Methods and Compositions for Degradation of Lignocellulosic Material

• US 20080076159A1
• Filed: 07/10/2007
• Published: 03/27/2008
• Est. Priority Date: 07/10/2006
• Status: Active Grant

• Current Assignee: Battelle Memorial Institute, Dyadic International (Usa), Inc., Iowa Corn Promotion Board
• Original Assignee: Dyadic International (Usa), Inc.
• Inventors: Burlingame, Richard, Sinitsyn, Arkady, Baez-Vasquez, Marco, Magnuson, Jon, Bradford, Marion
• Granted Patent: US 8,304,212 B2
• Time in Patent Office: Days
• US Class Current: 435/72
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12N 9/2445 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/20 produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/01021 Beta-glucosidase (3.2.1.21)
• CPC Class Codes: C12Y 302/01037 Xylan 1,4-beta-xylosidase (3.2.1.37)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Methods to convert lignocellulosic biomass to fermentable sugars with enzymes that degrade the lignocellulosic material are provided, as well as novel combinations of enzymes, including those that provide a synergistic release of sugars from plant biomass.

Claims

1. A multi-enzyme product comprising a product of a Chrysosporium including Chrysosporium lucknowense and exhibiting:

a glucoamylase activity of at least about 0.05 U per milligram of dry matter, a β

-glucosidase activity of at least about 22 nmol/min/mg of dry matter, and an α

-arabinofuranosidase activity of at least about 9 nmol/min/mg of dry matter.

2. The multi-enzyme product of claim 1, wherein the Chrysosporium lucknowense is selected from the group consisting of Strain C1 (Accession No. VKM F-3500-D);

Strain UV13-6 (Accession No. VKM F-3632 D);

Strain NG7C-19 (Accession No. VKM F-3633 D); and

/or Strain UV18-25 (Accession No. VKM F-3631 D) and mutants or derivatives thereof.

7. The multi-enzyme product of any one of claims 1, 2, 3, 4, 5, or 6, wherein the product is a crude fermentation product.

8. The multi-enzyme product of any one of claims 1, 2, 3, 4, 5, or 6, wherein the product has been subjected to a purification step.

9. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with an effective amount of a multi-enzyme product derived from one microorganism, whereby at least one fermentable sugar is produced, and wherein at least one of enzyme in the multi-enzyme product is selected from the group consisting of:

at least one enzyme in the multi-enzyme product of claim 1, at least one enzyme in the multi-enzyme product of claim 2, at least one enzyme in the multi-enzyme product of claim 3, at least one enzyme in the multi-enzyme product of claim 4, at least one enzyme in the multi-enzyme product of claim 5, and at least one enzyme in the multi-enzyme product of claim 6.

11. The method of claim 9, wherein at least one of enzyme in the multi-enzyme product is derived from Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens or solitum, Penicillium funiculosum (Talaromyces flavus), or mutants of any of the foregoing.

20. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 1.

27. The method of claim 9 or 10, wherein the multi-enzyme product comprises at least two of the products of claim 1, 3, 4, 5, or 6.

41. A method for degrading a distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles to sugars, the method comprising contacting the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles with a multi-enzyme product obtained from the fermentation of at least one microorganism, whereby at least 10% of the fermentable sugars are liberated, wherein at least one of enzyme in the multi-enzyme product is selected from the group consisting of:

at least one enzyme in the multi-enzyme product of claim 1, at least one enzyme in the multi-enzyme product of claim 2, at least one enzyme in the multi-enzyme product of claim 3, at least one enzyme in the multi-enzyme product of claim 4, at least one enzyme in the multi-enzyme product of claim 5, and at least one enzyme in the multi-enzyme product of claim 6.

42. The method of claim 41, whereby at least 15% of the sugars are liberated.

43. The method of claim 41, whereby at least 20% of the sugars are liberated.

44. The method of claim 41, whereby at least 23% of the sugars are liberated.

45. The method of claim 41, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles are derived from corn.

46. The method of claim 41, wherein the multi-enzyme product comprises at least one hemicellulase.

47. The method of claim 46, wherein the hemicellulase is selected from the group consisting of a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, an endo-galactanase, a mannanase, an endo-arabinase, an exo-arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xylogluconase, and mixtures thereof.

48. The method of claim 47, wherein the xylanase is selected from the group consisting of:

an endoxylanase, an exoxylanase, and a β

-xylosidase.

49. The method of claim 41, wherein the multi-enzyme product comprises at least one cellulase.

50. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 1.

51. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 2.

54. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 5.

55. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 6.

