CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a claims the benefit of U.S. Patent Application Serial No.

13/294,867, filed November 11 , 2011, the disclosure of which is incorporated herein by reference in its entirety.

FIELD

[0002] The present disclosure relates generally to preparing a hydrolysate from a lignocellulosic source, and more specifically to a preparing a solid hydrolysate or a concentrated hydrolysate from pretreated lignocellulosic biomass, including woody or herbaceous biomass, as well as waste or recycled paper.

BACKGROUND

[0003] Today, sugars (e.g., pure sucrose, pure fructose, pure glucose, pure dextrose) obtained from sugar cane, sugar beets, and starch are often supplied to the food industry. For example, table sugar is made up of pure sucrose, and corn syrup is made up of fructose and glucose.

These sugars may also be supplied to the biofuels and biochemicals industries; however, the sugars used for biofuel and biochemical production may be obtained from other sources such as cellulosic or lignocellulosic biomass.

[0004] Sugars used for biofuel and biochemical production are typically obtained by hydrolyzing biomass. Hydrolysis breaks down the polymeric sugars of biomass into monomeric sugars that can be used to produce biofuels, biochemicals and other bioproducts. Biomass can be hydrolyzed enzymatically or chemically.

[0005] Enzymatic hydrolysis of cellulose and hemicellulose in biomass produces a reaction mixture, known as a hydrolysate, which includes monomeric sugars. The sugar titer of the hydrolysate has been observed to depend, in part, on the solid loading of the biomass. For example, it has been observed that a biomass solid loading greater than 20% w/w can have an inhibitory effect on enzymatic hydrolysis. See Hodge DB et al., Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis oflignocellulose, Bioresource Technology 99 (2008) pp. 8940-8948.

[0006] The sugars obtained from hydrolysis of biomass can then be fermented or chemically processed to produce biofuels, biochemicals and other bioproducts. For example, Knauf and Kraus have shown that ethanol fermentation can produce up to 17% (v/v) ethanol. See Knauf M and Kraus K, Specific yeasts developed for modern ethanol production, Sugar Industry, vol. 131 (2006), pp. 753-758. To achieve the ethanol concentration described in Knauf and Kraus, however, a solid loading of biomass in an enzymatic hydrolysis and fermentation greater than the practical limit identified by Hodge et al. may be required. Thus, what is needed in the art is a hydrolysate that can be used for fermentation to produce biofuels, biochemicals and other bioproducts that has a high sugar content, without a high solid loading of the biomass in hydrolysis or in combined hydrolysis and fermentation.

[0007] Additionally, handling and storing a cellulosic sugar solution for biofuel and biochemical production can increase the risk of microorganism contamination due to the water content in sugar solution. What is also needed is a solid hydrolysate that can be used for fermentation or chemical processing to produce biofuels, biochemicals and other bioproducts, and that can be easily stored, handled and transported.

[0008] The hydrolysate can also be concentrated to improve ease of use, transportation and storage. However, current methods to concentrate hydrolysates typically result in significant sugar loss. See e.g., Clarke, M.A., et al., Adv. in Carbohydrate Chem. and Biochem., Vol. 52, pp. 441-470 (1997). Thus, what is also needed in the art is a method to concentrate a hydrolysate without significant sugar loss.

BRIEF SUMMARY

[0009] The present disclosure address a need in the art by providing a solid hydrolysate that has a higher sugar composition and improved bulk handling properties, including a faster rate of dissolution, in comparison with the sugar solutions or solid sugars currently used in the art for production of biofuels, biochemicals and other bioproducts. Provided herein are also methods for preparing such a solid hydrolysate from hgnocellulosic biomass. The present disclosure also provides methods for preparing a concentrated hydrolysate from hgnocellulosic biomass in a way that minimizes the amount of sugar lost from concentrating the hydrolysate. The hgnocellulosic biomass used in the methods described herein may include, for example, woody biomass (e.g., softwood, hardwood) or herbaceous biomass (e.g., switchgrass). Furthermore, the hgnocellulosic biomass may be pretreated, such as sulfite- or bisulfite-pretreated.

[0010] One aspect of the disclosure provides a solid Hgnocellulosic hydrolysate that includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose, in which the glucose, xylose, mannose, arabinose and galactose are at least 50% by weight of the total weight of the solid Hgnocellulosic hydrolysate. In one embodiment, the glucose, xylose, mannose, arabinose and galactose are at least 55% by weight of the total weight of the solid Hgnocellulosic hydrolysate.

[0011] In some embodiments, the solid Hgnocellulosic hydrolysate has a solubility of at least 0.25 g/mL in water. In other embodiments, the solid Hgnocellulosic hydrolysate has a solubility of at least 0.4 g/mL in water.

[0012] In some embodiments, the solid Hgnocellulosic hydrolysate has a rate of dissolution of at least 0.01 moles sugar/kg final solution/second.

[0013] In some embodiments, less than 10% by weight relative to the total weight of the solid hgnocellulosic hydrolysate is water. In certain embodiments, less than 1 % by weight relative to the total weight of the solid Hgnocellulosic hydrolysate is water.

[0014] In some embodiments, less than 10% by weight relative to the total weight of the solid hgnocellulosic hydrolysate is lignin. In certain embodiments, less than 10% by weight relative to the total weight of the solid Hgnocellulosic hydrolysate is one or more metals. The one or more metals may be selected from calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur. In some embodiments, less than 10% by weight relative to the total weight of the solid Hgnocellulosic hydrolysate is ash. In other embodiments, less than 10% by weight relative to the total weight of the solid Hgnocellulosic hydrolysate are lignosulfonates. [0015] In some embodiments, the solid lignocellulosic hydrolysate has a lignocellulosic hydrolysate source. The solid lignocellulosic hydrolysate has a total sugar composition of monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose. The lignocellulosic hydrolysate source has a total sugar composition of polymeric glucose, polymeric xylose, polymeric mannose, polymeric arabinose and polymeric galactose. In certain embodiments, the total sugar composition of the solid lignocellulosic hydrolysate is at least 70% of the total sugar composition of the lignocellulosic hydrolysate source.

[0016] In some embodiments, the solid lignocellulosic hydrolysate is a solid softwood hydrolysate, a solid hardwood hydrolysate, a solid herbaceous biomass hydrolysate, a solid agricultural waste hydrolysate, a solid waste or recycled paper hydrolysate, or a combination thereof. In certain embodiments, the lignocellulosic hydrolysate source is a softwood, a hardwood, an herbaceous biomass, agricultural waste, waste or recycled paper, or a combination thereof.

[0017] In some embodiments, the solid lignocellulosic hydrolysate has a total monomeric sugar weight, in which the total monomeric sugar weight is between 50% and 90%, or between 55% and 85% relative to the total weight of the solid lignocellulosic hydrolysate.

[0018] In one embodiment, the solid lignocellulosic hydrolysate is a solid softwood hydrolysate. The solid softwood hydrolysate has a total monomeric sugar weight. In certain embodiments, between 50% and 70% of the total monomeric sugar weight is monomeric glucose, between 1% and 5% of the total monomeric sugar weight is monomeric xylose, between 1% and 5% of the total monomeric sugar weight is monomeric galactose, between 0.5% and 1% of the total monomeric sugar weight is monomeric arabinose, and between 1% and 5% of the total monomeric sugar weight is monomeric mannose.

[0019] In another embodiment, the solid lignocellulosic hydrolysate is a solid hardwood hydrolysate. The solid hardwood hydrolysate has a total monomeric sugar weight. In certain embodiments, between 40% and 85% of the total monomeric sugar weight is monomeric glucose, between 5% and 10% of the total monomeric sugar weight is monomeric xylose, between 0.1% and 5% of the total monomeric sugar weight is monomeric galactose, between 0.1% and 1 % of the total monomeric sugar weight is monomeric arabinose, and between 1 % and 5% of the total monomeric sugar weight is monomeric mannose.

[0020] In yet another embodiment, the solid lignocellulosic hydrolysate is a solid herbaceous biomass hydrolysate. The solid herbaceous biomass hydrolysate has a total monomeric sugar weight. In certain embodiments, between 65% and 70% of the total monomeric sugar weight is monomeric glucose, between 1% and 5% of the total monomeric sugar weight is monomeric xylose, between 1% and 5% of the total monomeric sugar weight is monomeric galactose, between 0.5% and 1% of the total monomeric sugar weight is monomeric arabinose, and between 0.5% and 1% of the total monomeric sugar weight is monomeric mannose.

[0021] In some embodiments, the solid lignocellulosic hydrolysate has a bulk density between 400 kg/m 3 and 1600 kg/m 3. In other embodiments, the solid lignocellulosic hydrolysate has a bulk density between 400 kg/m 3 and 700 kg/m 3. In yet other embodiments, the solid lignocellulosic hydrolysate has a particle size between 2 microns and 500 microns.

[0022] In certain embodiments, the solid lignocellulosic hydrolysate described herein may be used in producing one or more biofuels, biochemicals, alcohols, organic acids, amino acids, diol products, protein products, gaseous products, or lipid compounds. In one embodiment, the solid lignocellulosic hydrolysate described herein may be used in fermentation to produce a biofuel or a biochemical. In one embodiment, the solid lignocellulosic hydrolysate described herein may be used in biofuel production. In another embodiment, the solid lignocellulosic hydrolysate described herein may be used in producing one or more alcohols, organic acids, amino acids, diol products, protein products, gaseous products, or lipid compounds.

[0023] Another aspect provides a solid lignocellulosic hydrolysate that includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose, in which the glucose, xylose, mannose, arabinose and galactose are at least 60% by weight of the total weight of the solid lignocellulosic hydrolysate, and in which the solid lignocellulosic hydrolysate has a solubility of at least 0.25 g/mL in water. In certain

embodiments, the solid lignocellulosic hydrolysate has a solubility of at least 0.4 g/mL in water. In other embodiments, less than 40% by weight relative to the total weight of the solid lignocellulosic hydrolysate is water and lignin. [0024] Yet another aspect provides a solid lignocellulosic hydrolysate that includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose, in which the glucose, xylose, mannose, arabinose and galactose are at least 60% by weight of the total weight of the solid lignocellulosic hydrolysate, and in which less than 40% by weight relative to the total weight of the solid lignocellulosic hydrolysate is water, ash, lignin lignosulfonates and one or more metals. The one or more metals may be selected from calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur.

[0025] Yet another aspect provides a solid lignocellulosic hydrolysate that includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose, in which the glucose, xylose, mannose, arabinose and galactose are at least 60% by weight of the total weight of the solid lignocellulosic hydrolysate, and in which less than 10% by weight relative to the total weight of the solid lignocellulosic hydrolysate is water. In certain embodiments, less than 30% by weight relative to the total weight of the solid

lignocellulosic hydrolysate is ash, lignin, lignosulfonates and one or more metals. The one or more metals may be selected from calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur.

[0026] Yet another aspect provides a solid lignocellulosic hydrolysate that includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose, in which the glucose, xylose, mannose, arabinose and galactose are at least 70% by weight of the total weight of the solid lignocellulosic hydrolysate, in which less than 10% by weight relative to the total weight of the solid lignocellulosic hydrolysate is water, and in which less than 20% by weight relative to the total weight of the solid lignocellulosic

hydrolysate is lignin. In certain embodiments, the solid lignocellulosic hydrolysate has a bulk density between 400 kg/m 3 and 1600 kg/m 3. In other embodiments, the solid lignocellulosic hydrolysate has a bulk density between 400 kg/m 3 and 700 kg/m 3.

[0027] Another aspect of the disclosure provides a method of obtaining a solid

lignocellulosic hydrolysate from a lignocellulosic biomass by: a) providing a lignocellulosic biomass; b) pretreating the lignocellulosic biomass to produce a pretreatment liquor and a pretreated biomass solid; c) separating the pretreated biomass solid from the pretreatment liquor; d) hydrolyzing the pretreated biomass solid to produce a lignocellulosic hydrolysate and residual solids; e) separating the residual solids from the lignocellulosic hydrolysate; and f)

concentrating, drying and/or crystallizing the lignocellulosic hydrolysate to obtain a solid lignocellulosic hydrolysate.

[0028] In some embodiments, the method further includes filtering the pretreatment liquor after separation from the pretreated biomass solid; and combining the filtered pretreatment liquor with the pretreated biomass solid for hydrolysis in step (d) of the method described above to produce the lignocellulosic hydrolysate and the residual solids.

[0029] In other embodiments, the method further includes purifying the lignocellulosic hydrolysate after separating the residual solids and before obtaining the solid lignocellulosic hydrolysate. The purifying of the lignocellulosic hydrolysate may reduce the amount of one or more metals present in the solid lignocellulosic hydrolysate. The one or more metals may be selected from calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur. In some embodiments, the lignocellulosic hydrolysate is purified by washing, ion exchange

chromatography, active carbon filtration, filter sterilization, ultra-violet irradiation, radiation, thermal sterilization, or a combination thereof.

[0030] In other embodiments, the method further includes sizing the solid lignocellulosic hydrolysate. The solid lignocellulosic hydrolysate may be sized, for example, by using a hammer mill, a solid crusher, a sieve, or a combination thereof.

[0031] In some embodiments, the pretreatment is a sulfur dioxide treatment, sulfite treatment, bisulfite treatment, dilute acid treatment, strong acid treatment, dilute alkaline treatment, strong alkaline treatment, oxidative delignification, ozonolysis, ammonia fiber explosion (AFEX), organosolvent treatment, hot water treatment, ionic liquid treatment, steam explosion, biological incubation, or a combination thereof.

[0032] In some embodiments, the solid lignocellulosic hydrolysate includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose. In certain embodiments, the lignocellulosic biomass includes polymeric glucose, polymeric xylose, polymeric mannose, polymeric arabinose and polymeric galactose. [0033] In certain embodiments, the pretreated biomass is hydrolyzed by enzymatic hydrolysis, dilute acid hydrolysis, strong acid hydrolysis, ionic liquid hydrolysis, or a combination thereof. In one embodiment, the enzymatic hydrolysis employs cellulase, xylanase, beta-glucosidase, or a combination thereof.

[0034] In some embodiments, the residual solids include lignin. In certain embodiments, the residual solids are separated from the lignocellulosic hydrolysate by filtration, centrifugation, or a combination thereof. In one embodiment, the filtration is ultra-filtration. In other

embodiments, a filter with a molecular weight cut-off (MWCO) size of between 5 kDa and 20 kDa is used for the ultra-filtration. In certain embodiments, a filter with a molecular weight cutoff (MWCO) size of 5 kDa, 10 kDa, 15 kDa or 20 kDa is used for the ultra-filtration.

[0035] In other embodiments, the lignocellulosic hydrolysate is dried by vacuum drying, spray drying, drum drying, fluidized bed drying, or a combination thereof.

