WO2014026154A1 - Optimized pretreatment of biomass - Google Patents

Abstract

Provided are methods and compositions for optimizing pretreatment processes using a two-stage pretreatment process for lignocellulosic biomass. Also provided are methods and compositions for decreasing the yield of one or more undesirable products during pretreatment.

Description

OPTIMIZED PRETREATMENT OF BIOMASS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/681,413, filed August 9, 2012, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] During the conversion of biomass into sugars and other products, the biomass normally goes through a pretreatment that extracts mostly pentose and hexose polymeric carbohydrates that are further enzymatically broken down or acid-hydro lyzed into monomers. Pretreatment systems are designed to roughly chop or hammer biomass feedstocks into smaller pieces that can be handled by mechanical systems that are used to treat the biomass with various physical and chemical modifications designed to provide access of the plant material to enzymes, pH, other chemicals, and various temperatures and pressures. The biomass can then subject to one or two stage treatments to free the carbohydrate fraction from other structural elements, and the carbohydrate fraction enzymatically hydrolyzed to produce sugar monomers. The object can be to extract as much sugar as possible from the biomass without producing breakdown products that interfere with and/or inhibit the fermentation of the sugars into desired products.

[0003] Pretreatment, in addition to requiring a significant amount of energy, can provide a poor yield of sugars. High heat and acid or alkali treatments can result in considerable breakdown products, reducing yields and increasing inhibitors of enzymes and fermentation. The viscosity of the materials can be considerable, even after steam and pressure treatments, making it difficult to move this matter through a mechanical system and further, making it difficult for enzymatic hydrolysis of biomass. Other engineered systems have been developed; however, the process remains problematic wherein inhibitor formation and yields are concerned. Further, the concentrations of enzyme required to reduce carbohydrate polymers and oligomers to monomers remains high and contributes to the elevated costs of producing cellulosic sugars.

SUMMARY OF THE INVENTION

[0004] In one aspect, disclosed herein are two stage methods of producing sugars from a biomass comprising: a) adding the biomass to a first liquid at a hydration temperature to produce a hydrated biomass; b) mechanical size reduction of the hydrated biomass to produce a mixture of size reduced solid particles; c) heating the mixture of size reduced solid particles at a first hydrolysis temperature for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction; d) heating the first solid fraction in an acidic medium comprising an acid at a second hydrolysis temperature for a second hydrolysis time of from about 1 minute to about 30 minutes to produce a mixture; and e) hydro lyzing the mixture with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solid fraction.

[0005] In some embodiments, the first liquid is water. In some embodiments, the C5 sugars of the first liquid fraction comprise soluble polysaccharides. In some embodiments, the methods further comprise hydrolyzing the first liquid fraction with one or more hemicellulase enzymes.

[0006] In some embodiments, the first liquid comprises from about 0.01% to about 10%> of an acid. In some embodiments, the first liquid comprises from about 0.01% to about 5% of an acid. In some embodiments, the first liquid comprises from about 0.01% to about 1% of an acid. In some embodiments, the first liquid comprises from about 0.1 % to about 0.5%> of the acid. In some embodiments, the first liquid comprises from about 0.1 % to about 0.3%> of the acid. In some embodiments, the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the first liquid is derived from S0₂ gas. In some embodiments, the first liquid is derived from H₂S0₄ gas. In some embodiments, the first liquid has a pH of from about 1.5 to about 3.5.

[0007] In some embodiments, the hydration temperature is from about 20 °C to about 110 °C. In some embodiments, the hydration temperature is from about 35 °C to about 70 °C. In some embodiments, the hydration temperature is from about 45 °C to about 55°C. In some

embodiments, the hydration temperature is about 50°C.

[0008] In some embodiments, the hydrated biomass comprises about 2% to about 12% solids (w/v). In some embodiments, the hydrated biomass comprises about 5-6% solids (w/v). In some embodiments, the hydrated biomass comprises about 10% to about 30% solids (w/v).

[0009] In some embodiments, the hydrated biomass is dewatered to about 30-32% solids (w/v) prior to heating at the first hydrolysis temperature.

[0010] In some embodiments, at least 50% of the solid particles in the mixture of size reduced solid particles are less than 10 mm in a dimension. In some embodiments, at least 50% of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension. In some embodiments, at least 50% of the solid particles in the mixture of size reduced solid particles are less than 5 mm in a dimension. In some embodiments, at least 50% of the solid particles in the mixture of size reduced solid particles are less than 2.5 mm in a dimension. In some embodiments, at least 50% of the solid particles in the mixture of size reduced solid particles are less than 1.5 mm in a dimension. In some embodiments, at least 50% the solid particles in the mixture of size reduced solid particles are from about 0.1 mm to about 1 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 1 mm in a dimension. In some embodiments, the dimension is diameter or width. In some embodiments, the dimension is diameter or width.

[0011] In some embodiments, the first hydrolysis temperature is about 125 °C to about 200 °C. In some embodiments, the first hydrolysis temperature is about 150 °C to about 170 °C. In some embodiments, heating the hydrated biomass is performed at a pressure of from about 100 psig to about 175 psig.

[0012] In some embodiments, the first hydrolysis time is from about 1 minute to about 120 minutes. In some embodiments, the first hydrolysis time is from about 5 minutes to about 60 minutes. In some embodiments, the first hydrolysis time is from about 20 minutes to about 40 minutes. In some embodiments, the first hydrolysis time is from about 5 minutes to about 15 minutes. In some embodiments, first hydrolysis time is less than about 20 minutes.

[0013] In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 140 °C. In some

embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 140 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 180 °C.

[0014] In some embodiments, the first liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural, hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof. [0015] In some embodiments, the method further comprises separating the first liquid fraction and the first solid fraction.

[0016] In some embodiments, the method further comprises concentrating the first liquid fraction.

[0017] In some embodiments, the acidic medium is an acidic solution. In some embodiments, the acidic medium comprises water. In some embodiments, the second hydrolysis temperature is from about 175 °C to about 275 °C. In some embodiments, the second hydrolysis temperature is from about 190 °C to about 240 °C.

[0018] In some embodiments, the second hydrolysis time is from about 1 minute to about 120 minutes. In some embodiments, the second hydrolysis time is from about 1 minute to about 60 minutes. In some embodiments, the second hydrolysis time is from about 5 minutes to about 15 minutes. In some embodiments, the second hydrolysis time is at least about 5 minutes.

[0019] In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 190°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 200°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 210°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 220°C.

[0020] In some embodiments, the acidic medium comprises from about 0.1% to about 10% of the acid. In some embodiments, the acidic medium comprises from about 0.1% to about 5% of the acid. In some embodiments, the acidic medium comprises from about 1% to about 3% of the acid. In some embodiments, the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the acidic medium is derived from S0₂ gas. In some embodiments, the acidic medium is derived from H₂S0₄ gas.

[0021] In some embodiments, the second liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural,

hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

[0022] In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 20% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 10% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 5% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.25% to about 1% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at about 0.5% based on total dry solids. [0023] In some embodiments, the method further comprises separating the second liquid fraction from the second solid fraction.

[0024] In some embodiments, the method further comprises concentrating the second liquid fraction.

[0025] In some embodiments, the first liquid fraction is combined with the second liquid fraction.

[0026] In some embodiments, the C5 sugars comprise xylose, arabinose, or a combination thereof.

[0027] In some embodiments, the C6 sugars comprise glucose.

[0028] In some embodiments, the biomass comprises cellulose, hemicellulose, or lignocellulose. In some embodiments, the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

[0029] In some embodiments, the method further comprises removing starch from the biomass prior to heating the hydrated biomass at the first hydrolysis temperature. In some embodiments, removing starch from the biomass comprises heating the hydrated biomass at greater than 100 °C. In some embodiments, the starch is hydrolyzed by one or more enzymes to produce glucose monomers. In some embodiments, the one or more enzymes comprise a-amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof. In some embodiments, the glucose monomers are combined with the second liquid fraction.

[0030] In some embodiments, the yield of C5 or C6 sugars is at least about 80% of a theoretical maximum. In some embodiments, the yield of C5 sugars or C6 sugars is at least about 90% of a theoretical maximum.

[0031] Also provided are compositions comprising the C5 sugars produced by the methods disclosed herein.

[0032] Also provided are compositions comprising the C6 sugars produced by the methods disclosed herein.

[0033] Also provided are compositions comprising the C5 sugars and the C6 sugars produced by the methods disclosed herein.

[0034] In another aspect, disclosed herein are systems for two stage production of sugars from a biomass comprising: a) a slurry mixer containing a first liquid at a hydration temperature; b) a rotary feeder that adds the biomass to the first liquid; c) a dewatering chamber that removes liquid from the biomass; d) a cutter pump that reduces the particle size of the biomass and pumps the biomass from the slurry mixer to the dewatering chamber; e) a microreactor that further reduces the particle size of the biomass to produce a mixture of size reduced solid particles; f) a hemicellulose reactor where the mixture of size reduced solid particles is heated at a first hydrolysis temperature and a first hydrolysis pressure for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction; g) a first flash tank for reducing temperature and pressure of the first liquid fraction and the first solid fraction; h) a first separator to separate the first liquid fraction from the first solid fraction; i) a retention module mixer that mixes the first solids fraction with an acidic medium comprising an acid, and heats the first solids fraction at a second hydrolysis temperature for a second hydrolysis time to produce a mixture; and j) a first enzyme reactor, wherein the mixture is hydrolyzed with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solids fraction.

[0035] In some embodiments, the system further comprises a second flash tank for reducing temperature and pressure of the mixture.

[0036] In some embodiments, the system further comprises a second separator to separate the second liquid fraction from the second solid fraction.

[0037] In some embodiments, the first liquid is water. In some embodiments, the C5 sugars of the first liquid fraction comprise soluble polysaccharides. In some embodiments, the system further comprises a second enzyme reactor, wherein the first liquid fraction is hydrolyzed with one or more hemicellulase enzymes.

[0038] In some embodiments, the first liquid comprises from about 0.01% to about 10% of an acid. In some embodiments, the first liquid comprises from about 0.01% to about 5% of an acid. In some embodiments, the first liquid comprises from about 0.01% to about 1% of an acid. In some embodiments, the first liquid comprises from about 0.1% to about 0.5% of an acid. In some embodiments, the first liquid comprises from about 0.1 % to about 0.3%> of the acid. In some embodiments, wherein the acid is S0₂ gas, sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the first liquid is derived from S0₂ gas. In some embodiments, the first liquid is derived from H₂S0₄ gas. In some embodiments, the first liquid has a pH of from about 1.5 to about 3.5.

[0039] In some embodiments, the hydration temperature is from about 20 °C to about 110 °C. In some embodiments, the hydration temperature is from about 35 °C to about 70 °C. In some embodiments, the hydration temperature is from about 45 °C to about 55°C. In some

embodiments, the hydration temperature is about 50°C. [0040] In some embodiments, the biomass is added to the first liquid at about 2% to about 12% solids (w/v). In some embodiments, the biomass is added to the first liquid at about 5-6% solids

(w/v).

[0041] In some embodiments, the dewatering chamber comprises one or more screw-type rotors. In some embodiments, the biomass is dewatered in the dewatering chamber to about 30-32% solids (w/v).

[0042] In some embodiments, at least 50%> the solid particles in the mixture of size reduced solid particles are less than 10 mm in a dimension. In some embodiments, at least 50% the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension. In some embodiments, at least 50%> the solid particles in the mixture of size reduced solid particles are less than 5 mm in a dimension. In some embodiments, at least 50% the solid particles in the mixture of size reduced solid particles are less than 2.5 mm in a dimension. In some

embodiments, at least 50%> the solid particles in the mixture of size reduced solid particles are less than 1.5 mm in a dimension. In some embodiments, at least 50% the solid particles in the mixture of size reduced solid particles are from about 0.1 mm to about 1 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 1 mm in a dimension. In some embodiments, the dimension is diameter or width.

[0043] In some embodiments, the hemicellulose reactor is a double-jacketed, screw type retention module.

[0044] In some embodiments, the first hydrolysis temperature is about 125 °C to about 200 °C. In some embodiments, the first hydrolysis temperature is about 150 °C to about 170 °C.

[0045] In some embodiments, first hydrolysis pressure is from about 100 psig to about 175 psig.

[0046] In some embodiments, the first hydrolysis time is from about 1 minute to about 120 minutes. In some embodiments, the first hydrolysis time is from about 5 minutes to about 60 minutes. In some embodiments, the first hydrolysis time is from about 20 minutes to about 40 minutes. In some embodiments, the first hydrolysis time is from about 5 minutes to about 15 minutes. In some embodiments, the first hydrolysis time is less than about 20 minutes.

[0047] In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 140 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about

5 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 140 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 180 °C.

[0048] In some embodiments, the first liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural, hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

[0049] In some embodiments, the acidic medium is an acidic solution. In some embodiments, the acidic medium comprises water.

[0050] In some embodiments, the second hydrolysis temperature is from about 175 °C to about 275 °C. In some embodiments, the second hydrolysis temperature is from about 190 °C to about 240 °C.

[0051] In some embodiments, the second hydrolysis time is from about 1 minute to about 120 minutes. In some embodiments, the second hydrolysis time is from about 1 minute to about 60 minutes. In some embodiments, the second hydrolysis time is from about 5 minutes to about 15 minutes. In some embodiments, the second hydrolysis time is at least about 5 minutes.

[0052] In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 190°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 200°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 210°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 220°C.

[0053] In some embodiments, the acidic medium comprises from about 0.1% to about 10 % of the acid. In some embodiments, the acidic medium comprises from about 0.1% to about 5 % of the acid. In some embodiments, the acidic medium comprises from about 1% to about 3% of the acid. In some embodiments, the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the acidic medium is derived from S0₂ gas. In some embodiments, the acidic medium is derived from H₂S0₄ gas. [0054] In some embodiments, the second liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural,

hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

[0055] In some embodiments, the one or more cellulase enzymes are at from about 0.251% to about 120% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 10% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 5% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.25% to about 1% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at about 0.5% based on total dry solids.

[0056] In some embodiments, the C5 sugars comprise xylose, arabinose, or a combination thereof.

[0057] In some embodiments, the C6 sugars comprise glucose.

[0058] In some embodiments, the biomass comprises cellulose, hemicellulose, or lignocellulose. In some embodiments, the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

[0059] In some embodiments, the system further comprises a microreactor for hydrolyzing starch with one or more enzymes to produce glucose monomers. In some embodiments, the hydration temperature is greater than 100 °C to remove starch from the biomass. In some embodiments, the one or more enzymes comprise a-amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof. In some embodiments, the system further comprises a separator to remove the glucose monomers from the biomass.

[0060] Provided herein are two stage methods of producing sugars from a biomass comprising: a) reducing the size of the biomass to smaller particles; b) adding a 0.01-0.5%) acid solution to the biomass to produce a slurry of 10-30%) w/v solids; c) treating the slurry for less than 20 minutes at 120-180°C to produce a first liquid fraction containing C5 sugars and a first solid fraction; d) separating and neutralizing the first liquid fraction; e) further treating the first solid fraction with a temperature greater than 190°C for greater than 5 minutes in a 0.5-10% acid solution to produce a mixture; f) neutralizing the mixture; g) hydrolyzing the mixture with at least one cellulase enzymes to produce a second liquid fraction and a second solid fraction; and h) separating the second liquid fraction from the second solid fraction.

[0061] In one embodiment, the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof. In one embodiment, the smaller particles are less than 10 mm in a dimension. In one embodiment, the smaller particles are less than 5 mm in a dimension. In one embodiment, the smaller particles are less than 2 mm in a dimension. In one embodiment, the smaller particles are less than 1 mm in a dimension. In one embodiment, the smaller particles are less than 0.2 mm in a dimension. In one embodiment, the smaller particles are uniform in size. In another embodiment, step c is carried out at a temperature of 120°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 130°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 140°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 150°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 160°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 170°C for 5 minutes. In another embodiment, step c is carried out at a temperature of 180°C for 5 minutes. In another embodiment, step e is carried out at a temperature of 190°C for greater than 5 minutes. In another embodiment, step e is carried out at a temperature of 200°C for greater than 5 minutes. In another embodiment, step e is carried out at a temperature of 210°C for greater than 5 minutes. In another embodiment, step e is carried out at a temperature of 220°C for greater than 5 minutes.

[0062] In a further embodiment, the acid solution of step b is derived from S0₂ gas. In another embodiment, the acid solution of step b is derived from H₂SO₄ gas. In a further embodiment, the acid solution for step e is derived from S0₂ gas. In another embodiment, the acid solution for step e is derived from H₂SO₄ gas. In another embodiment, the acid solution for step b is 0.1- 0.3% w/v. In a further embodiment, the acid solution for step e is 1-3% w/v. In another embodiment, the acid solution in step b or step e is selected from the group consisting of sulfurous acid, sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid and combinations thereof. [0063] In a further embodiment, the first liquid fraction of (d) is combined with the second liquid fraction of (h).

[0064] Provided also herein are two stage methods of producing sugars from a biomass comprising: a) reducing the size of the biomass to smaller pieces; b) adding water to the biomass to produce a slurry of 10-30% w/v solids; c) treating the 10-30% biomass (w/v) with water for no more than 20 minutes at 120-180°C to produce a first liquid fraction containing C5 sugars and a first solid fraction; d) removing and concentrating the first liquid fraction; e) hydrolyzing the first liquid fraction with at least one hemicellulase enzymes; f) treating the first solid fraction with a temperature greater than 190°C for greater than 5 minutes in a 0.5-10% acid solution to produce a mixture; g) neutralizing the mixture; h) hydrolyzing the mixture with cellulase enzymes to produce a second liquid fraction and a second solid fraction; and i) separating the second liquid fraction from the second solid fraction.

[0065] In one embodiment, the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof. In one embodiment, the smaller particles are less than 10 mm in a dimension. In one embodiment, the smaller particles are less than 5 mm in a dimension. In one embodiment, the smaller particles are less than 2 mm in a dimension. In one embodiment, the smaller particles are less than 1 mm in a dimension. In one embodiment, the smaller particles are less than 0.2 mm in a dimension. In one embodiment, the smaller particles are uniform in size. In another embodiment, step c is carried out at a temperature of 120°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 130°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 140°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 150°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 160°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 170°C for 5 minutes. In another embodiment, step c is carried out at a

temperature of 180°C for 5 minutes. In another embodiment, step f is carried out at a

temperature of 190°C for greater than 5 minutes. In another embodiment, step f is carried out at a temperature of 200°C for greater than 5 minutes. In another embodiment, step f is carried out at a temperature of 210°C for greater than 5 minutes. In another embodiment, step f is carried out at a temperature of 220°C for greater than 5 minutes.

[0066] In a further embodiment, the acid solution for step f is derived from S0₂ gas. In another embodiment, the acid solution for step f is derived from H₂SO₄ gas. In a further embodiment, the acid solution for step f is 1-3% w/v. In another embodiment, the acid in step f is selected from the group consisting of sulfurous acid, sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid and combinations thereof. In another embodiment, the first liquid fraction of d) is combined with the second liquid fraction of i).

[0067] Provided herein is also a process that is part of any of the methods above wherein starch is removed from a biomass prior to step (c). In a further embodiment, the starch is hydrolyzed to glucose monomers by enzymatic digestion. In another embodiment, the enzymatic digestion is carried out with enzymes selected from the group consisting of a-amylase, β-amylase, glucoamylase, pullulinase, and a combination thereof. In another embodiment, the glucose monomers derived from the starch are combined with the second liquid fraction i.

INCORPORATION BY REFERENCE

[0068] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0070] Figure 1 is a block diagram depicting the two-stage process of this invention, showing the lignocellulosic feedstock entering into the improved hydrolysis process system, thereby producing sugar hydrolysate products and a lignin residue solid product.

[0071] Figure 2 is a flow diagram of a two-stage pretreatment hydrolysis using a dilute acid hydrolysis in stage one. Illustrated are: a hopper or feeder conduit 1, a slurry-mixer 2, a cutter pump 3, a dewatering chamber 4, a microreactor 5, a hemicellulose hydrolysis reactor 6, a flash- tank 7, a separator 8, a retention module mixer 9, a flash tank 10, a collection tank 11, and a reactor 12.

[0072] Figure 3 is a flow diagram of a two-stage pretreatment hydrolysis using a hot water solution in stage one. Illustrated are: a hopper or feeder conduit 1, a slurry-mixer 2, a cutter pump 3, a dewatering chamber 4, a microreactor 5, a hemicellulose hydrolysis reactor 6, a flash- tank 7, a separator 8, a microreactor 13, a retention module mixer 9, a flash tank 10, a collection tank 11, and a reactor 12.

[0073] Figure 4 is a flow diagram of a two-stage pretreatment hydrolysis for a feedstock containing starch using a dilute acid hydrolysis in stage one. Illustrated are: a hopper or feeder conduit 1, a slurry-mixer 2, a cutter pump 3, a microreactor 14, a separator 15, a dewatering chamber 4, a microreactor 5, a hemicellulose hydrolysis reactor 6, a flash-tank 7, a separator 8, a retention module mixer 9, a flash tank 10, a collection tank 11, and a reactor 12.

[0074] Figure 5 is graph comparing the results of enzyme concentrations on the hydrolysis of carbohydrate yields from one-stage pretreatment versus yields from two-stage pretreatment. The combined C5 and C6 carbohydrate yields (Conversion Efficiency %) is plotted verses the Enzyme Dosage.

[0075] Figure 6 is a graph comparing the results of enzyme conversion efficiency extrapolated to 5% loading. The combined C5 and C6 carbohydrate yields (Conversion %) is plotted verses the Enzyme Dosage.

DETAILED DESCRIPTION OF THE INVENTION

[0076] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a purified monomer" includes mixtures of two or more purified monomers. The term "comprising" as used herein is synonymous with "including," "containing," or

"characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0077] "About" means a referenced numeric indication plus or minus 10% of that referenced numeric indication. For example, the term about 4 would include a range of 3.6 to 4.4. All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. [0078] Wherever the phrase "for example," "such as," "including" and the like are used herein, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise. Therefore, "for example ethanol production" means "for example and without limitation ethanol production."

[0079] In this specification and in the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings.

[0080] Definitions

[0081] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase "the medium can optionally contain glucose" means that the medium may or may not contain glucose as an ingredient and that the description includes both media containing glucose and media not containing glucose.