56. The method of claim 41, wherein the multi-enzyme product comprises at least two of the products of claim 2, 3, 4, 5, or 6.

57. The method of claim 41, wherein the multi-enzyme product comprises at least two of the products of claim 1, 3, 4, 5, or 6.

58. The method of claim 41, further comprising treating the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles with an effective amount of one or more accessory enzymes.

59. The method of claim 58, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

60. The method of claim 58, wherein the accessory enzyme is a glucoamylase.

61. The method of claim 58, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

62. The method claim 58, wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.

63. The method of claim 41, further comprising a pretreatment process.

64. The method of claim 63, wherein the pretreatment process is selected from the group consisting of physical treatment, metal ion, ultraviolet light, ozone, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment.

65. The method of claim 63, wherein the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.

66. The method of claim 65, wherein the heat treatment comprises heating the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles to at least about 121°

C. for at least about 15 minutes.

67. The method of claim 41, further comprising detoxifying the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

68. The method of claim 41, further comprising recovering the fermentable sugar.

69. The method of claim 41, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

70. The method of claim 41, further comprising recovering the residual distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles after degrading the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles to fermentable sugars.

71. A method for producing an organic substance, comprising:

saccharifying a lignocellulosic material with an effective amount of a multi-enzyme product obtained from at least one microorganism, wherein at least one of enzyme in the multi-enzyme product is selected from the group consisting of at least one enzyme in the multi-enzyme product of claim 1, at least one enzyme in the claim 2, at least one enzyme in the multi-enzyme product of claim 3, at least one enzyme in the multi-enzyme product of claim 4, at least one enzyme in the multi-enzyme product of claim 5, and at least one enzyme in the multi-enzyme product of claim 6;

fermenting the saccharified lignocellulosic material obtained with one or more fermenting microorganisms; and

recovering the organic substance from the fermentation.

72. The method of claim 71, wherein the lignocellulosic material is selected from the group consisting of consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.

73. The method of claim 71, wherein the lignocellulosic material is distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

74. The method of claim 73, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles are derived from corn.

75. The method of claim 71, wherein the multi-enzyme product comprises at least one hemicellulase.

76. The method of claim 75, wherein the hemicellulase is selected from the group consisting of a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, an endo-galactanase, a mannanase, an endo-arabinase, an exo-arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xylogluconase, and mixtures thereof.

77. The method of claim 76, wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β

-xylosidase.

78. The method of claim 71, wherein the multi-enzyme product comprises at least one cellulase.

79. The method of claim 71, further comprising treating the lignocellulosic material with an effective amount of one or more accessory enzymes.

80. The method of claim 79, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

81. The method of claim 80, wherein the accessory enzyme is a glucoamylase.

82. The method of claim 79, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

83. The method claim 81, wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.

84. The method of claim 71, further comprising a pretreatment process.

85. The method of claim 84, wherein the pretreatment process is selected from the group consisting of physical treatment, metal ion, ultraviolet light, ozone, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment.

86. The method of claim 84, wherein the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.

87. The method of claim 86, wherein the heat treatment comprises heating the lignocellulosic material to at least about 121°

C. for at least about 15 minutes.

88. The method of claim 71, further comprising detoxifying the lignocellulosic material.

89. The method of claim 71, wherein the steps are performed simultaneously in a simultaneous saccharification and fermentation.

90. The method of claim 71, wherein the organic substance is an alcohol, organic acid, ketone, amino acid, or gas.

91. The method of claim 90, wherein the organic substance is an alcohol.

92. The method of claim 91, wherein the alcohol is ethanol.

107. A genetically modified organism comprising components suitable for degrading a lignocellulosic material to fermentable sugars, wherein the organism has been genetically modified to overexpress at least one enzyme selected from the group consisting of at least one enzyme in the multi-enzyme product of claim 1, at least one enzyme in the multi-enzyme product of claim 2, at least one enzyme in the multi-enzyme product of claim 3, at least one enzyme in the multi-enzyme product of claim 4, at least one enzyme in the multi-enzyme product of claim 5, and at least one enzyme in the multi-enzyme product of claim 6.

108. The genetically modified organism of claim 107, wherein the genetically modified organism is a microorganism.

109. The genetically modified organism of claim 108, wherein the microorganism is a filamentous fungus.

110. The genetically modified organism of claim 109, wherein the filamentous fungus is selected from the group consisting of Trichoderma, Chrysosporium, Aspergillus, and Penicillium (Talaromyces).

111. The genetically modified organism of claim 109, wherein the filamentous fungus is selected from the group consisting of Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens or solitum, and Penicillium funiculosum (Talaromyces flavus).

