Method for reducing slurry viscosity after fermentation product production process

Abstract

The present invention relates to a method for reducing slurry viscosity at the back end of a fermentation product production (e.g., ethanol production from corn) process, comprising adding an oxidoreductase after a fermentation step prior to separation of the whole stillage, reducing the number of particles in the stillage water by increasing the size of 5 particles in the whole stillage, therefore, the viscosity of the slurry generated by evaporating the vinasse water is reduced.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a Sequence Listing in computer-readable form, which is hereby incorporated by reference herein. Field of the Invention

[0003] The present invention relates to a method for reducing the viscosity of a slurry at the back end of a process for producing a fermentation product (e.g., ethanol from corn), which comprises adding a redox enzyme after a fermentation step and before separating whole stillage, reducing the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water. Background Art

[0004] In a process for producing ethanol from corn, following an SSF or RSH process, the desired fermentation product (e.g., ethanol) is recovered from the fermented mash (often referred to as "beer mash") by distillation, separating it from other liquids and / or solids. The remaining fraction is called "whole stillage". Whole stillage typically contains about 10% to 20% solids. The whole stillage is separated into a solid fraction and a liquid fraction, for example, by centrifugation. The separated solid fraction is called "wet cake" (or "wet grains"), and the separated liquid fraction is called "stillage water". The wet cake and the stillage water contain about 35% and 7% solids, respectively. The wet cake (with optional additional dehydration) is used as a component in animal feed, or is dried to provide "dry distillers grains" (DDG) for use as a component in animal feed. Typically, the stillage water is evaporated to provide an evaporator condensate and a slurry, or alternatively can be recycled as a "countercurrent" to the slurry tank. The evaporator condensate can be sent to a methane converter before being discharged, and / or can be recycled as "cooking water" to the slurry tank. The slurry can be co-mixed into the DDG or added to the wet cake before or during a drying process (which can sequentially include one or more dryers) to produce DDGS (dry distillers grains with solubles). The slurry typically contains about 25% to 35% solids. Oil can also be extracted from the stillage water and / or the slurry as a by-product (for biodiesel production), as a feed or food additive or product, or other bioreproducible products.

[0005] Although decanting the whole stillage removes large particles into the wet cake, there are still smaller particles and soluble nutrients in the stillage water. The small particles in the stillage water contain valuable substances such as proteins, lipids, and carbohydrates. The small particles, including insoluble solids and some soluble nutrients, are typically concentrated into a slurry through a multi-step (e.g., seven evaporator cans) high-temperature evaporation process. As water evaporates to concentrate the stillage water into a slurry, the concentration of small particles in the stillage water becomes higher and higher, increasing the viscosity of the slurry.

[0006] Although improvements in the efficiency of corn kernel milling, chemical hydrolysis, and enzymatic hydrolysis have helped to maximize the conversion of starch to ethanol, they have also resulted in a greater concentration of small particles in the stillage after decantation, further increasing the slurry viscosity to a level that becomes a potential problem. Therefore, there is a need to improve methods for reducing the slurry viscosity at the back end of commercial ethanol plants. Summary of the Invention

[0007] The present invention provides a solution to the above problems by providing: a method for reducing the slurry viscosity at the back end of a method for producing a fermentation product (e.g., ethanol from corn), which comprises adding a redox enzyme after the fermentation step and before separating the whole stillage, reducing the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0008] Accordingly, in one aspect, a method for reducing the slurry viscosity in a method for producing a fermentation product from a starch-containing material comprises:

[0009] (a) liquefying the starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0010] (b) saccharifying the dextrin with glucoamylase to produce fermentable sugars;

[0011] (c) fermenting the sugars with a fermenting organism to produce a beer containing the fermentation product;

[0012] (d) distilling the beer to recover the fermentation product and produce whole stillage;

[0013] (e) processing the whole stillage to produce stillage water; and

[0014] (f) evaporating the stillage water to produce a slurry;

[0015] wherein a redox enzyme is added after the fermentation step, reducing the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0016] In one aspect, a method for reducing the slurry viscosity in a method for producing a fermentation product from a starch-containing material comprises:

[0017] (a) saccharifying the starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars;

[0018] (b) fermenting the sugars with a fermenting organism to produce a beer containing the fermentation product;

[0019] (c) distilling the beer to recover the fermentation product and produce whole stillage;

[0020] (d) Processing the spent grains to produce spent grain water; and

[0021] (e) Evaporating the spent grain water to produce a slurry;

[0022] Wherein a redox enzyme is added after the fermentation step, and the number of particles in the spent grain water is reduced by increasing the size of the particles in the spent grains, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0023] The redox enzyme can be added to the beer pool containing beer before the distillation step, during the distillation step, or to the spent grains before the processing step. The processing step may include a separation step by subjecting the spent grains to a filtration centrifuge, a sedimentation centrifuge, a pressure screen, or a paddle screen. In an embodiment, the centrifuge is a sedimentation centrifuge, particularly a horizontal sedimentation centrifuge.

[0024] The redox enzyme can be added to the spent grains and incubated for a period of time before performing the spent grain separation step.

[0025] The redox enzyme can be used at a temperature in the range from 30 °C to 100 °C, such as 40 °C to 90 °C, preferably 45 °C to 85 °C.

[0026] For particles in the range of 100 μm to 1000 μm in size, the cumulative passing is reduced by 10 vol% to 80 vol%.

[0027] The cumulative passing of particles with a diameter equal to 400 μm is reduced by 10 vol% to 80 vol%.

[0028] In an embodiment, the redox enzyme is laccase.

[0029] In an embodiment, the redox enzyme is peroxidase.

[0030] The saccharification and fermentation steps can be carried out simultaneously.

[0031] The starch-containing material may include sugar beet, maize, corn, wheat, rye, barley, oats, triticale, sorghum, sweet potato, rice, millet, pearl millet, and / or foxtail millet.

[0032] In an embodiment, the starch-containing material contains corn.

[0033] In an embodiment, the fermentation product contains ethanol, preferably fuel ethanol.

[0034] In an embodiment, the fermentation organism is yeast. Description of the Drawings

[0035] Figure 1It is shown that the particle size in the distillers' grains water is less than 400 μm, while the particle size of the whole distillers' grains ranges from 0 to 1820 μm.

[0036] Figure 2 It shows the effect of treatment with redox enzyme B at 65 °C in the presence and absence of hydrogen peroxide on the cumulative passage of the whole distillers' grains.

[0037] Figure 3 It shows the effect of treatment with redox enzyme A and redox enzyme B at 65 °C on the cumulative passage of the whole distillers' grains.

[0038] Figure 4 It shows the effect of treatment with redox enzyme A and redox enzyme B at 50 °C on the cumulative passage of the whole distillers' grains.

[0039] Figure 5 It shows the effect of treatment with redox enzyme A and redox enzyme B at 35 °C on the cumulative passage of the whole distillers' grains.

[0040] Definition

[0041] Auxiliary activity 1 (AA1) family: The AA1 family includes enzymes characterized as multi-copper oxidases that use diphenols and related substances as donors and oxygen as an acceptor. The AA1 family is divided into three subfamilies, including laccases, ferroxidases, and laccase-like multi-copper oxidases.

[0042] Auxiliary activity 2 (AA2) family: The AA2 family contains class II lignin-modifying peroxidases. AA2 enzymes are secreted heme-containing enzymes that use hydrogen peroxide or organic peroxides as electron acceptors to catalyze various oxidation reactions, where two electrons are derived from the substrate to reduce the enzyme while releasing two water molecules.

[0043] Cumulative passage: "Cumulative passage" means the volume percentage of particles with a particle size less than or equal to the particle size that passes through the whole distillers' grains and separates into the distillers' grains water.

[0044] Fermentation product: "Fermentation product" means a product produced by a process including the use of a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12, β-carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., neutral drinking alcohol; or industrial ethanol or products for the consumer alcohol industry (e.g., beer and liquor), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred types of beer include ale, stout, porter, lager, bitter, malt liquor, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In an embodiment, the fermentation product is ethanol.

[0045] Fermenting organism: "Fermenting organism" refers to any organism suitable for a fermentation process and capable of producing a desired fermentation product, including bacterial and fungal organisms, especially yeast.

[0046] GH3 β-xylosidase: "GH3 β-xylosidase" is an abbreviation for glycoside hydrolase family 3 β-xylosidase, which is xylan 1,4-β-xylosidase (EC 3.2.1.37), which catalyzes the hydrolysis of (1→4)-β-D-xylan to remove successive D-xylose residues from the non-reducing end.

[0047] Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. Starch heated in water begins to gelatinize between 50 °C and 75 °C; the exact temperature of gelatinization depends on the specific starch and can be readily determined by a person skilled in the art. Thus, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growth conditions. In the context of the present disclosure, the initial gelatinization temperature of a given starch-containing material is the temperature at which 5% of the starch granules lose birefringence using the method described by Gorinstein.S. and Lii.C., Starch / Starke [Starch], Vol. 44(12), pp. 461-466 (1992).

[0048] Laccase: "Laccase" refers to benzenediol:oxygen oxidoreductase (EC 1.10.3.2), which catalyzes the reaction 4 benzenediol + O2 = 4 benzoquinone + 2H2O. These laccases include low-specificity multi-copper proteins that act on o-quinols and p-quinols and generally act on aminophenols and phenylenediamines.

[0049] Laccase activity: Laccase (EC 1.10.3.2) activity can be determined based on its ability to catalyze the oxidation of syringaldazine (4,4′-[azobis(methylene)]bis(2,6-dimethoxyphenol)) to the corresponding quinone 4,4′-[azobis(methylene)]bis(2,6-dimethoxycyclohexa-2,5-dien-1-one), see Equation 1.

[0050]

[0051] The reaction was detected by the increase in absorbance at 530 nm.

[0052] One laccase unit is the amount of enzyme that catalyzes the conversion of 1 mmol of syringaldazine per minute under the given assay conditions. For measurements carried out at pH 5.5, the activity unit is labeled as LACU, and for measurements carried out at pH 7.5, the activity unit is labeled as LAMU.