[0082] Unless characterized otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0083] " Fermentive end-product" and "fermentation end-product" are used interchangeably herein to include biofuels, chemicals, compounds suitable as liquid fuels, gaseous fuels, triacylglycerols, reagents, chemical feedstocks, chemical additives, processing aids, food additives, bioplastics and precursors to bioplastics, and other products. Examples of fermentive end-products include but are not limited to 1,4 diacids (succinic, fumaric and malic), 2,5 furan dicarboxylic acid, 3 hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol/arabinitol, butanediol, butanol, methane, methanol, ethane, ethene, ethanol, n-propane, 1-propene, 1-propanol, propanal, acetone, propionate, n-butane, 1-butene, 1 -butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2-methylbutanol, 3-methylbutanal, 3-methylbutanol, 2-butene, 2- butanol, 2-butanone, 2,3-butanediol, 3-hydroxy-2-butanone, 2,3-butanedione, ethylbenzene, ethenylbenzene, 2-phenylethanol, phenylacetaldehyde, 1-phenylbutane, 4-phenyl- 1-butene, 4- phenyl-2-butene, 1 -phenyl-2-butene, 1 -phenyl -2 -butanol, 4-phenyl -2 -butanol, 1 -phenyl -2- butanone, 4-phenyl-2-butanone, l-phenyl-2,3-butandiol, l-phenyl-3-hydroxy-2-butanone, 4- phenyl-3-hydroxy-2-butanone, l-phenyl-2,3-butanedione, n-pentane, ethylphenol,

ethenylphenol, 2-(4-hydroxyphenyl)ethanol, 4-hydroxyphenylacetaldehyde, l-(4- hydroxyphenyl) butane, 4-(4-hydroxyphenyl)- 1-butene, 4-(4-hydroxyphenyl)-2-butene, l-(4- hydroxyphenyl)- 1-butene, l-(4-hydroxyphenyl)-2-butanol, 4-(4-hydroxyphenyl)-2 -butanol, l-(4- hydroxyphenyl)-2-butanone, 4-(4-hydroxyphenyl)-2-butanone, 1 -(4-hydroxyphenyl)-2,3- butandiol, 1 -(4-hydroxyphenyl)-3-hydroxy-2-butanone, 4-(4-hydroxyphenyl)-3-hydroxy-2- butanone, l-(4-hydroxyphenyl)-2,3-butanonedione, indolylethane, indolylethene, 2-(indole-3-

)ethanol, n-pentane, 1 -pentene, 1-pentanol, pentanal, pentanoate, 2-pentene, 2-pentanol, 3- pentanol, 2-pentanone, 3-pentanone, 4-methylpentanal, 4-methylpentanol, 2,3-pentanediol, 2- hydroxy-3-pentanone, 3-hydroxy-2-pentanone, 2,3-pentanedione, 2-methylpentane, 4-methyl-l- pentene, 4-methyl-2 -pentene, 4-methyl-3-pentene, 4-methyl-2-pentanol, 2-methyl-3-pentanol, 4- methyl-2-pentanone, 2-methyl-3-pentanone, 4-methyl-2,3-pentanediol, 4-methyl-2-hydroxy-3- pentanone, 4-methyl-3-hydroxy-2-pentanone, 4-methyl-2,3-pentanedione, 1-phenylpentane, 1- phenyl-1 -pentene, l-phenyl-2-pentene, l-phenyl-3 -pentene, l-phenyl-2-pentanol, l-phenyl-3- pentanol, l-phenyl-2-pentanone, l-phenyl-3 -pentanone, l-phenyl-2,3-pentanediol, 1 -phenyl -2- hydroxy-3-pentanone, l-phenyl-3 -hydroxy-2 -pentanone, 1 -phenyl -2,3-pentanedione, 4-methyl- 1-phenylpentane, 4-methyl-l -phenyl- 1 -pentene, 4-methyl-l-phenyl-2 -pentene, 4-methyl- 1- phenyl-3 -pentene, 4-methyl- l-phenyl-3 -pentanol, 4-methyl-l -phenyl-2-pentanol, 4-methyl-l- phenyl-3 -pentanone, 4-methyl- l-phenyl-2 -pentanone, 4-methyl-l -phenyl -2,3-pentanediol, 4- methyl- 1 -phenyl-2,3 -pentanedione, 4-methyl- 1 -phenyl-3 -hydroxy-2-pentanone, 4-methyl- 1 - phenyl-2-hydroxy-3 -pentanone, l-(4-hydroxyphenyl) pentane, l-(4-hydroxyphenyl)-l -pentene, 1 -(4-hydroxyphenyl)-2-pentene, 1 -(4-hydroxyphenyl)-3 -pentene, 1 -(4-hydroxyphenyl)-2- pentanol, l-(4-hydroxyphenyl)-3 -pentanol, l-(4-hydroxyphenyl)-2-pentanone, l-(4- hydroxyphenyl)-3-pentanone, 1 -(4-hydroxyphenyl)-2,3-pentanediol, 1 -(4-hydroxyphenyl)-2- hydroxy-3 -pentanone, 1 -(4-hydroxyphenyl)-3 -hydroxy-2 -pentanone, 1 -(4-hydroxyphenyl)-2,3- pentanedione, 4-methyl- l-(4-hydroxyphenyl) pentane, 4-methyl- l-(4-hydroxyphenyl)-2 -pentene, 4-methyl- 1 -(4-hydroxyphenyl)-3 -pentene, 4-methyl- 1 -(4-hydroxyphenyl)- 1 -pentene, 4-methyl- 1 -(4-hydroxyphenyl)-3-pentanol, 4-methyl- 1 -(4-hydroxyphenyl)-2-pentanol, 4-methyl- 1 -(4- hydroxyphenyl)-3-pentanone, 4-methyl- l-(4-hydroxyphenyl)-2 -pentanone, 4-methyl-l -(4- hydroxyphenyl)-2,3-pentanediol, 4-methyl- 1 -(4-hydroxyphenyl)-2,3-pentanedione, 4-methyl- 1 - (4-hydroxyphenyl)-3 -hydroxy-2 -pentanone, 4-methyl- 1 -(4-hydroxyphenyl)-2-hydroxy-3 - pentanone, l-indole-3 -pentane, l-(indole-3)-l -pentene, l-(indole-3)-2 -pentene, l-(indole-3)-3- pentene, l-(indole-3)-2 -pentanol, l-(indole-3)-3-pentanol, l-(indole-3)-2-pentanone, l-(indole- 3)-3-pentanone, 1 -(indole-3)-2,3-pentanediol, 1 -(indole-3)-2-hydroxy-3-pentanone, 1 -(indole-3 )- 3-hydroxy-2-pentanone, l-(indole-3)-2,3-pentanedione, 4-methyl-l-(indole-3-)pentane, 4- methyl- 1 -(indole-3)-2-pentene, 4-methyl- 1 -(indole-3)-3-pentene, 4-methyl- 1 -(indole-3)- 1 - pentene, 4-methyl-2-(indole-3)-3-pentanol, 4-methyl- l-(indole-3)-2 -pentanol, 4-methyl- 1- (indole-3)-3-pentanone, 4-methyl-l -(indole-3 )-2-pentanone, 4-methyl- l-(indole-3)-2, 3- pentanediol, 4-methyl-l-(indole-3)-2,3-pentanedione, 4-methyl-l-(indole-3)-3-hydroxy-2- pentanone, 4-methyl-l-(indole-3)-2-hydroxy-3-pentanone, n-hexane, 1-hexene, 1-hexanol, hexanal, hexanoate, 2-hexene, 3-hexene, 2-hexanol, 3-hexanol, 2-hexanone, 3-hexanone, 2,3- hexanediol, 2,3-hexanedione, 3,4-hexanediol, 3,4-hexanedione, 2-hydroxy-3-hexanone, 3- hydroxy-2-hexanone, 3-hydroxy-4-hexanone, 4-hydroxy-3-hexanone, 2-methylhexane, 3- methylhexane, 2-methyl-2-hexene, 2-methyl-3 -hexene, 5 -methyl- 1 -hexene, 5-methyl-2-hexene,

4- methyl-l -hexene, 4-methyl-2-hexene, 3 -methyl-3 -hexene, 3-methyl-2-hexene, 3-methyl-l- hexene, 2-methyl-3-hexanol, 5-methyl-2-hexanol, 5 -methyl-3 -hexanol, 2-methyl-3-hexanone, 5- methyl-2-hexanone, 5 -methyl-3 -hexanone, 2-methyl-3,4-hexanediol, 2-methyl-3,4-hexanedione,

5- methyl-2,3-hexanediol, 5-methyl-2,3-hexanedione, 4-methyl-2,3-hexanediol, 4-methyl-2,3- hexanedione, 2-methyl-3-hydroxy-4-hexanone, 2-methyl-4-hydroxy-3 -hexanone, 5-methyl-2- hydroxy-3 -hexanone, 5 -methyl-3 -hydroxy-2-hexanone, 4-methyl-2-hydroxy-3 -hexanone, 4- methyl-3-hydroxy-2-hexanone, 2,5-dimethylhexane, 2,5-dimethyl-2-hexene, 2,5-dimethyl-3- hexene, 2,5-dimethyl-3-hexanol, 2,5-dimethyl-3-hexanone, 2,5-dimethyl-3,4-hexanediol, 2,5- dimethyl-3,4-hexanedione, 2,5-dimethyl-3-hydroxy-4-hexanone, 5-methyl-l-phenylhexane, 4- methyl-l-phenylhexane, 5 -methyl- 1 -phenyl- 1 -hexene, 5-methyl-l-phenyl-2-hexene, 5-methyl-l- phenyl-3 -hexene, 4-methyl-l -phenyl- 1 -hexene, 4-methyl-l-phenyl-2-hexene, 4-methyl- 1- phenyl-3 -hexene, 5 -methyl- l-phenyl-2-hexanol, 5 -methyl- l-phenyl-3 -hexanol, 4-methyl- 1- phenyl-2 -hexanol, 4-methyl- l-phenyl-3 -hexanol, 5 -methyl- 1 -phenyl-2-hexanone, 5-methyl-l- phenyl-3 -hexanone, 4-methyl- l-phenyl-2-hexanone, 4-methyl- l-phenyl-3 -hexanone, 5-methyl-

1 -phenyl -2, 3-hexanediol, 4-methyl- 1 -phenyl-2,3-hexanediol, 5-methyl- 1 -phenyl-3-hydroxy-2- hexanone, 5 -methyl- 1 -phenyl-2-hydroxy-3 -hexanone, 4-methyl- 1 -phenyl-3 -hydroxy-2- hexanone, 4-methyl- l-phenyl-2-hydroxy-3 -hexanone, 5-methyl- l-phenyl-2,3-hexanedione, 4- methyl- 1 -phenyl-2,3 -hexanedione, 4-methyl- 1 -(4-hydroxyphenyl)hexane, 5 -methyl- 1 -(4- hydroxyphenyl)- 1 -hexene, 5 -methyl- 1 -(4-hydroxyphenyl)-2-hexene, 5 -methyl- 1 -(4- hydroxyphenyl)-3 -hexene, 4-methyl- 1 -(4-hydroxyphenyl)- 1 -hexene, 4-methyl- 1 -(4- hydroxyphenyl)-2-hexene, 4-methyl- 1 -(4-hydroxyphenyl)-3 -hexene, 5 -methyl- 1 -(4- hydroxyphenyl)-2-hexanol, 5-methyl- 1 -(4-hydroxyphenyl)-3 -hexanol, 4-methyl- 1 -(4- hydroxyphenyl)-2-hexanol, 4-methyl- 1 -(4-hydroxyphenyl)-3 -hexanol, 5-methyl- 1 -(4- hydroxyphenyl)-2-hexanone, 5-methyl-l-(4-hydroxyphenyl)-3-hexanone, 4-methyl-l -(4- hydroxyphenyl)-2-hexanone, 4-methyl-l -(4-hydroxyphenyl)-3-hexanone, 5 -methyl- 1 -(4- hydroxyphenyl)-2,3-hexanediol, 4-methyl-l -(4-hydroxyphenyl)-2, 3-hexanediol, 5 -methyl- 1 -(4- hydroxyphenyl)-3-hydroxy-2-hexanone, 5-methyl- 1 -(4-hydroxyphenyl)-2-hydroxy-3-hexanone, 4-methyl- 1 -(4-hydroxyphenyl)-3 -hydroxy-2-hexanone, 4-methyl- 1 -(4-hydroxyphenyl)-2- hydroxy-3 -hexanone, 5-methyl-l-(4-hydroxyphenyl)-2,3-hexanedione, 4-methyl-l -(4- hydroxyphenyl)-2,3-hexanedione, 4-methyl- 1 -(indole-3-)hexane, 5-methyl- 1 -(indole-3)- 1 - hexene, 5-methyl- l-(indole-3)-2-hexene, 5-methyl- l-(indole-3)-3-hex ene, 4-methyl- l-(indole- 3)-l-hexene, 4-methyl-l -(indole-3)-2-hexene, 4-methyl-l -(indole-3)-3-hexene, 5-methyl- 1- (indole-3)-2-hexanol, 5-methyl-l-(indole-3)-3-hexanol, 4-methyl-l-(indole-3)-2-hexanol, 4- methyl-l-(indole-3)-3-hexanol, 5-methyl-l-(indole-3)-2-hexanone, 5-methyl-l-(indole-3)-3- hexanone, 4-methyl-l-(indole-3)-2-hexanone, 4-methyl-l-(indole-3)-3-hexanone, 5-methyl-l-

(indole-3)-2,3-hexanediol, 4-methyl-l-(indole-3)-2,3-hexanediol, 5-methyl-l-(indole-3)-3- hydroxy-2-hexanone, 5-methyl-l-(indole-3)-2-hydroxy-3-hexanone, 4-methyl-l-(indole-3)-3- hydroxy-2-hexanone, 4-methyl-l-(indole-3)-2-hydroxy-3-hexanone, 5-methyl-l-(indole-3)-2,3- hexanedione, 4-methyl-l-(indole-3)-2,3-hexanedione, n-heptane, 1-heptene, 1-heptanol, heptanal, heptanoate, 2-heptene, 3-heptene, 2-heptanol, 3-heptanol, 4-heptanol, 2-heptanone, 3- heptanone, 4-heptanone, 2,3-heptanediol, 2,3-heptanedione, 3,4-heptanediol, 3,4-heptanedione,

2- hydroxy-3-heptanone, 3-hydroxy-2-heptanone, 3-hydroxy-4-heptanone, 4-hydroxy-3- heptanone, 2-methylheptane, 3-methylheptane, 6-methyl-2-heptene, 6-methyl-3-heptene, 2- methyl-3-heptene, 2-methyl-2-heptene, 5-methyl-2-heptene, 5-methyl-3-heptene, 3-methyl-3- heptene, 2-methyl-3-heptanol, 2-methyl-4-heptanol, 6-methyl-3-heptanol, 5-methyl-3-heptanol,

3- methyl-4-heptanol, 2-methyl-3-heptanone, 2-methyl-4-heptanone, 6-methyl-3-heptanone, 5- methyl-3-heptanone, 3-methyl-4-heptanone, 2-methyl-3,4-heptanediol, 2-methyl-3,4- heptanedione, 6-methyl-3,4-heptanediol, 6-methyl-3,4-heptanedione, 5-methyl-3,4-heptanediol,

5 - methyl-3 ,4-heptanedione, 2-methyl-3 -hydroxy-4-heptanone, 2 -methyl -4-hydroxy-3 -heptanone,

6- methyl-3 -hydroxy-4-heptanone, 6-methyl-4-hydroxy-3 -heptanone, 5 -methyl-3 -hydroxy-4- heptanone, 5-methyl-4-hydroxy-3-heptanone, 2,6-dimethylheptane, 2,5-dimethylheptane, 2,6- dimethyl-2-heptene, 2,6-dimethyl-3-heptene, 2,5-dimethyl-2-heptene, 2,5-dimethyl-3-heptene, 3,6-dimethyl-3-heptene, 2,6-dimethyl-3-heptanol, 2,6-dimethyl-4-heptanol, 2,5-dimethyl-3- heptanol, 2,5-dimethyl-4-heptanol, 2,6-dimethyl-3,4-heptanediol, 2,6-dimethyl-3,4- heptanedione, 2,5-dimethyl-3,4-heptanediol, 2,5-dimethyl-3,4-heptanedione, 2,6-dimethyl-3- hydroxy-4-heptanone, 2,6-dimethyl-4-hydroxy-3-heptanone, 2,5-dimethyl-3-hydroxy-4- heptanone, 2,5-dimethyl-4-hydroxy-3-heptanone, n-octane, 1-octene, 2-octene, 1-octanol, octanal, octanoate, 3-octene, 4-octene, 4-octanol, 4-octanone, 4,5-octanediol, 4,5-octanedione, 4- hydroxy-5-octanone, 2-methyloctane, 2-methyl-3-octene, 2-methyl-4-octene, 7-methyl-3-octene,

3 - methyl-3 -octene, 3-methyl-4-octene, 6-methyl-3-octene, 2-methyl-4-octanol, 7-methyl-4- octanol, 3 -methyl -4-octanol, 6-methyl-4-octanol, 2-methyl-4-octanone, 7-methyl-4-octanone, 3- methyl-4-octanone, 6-methyl-4-octanone, 2-methyl-4,5-octanediol, 2-methyl-4,5-octanedione, 3- methyl-4,5-octanediol, 3-methyl-4,5-octanedione, 2-methyl-4-hydroxy-5-octanone, 2-methyl-5- hydroxy-4-octanone, 3-methyl-4-hydroxy-5-octanone, 3-methyl-5-hydroxy-4-octanone, 2,7- dimethyloctane, 2, 7-dimethyl-3 -octene, 2,7-dimethyl-4-octene, 2,7-dimethyl-4-octanol, 2,7- dimethyl-4-octanone, 2,7-dimethyl-4,5-octanediol, 2,7-dimethyl-4,5-octanedione, 2,7-dimethyl-

4- hydroxy-5-octanone, 2,6-dimethyloctane, 2,6-dimethyl-3-octene, 2,6-dimethyl-4-octene, 3,7- dimethyl-3-octene, 2,6-dimethyl-4-octanol, 3,7-dimethyl-4-octanol, 2,6-dimethyl-4-octanone,

3.7- dimethyl-4-octanone, 2,6-dimethyl-4,5-octanediol, 2,6-dimethyl-4,5-octanedione, 2,6- dimethyl-4-hydroxy-5-octanone, 2,6-dimethyl-5-hydroxy-4-octanone, 3,6-dimethyloctane, 3,6- dimethyl-3-octene, 3,6-dimethyl-4-octene, 3,6-dimethyl-4-octanol, 3,6-dimethyl-4-octanone, 3,6-dimethyl-4,5-octanediol, 3,6-dimethyl-4,5-octanedione, 3,6-dimethyl-4-hydroxy-5-octanone, n-nonane, 1-nonene, 1-nonanol, nonanal, nonanoate, 2-methylnonane, 2-methyl-4-nonene, 2- methyl-5-nonene, 8-methyl-4-nonene, 2-methyl-5-nonanol, 8-methyl-4-nonanol, 2-methyl-5- nonanone, 8-methyl-4-nonanone, 8-methyl-4,5-nonanediol, 8-methyl-4,5-nonanedione, 8- methyl-4-hydroxy-5-nonanone, 8-methyl-5-hydroxy-4-nonanone, 2,8-dimethylnonane, 2,8- dimethyl-3-nonene, 2,8-dimethyl-4-nonene, 2,8-dimethyl-5-nonene, 2,8-dimethyl-4-nonanol,