[0036] In some embodiments, the method further includes filtering the pretreatment liquor. In certain embodiments where the method includes filtering of the pretreatment liquor, the method further includes purifying the pretreatment liquor to produce a purified pretreatment liquor; and concentrating, drying or crystallizing the purified pretreatment liquor to obtain a second solid lignocellulosic hydrolysate. The pretreatment liquor may be purified, for example, by ion exchange chromatography, active carbon filtration, filter sterilization, ultra-violet irradiation, radiation, thermal sterilization, or a combination thereof. In one embodiment, the pretreatment liquor is filtered by ultra-filtration. In some embodiments, the filtering of the pretreatment liquor separates lignosulfonates from the pretreatment liquor. In other

embodiments, a filter with a molecular weight cut-off (MWCO) size of between 5 kDa and 20 kDa is used for the ultra-filtration. In certain embodiments, a filter with a molecular weight cutoff (MWCO) size of 5 kDa, 10 kDa, 15 kDa or 20 kDa is used for the ultra-filtration.

[0037] Another aspect provides a method of obtaining a solid lignocellulosic hydrolysate from a lignocellulosic biomass by: a) providing a lignocellulosic biomass; b) pretreating the lignocellulosic biomass to produce a pretreatment liquor and a pretreated biomass solid; c) separating the pretreatment liquor from the pretreated biomass solid; d) filtering the pretreatment liquor; and e) concentrating, drying and/or crystallizing the filtered pretreatment liquor to obtain a solid lignocellulosic hydrolysate. In some embodiments, the method further includes purifying the filtered pretreatment liquor before obtaining the solid lignocellulosic hydrolysate. In some embodiments, the pretreatment liquor is filtered by ultra-filtration. In some embodiments, a filter with a molecular weight cut-off (MWCO) size of between 5 kDa and 20 kDa is used for the ultra-filtration. In certain embodiments, a filter with a molecular weight cut-off (MWCO) size of 5 kDa, 10 kDa, 15 kDa or 20 kDa is used for the ultra-filtration.

[0038] Yet another aspect provides a method of obtaining a solid lignocellulosic hydrolysate from a lignocellulosic biomass by: a) providing a lignocellulosic biomass; b) pretreating the lignocellulosic biomass to produce a pretreatment liquor and a pretreated biomass solid; c) separating the pretreated biomass solid from the pretreatment liquor; d) filtering the pretreatment liquor; e) hydrolyzing the pretreated biomass solid and the pretreatment liquor to produce a lignocellulosic hydrolysate and residual solids; f) separating the residual solids from the lignocellulosic hydrolysate; and g) concentrating, drying and/or crystallizing the lignocellulosic hydrolysate to obtain a solid lignocellulosic hydrolysate. In some embodiments, the method further includes purifying the lignocellulosic hydrolysate after separating the residual solids and before obtaining the solid lignocellulosic hydrolysate. In certain embodiments, the pretreatment liquor is filtered by ultra-filtration. In some embodiments, a filter with a molecular weight cutoff (MWCO) size of between 5 kDa and 20 kDa is used for the ultra-filtration. In certain embodiments, a filter with a molecular weight cut-off (MWCO) size of 5 kDa, 10 kDa, 15 kDa or 20 kDa is used for the ultra-filtration.

[0039] In another aspect, provided is a method for preparing a concentrated hydrolysate from sulfite- or bisulfite-pretreated lignocellulosic biomass, by:

a) providing sulfite- or bisulfite-pretreated lignocellulosic biomass solids;

b) enzymatically hydrolyzing the sulfite- or bisulfite-pretreated lignocellulosic biomass solids to produce a hydrolysate, wherein the hydrolysate includes one or more sugars and residual solids;

c) separating the residual solids from the hydrolysate to form a separated

hydrolysate;

d) ultra-filtering the separated hydrolysate using a filter with a molecular weight cutoff (MWCO) size of between 3 kDa and 20 kDa to form an ultra- filtered hydrolysate; and e) concentrating the ultra-filtered hydrolysate to obtain a concentrated hydrolysate, wherein the concentrated hydrolysate has a sugar titer of at least 25% by weight. In some embodiments, the filter has a MWCO size of 3 kDa, 5 kDa, 10 kDa, 15 kDa or 20 kDa. In one embodiment, the ultra-filtered hydrolysate is concentrated at a temperature of at least 50°C. In another embodiment, the ultra-filtered hydrolysate is concentrated under vacuum. In yet another embodiment, the ultra-filtered hydrolysate is concentrated by thermal concentration or thermal- vacuum concentration. In some embodiments, the concentrated hydrolysate has a sugar titer of at least 40% by weight. In yet other embodiments, the hydrolysate produced in step (b) has a sugar titer of less than 20% by weight. In certain embodiments, the sugar titer of the

concentrated hydrolysate is at least two times greater than the sugar titer of the hydrolysate produced in step (b). In one embodiment, the concentrated hydrolysate includes one or more sugars, wherein the one or more sugars are selected from monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose, and monomeric galactose. In some embodiments, the sulfite- or bisulfite-pretreated lignocellulosic biomass solids are unwashed or washed with water. In other embodiments, step (a) of the method described above includes: providing lignocellulosic biomass; pretreating the lignocellulosic biomass with a sulfite or bisulfite pretreatment to produce a pretreatment liquor and sulfite- or bisulfite-pretreated lignocellulosic biomass solids; and separating the sulfite- or bisulfite-pretreated lignocellulosic biomass solids from the pretreatment liquor. In one embodiment, the lignocellulosic biomass is selected from softwood, hardwood, herbaceous biomass, agricultural waste, waste paper, recycled paper, and any combination thereof. Additionally, in other embodiments, step (a) of the method may further include: ultra-filtering the pretreatment liquor using a filter with a molecular weight cut-off (MWCO) size of between 3 kDa and 20 kDa; and combining the ultra-filtered pretreatment liquor with the sulfite- or bisulfite-pretreated lignocellulosic biomass solids for enzymatic hydrolysis in step (b). In yet other embodiments, the residual solids are separated from the hydrolysate by coarse filtering, centrifugal separation, or a combination thereof. In yet other embodiments, the method may further include: purifying the ultra-filtered hydrolysate to remove color pigments and/or metal ions before concentrating. In yet other embodiments, the method may further include: concentrating, drying and/or crystallizing the concentrated hydrolysate to obtain a solid hydrolysate; and optionally sizing the solid hydrolysate. In some embodiments, the method further includes washing the separated residual solids from step (c) to recover one or more monomeric sugars, wherein the washing produces a residual sugar stream and a residual solid stream, wherein the residual solid stream has one or more monomeric sugars, and wherein the residual solid stream has residual lignin. In other embodiments, the method further includes ultra-filtering the residual sugar stream using a filter with a molecular weight cut-off (MWCO) size of between 3 kDa and 20 kDa to form an ultra-filtered hydrolysate; and concentrating the ultra-filtered residual sugar stream to obtain a concentrated residual sugar stream, wherein the concentrated residual sugar stream has a sugar titer of at least 25% by weight. In yet other embodiments, the method further includes combining the residual sugar stream with the separated hydrolysate; and ultra-filtering the combined residual sugar stream and the separated hydrolysate in step (d).

[0040] In yet another aspect, provided is a method of producing a concentrated hydrolysate, by:

a) providing a lignocellulosic biomass;

b) contacting the lignocellulosic biomass with a first dose of enzymes;

c) enzymatically hydrolyzing the lignocellulosic biomass to produce a first hydrolysate, wherein the first hydrolysate includes one or more sugars, residual solids, and the enzymes from the first dose;

d) adding additional lignocellulosic biomass to the first hydrolysate to produce a lignocellulosic mixture, wherein the lignocellulosic mixture includes the one or more sugars, the residual solids, the enzymes from the first dose, and the additional lignocellulosic biomass;

e) separating a solid fraction and a liquid fraction from the lignocellulosic mixture, wherein the solid fraction includes the residual solids, the enzymes from the first dose, and the additional lignocellulosic biomass, and wherein the liquid fraction includes the one or more sugars;

f) contacting the solid fraction with a second dose of enzymes;

g) enzymatically hydrolyzing the solid fraction to produce a second hydrolysate, wherein the second hydrolysate includes one or more additional sugars, additional residual solids, the enzymes from the first dose, and the enzymes from the second dose;

h) separating a solid fraction and a liquid fraction from the second hydrolysate, wherein the solid fraction includes the additional residual solids, the enzymes from the first dose, and the enzymes from the second dose, and wherein the liquid fraction includes the one or more additional sugars;

i) combining the liquid fraction separated from the lignocellulosic mixture in step (e) and the liquid fraction separated from the second hydrolysate to form a combined

hydrolysate;

j) ultra-filtering the combined hydrolysate using a filter with a molecular weight cut-off (MWCO) size of between 3 kDa and 20 kDa to form an ultra-filtered hydrolysate; and k) concentrating the ultra-filtered hydrolysate to obtain a concentrated hydrolysate, wherein the concentrated hydrolysate has a sugar titer of at least 25% by weight. In some embodiments, the second dose of enzymes is less than the first dose of enzymes. In certain embodiments, the second dose of enzymes is at least 20% less than the first dose of enzymes. In one embodiment, the lignocellulosic biomass is sulfite- or bisulfite-pretreated. In another embodiment, the additional lignocellulosic biomass is sulfite- or bisulfite-pretreated. In some embodiments, the sulfite- or bisulfite-pretreated lignocellulosic biomass is unwashed or washed with water. In other embodiments, the sulfite- or bisulfite-pretreated additional lignocellulosic biomass is unwashed or washed with water. In yet other embodiments, the filter has a MWCO size of 3 kDa, 5 kDa, 10 kDa, 15 kDa or 20 kDa. In one embodiment, the ultra-filtered hydrolysate is concentrated at a temperature of at least 50°C. In another embodiment, the ultra- filtered hydrolysate is concentrated under vacuum. In yet another embodiment, the ultra-filtered hydrolysate is concentrated by thermal concentration or thermal-vacuum concentration. In some embodiments, the concentrated hydrolysate has a sugar titer of at least 40% by weight. In yet other embodiments, the first hydrolysate has a sugar titer of less than 20% by weight. In certain embodiments, the sugar titer of the concentrated hydrolysate is at least two times greater than the sugar titer of the first hydrolysate. In one embodiment, the one or more sugars of the lignocellulosic mixture in step (d) are selected from monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose, and monomeric galactose. In another embodiment, the one or more sugars of the additional lignocellulosic mixture in step (g) are selected from monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose, and monomeric galactose. In some embodiments, the method further includes: coarse-filtering the combined hydrolysate before ultra-filtration in step (j). In other embodiments, the method further includes: purifying the ultra-filtered hydrolysate to remove color pigments and/or metal ions before concentrating. In yet other embodiments, the method further includes: concentrating, drying and/or crystallizing the concentrated hydrolysate to obtain a solid hydrolysate; and optionally sizing the solid hydrolysate. In some embodiments, the method further includes washing the solid fraction from step (h) to recover one or more monomeric sugars, wherein the washing produces a residual sugar stream and a residual solid stream, wherein the residual solid stream has one or more monomeric sugars, and wherein the residual solid stream has residual lignin. In other embodiments, the method further includes ultra-filtering the solid fraction from step (h) using a filter with a molecular weight cut-off (MWCO) size of between 3 kDa and 20 kDa to form an ultra-filtered hydrolysate; and concentrating the ultra-filtered residual sugar stream to obtain a concentrated residual sugar stream, wherein the concentrated residual sugar stream has a sugar titer of at least 25% by weight. In yet other embodiments, the method further includes combining the solid fraction from step (h) with the combined hydrolysate from step (i); and ultra-filtering the combined residual sugar stream and the separated hydrolysate in step j). In yet another aspect, provided is also a concentrated hydrolysate or a solid hydrolysate prepared according to any of the methods described above, for use in producing one or more products selected from biofuels, biochemicals, alcohols, organic acids, amino acids, diol products, protein products, gaseous products, and lipid compounds.

DESCRIPTION OF THE FIGURES

[0041] The present application can be best understood by reference to the following description taken in conjunction with the accompanying figures, in which like parts may be referred to by like numerals:

[0042] FIG. 1 depicts an exemplary method for converting solid pretreated lignocellulosic biomass into a solid hydrolysate;

[0043] FIG. 2 depicts an exemplary method for converting pretreatment liquor obtained from pretreatment of lignocellulosic biomass into a solid hydrolysate;

[0044] FIG. 3 depicts an exemplary method for converting solid pretreated lignocellulosic biomass and its pretreatment liquor into a solid hydrolysate; [0045] FIG. 4 depicts an exemplary method for converting solid pretreated lignocellulosic biomass into a concentrated hydrolysate;

[0046] FIG. 5 depicts an exemplary method for converting lignocellulosic biomass into a concentrated hydrolysate that involves enzyme recycling;

[0047] FIG. 6 is a graph depicting glucose titer and total sugar titer from hydrolysis of lignocellulosic biomass involving enzyme recycling;

[0048] FIG. 7 is a graph depicting sugar titers over time from ethanol fermentation of concentrated hydrolysate with Saccharomyces cerevisiae D5A; and

[0049] FIG. 8 is a graph depicting sugar titers over time from ethanol fermentation of concentrated hydrolysate with Saccharomyces cerevisiae Bio-Ferm® XR.

DETAILED DESCRIPTION

[0050] To provide a more thorough understanding of the present disclosure, the following description sets forth numerous specific details, such as specific configurations, parameters, examples, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is intended to provide a better description of the exemplary embodiments.

[0051] The following description relates to preparing hydrolysates from enzymatic hydrolysis of lignocellulosic biomass, such as woody biomass (e.g., softwood, hardwood), herbaceous biomass (e.g. , switchgrass), or waste or recycled paper. The hydrolysates may be in the form of a solid hydrolysate or a concentrated hydrolysate.

[0052] In some embodiments, the hydrolysate is a solid hydrolysate. With reference to FIG. 1, method 100 is an exemplary embodiment that depicts the conversion of pretreated

lignocellulosic biomass into a solid hydrolysate. The lignocellulosic biomass used in method 100 includes, for example softwood, hardwood, and/or switchgrass. In step 102, the

lignocellulosic biomass undergoes pretreatment to generate a pretreated biomass solid and a pretreatment liquor. Any pretreatment methods known in the art may be used in method 100, including for example sulfur dioxide treatment, sulfite treatment, bisulfite treatment, dilute acid treatment, strong acid treatment, dilute alkaline treatment, strong alkaline treatment, oxidative delignification, ozonolysis, ammonia fiber explosion (AFEX), organosolvent treatment, hot water treatment, ionic liquid treatment, steam explosion, biological incubation with fungi, yeast and/or bacteria, and enzymatic treatment. A combination of pretreatment methods may also be used.