[0053] Oxidoreductase: "Oxidoreductase" refers to an enzyme that catalyzes the transfer of electrons from an electron donor (reductant) to an electron acceptor (oxidant) in an oxidation-reduction reaction. "Oxidoreductase" encompasses any such enzyme that can increase the volume % of larger-sized particles in whole distiller's grains, including, for example, oxidoreductases (EC 1.10) acting on phenols and related substances as donors and oxidoreductases (EC 1.11) acting on peroxides as acceptors.

[0054] Peroxidase: "Peroxidase" refers to phenol donor:hydrogen peroxide oxidoreductase (EC 1.11.1.7), which catalyzes the reaction of 2 phenol donors + H2O2 = 2 donor phenoxy groups + 2H2O. These peroxidases include heme proteins having a histidine as a proximal ligand, where the iron in the resting enzyme is Fe(III).

[0055] Peroxidase activity: Peroxidase (EC 1.11.1.7) activity can be determined based on its ability to catalyze the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulfonate] to form a blue-green color in the presence of hydrogen peroxide. The reaction was detected by measuring the increase in absorbance at 418 nm, as depicted below in Equation 2 for the oxidation (catalyzing the conversion of 1 mmol of hydrogen peroxide per minute).

[0056]

[0057] One peroxidase unit (POXU) is the amount of enzyme that catalyzes the conversion of 1 mmol of hydrogen peroxide per minute under the given assay conditions.

[0058] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0059] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences, and the algorithm is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (e.g., version 5.0.0 or later). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the non-abbreviated (–nobrief) option) is used as the percentage of identity and is calculated as follows:

[0060] (Number of identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[0061] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two deoxyribonucleotide sequences, and the algorithm is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (e.g., version 5.0.0 or later). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The output of "longest identity" marked by Needle (obtained using the non-abbreviated (–nobrief) option) is used as the percentage of identity and is calculated as follows:

[0062] (Number of identical deoxyribonucleotides x 100) / (Alignment length - Total number of gaps in the alignment).

[0063] Thermostable: "Thermostable" means that when the enzyme is used in the liquefaction step of the method of the present invention, the enzyme is not denatured or inactivated. In other words, if the thermostable enzyme has a denaturation temperature (Td) compatible with the liquefaction temperature and retains its activity at this temperature, then the thermostable enzyme is suitable for liquefaction.

[0064] Thin Stillage: "Thin Stillage" refers to the separated liquid obtained from whole stillage, which is pumped to an evaporator to be concentrated into a slurry.

[0065] Whole Stillage: "Whole Stillage" includes the material remaining at the end of the distillation process after the recovery of a fermentation product such as ethanol. Detailed Description

[0066] The present invention relates to a method for reducing the viscosity of a slurry at the backend of a process for producing a fermentation product (e.g., ethanol from corn), which comprises adding a redox enzyme after the fermentation step and before separating the whole stillage, reducing the number of particles in the thin stillage by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the thin stillage.

[0067] Surprisingly, the work described herein shows that for particles in the size range of 100 μm to 1000 μm, the cumulative passage is reduced by 10 vol% to 80 vol%. The work herein further unexpectedly shows that the cumulative passage of particles with a diameter of 400 μm is reduced by 10 vol% to 80 vol%.

[0068] The cumulative passage of particles having a size equal to that which typically enters the thin stillage after decantation with a redox enzyme (e.g., about 400 μm) is significantly reduced, indicating that the redox enzyme can reduce the viscosity of the slurry, e.g., by increasing the volume % of larger-sized particles in the whole stillage while reducing the volume % of smaller-sized particles in the whole stillage, which otherwise would escape decantation and accumulate in the thin stillage that is evaporated to produce the slurry.

[0069] The present invention contemplates the use of a redox enzyme after fermentation in conventional starch-to-ethanol and raw starch hydrolysis (RSH) ethanol production processes.

[0070] Method for Producing a Fermentation Product from a Gelatinized Starch-Containing Material

[0071] One aspect of the present invention relates to a method for reducing the viscosity of a slurry at the backend of a process for producing a fermentation product (e.g., fuel ethanol) from a gelatinized starch-containing material, wherein a redox enzyme is added after the fermentation step, reducing the number of particles in the thin stillage by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the thin stillage.

[0072] In an embodiment, the method for reducing the viscosity of the slurry (e.g., at the backend of a process for producing a fermentation product from a starch-containing material) comprises the following steps:

[0073] (a) liquefying the starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0074] (b) Saccharifying the dextrin with glucoamylase to produce fermentable sugars; and

[0075] (c) Fermenting the sugar with a fermenting organism to produce the fermentation product;

[0076] (d) Distilling the fermentation product to recover the fermentation product and produce spent grains;

[0077] (e) Processing the spent grains to produce spent grain liquor; and

[0078] (f) Evaporating the spent grain liquor to produce a slurry;

[0079] Wherein a redox enzyme is added after the fermentation step to reduce the number of particles in the spent grain liquor by increasing the size of the particles in the spent grains, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0080] In an embodiment, the redox enzyme is added to a beer pool containing beer before the distillation step. In an embodiment, the redox enzyme is added during the distillation step. In an embodiment, the redox enzyme is added to the spent grains before the processing step.

[0081] In an embodiment, the processing step includes a separation step by subjecting the spent grains to a filter centrifuge, a sedimentation centrifuge, a pressure screen or a paddle screen. In an embodiment, the centrifuge is a sedimentation centrifuge, particularly a horizontal sedimentation centrifuge.

[0082] In an embodiment, the redox enzyme is added to the spent grains and incubated for a period of time before the spent grain separation step is carried out.

[0083] In an embodiment, the redox enzyme is laccase. In an embodiment, the redox enzyme is peroxidase.

[0084] Preferably, the redox enzyme used in the method has an optimal temperature compatible with the temperature range of the method step in which they are used. In an embodiment, the redox enzyme is used at a temperature in the range of 30 °C to 100 °C, such as 40 °C to 90 °C, preferably 45 °C to 85 °C.

[0085] In an embodiment, a thermostable glucoamylase is added during the liquefaction step (a). In an embodiment, a thermostable endoglucanase is added during the liquefaction step (a). In an embodiment, a thermostable lipase is added during the liquefaction step (a). In an embodiment, a thermostable phytase is added during the liquefaction step (a). In an embodiment, a thermostable protease is added during the liquefaction step (a). In an embodiment, a thermostable pullulanase is added during the liquefaction step (a). In an embodiment, a thermostable xylanase is added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase and a thermostable protease are added during the liquefaction step (a). In an embodiment, a thermostable α-amylase and a thermostable xylanase are added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase, a thermostable protease and a thermostable xylanase are added during the liquefaction step (a).

[0086] In an embodiment, an α-amylase is added during the step (b) and / or the step (c). In an embodiment, an α-glucosidase is added during the step (b) and / or the step (c). In an embodiment, a β-amylase is added during the step (b) and / or the step (c). In an embodiment, a β-glucanase is added during the step (b) and / or the step (c). In an embodiment, a β-glucosidase is added during the step (b) and / or the step (c). In an embodiment, a cellobiohydrolase is added during the step (b) and / or the step (c). In an embodiment, an endoglucanase is added during the step (b) and / or the step (c). In an embodiment, a lipase is added during the step (b) and / or the step (c). In an embodiment, a lytic polysaccharide monooxygenase (LPMO) is added during the step (b) and / or the step (c). In an embodiment, a maltogenic α-amylase is added during the step (b) and / or the step (c). In an embodiment, a pectinase is added during the step (b) and / or the step (c). In an embodiment, a peroxidase is added during the step (b) and / or the step (c). In an embodiment, a phytase is added during the step (b) and / or the step (c). In an embodiment, a protease is added during the step (b) and / or the step (c). In an embodiment, a trehalase is added during the step (b) and / or the step (c). In an embodiment, a xylanase is added during the step (b) and / or the step (c).

[0087] In an embodiment, the fermenting organism is yeast. In an embodiment, the yeast in-situ expresses an α-amylase during the step (b) and / or the step (c). In an embodiment, the yeast in-situ expresses a glucoamylase during the step (b) and / or the step (c).

[0088] Process parameters

[0089] Parameters for methods of producing fermentation products (such as ethanol from starch-containing materials such as corn) are well known in the art. See, for example, WO 2006 / 086792, WO 2013 / 082486, WO 2012 / 088303, WO 2013 / 055676, WO 2014 / 209789, WO 2014 / 209800, WO 2015 / 035914, WO 2017 / 112540, WO 2020 / 014407, WO 2021 / 126966, each of which is incorporated herein by reference.

[0090] Starch-containing material

[0091] Any suitable starch-containing starting material can be used. The material is selected based on the desired fermentation product. Examples of starch-containing materials include, but are not limited to, barley, beets, beans, cassava, cereals, corn, sorghum, oats, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. Starch-containing materials can also be waxy or non-waxy types of corn and barley. Commonly used commercial starch-containing materials include corn, sorghum, and / or wheat.

[0092] Reduction of the particle size of the starch-containing material

[0093] Before the liquefaction step (a), the particle size of the starch-containing material can be reduced, for example, by dry milling.

[0094] Slurry

[0095] Before the liquefaction step (a), a slurry comprising a starch-containing material (e.g., preferably ground) and water can be formed. α-Amylase and preferably protease can be added to the slurry. The slurry can be heated to between the initial gelatinization temperature of the starch-containing material and above this initial gelatinization temperature to initiate gelatinization of the starch.

[0096] Jet cooking

[0097] Before adding α-amylase during the liquefaction step (a), the slurry can optionally be jet cooked to further gelatinize the starch in the slurry. Jet cooking can be carried out at a temperature in the range from 100 °C to 120 °C for at least 15 minutes.

[0098] Liquefaction temperature

[0099] The temperature used during the liquefaction step (a) can be in the range from 70 °C to 110 °C, such as from 75 °C to 105 °C, from 80 °C to 100 °C, from 85 °C to 95 °C, or from 88 °C to 92 °C. Preferably, the temperature is at least 70 °C, at least 80 °C, at least 85 °C, at least 88 °C, or at least 90 °C.