2.8- dimethyl-5-nonanol, 2,8-dimethyl-4-nonanone, 2,8-dimethyl-5-nonanone, 2,8-dimethyl-4,5- nonanediol, 2,8-dimethyl-4,5-nonanedione, 2,8-dimethyl-4-hydroxy-5-nonanone, 2,8-dimethyl- 5-hydroxy-4-nonanone, 2,7-dimethylnonane, 3,8-dimethyl-3-nonene, 3,8-dimethyl-4-nonene, 3,8-dimethyl-5-nonene, 3,8-dimethyl-4-nonanol, 3,8-dimethyl-5-nonanol, 3,8-dimethyl-4- nonanone, 3,8-dimethyl-5-nonanone, 3,8-dimethyl-4,5-nonanediol, 3,8-dimethyl-4,5- nonanedione, 3,8-dimethyl-4-hydroxy-5-nonanone, 3,8-dimethyl-5-hydroxy-4-nonanone, n- decane, 1-decene, 1-decanol, decanoate, 2,9-dimethyldecane, 2,9-dimethyl-3-decene, 2,9- dimethyl-4-decene, 2,9-dimethyl-5-decanol, 2,9-dimethyl-5-decanone, 2,9-dimethyl-5,6- decanediol, 2,9-dimethyl-6-hydroxy-5-decanone, 2,9-dimethyl-5,6-decanedionen-undecane, 1- undecene, 1-undecanol, undecanal, undecanoate, n-dodecane, 1-dodecene, 1-dodecanol, dodecanal, dodecanoate, n-dodecane, 1-decadecene, n-tridecane, 1-tridecene, 1-tridecanol, tridecanal, tridecanoate, n-tetradecane, 1-tetradecene, 1-tetradecanol, tetradecanal,

tetradecanoate, n-pentadecane, 1-pentadecene, 1-pentadecanol, pentadecanal, pentadecanoate, n- hexadecane, 1-hexadecene, 1-hexadecanol, hexadecanal, hexadecanoate, n-heptadecane, 1- heptadecene, 1-heptadecanol, heptadecanal, heptadecanoate, n-octadecane, 1-octadecene, 1- octadecanol, octadecanal, octadecanoate, n-nonadecane, 1-nonadecene, 1-nonadecanol, nonadecanal, nonadecanoate, eicosane, 1-eicosene, 1-eicosanol, eicosanal, eicosanoate, 3- hydroxy propanal, 1,3 -propanediol, 4-hydroxybutanal, 1 ,4-butanediol, 3-hydroxy-2-butanone, 2,3-butandiol, 1,5-pentane diol, homocitrate, homoisocitorate, b-hydroxy adipate, glutarate, glutarsemialdehyde, glutaraldehyde, 2-hydroxy-l-cyclopentanone, 1 ,2-cyclopentanediol, cyclopentanone, cyclopentanol, (S)-2-acetolactate, (R)-2,3-Dihydroxy-isovalerate, 2- oxoisovalerate, isobutyryl-CoA, isobutyrate, isobutyraldehyde, 5-amino pentaldehyde, 1,10- diaminodecane, l,10-diamino-5-decene, l,10-diamino-5-hydroxydecane, l,10-diamino-5- decanone, 1 , 10-diamino-5 ,6-decanediol, 1 , 10-diamino-6-hydroxy-5-decanone,

phenylacetoaldehyde, 1,4-diphenylbutane, 1,4-diphenyl-l-butene, l,4-diphenyl-2-butene, 1,4- diphenyl-2-butanol, 1 ,4-diphenyl-2-butanone, l ,4-diphenyl-2,3-butanediol, l ,4-diphenyl-3- hydroxy-2-butanone, 1 -(4-hydeoxyphenyl)-4-phenylbutane, 1 -(4-hydeoxyphenyl)-4-phenyl- 1 - butene, l-(4-hydeoxyphenyl)-4-phenyl-2-butene, l-(4-hydeoxyphenyl)-4-phenyl-2-butanol, l-(4- hydeoxyphenyl)-4-phenyl-2-butanone, 1 -(4-hydeoxyphenyl)-4-phenyl-2,3-butanediol, 1 -(4- hydeoxyphenyl)-4-phenyl-3-hydroxy-2-butanone, 1 -(indole-3)-4-phenylbutane, 1 -(indole-3)-4- phenyl- 1 -butene, 1 -(indole-3)-4-phenyl-2 -butene, 1 -(indole-3)-4-phenyl-2-butanol, 1 -(indole-3)- 4-phenyl-2-butanone, l-(indole-3)-4-phenyl-2,3-butanediol, l-(indole-3)-4-phenyl-3-hydroxy-2- butanone, 4-hydroxyphenylacetoaldehyde, 1 ,4-di(4-hydroxyphenyl)butane, 1 ,4-di(4- hydroxyphenyl)- 1 -butene, 1 ,4-di(4-hydroxyphenyl)-2 -butene, 1 ,4-di(4-hydroxyphenyl)-2- butanol, l ,4-di(4-hydroxyphenyl)-2-butanone, l ,4-di(4-hydroxyphenyl)-2,3-butanediol, l ,4-di(4- hydroxyphenyl)-3 -hydro xy-2-butanone, 1 -(4-hydroxyphenyl)-4-(indole-3-)butane, 1 -(4- hydroxyphenyl)-4-(indole-3)- 1 -butene, 1 -di(4-hydroxyphenyl)-4-(indole-3)-2 -butene, 1 -(4- hydroxyphenyl)-4-(indole-3)-2-butanol, 1 -(4-hydroxyphenyl)-4-(indole-3)-2-butanone, 1 -(4- hydroxyphenyl)-4-(indole-3)-2,3 -butanediol, 1 -(4-hydroxyphenyl-4-(indole-3)-3 -hydroxy-2- butanone, indole-3-acetoaldehyde, l ,4-di(indole-3-)butane, l ,4-di(indole-3)-l -butene, 1 ,4- di(indole-3)-2 -butene, 1 ,4-di(indole-3)-2-butanol, 1 ,4-di(indole-3)-2-butanone, 1 ,4-di(indole-3)- 2,3-butanediol, l ,4-di(indole-3)-3-hydroxy-2-butanone, succinate semialdehyde, hexane-1 ,8- dicarboxylic acid, 3-hexene-l ,8-dicarboxylic acid, 3-hydroxy-hexane-l ,8-dicarboxylic acid, 3- hexanone-l ,8-dicarboxylic acid, 3,4-hexanediol-l ,8-dicarboxylic acid, 4-hydroxy-3-hexanone- 1 ,8-dicarboxylic acid, glycerol, fucoidan, iodine, chlorophyll, carotenoid, calcium, magnesium, iron, sodium, potassium, phosphate, lactic acid, acetic acid, formic acid, isoprenoids, and polyisoprenes, including rubber. Further, such products can include succinic acid, pyruvic acid, enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases and may be present as a pure compound, a mixture, or an impure or diluted form.

[0084] Fermentation end-products can include polyols or sugar alcohols; for example, methanol, glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, dulcitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, and/or polyglycitol.

[0085] The term "fatty acid comprising material" as used herein has its ordinary meaning as known to those skilled in the art and can comprise one or more chemical compounds that include one or more fatty acid moieties as well as derivatives of these compounds and materials that comprise one or more of these compounds. Common examples of compounds that include one or more fatty acid moieties include triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, lysophospholipids, free fatty acids, fatty acid salts, soaps, fatty acid comprising amides, esters of fatty acids and monohydric alcohols, esters of fatty acids and polyhydric alcohols including glycols (e.g. ethylene glycol, propylene glycol, etc.), esters of fatty acids and polyethylene glycol, esters of fatty acids and polyethers, esters of fatty acids and polyglycol, esters of fatty acids and saccharides, esters of fatty acids with other hydroxyl-containing compounds, etc. A fatty acid comprising material can be one or more of these compounds in an isolated or purified form. It can be a material that includes one or more of these compounds that is combined or blended with other similar or different materials. It can be a material where the fatty acid comprising material occurs with or is provided with other similar or different materials, such as vegetable and animal oils; mixtures of vegetable and animal oils; vegetable and animal oil byproducts; mixtures of vegetable and animal oil byproducts; vegetable and animal wax esters; mixtures, derivatives and byproducts of vegetable and animal wax esters; seeds; processed seeds; seed byproducts; nuts; processed nuts; nut byproducts; animal matter; processed animal matter; byproducts of animal matter; corn; processed corn; corn byproducts; distiller's grains; beans; processed beans; bean byproducts; soy products; lipid containing plant, fish or animal matter; processed lipid containing plant or animal matter; byproducts of lipid containing plant, fish or animal matter; lipid containing microbial material; processed lipid containing microbial material; and byproducts of lipid containing microbial matter. Such materials can be utilized in liquid or solid forms. Solid forms include whole forms, such as cells, beans, and seeds; ground, chopped, slurried, extracted, flaked, milled, etc. The fatty acid portion of the fatty acid comprising compound can be a simple fatty acid, such as one that includes a carboxyl group attached to a substituted or un- substituted alkyl group. The substituted or unsubstituted alkyl group can be straight or branched, saturated or unsaturated. Substitutions on the alkyl group can include hydroxyls, phosphates, halogens, alkoxy, or aryl groups. The substituted or unsubstituted alkyl group can have 7 to 29 carbons and preferably 1 1 to 23 carbons (e.g., 8 to 30 carbons and preferably 12 to 24 carbons counting the carboxyl group) arranged in a linear chain with or without side chains and/or substitutions. Addition of the fatty acid comprising compound can be by way of adding a material comprising the fatty acid comprising compound.

[0086] The term "pH modifier" as used herein has its ordinary meaning as known to those skilled in the art and can include any material that will tend to increase, decrease or hold steady the pH of the broth or medium. A pH modifier can be an acid, a base, a buffer, or a material that reacts with other materials present to serve to raise, lower, or hold steady the pH. In one embodiment, more than one pH modifier can be used, such as more than one acid, more than one base, one or more acid with one or more bases, one or more acids with one or more buffers, one or more bases with one or more buffers, or one or more acids with one or more bases with one or more buffers. In one embodiment, a buffer can be produced in the broth or medium or separately and used as an ingredient by at least partially reacting in acid or base with a base or an acid, respectively. When more than one pH modifiers are utilized, they can be added at the same time or at different times. In one embodiment, one or more acids and one or more bases are combined, resulting in a buffer. In one embodiment, media components, such as a carbon source or a nitrogen source serve as a pH modifier; suitable media components include those with high or low pH or those with buffering capacity. Exemplary media components include acid- or base- hydrolyzed plant polysaccharides having residual acid or base, ammonia fiber explosion (AFEX) treated plant material with residual ammonia, lactic acid, corn steep solids or liquor.

[0087] "Growth phase" is used herein to describe the type of cellular growth that occurs after the "Initiation phase" and before the "Stationary phase" and the "Death phase." The growth phase is sometimes referred to as the exponential phase or log phase or logarithmic phase.

[0088] The term "plant polysaccharide" as used herein has its ordinary meaning as known to those skilled in the art and can comprise one or more polymers of sugars and sugar derivatives as well as derivatives of sugar polymers and/or other polymeric materials that occur in plant matter. Exemplary plant polysaccharides include lignin, cellulose, starch, pectin, and hemicellulose. Others are chitin, sulfonated polysaccharides such as alginic acid, agarose, carrageenan, porphyran, furcelleran and funoran. Generally, the polysaccharide can have two or more sugar units or derivatives of sugar units. The sugar units and/or derivatives of sugar units can repeat in a regular pattern, or otherwise. The sugar units can be hexose units or pentose units, or combinations of these. The derivatives of sugar units can be sugar alcohols, sugar acids, amino sugars, etc. The polysaccharides can be linear, branched, cross-linked, or a mixture thereof. One type or class of polysaccharide can be cross-linked to another type or class of polysaccharide.

[0089] The term "saccharification" as used herein has its ordinary meaning as known to those skilled in the art and can include conversion of plant polysaccharides to lower molecular weight species that can be utilized by the organism at hand. For some organisms, this would include conversion to monosaccharides, disaccharides, trisaccharides, and oligosaccharides of up to about seven monomer units, as well as similar sized chains of sugar derivatives and

combinations of sugars and sugar derivatives.

[0090] The terms "SSF" and "SHF" are known to those skilled in the art; SSF meaning simultaneous saccharification and fermentation, or the conversion from polysaccharides or oligosaccharides into monosaccharides at the same time and in the same fermentation vessel wherein monosaccharides are converted to another chemical product such as ethanol. "SHF" indicates a physical separation of the polymer hydrolysis or saccharification and fermentation processes.

[0091] The term "biomass" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more biological materials that can be converted into a biofuel, chemical or other product. Biomass as used herein is synonymous with the term "feedstock" and includes corn syrup, molasses, silage, agricultural residues (corn stalks, grass, straw, grain hulls, bagasse, etc.), animal waste (manure from cattle, poultry, and hogs), Distillers Dried Solubles (DDS), Distillers Dried Grains (DDG), Condensed Distillers Solubles (CDS), Distillers Wet Grains (DWG), Distillers Dried Grains with Solubles (DDGS), woody materials (wood or bark, sawdust, timber slash, and mill scrap), municipal waste (waste paper, recycled toilet papers, yard clippings, etc.), and energy crops (poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, including macroalgae, etc.). One exemplary source of biomass is plant matter. Plant matter can be, for example, woody plant matter, non-woody plant matter, cellulosic material, lignocellulosic material, hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, sugar cane, grasses, switchgrass, sorghum, high biomass sorghum, bamboo, algae and material derived from these. Plants can be in their natural state or genetically modified, e.g., to increase the cellulosic or hemicellulosic portion of the cell wall, or to produce additional exogenous or endogenous enzymes to increase the separation of cell wall components. Plant matter can be further described by reference to the chemical species present, such as proteins, polysaccharides and oils. Polysaccharides include polymers of various

monosaccharides and derivatives of monosaccharides including glucose, fructose, lactose, galacturonic acid, rhamnose, etc. Plant matter also includes agricultural waste byproducts or side streams such as pomace, corn steep liquor, corn steep solids, distillers grains, peels, pits, fermentation waste, straw, lumber, sewage, garbage and food leftovers. Peels can be citrus which include, but are not limited to, tangerine peel, grapefruit peel, orange peel, tangerine peel, lime peel and lemon peel. These materials can come from farms, forestry, industrial sources, households, etc. Another non- limiting example of biomass is animal matter, including, for example milk, meat, fat, animal processing waste, and animal waste. Biomass can include cell or tissue cultures; for example, biomass can include plant cell culture(s) or plant tissue culture(s). "Feedstock" is frequently used to refer to biomass being used for a process, such as those described herein.

[0092] "Broth" is used herein to refer to inoculated medium at any stage of growth, including the point immediately after inoculation and the period after any or all cellular activity has ceased and can include the material after post-fermentation processing. It includes the entire contents of the combination of soluble and insoluble matter, suspended matter, cells and medium, as

appropriate.

[0093] The term "productivity" as used herein has its ordinary meaning as known to those skilled in the art and can include the mass of a material of interest produced in a given time in a given volume. Units can be, for example, grams per liter-hour, or some other combination of mass, volume, and time. In fermentation, productivity is frequently used to characterize how fast a product can be made within a given fermentation volume. The volume can be referenced to the total volume of the fermentation vessel, the working volume of the fermentation vessel, or the actual volume of broth being fermented. The context of the phrase will indicate the meaning intended to one of skill in the art. Productivity is different from "titer" in that productivity includes a time term, and titer is analogous to concentration. Titer and Productivity can generally be measured at any time during the fermentation, such as at the beginning, the end, or at some intermediate time, with titer relating the amount of a particular material present or produced at the point in time of interest and the productivity relating the amount of a particular material produced per liter in a given amount of time. The amount of time used in the productivity determination can be from the beginning of the fermentation or from some other time, and go to the end of the fermentation, such as when no additional material is produced or when harvest occurs, or some other time as indicated by the context of the use of the term.

"Overall productivity" refers to the productivity determined by utilizing the final titer and the overall fermentation time.

[0094] "Titer" refers to the amount of a particular material present in a fermentation broth. It is similar to concentration and can refer to the amount of material made by the organism in the broth from all fermentation cycles, or the amount of material made in the current fermentation cycle or over a given period of time, or the amount of material present from whatever source, such as produced by the organism or added to the broth. Frequently, the titer of soluble species will be referenced to the liquid portion of the broth, with insolubles removed, and the titer of insoluble species will be referenced to the total amount of broth with insoluble species being present, however, the titer of soluble species can be referenced to the total broth volume and the titer of insoluble species can be referenced to the liquid portion, with the context indicating the which system is used with both reference systems intended in some cases. Frequently, the value determined referenced to one system will be the same or a sufficient approximation of the value referenced to the other.

[0095] "Concentration" when referring to material in the broth or in solution generally refers to the amount of a material present from all sources, whether made by the organism or added to the broth or solution. Concentration can refer to soluble species or insoluble species, and is referenced to either the liquid portion of the broth or the total volume of the broth, as for "titer." When referring to a process or solution, such as "concentration of the sugar in solution", the term indicates increasing one or more components of the solution through evaporation, filtering, extraction, etc., by removal or reduction of a liquid portion. [0096] The term "biocatalyst" as used herein has its ordinary meaning as known to those skilled in the art and can include one or more enzymes and/or microorganisms, including solutions, suspensions, and mixtures of enzymes and microorganisms. In some contexts this word will refer to the possible use of either enzymes or microorganisms to serve a particular function, in other contexts the word will refer to the combined use of the two, and in other contexts the word will refer to only one of the two. The context of the phrase will indicate the meaning intended to one of skill in the art. For example, a biocatalyst can be a fermenting microorganism.

[0097] The terms "conversion efficiency" or "yield" as used herein have their ordinary meaning as known to those skilled in the art and can include the mass of product made from a mass of substrate. The term can be expressed as a percentage yield of the product from a starting mass of substrate. For the production of C5 and C6 sugars or saccharides (e.g., monosaccharides, e.g., glucose, xylose, arabinose, etc.) or soluble saccharide polymers (e.g., polymers comprising two or more saccharide units or residues), the yield is based upon the actual weight of the saccharides released compared to the weight of the oligosaccharides or-polysaccharides (e.g., cellulose, hemicellulose) in the input biomass. For the production of ethanol from glucose, the net reaction is generally accepted as:

C₆H₁₂0₆ -> 2 C₂H₅OH + 2 C0₂

and the theoretical maximum conversion efficiency, or yield, is 51% (wt.). Frequently, the conversion efficiency will be referenced to the theoretical maximum, for example, "80% of the theoretical maximum." In the case of conversion of glucose to ethanol, this statement would indicate a conversion efficiency of 41% (wt.). The context of the phrase will indicate the substrate and product intended to one of skill in the art.

[0098] For substrates (e.g., a biomass composition) comprising a mixture of different carbon sources (e.g., xylan, xylose, glucose, cellobiose, arabinose, cellulose, hemicellulose, etc.), the theoretical maximum conversion efficiency of the biomass to saccharides or ethanol can be calculated as an average of the maximum yields or conversion efficiencies of the individual carbon source constituents weighted by the relative concentration of each carbon source. In some cases, the theoretical maximum conversion efficiency can be calculated based on an assumed saccharification efficiency. By way of example only, given a carbon source comprising 10 g of cellulose, the theoretical maximum conversion efficiency can be calculated by assuming saccharification of the cellulose to the assimilable carbon source (glucose) of about 75% by weight. In this example, 10 g of cellulose can provide 7.5 g of glucose which can provide a maximum theoretical conversion efficiency of about 7.5 g*51% or 3.8 g of ethanol. In another aspect, the assimilable carbon source can be a sugar or saccharide polymer or oligomer containing multiple saccharide residues or units. In this aspect, assuming a maximum theoretical conversion efficiency of about 75% by weight, a carbon source comprising 10 g of a polysaccharide can provide 7.5 g of sugar polymers which can be further hydrolyzed and/or fermented using a biocatalyst and/or exogenous enzymes. In other cases, the efficiency of the saccharification step can be calculated or determined based upon a measurement of the sugars content of an input biomass, e.g., following hydrolysis with 72% sulfuric acid. See A Sluiter, et al, Determination of Structural Carbohydrates and Lignin in Biomass (NREL, revised June 2010), which is hereby incorporated by reference in its entirety. Saccharification efficiencies anticipated by the present invention include about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), 95%), 99%) or about 100% for any carbohydrate carbon sources larger than a single monosaccharide subunit.

[0099] "Pretreatment" or "pretreated" is used herein to refer to any mechanical, chemical, thermal, biochemical process or combination of these processes whether in a combined step or performed sequentially, that achieves disruption or expansion of the biomass so as to render the biomass more susceptible to attack by enzymes and/or microbes, and can include the enzymatic hydrolysis of released carbohydrate polymers or oligomers to monomers. In one embodiment, pretreatment includes removal or disruption of lignin so as to make the cellulose and

hemicellulose polymers in the plant biomass more available to cellulolytic enzymes and/or microbes, for example, by treatment with acid or base. In one embodiment, pretreatment includes disruption or expansion of cellulosic and/or hemicellulosic material. In another embodiment, it can refer to starch release and/or enzymatic hydrolysis to glucose. Steam explosion, and ammonia fiber expansion (or explosion) (AFEX) are well known thermal/chemical techniques. Hydrolysis, including methods that utilize acids, bases, and/or enzymes can be used. Other thermal, chemical, biochemical, enzymatic techniques can also be used.

[00100] "Fed-batch" or "fed-batch fermentation" is used herein to include methods of culturing microorganisms where nutrients, other medium components, or biocatalysts (including, for example, enzymes, fresh organisms, extracellular broth, genetically modified plants and/or organisms, etc.) are supplied to the fermentor during cultivation, but culture broth is not harvested from the fermentor until the end of the fermentation, although it can also include "self seeding" or "partial harvest" techniques where a portion of the fermentor volume is harvested and then fresh medium is added to the remaining broth in the fermentor, with at least a portion of the inoculum being the broth that was left in the fermentor. During a fed-batch fermentation, the broth volume can increase, at least for a period, by adding medium or nutrients to the broth while fermentation organisms are present. Suitable nutrients which can be utilized include those that are soluble, insoluble, and partially soluble, including gasses, liquids and solids. In one embodiment, a fed-batch process is referred to with a phrase such as, "fed-batch with cell augmentation." This phrase can include an operation where nutrients and cells are added or one where cells with no substantial amount of nutrients are added. The more general phrase "fed- batch" encompasses these operations as well. The context where any of these phrases is used will indicate to one of skill in the art the techniques being considered.

[00101] "Sugar compounds" or "sugar streams" is used herein to indicate mostly monosaccharide sugars, dissolved, crystallized, evaporated, or partially dissolved, including but not limited to hexoses and pentoses; sugar alcohols; sugar acids; sugar amines; compounds containing two or more of these linked together directly or indirectly through covalent or ionic bonds; and mixtures thereof. Included within this description are disaccharides; trisaccharides; oligosaccharides; polysaccharides; and sugar chains, branched and/or linear, of any length. A sugar stream can consist of primarily or substantially C6 sugars, C5 sugars, or mixtures of both C6 and C5 sugars in varying ratios of said sugars. C6 sugars have a six-carbon molecular backbone and C5 sugars have a five-carbon molecular backbone. The term "saccharide compounds" can be used interchangeably with "sugar compounds." The term "saccharide stream" can be used interchangeably with "sugar stream."

[00102] "Saccharide oligomer," "sugar oligomer," or "oligosaccharide" are used herein to indicate a saccharide that contains two to ten saccharide residues or units or derivatives of saccharide units. In one embodiment, a saccharide oligomer can be soluble. In one embodiment, a saccharide oligomer can be soluble in an aqueous medium. In some embodiments, the saccharide oligomer comprise 2 to 10 or 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 saccharide residues or units, or between 2 to 5 saccharide units. In some embodiments, the saccharide oligomer comprises more than 2 saccharide residues. In some embodiments, the saccharide oligomers comprise 2 saccharide residues. In some embodiments, the saccharide oligomers comprise less than 10 saccharide residues. In some embodiments, the saccharide oligomers comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 saccharide residues or units.

