherein the recombinant Prototheca cells comprise one or more polynucleotides encoding a sucrose invertase and/or a lipid pathway enzyme.

19. The method of claim 18, wherein the lipid pathway enzyme is a fatty acyl-ACP thioesterase.

20. The method of claim 16, wherein the Prototheca cells are Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, or Prototheca zopfii.