[0100] Liquefaction pH

[0101] The pH used during the liquefaction step (a) can be in the range from 4 to 6, from 4.5 to 5.5, or from 4.8 to 5.2. Preferably, the pH is at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0, or at least 5.1.

[0102] Liquefaction time

[0103] The time for performing the liquefaction step (a) can be in the range from 30 minutes to 5 hours, from 1 hour to 3 hours, or from 90 minutes to 150 minutes. Preferably, the time is at least 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 2 hours.

[0104] Liquefying enzyme

[0105] The present invention contemplates the use of thermostable enzymes during the liquefaction step (a). The use of various thermostable enzymes during the liquefaction step (a) is well known in the art and includes, for example, thermostable α - amylase, thermostable glucoamylase, thermostable endoglucanase, thermostable lipase, thermostable phytase, thermostable protease, thermostable pullulanase, and / or thermostable xylanase. The present invention contemplates the use of any thermostable enzyme in the liquefaction step (a). Guidance for determining the denaturation temperature of candidate thermostable enzymes for use in the liquefaction step (a) is provided in the following Materials and Methods section. The following listed published patent applications describe activity assays for determining whether candidate thermostable enzymes contemplated for use in the liquefaction step (a) will be inactivated at the temperatures contemplated for use in the liquefaction step (a).

[0106] Examples of suitable thermostable α - amylases and guidance for their use in the liquefaction step (a) include, but are not limited to, the α - amylases described in the following: WO 94 / 18314, WO 94 / 02597, WO 96 / 23873, WO 96 / 23874, WO 96 / 39528, WO 97 / 41213, WO 97 / 43424, WO 99 / 19467, WO 00 / 60059, WO 2002 / 010355, WO2002 / 092797, WO 2009 / 149130, WO 2009 / 61378, WO 2009 / 061379, WO 2009 / 061380, WO2009 / 061381, WO 2009 / 098229, WO 2009 / 100102, WO 2010 / 115021, WO 2010 / 115028, WO2010 / 036515, WO 2011 / 082425, WO 2013 / 096305, WO 2013 / 184577, WO 2014 / 007921, WO2014 / 164777, WO 2014 / 164800, WO 2014 / 164834, WO 2019 / 113413, WO 2019 / 113415, WO2019 / 197318 (each of which is incorporated herein by reference).

[0107] Examples of suitable thermostable glucoamylases include, but are not limited to, the glucoamylases described in WO 2011 / 127802, WO 2013 / 036526, WO 2013 / 053801, WO 2018 / 164737, WO 2020 / 010101, and WO 2022 / 090564 (each of which is incorporated herein by reference).

[0108] Examples of suitable thermostable endoglucanases include, but are not limited to, the endoglucanases described in WO 2015 / 035914 (which is incorporated herein by reference).

[0109] Examples of suitable thermostable lipases include, but are not limited to, the lipases described in WO 2017 / 112542 and WO 2020 / 014407 (both of which are incorporated herein by reference).

[0110] Examples of suitable thermostable phytases include, but are not limited to, phytases described in the following: WO 1996 / 28567, WO 1997 / 33976, WO 1997 / 38096, WO 1997 / 48812, WO 1998 / 05785, WO 1998 / 06856, WO 1998 / 13480, WO 1998 / 20139, WO 1998 / 028408, WO 1999 / 48330, WO 1999 / 49022, WO2003 / 066847, WO 2004 / 085638, WO 2006 / 037327, WO 2006 / 037328, WO 2006 / 038062, WO2006 / 063588, WO 2007 / 112739, WO 2008 / 092901, WO 2008 / 116878, WO 2009 / 129489 and WO2010 / 034835 (each incorporated herein by reference). Commercially available phytase-containing products include BIO-FEED PHYTASE TM , PHYTASE NOVO TM CT or L, LIQMAX or RONOZYME TM NP, HIPHOS, P5000(CT), NATUPHOS TM NG 5000.

[0111] Examples of suitable thermostable proteases include, but are not limited to, proteases described in the following: WO 1992 / 02614, WO 98 / 56926, WO 2001 / 151620, WO 2003 / 048353, WO 2006 / 086792, WO 2010 / 008841, WO 2011 / 076123, WO 2011 / 087836, WO 2012 / 088303, WO 2013 / 082486, WO 2014 / 209789, WO 2014 / 209800, WO 2018 / 098124, WO 2018 / 118815A1 and WO 2018 / 169780 A1 (each incorporated herein by reference).

[0112] Examples of suitable commercially available protease-containing products include AVANTEC FORTIVA FORTIVA

[0113] Examples of suitable thermostable pullulanases include, but are not limited to, the pullulanases described in WO 2015 / 007639, WO 2015 / 110473, WO 2016 / 087327, WO 2017 / 014974, and WO 2020 / 187883 (each incorporated herein by reference in its entirety). Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYME TM D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

[0114] Examples of suitable thermostable xylanases include, but are not limited to, the xylanases described in WO 2017 / 112540 and WO 2021 / 126966 (each incorporated herein by reference). Suitable commercially available products containing thermostable xylanases include FORTIVA

[0115] One or more of the enzymes described above will be used in an effective amount in the methods of the present invention. Guidance for determining the effective amount of the enzymes to be used in the liquefaction step (a) and guidance for performing the activity assays for determining the activity of those enzymes can be found in the published patent applications cited for each different thermostable liquefying enzyme.

[0116] Saccharification temperature

[0117] Saccharification can be carried out at a temperature ranging from 20°C to 75°C, 30°C to 70°C, or 40°C to 65°C. Preferably, the saccharification temperature is at least about 50°C, at least about 55°C, or at least about 60°C.

[0118] Saccharification pH

[0119] Saccharification can occur at a pH ranging from 4 to 5. Preferably, the pH is about 4.5.

[0120] Saccharification time

[0121] Saccharification can last from about 24 hours to about 72 hours.

[0122] Fermentation time

[0123] Fermentation can last from 6 to 120 hours, from 24 hours to 96 hours, or from 35 hours to 60 hours.

[0124] Simultaneous saccharification and fermentation

[0125] The SSF can be carried out at a temperature from 25 °C to 40 °C, from 28 °C to 35 °C, or from 30 °C to [temperature value not provided] °C, at a pH from 3.5 to 5 or from 3.8 to 4.3 for 24 to 96 hours, 36 to 72 hours, or 48 to 60 hours. Preferably, the SSF is carried out at about 32 °C, at a pH from 3.8 to 4.5 for 48 to 60 hours.

[0126] Saccharification and / or fermentation enzyme

[0127] The present invention contemplates the use of enzymes during the saccharification step (b) and / or the fermentation step (c). The use of various enzymes during the saccharification step (b) and / or the fermentation step (c) is well known in the art and includes, for example, α-amylase, α-glucosidase, β-amylase, β-glucanase, β-glucosidase, cellobiohydrolase, endoglucanase, glucoamylase, lipase, lytic polysaccharide monooxygenase (LPMO), maltogenic α-amylase, pectinase, peroxidase, phytase, protease, trehalase, and xylanase.

[0128] The enzymes used in the saccharification step (b) and / or the fermentation step (c) can be added exogenously as a single component or formulated as a composition containing the enzyme. The enzymes used in the saccharification step (b) and / or the fermentation step (c) can also be added via in situ expression from a fermenting organism (e.g., yeast).

[0129] Examples of suitable α-amylases include, but are not limited to, the α-amylases described in the following: WO 2004 / 055178, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2013 / 044867, WO 2021 / 163011, and WO 2021 / 163030 (each of which is incorporated herein by reference).

[0130] Examples of suitable glucoamylases include, but are not limited to, the glucoamylases described in the following: WO 1984 / 02921, WO 1992 / 00381, WO 1999 / 28448, WO 2000 / 04136, WO 2001 / 04273, WO 2006 / 069289, WO 2011 / 066560, WO 2011 / 066576, WO 2011 / 068803, WO 2011 / 127802, WO 2012 / 064351, WO2013 / 036526, WO 2013 / 053801, WO 2014 / 039773, WO 2014 / 177541, WO 2014 / 177546, WO2016 / 062875, WO 2017 / 066255, and WO 2018 / 191215 (each of which is incorporated by reference).

[0131] Examples of suitable compositions comprising α - amylase and glucoamylase include, but are not limited to, the compositions described in WO 2006 / 069290, WO 2009 / 052101, WO 2011 / 068803, and WO 2013 / 006756 (each incorporated herein by reference). Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN TM SUPER, SAN TM EXTRA L, SPIRIZYME TM PLUS, SPIRIZYME TM FUEL, SPIRIZYME TM B4U, SPIRIZYME TM ULTRA, SPIRIZYME TM EXCEL, SPIRIZYME ACHIEVE, and AMG TM E (from Novozymes); OPTIDEX TM 300, GC480, GC417 (from DuPont - Genencor); AMIGASE TM and AMIGASE TM PLUS (from DSM); G - ZYME TM G900, G - ZYME TM and G990 ZR (from DuPont - Genencor).

[0132] Examples of suitable β - glucanases include, but are not limited to, the β - glucanases described in WO 2021 / 055395 (incorporated herein by reference).

[0133] Examples of suitable β - glucosidases include, but are not limited to, the β - glucosidases described in WO 2005 / 047499, WO 2013 / 148993, WO2014 / 085439, and WO 2012 / 044915 (each incorporated herein by reference).

[0134] Examples of suitable cellobiohydrolases include, but are not limited to, the cellobiohydrolases described in WO 2013 / 148993, WO 2014 / 085439, WO 2014 / 138672, and WO 2016 / 040265 (each incorporated herein by reference).

[0135] Examples of suitable endoglucanases include, but are not limited to, the endoglucanases described in WO 2013 / 148993 and WO 2014 / 085439, both of which are incorporated herein by reference.

[0136] Examples of suitable maltogenic α - amylases are described in U.S. Patent Nos. 4,598,048, 4,604,355, and 6,162,628, which are hereby incorporated by reference.