[00103] "Saccharide polymer" or "sugar polymer" is used herein to indicate a saccharide that contains two or more saccharide residues or units or derivatives of saccharide units. In one embodiment, a saccharide polymer can be soluble. In one embodiment, a saccharide polymer can be soluble in an aqueous medium. In some embodiments, the saccharide polymer comprises 2 to 10 saccharide residues or units. In some embodiments, the saccharide polymers comprise 2 to 10 or 2 to 20, 2 to 30, 2 to 40, 2 to 50, 2 to 60, 2 to 70, 2 to 80, 2 to 90, or 2 to 100 saccharide residues or units. In some embodiments, the saccharide polymers comprise more than 2 saccharide residues. In some embodiments, the saccharide polymers comprise 2 saccharide residues. In some embodiments, the saccharide polymers comprise less than 10 saccharide residues. In some embodiments, the saccharide polymers comprise more than 10 saccharide residues. In some embodiments, the saccharide polymers comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 saccharide residues or units. In some embodiments, the saccharide polymers comprise disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides,

heptasaccharides, octasaccharides, enneasaccharides, and/or decasaccharides. In some embodiments, the saccharide polymers comprise less than 100 saccharide residues. In some embodiments, the saccharide polymers comprise less than 200 saccharide residues. In some embodiments, the saccharide polymers comprise less than 300 saccharide residues. In some embodiments, the saccharide polymers comprise more than 100 saccharide residues. In some embodiments, the saccharide polymers comprise more than 200 saccharide residues. In some embodiments, the saccharide polymers comprise more than 300 saccharide residues. In some embodiments, the saccharide polymers comprise more than 10 and less than 100 saccharide residues. In some embodiments, the saccharide polymers comprise from 10 to 100 saccharide residues. In some embodiments, the saccharide polymers comprise between 10 to 100 saccharide residues. In some embodiments, the saccharide polymers comprise 10 to 100 saccharide residues. In some embodiments, the saccharide polymers comprise more than 10 and less than 100 saccharide residues. In some embodiments, the saccharide polymers comprise from 10 to 100 saccharide residues. In some embodiments, the saccharide polymers comprise between 10 to 100 saccharide residues. In some embodiments, the saccharide polymers comprise 10 to 100 saccharide residues.

[00104] "C5-rich" composition means that one or more steps have been taken to remove at least some of the C6 sugars originally in the composition. For example, a C5-rich composition can include no more than about 50% C6 sugars, nor more than about 40% C6 sugars, no more than about 30% C6 sugars, no more than about 20% C6 sugars, no more than about 10% C6 sugars, no more than about 5%> C6 sugars, or it can include from about 2%> to about 10%> C6 sugars by weight. Likewise, a "C6-rich" composition is one in which at least some of the originally-present C5 sugars have been removed. For example, a C6-rich composition can include no more than about 50% C5 sugars, nor more than about 40% C5 sugars, no more than about 30%) C5 sugars, no more than about 20%> C5 sugars, no more than about 10%> C5 sugars, no more than about 5%> C5 sugars, or it can include from about 2%> to about 10%> C5 sugars by weight. [00105] A "liquid" composition may contain solids and a "solids" composition may contain liquids. A liquid composition refers to a composition in which the material is primarily liquid, and a solids composition is one in which the material is primarily solid.

[00106] The terms "non-cellulosic" and "sugar- or starch- based" are used interchangeably and have the same meaning. For example "non-cellulosic fermentation process" is used interchangeably and means the same thing as "sugar- and starch-based fermentation process."

Starch is a carbohydrate consisting of consisting of a large number of glucose units joined by glycosidic bonds. The glycosidic bonds are typically the easily hydrolysable alpha glycosidic bonds. This polysaccharide can be produced by all green plants as an energy store. There can be two types of starch molecules: the linear and helical amylose and the branched amylopectin, although amylase can also contain branches.

Description

[00107] The following description and examples illustrate some exemplary embodiments of the disclosure in detail. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure that are encompassed by its scope. Accordingly, the description of a certain exemplary embodiment should not be deemed to limit the scope of the present disclosure.

[00108] Acid hydrolysis of lignocellulosic biomass to produce sugars can be costly and requires special equipment. The process, especially under high temperatures and pressure, can release structural carbohydrates in cellulosic biomass and can expose crystalline cellulose to enzymatic degradation. The hydrolyzed sugars produced through this pretreatment process themselves can be labile to the harsh hydrolysis conditions and can be degraded to unwanted or toxic byproducts. If exposed to acid too long, especially under high temperatures, the glucose derived from cellulose can degrade into hydroxymethylfurfural, which can be further degraded into levulinic acid and formic acid. Xylose, a hemicellulose sugar, can be degraded into furfural and further to tars and other degradation products.

[00109] For acid to completely hydro lyze the cellulose and hemicellulose in a

lignocellulosic substrate, degradation of the desirable sugars and formation of the toxic byproducts may be unavoidable due to kinetic constraints. Too gentle a process, so that significant degradation of sugars is avoided, may not result in complete hydrolysis of substrate. Furthermore, the acid can be corrosive and can require special handling and equipment.

Accordingly, in the last twenty years attention pretreatment has focused on enzymatic hydrolysis of cellulose with cellulase followed by fermentation of the resulting sugars to produce ethanol which in turn can be distilled to purify it sufficiently for fuel uses. [00110] Cellulase is an enzyme complex that can include, for example, three different types of enzymes involved in the saccharification of cellulose. The cellulase enzyme complex produced by Trichoderma reesei QM 9414 contains the enzymes endoglucanase (E.C. 3.2.1.4), cellobiohydrolase (E.C.3.2.1.91) and 13-glucosidase (E.C.3.2.1.21). Gum et al,

Biochem.Biophys.Acta, 446:370-86 (1976). The combined synergistic actions of these three enzymes in a mixed cellulose preparation can completely hydrolyze cellulose to D-glucose. However, cellulase may not be able to completely degrade the cellulose found in native, unpretreated lignocellulose. It appears that the hemicellulose and lignin can interfere with the access of the enzyme complex to the cellulose, probably due to their coating and binding of the cellulose fibers. Furthermore, lignin itself can bind cellulase thereby rendering it inactive or less effective for digesting cellulose. For example, raw ground hardwood can be only about 10 to 20% digestible into sugars using a cellulase preparation.

[00111] In one embodiment, provided herein is an optimized two-stage hydrolysis process for the continuous dilute-acid saccharification of lignocellulosics in biomass, or of other cellulosic materials, to produce hydrolysate sugars in a single-solution, of moderate

concentration, and a solid lignin residue, and including cellulose hydrolysis process

improvements. In another embodiment, an optimized two-stage pretreatment is provided wherein hemicellulose is removed at high concentrations without acid and with reduced formation of degradation products. In a further embodiment, starch-containing lignocellulosic materials are pretreated prior to an optimized two-stage pretreatment to maximize the extraction of almost all of the C6 sugars in the biomass. A further embodiment of a method disclosed herein is the reduction or elimination of the use of hemicellulase enzymes and a reduction in cellulase enzymes for enzymatic hydrolysis of the pretreated materials resulting from one or more of these processes.

[00112] A challenge in processing biomass is to optimize and precook the material such that one can get a) low temp that catalyzes and focuses on hemicellulose hydrolysis 2) disturbs and opens up the crystalline alpha cellulose structure by explosion process, and does not lead to over runaway reaction, thereby controlling degradation products. Such a maximized process would require only a minimum or reduced amount of enzyme to get maximum sugar yield with a short saccharification time rendering it easy and efficient to recover C5and C6 at the highest possible yields as monomeric sugars. A two-stage acid thermal hydrolysis process as disclosed herein can enable these yields compared to a one-stage process, as it enables and maximizes the sugar recovery while minimizing enzyme levels and inhibitor formation in processing lignocellulosic biomass. [00113] An advantage of the two-stage process is that it can be more efficient than a one stage pretreatment. Without being bound by theory, this can be because a single stage hydrolysis is geared to only C5 or C6 catalysis, or a poor combination of hydrolysis conditions. Under gentle conditions, e.g., 160°C, the crystalline structure of cellulose may not be fully "exploded", e.g. , it may be only partially opened up. It can require much higher temperature and pressure to achieve separation of microfibrils of cellulose, generally 200°C or higher. At higher

temperature, however, a single step process can lead to production of furfural, loss of C5 sugars, suboptimum separation of lignin from cellulose microfibrils, and further creation of toxic breakdown products and leaching.

[00114] In one embodiment of the methods disclosed herein, a two-stage hydrolysis is optimized so that the structural cellulose is fully opened and the surface area is fully exposed, reducing the enzyme requirement and hydrolysis time necessary to convert polymeric carbohydrate to monomers. See Figure 1. Removing the C5 upfront with particle size reduction can allow for a higher surface area exposure to higher temperature and steam explosion to more efficiently break apart the very resilient cellulose fibers without massive inhibitor production, thereby facilitating C6 monomer formation at low enzyme dose. Other methods, including two- stage hydrolysis methods previously attempted, can result in relatively heterogeneous material with a significant portion of the cellulose still intact with lignin, and the material is not uniformly treated, thus not fully accessible, which requires additional enzyme and time to enzymatically hydrolyze the C6 polymers. Even if the temperature and acid treatments are prolonged, not all of the cellulose is recovered and the inhibitor concentration is high due to the extreme processing and high acid concentrations.

[00115] A first stage of the methods disclosed herein can comprise a gentle uniform pretreatment with or without acid and the subsequent removal of the C5 polymer or monomer that is freed from lignin and cellulose. In stage one, the fresh cellulosic feedstock can be admixed with hot, pressurized dilute-acid water solution or just hot water at, e.g., about: 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, or 190°C. The resulting heated aqueous feedstock slurry can be maintained within 10°C for, e.g., 5, 10, 15, or 20 minutes to extract the hemicellulose fraction of the biomass. It can then be further heated, by additional rapid surplus process heat as it is flashed out of the reactor.

[00116] The temperatures applied in both stages can be determined for the particular type of biomass, e.g., corn stover or sorghum, other grasses, softwood or hardwood, depending on the ratio of hemicellulose, cellulose and lignin present in the biomass. For example, poplar

(classified as a hardwood), has a high cellulose to lignin ratio, about 4:3, and a low

cellulose:hemicellulose content (about 4:2). Wheat straw is roughly about 4:3 cellulose to hemicellulose and lower cellulose to lignin (2: 1) as percent of dry matter. With an understanding of the cell wall content of the biomass to be processed, optimal protocols can be designed to extract the C5 content of the feedstock and subsequent extraction of that product prior to the second stage wherein the cellulose can be separated from the lignin portion. Knowing the optimal temperature and/or acid to extract the C5 content and, importantly, maintaining the proper extraction conditions for a uniformly particulate matter, can result in a first stage process that extracts the maximum content of C5 without the degradation that produces inhibitors and without loss of the sugars. Even at high solids content, the uniformity of the time, temperature and/or acid can result in maximum product.

[00117] If a dilute acid solution is used during the first stage, it should have a pH value from about 1.5 to about 3.5, and the weight ratio of acid solution to the dry biomass should be about 0.1-3%, depending on the feedstock. A dilute aqueous acid solution which has been found to be satisfactory for the first hydrolysis step contains by weight about 0.2% of hydrochloric acid, about 1.3% of formic acid and about 2.7% of acetic acid, 0.5-3% H₂SO₃, or 0.1- 3 % H2SO4.

[00118] The C5 carbohydrate in solution can be removed following the first stage of treatment and the solids recycled into the second stage to separate the cellulose from the lignin. The C5 carbohydrate can then be further enzymatically hydrolyzed to monomers with

hemicellulases without the interference of cellulase enzymes. The amount of enzyme required for this hydrolysis can be less than one-half, often one-quarter of the normal concentration due the reduction of inhibitor creation and the accessibility of the substrate. With some biomass, a small amount of C6 carbohydrate will be captured with the C5 stream. Hydrolyzing C5 upfront into monomer form with dilute acid can require only cellulose enzyme not a mixture

hemicellulase and cellulase enzyme.

[00119] During the second stage, the lignocellulosic solids portion can be processed at high solids concentration with a dilute and/or weak acid like S0₂ or gas that can diffuse better than liquid, due to uniform particle size and treatment, and leading to fully exposed and exploded biomass and reduced enzyme loading. The enzymatic hydrolysis can be carried out with cellulases alone, thus reducing interference from hemicellulases. Further, the amount of enzyme can be reduced to 25-50% of a normal enzyme load used for standard enzymatic hydrolysis since hemicellulases are absent and inhibitors are greatly reduced.

[00120] In one embodiment, at either stage, steam and/or acid hydrolysis process can be carried out by means of suitable conventional apparatus (reactor), e.g., a closable vessel that retains steam at a pressure of at least 50 psig (150°C) and is connected to a source of steam and a source of dilute acid. Preferably, the reactor is capable of uniformly applying the steam and/or acid to the biomass as it is mixed. For these purposes, a microreactor and retention module attached to a flash tank such as that described in U.S. Patent Applications Nos. 2011/ 275860A and 2012/037325A is preferable for its ability to apply steam and pressure consistently, maintaining optimal conditions within +/- 5°C and +1-2 psig while conveying the biomass to the flash tank.

[00121] In one embodiment, the efficiency of this process results in more than 95% recovery of total sugars in the biomass. Further, the C5 and C6 streams are concentrated, reducing the costs of further concentration. In earlier art, a plug- flow-reactor was used to gain higher hydrolysis conversion of cellulose to glucose, by using extremely high hydrolysis rates, achieved by high temperatures of reaction, as provided by direct injection of high pressure steam into high solids density slurries. Under these circumstances, hemicellulose hydrolysate sugars are all degraded by the single stage, high temperature hydrolysis process. Further, high dilution of the hydrolysate sugars by steam condensation causes the resulting single solution of glucose at low concentration and large volume with a high cost of acid neutralization, and still inefficient fermentation.

[00122] In one embodiment, the combined ratio of the C5 and C6 streams can be adjusted for manufacturer's needs. In another embodiment, the streams can be processed separately into products such as those described supra.

[00123] In one instance, U.S. Pat. No. 4,201,596 suggests a continuous process for affecting the acid-hydrolysis of cellulosic waste materials in high-solids density slurries. By control of high temperature, through direct steam injection, the high density slurry solids may be converted to yields of about fifty percent of the potential glucose in cellulose in seconds. This chemical processing method, for converting polysaccharides into pentose and/or hexose sugars, is by a known use of a tubular-type plug- flow-reactor (PFR) for dilute-acid cellulose hydrolysis. Unfortunately, relatively low conversions, negative byproduct formations, high energy in to pressure over 500 psi, have limited the commercial use of that cellulose conversion by PFR method to research and development investigations.

[00124] U.S. Pat. No. 4,615,742 shows a processing batch percolation-type hydrolysis reactor. In this counter-current hydrolysis, a flow of dilute-acid solution contacts a body of particulate wood that is moving in a direction opposite to the flow of the dilute acid solution. The counter-current flow of the dilute acid solution and the particulate wood results in a much higher yield of sugars from the wood, a minimal degradation and a relatively high concentration of glucose, but the process conditions result in a low xylose in the dilute-acid hydrolysate solution. The primary disadvantage of this particular approach, for counter-current hydrolysis, is the extreme mechanical complexity and expense of moving by conveying the solids and pumping the liquids in the opposite directions. [00125] Yield and operability are improved by conducting a lignocellulose pre-hydrolysis first and then a hydrolysis of the residue. See, e.g., U.S. Pat. No. 4,070,232. By pre-hydrolysis of the fresh feedstock, at below 150° C, the hemicellulose can be hydrolyzed at temperatures where sugar degradation is relatively insignificant. This allowed reasonable yields and recovery, by separation of sugars from hemicellulose hydrolysis. It also opens up the structure of the wood particles so that infusion of acid and diffusion of cellulose hydrolysate-sugars are enhanced, with minimum tar fouling of the pipes and fewer degradation products. In this process, however, subsequent hydrolysis of the cellulose lignin fibers was performed in alkali at low temperature, a process that is expensive because it requires so much water to neutralize and remove the alkali.

[00126] Two-stage hydrolysis of a poplar feedstock has been described to enhance yield of sugars by separating a volatile stream of components and a low viscosity effluent of the feedstock which are then separately subjected to enzymatic hydrolysis (U.S. patent application No. 12/247,554). Other methods that attempt to break pretreatment into separate hydrolysis steps sometimes involve three steps including fermentation during hydrolysis and separate removal of inhibitors as well as continued fermentation with hemicelluloses following combined cellulase hydrolysis and fermentation in an effort to reduce the inhibitory effect of

hemicellulases on cellulase activity. (See, e.g., PCT/FI05/000261). This is time-consuming and involves a complex system, increased energy costs and manpower. Still other two-step pretreatment separations involve the separation of lignin from cellulose and hemicellulose. The initial steps in these pretreatments often involve very high pressures and heat that can produce considerable amounts of inhibitors. See, e.g., Canadian patent No. 1,267,407. This type of treatment is often used with woody biomass.

[00127] Figure 1 exemplifies the overall two-stage process disclosed herein. The high density slurries and gentle hydrolysis reaction temperatures of hemicellulose in stage one and the improvement of a quicker, but more even high temperature hydrolysis of the fraction of unhydrolyzed cellulose residue in stage-two, provides higher cellulose to glucose conversion, compared to known processes. In addition, other process improvements in these embodiments result in much less inhibitor formation and reduced enzyme loads during enzymatic hydrolysis than known in one stage pretreatment or in conventional two stage pretreatments.

[00128] The optimized cellulose hydrolysis process improvements of the herein described pretreatment processes can be the result of the uniform small particle size of the biomass, reduced stage one temperature and mild acid treatment or reduced temperature and hot water treatment, shorter retention times during hydrolysis, and reduced enzyme loads. This can result in reduction of pretreatment costs and less expensive sugars for bio fuel and chemical producers. [00129] The process improvement disclosed herein can provide for the production of a product of separate streams of C5 or C6 sugars, or a product comprised of a single solution of the two streams combined which has a relative high concentration of all of the pentose and hexose sugars. The hydrolysis conditions of both stages can be prearranged to maximize the yields of the various sugars produced into a single solution, as a result of the improved process disclosed herein. The resulting high concentration of pentose and hexose sugars in the single solution product can be valuably used by a variety of yeast fermentation and chemical processing methods.

[00130] The methods disclosed herein can provide for sequestering of glucose from a lignocellulosic biomass that also comprises starch. Examples of such feedstocks include, but are not limited to seed hulls, such as oat or rice hulls, corn and corn cobs, or any other seed- producing plant. Often such feedstocks are obtained from grain or tuber starch processing plants wherein the slip streams rich in starch are only partially or separately removed from the liquor or the residual starch in seed grain from beer producers The two-stage processes described herein can be adapted to remove starch prior to the processing of the lignocellulosic portion of the biomass, and the C6 sugars obtained from the starch can be combined with the C6 sugar product of the resulting two-stage processing thus increasing yields of product.

[00131] Figure 2 exemplifies an embodiment of the methods disclosed herein wherein a dilute acid hydrolysis is used in stage one. As shown in Figure 2, the preferred embodiment of the improved process is a two-stage system, made up of two heat-exchanger flow-reactor and flash tank sub-systems, in series with an separation-extraction mechanism set between them to remove solubilized C5 carbohydrates. Any numbers recited in the following description are exemplary embodiments and not intended to be limiting.

[00132] In the exemplary embodiment illustrated in Figure 2, the biomass is first cleaned, debaled if necessary, partially reduced in size for handling by any means, e.g., chopping, shredding, hammer mill, or the like. It is expected, if conventional silage is used, that prewashing may be necessary to remove lactic acid or other fermentation inhibitors. It is then conveyed to a hopper or feeder conduit 1 , where it is weighed and conveyed through conduit 1 , and fed by a rotary-feeder to slurry-mixer 2 (presoak), where it is admixed with a solution of a hot and dilute-acid catalyst solution through a cutter pump 3 which reduces the size of the solids to, e.g., about 3-4 mm.

[00133] The resulting uniformly preheated, dilute-acid slurry, containing about 5-6% biomass solids (w/v) lignocellulosic feedstock solids, is then pumped to a dewatering chamber whereby the extra water and acid are removed and the solids containing about 30-32% solids (w/v) lignocellulosic feedstock solids, is pumped evenly and with positive pressure for a preferred time through tube 4 with a screw-type rotor where a temperature of 50°C and pressure is maintained evenly for a preferred time in this heat exchanger. At the end of dewatering processing through tube 4, the water is drained and recirculated to chamber 2 (conduit not shown). The solids plug that is formed enters a microreactor 5 that further reduces the size of the biomass to uniform pieces averaging 1 mm in size, and then is conveyed by gravity to a first double-jacketed, screw-type, retention module (1^st stage) wherein additional process steam heat with acid is conveyed to raise the temperature to 150-170 °C and pressure 100-175 psig in about 0.1-0.5% acid for a retention time of 20-40 minutes. The flow-rate of the slurry, in

hemicellulose hydrolysis reactor 6 is controlled by a preselected pumping rate, to be compatible and provide the required detention-time. The uniform size of the particles, the fine steam and rotor control keeping uniform temperature and pressure drop constant throughout the chamber results in optimum hemicellulose hydrolysis of the feedstock for production of hemicellulose hydrolysate sugars, dissolved in solution of the slurry with minimum inhibitor formation.

[00134] From reactor 6, the reacted slurry is continuously blown into the flash-tank 7, for reduced pressure, steam production; also the temperature is dropped to interrupt degradation of the hydrolysate sugars. The flashed slurry is conveyed from 7 to a first stage separator 8, for separation of unhydrolyzed cellulosic residue from the solution containing acid-hydrolyzed hemicellulose in the form of C5 monomeric sugars. It is neutralized and concentrated, further hydrolyzed, if necessary, or processed as C5 rich hemicellulose syrup.

[00135] The recovered, unhydrolyzed, cellulose-lignin residue is conveyed from stage-one separator 8, by conduit into stage two. Therein, it is conveyed by conduit to the stage-two retention module mixer 9, along with the fresh pre-heated 1-3% acid solution and uniform high- temperature process heat at 190-240°C adequate to expose the microcrystalline structure bound to the lignin in the solids. The relatively high-temperature process-heat, which would separate microfibrils from the bound crystalline structure separating the cellulose polymer and randomly degrade cellulose, is effectively and uniformly applied to the rotating slurry for 5-15 minutes to ensure minimum sugar /lignin degradation product with a maximum release of cellulose to depolymerize and solubilize the cellulose sugars to maximize oligosaccharides to its soluble form and is transferred to flash tank 10 where the temperature is dropped and the slurry is conveyed to a collection tank 11 as solid liquid slurry to recover C6 sugars solids and partially solubilized cellulose.

[00136] Material rich in partially hydrolyzed cellulose with some intact but loosely bound lignin is subjected to final enzyme hydrolysis to produce and recover monomer C6 sugar.

Enzymatic hydrolysis of the cellulose is carried out in reactor 12 after the pH is raised to about 5.5-7.0 depending on the pKI of the cellulases used to convert the cellulose polymers to C6 monomeric sugars. In some instances, depending on the desired product, cellulase enzymes can be used to produce oligomers as well. The remaining fraction of solid lignin residue is separated and collected for various purposes.