[0053] In step 104, the pretreated biomass solid is enzymatically hydrolyzed to breakdown the polymeric sugars into monomeric sugars. For example, in one embodiment, a suitable pH for enzymatic hydrolysis is around pH 5.3. It should be understood, however, that the pH for enzymatic hydrolysis may vary in other embodiments. Additionally, in other exemplary embodiments, the hydrolysis of the pretreated biomass solid may be accomplished by dilute or strong acid hydrolysis, or by hot water hydrolysis.

[0054] It should be understood that prior to hydrolysis, several optional steps may be performed to improve hydrolysis. In other embodiments, the pH of the pretreated biomass solid obtained from step 102 may be adjusted, and/or the pretreated biomass solid may be washed with water to reduce the levels of inhibitors that may affect hydrolysis or the quality of the hydrolysate product. For example, the pretreated biomass solids may be washed with water, in an amount about 1 to 4 times the solid weight of the pretreated biomass solids.

[0055] Hydrolysis in step 104 produces a hydrolysate, which may include one or more sugars as well as unhydrolyzed or undigested cellulose fiber residuals and lignin. In step 106, the residuals and lignin are separated from the hydrolysate by filtration and/or clarification. By using a sufficiently small screen size, filtration can remove lignosulfonates and/or other high molecular weight components, which may act as potential inhibitors downstream in fermentation to produce biofuels (e.g., ethanol) or biochemicals. Clarification can also remove larger residuals from the hydrolysate. The residuals and lignin exiting step 106 may also be pressed and counter-current-washed or sequentially washed with water to remove excessive monomeric sugars from the residuals. The recovered permeate from residual pressing and washing can be recycled back to the filtration step 106, followed by the purification 108 and concentration step 110. Thus, more sugar can be recovered from the residuals and the resulting residuals are richer in lignin. [0056] In step 108, the separated hydrolysate is purified to remove color pigment and/or metal ions. Any methods known in the art to purify a hydrolysate may be used, including for example an active carbon column and an ion exchange column. A combination of methods to purify the separated hydrolysate may also be used.

[0057] In steps 110 and 112, the purified hydrolysate is concentrated and then dried to produce a solid hydrolysate. Any suitable methods known in the art to concentrate a hydrolysate may be used. For example, a hydrolysate can be concentrated using a multi-effect evaporator. Any suitable methods known in the art to dry the concentrated hydrolysate may be used, including for example vacuum drying, spray drying, drum drying, and fluidized bed drying. A combination of drying methods may also be used.

[0058] It should be understood that, in other exemplary embodiments, the concentrated hydrolysate may be crystallized rather than dried to produce a solid hydrolysate. In yet other exemplary embodiments, the hydrolysate is concentrated, crystallized and dried to produce a solid hydrolysate.

[0059] In step 114, the solid hydrolysate is sized to a particular particle size range that may be needed for subsequent processing (e.g. , fermentation to produce biofuels or biochemicals). It should be understood, however, that one or more steps may be omitted or added from method 100. For example, in other exemplary embodiments, step 114 may be omitted if the particle size of the solid hydrolysate obtained from steps 102-112 is suitable for subsequent processing (e.g., fermentation to produce biofuels or biochemicals).

[0060] With reference again to FIG. 1, in step 102, the pretreatment liquor obtained from the pretreatment of the biomass can be further processed to also obtain a solid hydrolysate.

[0061] With reference to FIG. 2, process 200 is an exemplary embodiment that depicts the conversion of the hydrolyzed sugars in the pretreatment liquor into a solid hydrolysate. In step 202, lignocellulosic biomass undergoes pretreatment to generate a pretreated biomass solid and a pretreatment liquor. In step 204, the pretreatment liquor is ultra-filtered to remove

lignosulfonates and other high molecular weight components. The filtered pretreatment liquor is then purified in step 206 to remove color pigment and/or metal ions. In steps 208 and 210, respectively, the purified pretreatment liquor is concentrated and dried to produce a solid hydrolysate that can be subsequently sized (step 212).

[0062] It should be understood, however, that one or more steps may be omitted or added from method 200. For example, in other exemplary embodiments, crystallization may be an additional step in combination with concentration and drying to produce a solid hydrolysate, or crystallization may replace drying in step 210.

[0063] With reference to FIG. 3, method 300 depicts an exemplary embodiment in which pretreated biomass and pretreatment liquor are combined and converted into a solid hydrolysate. In step 302, the lignocellulosic biomass undergoes pretreatment to generate a pretreated biomass solid and a pretreatment liquor. The pretreatment liquor is separated from the pretreated biomass solids, and is ultra-filtered in step 304. Both the pretreated biomass solids and the filtered pretreatment liquor are combined for hydrolysis in step 306. While step 306 involves enzymatic hydrolysis, it should be understood that in other exemplary embodiments, the pretreated biomass solids and filtered pretreatment liquor may be hydrolyzed by dilute or strong acid hydrolysis, or by hot water hydrolysis.

[0064] It should be understood, however, that one or more steps may be omitted or added from method 300. For example, in some embodiments, several optional steps may be performed prior to hydrolysis to improve hydrolysis. For example, in one embodiment, the pH of the pretreatment liquor before and/or after ultra-filtration (step 304) may be adjusted. In another embodiment, the pH of the pretreatment liquor obtained from step 302 is adjusted to

approximately pH 5, and the pretreatment liquor is ultra- filtered to remove lignosulfonates in step 304. In yet other embodiments, the pH of the filtered pretreatment liquor may be further adjusted to approximately pH 10, and mixed with the pretreated biomass solid obtained from step 302 to give a final pH of 5.3 for hydrolysis in step 306.

[0065] Hydrolysis in step 306 produces a hydrolysate, which includes one or more sugars as well as unhydrolyzed or undigested cellulose fiber residuals and lignin. In step 308, filtration removes the residual cellulose and lignin from the hydrolysate, as well as lignosulfonates and/or other high molecular weight components. In step 310, the separated hydrolysate is then purified to remove color pigment and/or metal ions. In steps 312 and 314, the purified hydrolysate is then concentrated and dried to produce a solid hydrolysate. In addition, the residuals exiting step 308 can be pressed and counter-current-washed or sequentially washed with water to remove excessive monomeric sugars from the residuals. The recovered permeate from residual pressing and washing can be recycled back to the filtration step 308, followed by the purification step 310 and concentration step 312. Thus, more sugar can be recovered from the residuals and the resulting residuals are richer in lignin.

[0066] It should be understood that, in other exemplary embodiments, crystallization can replace drying or be an additional step in combination with concentration and drying to produce a solid hydrolysate.

[0067] In other embodiments, the hydrolysate is a concentrated hydrolysate. With reference to FIG. 4, method 400 is an exemplary embodiment that depicts the conversion of pretreated lignocellulosic biomass into a concentrated hydrolysate. The lignocellulosic biomass used in method 400 may include, for example, woody biomass (e.g. , softwood, hardwood), herbaceous biomass (e.g., switchgrass), or waste or recycled paper. In step 402, the lignocellulosic biomass undergoes pretreatment to generate pretreated biomass solids and a pretreatment liquor.

Pretreatment methods suitable for step 402 may include, for example, sulfite or bisulfite pretreatment. A combination of pretreatment methods may also be used.

[0068] In step 404, the pretreated biomass solids are enzymatically hydrolyzed to breakdown the polymeric sugars into monomeric sugars. It should be understood that prior to hydrolysis, several optional steps may be performed to improve hydrolysis. In other embodiments, the pH of the pretreated biomass solids obtained from step 402 may be adjusted, and/or the pretreated biomass solids may be washed with water to reduce the levels of inhibitors that may affect hydrolysis or the quality of the hydrolysate product. For example, the pretreated biomass solids may be washed with water, in an amount about 1 to 4 times the solid weight of the pretreated biomass solids.

[0069] Hydrolysis in step 404 produces a hydrolysate, which includes one or more sugars, as well as unhydrolyzed or undigested cellulose fiber residuals and lignin. In step 406, residual solids are separated from the hydrolysate. This separation step may involve coarse filtration and/or a centrifugal solid separation to remove any insoluble coarse particles or solids before ultrafiltration in step 408. The ultra-filter may have a MWCO size of between 3 kDa and 20 kDa. Ultra-filtration removes high molecular weight components, extractives and lignin derivatives from the hydrolysate. In step 410, the ultra-filtered hydrolysate is concentrated to obtain a concentrated hydrolysate. Concentration may be performed at elevated temperatures, for example, at temperatures of at least 50°C. Additionally, concentration may also be performed under vacuum. The concentrated hydrolysate obtained from method 400 has a sugar titer of at least 25% by weight. In addition, the residual solids exiting step 406 can be pressed and counter-current-washed or sequentially washed with water to remove excessive monomeric sugars from the residuals. The recovered permeate from residual pressing and washing can be recycled back to the ultra-filtration step 408, followed by the concentration step 410. Thus, more sugar can be recovered from the residuals and the resulting residual solids are richer in lignin.

[0070] It should be understood, however, that one or more steps may be omitted or added from method 400. As depicted in optional step 412 (denoted by the dotted lines), the

pretreatment liquor obtained from pretreatment in step 402 may be ultra- filtered and combined with the pretreated biomass solids for hydrolysis in step 404. However, it should be understood that enzymatic hydrolysis of the pretreated biomass solids can be with or without combining the ultra-filtered pretreatment liquor.

[0071] Additionally, the hydrolysate may be coarse-filtered before ultra-filtration in step 408. The ultra-filtered hydrolysate may also be purified before concentration in step 410 to remove color pigments and/or metal ions. In other exemplary embodiments, the concentrated

hydrolysate obtained according to method 400 may be concentrated, dried and/or crystallized to obtain a solid hydrolysate, which may be optionally sized. The solid hydrolysate can be transported or stored for later use (e.g. , in a subsequent fermentation process).

[0072] In other exemplary embodiments, the methods described herein to convert biomass into a solid hydrolysate or a concentrated hydrolysate may employ enzyme recycling to maximize the efficiency of each enzyme dose. With reference to FIG. 5, method 500 depicts an exemplary method that involves enzyme recycling to produce a concentrated hydrolysate.

[0073] The lignocellulosic biomass used in method 500 may include, for example, woody biomass (e.g., softwood, hardwood), herbaceous biomass (e.g. , switchgrass), or waste or recycled paper. In step 510, the lignocellulosic biomass is pretreated. In one embodiment, for example, the lignocellulosic biomass may be pretreated using a sulfite or bisulfite pretreatment. A combination of pretreatment methods may also be used.

[0074] In step 512, a first dose of enzymes is mixed with the pretreated lignocellulosic biomass for enzymatic hydrolysis to breakdown the polymeric sugars in the lignocellulosic biomass into monomeric sugars. It should be understood that prior to hydrolysis, several optional steps may be performed to improve hydrolysis or the quality of the hydrolysate product. In other embodiments, the pH of the pretreated lignocellulosic biomass may be adjusted, and/or the pretreated lignocellulosic biomass may be washed with water to reduce the levels of inhibitors that may affect hydrolysis. For example, the pretreated lignocellulosic biomass may be washed with water, in an amount about 1 to 4 times the solid weight of the pretreated lignocellulosic biomass.

[0075] Hydrolysis in step 512 produces a first hydrolysate, which includes one or more sugars, as well as unhydrolyzed or undigested cellulose fiber residuals and lignin. Additional lignocellulosic biomass is added to and mixed with the first hydrolysate in step 514 to produce a lignocellulosic mixture. The additional lignocellulosic biomass may be the same or different type as the initial lignocellulosic biomass, and the amount of additional lignocellulosic biomass added may be the same or different as the initial lignocellulosic biomass added. The additional lignocellulosic biomass may be unwashed or washed with water. Mixing helps promote the binding of enzymes in the hydrolysis mixture to the additional biomass. Mixing may also be followed by an incubation period to allow binding of the enzymes from the first dose to the additional lignocellulosic biomass.

[0076] In step 516, the lignocellulosic mixture is then separated into a liquid fraction and a solid fraction. The liquid fraction includes the one or more sugars, and the solids fraction includes the residual solids, the additional lignocellulosic biomass and at least a portion of the enzymes from the first dose.

[0077] In step 518, a second dose of enzymes is added to the solids fraction for enzymatic hydrolysis to breakdown the polymeric sugars in the solid fraction of the lignocellulosic biomass into monomeric sugars. In this exemplary embodiment, the second dose of enzymes is less than the first dose of enzymes. In other exemplary embodiments, the second dose of enzymes may be the same as the first dose of enzymes. Hydrolysis in step 518 produces a second hydrolysate, which includes one or more additional sugars as well as residual solids of unhydrolyzed materials. In step 520, the residual solids are separated from the second hydrolysate. In addition, the residual solids exiting step 520 can be pressed and counter-current-washed or sequentially washed with water to remove excessive monomeric sugars from the residuals. The recovered permeate from residual pressing and washing can be recycled back to the ultrafiltration step 522, followed by the concentration step 524. Thus, more sugar can be recovered from the residuals and the resulting residual solids are richer in lignin.

[0078] The first and second hydrolysates separated from steps 516 and 520, respectively, are combined and then ultra- filtered in step 522. The ultra-filter may have a MWCO size of between 3 kDa and 20 kDa. As described above, ultra-filtration removes high molecular weight extractives and lignin derivatives from the hydrolysate. In step 524, the ultra-filtered hydrolysate is concentrated to obtain a concentrated hydrolysate. Concentration may be performed at elevated temperatures of at least 50°C. Additionally, concentration may be performed under vacuum. The concentrated hydrolysate obtained from method 500 has a sugar titer of at least 25% by weight.

[0079] It should be understood, however, that one or more steps may be omitted or added from method 500. For example, the ultra-filtered hydrolysate may also be purified before concentration in step 524 to remove color pigments and/or metal ions. In other exemplary embodiments, the concentrated hydrolysate obtained according to method 500 may be concentrated, dried and/or crystallized to obtain a solid hydrolysate, which may be optionally sized. The solid hydrolysate can be transported or stored for later use (e.g., in a subsequent fermentation process).

[0080] As used herein, the term "about" refers to an approximation of a stated value within an acceptable range. Preferably, the range is +/- 10% of the stated value. Lignocellulosic Biomass

[0081] The lignocellulosic biomass used to prepare a solid or concentrated hydrolysate in the methods described herein can be plant material that is made up of organic compounds relatively high in oxygen, such as carbohydrates, and may also contain a wide variety of other organic compounds.