[0137] Examples of suitable lipases include, but are not limited to, the lipases described in WO 2017 / 112533, WO 2017 / 112539, and WO 2020 / 076697, each of which is incorporated herein by reference.

[0138] Examples of suitable LPMOs include, but are not limited to, the LPMOs described in WO 2013 / 148993, WO 2014 / 085439, and WO 2019 / 083831, each of which is incorporated herein by reference.

[0139] Examples of suitable phytases include, but are not limited to, the phytases described in WO 2001 / 62947, which is incorporated herein by reference.

[0140] Examples of suitable pectinases include, but are not limited to, the pectinases described in WO 2022 / 173694, which is incorporated herein by reference.

[0141] Examples of suitable peroxidases include, but are not limited to, the peroxidases described in WO 2019 / 231944, which is incorporated herein by reference.

[0142] Examples of suitable proteases include, but are not limited to, the proteases described in WO 2017 / 050291, WO 2017 / 148389, WO 2018 / 015303, and WO 2018 / 015304, each of which is incorporated herein by reference.

[0143] Examples of suitable trehalases include, but are not limited to, the trehalases described in WO 2016 / 205127, WO 2019 / 005755, WO2019 / 030165, and WO 2020 / 023411, each of which is incorporated herein by reference.

[0144] Examples of suitable xylanases include, but are not limited to, the xylanases described in WO 2016 / 005521, WO 2019 / 055455, WO2020 / 160126, and WO 2021 / 026201, each of which is incorporated herein by reference.

[0145] Commercially available xylanase-containing products are sold under the trademark .

[0146] Method for producing a fermentation product from an ungelatinized starch-containing material

[0147] One aspect of the present invention relates to a method for reducing the viscosity of a slurry at the backend of a method for producing a fermentation product (e.g., fuel ethanol) from an ungelatinized starch-containing material (i.e., granular starch - commonly referred to as the "raw starch hydrolysis" process), wherein laccase and / or peroxidase is added after the fermentation step to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0148] In an embodiment, a method for producing a fermentation product from an ungelatinized starch-containing material comprises the following steps:

[0149] (a) saccharifying the starch-containing material using glucoamylase and α-amylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; and

[0150] (b) fermenting the sugar using a fermenting organism to produce a fermentation product;

[0151] (c) distilling the fermentation product to recover the fermentation product and produce whole stillage;

[0152] (d) processing the whole stillage to produce stillage water; and

[0153] (e) evaporating the stillage water to produce a slurry;

[0154] wherein a redox enzyme is added after the fermentation step to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0155] In an embodiment, the redox enzyme is added to the beer pool containing beer before the distillation step. In an embodiment, the redox enzyme is added during the distillation step. In an embodiment, the redox enzyme is added to the whole stillage before the processing step.

[0156] In an embodiment, the processing step includes a separation step by subjecting the whole stillage to a filtration centrifuge, a sedimentation centrifuge, a pressure screen or a paddle screen. In an embodiment, the centrifuge is a sedimentation centrifuge, particularly a horizontal sedimentation centrifuge.

[0157] In an embodiment, the redox enzyme is added to the whole stillage and incubated for a period of time before the whole stillage separation step is carried out.

[0158] In an embodiment, the redox enzyme is laccase. In an embodiment, the redox enzyme is peroxidase.

[0159] Preferably, the redox enzymes used in the method have an optimal temperature compatible with the temperature range of the method steps in which they are used. In the examples, the redox enzymes are used at temperatures in the range of 30 °C to 100 °C, such as 40 °C to 90 °C, preferably 45 °C to 85 °C.

[0160] Raw starch hydrolysis (RSH) processes are well known in the art. Those skilled in the art will understand that the process parameters described in Part II above apply to the methods described in this part, including the selection of starch-containing materials, reducing the grain particle size, saccharification temperature, time and pH, simultaneous saccharification and fermentation conditions, and saccharifying enzymes, except for the process parameters related to the liquefaction step (a) not carried out in the RSH process. The process parameters of exemplary raw starch hydrolysis processes are described in further detail in WO 2004 / 106533 (which is incorporated herein by reference).

[0161] Examples of α-amylases preferably used in step (a) and / or step (b) include, but are not limited to, those described in WO2004 / 055178, WO 2005 / 003311, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO2021 / 163015, and WO 2021 / 163036 (each of which is incorporated herein by reference).

[0162] Examples of glucoamylases preferably used in step (a) and / or step (b) include, but are not limited to, WO 1999 / 28448, WO 2005 / 045018, WO 2005 / 069840, WO 2006 / 069289 (each of which is incorporated herein by reference).

[0163] Examples of compositions containing α-amylase and glucoamylase preferably used in step (a) and / or step (b) include, but are not limited to, the compositions described in WO 2015 / 031477 (which is incorporated herein by reference).

[0164] Back-end or downstream processing

[0165] A. Recovery of fermentation products and production of whole stillage

[0166] After fermentation or SSF, the fermentation product can be separated from the fermentation medium. Any method known in the art can be used to optionally recover the fermentation product (e.g., ethanol) from the fermentation medium, including but not limited to: chromatography, electrophoresis, differential solubility, distillation, or extraction. For example, alcohols are separated and purified from fermented starch-containing materials by conventional distillation methods.

[0167] Thus, in one embodiment, the method of the present invention further includes distillation to obtain a fermentation product, e.g., ethanol. Fermentation and distillation can be carried out simultaneously and / or separately / sequentially; optionally, followed by one or more process steps for further refining the fermentation product. After the distillation process is completed, the remaining material is regarded as whole stillage.

[0168] As another example, the desired fermentation product can be extracted from the fermentation broth by microfiltration or membrane filtration techniques. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol (i.e., neutral drinking alcoholic beverages), or industrial ethanol.

[0169] In some embodiments of these methods, the recovered fermentation product is substantially pure. With respect to these methods herein, "substantially pure" means that the recovered preparation contains no more than 15% impurities, where impurities mean compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure preparation is provided, wherein the preparation contains no more than 25% impurities, or no more than 20% impurities, or no more than 10% impurities, or no more than 5% impurities, or no more than 3% impurities, or no more than 1% impurities, or no more than 0.5% impurities.

[0170] Suitable assays known in the art can be used to test for ethanol and contaminant production as well as sugar consumption. For example, the ethanol product and other organic compounds can be analyzed by methods such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography - mass spectrometry), and LC-MS (liquid chromatography - mass spectrometry) or other suitable analytical methods using conventional procedures well known in the art. The release of ethanol in the fermentation broth can also be tested with the culture supernatant. By HPLC (Lin et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775 - 779 (2005)) or other suitable assays and detection methods well known in the art, the by-products and residual sugars (e.g., glucose or xylose) in the fermentation medium can be quantified, for example, using a refractive index detector for glucose and alcohols, and a UV detector for organic acids.

[0171] B. Processing of Whole Stillage

[0172] In one embodiment, the whole distillers grains are processed into two streams - a wet cake and a separated liquid. The whole distillers grains are separated or partitioned into a solid phase and a liquid phase by one or more methods of separating the separated liquid from the wet cake. The separated liquid is divided into two streams - distillers' solubles, which enters an evaporator, and a countercurrent, which is recycled to the front of the apparatus. Any suitable separation technique (including centrifugation, pressing, and filtration) can be used to separate the whole distillers grains into a separated liquid (e.g., distillers' solubles when pumped to the evaporator rather than the front end of the apparatus) and a wet cake to remove a large portion of the liquid / water. In a preferred embodiment, separation / dehydration is carried out by centrifugation. In the industry, a preferred centrifuge is a sedimentation centrifuge, preferably a high-speed sedimentation centrifuge. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval, which is a high-performance sedimentation centrifuge. Similar sedimentation centrifuges can also be purchased from Flottweg. In another preferred embodiment, other conventional separation equipment (such as plate / frame filter presses, belt filter presses, screw presses, gravity thickeners, and dewaterers) or similar equipment is used for separation.

[0173] C. Processing of Distillers' Solubles

[0174] Distillers' solubles is the term for the supernatant liquid from the centrifugation of whole distillers grains. Typically, distillers' solubles contains 4% - 8% dry solids (DS) (mainly proteins, soluble fibers, fats, fines, and cell wall components) and has a temperature of about 60°C - 90°C. The distillers' solubles stream can be condensed by evaporation to provide two process streams, including: (i) an evaporator condensate stream, which contains the condensed water removed from the distillers' solubles during evaporation; and (ii) a slurry stream, which contains a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils remaining in the distillers' solubles due to the removal of the evaporated water.

[0175] Optionally, oil can be removed from the distillers' solubles, or oil can be removed as an intermediate step in the evaporation process, which typically uses a series of several evaporation stages.

[0176] The slurry and / or deoiled slurry can be introduced into a dryer together with the wet cake (from the whole distillers grains separation step) to provide a product called dried distillers grains with solubles, which can also be used as animal feed. In an embodiment, the slurry and / or deoiled slurry is sprayed into one or more dryers to combine the slurry and / or deoiled slurry with the whole distillers grains to produce dried distillers grains with solubles.

[0177] Distillers' slops (e.g., optionally hydrolyzed) in the range of 5 vol.-% - 90 vol.-%, such as in the range of 10% - 80%, such as in the range of 15% - 70%, such as in the range of 20% - 60% can be recycled (as a countercurrent) to step (a). The recycled distillers' slops (i.e., the countercurrent) can account for about 1 vol.-% - 70 vol.-%, preferably 15 vol.-% - 60 vol.-%, especially from about 30 vol.-% - 50 vol.-% of the slurry formed in step (a). In an embodiment, the method further includes recycling at least a portion of the distillers' slops stream to the slurry, optionally after oil has been extracted from the distillers' slops stream.