[00137] Figure 3 exemplifies an embodiment of the methods disclosed herein wherein a hot water hydrolysis is used in stage one. As shown in Figure 3, the preferred embodiment of the improved process is a two-stage system, made up of two heat-exchanger flow-reactor and flash tank sub-systems, in series with an separation-extraction mechanism set between them to remove solubilized C5 carbohydrates. Any numbers recited in the following description are exemplary embodiments and not intended to be limiting.

[00138] The biomass is first cleaned, debaled if necessary, partially reduced in size for handling, by any means, e.g., chopping, shredding, hammer mill, or the like. It is expected, if conventional silage is used, that prewashing may be necessary to remove lactic acid or other fermentation inhibitors. It is then conveyed to a hopper or feeder conduit 1, where it is weighed and conveyed through conduit 1 , and fed by a rotary- feeder to slurry-mixer 2 (presoak), where it is admixed with a solution of a hot water solution through a cutter pump 3 which reduces the size of the solids to 3-4 mm.

[00139] The resulting uniformly preheated, slurry, containing about 5-6% biomass solids

(w/v) Hgnocellulosic feedstock solids, is then pumped to a dewatering chamber whereby the extra water and acid are removed and the solids containing about 30-32% solids (w/v) hgnocellulosic feedstock solids, is pumped evenly and with positive pressure for a preferred time through tube 4 with a screw-type rotor where a temperature of 50°C and pressure is maintained evenly for a preferred time in this heat exchanger. At the end of dewatering processing through tube 4, the water is drained and recirculated to chamber 2 (conduit not shown). The solids plug that is formed enters a microreactor 5 that further reduces the size of the biomass to uniform pieces averaging 1 mm in size, and then is conveyed by gravity to a first double-jacketed, screw- type, retention module (1^st stage) 6 wherein additional process steam heat is conveyed to raise the temperature to 150-170°C and pressure 100-175 psig for a retention time of 20-40 minutes. The flow-rate of the slurry, in hemicellulose hydrolysis reactor 6 is controlled by a preselected pumping rate to be compatible and provide the required detention-time. The uniform size of the particles, the fine steam and rotor control keeping uniform temperature and pressure drop constant throughout the chamber results in optimum hemicellulose separation from the solids for production of hemicellulose carbohydrates, dissolved in solution of the slurry with minimum inhibitor formation.

[00140] From reactor 6, the reacted slurry is continuously blown into the flash-tank 7, for reduced pressure and steam production, and also the temperature is dropped to interrupt degradation of the hydro lysate sugars. The flashed slurry is conveyed from 7 to a first stage separator 8, for separation of unhydrolyzed cellulosic residue from the solution containing solubilized separated hemicellulose in the form of C5 polymers and oligomers. The C5 carbohydrate is concentrated, and conveyed to microreactor 13 where it is enzymatically hydrolyzed to monomers with hemicellulases and, if necessary, concentrated as C5 rich hemicellulose syrup.

[00141] The recovered, unhydrolyzed, cellulose-lignin residue is conveyed from stage-one separator 8, by conduit into stage two. Therein, it enters the stage-two retention module mixer 9, along with the pre-heated 1-3% acid solution and uniform high-temperature process heat at 190- 240°C adequate to expose the microcrystalline structure bound to the lignin in the solids. The relatively high-temperature process-heat, which would separate microfibrils from the bound crystalline structure separating the cellulose polymer and randomly degrade cellulose, is effectively and uniformly applied to the rotating slurry for 5-15 minutes to ensure minimum sugar /lignin degradation product with a maximum release of cellulose to depolymerize and solubilize the cellulose sugars to maximize oligosaccharides to its soluble form and is transferred to flash tank 10 where the temperature is dropped and the slurry is conveyed to a collection tank 11 as solid liquid slurry to recover C6 sugars solids and partially solubilized cellulose.

[00142] Material rich in partially hydrolyzed cellulose with some intact but loosely bound lignin is subjected to final enzyme hydrolysis to produce and recover monomer C6 sugar.

Enzymatic hydrolysis of the cellulose is carried out in reactor 12 after the pH is raised to about 5.5-7.0 depending on the pKI of the cellulases used to convert the cellulose polymers to C6 monomeric sugars. In some instances, depending on the desired product, cellulase enzymes can be used to produce oligomers as well. The remaining fraction of solid lignin residue is separated and collected for various purposes.

[00143] Figure 4 is a diagrammatic representation of the processing of a starch-containing feedstock. Basically, the process is the same as that shown in Figure 2 or Figure 3 with the exception that the starch is removed and enzymatically hydrolyzed prior to the two stage process that extracts C5 and C6 monomeric sugars. This embodiment is used with feedstocks such as corn, hulls, or any type of seed or solid or liquid material containing starch. Any numbers recited in the following description are exemplary embodiments and not intended to be limiting.

[00144] In a pre-step, after the slurry is formed in 2, the biomass is heated in hot water at

>100°C to gelatinize the starch, and then further enzymatically-hydrolyzed with a-amylase or β- amylase (in those cases where some starch having an a- 1-6 linkage of branched chain glucose polymer is present), for 20-60 min. in a microreactor 14. The partially hydrolyzed starch is further hydrolyzed at 50°C with glucoamylase for 30-50 min and/or pullulinase enzyme. The solubilized glucose monomer solution is separated 15 from the solids containing the structural

C5 and C6 polymers, concentrated and collected. The solids are then processed through stage one and stage two as described in Figure 2 or Figure 3.

[00145] For example, the solids can be admixed with a solution of a hot and dilute-acid catalyst solution. The resulting uniformly preheated, dilute-acid slurry, containing about 5-6% biomass solids (w/v) lignocellulosic feedstock solids, is then pumped to a dewatering chamber whereby the extra water and acid are removed and the solids containing about 30-32% solids (w/v) lignocellulosic feedstock solids, is pumped evenly and with positive pressure for a preferred time through tube 4 with a screw-type rotor where a temperature of 50°C and pressure is maintained evenly for a preferred time in this heat exchanger. At the end of dewatering processing through tube 4, the water is drained and recirculated to chamber 2 (conduit not shown). The solids plug that is formed enters a microreactor 5 that further reduces the size of the biomass to uniform pieces averaging 1 mm in size, and then is conveyed by gravity to a first double-jacketed, screw-type, retention module (1^st stage) wherein additional process steam heat with acid is conveyed to raise the temperature to 150-170 °C and pressure 100-175 psig in about 0.1-0.5% acid for a retention time of 20-40 minutes. The flow-rate of the slurry, in

hemicellulose hydrolysis reactor 6 is controlled by a preselected pumping rate, to be compatible and provide the required detention-time. The uniform size of the particles, the fine steam and rotor control keeping uniform temperature and pressure drop constant throughout the chamber results in optimum hemicellulose hydrolysis of the feedstock for production of hemicellulose hydrolysate sugars, dissolved in solution of the slurry with minimum inhibitor formation.

[00146] From reactor 6, the reacted slurry is continuously blown into the flash-tank 7, for reduced pressure, steam production; also the temperature is dropped to interrupt degradation of the hydrolysate sugars. The flashed slurry is conveyed from 7 to a first stage separator 8, for separation of unhydrolyzed cellulosic residue from the solution containing acid-hydrolyzed hemicellulose in the form of C5 monomeric sugars. It is neutralized and concentrated, further hydrolyzed, if necessary, or processed as C5 rich hemicellulose syrup.

[00147] The recovered, unhydrolyzed, cellulose-lignin residue is conveyed from stage-one separator 8, by conduit into stage two. Therein, it is conveyed by conduit to the stage-two retention module mixer 9, along with the fresh pre-heated 1-3% acid solution and uniform high- temperature process heat at 190-240°C adequate to expose the microcrystalline structure bound to the lignin in the solids. The relatively high-temperature process-heat, which would separate microfibrils from the bound crystalline structure separating the cellulose polymer and randomly degrade cellulose, is effectively and uniformly applied to the rotating slurry for 5-15 minutes to ensure minimum sugar /lignin degradation product with a maximum release of cellulose to depolymerize and solubilize the cellulose sugars to maximize oligosaccharides to its soluble form and is transferred to flash tank 10 where the temperature is dropped and the slurry is conveyed to a collection tank 11 as solid liquid slurry to recover C6 sugars solids and partially solubilized cellulose.

[00148] Material rich in partially hydrolyzed cellulose with some intact but loosely bound lignin is subjected to final enzyme hydrolysis to produce and recover monomer C6 sugar.

Enzymatic hydrolysis of the cellulose is carried out in reactor 12 after the pH is raised to about 5.5-7.0 depending on the pKI of the cellulases used to convert the cellulose polymers to C6 monomeric sugars. In some instances, depending on the desired product, cellulase enzymes can be used to produce oligomers as well. The remaining fraction of solid lignin residue is separated and collected for various purposes.

[00149] In one aspect, disclosed herein are two stage methods of producing sugars from a biomass. Many methods are disclosed herein. Some methods comprise one or more of the following steps: a) adding the biomass to a first liquid at a hydration temperature to produce a hydrated biomass; b) mechanical size reduction of the hydrated biomass to produce a mixture of size reduced solid particles; c) heating the mixture of size reduced solid particles at a first hydrolysis temperature for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction; d) heating the first solid fraction in an acidic medium

comprising an acid at a second hydrolysis temperature for a second hydrolysis time to produce a mixture; and e) hydrolyzing the mixture with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solid fraction.

[00150] In some embodiments, the method comprises adding a biomass to a first liquid at a hydration temperature to produce a hydrated biomass. In some embodiments, the first liquid is water. In other embodiments, the first liquid comprises from about 0.01% to about 10% of an acid. For example, the first liquid can comprise about: 0.01-10%, 0.01-5%, 0.01-2.5%, 0.01-1%, 0.01-0.75%, 0.01-0.5%, 0.01-0.3%, 0.01-0.1%, 0.01-0.05%, 0.05-10%, 0.05-5%, 0.05-2.5%, 0.05-1%, 0.05-0.75%, 0.05-0.5%, 0.05-0.3%, 0.05-0.1%, 0.1-10%, 0.1-5%, 0.1-2.5%,0.1-1%, 0.1-0.75%, 0.1-0.5%, 0.1-0.3%, 0.3-10%, 0.3-5%, 0.3-2.5%, 0.3-1%, 0.3-0.75%, 0.3-0.5%, 0. 5- 10%, 0.5-5%, 0.5-2.5%, 0.5-1%, 0.5-0.75%, 0.75-10%, 0.75-5%, 0.75-2.5%, 0.75-1%, 1-10%, 1- 5%, 1-2.5%, 2.5-10%, 2.5-5%, 5-10%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, or 10% of the acid. In some embodiments, the first liquid comprises from about 0.01% to about 10% of the acid. In some embodiments, the first liquid comprises from about 0.01% to about 5% of the acid. In some embodiments, the first liquid comprises from about 0.01% to about 1% of the acid. In some embodiments, the first liquid comprises from about 0.1% to about 1% of the acid. In some embodiments, the first liquid comprises from about 0.1 % to about 0.5%> of the acid. In some embodiments, the first liquid comprises from about 0.1 % to about 0.3%> of the acid. In some embodiments, the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the first liquid is derived from S0₂ gas. In some embodiments, the first liquid is derived from H₂S0₄ gas.

[00151] In some embodiments, the first liquid has a pH of from about 0.1 to about 5.5. For example, the first liquid can have a pH of about: 0.1-5.5, 0.1-3.5, 0.1-2, 0.1-1.5, 0.1-1, 0.1-0.5, 0.5-5.5, 0.5-3.5, 0.5-2, 0.5-1.5, 0.5-1, 1-5.5, 1-3.5, 1-2, 1-1.5, 1.5-5.5, 1.5-3.5, 1.5-2, 2-5.5, 2-3.5, 3.5-5.5, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5. In one embodiment, the first liquid has a pH of from about 1.5 to about 3.5.

[00152] In some embodiments, the hydration temperature is from about 20 °C to about

110 °C. For example, the hydration temperature can be about: 20-110 °C, 20-75 °C, 20-60 °C, 20-55 °C, 20-50 °C, 20-45 °C, 20-35 °C, 20-25 °C, 25-110 °C, 25-75 °C, 25-60 °C, 25-55 °C, 25-50 °C, 25-45 °C, 25-35 °C, 35-110 °C, 35-75 °C, 35-60 °C, 35-55 °C, 35-50 °C, 50-110 °C, 50-75 °C, 50-60 °C, 50-55 °C, 55-110 °C, 55-75 °C, 55-60 °C, 60-110 °C, 60-75 °C, 75-110 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 52.5 °C, 55 °C, 57.5 °C, 60 °C, 62.5 °C, 65 °C, 67.5 °C, 70 °C, 72.5 °C, 75 °C, 80 °C, 85 °C, 90 °C, 95 °C, 100 °C, 105 °C, or 110 °C. In some embodiments, the hydration temperature is from about 35 °C to about 70 °C. In some embodiments, the hydration temperature is from about 45 °C to about 55°C. In some embodiments, the hydration temperature is about 50°C.

[00153] In some embodiments, the biomass is added to the first liquid to produce a hydrated biomass comprising from about 2%> to about 35 > solids (w/v). For example, the hydrated biomass can comprise about: 2-35%, 2-32%, 2-30%, 2-25%, 2-15%, 2-12%, 2-10%, 2- 6%, 2-5%, 5-35%, 5-32%, 5-30%, 5-25%, 5-15%, 5-12%, 5-10%, 5-6%, 6-35%, 6-32%, 6-30%, 6-25%, 6-15%, 6-12%, 6-10%, 10-35%, 10-32%, 10-30%, 10-25%, 10-15%, 10-12%, 12-35%, 12-32%, 12-30%, 12-25%, 12-15%, 15-35%, 15-32%, 15-30%, 15-25%, 25-35%, 25-32%, 25- 30%), 30-35%), 30-32%), or 32-35%> solids (w/v). In some embodiments, the hydrated biomass comprises about 2%> to about 12%> solids (w/v). In some embodiments, the hydrated biomass comprises about 5-6% solids (w/v). In some embodiments, the hydrated biomass comprises about 10%) to about 30%> solids (w/v).

[00154] In some embodiments, the method comprises mechanical size reduction of the hydrated biomass to produce a mixture of size reduced solid particles. In some embodiments, the particles in the mixture of size reduced solid particles are uniform in size, or substantially uniform in size. In some embodiments, at least 50%> (e.g., at least: 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% ) of the solid particles are less than 50 mm in a dimension. For example, at least 50% (e.g., at least: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%), or 100%) ) of the solid particles can be less than about: 50 mm, 40mm, 30 mm, 20 mm, 15 mm, 10 mm, 9 mm, 8 mm, 7.5 mm, 7 mm, 6.5 mm, 6 mm, 5.5 mm, 5 mm, 4.5 mm, 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.1 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.15 mm, or 0.1 mm in a dimension. In some embodiments, at least 50%> of the solid particles in the mixture of size reduced solid particles are less than 1.5 mm in a dimension. In some embodiments, at least 50% the solid particles in the mixture of size reduced solid particles are from about 0.1 mm to about 1 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension. In some embodiments, all of the solid particles in the mixture of size reduced solid particles are less than 1 mm in a dimension. In some

embodiments, the dimension is diameter or width.

[00155] In some embodiments, the method comprises heating the mixture of size reduced solid particles at a first hydrolysis temperature for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction. In some embodiments, the C5 sugars comprise xylose, arabinose, or a combination thereof. In some embodiments, the first liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural, hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof. In some embodiments, the method further comprises separating the first liquid fraction and the first solid fraction. In some embodiments, the method further comprises concentrating the first liquid fraction.

[00156] In some embodiments, the first hydrolysis temperature is from about 125 °C to about 200 °C. For example, the first hydrolysis temperature can be about: 125-200 °C, 125-190 °C, 125-180 °C, 125-170 °C, 125-160 °C, 125-150 °C, 125-140 °C, 125-130 °C, 130-200 °C, 130-190 °C, 130-180 °C, 130-170 °C, 130-160 °C, 130-150 °C, 130-140 °C, 140-200 °C, 140- 190 °C, 140-180 °C, 140-170 °C, 140-160 °C, 140-150 °C, 150-200 °C, 150-190 °C, 150-180 °C, 150-170 °C, 150-160 °C, 160-200 °C, 160-190 °C, 160-180 °C, 160-170 °C, 170-200 °C, 170-190 °C, 170-180 °C, 180-200 °C, 180-190 °C, or 190-200 °C. In some embodiments, the first hydrolysis temperature is about 150 °C to about 170 °C.

[00157] In some embodiments, the first hydrolysis time is from about 1 minute to about

120 minutes. For example, the first hydrolysis time can be about: 1-120 min., 1-60 min., 1-45 min., 1-30 min., 1-15 min., 1-10 min., 1-5 min., 5-120 min., 5-60 min., 5-45 min., 5-30 min., 5- 15 min., 5-10 min., 10-120 min., 10-60 min., 10-45 min., 10-30 min., 10-15 min., 15-120 min., 15-60 min., 15-45 min., 15-30 min., 30-120 min., 30-60 min., 30-45 min., 45-120 min., 45-60 min., 60-120 min., 1 min., 2 min., 3 min., 4 min., 5 min., 6 min., 7 min., 8 min., 9 min., 10 min., 11 min., 12 min., 13 min., 14 min., 15 min., 17.5 min., 20 min., 22.5 min., 25 min., 27.5 min., 30 min., 35 min., 40 min., 45 min., 50 min., 55 min., 60 min., 75 min., 90 min., or 120 min. In some embodiments, the first hydrolysis time is from about 20 minutes to about 40 minutes. In some embodiments, the first hydrolysis time is from about 5 min. to about 15 min. In some

embodiments, first hydrolysis time is less than about 20 minutes.

[00158] In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 140 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 120 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 130 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 140 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 150 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 160 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 170 °C. In some embodiments, the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 180 °C.

[00159] In some embodiments, heating the hydrated biomass is performed at a pressure of from about 0 psig to about 250 psig. For example, the hydrated biomass can be heated at a pressure of about: 0-250 psig, 0-225 psig, 0-200 psig, 0-175 psig, 0-150 psig, 0-125 psig, 0-100 psig, 0-75 psig, 0-50 psig, 0-25 psig, 25-250 psig, 25-225 psig, 25-200 psig, 25-175 psig, 25-150 psig, 25-125 psig, 25-100 psig, 25-75 psig, 25-50 psig, 50-250 psig, 50-225 psig, 50-200 psig, 50-175 psig, 50-150 psig, 50-125 psig, 50-100 psig, 50-75 psig, 75-250 psig, 75-225 psig, 75-

200 psig, 75-175 psig, 75-150 psig, 75-125 psig, 75-100 psig, 100-250 psig, 100-225 psig, 100-

200 psig, 100-175 psig, 100-150 psig, 100-125 psig, 125-250 psig, 125-225 psig, 125-200 psig,

125-175 psig, 125-150 psig, 150-250 psig, 150-225 psig, 150-200 psig, 150-175 psig, 175-250 psig, 175-225 psig, 175-200 psig, 200-250 psig, 200-225 psig, 225-250 psig. In some

embodiments, heating the hydrated biomass is performed at a pressure of from about 100 psig to about 175 psig.

[00160] Some embodiments comprise removing water and/or acid from the hydrated biomass. In some embodiments, the hydrated biomass is dewatered to about 30-32% solids (w/v) prior to heating at the first hydrolysis temperature.

[00161] In some embodiments, the C5 sugars of the first liquid fraction comprise soluble polysaccharides. In some embodiments, the methods further comprise hydrolyzing the first liquid fraction with one or more hemicellulase enzymes.

[00162] In some embodiments, the method comprises heating the first solid fraction in an acidic medium comprising an acid at a second hydrolysis temperature for a second hydrolysis time to produce a mixture. In some embodiments, the acidic medium is an acidic solution. In some embodiments, the acidic medium comprises water.

[00163] In some embodiments, the acidic medium comprises from about 0.1 % to about

10%) of the acid. For example, the acidic medium can comprise about: 0.1-10%), 0.1-5%, 0.1-4%, 0.1-3%, 0.1-2%, 0.1-1%, 0.1-0.5%, 0.5-10%, 0.5-5%, 0.5-4%, 0.5-3%, 0.5-2%, 0.5-1%, 1-10%,

1- 5%, 1-4%, 1-3%, 1-2%, 2-10%, 2-5%, 2-4%, 2-3%, 3-10%, 3-5%, 3-4%, 4-10%, 4-5%, 5- 10%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.5%, 6%, 6.5%, 7%), 7.5%), 8%), 8.5%), 9%), 9.5%>, or 10%> of the acid. In some embodiments, the acidic medium comprises from about 1% to about 3% of the acid. In some embodiments, the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof. In some embodiments, the acidic medium is derived from S0₂ gas. In some

embodiments, the acidic medium is derived from H₂S0₄ gas.

[00164] In some embodiments, the acidic medium has a pH of from about 0.1 to about 5.5.

For example, the first liquid can have a pH of about: 0.1-5.5, 0.1-3.5, 0.1-2, 0.1-1.5, 0.1-1, 0.1- 0.5, 0.5-5.5, 0.5-3.5, 0.5-2, 0.5-1.5, 0.5-1, 1-5.5, 1-3.5, 1-2, 1-1.5, 1.5-5.5, 1.5-3.5, 1.5-2, 2-5.5,

2- 3.5, 3.5-5.5, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5. [00165] In some embodiments, the second hydrolysis temperature is from about 175 °C to about 275 °C. For example, the second hydrolysis temperature can be about: 175-275 °C, 175-

240 °C, 175-220 °C, 175-210 °C, 175-200 °C, 175-190 °C, 190-275 °C, 190-240 °C, 190-220

°C, 190-210 °C, 190-200 °C, 200-275 °C, 200-240 °C, 200-220 °C, 200-210 °C, 210-275 °C,

210-240 °C, 210-220 °C, 220-275 °C, 220-240 °C, 240-275 °C, 175 °C, 180 °C, 185 °C, 190 °C,

195 °C, 200 °C, 205 °C, 210 °C, 215 °C, 220 °C, 225 °C, 230 °C, 235 °C, 240 °C, 245 °C, 250

°C, 255 °C, 260 °C, 265 °C, 270 °C, or 275 °C. In some embodiments, the second hydrolysis temperature is from about 190 °C to about 240 °C. In some embodiments, the second hydrolysis temperature is from about 190 °C to about 220 °C.