[0082] Lignocellulosic biomass is a type of biomass that is made up of cellulose and hemicellulose bonded to lignin in plant cell walls. Lignocellulosic biomass can be grouped into four main categories: agricultural residues (e.g. , corn stover, wheat straw, rice straw, sugarcane bagasse), dedicated energy crops (e.g. , sugarcane, switchgrass), wood and wood residues (e.g., sawmill residuals, urban wastewood, pulp or paper mill screen rejects or fines, softwood chips, hardwood chips), and municipal paper waste. Any source of lignocellulosic biomass can be used, and some typical examples are described herein. Lignocellulosic biomass may originate from a woody biomass (e.g. softwood, hardwood) or an herbaceous biomass (e.g., switchgrass). Wood chips and bark materials from these sources can be used as a suitable biomass for the methods described herein.

[0083] As used herein, "a hydrolysate source" refers to the biomass source from which a solid or concentrated hydrolysate may be obtained. In some embodiments, the hydrolysate source is lignocellulosic biomass, which has a total sugar composition of polymeric glucose, polymeric xylose, polymeric mannose, polymeric arabinose and polymeric galactose. The total sugar composition may vary from one type of lignocellulosic biomass to another.

Pretreatment

[0084] Pretreatment of lignocellulosic biomass refers to one or more physical, chemical, physicochemical or biological methods to make cellulose and/or hemicellulose in the biomass more available for hydrolysis to produce monomeric sugars. Digestibility of cellulose in lignocellulosic biomass is hindered by various physicochemical, structure and compositional factors. As such, pretreatment of lignocellulosic biomass can help facilitate hydrolysis for sugar production. Pretreatment of lignocellulosic biomass can expose the cellulose and/or

hemicellulose in the plant fibers by breaking down the lignin structure and disrupting the crystalline structure of cellulose and/or hemicellulose, thereby making the biomass more accessible for hydrolysis.

[0085] Unless indicated otherwise, a pretreatment does not include further processing steps such as separation of solid and liquid phases of the pretreatment product, or rinsing or conditioning of the solid or liquid product phases.

[0086] Physical pretreatment methods often involve size reduction to reduce the physical size of biomass. Numerous physical pretreatment methods are known in the art. Examples include chipping, grinding, shredding, chopping, milling, and pyrolysis. In one embodiment, biomass sizing may be employed as a physical pretreatment method to reduce the size of the wood chip to improve the time or temperature for hydrolysis. For woody feedstock in particular, biomass sizing is an effective practice for reducing inhibitors. Biomass sizing may reduce any conditioning requirement of the pretreatment liquor, better enabling it to serve as a diluent for hydrolysis.

[0087] Chemical pretreatment methods often involve removing chemical barriers to allow enzymes to access the cellulose for microbial destruction. Numerous chemical pretreatment methods are known in the art. Examples include acid hydrolysis, alkaline hydrolysis, ozonolysis, oxidative delignification, organic solvents, ionic liquids (IL), electrolyzed water, sulfite or bisulfite pulping, kraft pulping, and green liquor. In certain embodiments, the lignocellulosic biomass used in the methods described herein has been sulfite or bisulfite pretreated.

[0088] Physicochemical pretreatment methods include, for example, steam explosion with or without sulfur dioxide, ammonia fiber explosion (AFEX), and carbon dioxide explosion.

[0089] Biological pretreatment methods include, for example, various types of rot fungi (e.g. , brown-, white-, and soft-rot fungi). Examples of other pretreatment methods include pulsed- electric-field pretreatment (PEF).

[0090] Applying one or more of the pretreatment methods described above to lignocellulosic biomass produces a pretreatment biomass composition, which can be separated into pretreatment liquor and pretreated biomass solids. a) Pretreated biomass solids

[0091] Pretreated biomass solids make up the solid fraction produced from pretreatment of lignocellulosic biomass. Pretreated biomass solids are typically rich in cellulose. The pretreated biomass solids may contain inhibitors and have a different pH from the enzymatic hydrolysis pH and the fermentation pH. As a result, the pretreated biomass solids may be conditioned before hydrolysis or fermentation.

[0092] With reference to FIGS. 1 and 4, in some exemplary embodiments, the pretreated biomass solids are separated from the pretreatment liquor, and then hydrolyzed to convert the polymeric sugars in the pretreated biomass solids into monomeric sugars in the hydrolysate. It should be understood that the biomass used in any of the methods described herein may be conditioned before hydrolysis or fermentation.

[0093] In some embodiments, the pH of pretreated biomass solids is adjusted prior to hydrolysis. Any suitable techniques to adjust the pH of the pretreated biomass solids to a suitable condition for hydrolysis may be employed. Examples include the use of buffers. In some embodiments, the pH of pretreated biomass solids may be adjusted to a range of 4-8. In certain embodiments, the pH of the pretreated biomass solids may be adjusted to 4-6.5. In certain embodiment, the pH of pretreated biomass solids is adjusted to a pH of about 5.0-5.3. In one embodiment, the pH of pretreated biomass solids is adjusted to a pH of about 5.3.

[0094] In other embodiments, the pretreated biomass solids are washed to remove hydrolysis and fermentation inhibitors or inhibitors that may affect the quality of the hydrolysate product. For example, the pretreated biomass solids may be washed with water. In instances where the pretreated biomass solids are transported or stored before hydrolysis, washing can also promote safer material storage and transportation. Pretreated biomass solids may be washed with various solvents, including for example water. If pretreated biomass solids are not washed, pretreated biomass solids may be mixed with the pretreatment liquor for safer material storage and transportation. In other embodiments, the pretreated biomass solids are unwashed. b) Pretreatment liquor

[0095] Pretreatment liquor, also known as prehydrolysate, is the liquid fraction produced from pretreatment of lignocellulosic biomass. The pretreatment liquor is typically rich in hemicellulose sugars and/or hemicellulose oligomers, along with lignin (and/or lignosulfonate in the case of sulfite pulping), extractives, furans, aldehydes, acetic acid, or other inhibitors that may restrict the growth and productivity of a fermenting organism.

[0096] The pretreatment liquor may have a pH range outside of the typical enzymatic hydrolysis pH range or typical fermentation pH range. As a result, in some embodiments, the pH of the pretreatment liquor may be adjusted prior to hydrolysis or fermentation. Moreover, in other embodiments, pretreatment liquor may be isolated from the pretreated biomass solids and used in a separate process for biofuel production, bioproduct production, or biogas production.

[0097] With reference to FIG. 2, in one exemplary embodiment, the pretreatment liquor can be separated from the pretreated biomass solids in the pretreatment process, and further processed to produce a solid hydrolysate. In some embodiments, the pretreatment liquor is filtered (e.g. , by ultra-filtration) and purified to remove non-sugar constituents such as

Hgnosulfonates, other high molecular weight components and metals, and then concentrated, dried and/or crystallized to form a solid hydrolysate.

[0098] With reference to FIGS. 3 and 4, in other exemplary embodiments, the pretreatment liquor may be filtered (e.g. , by ultra-filtration) and combined with the pretreated biomass solids for hydrolysis. The hydrolysate produced from the hydrolysis of the pretreatment liquor and pretreated biomass solids can then be filtered and purified to remove non-sugar constituents such as Hgnosulfonates, other high molecular weight components and metals, and then concentrated to form a concentrated hydrolysate, or further dried and/or crystallized to form a solid hydrolysate.

Hydrolysis

[0099] Hydrolysis breaks down the polymeric sugars of the lignocellulosic biomass into monomeric sugars that can be used to prepare biofuels, biochemicals or other bioproducts. [0100] "Hydrolysate" refers to the reaction mixture formed from enzymatic hydrolysis of biomass, which includes one or more monomeric sugars resulting from breaking down at least a portion of oligosaccharides in the biomass and residual solids. The residual solids may include unhydrolyzed or undigested cellulose fibers and lignin.

[0101] "Lignocellulosic hydrolysate" refers a hydrolysate obtained from lignocellulosic biomass. The lignocellulosic hydrolysate is typically rich in monomeric sugars.

[0102] Any suitable methods known in the art to hydrolyze biomass may be used. For example, hydrolysis may be performed enzymatically or chemically. a) Enzymatic hydrolysis

[0103] In some embodiments, hydrolysis is performed enzymatically. Hydrolysis enzymes catalyze the conversion of biomass into monomeric and/or oligomeric sugars. Any suitable hydrolysis enzymes may be used in the methods described herein, including for example cellulases, beta-glucosidases, xylanases, endoxylanases, β-xylosidases, arabinofuranosidases, glucuronidases, and acetyl xylan esterases. Combinations of enzymes (i.e., enzyme cocktails) can also be tailored to the structure of a specific biomass feedstock to increase the level of hydrolysis and degradation.

[0104] In some embodiments, the hydrolysis enzyme(s) are applied to pretreated biomass solids that are washed or unwashed. In other embodiments, the hydrolysis enzyme(s) are applied to the pretreated biomass solids with or without the pretreatment liquor. In yet another embodiment where the pretreated biomass solids are in the form of a pulp cake or sheet, a concentrated enzyme is sprayed or spread onto the pulp cake or sheet.

[0105] In some embodiments, the hydrolysis enzyme(s) are applied to pretreated biomass in a way that achieves a roughly uniform distribution of enzymes. For example, the hydrolysis enzyme(s) may be sprayed onto the pretreated biomass to achieve uniform application. The methods described in U.S. Application Serial No. 12/816999 (filed June 16, 2010) may be used to spray one or more hydrolysis enzymes onto pretreated biomass. In other embodiments, the hydrolysis enzyme(s) may be applied to pretreated biomass in a mixing tank with agitation and circulation by an agitator or a circulation pump. [0106] In applying one or more hydrolysis enzymes to pretreated biomass, various enzyme doses may be used. As used herein, enzyme dose (or enzyme dosage) refers to the amount of enzyme protein or enzyme product used. Enzyme dose is typically expressed as milligram (mg) or gram (g) of enzyme protein or enzyme product per dry gram of pretreated biomass. In one embodiment, 0.14 g of enzyme product per gram of pretreated biomass (dry basis) may be applied for the enzymatic hydrolysis. In another embodiment, 0.06 g of enzyme product per gram of pretreated biomass (dry basis) may be applied for the enzymatic hydrolysis. Further in another embodiment, 0.04 g of enzyme product per gram of pretreated biomass (dry basis) may be applied for the enzymatic hydrolysis. In another embodiment, 0.02 g of enzyme product per gram of pretreated biomass (dry basis) may be applied for the enzymatic hydrolysis. It should be understood that the enzyme product can vary depending on source and enzyme product concentration.

[0107] One or more enzyme doses may be used in enzymatic hydrolysis. For example, multiple enzyme doses may be used when enzyme recycling is employed in the methods described herein to maximize the efficiency of each enzyme dose. Enzyme recycling typically involves first adding biomass to the hydrolysate after an initial hydrolysis step. This additional biomass is mixed and/or incubated with the hydrolysate, which allows for recycling of the enzymes present in the hydrolysate. The amount of time that the additional biomass and the hydrolysate may be incubated for may vary depending on the amount and type of biomass added, the enzyme dose, and the conditions for mixing and/or incubation. For example, in certain embodiments, the incubation time may be 5 minutes to 50 minutes, 10 minutes to 30 minutes, or about 20 minutes. After mixing and/or incubation of the hydrolysate with the additional biomass, the solid and liquid fractions are separated. The liquid fraction typically includes the one or more sugars, whereas the solid fraction includes any residual undigested biomass materials, at least a portion of the enzymes initially added, and the additional biomass that was added. An additional dose of enzymes is then added to the solid fraction to further hydrolyze the biomass in the solid fraction.

[0108] This process of adding biomass to the hydrolysate to form a lignocellulosic mixture, separating the liquid and solid fractions from the lignocellulosic mixture, and adding another dose of enzymes to the solid fraction for further hydrolysis may be repeated any number of times. For example, in some embodiments, enzymatic recycling may be repeated once, twice, three times, four times, or five or more times.

[0109] In certain embodiments, the one or more additional doses of enzymes may be less than the first dose. For example, in one embodiment, the one or more additional doses is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% less than the first dose of the hydrolysis enzymes. In other embodiments, the additional doses of enzymes may be the same as the first dose.

[0110] Enzymatic hydrolysis may be carried out at a pH of 4.0 to 8.0, or between 4.0 to 6.5, or between 5.0 to 5.3. When enzymatic recycling is employed, each of the subsequent hydrolyses may occur at the same or similar pH as the initial hydrolysis.

[0111] The duration for enzymatic hydrolysis may vary depending on the lignocellulosic source, the enzyme(s) used, the enzyme dosage, and the solid loading of pretreated biomass. In some embodiments, enzymatic hydrolysis may be performed in 24 hours, 48 hours, 72 hours, or 96 hours. When enzymatic recycling is employed, each of the subsequent hydrolyses may occur at a duration that is the same or similar duration as the initial hydrolysis. b) Chemical hydrolysis

[0112] In other embodiments, hydrolysis is performed chemically. Suitable chemical hydrolysis methods may include, for example, dilute acid hydrolysis (using organic acids or inorganic acids), strong acid hydrolysis (using organic acids or inorganic acids), hot water hydrolysis, or hydrolysis mediated with ionic liquid.

Separation

[0113] After hydrolyzing the biomass, residual solids may be separated from the

hydrolysated to form a separated hydrolysate. "Separated hydrolysate" refers to the hydrolysate that remains after residual solids have been removed.

[0114] It should be understood that the residual solids may be further washed to recover sugars. For example, the residual solids may be washed by sequential washing, or counter- current washing. Additionally, in certain embodiments, the residual solids may be washed with water. Washing may generate a residual sugar stream, as well as a residual lignin-rich solid stream. The residual sugar stream may be ultra- filtered and concentrated to produce a concentrated sugar stream, or may also be combined with the separated hydrolysate for ultrafiltration and concentration.

[0115] In some embodiments, filtration may be used in the methods described herein to remove undigested pretreated biomass, insoluble lignin and other materials. Filtration may be applied to the hydrolysate, the pretreatment liquor, and/or washing eluent obtained from washing pretreated lignocellulosic biomass solids.

[0116] Any suitable methods known in the art, or a combination of methods, may be used for filtration. For example, a coarse or fine filter may be used. A coarse filter may be used to remove the larger insoluble particles, whereas a fine filter may be used to remove the smaller insoluble particles. In one embodiment, an ultra-filter is used. An ultra-filter may be used to remove impurities, such as lignosulfonates. The ultra-filter may have a molecular-weight-cut-off (MWCO) size of 3 kDa, 5 kDa, 10 kDa, 15 kDa or 20 kDa. In one embodiment, an ultra-filter with a MWCO of about 10 kDa is used.

[0117] Ultra-filtration removes certain compounds that may contribute to sugar

decomposition under the conditions described herein for concentrating the hydrolysate. For example, in some embodiments, ultra-filtration of a hydrolysate obtained from enzymatic hydrolysis of sulfite- or bisulfite-pretreated biomass removes calcium impurities formed from pretreatment that may catalyze sugar decomposition, affecting sugar titer of the concentrated product. Such calcium impurities may include calcium bisulfite and calcium lignosulfonate. Additionally, ultra-filtration removes some of the large molecular components, extractives and lignosulfonate in the hydrolysate that may increase viscosity in the end product. Thus, the resulting concentrated hydrolysate is less viscous, while having a higher sugar titer than a hydrolysate concentrated without ultra-filtration.