[0178] D. Drying of the wet cake and production of dried distillers grains and dried distillers grains with solubles

[0179] After separating a wet cake containing about 25 wt% - 40 wt%, preferably 30 wt% - 38 wt% dry solids from the distillers' slops (e.g., by dehydration), it can be dried on a drum dryer, spray dryer, ring dryer, fluidized bed dryer, etc. to produce "dried distillers grains (DDG)". DDG is a valuable feed ingredient for animals such as livestock, poultry, and fish. Preferably, DDG is provided with a moisture content of less than about 10 wt% - 12 wt% to avoid mold and microbial decomposition and to increase the shelf life. Additionally, a high moisture content also makes transporting DDG more expensive. Preferably, the wet cake is dried under conditions that do not denature the proteins in the wet cake. The wet cake can be blended with the slurry separated from the distillers' slops and dried to produce dried distillers grains with solubles (DDGS). A partially dried intermediate product, such as sometimes referred to as modified wet distillers' grains, can be produced by partially drying the wet cake and optionally adding the slurry before, during, or after the drying process.

[0180] Exemplary oxidoreductases suitable for the method of the present invention

[0181] Aspects of the present invention relate to adding an oxidoreductase after the fermentation step in a method for producing a fermentation product to reduce the viscosity of the slurry, e.g., by reducing the number of smaller-sized particles in the whole distillers' slops, thereby reducing the number of small particles entering the distillers' slops water through the whole distillers' slops processing step (e.g., decantation), thereby reducing the viscosity of the slurry produced by evaporating the distillers' slops water.

[0182] The present invention contemplates the use of any redox enzyme that is capable of increasing the number of larger-sized particles in the total distillers grains while reducing the number of smaller-sized particles. Preferably, the redox enzyme reduces the accumulation of particles in the range of 100 μm to 1000 μm in size by at least 10 vol%, at least 15 vol%, at least 20 vol%, at least 25 vol%, at least 30 vol%, at least 35 vol%, at least 40 vol%, at least 45 vol%, at least 50 vol%, at least 55 vol%, at least 60 vol%, at least 65 vol%, at least 70 vol%, at least 75 vol% or at least 80 vol%. Similarly, the preferred redox enzyme increases the distribution of particles with a diameter greater than or equal to 400 μm in the total distillers grains by at least 15 vol%, at least 15 vol%, at least 20 vol%, at least 25 vol%, at least 30 vol%, at least 35 vol%, at least 40 vol%, at least 45 vol%, at least 50 vol%, at least 55 vol%, at least 60 vol%, at least 65 vol%, at least 70 vol%, at least 75 vol% or at least 80 vol%.

[0183] In an embodiment, the redox enzyme is laccase.

[0184] In an embodiment, the laccase is AA1 laccase (EC 1.10.3.2).

[0185] The laccase applicable to the method of the present invention can be obtained from Thermothelomyces.

[0186] The laccase applicable to the method of the present invention can be obtained from the following species: Thermothelomyces fergusii, Thermothelomyces guttulatus, Thermothelomyces heterothallicus, Thermothelomyces hinnuleus, Thermothelomyces myriococcoides or Thermothelomyces thermophilus.

[0187] Exemplary laccases have the amino acid sequence of SEQ ID NO: 1. In embodiments, the laccase has the amino acid sequence of SEQ ID NO: 1 with 0 to 10 conservative amino acid substitutions and has laccase activity. In embodiments, the laccase is a laccase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 and having laccase activity.

[0188] Laccases suitable for the methods of the present invention can be obtained from Coprinopsis.

[0189] The laccase applicable to the method of the present invention can be obtained from the following species: Coprinus alcobae, Coprinus arachnoideus, Coprinus asterophoroides, Coprinus cinerea, Coprinus clastophyllus, Coprinus colosseus, Coprinus comatus, Coprinus coniophorus, Coprinus cordisporus, Coprinus cortinatus, Coprinus ephemerus, Coprinus fissolanatus, Coprinus foetidellus, Coprinus goudensis, Coprinus latisporus, Coprinus littoralis, Coprinus maysoidisporus, Coprinus myceliocephalus, Coprinus palmeranus, Coprinus patouillardii, Coprinus phaeopunctatus, Coprinus pinetorum, Coprinus aff. radians PP63, Coprinus roseistipitatus, Coprinus rufopruinatus, Coprinus simulans, Coprinus spadiceisporus, Coprinus sterquilinus, Coprinus subdomesticus, Coprinus trigonosporus, Coprinus vosoustii or Coprinus xerophilus.

[0190] Exemplary laccases have the amino acid sequence of SEQ ID NO:2. In an embodiment, the laccase has the amino acid sequence of SEQ ID NO:2 carrying 0 to 10 conservative amino acid substitutions and has laccase activity. In an embodiment, the laccase is a laccase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2 and having laccase activity.

[0191] The laccase applicable to the method of the present invention can be obtained from Polyporus.

[0192] The laccase applicable to the method of the present invention can be obtained from the following species: Polyporus americanus, Polyporus anthracophilus, Polyporus arcularioides, Polyporus arcularius, Polyporus auratus, Polyporus austrosinensis, Polyporus brasiliensis, Polyporus brevibasidiosus, Polyporus brumalis, Polyporus chozeniae, Polyporus ciliates, Polyporus corylinus, Polyporus cryptopus, Polyporus curtipes, Polyporus cuticulatus, Polyporus decurrens, Polyporus dictyopus, Polyporus elongoporus, Polyporus foedatus, Polyporus fraxineus, Polyporus frondosus, Polyporus gayanus, Polyporus grammocephalus, Polyporus guianensis, Polyporus hapalopus, Polyporus hartmannii, Polyporus hemicapnodes, Polyporus hypomelanus, Polyporus koreanus, Polyporus lamelliporus, Polyporus lepideus, Polyporus leprieurii, Polyporus leptocephalus, Polyporus longiporus, Polyporus mangshanensisPolyporus mangshanensis, Polyporus marianiae, Polyporus mcmurphyi, Polyporus melanopus, Polyporus meridionalis, Polyporus minutosquamosus, Polyporus orientivarius, Polyporus parvovarius, Polyporus philippinensis, Polyporus pinsitus, Polyporus plorans, Polyporus pseudobetulinus, Polyporus radicatus, Polyporus roseofuscus, Polyporus sagranus, Polyporus squamulosus, Polyporus subvarius, Polyporus tessellatus, Polyporus thailandensis, Polyporus tricholoma, Polyporus tsugae, Polyporus tuberaster, Polyporus tucumanensis, Polyporus tumulosus, Polyporus ulleungus, Polyporus umbellatus or Polyporus varius.

[0193] Exemplary laccases have the amino acid sequence of SEQ ID NO:3. In an embodiment, the laccase has the amino acid sequence of SEQ ID NO:3 with 0 to 10 conservative amino acid substitutions and has laccase activity. In an embodiment, the laccase is a laccase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:3 and having laccase activity.

[0194] In an embodiment, the oxidoreductase is peroxidase.

[0195] In an embodiment, the peroxidase is AA2 peroxidase (EC 1.11.1.7).

[0196] The peroxidase applicable to the method of the present invention can be obtained from Coprinus.

[0197] The peroxidase applicable to the method of the present invention can be obtained from the following species: Coprinus alcoberensis, Coprinus arachnoideus, Coprinus asterophorus, Coprinus cinereus, Coprinus fissilis, Coprinus giganteus, Coprinus comatus, Coprinus griseoconus, Coprinus cordisporus, Coprinus filamentosus, Coprinus ephemerus, Coprinus fimbriatus, Coprinus foetens, Coprinus hoogdajensis, Coprinus latisporus, Coprinus marinus, Coprinus marzuolus, Coprinus myceliofibrosus, Coprinus palmeri, Coprinus radians, Coprinus spadiceus, Coprinus pinetorum, Coprinus radians-like PP63, Coprinus roseipes, Coprinus rubrobrunneus, Coprinus simulans, Coprinus sordidus, Coprinus sterquilinus, Coprinus subhumanus, Coprinus trigonosporus, Coprinus vossoensis or Coprinus xerophilus.

[0198] The exemplary peroxidase has the amino acid sequence of SEQ ID NO:4. In an embodiment, the peroxidase has the amino acid sequence of SEQ ID NO:4 with 0 to 10 conservative amino acid substitutions and has peroxidase activity. In an embodiment, the peroxidase is a peroxidase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:4 and having peroxidase activity.

[0199] The peroxidase applicable to the method of the present invention can be obtained from Glycine.

[0200] The peroxidase applicable to the method of the present invention can be obtained from the following species: Glycine gracilis, Glycine max or Glycine soja.

[0201] Exemplary peroxidases have the amino acid sequence of SEQ ID NO:5. In embodiments, the peroxidase has the amino acid sequence of SEQ ID NO:5 with 0 to 10 conservative amino acid substitutions and has peroxidase activity. In embodiments, the peroxidase is a peroxidase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:5 and having peroxidase activity.

[0202] The peroxidase suitable for the method of the present invention can be obtained from the genus Armoracia.

[0203] The peroxidase suitable for the method of the present invention can be obtained from the species Armoracia rusticana.

[0204] Exemplary peroxidases have the amino acid sequence of SEQ ID NO:6. In embodiments, the peroxidase has the amino acid sequence of SEQ ID NO:6 with 0 to 10 conservative amino acid substitutions and has peroxidase activity. In embodiments, the peroxidase is a peroxidase having an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 and having peroxidase activity.

[0205] Preferably, the optimum temperature range of the oxidoreductase is from 30 °C to 100 °C, such as from 40 °C to 90 °C, preferably from 45 °C to 85 °C.

[0206] The oxidoreductase can be administered in beer, beer pools, distillation and / or whole distillers grains at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS, such as 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS.

[0207] Exemplary fermenting organisms

[0208] Aspects of the present invention relate to the use of fermenting organisms for the production of fermentation products. Particularly suitable fermenting organisms are capable of directly or indirectly fermenting (i.e., converting) sugars such as arabinose, glucose, maltose and / or xylose into the desired fermentation product such as ethanol. Examples of fermenting organisms include fungal organisms such as yeasts. Preferred yeasts include strains of the species Saccharomyces, particularly Saccharomyces cerevisiae.