[00166] In some embodiments, the second hydrolysis time is from about 1 minute to about

120 minutes. For example, the second hydrolysis time can be about: 1-120 min., 1-60 min., 1-45 min., 1-30 min., 1-15 min., 1-10 min., 1-5 min., 5-120 min., 5-60 min., 5-45 min., 5-30 min., 5- 15 min., 5-10 min., 10-120 min., 10-60 min., 10-45 min., 10-30 min., 10-15 min., 15-120 min., 15-60 min., 15-45 min., 15-30 min., 30-120 min., 30-60 min., 30-45 min., 45-120 min., 45-60 min., 60-120 min., 1 min., 2 min., 3 min., 4 min., 5 min., 6 min., 7 min., 8 min., 9 min., 10 min., 11 min., 12 min., 13 min., 14 min., 15 min., 17.5 min., 20 min., 22.5 min., 25 min., 27.5 min., 30 min., 35 min., 40 min., 45 min., 50 min., 55 min., 60 min., 75 min., 90 min., or 120 min. In some embodiments, the second hydrolysis time is from about 1 minute to about 30 minutes. In some embodiments, the second hydrolysis time is from about 5 minutes to about 15 minutes. In some embodiments, the second hydrolysis time is at least about 5 minutes.

[00167] In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 190°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 200°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 210°C. In some embodiments, the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 220°C.

[00168] In some embodiments, the method comprises hydrolyzing the mixture with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solid fraction. In some embodiments, the C6 sugars comprise glucose. In some embodiments, the second liquid fraction further comprises low levels of an inhibitor compound. In some embodiments, the inhibitor compound is furfural, hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof. In some embodiments, the method further comprises separating the second liquid fraction from the second solid fraction. In some embodiments, the method further comprises concentrating the second liquid fraction. In some embodiments, the first liquid fraction is combined with the second liquid fraction. [00169] In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 20 % based on total dry solids. For example, the one or more cellulase enzymes can be at a concentration of about: 0.1-20%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-2.5%, 0.1-1%, 0.1-0.75%,

0.1-0.5%, 0.1-0.25%, 0.25-20%, 0.25-15%, 0.25-10%, 0.25-5%, 0.25-2.5%, 0.25-1%, 0.25-

0.75%, 0.25-0.5%, 0.5-20%, 0.5-15%, 0.5-10%, 0.5-5%, 0.5-2.5%, 0.5-1%, 0.5-0.75%, 0.75-

20%, 0.75-15%, 0.75-10%, 0.75-5%, 0.75-2.5%, 0.75-1%, 1-20%, 1-15%, 1-10%, 1-5%, 1-

2.5%, 2.5-20%, 2.5-15%, 2.5-10%, 2.5-5%, 5-20%, 5-15%, 5-10%, 10-20%, 10-15%, 15-20%,

0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%,

0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,

13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 10% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about

5% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.1% to about 1% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at from about 0.25% to about 1% based on total dry solids. In some embodiments, the one or more cellulase enzymes are at about 0.5% based on total dry solids.

[00170] In some embodiments, the biomass comprises cellulose, hemicellulose, or lignocellulose. In some embodiments, the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

[00171] In some embodiments, the method further comprises removing starch from the biomass prior to heating the hydrated biomass at the first hydrolysis temperature. In some embodiments, removing starch from the biomass comprises heating the hydrated biomass at greater than 100 °C. In some embodiments, the starch is hydro lyzed by one or more enzymes to produce glucose monomers. In some embodiments, the one or more enzymes comprise a- amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof. In some embodiments, the glucose monomers are combined with the second liquid fraction.

[00172] In some embodiments, the yield of C5 sugars or C6 sugars is at least about 50%> of a theoretical maximum. In some embodiments, the yield of C5 or C6 sugars is at least about 60% of a theoretical maximum. In some embodiments, the yield of C5 sugars or C6 sugars is at least about 70% of a theoretical maximum. In some embodiments, the yield of C5 or C6 sugars is at least about 80% of a theoretical maximum. In some embodiments, the yield of C5 sugars or C6 sugars is at least about 90% of a theoretical maximum.

[00173] In another aspect, disclosed herein are systems for two stage production of sugars from a biomass. Many systems are disclosed herein. Some systems comprise one or more of the following components: a) a slurry mixer containing a first liquid at a hydration temperature; b) a rotary feeder that adds the biomass to the first liquid; c) a dewatering chamber that removes liquid from the biomass; d) a cutter pump that reduces the particle size of the biomass and pumps the biomass from the slurry mixer to the dewatering chamber; e) a microreactor that further reduces the particle size of the biomass to produce a mixture of size reduced solid particles; f) a hemicellulose reactor where the mixture of size reduced solid particles is heated at a first hydrolysis temperature and a first hydrolysis pressure for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction; g) a first flash tank for reducing temperature and pressure of the first liquid fraction and the first solid fraction; h) a first separator to separate the first liquid fraction from the first solid fraction; i) a retention module mixer that mixes the first solids fraction with an acidic medium comprising an acid, and heats the first solids fraction at a second hydrolysis temperature for a second hydrolysis time to produce a mixture; and j) a first enzyme reactor, wherein the mixture is hydrolyzed with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solids fraction.

[00174] In some embodiments, the system further comprises a second flash tank for reducing temperature and pressure of the mixture.

[00175] In some embodiments, the system further comprises a second separator to separate the second liquid fraction from the second solid fraction.

[00176] In some embodiments, the system further comprises a second enzyme reactor, wherein the first liquid fraction is hydrolyzed with one or more hemicellulase enzymes.

[00177] In some embodiments, the dewatering chamber comprises one or more screw-type rotors. For example, the dewatering chamber can comprise 1, 2, 3, 4, or 5 screw-type rotors. In embodiments where there are more than one screw-type rotors, the screw-type rotors can be in a sequence. The use of more than one screw-type rotor can reduce torsional strain on the rotor as the solids percentage of the biomass increases (e.g., as water is removed).

[00178] In some embodiments, the system comprises more than one dewatering chamber.

In some embodiments, each of the dewatering chambers has a screw-type rotor.

[00179] In some embodiments, the hemicellulose reactor is a double-jacketed, screw type retention module. [00180] In some embodiments, the system further comprises a microreactor for hydrolyzing starch with one or more enzymes to produce glucose monomers. In some embodiments, the system further comprises a separator to remove the glucose monomers from the biomass.

[00181] Feedstock and Pretreatment of Feedstock

[00182] Disclosed herein are methods to optimize the pretreatment of lignocellulosic biomass to increase yields of monomeric sugars from cellulose and hemicellulose in biomass and minimize the production of inhibitors of hydrolysis and fermentation in the process. In one embodiment, the use of a two-stage hydrolysis of biomass to separate and hydro lyze

hemicellulosic sugars from cellulosic sugars is performed within narrow parameters chosen to work with different feedstocks.

[00183] In one embodiment, the feedstock (biomass) contains cellulosic, hemicellulosic, and/or lignocellulosic material. The feedstock can be derived from agricultural crops, crop residues, trees, woodchips, sawdust, paper, cardboard, grasses, algae, municipal waste and other sources.

[00184] Cellulose is a linear polymer of glucose where the glucose units are connected via β(1→4) linkages. Hemicellulose is a branched polymer of a number of sugar monomers including glucose, xylose, mannose, galactose, rhamnose and arabinose, and can have sugar acids such as mannuronic acid and galacturonic acid present as well. Lignin is a cross-linked, racemic macromolecule of mostly /?-coumaryl alcohol, conferyl alcohol and sinapyl alcohol. These three polymers occur together in lignocellulosic materials in plant biomass. The different characteristics of the three polymers can make hydrolysis of the combination difficult as each polymer tends to shield the others from enzymatic attack.

[00185] In one embodiment, methods are provided for the pretreatment of feedstock used in the fermentation and production of the bio fuels and chemicals. The pretreatment steps can include mechanical, thermal, pressure, chemical, thermochemical, and/or biochemical tests pretreatment prior to being used in a bioprocess for the production of fuels and chemicals, but untreated biomass material can be used in the process as well. Mechanical processes can reduce the particle size of the biomass material so that it can be more conveniently handled in the bioprocess and can increase the surface area of the feedstock to facilitate contact with chemicals/biochemicals/biocatalysts. Mechanical processes can also separate one type of biomass material from another. The biomass material can also be subjected to thermal and/or chemical pretreatments to render plant polymers more accessible. Multiple steps of treatment can also be used. [00186] Mechanical processes include, are not limited to, washing, soaking, milling, size reduction, screening, shearing, size classification and density classification processes. Chemical processes include, but are not limited to, bleaching, oxidation, reduction, acid treatment, base treatment, sulfite treatment, acid sulfite treatment, basic sulfite treatment, ammonia treatment, and hydrolysis. Thermal processes include, but are not limited to, sterilization, ammonia fiber expansion or explosion ("AFEX"), steam explosion, holding at elevated temperatures, pressurized or unpressurized, in the presence or absence of water, and freezing. Biochemical processes include, but are not limited to, treatment with enzymes, including enzymes produced by genetically-modified plants, and treatment with microorganisms. Various enzymes that can be utilized include cellulase, amylase, β-glucosidase, xylanase, gluconase, and other

polysaccharases; lysozyme; laccase, and other lignin-modifying enzymes; lipoxygenase, peroxidase, and other oxidative enzymes; proteases; and lipases. One or more of the mechanical, chemical, thermal, thermochemical, and biochemical processes can be combined or used separately. Such combined processes can also include those used in the production of paper, cellulose products, microcrystalline cellulose, and cellulosics and can include pulping, kraft pulping, acidic sulfite processing. The feedstock can be a side stream or waste stream from a facility that utilizes one or more of these processes on a biomass material, such as cellulosic, hemicellulosic or lignocellulosic material. Examples include paper plants, cellulosics plants, distillation plants, cotton processing plants, and microcrystalline cellulose plants. The feedstock can also include cellulose-containing or cellulosic containing waste materials. The feedstock can also be biomass materials, such as wood, grasses, corn, starch, or sugar, produced or harvested as an intended feedstock for production of ethanol or other products such as by biocatalysts.

[00187] In another embodiment, a method can utilize a pretreatment process disclosed in

U.S. Patents and Patent Applications US20040152881, US20040171136, US20040168960, US20080121359, US20060069244, US20060188980, US20080176301, 5693296, 6262313, US20060024801, 5969189, 6043392, US20020038058, US5865898, US5865898, US6478965, 5986133, or US20080280338, each of which is incorporated by reference herein in its entirety.

[00188] In another embodiment, the AFEX process is be used for pretreatment of biomass.

In one embodiment, the AFEX process is used in the preparation of cellulosic, hemicellulosic or lignocellulosic materials for fermentation to ethanol or other products. The process generally includes combining the feedstock with ammonia, heating under pressure, and suddenly releasing the pressure. Water can be present in various amounts. The AFEX process has been the subject of numerous patents and publications.

[00189] In another embodiment, the pretreatment of biomass comprises the addition of calcium hydroxide to a biomass to render the biomass susceptible to degradation. Pretreatment comprises the addition of calcium hydroxide and water to the biomass to form a mixture, and maintaining the mixture at a relatively high temperature. Alternatively, an oxidizing agent, selected from the group consisting of oxygen and oxygen-containing gasses, can be added under pressure to the mixture. Examples of carbon hydroxide treatments are disclosed in U.S. Patent No. 5,865,898 to Holtzapple and S. Kim and M. T. Holtzapple, Bioresource Technology, 96, (2005) 1994, incorporated by reference herein in its entirety.

[00190] In one embodiment, pretreatment of biomass comprises dilute acid hydrolysis.

Example of dilute acid hydrolysis treatment are disclosed in T. A. Lloyd and C. E Wyman, Bioresource Technology, (2005) 96, 1967), incorporated by reference herein in its entirety.

[00191] In another embodiment, pretreatment of biomass comprises pH controlled liquid hot water treatment. Examples of pH controlled liquid hot water treatments are disclosed in N. Mosier et ah, Bioresource Technology, (2005) 96, 1986, incorporated by reference herein in its entirety.

[00192] In one embodiment, pretreatment of biomass comprises aqueous ammonia recycle process (ARP). Examples of aqueous ammonia recycle process are described in T. H. Kim and Y. Y. Lee, Bioresource Technology, (2005) 96, 2007, incorporated by reference herein in its entirety.

[00193] In one embodiment, the above-mentioned methods have two steps: a pretreatment step that leads to a wash stream, and an enzymatic hydrolysis step of pretreated-biomass that produces a hydrolysate stream. In the above methods, the pretreatment step can include acid hydrolysis, hot water pretreatment, steam explosion or alkaline reagent based methods (AFEX, ARP, and lime pretreatments). Dilute acid and hot water treatment methods can be used to solubilize all or a portion of the hemicellulose. Methods employing alkaline reagents can be used remove all, most, or a portion of the lignin during the pretreatment step. As a result, the wash stream from the pretreatment step in the former methods contains mostly hemicellulose-based sugars, whereas this stream has mostly lignin for the high-pH methods. The subsequent enzymatic hydrolysis of the residual biomass leads to mixed sugars (C5 and C6) in the alkali based pretreatment methods, while glucose is the major product in the hydrolyzate from the low and neutral pH methods. In one embodiment, the treated material is additionally treated with catalase or another similar chemical, chelating agents, surfactants, and other compounds to remove impurities or toxic chemicals or further release polysaccharides.

[00194] The biomass can be pretreated according to any of the methods disclosed herein; for example, by dilute acid, hot water treatment, stream explosion, or an alkaline pretreatment. The biomass can be pretreated using a combination of techniques; for example, the biomass can be pretreated using hot water or stream explosion followed by alkaline treatment. [00195] In one embodiment, pretreatment of biomass comprises ionic liquid (IL) pretreatment. Biomass can be pretreated by incubation with an ionic liquid, followed by IL extraction with a wash solvent such as alcohol or water. The treated biomass can then be separated from the ionic liquid/wash-solvent solution by centrifugation or filtration, and sent to the saccharification reactor or vessel. Examples of ionic liquid pretreatment are disclosed in US publication No. 2008/0227162, incorporated herein by reference in its entirety.

[00196] In another embodiment, a method can utilize a pretreatment process disclosed in

U.S. Patent No. 4600590 to Dale, U.S. Patent No. 4644060 to Chou, U.S. Patent No. 5037663 to Dale. U.S. Patent No. 5171592 to Holtzapple, et al, U.S. Patent No. 5939544 to Karstens, et al, U.S. Patent No. 5473061 to Bredereck, et al, U.S. Patent No. 6416621 to Karstens., U.S. Patent No. 6106888 to Dale, et al, U.S. Patent No. 6176176 to Dale, et al, PCT publication

WO2008/020901 to Dale, et al, Felix, A., et al, Anim. Prod. 51, 47-61 (1990), Wais, A.C., Jr., et al, Journal of Animal Science, 35, No. 1,109-1 12 (1972), which are incorporated herein by reference in their entireties.

[00197] Alteration of the pH of a pretreated feedstock can be accomplished by washing the feedstock {e.g. , with water) one or more times to remove an alkaline or acidic substance, or other substance used or produced during pretreatment. Washing can comprise exposing the pretreated feedstock to an equal volume of water 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more times. In another embodiment, a pH modifier can be added. For example, an acid, a buffer, or a material that reacts with other materials present can be added to modulate the pH of the feedstock. In one embodiment, more than one pH modifier can be used, such as one or more bases, one or more bases with one or more buffers, one or more acids, one or more acids with one or more buffers, or one or more buffers. When more than one pH modifiers are utilized, they can be added at the same time or at different times. Other non- limiting exemplary methods for neutralizing feedstocks treated with alkaline substances have been described, for example in U.S. Patent Nos. 4,048,341; 4,182,780; and 5,693,296.

[00198] In one embodiment, one or more acids can be combined, resulting in a buffer.

Suitable acids and buffers that can be used as pH modifiers include any liquid or gaseous acid that is compatible with the microorganism. Non-limiting examples include S0₂ or sulfurous acid, peroxyacetic acid, sulfuric acid, lactic acid, citric acid, oxalic acid, phosphoric acid, and hydrochloric acid. In some instances, the pH can be lowered to neutral pH or acidic pH, for example a pH of 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, or lower. In some embodiments, the pH is lowered and/or maintained within a range of about pH 4.5 to about 7.1, or about 4.5 to about 6.9, or about pH 5.0 to about 6.3, or about pH 5.5 to about 6.3, or about pH 6.0 to about 6.5, or about pH 5.5 to about 6.9 or about pH 6.2 to about 6.7. [00199] In another embodiment, biomass can be pre-treated at an elevated temperature and/or pressure. In one embodiment, at stage one or stage two, biomass is pre treated at a temperature range of 20°C to 400°C. In another embodiment, biomass is pretreated at a temperature of about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 80°C, 90°C, 100°C, 120°C, 150°C, 200°C, 250°C, 300°C, 350°C, 400°C or higher. In another embodiment, elevated temperatures are provided by the use of steam, hot water, or hot gases. In one embodiment, steam can be injected into a biomass containing vessel. In another

embodiment, the steam, hot water, or hot gas can be injected into a vessel jacket such that it heats, but does not directly contact the biomass.

[00200] In another embodiment, a biomass can be treated at an elevated pressure. In one embodiment, biomass is pre treated at a pressure range of about lpsi to about 30psi. In another embodiment, biomass is pre treated at a pressure or about lpsi, 2psi, 3psi, 4psi, 5psi, 6psi, 7psi, 8psi, 9psi, lOpsi, 12psi, 15psi, 18psi, 20psi, 22psi, 24psi, 26psi, 28psi, 30psi or more. In some embodiments, biomass can be treated with elevated pressures by the injection of steam into a biomass containing vessel. In one embodiment, the biomass can be treated to vacuum conditions prior or subsequent to alkaline or acid treatment or any other treatment methods provided herein.

[00201] In one embodiment, alkaline or acid pretreated biomass is washed (e.g. with water

(hot or cold) or other solvent such as alcohol (e.g. ethanol)), pH neutralized with an acid, base, or buffering agent (e.g. phosphate, citrate, borate, or carbonate salt) or dried prior to fermentation. In one embodiment, the drying step can be performed under vacuum to increase the rate of evaporation of water or other solvents. Alternatively, or additionally, the drying step can be performed at elevated temperatures such as about 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 80°C, 90°C, 100°C, 120°C, 150°C, 200°C, 250°C, 300°C or more.

[00202] In one embodiment, the pretreatment step includes a step of solids recovery at each stage. The solids recovery step can be during or also after pretreatment (e.g., acid or alkali pretreatment), or before the drying step. In one embodiment, the solids recovery step can include the use of a sieve, filter, screen, or a membrane for separating the liquid and solids fractions. In one embodiment, a suitable sieve pore diameter size ranges from about 0.001 microns to 8 mm, such as about 0.005 microns to 3 mm or about 0.01 microns to 1 mm. In one embodiment, a sieve pore size has a pore diameter of about O.Olmicrons, 0.02 microns, 0.05 microns, 0.1 microns, 0.5 microns, 1 micron, 2 microns, 4 microns, 5 microns, 10 microns, 20 microns, 25 microns, 50 microns, 75 microns, 100 microns, 125 microns, 150 microns, 200 microns, 250 microns, 300 microns, 400 microns, 500 microns, 750 microns, 1mm or more. In one embodiment, biomass (e.g. corn stover) is processed or pretreated prior to fermentation. In one embodiment, a method of pre-treatment includes but is not limited to, biomass particle size reduction, such as for example shredding, milling, chipping, crushing, grinding, or pulverizing. In one embodiment, biomass particle size reduction can include size separation methods such as sieving, or other suitable methods known in the art to separate materials based on size. In one embodiment, size separation can provide for enhanced yields. In one embodiment, separation of finely shredded biomass (e.g. particles smaller than about 8 mm in diameter, such as, 8, 7.9, 7.7, 7.5, 7.3, 7, 6.9, 6.7, 6.5, 6.3, 6, 5.9, 5.7, 5.5, 5.3, 5, 4.9, 4.7, 4.5, 4.3, 4, 3.9, 3.7, 3.5, 3.3, 3, 2.9, 2.7, 2.5, 2.3, 2, 1.9, 1.7, 1.5, 1.3, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm) from larger particles allows the recycling of the larger particles back into the size reduction process, thereby increasing the final yield of processed biomass. In one embodiment, a fermentative mixture is provided which comprises a pretreated lignocellulosic feedstock comprising less than about 50% of a lignin component present in the feedstock prior to pretreatment and comprising more than about 60% of a hemicellulose component present in the feedstock prior to pretreatment; and a microorganism capable of fermenting a five-carbon sugar, such as xylose, arabinose or a combination thereof, and a six-carbon sugar, such as glucose, galactose, mannose or a combination thereof. In some instances, pretreatment of the lignocellulosic feedstock comprises adding an alkaline substance which raises the pH to an alkaline level, for example NaOH. In one embodiment, NaOH is added at a concentration of about 0.5% to about 2% by weight of the feedstock. In one embodiment, pretreatment also comprises addition of a chelating agent.

[00203] Hydrolysis

[00204] In one embodiment, a biomass hydrolyzing unit provides useful advantages for the conversion of biomass to bio fuels and chemical products. One advantage of this unit is its ability to produce monomeric sugars from multiple types of biomass, including mixtures of different biomass materials, and is capable of hydro lyzing polysaccharides and higher molecular weight saccharides to lower molecular weight saccharides. In one embodiment, the hydro lyzing unit utilizes a pretreatment process and a hydrolytic enzyme which facilitates the production of a sugar stream containing a concentration of a monomeric sugar or several monomeric sugars derived from cellulosic and/or hemicellulosic polymers. Examples of biomass material that can be pretreated and hydrolyzed to manufacture sugar monomers include, but are not limited to, cellulosic, hemicellulosic, lignocellulosic materials; pectins; starches; wood; paper; agricultural products; forest waste; tree waste; tree bark; leaves; grasses; sawgrass; woody plant matter; non- woody plant matter; carbohydrates; starch; inulin; fructans; glucans; corn; hulls; sugar cane; sorghum, other grasses; bamboo, algae, and material derived from these materials. This ability to use a very wide range of pretreatment methods and hydrolytic enzymes gives distinct advantages in biomass fermentations. Various pretreatment conditions and enzyme hydrolysis can enhance the extraction of sugars from biomass, resulting in higher yields, higher productivity, greater product selectivity, and/or greater conversion efficiency.