Purification

[0118] In some embodiments, purification may be used in the methods described herein to remove color pigments and metal ions. Purification may be applied to the hydrolysate, the pretreatment liquor, and/or washing eluent obtained from washing pretreated lignocellulosic biomass solids. In certain embodiments, the hydrolysate, the pretreatment liquor, and/or washing eluent obtained from washing pretreated lignocellulosic biomass solids may be purified after filtration.

[0119] Any suitable methods known in the art, or a combination of methods, may be used for purification. For example, in some embodiments, a wash, an ion exchange column, an active carbon column, filter sterilization, thermal sterilization, UV irradiation, and radiation may be used for purification.

Concentration, Drying and/or Crystallization

[0120] Concentration may be used in the methods described herein to concentrate a hydrolysate, which may be further dried and/or crystallized to produce a solid hydrolysate.

[0121] With respect to produce a solid hydrolysate, a combination of concentration, drying and crystallization may be used. In one embodiment, a hydrolysate can be concentrated before drying. In another embodiment, a hydrolysate can be concentrated before crystallization. In yet another embodiment, concentration is performed before crystallization, followed by drying. a) Concentration

[0122] Concentration of a hydrolysate allows for the control of the sugar titer or solid content in the hydrolysate. Any suitable methods known in the art may be used for concentration, including for example a vacuum evaporator, a multi-effect evaporator, or a membrane-based separation (e.g., pervaporation).

[0123] In some embodiments, thermal concentration or thermal-vacuum concentration may be used to obtain a concentrated hydrolysate with a high sugar titer. The hydrolysate may be concentrated, with or without vacuum, at a temperature of at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, or between 50°C and 100°C, between 60°C and 70°C, between 65°C and 75°C, between 65°C and 80°C, between 70°C and 80°C, between 75°C and 85°C, or between 80°C and 95°C. [0124] In some instances, concentration may not be needed to produce a solid hydrolysate if the sugar titer or solid content of the hydrolysate is high enough for drying. b) Drying

[0125] As discussed above, drying may be employed to produce a solid hydrolysate. Any suitable methods known in the art may be used for drying, including for example vacuum drying, spray drying, drum drying, and fluidized bed drying.

[0126] It should be understood that a combination of drying methods may be used. For example, in some embodiments, the hydrolysate is drum dried before spray drying to obtain a solid hydrolysate.

[0127] It should also be understood that a combination of drying methods may be used with the concentration methods described above. For example, in some embodiments, the hydrolysate undergoes multi-effect evaporation before drum drying to obtain a solid hydrolysate. In yet other embodiments, the hydrolysate undergoes multi-effect evaporation before spray drying to obtain a solid hydrolysate. c) Crystallization

[0128] As discussed above, crystallization may be employed to produce a solid hydrolysate. Any suitable methods known in the art may be used for crystallization. In some embodiments, the hydrolysate is first concentrated to a higher sugar concentration (e.g., using a vacuum evaporator or a multi-effect evaporator), and then seeded with a small amount of fine sugar to initiate the sugar crystallization process. The hydrolysate crystals are grown, and then separated and harvested by filtration or centrifugation. The uncrystalhzed hydrolysate may be recycled, purified, and recrystallized upon further concentration in a vacuum evaporator or a multi-effect evaporator. It should be understood that, if the reactor is run under vacuum, the crystallization reactor may serve as a vacuum evaporator to further concentrate the hydrolysate before the crystal seeding process. Sizing

[0129] A solid hydrolysate obtained from lignocellulosic biomass according to any of the methods described herein may be sized for subsequent fermentation processes to produce biofuels, biochemicals, or other bioproducts. In some embodiments, the solid hydrolysate is sized between 2 microns and 500 microns. In other embodiments, the solid hydrolysate is sized between 10 microns and 250 microns, between 50 microns and 200 microns, or between 100 microns and 150 microns. In yet other embodiments, the solid hydrolysate is sized less than 500 microns, less than 400 microns, less than 300 microns, less than 200 microns, or less than 100 microns.

[0130] Any suitable methods known in art the art may be used to size the solid hydrolysate, including for example a hammer mill, a solid crusher, and a sieve. A combination of sizing methods may be also used.

Concentrated hydrolysate

[0131] The concentrated hydrolysate obtained according to the methods described herein has a high sugar titer, without significant sugar loss from the concentrating process.

[0132] The concentrated hydrolysate may include monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose.

[0133] In certain embodiments, the sugar titer is at least 25%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or between 25% and 35%, between 30% and 50%, between 40% and 60%, or between 50% and 70% by weight of the total concentrated hydrolysate.

[0134] In other embodiments, the sugar titer of the concentrated hydrolysate is at least two times, at least three times, at least four times, at least five times, or at least ten times greater than the sugar titer of the initial hydrolysate. Solid Hydrolysate

[0135] The solid hydrolysate obtained from lignocellulosic biomass according to any of the methods described herein has a total sugar composition of monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose. The

lignocellulosic source from which the solid hydrolysate is obtained has a total sugar composition of polymeric glucose, polymeric xylose, polymeric mannose, polymeric arabinose and polymeric galactose. The methods described herein can produce a solid hydrolysate, which has a total sugar composition. In some embodiments, the total sugar composition of the solid hydrolysate is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total sugar composition of the lignocellulosic hydrolysate source. a) Sugar content

[0136] With reference to FIG. 1, the solid hydrolysate is the solid product obtained from the hydrolysate. The solid hydrolysate includes monomeric glucose, monomeric xylose, monomeric mannose, monomeric arabinose and monomeric galactose. In some embodiments, the glucose, xylose, mannose, arabinose and galactose are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 85% by weight of the total weight of the solid hydrolysate. In other embodiments, the glucose, xylose, mannose, arabinose and galactose are between 55% and 95%, between 60% and 90%, between 65% and 85%, between 70% and 80%, between 70% and 90%, or between 80% and 95% by weight of the total weight of the solid hydrolysate.

[0101] The total monomeric sugar composition may vary depending on the method used to obtain the solid hydrolysate. For example, the total monomeric sugar composition may be affected by the amount of lignin solubilized during pretreatment, or the purity of the cellulose before hydrolysis.

[0102] In some embodiments, the solid hydrolysate has a total monomeric sugar content between 50% and 90%, between 50% and 80%, between 55% and 85%, between 60% and 85%, between 65% and 90% or between 70% and 95% by weight of the total weight of the solid hydrolysate. [0103] In certain embodiments, the glucose is between 50% and 90%, between 40% and 91%, between 65% and 92% by weight of the total weight of the solid hydrolysate. The monomeric glucose content of the solid hydrolysate may vary depending, for example, on the enzymes used for hydrolysis (e.g. , cellulase, beta-glucosidase, with or without xylanase and mannanase and other enzymes). The xylose, mannose, arabinose and galactose sugar contents may vary depending on the amount of pretreatment liquor included in the sugar solution to be dried and the amount of lignosulfonate removed.

[0104] Additionally, in certain embodiments, the hemicellulose sugar content may vary depending on the lignocellulosic source and the amount of lignosulfonates present in the hydrolysate. In some embodiments, the hemicellulose sugar content is between 7% and 10% or between 3% and 7% by weight of the total weight of the solid hydrolysate.

[0105] The relative monomeric sugar ratios of the solid hydrolysate may vary depending on the method used to obtain the solid hydrolysate and the lignocellulosic source used.

[0106] In some embodiments where the solid hydrolysate is a solid softwood hydrolysate, between 50% and 70% of the total monomeric sugar weight is monomeric glucose, between 1% and 5% of the total monomeric sugar weight is monomeric xylose, between 1% and 5% of the total monomeric sugar weight is monomeric galactose, between 0.5% and 1 % of the total monomeric sugar weight is monomeric arabinose, and between 1 % and 5% of the total monomeric sugar weight is monomeric mannose.

[0107] In some embodiments where the solid hydrolysate is a solid hardwood hydrolysate, between 40% and 85% of the total monomeric sugar weight is monomeric glucose, between 5% and 10% of the total monomeric sugar weight is monomeric xylose, between 0.1 % and 5% of the total monomeric sugar weight is monomeric galactose, between 0.1 % and 1 % of the total monomeric sugar weight is monomeric arabinose, and between 1 % and 5% of the total monomeric sugar weight is monomeric mannose.

[0108] In some embodiments where the solid hydrolysate is a solid herbaceous biomass hydrolysate (e.g., a solid switchgrass hydrolysate), between 65% and 70% of the total monomeric sugar weight is monomeric glucose, between 1% and 5% of the total monomeric sugar weight is monomeric xylose, between 1% and 5% of the total monomeric sugar weight is monomeric galactose, between 0.5% and 1% of the total monomeric sugar weight is monomeric arabinose, and between 0.5% and 1% of the total monomeric sugar weight is monomeric mannose.

[0109] As discussed above, with reference to FIG. 2, a solid hydrolysate can also be obtained from the pretreatment liquor. In some embodiments, the glucose is between 5% and 40%, or between 10% and 35% by weight of the total weight of the solid hydrolysate. In other embodiments, the hemicellulose is between 60% and 95%, or between 65% and 90% by weight of the total weight of the solid hydrolysate.

[0110] As discussed above, with reference to FIG. 3, a solid hydrolysate can also be obtained from the pretreated biomass solids and the pretreatment liquor combined. In some embodiments, the glucose is between 40% and 90% by weight of the total weight of the solid hydrolysate. In other embodiments, the hemicellulose is between 5% and 20% by weight of the total weight of the solid hydrolysate. b) Water content and content of other non-sugar constituents

[0111] The methods described herein produce a solid hydrolysate from lignocellulosic biomass that has a reduced amount of water and other non-sugar constituents, including for example ash, lignin, lignosulfonates, and one or more metals.

[0112] In some embodiments, less than 10%, less than 8%, less than 5%, less than 1%, less than 0.01% by weight relative to the total weight of the solid lignocellulosic hydrolysate is water.

[0113] In some embodiments, less than 10%, less than 5% or less than 1% by weight relative to the total weight of the solid lignocellulosic hydrolysate is ash.

[0114] In some embodiments, less than 10%, less than 5% or less than 1% by weight relative to the total weight of the solid lignocellulosic hydrolysate is lignin.

[0115] In some embodiments, less than 10%, less than 5% or less than 1% by weight relative to the total weight of the solid lignocellulosic hydrolysate are lignosulfonates. [0116] In some embodiments, less than 10%, less than 5% or less than 1 % by weight relative to the total weight of the solid lignocellulosic hydrolysate is one or more metals. The one or more metals may include, for example, calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur.

[0117] In some embodiments, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1 % by weight relative to the total weight of the solid lignocellulosic hydrolysate are water, ash, lignin, lignosulfonates and one or more metals (e.g. , calcium, potassium, magnesium, manganese, sodium, silicon, and sulfur). c) Solubility and rate of dissolution

[0118] The solid lignocellulosic hydrolysate is soluble in water, which makes this solid lignocellulosic hydrolysate useful for subsequent fermentation to produce a biofuel, a biochemical, or other bioproducts.

[0119] In some embodiments, the solid lignocellulosic hydrolysate has a solubility of at least 0.25 g/mL, at least 0.3 g/mL, at least 0.35 g/mL, at least 0.4 g/mL, at least 0.5 g/mL, at least 0.6 g/mL, at least 0.7 g/mL, or at least 0.75 g/mL in water.

[0120] The solid lignocellulosic hydrolysate prepared according to the methods described herein typically has a higher rate of dissolution in water compared to other solid sugars that are currently commercially available. In some instances, the solid lignocellulosic hydrolysate dissolves at least 1 , 2, 3 or 4 times faster than other solid sugars that are currently commercially available.

[0121] In some embodiments, the solid lignocellulosic hydrolysate has a rate of dissolution of at least 0.01 moles sugar/kg final solution/second, at least 0.02 moles sugar/kg final solution/second, at least 0.03 moles sugar/kg final solution/second, or at least 0.04 moles sugar/kg final solution/second. In other embodiments, the solid lignocellulosic hydrolysate has a rate of dissolution of between 0.01-1 moles sugar/kg final solution/second, between 0.01-0.1 moles sugar/kg final solution/second, between 0.02-0.1 moles sugar/kg final solution/second, between 0.03-0.1 moles sugar/kg final solution/second, between 0.04-0.1 moles sugar/kg final solution/second, between 0.04-0.08 moles sugar/kg final solution/second, or between 0.04-0.65 moles sugar/kg final solution/second.

[0122] The rate of dissolution may be determined or measured according to any methods known in the art. It should be understood that the rate of dissolution may be affected by various factors including, for example, the shaking or agitation conditions and the temperature. In some embodiments, the rate of dissolution can be measured under a shaking condition used in a fermentation process (e.g. , at a shaking or agitation speed of 120 r.p.m.). Such shaking conditions used in fermentation are, for example, described in Watanabe et al., Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol, Bioresource Technology 101 (2010) pp. 9710-9714). In some embodiments, dissolution occurs when the solid lignocellulosic hydrolysate added to a medium (e.g. , water) is no longer visible as a solid.

[0123] In other embodiments, the solid lignocellulosic hydrolysate dissolves in water in less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, less than 20 seconds, less than 10 seconds, or less than 5 seconds. In one embodiment, the solid lignocellulosic hydrolysate dissolves in a fermentation medium in 20 seconds to 30 seconds. In another embodiment, the solid lignocellulosic hydrolysate with a 30 -35 solid loading dissolves in a fermentation medium in less than 30 seconds or less than 20 seconds. It should be understood, however, that the solid loading in water may affect the amount it takes to dissolve in water. For example, a solid loading of the solid lignocellulosic hydrolysate greater than 35% may take a longer time to dissolve.

[0124] Further, it should be understood that the solid lignocellulosic hydrolysate may dissolve in solvents that may be suitable for fermentation to produce a biofuel, biochemical or other bioproduct. The solvents may include water, or combinations of one or more solvents and water. d) Bulk density

[0125] The bulk density of the solid lignocellulosic hydrolysate may affect its rate of dissolution. In certain embodiments, a lower bulk density of the solid lignocellulosic hydrolysate may result in faster dissolution of the solid lignocellulosic hydrolysate in water. Bulk density refers to the mass of particles compared to the total volume occupied by the particles. The total volume may include particle volume, inter-particle volume and internal pore volume. As used herein, the bulk densities provided herein are the values before compression of the solid lignocellulosic hydrolysate to remove any voids.

