Pentose fermentation of normally toxic lignocellulose prehydrolysate with strain of Pichia stipitis yeast using air

• US 6,498,029 B2
• Filed: 06/21/2001
• Issued: 12/24/2002
• Est. Priority Date: 11/17/1999
• Status: Expired due to Fees

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Midwest Research Institute
• Inventors: Keller, Fred A. Jr., Nguyen, Quang A.
• Primary Examiner(s): Marx, Irene
• Assistant Examiner(s): Afremova, Vera
• Application Number:
  US09/886,581
• Publication Number:
  US 20010036659A1
• Time in Patent Office: 551 Days
• Field of Search: 435/255.5, 435/255.1, 435/938
• US Class Current: 435/255.5
• CPC Class Codes: C12N 1/145 Fungal isolates
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12P 7/10 substrate containing cellulosic material
• CPC Class Codes: C12R 2001/645 Fungi ; Processes using fungi
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y10S 435/938 Pichia

Abstract

Strains of the yeast Pichia stipitis NPw9 (ATCC PTA-3717) useful for the production of ethanol using oxygen for growth while fermenting normally toxic lignocellulosic prehydrolysates.

Claims

1. A biologically pure culture of Pichia stipitis strain NPw9 having ATCC accession No. PTA-3717 useful for production of ethanol using oxygen for growth while fermenting toxic lignocellulosic prehydrolysates in excess of 17% equivalent total solids and/or municipal solid waste (MSW) without detoxifying said lignocellulosic prehydrolysates.

2. A process for the preparation of Pichia stipitis strain NPw9 having ATCC accession No. PTA-3717 comprising:

a) growing a culture of Pichia stipitis yeast on toxic prehydrolysates of agricultural residues, grain processing intermediates and residues, softwoods, hardwoods or mixtures thereof;

b) maximizing the number of cell generations and using all generated cells by splitting and transferring culture progeny into additional cultures while said yeast have active mitochondria by gradually increasing prehydrolysate concentration while increasing aeration and reducing nutrient concentration in an amount sufficient to obligate yeast dependency on oxygen at an oxygen absorption rate of 0.001 to ≦

0.7 mM of 0₂ per liter of culture per minute;

c) gradually substituting for yeast extract a lesser amount of corn-steep liquor;

d) replacing a nitrogen source of peptone with a lesser amount of ammonium sulfate, or an inexpensive nitrogen source; and

e) collecting cells of Pichia stipitis strain NPw9 having accession No. PTA-3717.

3. The process of claim 2 wherein said toxic prehydrolysate is debarked softwood Douglas fir.

4. The process of claim 2 wherein said toxic prehydrolysate is a blend of whole tree softwoods of White Fir and Ponderosa Pine.

5. The process of claim 2, wherein said toxic prehydrolysate is a blend of mixed softwood or hardwood, or a blend of mixed woods.

6. The process of claim 2, wherein said prehydrolysate is derived from agricultural residues grain processing intermediates and residues, and/or MSW, or blends thereof.

7. The process of claims 3, 4, 5, or 6 wherein said toxic prehydrolysate is in the form of a slurry.

8. The process of claims 4, 5, or 6 wherein said toxic prehydrolysate is in the form of a filtrate.

9. The process of claim 8 wherein said toxic prehydrolysate has a pH of about 6.0 to about 3.0.

20. The process of claim 6 wherein said grain processing intermediate and residues are selected from the group consisting of corn fiber, distiller'"'"'s grains and stillage.

10. A process for preparation of Pichia stipitis strain NPw9 having ATCC accession No. PTA-3717 comprising:

a) growing a culture of Pichia stipitis;

derived from Northern Regional Research Lab (NRRL), Agricultural Research Service, USDA, on toxic prehydrolysate of a blend of whole tree softwoods, hardwoods, agricultural residues, grain processing intermediates and residues, or mixtures or blends thereof;

b) maximizing the number of cell generations and using all generated cells by splitting and transferring culture progeny into additional cultures while said yeast have active mitochondria by gradually increasing prehydrolysate concentration while increasing aeration and reducing nutrient concentration in an amount sufficient to obligate yeast dependency on oxygen at an oxygen absorption rate of 0.001 to ≦

0.7 mM of 0₂ per liter of culture per minute;

c) gradually substituting for yeast extract a lesser amount of corn-steep liquor;

d) replacing a nitrogen source of peptone with a lesser amount of ammonium sulfate; and

e) collecting cells of Pichia stipitis strain NPw9 having ATCC accession No. PTA3717.

11. The process of claim 10 wherein said prehydrolysate is debarked softwood Douglas Fir.

12. The process of claim 10 wherein said toxic prehydrolysate is a blend of whole tree softwoods such as White Fir and Ponderosa pine.

13. The process of claim 10 wherein said toxic prehydrolysate is a blend of mixed forest softwood and hardwood waste.

14. The process of claim 10 wherein said toxic prehydrolysate is a blend of agricultural wastes, residues, grain processing internediates or residues, or MSW, or mixtures thereof.

15. The process of claim 10 wherein said toxic prehydrolysate is a blend of hardwoods and agricultural waste.

16. The process of claim 10 wherein said toxic prehydrolysate is a blend of hardwoods, softwoods, agricultural waste and MSW.

17. The process of claim 11 wherein said toxic prehydrolysate is in the form of a slurry.

18. The process of claim 12 wherein said toxic prehydrolysate is in the form of a filtrate.

19. The process of claim 16 wherein said toxic prehydrolysate has a pH of about 7.5 to about 3, but preferably about 5.0.