[00205] In one embodiment, the enzyme treatment is used to hydrolyze various higher saccharides (higher molecular weight) present in biomass to lower saccharides (lower molecular weight), such as in preparation for fermentation by biocatalysts such as yeasts to produce ethanol, hydrogen, or other chemicals such as organic acids including succinic acid, formic acid, acetic acid, and lactic acid. These enzymes and/or the hydrolysate can be used in fermentations to produce various products including fuels, and other chemicals.

[00206] In one example, the process for converting biomass material into ethanol includes pretreating the biomass material (e.g., "feedstock"), hydro lyzing the pretreated biomass to convert polysaccharides to oligosaccharides, further hydrolyzing the oligosaccharides to monosaccharides, and converting the monosaccharides to bio fuels and chemical products. This process is repeated in the second stage. Enzymes such as cellulases, polysaccharases, lipases, proteases, ligninases, and hemicellulases, help produce the monosaccharides can be used in the biosynthesis of fermentation end-products. Biomass material that can be utilized includes woody plant matter, non- woody plant matter, cellulosic material, lignocellulosic material,

hemicellulosic material, carbohydrates, pectin, starch, inulin, fructans, glucans, corn, hulls, distiller's grains, algae, sugarcane, other grasses, switchgrass, bagasse, wheat straw, barley straw, rice straw, corncobs, bamboo, citrus peels, sorghum, high biomass sorghum, seed hulls, and material derived from these. The final product can then be separated and/or purified, as indicated by the properties for the desired final product. In some instances, compounds related to sugars such as sugar alcohols or sugar acids can be utilized as well.

[00207] Chemicals that can be used in the methods disclosed herein can be purchased from a commercial supplier, such as Sigma- Aldrich. Additionally, commercial enzyme cocktails (e.g. Accellerase™ 1000, CelluSeb-TL, CelluSeb-TS, Cellic™' CTec, STARGEN™,

Maxalig™, Spezyme.R™, Distillase.R™, G-Zyme.R™, Fermenzyme.R™, Fermgen™, GC 212, or Optimash™) or any other commercial enzyme cocktail can be purchased from vendors such as Specialty Enzymes & Biochemicals Co., Genencor, or Novozymes. Alternatively, enzyme cocktails can be prepared by growing one or more organisms such as for example a fungi (e.g. a Trichoderma, a Saccharomyces, a Pichia, a White Rot Fungus etc.), a bacteria (e.g. a Clostridium, or a coliform bacterium, a Zymomonas bacterium, Sacharophagus degradans etc.) in a suitable medium and harvesting enzymes produced therefrom. In some embodiments, the harvesting can include one or more steps of purification of enzymes.

[00208] In one embodiment, treatment of biomass comprises enzyme hydrolysis. In one embodiment, a biomass is treated with an enzyme or a mixture of enzymes, e.g., endoglucanases, exoglucanases, cellobiohydrolases, cellulase, beta-glucosidases, glycoside hydrolases, glycosyltransferases, lyases, esterases, amylases, glucoamylases, and proteins containing carbohydrate-binding modules. In one embodiment, the enzyme or mixture of enzymes is one or more individual enzymes with distinct activities. In another embodiment, the enzyme or mixture of enzymes can be enzyme domains with a particular catalytic activity. For example, an enzyme with multiple activities can have multiple enzyme domains, including for example glycoside hydrolases, glycosyltransferases, lyases and/or esterases catalytic domains. In another aspect, provided is a method for producing fermentation end products by: reducing a biomass material to a high percentage of homogeneous particles less than 1.5 mm in size; pretreating the biomass to release a C5 fraction and a C6 fraction; separating said C5 fraction from said C6 fraction; and hydrolyzing the C5 and C6 fractions with enzymes wherein the C5 fraction is hydrolyzed with one or more hemicellulase and the C6 fraction is hydrolyzed with one or more cellulase. In one embodiment, the percentage of homogeneous particles is 95% or greater, 90%> or greater, 85% or greater, 80%> or greater, 75% or greater, 70%> or greater, 65%> or greater, 60% or greater, 55% or greater, 50% or greater, 45% or greater, 40% or greater, 35% or greater, or 30% or greater.

[00209] In a further aspect, provided is a method of producing sugar polymers and oligomers and hydrolyzing this material with a 0.25-0.90% v/w enzyme addition; collecting enzymatically-released sugars in the solution; and fermenting the sugars with a biocatalyst to produce a fermentation end product. In one embodiment, the C5 fraction is separated from the C6 fraction during pretreatment. In another embodiment, the C5 fraction is enzymatically hydrolyzed separately from the C6 fraction. In another embodiment, the total enzyme added to hydrolyze the C5 fraction is 0.25-0.9% of the normal volume of enzymes. In another embodiment, the total enzyme added to hydrolyze the C6 fraction is 0.25-0.9% of the normal volume of enzymes. In one embodiment, the total enzyme added to hydrolyze the C5 fraction is 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90% of the normal volume of enzymes. In one embodiment, the total enzyme added to hydrolyze the C6 fraction is 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90% of the normal volume of enzymes.

[00210] In one embodiment, enzymes that degrade polysaccharides are used for the hydrolysis of biomass and can include enzymes that degrade cellulose, namely, cellulases.

Examples of some cellulases include endocellulases and exo-cellulases that hydrolyze beta- 1,4- glucosidic bonds.

[00211] In one embodiment, enzymes that degrade polysaccharides are used for the hydrolysis of biomass and can include enzymes that have the ability to degrade hemicellulose, namely, hemicellulases. Hemicellulose can be a major component of plant biomass and can contain a mixture of pentoses and hexoses, for example, D-xylopyranose, L-arabinofuranose, D- mannopyranose, D-glucopyranose, D-galactopyranose, D-glucopyranosyluronic acid and other sugars. In one embodiment, enzymes that degrade polysaccharides are used for the hydrolysis of biomass and can include enzymes that have the ability to degrade pectin, namely, pectinases. In plant cell walls, the cross-linked cellulose network can be embedded in a matrix of pectins that can be covalently cross-linked to xyloglucans and certain structural proteins. Pectin can comprise homogalacturonan (HG) or rhamnogalacturonan (RH).

[00212] In one embodiment, hydrolysis of biomass includes enzymes that can hydrolyze starch. Enzymes that hydrolyze starch include alpha-amylase, glucoamylase, beta-amylase, exo- alpha-l,4-glucanase, and pullulanase.

[00213] In one embodiment, hydrolysis of biomass comprises hydrolases that can include enzymes that hydrolyze chitin, such as chitinases. In another embodiment, hydrolases can include enzymes that hydrolyze lichen, namely, lichenase.

[00214] In one embodiment, after pretreatment and/or hydrolysis by any of the above methods the feedstock contains cellulose, hemicellulose, soluble oligomers, simple sugars, lignin, volatiles and ash. The parameters of the hydrolysis can be changed to vary the concentration of the components of the pretreated feedstock. For example, a hydrolysis can be chosen so that the concentration of soluble C5 saccharides is high and the concentration of lignin is low after hydrolysis. Examples of parameters of the hydrolysis include temperature, pressure, time, concentration, composition and pH.

[00215] In one embodiment, the parameters of the pretreatment and hydrolysis are changed to vary the concentration of the components of the pretreated feedstock such that concentration of the components in the pretreated and hydrolyzed feedstock is optimal for fermentation with a microbe such as a yeast or bacterium microbe.

[00216] In one embodiment, the parameters of the pretreatment are changed to encourage the release of the components of a genetically modified feedstock such as enzymes stored within a vacuole to increase or complement the enzymes synthesized by biocatalyst to produce optimal release of the fermentable components during hydrolysis and fermentation.

[00217] In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of accessible cellulose in the pretreated feedstock is 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. In one embodiment, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 5% to 30%. In one embodiment, the parameters of the pretreatment are changed such that concentration of accessible cellulose in the pretreated feedstock is 10%> to 20%. [00218] In one embodiment, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40% or 50%. In one embodiment, the parameters of the pretreatment are changed such that

concentration of hemicellulose in the pretreated feedstock is 5% to 40%. In one embodiment, the parameters of the pretreatment are changed such that concentration of hemicellulose in the pretreated feedstock is 10%> to 30%>.

[00219] In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of soluble oligomers in the pretreated feedstock is 1%, 10%>, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), or 99%). Examples of soluble oligomers include, but are not limited to, cellobiose and xylobiose. In one embodiment, the parameters of the pretreatment are changed such that concentration of soluble oligomers in the pretreated feedstock is 30% to 90%. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that concentration of soluble oligomers in the pretreated feedstock is 45% to 80%.

[00220] In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of simple sugars in the pretreated feedstock is 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 20%. In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of simple sugars in the pretreated feedstock is 0% to 5%. Examples of simple sugars include, but are not limited to, C5 and C6 monomers and dimers.

[00221] In one embodiment, the parameters of the pretreatment are changed such that concentration of lignin in the pretreated and/or hydrolyzed feedstock is 1%, 5%, 10%, 12%, 13%, 14%, 15%, 16%, 17%, 19%, 20%, 30%, 40% or 50%. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that concentration of lignin in the pretreated feedstock is 0% to 20%. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that concentration of lignin in the pretreated feedstock is 0% to 5%. In one embodiment, the parameters of the pretreatment and hydrolysis are changed such that concentration of lignin in the pretreated and/or hydrolyzed feedstock is less than 1% to 2%. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that the concentration of phenolics is minimized.

[00222] In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that concentration of furfural and low molecular weight lignin in the pretreated and/or hydrolyzed feedstock is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that concentration of furfural and low molecular weight lignin in the pretreated and/or hydrolyzed feedstock is less than 1% to 2%.

[00223] In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed such that the concentration of simple sugars is at least 75% to 85%, and the

concentration of lignin is 0% to 5% and the concentration of furfural and low molecular weight lignin in the pretreated feedstock is less than 1% to 2%.

[00224] In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed to obtain a high concentration of hemicellulose and a low concentration of lignin. In one embodiment, the parameters of the pretreatment and/or hydrolysis are changed to obtain a high concentration of hemicellulose and a low concentration of lignin such that concentration of the components in the pretreated stock is optimal for fermentation with a microbe such as biocatalyst.

[00225] In one embodiment, more than one of these steps can occur at any given time.

For example, hydrolysis of the pretreated feedstock and hydrolysis of the oligosaccharides can occur simultaneously, and one or more of these can occur simultaneously to the conversion of monosaccharides to a fuel or chemical.

[00226] In another embodiment, an enzyme can directly convert the polysaccharide to monosaccharides. In some instances, an enzyme can hydrolyze the polysaccharide to

oligosaccharides and the enzyme or another enzyme can hydrolyze the oligosaccharides to monosaccharides.

[00227] In another embodiment, the enzymes can be added to the fermentation or they can be produced by microorganisms present in the fermentation. In one embodiment, the

microorganism present in the fermentation produces some enzymes. In another embodiment, enzymes are produced separately and added to the fermentation.

[00228] For the overall conversion of pretreated biomass to final product to occur at high rates, the enzymes for each conversion step can be present with sufficiently high activity. If one of these enzymes is missing or is present in insufficient quantities, the production rate of an end product can be reduced. The production rate can also be reduced if the microorganisms responsible for the conversion of monosaccharides to product only slowly take up

monosaccharides and/or have only limited capability for translocation of the monosaccharides and intermediates produced during the conversion to end product. Additions of fractions obtained from pretreatment and/or pretreatment and hydrolysis can increase initial or overall growth rates. In another embodiment, oligomers are taken up slowly by a biocatalyst, necessitating an almost complete conversion of polysaccharides and oligomers to monomeric sugars.

[00229] In another embodiment, the enzymes of the method are produced by a biocatalyst, including a range of hydro lytic enzymes suitable for the biomass materials used in the fermentation methods. In one embodiment, a biocatalyst is grown under conditions appropriate to induce and/or promote production of the enzymes needed for the saccharification of the polysaccharide present. The production of these enzymes can occur in a separate vessel, such as a seed fermentation vessel or other fermentation vessel, or in the production fermentation vessel where ethanol production occurs. When the enzymes are produced in a separate vessel, they can, for example, be transferred to the production fermentation vessel along with the cells, or as a relatively cell free solution liquid containing the intercellular medium with the enzymes. When the enzymes are produced in a separate vessel, they can also be dried and/or purified prior to adding them to the hydrolysis or the production fermentation vessel. The conditions appropriate for production of the enzymes are frequently managed by growing the cells in a medium that includes the biomass that the cells will be expected to hydrolyze in subsequent fermentation steps. Additional medium components, such as salt supplements, growth factors, and cofactors including, but not limited to phytate, amino acids, and peptides can also assist in the production of the enzymes utilized by the microorganism in the production of the desired products.

[00230] Fermentation

[00231] Provided herein are methods and compositions for a two-stage process of producing one or more fermentation products from feedstocks comprising one or more cellulosic and/or hemicellulosic polysaccharides. The source of the one or more polysaccharides can be a lignocellulosic feedstock. The fermentation product can be produced by two-stage pretreating and/or hydrolyzing a biomass comprising cellulose, hemicellulose, or lignocellulose.

[00232] Provided herein are methods and compositions for a two-stage process of producing one or more fermentation products from feedstocks comprising a mixture of non- cellulosic polysaccharides (e.g., starch) and one or more cellulosic and/or hemicellulosic polysaccharides. The source of the one or more polysaccharides can be a lignocellulosic feedstock. The fermentation product can be produced by two-stage pretreating and/or hydrolyzing a biomass comprising cellulose, hemicellulose, or lignocellulose.

[00233] Enhanced rates of fermentation can be achieved using a first stage process to hydrolyze hemicellulose and a second stage to hydrolyze cellulose in comparison to a one-stage pretreatment hydrolysis. The enhanced rates of fermentation can be from about 1% higher to about 100% higher; for example, about 1-100%, 1-75%, 1-50%, 1 -25%, 1-10%, 10-100%, 10- 75%, 10-50%, 10-25%, 25-100%, 25-75%, 25-50%, 50-100%, 50-75%, 75-100%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,

22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher.

[00234] Increased yields of one or more fermentation end-products can be achieved using lignocellulosic feedstocks pretreated in a two-stage process in comparison to fermentation of lignocellulosic feedstock pretreated in a one-stage process. The increased yields of one or more fermentation end-products can be from about 1% higher to about 100% higher; for example, about 1-100%, 1-75%, 1-50%, 1-25%, 1-10%, 10-100%, 10-75%, 10-50%, 10-25%, 25-100%, 25-75%, 25-50%, 50-100%, 50-75%, 75-100%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher.

[00235] In one embodiment, the concentration of monosaccharides at the start of a fermentation or simultaneous saccharification and fermentation reaction can be less than about 100 g/L; for example, less than about 100 g/L, 90 g/L, 80 g/L, 70 g/L, 60 g/L, 50 g/L, 40 g/L, 30 g/L, 25 g/L, 20 g/L, 15 g/L, 10 g/L, 9 g/L, 8 g/L, 7 g/L, 6 g/L, 5 g/L, 4 g/L, 3 g/L, 2 g/L, or 1 g/L. In another embodiment, the concentration of monosaccharides at the start of a fermentation or simultaneous saccharification and fermentation reaction can be from about 1 g/L to about 100 g/L; for example, about 1-100 g/L, 1-75 g/L, 1-50 g/L, 1-25 g/L, 1-10 g/L, 10-100 g/L, 10-75 g/L, 10-50 g/L, 10-25 g/L, 25-100 g/L, 25-75 g/L, 25-50 g/L, 50-100 g/L, 50-75 g/L, or 75-100 g/L.

[00236] The present disclosure also provides a fermentative mixture comprising: a cellulosic feedstock pre-treated with an acid substance and at a temperature of from about 80°C to about 120°C; subsequently hydro lyzed with an enzyme mixture, and a microorganism capable of fermenting a five-carbon sugar and/or a six-carbon sugar. In one embodiment, the five-carbon sugar is xylose, arabinose, or a combination thereof. In one embodiment, the six-carbon sugar is glucose, galactose, mannose, or a combination thereof. In one embodiment, the acid is equal to or less than 2% HC1 or S0₂ or H₂S0₄. In one embodiment, the microorganism is a Rhodococcus strain, a Clostridium strain, a Trichoderma strain, a Saccharomyces strain, a Zymomonas strain, or another microorganism suitable for fermentation of biomass. In another embodiment, the fermentation process comprises fermentation of the biomass using a microorganism that is Clostridium phytofermentans, Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae,

Clostridium celerecrescens, Clostridium polys accharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor s accharolyticum, Rhodococcus opacus, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter

succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum

thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum, Sacharophagus degradans, or Thermoanaerobacterium saccharolyticum. In still another embodiment, the microorganism is genetically modified to enhance activity of one or more hydrolytic enzymes, such as a genetically-modified Saccaromyces cerevisae.

[00237] In one embodiment, a wild type or a genetically-improved microorganism can be used for chemical production by fermentation. Methods to produce a genetically-improved strain can include genetic modification, mutagenesis, and adaptive processes, such as directed evolution. For example, yeasts can be genetically-modified to ferment C5 sugars. Other useful yeasts are species of Candida, Cryptococcus, Debaryomyces, Deddera, Hanseniaspora,

Kluyveromyces, Pichia, Schizosaccharomyces, and Zygosaccharomyces . Rhodococus strains, such as Rhodococcus opacus variants are a source of triacylglycerols and other storage lipids. (See, e.g., Waltermann, et al., Microbiology 146: 1143-1149 (2000)). Other useful organisms for fermentation include, but are not limited to, yeasts, especially Saccaromyces strains and bacteria such as Clostridium phytofermentans, Thermoanaerobacter ethanolicus, Clostridium

thermocellum, Clostridium beijerinickii, Clostridium acetobutylicum, Clostridium tyrobutyricum, Clostridium thermobutyricum, Thermoanaerobacterium saccharolyticum, Thermoanaerobacter thermohydrosulfuricus, Clostridium acetobutylicum, Moorella ssp., Carboxydocella ssp., Zymomonas mobilis, recombinant E. Coli, Klebsiella oxytoca, Rhodococcus opacus and

Clostridium beijerickii.

[00238] An advantage of yeasts are their ability to grow under conditions that include elevated ethanol concentration, high sugar concentration, low sugar concentration, and/or operate under anaerobic conditions. These characteristics, in various combinations, can be used to achieve operation with long or short fermentation cycles and can be used in combination with batch fermentations, fed batch fermentations, self-seeding/partial harvest fermentations, and recycle of cells from the final fermentation as inoculum.

[00239] Examples of yeasts that can be used as a biocatalyst or fermentive microorganism in the methods disclosed herein include but are not limited to, species found in the genus

Ascoidea, Brettanomyces, Candida, Cephaloascus, Coccidiascus, Dipodascus, Eremothecium, Galactomyces, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces,

Sporopachydermia, Torulaspora, Yarrowia, or Zygosaccharomyces; for example, Ascoidea rebescens, Brettanomyces anomalus, Brettanomyces bruxellensis, Brettanomyces claussenii, Brettanomyces custersianus, Brettanomyces lambicus, Brettanomyces naardenensis,

Brettanomyces nanus, Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida blattae, Candida carpophila, Candida cerambycidarum, Candida chauliodes, Candida corydali, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida insectamens, Candida insectorum, Candida intermedia, Candida jejfresii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida lyxosophila, Candida maltosa, Candida marina, Candida membranifaciens, Candida milleri, Candida oleophila, Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candida rugosa, Candida sake, Candida shehatea, Candida temnochilae, Candida tenuis, Candida tropicalis, Candida tsuchiyae, Candida sinolaborantium, Candida sojae, Candida subhashii, Candida viswanathii, Candida utilis, Cephaloascus fragrans, Coccidiascus legeri, Dypodascus albidus, Eremothecium cymbalariae, Galactomyces candidum, Galactomyces geotrichum, Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces bacillisporus, Kluyveromyces blattae, Kluyveromyces dobzhanskii, Kluyveromyces hubeiensis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Kluyveromyces nonfermentans, Kluyveromyces piceae, Kluyveromyces sinensis, Kluyveromyces thermotolerans, Kluyveromyces waltii, Kluyveromyces wickerhamii, Kluyveromyces yarrowii, Pichia anomola, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia

norvegensis, Pichia ohmeri, Pichia pastoris, Pichia subpelliculosa, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguus,

Saccharomyces florentinus, Saccharomyces kluyveri, Saccharomyces martiniae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus,

Saccharomyces uvarum, Saccharomyces zonatus, Schizosaccharomyces cryophilus,

Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, Sporopachydermia cereana, Sporopachydermia lactativora, Sporopachydermia quercuum, Torulaspora delbrueckii, Torulaspora franciscae, Torulaspora globosa, Torulaspora pretoriensis, Yarrowia lipolytica, Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces cidri, Zygosaccharomyces fermentati, Zygosaccharomyces florentinus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces mellis,

Zygosaccharomyces microellipsoides, Zygosaccharomyces mrakii, Zygosaccharomyces pseudorouxii, or Zygosaccharomyces rouxii, or a variant or genetically modified version thereof.