112. The genetically modified organism of claim 107, wherein the organism has been genetically modified to overexpress at least one accessory enzyme.

113. The genetically modified organism of claim 107, wherein the genetically modified organism is a plant.

114. A recombinant enzyme isolated from the genetically modified microorganism of claim 108.

115. The recombinant enzyme of claim 114, wherein the enzyme is part of a crude fermentation product.

116. The recombinant enzyme of claim 114, wherein the enzyme has been subjected to a purification step.

117. A recombinant enzyme derived from an enzyme of a multi-enzyme product of any one of claims 1, 2, 3, 4, 5, or 6.

118. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with an effective amount of a multi-enzyme product derived from the genetically modified microorganism of claim 108, whereby at least one fermentable sugar is produced.

123. A genetically modified organism comprising components suitable for degrading a lignocellulosic material to fermentable sugars, wherein the microorganism is selected from the group consisting of Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens, and Penicillium funiculosum, and wherein the organism has been genetically modified to overexpress:

a) at least one enzyme selected from the group consisting of;

at least one enzyme in the multi-enzyme product of claim 1, at least one enzyme in the multi-enzyme product of claim 2, at least one enzyme in the multi-enzyme product of claim 3, at least one enzyme in the multi-enzyme product of claim 4, at least one enzyme in the multi-enzyme product of claim 5, and at least one enzyme in the multi-enzyme product of claim 6;

and

b) at least one additional enzyme selected from the group consisting of a cellulase, a hemicellulase, a glucoamylase, a pectinase, and a ligninase.

3. A multi-enzyme product comprising a product of a Trichoderma including Trichoderma reesei and exhibiting:

a glucoamylase activity of at least about 0.001 U per milligram of dry matter, a β

-xylosidase activity of at least about 280 nmol/min/mg of dry matter, a β

-glucosidase activity of at least about 454 nmol/min/mg of dry matter, and an α

-arabinofuranosidase activity of at least about 100 nmol/min/mg of dry matter.

52. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 3.

4. A multi-enzyme product comprising a product of a Aspergillus including Aspergillus japonicus and exhibiting:

a glucoamylase activity of at least about 0.65 U per milligram of dry matter, a β

-xylosidase activity of at least about 553 nmol/min/mg of dry matter, a β

-glucosidase activity of at least about 167 nmol/min/mg of dry matter, and an α

-arabinofuranosidase activity of at least about 62 nmol/min/mg of dry matter.

53. The method of claim 41, wherein the multi-enzyme product comprises the product of claim 4.

5. A multi-enzyme product comprising a product of a Penicillium including Penicillium funiculosum (Talaromyces including Talaromyces flavus) and exhibiting:

a glucoamylase activity of at least about 0.18 U per milligram of dry matter, a β

-xylosidase activity of at least about 8 nmol/min/mg of dry matter, a β

-glucosidase activity of at least about 691 nmol/min/mg of dry matter, and an α

-arabinofuranosidase activity of at least about 29 nmol/min/mg of dry matter.

6. A multi-enzyme product comprising a product of a Penicillium including Penicillium canescens (P. solitum) and exhibiting:

a glucoamylase activity of at least about 0.08 U per milligram of dry matter, a β

-xylosidase activity of at least about 15 nmol/min/mg of dry matter, a β

-glucosidase activity of at least about 40 nmol/min/mg of dry matter, and an α

-arabinofuranosidase activity of at least about 956 nmol/min/mg of dry matter.

10. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with an effective amount of a multi-enzyme product derived from one microorganism, whereby at least one fermentable sugar is produced, and wherein at least one of enzyme in the multi-enzyme product is derived from Trichoderma, Chrysosporium, Aspergillus, Penicillium (Talaromyces), or mutants of any of the foregoing.

13. The method of claim 9, 10 or 12, wherein the lignocellulosic material is selected from the group consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.

14. The method of claim 9, 10 or 12, wherein the lignocellulosic material is distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

15. The method of claim 14, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles are derived from corn.

16. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises at least one hemicellulase.

17. The method of claim 16, wherein the hemicellulase is selected from the group consisting of a xylanase, an arabinofuranosidase, an acetyl xylan esterase, a glucuronidase, an endo-galactanase, a mannanase, an endo-arabinase, an exo-arabinase, an exo-galactanase, a ferulic acid esterase, a galactomannanase, a xylogluconase, and mixtures thereof.

18. The method of claim 17, wherein the xylanase is selected from the group consisting of endoxylanases, exoxylanase, and β

-xylosidase.

19. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises at least one cellulase.

21. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 2.

22. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 3.

23. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 4.

24. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 5.

25. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises the product of claim 6.

26. The method of claim 9, 10 or 12, wherein the multi-enzyme product comprises at least two of the products of claim 2, 3, 4, 5, or 6.

28. The method of claim 9, 10 or 12, further comprising treating the lignocellulosic material with an effective amount of one or more accessory enzymes.

29. The method of claim 28, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

30. The method of claim 28, wherein the accessory enzyme is a glucoamylase.

31. The method of claim 28, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

32. The method claim 28, wherein the accessory enzyme is produced by culturing at least one organism on a substrate to produce the enzyme.

33. The method of claim 9, 10 or 12, further comprising a pretreatment process.

34. The method of claim 33, wherein the pretreatment process is selected from the group consisting of physical treatment, metal ion, ultraviolet light, ozone, organosolv treatment, steam explosion treatment, lime impregnation with steam explosion treatment, hydrogen peroxide treatment, hydrogen peroxide/ozone (peroxone) treatment, acid treatment, dilute acid treatment, and base treatment.

35. The method of claim 33, wherein the pretreatment process is selected from the group consisting of organosolv, steam explosion, heat treatment and AFEX.

36. The method of claim 35, wherein the heat treatment comprises heating the lignocellulosic material to at least about 121°

C. for at least about 15 minutes.

37. The method of claim 9, 10 or 12, further comprising detoxifying the lignocellulosic material.

38. The method of claim 9, 10 or 12, further comprising recovering the fermentable sugar.

39. The method of claim 9, 10 or 12, wherein the sugar is selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, rhamnose, sucrose and fructose.

40. The method of claim 9, 10 or 12, further comprising recovering the residual lignocellulosic material after degrading the lignocellulosic material to fermentable sugars.

119. A feed additive comprising the recovered lignocellulosic material of claim 40 or claim 70.

120. The feed additive of claim 119, wherein the protein content of the recovered lignocellulosic material is higher than that of the starting lignocellulosic material.

121. A method of improving the performance of an animal, comprising administering to the animal the feed additive of claim 119.

122. A method for improving the nutritional quality of an animal feed, comprising adding the feed additive of claim 119 to an animal feed.

12. A method for degrading a lignocellulosic material to fermentable sugars, comprising contacting the lignocellulosic material with an effective amount of a multi-enzyme product derived from one microorganism, whereby at least one fermentable sugar is produced, and wherein at least one of enzyme in the multi-enzyme product is derived from Penicillium funiculosum or a mutant derived therefrom.

93. A method for producing a multi-enzyme product capable of producing fermentable sugars, comprising cultivating a microorganism selected from the group consisting of Trichoderma, Chrysosporium, Aspergillus, Penicillium (Talaromyces) and mutants thereof in a nutrient medium, and recovering the multi-enzyme product from the fermentation broth.

94. The method of claim 93, wherein the microorganism is selected from the group consisting of Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens or solitum, Penicillium funiculosum (Talaromyces flavus) and mutants thereof.

95. A method for producing fermentable sugars, comprising cultivating a microorganism selected from the group consisting of Trichoderma, Chrysosporium, Aspergillus, Penicillium (Talaromyces) and mutants thereof in a nutrient medium comprising a lignocellulosic material, whereby fermentable sugars are produced.

96. The method of claim 95, wherein the microorganism is selected from the group consisting of Trichoderma reesei, Chrysosporium lucknowense, Aspergillus japonicus, Penicillium canescens or solitum, Penicillium funiculosum (Talaromyces flavus) and mutants thereof.

97. The method of claim 95, wherein the lignocellulosic material is selected from the group consisting of herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue.

98. The method of claim 95, wherein the lignocellulosic material is distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles.

99. The method of claim 98, wherein the distiller'"'"'s dried grains or distiller'"'"'s dried grains with solubles are derived from corn.

100. The method of claim 95, wherein the nutrient medium further comprises one or more accessory enzymes.

101. The method of claim 100, wherein the accessory enzyme is selected from the group consisting of a glucoamylase, a pectinase, and a ligninase.

102. The method of claim 100, wherein the accessory enzyme is a glucoamylase.

103. The method of claim 100, wherein the accessory enzyme is added as a crude or a semi-purified enzyme mixture.

104. The method of claim 95, wherein the nutrient medium further comprises an agent for detoxifying the lignocellulosic material.

105. The method of claim 95, further comprising recovering the fermentable sugar.

106. The method of claim 95, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.