[0126] In some embodiments, the solid lignocellulosic hydrolysate has a bulk density between 400 kg/m ³ and 1600 kg/m ³ , between 600 kg/m ³ and 1600 kg/m ³ , between 800 kg/m ³ and 1600 kg/m ³ , between 400 kg/m ³ and 900 kg/m ³ , between 600 kg/m ³ and 900 kg/m ³ , between 800 kg/m ³ and 900 kg/m ³ , between 400 kg/m ³ and 700 kg/m ³ , between 500 kg/m ³ and 700

3 3 3 3 3

kg/m , between 600 kg/m and 700 kg/m , between 400 kg/m and 600 kg/m , or between 400

3 3

kg/m and 500 kg/m . In other embodiments, the solid lignocellulosic hydrolysate has a bulk

3 3 3 3 density less than 1600 kg/m , less than 1200 kg/m , less than 900 kg/m , less than 800 kg/m ,

3 3 3 3

less than 700 kg/m , less than 650 kg/m , less than 600 kg/m , less than 550 kg/m , less than 500

3 3 3

kg/m , less than 450 kg/m , or less than 400 kg/m .

[0127] In certain embodiments, the solid lignocellulosic hydrolysate has a crystalline bulk

3 3 3 3 density less than 900 kg/m , less than 800 kg/m , less than 700 kg/m , less than 650 kg/m , less

3 3 3 3

than 600 kg/m , less than 550 kg/m , less than 500 kg/m , less than 450 kg/m , or less than 400 kg/m ³ .

Uses for the Concentrated or Solid Hydrolysate a) Biological conversion or fermentation

[0128] The concentrated or solid hydrolysate obtained from lignocellulosic biomass according to the methods described herein can be used to prepare one or more biofuels (e.g. , ethanol, propanol, butanol) or a bioproduct (e.g. , amino acids, organic acids, pharmaceuticals, specialty chemicals). In some embodiments, the concentrated or solid hydrolysate can be used to prepare alcohol compounds (e.g., ethanol, butanol, isobutanol), organic acids (e.g., acetic acid, lactic acid, citric acid), amino acids (e.g. , lysine, methionine, alanine, glutamic acid), diols (e.g., propanediol and butanediol), protein products (e.g. , enzymes, polypeptides), gaseous products (e.g., as biogas, methane, hydrogen, carbon dioxide) and lipids. [0129] The concentrated or solid hydrolysate can be used for subsequent fermentation with one or more fermenting organisms to produce a fermentation product, e.g., a biofuel, a biochemical, or other bioproducts. The fermentation process may use fermentation organisms such as yeast, fungi, mold, algae, bacteria (e.g., Escherichia coli and Clostridium), or a mixture of these organisms.

[0130] The methods and conditions suitable for sugar fermentation into a biofuel or a bioproduct are well known in the art. For example, Sedlak and Ho teach one way to produce ethanol from sugar fermentation of cellulosic biomass, such as corn stover. See e.g., Miroslav Sedlak and Nancy W. Y. Ho, Production of ethanol from cellulosic biomass hydrolysates using genetically engineered Saccharomyces yeast capable of cofermenting glucose and xylose, Applied Biochemistry and Biotechnology, 113-116: 403-416 (2004); Watanabe et al., Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol, Bioresource Technology 101 (2010) pp. 9710-9714.

[0131] In some embodiments, fermentation may occur in less than 24 hours, or for 24 hours to 72 hours, or for 36 hours to 60 hours.

[0132] In some embodiments, fermentation converts 60% to 100% of the solid

lignocellulosic hydrolysate to the fermentation product. In other embodiments, fermentation converts at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the solid hydrolysate to the fermentation product. b) Chemical catalysis

[0133] The concentrated or solid hydrolysate prepared according to the methods described herein can also be used in various chemical catalysis reactions to produce alcohols, ketones, aldehydes, alkanes, alkenes, organic acids, polyols, furans (e.g., hydroxymethylfurfural, furandicarboxylic acid, dimethylfuran), gasoline, diesel, jet fuel, and gaseous products (e.g., hydrogen, carbon dioxide). c) Food products

[0134] The concentrated or solid hydrolysate prepared according to the methods described herein can also be used to produce food products, including for example soft drinks, beer, wine and vinegar.

[0135] Although individual features of the compositions and methods described herein may be included in different claims, these may be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or

advantageous. Also, the inclusion of a feature in one category of claims does not imply a limitation to this category, but rather the feature may be equally applicable to other claim categories, as appropriate. Where a composition or method 'comprises' one or more specified items or steps, others can also be included. The invention also contemplates, however, that the described composition or method may be used without other items or steps and thus it includes the recited composition or method 'consisting of or 'consisting essentially of the recited items, materials or steps, as those terms are commonly understood in patent law.

EXAMPLES

[0136] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

Materials and Reagents a) Biomass

[0137] Softwood thinnings (early harvest of pine softwood 12-15 years after planting and before final harvest that may be sourced to a pulp mill), hardwood (maple chips) and switchgrass (Alamo variety) were used as the biomass in Examples 1-15 below. Table 1 below summarizes the carbohydrate composition of the biomass used. The carbohydrate composition of each biomass was determined by converting the polymeric sugars in the feedstock into monomeric sugars such as glucose, xylose, mannose, arabinose and galactose. Results are reported as the original polymeric composition of the biomass. As seen in Table 1 below, the total polymeric sugar composition observed for softwood, hardwood and switchgrass was 56%, 57% and 59%, respectively.

Table 1. Carbohydrate composition of biomass used

b) Biomass pretreatment reagents

[0138] Sulfonation using calcium bisulfite was used for biomass pretreatment. Calcium bisulfite was produced by constantly purging pure sulfur dioxide into a calcium oxide solution. The final calcium bisulfite concentration contained about 3-4% total sulfur dioxide, of which about 1 % was free sulfur dioxide. The pH of this calcium bisulfite solution was about 1.4. c) Hydrolysis reagents

[0139] Cellulase (Celluclast, Sigma Catalog # C-2730), Cellic® CTec2 enzyme product (Novozymes), beta-glucosidase (Novozymes-188, Sigma Catalog # C-6105), and xylanase (Sigma Catalog # X2753) were used accordingly in the enzymatic hydrolysis experiments described below after biomass pretreatment.

[0140] For enzymatic hydrolysis, a pre- mixed enzyme cocktail (Sigma mixture) containing cellulase (99.5 mg/niL), beta-glucosidase (42.5 mg/niL) and xylanase (3.4 mg/niL) was used. The total enzyme protein titer was 145.5 mg/niL. In some of the Examples described below, Cellic® CTec2 enzyme product was used instead of cellulase. d) Ethanol fermentation reagents

[0141] A yeast strain Saccharomyces cerevisiae T2 was obtained from Dr. Sheldon Duff at the University of British Columbia. This yeast strain was used for ethanol fermentation described in some of the Examples described below.

Pretreatment Procedures

[0142] Before pretreating the biomass used in the Examples below, the softwood, hardwood and switchgrass were fractured with a BearCat garden chipper with a ¾" screen to obtain the chipped materials. The 3-mm round-hole fines from the chipped materials were removed to avoid circulation problems in the pretreatment reactor.

[0143] The chipped softwood and hardwood were individually pretreated in a one cubic foot reactor with an acid sulfite pretreatment consisting of 12.5% calcium bisulfite on wood with a single step temperature schedule: ramped from 90°C to 155°C in 15 minutes and held at 155°C for 120 minutes. After cooking, the liquor was drained and the cooked chips were collected. The cooked chips were then sent to an Alpine grinder, without any water, to refine the chips into a pulp. The pulp batch numbers were CS10220A for the pretreated hardwood and CS 10228 A for the pretreated softwood.

[0144] The chipped switchgrass was pretreated with an 18.4% calcium bisulfite loading on dry biomass with a single step temperature schedule: ramped from 90°C to 155°C in 15 minutes and held at 155°C for 75 minutes. After cooking, the liquor was drained and the cooked switchgrass was collected. The cooked switchgrass was not further refined. The pulp batch number was CS10226A for the pretreated switchgrass.

[0145] Under the same temperature scheme described above at 12.5% calcium bisulfite loading, another batch (CS 10222 A) was also run for hardwood to provide additional materials for enzymatic hydrolysis and sugar production described in the Examples below. The pretreatment temperature at 155°C was close to 160°C to 170°C, which were temperatures reported in some acid sulfite pulp processes by Seaman in 1954 (US Patent 2,698,234) and by Wolfinger et al. in 2004 (Martin G. Wolfinger & Herbert Sixta, Modeling of the acid sulfite pulping process - Problem definition and theoretical approach for a solution with the main focus on the recovery of cooking chemicals, Lenzinger Berichte, 83: 35-45 (2004)).

[0146] Table 2 below summarizes the carbohydrate compositions of the pretreated biomass. The carbohydrate composition of each pretreated biomass was determined by converting the polymeric sugars in the pretreated biomass into monomeric sugars such as glucose, xylose, mannose, arabinose and galactose. As seen in Table 2 below, the total polymeric sugar composition observed for softwood, hardwood and switchgrass was 43%, 62% and 60%, respectively. These pretreated biomass materials were used for the enzymatic hydrolysis and for the solid hydrolysate production.

Carbohydrate composition of pretreated biomass

[0147] A pretreatment liquor (or prehydrolysate) was also produced for each biomass. Table 3 below summarizes the carbohydrate composition of the pretreatment liquor sugar composition. In some Examples described below, these pretreatment liquor streams were used along with the pretreated biomass solid in the enzymatic hydrolysis process. Carbohydrate composition of pretreatment liquor

Example 1. Hydrolysis of pretreated softwood without its pretreatment liquor

[0148] The pretreated softwood was washed with 4x water, and the pH of the pretreated softwood was adjusted to about 5.3 using calcium oxide. After washing and pH adjustment, the pretreated softwood was pressed to achieve a solid content of about 40%, and then hydrolyzed in a shake flask at a solid concentration of 18.8% and an enzyme dosage (Sigma mixture) of 30 mg enzyme protein/g (glucan and xylan). The enzyme dosage also corresponded to 0.111 g enzyme product/dry gram of pulp materials.

[0149] A 50 mmol sodium citrate buffer was used during the hydrolysis. The total hydrolysis volume was 500 mL. The hydrolysis temperature was controlled at 50°C and the shaking speed was 200 rpm. Most of the hydrolysis was completed in about 72 hours. The hydrolysis was taken out at about 120 hours to ensure a more complete hydrolysis.

[0150] Enzymatic hydrolysis of the pretreated softwood produced a softwood hydrolysate. Table 4 below summarizes the sugar titers of the softwood hydrolysate at 72 hours, 96 hours and 120 hours. A total sugar titer of 8.3% was observed in the softwood hydrolysate after 120 hours. Table 4. Sugar Titer in Softwood Hydrolysate

Example 2. Hydrolysis of pretreated hardwood with its pretreatment liquor

[0151] Unwashed pretreated hardwood and its pretreatment liquor were combined and used in hydrolysis. The combination of the pretreatment biomass with its pretreatment liquor increased sugar titer, while minimizing hydrolysis water usage.

[0152] Before hydrolysis, the pH of the pretreatment liquor was first adjusted to 7.5 using potassium hydroxide in the presence of a sodium citrate buffer. The pretreated hardwood was sterilized in an autoclave, and then was combined with the pretreatment liquor to achieve a final solid content of about 18%. The pH of the hydrolysis was controlled at around a pH of 5.3 using a 50 mmol sodium citrate buffer.

[0153] The CTec2 enzyme dosage for hydrolysis was 0.133 g enzyme product/dry gram of pulp materials. The total hydrolysis volume was 424 mL. The hydrolysis temperature was controlled at 50°C and the shaking speed was 200 rpm. The hydrolysis was completed in about 72 hours.

[0154] Enzymatic hydrolysis of the pretreated hardwood produced a hardwood hydrolysate. Table 5 below summarizes the sugar titers in the hardwood hydrolysate at 24 hours and 76 hours. A total sugar titer of 15.8%, which included sugar contribution from the pretreatment liquor, was observed after 76 hours. Table 5. Sugar Titer in Hardwood Hydrolysate

Example 3. Hydrolysis of pretreated switchgrass without pretreatment liquor

[0155] The pretreated switchgrass was washed with 4x water, and the pH of the pretreated switchgrass was adjusted to about 5.3 using calcium oxide. After washing and pH adjustment, the pretreated switchgrass was pressed to achieve a solid content of about 41%, and then hydrolyzed in a shake flask at a solid concentration of 18.8% at an enzyme dosage (Sigma mixture) of 30 mg enzyme protein/g (glucan + xylan). The enzyme dosage also corresponded to 0.133 g enzyme product/dry gram of pulp materials.

[0156] A 50 mmol sodium citrate buffer was used during the hydrolysis. The total hydrolysis volume was 500 mL. The hydrolysis temperature was controlled at 50°C and the shaking speed was 200 rpm. Most of the hydrolysis was completed in about 72 hours; however, the hydrolysis was stopped at 120 hours to ensure a more complete hydrolysis.

[0157] Enzymatic hydrolysis of the pretreated switchgrass produced a switchgrass hydrolysate. Table 6 below summarizes the sugar titers in the switchgrass hydrolysate at 72 hours, 96 hours and 120 hours. A total sugar titer of 13.6% was observed after 120 hours.

Table 6. Sugar Titer in Switchgrass Hydrolysate

Total

Glucose Xylose Galactose Arabinose Mannose

Time (hr) Sugar

(%) (%) (%) (%) (%)

Titer (%)

72 12.06 0.35 0.00 0.06 0.06 12.53

96 12.55 0.43 0.12 0.03 0.03 13.17

120 12.74 0.48 0.22 0.09 0.07 13.59 Example 4. Hydrolysis of pretreated softwood and ultra-filtered softwood pretreatment liquor

[0158] The pretreatment liquor obtained after softwood pretreatment was ultra- filtered using a 10 kDa molecular- weight-cut-off (MWCO) filter to the hgnosulfonates. The filtered softwood pretreatment liquor was combined with the pretreated softwood to achieve a final solid content of 17%. The combined pretreated softwood and ultra-filtered softwood pretreatment liquor was then hydrolyzed using CTec2 enzymes. The enzyme dosages used in this Example are summarized in Table 7 below. The hydrolysis temperature was controlled at 50°C and the shaking speed was 200 rpm. The hydrolysis time was completed in 96 hours. Table 7 also compares the effect of the buffer and ultra-filtration of the pretreatment liquor on glucose yields after hydrolysis.

Table 7. Effects of Buffer and Ultra-Filtration of Softwood Pretreatment Liquor on Enzymatic Hydrolysis of Pretreated Softwood

[0159] The normalized yield of glucan conversion to glucose was calculated by dividing the glucose amount of each test with filtered or unfiltered liquor by the maximum amount of glucose, at an excessive enzyme dosage at 0.140 g enzyme product/dry gram of pulp materials in the citrate buffer control test.