[0209] Examples of commercially available yeasts include, for example, RED STAR TM and ETHANOL RED TM yeast (available from Fermentis / Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC TM fresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other available yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ), such as, for example, BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB-1952 (ARS Culture Collection). There are also other Saccharomyces cerevisiae strains suitable as host cells, such as DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives, as well as yeast species 1400, 424A (LNH-ST), 259A (LNH-ST) and their derivatives.

[0210] As used herein, a "derivative" of a strain is derived from a reference strain, such as by mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Those skilled in the art will understand that genetic alterations (including the metabolic modifications exemplified herein) can be described with reference to a suitable host organism and its corresponding metabolic reactions or a suitable source organism for the desired genetic material (such as genes of a desired metabolic pathway). However, given the whole-genome sequencing of a wide variety of organisms and the high level of skill in the field of genomics, those skilled in the art can apply the teachings and guidance provided herein to other organisms. For example, the metabolic alterations exemplified herein can be readily applied to other species by incorporating the same or similar coding nucleic acids from a species different from the reference species.

[0211] The fermenting organism can be a strain of the genus Saccharomyces, such as a Saccharomyces cerevisiae strain produced using the methods described and involved in U.S. Patent No. 8,257,959 - BB. In one embodiment, the recombinant cell is a derivative of the strain Saccharomyces cerevisiae CIBTS1260 (deposited under accession number NRRL Y - 50973 at the Agricultural Research Service Culture Collection (NRRL), 61604, Illinois, USA).

[0212] The fermenting organism can also be a derivative of the Saccharomyces cerevisiae strain NMIV14 / 004037 (see WO 2015 / 143324 and WO2015 / 143317, each incorporated herein by reference), strain numbers V15 / 004035, V15 / 004036, and V15 / 004037 (see WO 2016 / 153924, incorporated herein by reference), strain numbers V15 / 001459, V15 / 001460, V15 / 001461 (see WO 2016 / 138437, incorporated herein by reference), strain number NRRL Y67342 (see WO 2018 / 098381, incorporated herein by reference), strain numbers NRRL Y67549 and NRRL Y67700 (see WO 2019 / 161227, incorporated herein by reference), or any strain described in WO 2017 / 087330 (incorporated herein by reference).

[0213] The fermenting organism can comprise one or more heterologous polynucleotides encoding α - amylase, glucoamylase, protease, and / or cellulase. Examples of α - amylase, glucoamylase, protease, and cellulase suitable for expression in the fermenting organism are known in the art (see WO 2021 / 231623, which is incorporated herein by reference).

[0214] The fermenting organism can be in the form of a composition that comprises the fermenting organism and natural and / or non - natural components.

[0215] The fermented organism can be in any viable form, including crushed, dried, including active drying and ready-to-eat, compressed, paste (liquid) form, etc. In one embodiment, the fermented organism (e.g., a Saccharomyces cerevisiae strain) is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the fermented organism is crushed yeast. In one embodiment, the fermented organism is compressed yeast. In one embodiment, the fermented organism is paste yeast.

[0216] In one embodiment, it is a composition that comprises the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0217] The composition described herein may comprise the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0218] The composition described herein may comprise the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citric acid mono- and diglycerides, polyglycerol esters, propylene glycol fatty acid esters.

[0219] In one embodiment, the composition comprises the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and Olindronal SMS, Olindronal SK, or Olindronal SPL, including the compositions described in European Patent No. 1,724,336, which is hereby incorporated by reference. For active dry yeast, these products are commercially available from Bussetti of Austria.

[0220] The composition described herein may comprise the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable gum. In one embodiment, the gum is selected from the group consisting of: locust bean gum, guar gum, tragacanth gum, gum arabic, xanthan gum, and gum acacia, particularly for paste, compressed, and dry yeast.

[0221] The composition described herein may comprise the fermented organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0222] The compositions described herein may comprise a fermenting organism (e.g., a strain of Saccharomyces cerevisiae) described herein and any suitable antioxidant. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particularly for active dry yeast.

[0223] During fermentation (e.g., SSF), suitable concentrations of viable fermenting organisms are well known in the art or can be readily determined by one skilled in the art. In one embodiment, a fermenting organism (such as an ethanol-fermenting yeast (e.g., Saccharomyces cerevisiae)) is added to a fermentation medium such that the viable fermenting organism (such as yeast) count per mL of the fermentation medium is between 10 5 and 10 12 , preferably between 10 7 and 10 10 , especially about 5 x 10 7 .

[0224] The invention is further summarized in the following paragraphs:

[0225] Claims:

[0226] 1. A method for reducing the viscosity of a slurry, the method comprising:

[0227] (a) liquefying a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0228] (b) saccharifying the dextrin with glucoamylase to produce fermentable sugars;

[0229] (c) fermenting the sugars with a fermenting organism to produce beer comprising the fermentation product;

[0230] (d) distilling the beer to recover the fermentation product and produce whole stillage;

[0231] (e) processing the whole stillage to produce stillage water; and

[0232] (f) evaporating the stillage water to produce a slurry;

[0233] wherein a redox enzyme is added after the fermentation step to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0234] 2. A method for reducing the viscosity of a slurry in a method for producing a fermentation product from a starch-containing material, the method comprising:

[0235] (a) Saccharify a starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars;

[0236] (b) Ferment the sugars with a fermenting organism to produce beer containing the fermentation product;

[0237] (c) Distill the beer to recover the fermentation product and produce whole stillage;

[0238] (d) Process the whole stillage to produce stillage water; and

[0239] (e) Evaporate the stillage water to produce a slurry;

[0240] wherein after the fermentation step, a redox enzyme is added to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0241] 3. A method for reducing the viscosity of a slurry, the method comprising:

[0242] (a) Liquefy a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0243] (b) Saccharify the dextrin with glucoamylase to produce fermentable sugars;

[0244] (c) Ferment the sugars with a fermenting organism to produce beer containing the fermentation product;

[0245] (d) Distill the beer to recover the fermentation product and produce whole stillage;

[0246] (e) Process the whole stillage to produce stillage water; and

[0247] (f) Evaporate the stillage water to produce a slurry;

[0248] wherein after the fermentation step and before the distillation step, a redox enzyme is added to the beer to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0249] 4. A method for reducing the viscosity of a slurry in a method for producing a fermentation product from a starch-containing material, the method comprising:

[0250] (a) Saccharify a starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars;

[0251] (b) Fermenting the sugar with a fermenting organism to produce beer containing the fermentation product;

[0252] (c) Distilling the beer to recover the fermentation product and produce whole stillage;

[0253] (d) Processing the whole stillage to produce stillage water; and

[0254] (e) Evaporating the stillage water to produce a slurry;

[0255] wherein after the fermentation step and before the distillation step, an oxidoreductase is added to the beer to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0256] 5. A method for reducing the viscosity of a slurry, the method comprising:

[0257] (a) Liquefying a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0258] (b) Saccharifying the dextrin with glucoamylase to produce fermentable sugar;

[0259] (c) Fermenting the sugar with a fermenting organism to produce beer containing the fermentation product;

[0260] (d) Distilling the beer to recover the fermentation product and produce whole stillage;

[0261] (e) Processing the whole stillage to produce stillage water; and

[0262] (f) Evaporating the stillage water to produce a slurry;

[0263] wherein after the fermentation step, during the distillation step and before the processing step, an oxidoreductase is added to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

[0264] 6. A method for reducing the viscosity of a slurry in a method for producing a fermentation product from a starch-containing material, the method comprising:

[0265] (a) Saccharifying a starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugar;

[0266] (b) Fermenting the sugar with a fermenting organism to produce beer containing the fermentation product;

[0267] (c) Distill the beer to recover the fermentation product and produce whole distillers grains;

[0268] (d) Process the whole distillers grains to produce distillers grains water; and

[0269] (e) Evaporate the distillers grains water to produce a slurry;

[0270] wherein after the fermentation step, during the distillation step and before the processing step, an oxidoreductase is added to reduce the number of particles in the distillers grains water by increasing the size of the particles in the whole distillers grains, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0271] 7. A method for reducing the viscosity of a slurry, the method comprising:

[0272] (a) Liquefy a starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch to produce dextrin;

[0273] (b) Saccharify the dextrin with glucoamylase to produce fermentable sugars;

[0274] (c) Ferment the sugars with a fermenting organism to produce beer containing the fermentation product;

[0275] (d) Distill the beer to recover the fermentation product and produce whole distillers grains;

[0276] (e) Process the whole distillers grains to produce distillers grains water; and

[0277] (f) Evaporate the distillers grains water to produce a slurry;

[0278] wherein after the distillation step and before the processing, an oxidoreductase is added to the whole distillers grains to reduce the number of particles in the distillers grains water by increasing the size of the particles in the whole distillers grains, thereby reducing the viscosity of the slurry produced by evaporating the distillers grains water.

[0279] 8. A method for reducing the viscosity of a slurry in a method for producing a fermentation product from a starch-containing material, the method comprising:

[0280] (a) Saccharify a starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars;

[0281] (b) Ferment the sugars with a fermenting organism to produce beer containing the fermentation product;

[0282] (c) Distill the beer to recover the fermentation product and produce whole distillers grains;

[0283] (d) Process the whole distillers grains to produce distillers grains water; and

[0284] (e) Evaporating the distillers' grains water to produce a slurry;

[0285] Wherein after the distillation step and before the processing, an oxidoreductase is added to the whole distillers' grains, and the number of particles in the distillers' grains water is reduced by increasing the size of the particles in the whole distillers' grains, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

[0286] 9. The method according to any one of paragraphs 1 to 8, wherein the processing step comprises a separation step by subjecting the whole distillers' grains to a filtration centrifuge, a sedimentation centrifuge, a pressure screen or a paddle screen.

[0287] 10. The method according to paragraph 9, wherein the centrifuge is a sedimentation centrifuge, particularly a horizontal sedimentation centrifuge.

[0288] 11. The method according to any one of paragraphs 1 to 10, wherein the laccase and / or the oxidoreductase is added to the whole distillers' grains and incubated for a period of time before performing the separation step of the whole distillers' grains.

