Secondary liquefaction in ethanol production

• US 20040091983A1
• Filed: 05/09/2003
• Published: 05/13/2004
• Est. Priority Date: 11/10/2000
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes), Novozymes North America Incorporated (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes), Novozymes North America Incorporated (Novozymes)
• Inventors: Fuglsang, Claus Crone, Felby, Claus, Veit, Christopher
• Granted Patent: US 7,244,597 B2
• Time in Patent Office: Days
• US Class Current: 435/161
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The invention relates to a method of producing ethanol by fermentation, said method comprising a secondary liquefaction step in the presence of a thermostable acid alpha-amylase or, a thermostable maltogenic acid alpha-amylase.

Claims

1. A method of producing ethanol by fermentation, said method comprising a secondary liquefaction step in the presence of a thermostable acid alpha-amylase.

3. The method of any of claims 1-2, further comprising recovery of the ethanol.

4. The method of any of claims 1-3, further comprising a pre-saccharification step which is performed after the secondary liquefaction step (c) and before step (d).

5. The method of any of claims 1-4, wherein the starch containing material is selected from the group consisting of:

tubers, roots and whole grain; and

any combinations of these.

6. The method of any of claims 1-5, wherein the starch containing material is obtained from cereals.

7. The method of any of the claims 1-5, wherein the starch containing material is selected from the groups consisting of corns, cobs, wheat, barley, rye, milo and potatoes;

or any combination of these.

8. The method of claim 5, wherein the starch containing material is whole grain selected from the group consisting of corn, wheat or barley or any combinations of these.

9. The method of any of the claims 1-8, wherein the starch containing material is whole grain and said method comprises a step of milling the whole grain before step (a).

10. The method of any of the claims 1-5, wherein the starch containing material is obtainable by a process comprising milling of whole grain.

11. The method of any of the claims 1-5, further comprising prior to step (a) the steps of;

i) milling of whole grain;

ii) forming a slurry comprising the milled grain and water to obtain the starch containing material.

12. The method of any of the claims 9-11, wherein the milling step is a dry milling step.

13. The method of any of the claims 9-11, wherein the milling step is a wet milling step.

14. The method of any of the claims 1-5, wherein the starch-containing material is a side stream from starch processing.

15. The method of any of the claims 1-14, further comprising a step of;

(f) distillation to obtain the ethanol;

wherein the fermentation in step (e) and the distillation in step (f) is carried out simultaneously or separately/sequential;

optionally followed by one or more process steps for further refinement of the ethanol.

16. The method of claim 15;

wherein the starch containing material is milled whole grain, said method further comprising the steps of;

(g) separation of Whole stillage produced by of the distillation in step (f), into wet grain and Thin stillage; and

(h) recycling Thin stillage to the starch containing material prior to step (a).

17. The method of any of the claims 1-16 wherein the fermentation in step (e) is performed using a micro-organism, such as bacteria and fungi, e.g. Zymomonas species and Sacharomyces species.

18. The method of claim 17, wherein the micro-organism is capable of fermenting sugars to ethanol.

19. The method of claim 17 or 18, wherein the micro-organism is a yeast, such as e.g. Saccharomyces cerevisiae.

20. The method of any of the claims 1-19, wherein the fermentation is carried out in the presence of glucoamylase, and/or protease.

21. The method of claim 20, wherein the protease is a microbial protease, e.g., selected from the group of fungal proteases, such as e.g. an acid fungal protease derived from a strain of Aspergillus, preferably A. niger.

22. The method of claim 20, wherein the protease is a neutral or alkaline protease, such as a protease derived from a strain of Bacillus.

23. The method of claims 1-22 wherein is added a glucoamylase and/or phytase in order to promote the fermentation.

24. The method of claim 23, wherein the phytase is microbial, such as, e.g., derived from a strain of Peniophra lycii or Aspergillus oryzae.

25. The method of claim 23, wherein the glucoamylase is microbial, such as, e.g., derived from a strain of Aspergillus niger or Talaromyces emersonii.

26. The method of claims 1-25, wherein the alpha-amylase in step (a) is microbial, such as e.g. a bacterial alpha-amylase, including e.g. a thermostable bacterial alpha-amylase.

27. The method of claim 26, wherein the alpha-amylase is derived from a strain of the genus Bacillus or a strain of Aspergillus.

28. The method of claims 1-27 wherein the thermostable acid alpha amylase in step (c) is fungal, e.g. derived from Aspergillus niger.

29. The method of claims 1-28, wherein the liquefied material in step (c) has been liquefied to a DE of 5-15, e.g. 8-12, such as, e.g. 10-12.

30. The method of claim 29, comprising prior to step (a) the steps of;

i) milling of whole grain; and

ii) forming a slurry comprising the milled grain and water.

31. The method of claims 1-30, wherein the liquefaction in step (a) is performed at 60-95°

C. for 10-120 min, preferably at 75-90°

C. for 15-40 min.

32. The method of claims 1-31, wherein the liquefaction in step (c) is performed at 60-95°

C. for 10-120 min, preferably at 70-85°

C. for 15-80 min.

33. The method of claims 1-32, wherein in step (c) in addition to the thermostable acid alpha-amylase is also added an alpha-amylase which is not a thermostable acid alpha-amylase, i.e. e.g. the alpha-amylase TTC.

34. The method of claims 1-33 wherein the thermostable acid alpha-amylase is a thermostable maltogenic acid alpha-amylase

35. The method of any of claims 1-34, wherein the thermostable maltogenic acid alpha-amylase is an alpha-amylase having an amino acid sequence which has at least 70% identity to SEQ ID NO:

1 preferably at least 75%, 80%, 85% or at least 90%, e.g. at least 95%, 97%, 98%, or at least 99% identity to SEQ ID NO;

1.