[0160] As seen in Table 7 above, ultra-filtration of the softwood pretreatment liquor increased the net glucan hydrolysis yield by 6.5% (i.e., comparing the average normalized yield of tests 3 and 4 with and without ultra-filtration of liquor), 6.8% (i.e., comparing the average normalized yield of tests 5 and 6 with and without ultra-filtration), and 7.6% (i.e. , comparing the average normalized yield of tests 7 and 8 with and without ultra-filtration), respectively for CTec2 enzyme dosages 0.061, 0.044 and 0.021 g enzyme product/dry gram of pulp materials. These results demonstrate that ultra-filtration of the softwood pretreatment liquor increased sugar yield, especially at lower enzyme dosages where enzyme dosage may limit the overall extent of hydrolysis.

[0161] As seen again in Table 7 above, hydrolysis of the control tests with the citrate buffer increased the net glucan hydrolysis yield compared to tests with unfiltered softwood pretreatment liquor by 2.7% (i.e., comparing the average normalized yield of control tests 1 and 2 with tests 1 and 2 without ultra-filtration), 7.3% (i.e., comparing the average normalized yield of control tests 3 and 4 with tests 3 and 4 without ultra-filtration), 9.0% (i.e. , comparing the average normalized yield of control tests 5 and 6 with tests 5 and 6 without ultra-filtration) and 13.1 % (i.e., comparing the average normalized yield of control tests 7 and 8 with tests 7 and 8 without ultrafiltration), respectively for enzyme dosages 0.140, 0.061, 0.044 and 0.021 g enzyme product/dry gram of pulp materials. These results demonstrate that ultra filtration improves overall glucan hydrolysis nearly as much as is achieved with a clean citrate buffer. Example 5. Vacuum drying of an unfiltered softwood hydrolysate to produce a solid softwood hydrolysate

[0162] The softwood hydrolysate from Example 1 was vacuum dried at 65 °C to produce a solid softwood hydrolysate. A similar softwood hydrolysate was also prepared according to the procedures described in Example 1 using CTec2 at an enzyme dosage of 0.061 g enzyme product/dry gram of pulp materials. The total monomeric sugar compositions of the two solid softwood hydrolysates are summarized in Table 8 below. As seen in Table 8 below, the total sugar compositions of the solid softwood hydrolysate obtained from the Sigma mixture was 58% and from CTec2 was 69%.

Table 8. Total sugar composition of solid softwood hydrolysate (unfiltered)

Example 6. Vacuum drying of an ultra-filtered softwood hydrolysate to produce a solid softwood hydrolysate

[0163] The softwood hydrolysate from Example 1 was first filtered through an ultra-filter with a MWCO of 10 kDa. The filtered softwood hydrolysate was then vacuum dried at 65°C to produce a solid softwood hydrolysate. A similar softwood hydrolysate was also prepared according to the procedures described in Example 1 using Sigma enzyme mixture at an enzyme dosage of 0.111 g enzyme product/dry dram of pulp materials and using CTec2 at an enzyme dosage of 0.061 g enzyme product/dry gram of pulp materials. This softwood hydrolysate prepared using CTec2 was also ultra-filtered with a 10 kDa MWCO filter and dried under vacuum at 65°C. The total monomeric sugar compositions of the two solid softwood hydrolysates are summarized in Table 9 below. As seen in Table 9 below, the total sugar compositions of the solid softwood hydrolysate obtained from the Sigma mixture was 74% and from CTec2 was 78%. Table 9. Total sugar composition of solid softwood hydrolysate (ultra-filtered)

Example 7. Vacuum drying of an unfiltered switchgrass hydrolysate to produce a solid switchgrass hydrolysate

[0164] The switchgrass hydrolysate from Example 3 was vacuum dried at 65 °C to produce a solid switchgrass hydrolysate. A similar switchgrass hydrolysate was also prepared according to the procedures described in Example 3 using Sigma enzyme mixture at an enzyme dosage of 0.133 g enzyme product/dry dram of pulp materials and using and using CTec2 at an enzyme dosage of 0.073 g enzyme product/dry gram of pulp materials. The total monomeric sugar compositions of the two solid switchgrass hydrolysates are summarized in Table 10 below. As seen in Table 10 below, the total sugar compositions of the solid switchgrass hydrolysate obtained from the Sigma mixture was 70% and from CTec2 was 71%.

Table 10. Total sugar composition of solid switchgrass hydrolysate (unfiltered)

switchgrass hydrolysate

[0165] The switchgrass hydrolysate from Example 3 was first filtered through an ultra-filter with a MWCO of 10 kDa. The filtered switchgrass hydrolysate was then vacuum dried at 65°C to produce a solid switchgrass hydrolysate. A similar pretreated switchgrass hydrolysate was also prepared according to the procedures described in Example 3 using Sigma enzyme mixture at an enzyme dosage of 0.133 g enzyme product/dry gram of pulp materials and using CTec2 at an enzyme dosage of 0.073 g enzyme product/dry gram of pulp materials. This switchgrass hydrolysate prepared using CTec2 was ultra-filtered with a 10 kDa MWCO filter and dried under vacuum at 65 °C. The total monomeric sugar compositions of the two solid switchgrass hydrolysates are summarized in Table 11 below. As seen in Table 11 below, the total sugar compositions of the solid switchgrass hydrolysate obtained from the Sigma mixture was 71% and from CTec2 was 72%.

Table 11. Total sugar composition of solid switchgrass hydrolysate (ultra-filtered)

Example 9. Metal and sulfur contents in solid softwood and switchgrass hydrolysates

[0166] The major metal ions and sulfur contents of the solid softwood and switchgrass hydrolysates were analyzed. Table 12 below summarizes the metal content of the solid softwood and switchgrass hydrolysates, obtained from the ultra-filtered hydrolysate samples. As seen in Table 12 below, the total major metal contents were approximately 4.56% and 3.55%, respectively, for the solid softwood hydrolysate and for the solid switchgrass hydrolysate.

Table 12. Major metal content

[0167] It should be noted that during hydrolysis, the use of the 50 mmol sodium citrate buffer for pH control added sodium (Na) to the final ash content. Eliminating the use of the sodium citrate buffer in an automated pH control process may reduce the sodium content observed in Table 12 above. [0168] The sulfur contents of the solid softwood and switchgrass hydrolysates are shown in Table 13 below. It should be noted that most of the sulfur may have come from calcium bisulfite residuals from the pretreatment.

Table 13. Sulfur content

Example 10. Spray drying of an ultra-filtered softwood hydrolysate to produce a solid softwood hydrolysate

[0169] Softwood hydrolysate prepared according to the procedure set forth in Example 1 was filtered through an ultra-filter with a MWCO of 10 kDa. The filtered softwood hydrolysate was then spray-dried at 150°C with a product outlet temperature of 84°C to produce a solid softwood hydrolysate. The total monomeric sugar composition of the solid softwood hydrolysate is summarized in Table 13 below. As seen in Table 14 below, the total sugar composition of the solid softwood hydrolysate was 74%. The weighted average C5/C6 sugar molecular weight was observed to be 178.5 g/mole.

Table 14. Total sugar composition of solid softwood hydrolysate (ultra- filtered)

Example 11. Spray drying of an ultra-filtered switchgrass hydrolysate to produce a solid switchgrass hydrolysate

[0170] Switchgrass hydrolysate prepared according to the procedure set forth in Example 3 was filtered through an ultra-filter with a MWCO of 10 kDa. The filtered hydrolysate was then spray-dried at 150°C with a product outlet temperature of 84°C to produce a solid switchgrass hydrolysate. The total monomeric sugar composition of the solid switchgrass hydrolysate is summarized in Table 15 below. As seen in Table 15 below, the total sugar composition of the solid switchgrass hydrolysate was 72%. The weighted average C5/C6 sugar molecular weight was observed to be 178.8 g/mole.

Table 15. Total sugar composition of solid switchgrass hydrolysate (ultra-filtered)

Example 12. Spray drying of an unfiltered hardwood hydrolysate and hardwood pretreatment liquor to produce a solid hardwood hydrolysate

[0171] Hardwood hydrolysate prepared according to the procedure set forth in Example 2 was combined with hardwood pretreatment liquor. The hardwood hydrolysate and hardwood pretreatment liquor were spray-dried at 150°C with a product outlet temperature of 82-85°C to produce a solid hardwood hydrolysate. Table 16 below summarizes the total monomeric sugar composition of the solid hardwood hydrolysate. As seen in Table 16 below, the total sugar composition of the solid hardwood hydrolysate was 58%.

Table 16. Total sugar composition of solid hardwood hydrolysate (unfiltered)

Example 13. Ethanol fermentation using solid softwood hydrolysate

[0172] Before ethanol fermentation, the Saccharomyces cerevisiae T2 yeast seed was first grown in a complex medium containing 1% yeast extract and 2% peptone, supplemented with 3% glucose. The stock seed culture was incubated in a shake flask on an orbital shaking incubator controlled at 38°C and 200 rpm for 18-20 hours. The yeast seed culture was then centrifuged and the yeast seed pellet was dissolved in a small volume of 100 mmol sodium citrate buffer. The yeast seed was inoculated at 2 g/L dry cell weight in the fermentation tests. [0173] The solid softwood hydrolysate from Example 10 was added directly to a sterilized complex medium containing 0.5% yeast extract and 1% peptone. The solid softwood hydrolysate dissolved almost instantly in 20 seconds at 120 rpm shaking agitation.

[0174] After the yeast seed was inoculated, the fermentation was conducted in a total 5-mL volume in 25 mL Erlenmeyer flasks shaking at 120 rpm. The initial glucose concentration was 22.8%. The fermentation temperature was controlled at 38°C and the pH was controlled at around 5 by daily pH adjustment.

[0175] Table 17 below summarizes the results of the solid softwood hydrolysate

fermentation. As seen in Table 17 below, the ethanol titer reached 8.04% within 48 hours, and most of the fermentation was observed to be completed.

Table 17. Ethanol fermentation of solid softwood hydrolysate

[0176] The ethanol yield produced from the solid softwood hydrolysate was observed to be 87.5%. The total ethanol yield in this Example was calculated as the total ethanol produced divided by the total sugar used in the fermentation and divided by 0.511, which is the factor that corresponds to a theoretical yield at 100% metabolic conversion. Since the current yeast strain primarily uses glucose, the ethanol yield is mainly from glucose.

Example 14. Ethanol fermentation using solid switchgrass hydrolysate

[0177] Before ethanol fermentation, the Saccharomyces cerevisiae T2 yeast seed was first prepared in a complex medium according to the procedure set forth in Example 13.

[0178] The solid switchgrass hydrolysate powder from Example 10 was added directly to a sterilized complex medium containing 0.5% yeast extract and 1% peptone. The solid switchgrass hydrolysate dissolved almost instantly in 20 seconds at 120 rpm shaking agitation. [0179] After the yeast seed was inoculated, the fermentation was conducted in a total 5-mL volume in 25 mL Erlenmeyer shake flasks shaking at 120 rpm. The initial glucose concentration was 22.4%. The fermentation temperature was controlled at 38°C and the pH was controlled at around 5 by daily pH adjustment.

[0180] Table 18 below summarizes the results of solid switchgrass hydrolysate fermentation. As seen in Table 18 below, the ethanol titer reached 8.93% within 48 hours, and most of the fermentation was observed to be completed.

Table 18. Ethanol fermentation of solid switchgrass hydrolysate

[0181] The ethanol yield produced from the solid spray-dried switchgrass hydrolysate was observed to be 85.3%. The total ethanol yield in this Example was calculated as the total ethanol produced divided by the total sugar used in the fermentation and divided by 0.511, which is the factor that corresponds to a theoretical yield at 100% metabolic conversion. Since the current yeast strain primarily uses glucose, the ethanol yield is mainly from glucose.

Example 15. Comparing rate of dissolution of solid lignocellulosic hydrolysate and Sigma glucose crystals

[0182] Solid softwood hydrolysate (LH3S) prepared according to the procedure set forth in Example 10 above, solid switchgrass hydrolysate (LH1S) prepared according to the procedure set forth in Example 11 above, and glucose crystals purchased from Sigma (product # G8270) were used in this Example.

[0183] To achieve a glucose titer of about 18.9% wt/wt, 1.52 g of solid softwood hydrolysate (33.0% wt/wt solid loading) were added to 3.08 ml of a standard fermentation complex medium; 1.45g of solid switchgrass hydrolysate (31.5% wt/wt solid loading) were added to 3.15 ml of a standard fermentation complex medium; and 0.95 g of Sigma glucose crystals (20.6% wt/wt solid loading) were added to 3.65 niL of a standard fermentation complex medium. The complex medium used was prepared according to the procedure set forth in Example 13.

[0184] The tests were conducted in a 25 ml Erlenmeyer flask on an orbital shaking incubator at 120 r.p.m., at room temperature. The amount of time it took for the solid sample to dissolve in the complex medium was measured. The solid sample was considered to be dissolved when solids were no longer visible.

[0185] The solid softwood hydrolysate was observed to dissolve in about 20-31 seconds (rate of dissolution of 0.044-0.068 moles sugar/kg final solution/second). The solid switchgrass hydrolysate was observed to dissolve in about 20-30 seconds (rate of dissolution of 0.042-0.064 moles sugar/kg final solution/second). In contrast, the Sigma glucose crystals were observed to dissolve in about 215 to 240 seconds (rate of dissolution of 0.0047 to 0.0053 moles sugar/kg final solution/second).

[0186] Thus, the solid softwood and switchgrass hydrolysates were observed to dissolve about an order of magnitude faster than the commercially available solid sugar.

Example 16. Thermal and vacuum concentrating a hydrolysate in a rotary evaporator

[0187] The biomass used in this Example was softwood with bark (whole tree materials). The carbohydrate composition of this biomass is summarized in Table 19 below.

Table 19. Carbohydrate composition of biomass used

Polymer Sugar of Total Biomass Softwood w/ Bark

Arabinan ( ) 1.89

Galactan ( ) 3.01

Glucan ( ) 33.2

Xylan (%) 5.90

Mannan ( ) 7.81

Total (%) 51.81 [0188] The biomass was fractured with a chipper and then pretreated using calcium bisulfite according to the pretreatment methods and reagents described above. The pretreatment conditions were ramped from 90°C to 165°C in 15 minutes and held at 165°C for 75 minutes. The pulp batch number was CS 10221 A for the softwood with bark used in this Example.

[0189] After pretreatment, the pretreated softwood with bark had the carbohydrate composition summarized in Table 20 below.

Table 20. Carbohydrate of pretreated softwood with bark

[0190] The pretreated biomass softwood without bark was enzymatic hydrolyzed, without its pretreatment liquor, according to the procedure described in Example 1 above.