[0289] 12. The method according to any one of paragraphs 1 to 11, wherein the laccase and / or oxidoreductase is used at a temperature in the range of 30 °C to 100 °C, such as 40 °C to 90 °C, preferably 45 °C to 85 °C.

[0290] 13. The method according to any one of paragraphs 1 to 12, wherein for particles in the size range of 100 μm to 1000 μm, the cumulative passage is reduced by 10% to 80% by volume.

[0291] 14. The method according to any one of paragraphs 1 to 13, wherein the cumulative passage of particles with a diameter of 400 μm is reduced by 10% to 80% by volume.

[0292] 15. The method according to any one of paragraphs 1 to 14, wherein the oxidoreductase is laccase.

[0293] 16. The method according to paragraph 15, wherein the laccase is AA1 laccase (EC 1.10.3.2).

[0294] 17. The method according to any one of paragraphs 15 to 16, wherein the laccase is from the genus Thermomyces.

[0295] 18. The method according to any one of paragraphs 15 to 17, wherein the laccase is from the following species: Thermomyces fergusii, Thermomyces lanuginosus, Thermomyces heterothallicus, Thermomyces aurantiacus, Thermomyces polysporus or Thermomyces thermophilus.

[0296] 19. The method according to any one of paragraphs 15 to 18, wherein the laccase has an amino acid sequence of SEQ ID NO:1 with 0 to 10 conservative amino acid substitutions, or is a laccase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1 and having laccase activity.

[0297] 20. The method according to paragraph 15, wherein the laccase is from the genus Coprinopsis.

[0298] 21. The method according to paragraph 15 or 20, wherein the laccase is from the following species: Coprinus alcoberensis, Coprinus arachnoideus, Coprinus asterophorus, Coprinus cinereus, Coprinus fissilis, Coprinus giganteus, Coprinus comatus, Coprinus griseoconus, Coprinus cordisporus, Coprinus filamentosus, Coprinus ephemerus, Coprinus fimbriatus, Coprinus foetens, Coprinus hoogdajensis, Coprinus latemarginatus, Coprinus marinus, Coprinus marzuolus, Coprinus myceliofibulatus, Coprinus palmeri, Coprinus radiatus, Coprinus spadiceus, Coprinus pinetorum, Coprinus radians PP63, Coprinus roseipes, Coprinus rubrobrunneus, Coprinus simulans, Coprinus castaneosporus, Coprinus sterquilinus, Coprinus subhomincola, Coprinus trigonosporus, Coprinus vossoesterianus or Coprinus xerophilus.

[0299] 22. The method according to any one of paragraphs 15 or 21 to 22, wherein the laccase has an amino acid sequence of SEQ ID NO:2 with 0 to 10 conservative amino acid substitutions, or is a laccase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2 and having laccase activity.

[0300] 23. The method according to paragraph 15, wherein the laccase is from the genus Polyporus.

[0301] 24. The method as described in paragraph 15 or 23, wherein the laccase is from the following species: Polyporus americanus, Polyporus carbonarius, Polyporus pseudofelleus, Polyporus felleus, Polyporus chrysoloma, Polyporus austrosinensis, Polyporus brasilianus, Mycoleptodonoides aitchisonii, Mycoleptodon mutabilis, Polyporus chioneus, Polyporus ciliatus, Polyporus dryophilus, Polyporus cryptopus, Polyporus brevipes, Polyporus corticatus, Polyporus elongatus, Polyporus reticulatus, Polyporus tubaeformis, Polyporus sordidus, Polyporus fraxineus, Polyporus badius, Polyporus guyanensis, Polyporus tener, Polyporus harmani, Polyporus semisordidus, Polyporus subfuscus, Polyporus coreanus, Polyporus alveolarius, Polyporus squamosus, Polyporus leprius, Polyporus leptocephalus, Polyporus longiporus, Polyporus mangshanensis, Polyporus marianus, Polyporus murphyi, Polyporus melanopus, Polyporus meridionalis, Polyporus microlepideus, Polyporus varius var. orientalis, Polyporus varius var. minor, Polyporus philippinensis, Polyporus epiphyllus, Polyporus populinus, Polyporus pseudobetulinus, Polyporus rhizophyllus, Polyporus roseo-badia, Polyporus sagaranensis, Polyporus microlepidotus, Polyporus subvarius, Polyporus tessellatus, Polyporus siamensis, Polyporus tricholomatoides, Polyporus pini, Polyporus tuberaster, Polyporus tucumanensis, Polyporus gibbous, Polyporus ulleungdoensis, Polyporus umbellatus or Polyporus versipellis.

[0302] 25. The method as described in any one of paragraphs 15 or 23 to 24, wherein the laccase has an amino acid sequence of SEQ ID NO:3 with 0 to 10 conservative amino acid substitutions, or is a laccase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3 and having laccase activity.

[0303] 26. The method as described in any one of paragraphs 1 to 14, wherein the oxidoreductase is peroxidase.

[0304] 27. The method as described in paragraph 26, wherein the peroxidase is AA2 peroxidase (EC 1.11.1.7).

[0305] 28. The method as described in paragraph 26 or 27, wherein the peroxidase is from the genus Coprinus.

[0306] 29. The method according to any one of paragraphs 26 to 28, wherein the peroxidase is from the following species: *Coprinopsis alcobaensis*, *Coprinopsis arachnoidea*, *Coprinopsis asterophora*, *Coprinopsis cinerea*, *Coprinopsis fissilis*, *Coprinopsis gigas*, *Coprinopsis comatus*, *Coprinopsis griseoconicus*, *Coprinopsis cordispora*, *Coprinopsis filamentosa*, *Coprinopsis ephemerella*, *Coprinopsis fimbriata*, *Coprinopsis foetens*, *Coprinopsis hoogdensis*, *Coprinopsis latispora*, *Coprinopsis maritima*, *Coprinopsis marzuolus*, *Coprinopsis mycelialis*, *Coprinopsis palmeri*, *Coprinopsis radians*, *Coprinopsis rubrobrunnea*, *Coprinopsis simulans*, *Coprinopsis spadiceospora*, *Coprinopsis sterquilinus*, *Coprinopsis subhumanus*, *Coprinopsis trigonospora*, *Coprinopsis vossoensis* or *Coprinopsis xerophila*.

[0307] 30. The method according to any one of paragraphs 26 to 29, wherein the peroxidase has an amino acid sequence of SEQ ID NO: 4 with 0 to 10 conservative amino acid substitutions, or is a peroxidase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 4 and having peroxidase activity.

[0308] 31. The method according to paragraph 26, wherein the peroxidase is from the genus *Glycine*.

[0309] 32. The method according to paragraph 31, wherein the peroxidase is from the following species: *Glycine gracilis*, *Glycine max* or *Glycine soja*.

[0310] 33. The method according to paragraph 31 or 32, wherein the peroxidase has an amino acid sequence of SEQ ID NO: 5 with 0 to 10 conservative amino acid substitutions, or is a peroxidase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 and having peroxidase activity.

[0311] 34. The method according to paragraph 26, wherein the peroxidase is from the genus *Armoracia*.

[0312] 35. The method according to paragraph 31, wherein the peroxidase is from the species *Armoracia rusticana*.

[0313] 36. The method according to paragraph 34 or 35, wherein the peroxidase has an amino acid sequence of SEQ ID NO: 6 with 0 to 10 conservative amino acid substitutions, or is a peroxidase having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 6 and having peroxidase activity.

[0314] 37. The method according to any one of claims 1 to 36, wherein the saccharification and fermentation steps are carried out simultaneously.

[0315] 38. The method according to any one of claims 1 to 37, wherein the starch-containing material comprises sugar beet, maize, corn, wheat, rye, barley, oats, triticale, sorghum, sweet potato, rice, millet, pearl millet and / or foxtail millet.

[0316] 39. The method according to any one of claims 1 to 38, wherein the fermentation product is ethanol, preferably fuel ethanol.

[0317] 40. The method according to any one of claims 1 to 39, wherein the fermenting organism is yeast.

[0318] The invention described and claimed herein is not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended to be illustrative of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control. Various references are cited herein, the disclosures of which are incorporated herein by reference in their entireties. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0319] Materials and Methods

[0320] Enzymes Used in the Examples

[0321] Reductase A: An exemplary laccase from Thermotoga maritima as disclosed in SEQ ID NO: 1

[0322] Reductase B: An exemplary peroxidase from Coprinopsis cinerea as disclosed in SEQ ID NO: 4

[0323] Determination of the Td of the Liquefying Enzyme by Differential Scanning Calorimetry

[0324] The thermal stability of the enzyme was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). In the thermogram (Cp vs. T) obtained after heating an enzyme solution (about 0.5 mg / ml) in buffer (50 mM acetate, pH 5.0) at a constant programmed heating rate of 200 K / hour, the thermal denaturation temperature Td (°C) was taken as the top of the denaturation peak (the main endothermic peak).

[0325] The sample solution and the reference solution (about 0.2 ml) were loaded into the calorimeter from storage at 10 °C (reference solution: buffer without enzyme), and thermally pre-equilibrated at 20 °C for 20 minutes, followed by a DSC scan from 20 °C to 120 °C. The denaturation temperature was determined with an accuracy of about + / - 1 °C.

[0326] Example

[0327] Example 1: Characterization of Solids in Stillage and Whole Stillage from a Commercial Ethanol Plant

[0328] Whole stillage and stillage were obtained from a commercial ethanol plant in the Midwestern United States. In the particle size distribution test, about 4.5 ml of well-mixed stillage was diluted with 20 ml of deionized water. The diluted stillage was analyzed using an Ls13 320 laser diffraction particle size analyzer (Beckman Coulter), and the particle size measurements were performed in duplicate. In the solids content measurement, 9 ml of stillage was mixed with 20 ml of deionized water in a 50 ml tube. After centrifuging at 5300 rpm for 10 minutes using a floor-standing centrifuge AVANTI J-E (Beckman Coulter), the supernatant was removed. The solids were frozen in a -20 °C freezer and then dried in an AssetRD-184 freeze dryer (LABCONCO). The solids content measurements were performed in triplicate. The solids content % was obtained by dividing the dry solids by the weight of 9 ml of stillage.