36. The method of claims 1-34, wherein the thermostable maltogenic acid alpha-amylase is an alpha-amylase having the amino acid sequence set forth in SEQ ID NO:

1.

37. The method of any of the claims 1-36, wherein the thermostable maltogenic acid alpha-amylase is as defined in claims 41-46.

47. The method of any of the claims 38-45, wherein the starch-containing raw material used in the process for production of the ethanol, is as defined in any of the claims 5-10.

48. The method of any of the claims 38-45, further comprising the process steps as defined in any of the claims 3-4 or 11-33.

49. A method as defined in any of claim 1-48, wherein the secondary liquefaction and the fermentation is perform essentially simultaneously and wherein the alpha-amylase for the secondary liquefaction is expressed by the microorganism, preferably yeast, the microorganism being genetically modified so as to express the alpha-amylase.

50. The method of any of the claims 1-49, further comprising addition of one or more additional enzyme(s) in one or more of the process steps.

51. The method of claim 50, wherein the additional enzyme is not an alpha-amylase.

52. The method of claim 51, wherein the additional enzyme is not a thermostable maltogenic acid alpha-amylase.

53. The method of any of the claims 1-52 where all process steps are performed batch wise, or where all process steps are performed as a continuous flow, or where one or more process step(s) is(are) performed batch wise and one or more process step(s) is(are) performed as a continuous flow.

54. A yeast genetically modified to express the alpha-amylase defined in any of the claims 35-36 or any of the claims 41-47.

57. Use according to claim 55 or 56 wherein the starch-containing raw material used in the process for production of the ethanol, is as defined in any of the claims 5-10.

2. A method of producing ethanol from a starch containing material, said method comprising the steps of;

a) liquefaction of a starch containing material in the presence of an alpha-amylase;

b) jet cooking;

c) liquefaction in the presence of a thermostable acid alpha-amylase;

d) saccharification; and

e) fermentation to produce ethanol;

wherein the steps (a), (b), (c) and (d) is performed in the order (a), (b), (c), (d) and wherein (e) is performed simultaneously to or following (d).

38. A method of producing ethanol by fermentation, said method comprising a secondary liquefaction step in the presence of an alpha-amylase having an amino acid sequence which has at least 70% identity to SEQ ID NO:

1 preferably at least 75%, 80%, 85% or at least 90%, e.g. at least 95%, 97%, 98%, or at least 99% identity to SEQ ID NO;

1.

40. The method of claim 38 or 39, wherein said alpha-amylase has the amino acid sequence set forth in SEQ ID NO:

1.

41. The method of any of the claims 38-40, wherein the alpha-amylase in the secondary liquefaction step is maltogenic such that when using a DE 12 alpha-amylase TTC liquefied corn starch at 30% dry substance at 60°

C., pH 4,5 and dosing the enzyme at 1 AFAU/g dry substance, said enzyme will in 24 hours catalyze the formation of at least 15%, or at least 20%, at least 25%, at least 30 w/w maltose as based on the total amount of starch.

42. The method of any of the claims 38-41, wherein the alpha-amylase used in the secondary liquefaction step is thermostable, such that the enzyme will maintain more than 90% of its activity for 1 hour at 70°

C. using a DE 12 alpha-amylase TTC liquefied corn starch at 30% dry substance as substrate, pH 5.5;

0.1 M citrate buffer and 4.3 mM Ca²⁺.

44. The method of any of the claims 38-43, wherein the alpha-amylase used in the secondary liquefaction step is acid, such that the enzyme maintain will more than 70% of its activity in the range from pH 3.5-5.0 (e.g. at pH 4) at the conditions:

substrate DE 12 alpha-amylase TTC liquefied corn starch at 30% dry substance, Temperature 40°

C., and 0.1 M citrate buffer and 4.3 mM Ca²⁺.

45. The method of claim 38-39, wherein the alpha-amylase used in the secondary liquefaction step is;

i) thermostable as defined in claim 42 and/or claim 43;

ii) acid as defined in claim 44.

46. The method of claim 45, wherein said thermostable acid alpha-amylase is also iii) maltogenic as defined in claim 41.

39. A method of producing ethanol from a starch containing material, said method comprising the steps of;

a) liquefaction of a starch containing material in the presence of an alpha-amylase;

b) jet cooking;

c) liquefaction in the presence of an alpha-amylase having an amino acid sequence which has at least 70% identity to SEQ ID NO;

1, preferably at least 75%, 80%, 85% or at least 90%, e.g. at least 95%, 97%, 98%, or at least 99% identity to SEQ ID NO;

1; and

d) saccharification; and

e) fermentation to produce ethanol;

wherein the steps (a), (b), (c) and (d) is performed in the order (a), (b), (c), (d) and wherein (e) is performed simultaneously to or following (d).

43. The method of any of the claims 3842, wherein the alpha-amylase used in the secondary liquefaction step is thermostable, such the enzyme maintain will more than 80% of its activity for 15 min in the range from 50-80°

C. using a DE 12 alpha-amylase TTC liquefied corn starch at 30% dry substance as substrate. pH 5.5, 0.1 M citrate buffer and 4.3 mM Ca²⁺.

55. Use of a thermostable acid alpha-amylase in the secondary liquefaction step in a process for production of ethanol.

58. Use according to claim 55-56, wherein the ethanol is to be used as a fuel alcohol and/or a fuel additive.

56. Use of a thermostable maltogenic acid alpha-amylase in the secondary liquefaction step in a process for production of ethanol.