[0191] After enzymatic hydrolysis, a hydrolysate was obtained. The coarse residuals in the hydrolysate were removed by centrifugation at 7500 rpm. A coarse-clarified hydrolysate was collected, and then concentrated using the thermal vacuum concentration at 75+5°C in a rotary evaporator (Model RE111 Rotavapor, Buchi, Switzerland). The initial total sugar titer of the coarse-clarified hydrolysate was 7.8% before the thermal vacuum concentration. A total of 2643 g hydrolysate was concentrated into 229 g through semi-batch sample addition to the evaporation flask. The results from concentration the coarse-clarified hydrolysate are summarized in Table 21 below. Table 21. Summary of thermal vacuum concentration

[0192] The final concentrated hydrolysate syrup was observed to be extremely viscous. The syrup was molten at 75°C, but not at room temperature. The final concentrated hydrolysate syrup had a sugar titer of 28.9%, which was much lower than the expected sugar titer.

Example 17. Thermal and vacuum-oven concentrating a hydrolysate without and with a 5 kDa molecular filter

[0193] The biomass used in this Example was Douglas-fir without bark. The carbohydrate composition of this biomass is summarized in Table 22 below.

Table 22. Carbohydrate composition of biomass used

[0194] The biomass was fractured with a chipper and then pretreated using calcium bisulfite according to the pretreatment methods and reagents described above. The pretreatment conditions were ramped from 90°C to 165°C in 15 minutes and held at 165°C for 75 minutes. The pulp batch numbers were CS10229A and CS 10231 A for the Douglas-fir without bark.

[0195] After pretreatment, the pretreated softwood with bark had the carbohydrate composition summarized in Table 23 below.

Table 23. Carbohydrate of pretreated Douglas fir without bark

[0196] The pretreated biomass softwood without bark was enzymatic hydrolyzed, without its pretreatment liquor, according to the procedure described in Example 1 above.

[0197] After enzymatic hydrolysis, a hydrolysate was obtained from the pretreated biomass batch CS10231A under same pretreatment conditions as the batch CS10229A. The coarse residuals in the hydrolysate were removed by centrifugation at 7500 rpm. This coarse-clarified hydrolysate was collected, and was observed to have an initial total sugar titer of 17.17%. Two samples were taken from the coarse-clarified hydrolysate. One sample was filtered by a 5 kDa molecular filter to remove any compounds larger than 5 kDa, and the other sample was used as is without the 5 kDa molecular filtration. Each sample was concentrated with a thermal vacuum- oven at 75+5°C. The results from concentration the coarse-clarified hydrolysate are summarized in Table 24 below. Table 24. Summary of thermal vacuum concentration

[0198] The results in Table 24 showed that a high sugar titer syrup of 47.8% could be achieved by removing compounds with molecular weight greater than 5 kDa. In contrast, absent removing compounds with molecular weight greater than 5 kDa, the concentrated hydrolysate only had a sugar titer of 29.6%. In addition, the concentrated hydrolysate with ultra-filtration was observed to have a light amber color, while the concentrated hydrolysate without ultrafiltration was observed to have a black or very dark color.

Example 18. Thermal and vacuum-oven concentrating hydrolysate samples without a 5 kDa molecular filter

[0199] Pretreated biomass samples (switchgrass and Loblolly pine softwood) were used in this Example to compare the sugar loss behavior at thermal and vacuum concentrating process of a clarified hydrolysate without a molecular filter. These samples were pretreated by different methods.

[0200] A steam-exploded switchgrass sample was provided by Dr. Renata Bura of University of Washington. Steam explosion involves a biomass sample pressurized under hot steam and exploded quickly to an expansion vessel, creating a fine and pretreated biomass ready for enzymatic hydrolysis. Steam explosion is typically effective for herbaceous biomass

pretreatment, such as switchgrass. [0201] A green-liquor pretreated loblolly pine sample was provided by Dr. Hasan Jameel of North Carolina State University. A green-liquor pretreatment method follows a similar conventional pulping method, using sodium carbonate and sodium sulfide in pretreating the woody chips.

[0202] Additionally, a calcium bisulfite pretreated switchgrass sample was also used and obtained from the procedure described in Example 3. A calcium bisulfite pretreated loblolly pine softwood sample was also used and obtained from the procedure described in Example 1.

[0203] These pretreated samples were previously washed with deionized water and then hydrolyzed enzymatically. After enzymatic hydrolysis, the insoluble or undigested biomass was removed by centrifuging the sample at 7500 rpm. Coarse-clarified hydrolysate samples of 5 mL were used and tested at the same time for the thermal vacuum concentrating at 75+5 °C in 25-mL flat bottom flasks in a thermal vacuum oven. Weights of samples and evaporated water were analyzed for concentrating factors and expected sugar titers after the thermal vacuum concentration process. After the concentrating process, the sample was re-dissolved back similarly to its original volume and the sugar titer was analyzed. Table 25 shows the sugar concentrating results, indicating that the coarse-clarified hydrolysate samples from the steam- exploded switchgrass sample and the green-liquor pretreated softwood sample had only 9.9% and 6.8% sugar loss, respectively during the thermal vacuum concentrating process. However, the coarse-clarified hydrolysate samples from the calcium bisulfite pretreated switchgrass and softwood samples had a significant sugar loss of 32.5% and 39.4%, respectively during the thermal vacuum concentrating process.

Table 25. Sugar loss during thermal vacuum concentrating process of various coarse-clarified hydrolysate samples without ultra-filtration

[0204] Thus, the observations from this Example, as well as observations from Examples 16 and 17 above, exhibited a significant sugar loss only with hydrolysate samples of bisulfite pretreated switchgrass and softwood materials that were not ultra-filtered. With ultra-filtration, the sugar loss was greatly reduced during the thermal vacuum concentrating process for the hydrolysate samples from the calcium bisulfite pretreated biomass samples.

Example 19. Hydrolysis of pretreated switchgrass solids using various enzyme dosages

[0205] This Example was performed to determine suitable enzyme dosage on pretreated switchgrass CS 10226 A and its pretreatment liquor.

[0206] The pH of the pretreatment liquor was first adjusted to 12 using calcium oxide. Both the pretreated switchgrass and the pretreatment liquor were autoclaved before use. The pH pre- adjusted and autoclaved pretreatment liquor was clarified by settling to remove any precipitates. The clarified pretreatment liquor was used in the enzymatic hydrolysis tests. The pretreated switchgrass solid loading was 15.6% in enzymatic hydrolysis tests. The enzymatic hydrolysis tests were conducted in a 50-mL volume in 125-mL shake flasks. The hydrolysis temperature was set at 50°C, and the shaking speed was controlled at 200 rpm. During hydrolysis, the pH was controlled at about 5.0 using a 50 mmol sodium citrate buffer. The pH was adjusted daily. Table 26 below summarizes the glucose titer and conversion yields at three different enzyme dosages after 3 days of hydrolysis.

Table 26. Summary of glucose titer and conversion yields at three different enzyme dosages

[0207] As the control case, the first enzyme dose was 0.098 g CTec2 enzyme product/g dry pulp in the hydrolysis without any pretreatment pretreatment liquor to convert glucan to glucose. The second and the third enzyme dosages were 0.042 and 0.032 g CTec2 enzyme product/g dry pulp, respectively, in the hydrolysis with the pretreatment liquor. The results in Table 25 above show that at a low enzyme dosage of 0.032 g enzyme product/g dry pulp, glucan in the pretreated switchgrass was converted into glucose at a yield of 81.7%. When 30% more enzyme was used at 0.042 g enzyme product/g dry pulp, the conversion yield was increased to 88.3%. When two times more enzymes were used at 0.098 g/g dry pulp, glucan was converted into glucose at a yield of 94.2%. The yield was calculated on the pretreated switchgrass solid and prehydrolysate sugar contribution was excluded.

Example 20. Batch hydrolysis of pretreated switchgrass

[0208] In batch hydrolyses of pretreated switchgrass, 8.3 g dry pulp (wet in test) or a dry pulp loading of 18.1% in the hydrolysis was used. An enzyme dose was 0.027 g enzyme product/g dry pulp was used in the hydrolysis. The hydrolysis was conducted in a 50-mL volume in a 250-mL Erlenmeyer flask on an orbital shaking incubator. The hydrolysis temperature was set at 50°C, and the shaking speed was controlled at 200 rpm. The hydrolysis pH was controlled at about 5.0 using a 50 mmol sodium citrate buffer. The pH was adjusted daily. Table 27 below summarizes the hydrolysis results. Table 27. Summary of hydrolysis yields at 4 days and 5 days

[0209] At the enzyme dose of 0.027 g enzyme product/g dry pulp, the 4-day hydrolysis was observed to have 75.9% total sugar yield, while the 5-day hydrolysis was observed to have a 84.4% yield. Both hydrolysis was observed to produce sugars even after two days of hydrolysis.

Example 21. Hydrolysis of pretreated switchgrass solids using enzyme recycling

[0210] This Example demonstrates the use of enzyme recycling in the hydrolysis of pretreated biomass, while maintaining a high glucose yield but not overdosing in enzyme usage.

[0211] In light of the results observed in Examples 19 and 20 above, an enzyme dosage of 0.027 g enzyme product/g dry pulp was selected to achieve at sugar conversion yield of at least 75%. Enzyme doses below this (e.g., 0.019 g enzyme product/g dry pulp) were not observed to produce sufficient sugar conversion yields 18% solid loading within 3 days. Thus, in this Example, an initial enzyme dose of 0.027 g enzyme product/g dry pulp was used.

[0212] At the initial enzymatic hydrolysis, this enzyme dose was added to an enzymatic hydrolysis containing 8.3 g of sterilized dry pulp (wet in test). The solid dry pulp loading was 18.1% in the hydrolysis, and hydrolysis was conducted in a 50-mL volume in a 250-mL

Erlenmeyer flask on an orbital shaking incubator. The hydrolysis temperature was set at 50°C, and the shaking speed was controlled at 200 rpm. The hydrolysis pH was controlled at about 5.0 using a 50 mmol sodium citrate buffer. The pH was adjusted daily.

[0213] After 3 days of hydrolysis, most biomass was observed to be hydrolyzed, and a hydrolysate was observed to be formed. A glucose titer of 9.2% was observed from the hydrolysis after 3 days, which was not much different from the glucose titer of 9.9% observed after 4 days of hydrolysis.

[0214] Enzyme recycling was performed on this hydrolysis mixture. First, 8.3 g of fresh and sterilized dry pulp was added to the hydrolysis flask and mixed well with the hydrolysate. The mixed fresh pulp and hydrolysate slurry was left sitting for 20 minutes to allow the binding of the used enzyme product to the freshly added pretreated pulp materials. After enzyme binding, the entire mixture was filtered and pressed under sterile conditions to separate: (1) the filtrate or the hydrolysate, which mostly contained the lignocellulosic monomeric sugars; and (2) the filtered cake, which mostly contained the freshly added pulp materials, the undigested pulp or residual pulp from the initial enzymatic hydrolysis, and the insoluble lignin residuals, along with the recycle enzymes.

[0215] Enzymatic hydrolysis was continued by adding 0.019 g enzyme product/g dry fresh pulp and buffer to the filtered cake. The solid content was approximately 18%. The newly added enzyme dosage was 30% less than the initial enzyme dose. The enzymatic hydrolysis conditions were controlled as previously described above for this Example.

[0216] After 4 additional days of hydrolysis, the first enzyme recycling process was observed to have a glucose titer of 10.7%, which was not much different from the glucose titer of 10.9% after two additional days of hydrolysis.

[0217] The second enzyme recycling process was initiated by adding another 8.3 g of fresh and sterilized dry pulp to the hydrolysis slurry. The mixed fresh pulp and hydrolysate slurry was left to incubate for 20 minutes to allow enzyme binding to the fresh pulp materials. After enzyme binding, the entire mixture was filtered and pressed under sterile conditions to separate the enzyme bounded pulp cake and the hydrolysate. Enzymatic hydrolysis was continued by adding 0.019 g enzyme product/g dry fresh pulp in the buffer. The solid content was about 18%. Once again, the newly added enzyme dosage was 30% less enzyme than the initial enzyme dose. Enzymatic hydrolysis was continued.

[0218] The glucose and total sugar titers are shown in FIG. 6. The final glucose titer in the filtered hydrolysate was observed to be 11.0%, and the total sugar titer was observed to be 13.1%. After the hydrolysis with enzyme recycling, the unhydrolyzed residuals were collected by centrifugation, and washed two times with about 6x deionized water volume of the residual volume each time. The residuals were analyzed for its polymeric sugar content.

[0219] The mass balance of sugar production and total sugar yield of the entire process (i.e., the initial hydrolysis, first enzyme recycled hydrolysis and second enzyme recycled hydrolysis) are shown in Table 28 below.

Table 28. Mass balance summary

[0220] For the initial hydrolysis and the first enzyme recycled hydrolysis, the sugar in the filtrate represented the sugar in the hydrolysate that was removed. In the second enzyme recycled hydrolysis, the sugar in the filtrate included the sugar in the hydrolysate, but also the sugar in the hydrolysate entrapped in the residual solids.

[0221] The overall effectiveness of the enzyme recycling strategy can be understood by comparing the total average sugar produced per equivalent batch and total average enzyme used per batch in these three stages to the total sugar produced and total enzyme used per batch in Example 19. The initial hydrolysis and first recycled hydrolysis each took place over 4 days. The second recycled hydrolysis took place over 6 days. From the results of Example 19, one of skill in the art would have expected 3 loadings of pulp with 0.027 g enzyme loading and 4 days of hydrolysis to produce 12.54g of sugar (i.e., 3 x 4.18 = 12.54g). Instead, this Example demonstrated the production of 14.71g of sugar, while using 20% lower enzyme dosages. Thus, this Example demonstrated that enzyme recycling resulted in unexpected efficiencies in hydrolysis of pretreated biomass.

Example 22. Ethanol fermentation with concentrated hydrolysate from pretreated softwood

[0222] This Example demonstrates the use of concentrated hydrolysate for the fermentation of cellulosic ethanol. After ultra-filtration of the hydrolysate, the concentrated hydrolysate is produced from a thermal-vacuum concentrating process described from Example 16. A standard yeast Saccharomyces cerevisiae D5A (American Type Culture Collections, Manassas, VA) and Bio-Ferm® XR (North American Bioproducts Corp, Duluth, GA) were used in the fermentation.

[0223] With initial concentrated hydrolysate glucose titer of 27% (wt/vol) and D5A yeast 2 g/L, the ethanol can achieve as high as 11% (wt/vol) at 48 hours, as shown in FIG. 7. With initial concentrated hydrolysate glucose titer of 32% (wt/vol) and Bio-Ferm® XR yeast 2 g/L, the ethanol can achieve as high as 13% (wt/vol) at 88 hours, as shown in FIG. 8.