[0329] Figure 1 It was shown that the particle size in stillage was below 400 μm, while the particle size of whole stillage was in the range of 0 to 1820 μm. The data indicate that decantation in this ethanol production plant retained particles with a diameter above 400 μm in the wet cake. Although the cut-off particle size may vary from plant to plant due to equipment settings, in an ethanol plant, the cut-off particle size should not deviate significantly from 400 μm. The average + / - standard deviation of the solids content in stillage was 3.91% + / - 0.15%.

[0330] Example 2: Increasing Particle Size by Treating Whole Stillage with Redox Enzyme at 65 °C

[0331] The whole distillers grains used in this example were obtained from a commercial ethanol plant in the Midwestern United States. Measured using a moisture analyzer, the average dry solids % of the whole distillers grains was 12.25% and the pH was approximately 5.1. Approximately 10 g of whole distillers grains were weighed into each tube. Reductase A and reductase B were used in this experiment. Hydrogen peroxide was diluted to 0.06% and added at 100 μl / tube according to Table 1 below. During incubation in a 65 °C water bath, all samples were vortexed every 10 minutes. After 60 minutes of treatment, all samples were cooled and stored in a refrigerator before particle size distribution testing.

[0332] Table 1: Treatment of whole distillers grains with laccase and peroxidase at 65 °C for 1 hour

[0333]

[0334] The treated whole distillers grains (WS) samples were diluted by adding 2 ml of slurry to 10 ml of deionized water. Each diluted sample was analyzed using an Ls13 320 laser diffraction particle size analyzer (Beckman Coulter), and the average value of WS particle size measurements was used in the figure. Figure 2 and Figure 3 show that treatment with reductase results in an increase in the WS particle size. In each figure, the percentage (y-axis) is the cumulative volume % of all particles less than or equal to a specific WS particle size (x-axis). At a particle size of 400 μm in diameter, treatment with 10 and 40 μg / g dry solids (DS) of reductase A reduced the cumulative passage of particles from 81 volume % (enzyme-free control) to 35 volume % and 22 volume %, as Figure 2 shown. For treatment with reductase B, a similar trend of cumulative passage reduction was observed for particles in the whole distillers grains with a size of 400 μm in diameter, as Figure 3 shown.

[0335] The effect of reductase treatment on particle size is shown in Table 2 below. Particles with a size equal to or less than a specific diameter (μm) are shown as different levels of particle volume percentage. At 50% particle volume, the particle sizes of the enzyme-free control and the whole distillers grains treated with 10 μg of reductase A were ≤133.1 μm and ≤676.5 μm, respectively, indicating that there were more small particles in the enzyme-free control sample than in the 10 μg reductase A-treated sample. In other words, treatment with 10 μg of reductase A reduced the number of smaller-sized particles and increased the number of larger-sized particles. Similarly, with or without peroxide, treatment with reductase B also reduced the volume % of smaller-sized particles in the whole distillers grains and increased the volume % of larger-sized particles.

[0336] Table 2: Effect of laccase and peroxidase treatment on particle size at 65 °C

[0337]

[0338] Example 3: Increasing the particle size by treating whole distillers grains with redox enzymes at 50 °C

[0339] The whole distillers grains from Example 2 were used at 10 g / tube in this example. Redox enzyme A and redox enzyme B from Example 2 were used and given at 10 and 100 μg / gDS. 100 μl of 0.06% hydrogen peroxide was added to each tube of whole distillers grains treated with peroxidase. The treatment was carried out at 50 °C for 2 hours in a temperature-controlled water bath. After treatment, 100 μl of the treated whole distillers grains sample was taken and diluted with 4.9 ml of deionized water before particle size analysis. Analysis was carried out in the same manner as in Example 2.

[0340] Figure 4 The effect of treating whole distillers grains with redox enzyme A and redox enzyme B at 50 °C on the cumulative passing is shown. Both redox enzymes shifted the particle size distribution towards larger diameter particle sizes. Regardless of the redox enzyme, a higher dose of 100 μg / gDS produced a more pronounced increase in particle size than a lower dose of 10 μg / gDS.

[0341] The effect of redox enzyme treatment on particle size is shown in Table 3 below. Particles with a size equal to or less than a specific diameter (μm) are shown as different levels of particle volume percentage. At 50% particle volume, the particle sizes of the enzyme-free control and whole distillers grains treated with 10 μg of redox enzyme A were ≤162.8 μm and ≤227.5 μm, respectively. This indicates that there was a greater number of smaller-sized particles in the control sample without redox enzyme compared to the sample treated with 10 μg of redox enzyme A. In other words, treatment with 10 μg of redox enzyme A decreased the volume % of smaller-sized particles in the whole distillers grains and increased the volume % of larger-sized particles.

[0342] Table 3: Effect of redox enzyme treatment on particle size at 50 °C

[0343]

[0344] Example 4: Increasing the particle size by treating whole distillers grains with redox enzymes at 35 °C

[0345] The whole distillers grains from Example 2 were used at 10 g / tube in this example. Redox enzyme A and redox enzyme B from Example 2 were used in this example and given at 10 μg / gDS. 100 μl of 0.06% hydrogen peroxide was added in a single peroxidase treatment. The treatment was carried out at 35 °C for 16 hours in a temperature-controlled water bath. After treatment, sample preparation and particle size analysis were carried out in the same manner as in Example 2.

[0346] The effect of redox enzyme treatment on the particle size distribution is shown in Table 4 below. Particles equal to or smaller than a specific diameter (μm) are shown as different levels of percentage of particle volume. At 50% particle volume, the particle sizes of the enzyme-free control and the spent grains treated with 10 μg of redox enzyme A were ≤763.6 μm and ≤843.8 μm, respectively. This indicates that there were a greater number of smaller-sized particles in the control sample without enzyme compared to the sample treated with 10 μg of redox enzyme A. In other words, the treatment with 10 μg of redox enzyme A decreased the volume % of smaller-sized particles and increased the volume % of larger-sized particles. Similarly, the sample treated with redox enzyme B decreased the volume % of smaller-sized particles in the spent grains and increased the volume % of larger-sized particles.

[0347] Table 4: Effect of laccase and peroxidase treatment on particle size at 35 °C

[0348]

[0349] Figure 5 The effect of treatment with redox enzymes A and B on the cumulative passage of the spent grains at 35 °C is shown. The redox enzyme treatment shifted the particle size towards larger diameters. The increase in particle size was smaller compared to the treatment at 65 °C in Example 2, which may be due to the relatively lower activity of the redox enzymes at 35 °C.

Claims

1. A method for reducing the viscosity of a slurry, the method comprising: (a) liquefying the starch-containing material with a thermostable α-amylase at a temperature above the initial gelatinization temperature of the starch-containing material to produce dextrin; (b) saccharifying the dextrin with glucoamylase to produce fermentable sugars; (c) fermenting the sugars with a fermenting organism to produce beer containing the fermentation product; (d) distilling the beer to recover the fermentation product and produce whole stillage; (e) processing the whole stillage to produce stillage water; and (f) evaporating the stillage water to produce a slurry; wherein a redox enzyme is added after the fermentation step to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the stillage water.

2. A method for reducing the viscosity of a slurry in a method for producing a fermentation product from a starch-containing material, the method comprising: (a) saccharifying the starch-containing material with α-amylase and glucoamylase at a temperature below the initial gelatinization temperature of the starch to produce fermentable sugars; (b) fermenting the sugars with a fermenting organism to produce beer containing the fermentation product; (c) distilling the beer to recover the fermentation product and produce whole stillage; (d) processing the whole stillage to produce stillage water; and (e) evaporating the stillage water to produce a slurry; wherein a redox enzyme is added after the fermentation step to reduce the number of particles in the stillage water by increasing the size of the particles in the whole stillage, thereby reducing the viscosity of the slurry produced by evaporating the slurry.

3. The method according to claim 1 or 2, wherein the redox enzyme is added to a beer tank containing the beer before the distillation step.

4. The method according to any one of claims 1 to 3, wherein the redox enzyme is added during the distillation step.

5. The method according to any one of claims 1 to 4, wherein the redox enzyme is added to the whole stillage before the processing step.

6. The method according to any one of claims 1 to 5, wherein the processing step comprises a separation step by subjecting the whole stillage to a filtration centrifuge, a sedimentation centrifuge, a pressure screen or a paddle screen.

7. The method according to claim 6, wherein the centrifuge is a sedimentation centrifuge, particularly a horizontal sedimentation centrifuge.

8. The method according to any one of claims 1 to 7, wherein the redox enzyme is added to the whole stillage and incubated for a period of time before performing the separation step of the whole stillage.

9. The method according to any one of claims 1 to 8, wherein the redox enzyme is used at a temperature in the range of 30 °C to 100 °C, such as 40 °C to 90 °C, preferably 45 °C to 85 °C.

10. The method according to any one of claims 1 to 9, wherein for particles in the range of 100 μm to 1000 μm in size, the cumulative reduction is 10 vol% to 80 vol%.

11. The method according to any one of claims 1 to 10, wherein the cumulative passage of particles with a diameter of 400 μm is reduced by 10% to 80% by volume.

12. The method according to any one of claims 1 to 11, wherein the oxidoreductase is laccase.

13. The method according to any one of claims 1 to 11, wherein the oxidoreductase is peroxidase.

14. The method according to any one of claims 1 to 13, wherein the saccharification and fermentation steps are carried out simultaneously.

15. The method according to any one of claims 1 to 14, wherein the starch-containing material comprises sugar beet, maize, corn, wheat, rye, barley, oats, triticale, sorghum, sweet potato, rice, millet, pearl millet and / or foxtail millet.

16. The method according to any one of claims 1 to 15, wherein the fermentation product is ethanol, preferably fuel ethanol.

17. The method according to any one of claims 1 to 16, wherein the fermentation organism is yeast.

Images