[00240] Examples of bacteria that can be used as a biocatalyst or fermentive

microorganism in the methods disclosed herein include but are not limited to any bacterium found in the genus of Butyrivibrio , Ruminococcus, Eubacterium, Bacteroides, Acetivibrio, Caldibacillus, Acidothermus, Cellulomonas, Curtobacterium, Micromonospora, Actinoplanes, Streptomyces, Thermobifida, Thermomonospora, Microbispora, Fibrobacter, Sporocytophaga, Cytophaga, Flavobacterium, Achromobacter, Xanthomonas, Cellvibrio, Pseudomonas,

Myxobacter, Escherichia, Klebsiella, Thermoanaerobacterium, Thermoanaerobacter,

Geobacillus, Saccharococcus, Paenibacillus, Bacillus, Caldicellulosiruptor, Anaerocellum, Anoxybacillus, Zymomonas, Clostridium; for example, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Ruminococcus succinogenes, Ruminococcus albus, Eubacterium cellulolyticum, Bacteroides cellulosolvens, Acetivibrio cellulolyticus, Acetivibrio cellulosolvens, Caldibacillus cellulovorans, Bacillus circulans, Acidothermus cellulolyticus, Cellulomonas cartae,

Cellulomonas cellasea, Cellulomonas cellulans, Cellulomonas fimi, Cellulomonas flavigena, Cellulomonas gelida, Cellulomonas iranensis, Cellulomonas persica, Cellulomonas uda, Curtobacterium falcumfaciens, Micromonospora melonosporea, Actinoplanes aurantiaca, Streptomyces reticuli, Streptomyces alboguseolus, Streptomyces aureofaciens, Streptomyces cellulolyticus, Streptomyces flavogriseus, Streptomyces lividans, Streptomyces nitrosporeus, Streptomyces olivochromogenes, Streptomyces rochei, Streptomyces thermovulgaris,

Streptomyces viridosporus, Thermobifida alba, Thermobifida fusca, Thermobifida cellulolytica, Thermomonospora curvata, Microbispora bispora, Fibrobacter succinogenes, Sporocytophaga myxococcoides, Cytophaga sp., Flavobacterium johnsoniae, Achromobacter piechaudii, Xanthomonas sp., Cellvibrio vulgaris, Cellvibrio fiulvus, Cellvibrio gilvus, Cellvibrio mixtus, Pseudomonas fluorescens, Pseudomonas mendocina, Myxobacter sp. AL-1, Escherichia albertii, Escherichia blattae, Escherichia coli, Escherichia fergusonii, Escherichia hermannii,

Escherichia vulneris, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella terrigena, Thermoanaerobacterium thermo sulfur igenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum,

Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum,

Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus,

Thermoanaerobacter brocki, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis,

Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus,

Anaerocellum thermophilum, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium straminosolvens, Clostridium acetobutylicum, Clostridium aerotolerans, Clostridium

beijerinckii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum,

Clostridium cadaveric, Clostridium chauvoei, Clostridium clostridioforme, Clostridium colicanis, Clostridium difficile, Clostridium fallax, Clostridium formicaceticum, Clostridium histolyticum, Clostridium innocuum, Clostridium ljungdahlii, Clostridium laramie, Clostridium lavalense, Clostridium novyi, Clostridium oedematiens, Clostridium paraputrificum, Clostridium perfringens, Clostridium phytofermentans, Clostridium piliforme, Clostridium ramosum, Clostridium scatologenes, Clostridium septicum, Clostridium sordellii, Clostridium sporogenes, Clostridium tertium, Clostridium tetani, Clostridium tyrobutyricum, Clostridium

thermobutyricum, Zymomonas mobilis, or a variant or genetically modified version thereof.

[00241] In one embodiment, fed-batch fermentation is performed on the treated biomass to produce a fermentation end-product, such as alcohol, ethanol, organic acid, succinic acid, TAG, or hydrogen. In one embodiment, the fermentation process comprises simultaneous hydrolysis and fermentation (SSF) of the biomass using one or more microorganisms such as a Rhodococcus strain, a Clostridium strain, a Trichoderma strain, a Saccharomyces strain, a Zymomonas strain, or another microorganism suitable for fermentation of biomass. In another embodiment, the fermentation process comprises simultaneous hydrolysis and fermentation of the biomass using a microorganism that is Clostridium algidixylanolyticum, Clostridium xylanolyticum, Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium

thermocellum, Clostridium josui, Clostridium papyrosolvens, Clostridium cellobioparum, Clostridium hungatei, Clostridium cellulosi, Clostridium stercorarium, Clostridium termitidis, Clostridium thermocopriae, Clostridium celerecrescens, Clostridium polys accharolyticum, Clostridium populeti, Clostridium lentocellum, Clostridium chartatabidum, Clostridium aldrichii, Clostridium herbivorans, Clostridium phytofermentans, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, Caldicellulosiruptor saccharolyticum, Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes, Eubacterium cellulosolvens, Butyrivibrio fibrisolvens, Anaerocellum thermophilum, Halocella cellulolytica, Thermoanaerobacterium thermosaccharolyticum, Sacharophagus degradans, or Thermoanaerobacterium

saccharolyticum .

[00242] In one embodiment, the fermentation process can include separate hydrolysis and fermentation (SHF) of a biomass with one or more enzymes, such as a xylanases, endo-l,4-beta- xylanases, xylosidases, beta-D-xylosidases, cellulases, hemicellulases, carbohydrases, glucanases, endoglucanases, endo-l,4-beta-glucanases, exoglucanases, glucosidases, beta-D- glucosidases, amylases, cellobiohydrolases, exocellobiohydrolases, phytases, proteases, peroxidase, pectate lyases, galacturonases, or laccases. In one embodiment, one or more enzymes used to treat a biomass is thermostable. In another embodiment, a biomass is treated with one or more enzymes, such as those provided herein, prior to fermentation. In another embodiment, a biomass is treated with one or more enzymes, such as those provided herein, during

fermentation. In another embodiment, a biomass is treated with one or more enzymes, such as those provided herein, prior to fermentation and during fermentation. In another embodiment, an enzyme used for hydrolysis of a biomass is the same as those added during fermentation. In another embodiment, an enzyme used for hydrolysis of biomass is different from those added during fermentation.

[00243] In some embodiments, fermentation can be performed in an apparatus such as bioreactor, a fermentation vessel, a stirred tank reactor, or a fluidized bed reactor. In one embodiment, the treated biomass can be supplemented with suitable chemicals to facilitate robust growth of the one or more fermenting organisms. In one embodiment, a useful supplement includes but is not limited to, a source of nitrogen and/or amino acids such as yeast extract, cysteine, or ammonium salts (e.g. nitrate, sulfate, phosphate etc.); a source of simple carbohydrates such as corn steep liquor, and malt syrup; a source of vitamins such as yeast extract; buffering agents such as salts (including but not limited to citrate salts, phosphate salts, or carbonate salts); or mineral nutrients such as salts of magnesium, calcium, or iron. In some embodiments redox modifiers are added to the fermentation mixture including but not limited to cysteine or mercaptoethanol.

[00244] In one embodiment, the titer and/or productivity of fermentation end-product production by a microorganism is improved by culturing the microorganism in a medium comprising one or more compounds comprising hexose and/or pentose sugars. In one

embodiment, a process comprises conversion of a starting material (such as a biomass) to a biofuel, such as one or more alcohols. In one embodiment, methods can comprise contacting substrate comprising both hexose (e.g. glucose, cellobiose) and pentose (e.g. xylose, arabinose) saccharides with a microorganism that can hydrolyze C5 and C6 saccharides to produce ethanol. In another embodiment, methods can comprise contacting substrate comprising both hexose (e.g. glucose, cellobiose) and pentose (e.g. xylose, arabinose) saccharides with R. opacus to produce TAG.

[00245] In some embodiments, batch fermentation with a microorganism of a mixture of hexose and pentose saccharides using the methods disclosed herein can provide uptake rates of about 0.1-8 g/L/h or more of hexose and about 0.1-8 g/L/h or more of pentose (xylose, arabinose, etc.). In some embodiments, batch fermentation with a microorganism of a mixture of hexose and pentose saccharides using the methods disclosed herein can provide uptake rates of about

0.1, 0.2, 0.4, 0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or 6 g/L/h or more of hexose and about 0.1, 0.2, 0.4,

0.5, 0.6 0.7, 0.8, 1, 2, 3, 4, 5, or 6 g/L/h or more of pentose.

[00246] In one embodiment, a method for production of ethanol or another alcohol produces about 10 g/1 to 120 gain 40 hours or less. In another embodiment, a method for production of ethanol produces about 10 g/1, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25 g/L, 26 g/L, 27 g/L, 28 g/L, 29 g/L, 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69 g/L, 70 g/L, 71 g/L, 72 g/L, 73 g/L, 74 g/L, 75 g/L, 76 g/L, 77 g/L, 78 g/L, 79 g/L, 80 g/L, 81 g/L, 82 g/L, 83 g/L, 84 g/L, 85 g/L, 86 g/L, 87 g/L, 88 g/L, 89 g/L, 90 g/L, 91 g/L, 92 g/L, 93 g/L, 94 g/L, 95 g/L, 96 g/L, 97 g/L, 98 g/L, 99 g/L, 100 g/L, 110 g/1, 120 g/1, or more alcohol in 40 hours by the fermentation of biomass. In another embodiment, alcohol is produced by a method comprising simultaneous fermentation of hexose and pentose saccharides. In another embodiment, alcohol is produced by a microorganism comprising simultaneous fermentation of hexose and pentose saccharides.

[00247] In another embodiment, the level of a medium component is maintained at a desired level by adding additional medium component as the component is consumed or taken up by the organism. Examples of medium components included, but are not limited to, carbon substrate, nitrogen substrate, vitamins, minerals, growth factors, cofactors, and biocatalysts. The medium component can be added continuously or at regular or irregular intervals. In one embodiment, additional medium component is added prior to the complete depletion of the medium component in the medium. In one embodiment, complete depletion can effectively be used, for example to initiate different metabolic pathways, to simplify downstream operations, or for other reasons as well. In one embodiment, the medium component level is allowed to vary by about 10% around a midpoint, in one embodiment, it is allowed to vary by about 30% around a midpoint, and in one embodiment, it is allowed to vary by 60% or more around a midpoint. In one embodiment, the medium component level is maintained by allowing the medium

component to be depleted to an appropriate level, followed by increasing the medium component level to another appropriate level. In one embodiment, a medium component, such as vitamin, is added at two different time points during fermentation process. For example, one-half of a total amount of vitamin is added at the beginning of fermentation and the other half is added at midpoint of fermentation.

[00248] In another embodiment, the nitrogen level is maintained at a desired level by adding additional nitrogen-containing material as nitrogen is consumed or taken up by the organism. The nitrogen-containing material can be added continuously or at regular or irregular intervals. Useful nitrogen levels include levels of about 5 to about 10 g/L. In one embodiment, levels of about 1 to about 12 g/L can also be usefully employed. In another embodiment, levels, such as about 0.5, 0.1 g/L or even lower, and higher levels, such as about 20, 30 g/L or even higher are used. In another embodiment, a useful nitrogen level is about 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 23, 24, 25, 26, 27, 28, 29 or 30 g/L. Nitrogen can be supplied as a simple nitrogen-containing material, such as an ammonium compounds (e.g. ammonium sulfate, ammonium hydroxide, ammonia, ammonium nitrate, or any other compound or mixture containing an ammonium moiety), nitrate or nitrite compounds (e.g. potassium, sodium, ammonium, calcium, or other compound or mixture containing a nitrate or nitrite moiety), or as a more complex nitrogen- containing material, such as amino acids, proteins, hydrolyzed protein, hydrolyzed yeast, yeast extract, dried brewer's yeast, yeast hydrolysates, distillers' grains, soy protein, hydrolyzed soy protein, fermentation products, and processed or corn steep powder or unprocessed protein-rich vegetable or animal matter, including those derived from bean, seeds, soy, legumes, nuts, milk, pig, cattle, mammal, fish, as well as other parts of plants and other types of animals. Nitrogen- containing materials useful in various embodiments also include materials that contain a nitrogen-containing material, including, but not limited to mixtures of a simple or more complex nitrogen-containing material mixed with a carbon source, another nitrogen-containing material, or other nutrients or non-nutrients, and AFEX treated plant matter.

[00249] In another embodiment, the carbon level is maintained at a desired level by adding sugar compounds or material containing sugar compounds ("Sugar-Containing Material") as sugar is consumed or taken up by the organism. The sugar-containing material can be added continuously or at regular or irregular intervals. In one embodiment, additional sugar-containing material is added prior to the complete depletion of the sugar compounds available in the medium. In one embodiment, complete depletion can effectively be used, for example to initiate different metabolic pathways, to simplify downstream operations, or for other reasons as well. In one embodiment, the carbon level (as measured by the grams of sugar present in the sugar- containing material per liter of broth) is allowed to vary by about 10% around a midpoint, in one embodiment, it is allowed to vary by about 30% around a midpoint, and in one embodiment, it is allowed to vary by 60% or more around a midpoint. In one embodiment, the carbon level is maintained by allowing the carbon to be depleted to an appropriate level, followed by increasing the carbon level to another appropriate level. In some embodiments, the carbon level can be maintained at a level of about 5 to about 120 g/L. However, levels of about 30 to about 100 g/L can also be usefully employed as well as levels of about 60 to about 80 g/L. In one embodiment, the carbon level is maintained at greater than 25 g/L for a portion of the culturing. In another embodiment, the carbon level is maintained at about 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, 25 g/L, 26 g/L, 27 g/L, 28 g/L, 29 g/L, 30 g/L, 31 g/L, 32 g/L, 33 g/L, 34 g/L, 35 g/L, 36 g/L, 37 g/L, 38 g/L, 39 g/L, 40 g/L, 41 g/L, 42 g/L, 43 g/L, 44 g/L, 45 g/L, 46 g/L, 47 g/L, 48 g/L, 49 g/L, 50 g/L, 51 g/L, 52 g/L, 53 g/L, 54 g/L, 55 g/L, 56 g/L, 57 g/L, 58 g/L, 59 g/L, 60 g/L, 61 g/L, 62 g/L, 63 g/L, 64 g/L, 65 g/L, 66 g/L, 67 g/L, 68 g/L, 69 g/L, 70 g/L, 71 g/L, 72 g/L, 73 g/L, 74 g/L, 75 g/L, 76 g/L, 77 g/L, 78 g/L, 79 g/L, 80 g/L, 81 g/L, 82 g/L, 83 g/L, 84 g/L, 85 g/L, 86 g/L, 87 g/L, 88 g/L, 89 g/L, 90 g/L, 91 g/L, 92 g/L, 93 g/L, 94 g/L, 95 g/L, 96 g/L, 97 g/L, 98 g/L, 99 g/L, 100 g/L, 101 g/L, 102 g/L, 103 g/L, 104 g/L, 105 g/L, 106 g/L, 107 g/L, 108 g/L, 109 g/L, 110 g/L, 111 g/L, 112 g/L, 113 g/L, 114 g/L, 115 g/L, 116 g/L, 117 g/L, 118 g/L, 119 g/L, 120 g/L, 121 g/L, 122 g/L, 123 g/L, 124 g/L, 125 g/L, 126 g/L, 127 g/L, 128 g/L, 129 g/L, 130 g/L, 131 g/L, 132 g/L, 133 g/L, 134 g/L, 135 g/L, 136 g/L, 137 g/L, 138 g/L, 139 g/L, 140 g/L, 141 g/L, 142 g/L, 143 g/L, 144 g/L, 145 g/L, 146 g/L, 147 g/L, 148 g/L, 149 g/L, or 150 g/L.

[00250] The carbon substrate, like the nitrogen substrate, can be used for cell production and enzyme production, but unlike the nitrogen substrate, the carbon substrate can serve as the raw material for production of fermentation end-products. Frequently, more carbon substrate can lead to greater production of fermentation end-products. In another embodiment, it can be advantageous to operate with the carbon level and nitrogen level related to each other for at least a portion of the fermentation time. In one embodiment, the ratio of carbon to nitrogen is maintained within a range of about 30: 1 to about 10: 1. In another embodiment, the ratio of carbon nitrogen is maintained from about 20: 1 to about 10: 1 or more preferably from about 15: 1 to about 10: 1. In another embodiment, the ratio of carbon nitrogen is about 30: 1, 29: 1, 28: 1, 27:1, 26: 1, 25: 1, 24: 1, 23: 1, 22: 1, 21 : 1, 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11 : 1, 10:1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, or 1 : 1.

[00251] Maintaining the ratio of carbon and nitrogen ratio within particular ranges can result in benefits to the operation such as the rate of metabolism of carbon substrate, which depends on the amount of carbon substrate and the amount and activity of enzymes present, being balanced to the rate of end product production. Balancing the carbon to nitrogen ratio can, for example, facilitate the sustained production of these enzymes such as to replace those which have lost activity.

[00252] In another embodiment, the amount and/or timing of carbon, nitrogen, or other medium component addition can be related to measurements taken during the fermentation. For example, the amount of monosaccharides present, the amount of insoluble polysaccharide present, the polysaccharase activity, the amount of product present, the amount of cellular material (for example, packed cell volume, dry cell weight, etc.) and/or the amount of nitrogen (for example, nitrate, nitrite, ammonia, urea, proteins, amino acids, etc.) present can be measured. The concentration of the particular species, the total amount of the species present in the fermentor, the number of hours the fermentation has been running, and the volume of the fermentor can be considered. In various embodiments, these measurements can be compared to each other and/or they can be compared to previous measurements of the same parameter previously taken from the same fermentation or another fermentation. Adjustments to the amount of a medium component can be accomplished such as by changing the flow rate of a stream containing that component or by changing the frequency of the additions for that component. For example, the amount of saccharide can be increased when the cell production increases faster than the end product production. In another embodiment, the amount of nitrogen can be increased when the enzyme activity level decreases.

[00253] In another embodiment, a fed batch operation can be employed, wherein medium components and/or fresh cells are added during the fermentation without removal of a portion of the broth for harvest prior to the end of the fermentation. In one embodiment, a fed-batch process is based on feeding a growth limiting nutrient medium to a culture of microorganisms. In one embodiment, the feed medium is highly concentrated to avoid dilution of the bioreactor. In another embodiment, the controlled addition of the nutrient directly affects the growth rate of the culture and avoids overflow metabolism such as the formation of side metabolites. In one embodiment, the growth limiting nutrient is a nitrogen source or a saccharide source.

[00254] In various embodiments, particular medium components can have beneficial effects on the performance of the fermentation, such as increasing the titer of desired products, or increasing the rate that the desired products are produced. Specific compounds can be supplied as a specific, pure ingredient, such as a particular amino acid, or it can be supplied as a component of a more complex ingredient, such as using a microbial, plant or animal product as a medium ingredient to provide a particular amino acid, promoter, cofactor, or other beneficial compound. In some cases, the particular compound supplied in the medium ingredient can be combined with other compounds by the organism resulting in a fermentation-beneficial compound. One example of this situation would be where a medium ingredient provides a specific amino acid which the organism uses to make an enzyme beneficial to the fermentation.

Other examples can include medium components that are used to generate growth or product promoters, etc. In such cases, it can be possible to obtain a fermentation-beneficial result by supplementing the enzyme, promoter, growth factor, etc. or by adding the precursor. In some situations, the specific mechanism whereby the medium component benefits the fermentation is not known, only that a beneficial result is achieved.

[00255] In one embodiment, a fermentation to produce a fuel is performed by culturing a strain of R. opacus in a medium having a supplement of lignin component and a concentration of one or more carbon sources. The resulting production of end product such as TAG can be up to 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and in some cases up to 10- fold and higher in volumetric productivity than a process using only the addition of a relatively pure saccharide source, and can achieve a carbon conversion efficiency approaching the theoretical maximum. The theoretical maximum can vary with the substrate and product. For example, the generally accepted maximum efficiency for conversion of glucose to ethanol is 0.51 g ethanol/g glucose. In one embodiment, a biocatalyst can produce about 40-100% of a theoretical maximum yield of ethanol. In another embodiment, a biocatalyst can produce up to about 40%, 50%, 60%, 70%, 80%, 90%, 95% and even 100% of the theoretical maximum yield of ethanol. In one embodiment, a biocatalyst can produce up to about 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, 50 %, 51 %, 52 %, 53 %, 54 %, 55 %, 56 %, 57 %, 58 %, 59 %, 60 %, 61 %, 62 %, 63 %, 64 %, 65 %, 66 %, 67 %, 68 %, 69 %, 70 %, 71 %, 72 %, 73 %, 74 %, 75 %, 76 %, 77 %, 78 %, 79 %, 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, 99.99 %, orlOO % of a theoretical maximum yield of a fuel. It can be possible to obtain a fermentation-beneficial result by supplementing the medium with a pretreatment or hydrolysis component. In some situations, the specific mechanism whereby the medium component benefits the fermentation is not known, only that a beneficial result is achieved.

[00256] Various embodiments offer benefits relating to improving the titer and/or productivity of fermentation end-product production by a biocatalyst by culturing the organism in a medium comprising one or more compounds comprising particular fatty acid moieties and/or culturing the organism under conditions of controlled pH.

[00257] In one embodiment, the pH of the medium is controlled at less than about pH 7.2 for at least a portion of the fermentation. In one embodiment, the pH is controlled within a range of about pH 3.0 to about 7.1 or about pH 4.5 to about 7.1 , or about pH 5.0 to about 6.3, or about pH 5.5 to about 6.3, or about pH 6.0 to about 6.5, or about pH 5.5 to about 6.9 or about pH 6.2 to about 6.7. The pH can be controlled by the addition of a pH modifier. In one embodiment, a pH modifier is an acid, a base, a buffer, or a material that reacts with other materials present to serve to raise of lower the pH. In one embodiment, more than one pH modifier can be used, such as more than one acid, more than one base, one or more acid with one or more bases, one or more acids with one or more buffers, one or more bases with one or more buffers, or one or more acids with one or more bases with one or more buffers. When more than one pH modifiers are utilized, they can be added at the same time or at different times. In one embodiment, one or more acids and one or more bases can be combined, resulting in a buffer. In one embodiment, media components, such as a carbon source or a nitrogen source can also serve as a pH modifier; suitable media components include those with high or low pH or those with buffering capacity.

Exemplary media components include acid- or base-hydrolyzed plant polysaccharides having with residual acid or base, AFEX treated plant material with residual ammonia, lactic acid, corn steep solids or liquor.

[00258] In one embodiment, a constant pH can be utilized throughout the fermentation. In one embodiment, the timing and/or amount of pH reduction can be related to the growth conditions of the cells, such as in relation to the cell count, the end product produced, the end product present, or the rate of end product production. In one embodiment, the pH reduction can be made in relation to physical or chemical properties of the fermentation, such as viscosity, medium composition, gas production, off gas composition, etc.

[00259] Recovery of Fermentation End Products

[00260] In another aspect, methods are provided for the recovery of the fermentive end products, such as an alcohol (e.g. ethanol, propanol, methanol, butanol, etc.) another biofuel or chemical product. In one embodiment, broth will be harvested at some point during of the fermentation, and fermentive end product or products will be recovered. The broth with end product to be recovered will include both end product and impurities. The impurities include materials such as water, cell bodies, cellular debris, excess carbon substrate, excess nitrogen substrate, other remaining nutrients, other metabolites, and other medium components or digested medium components. During the course of processing the broth, the broth can be heated and/or reacted with various reagents, resulting in additional impurities in the broth.

[00261] In one embodiment, the processing steps to recover end product frequently includes several separation steps, including, for example, distillation of a high concentration alcohol material from a less pure alcohol-containing material. In one embodiment, the high concentration alcohol material can be further concentrated to achieve very high concentration alcohol, such as 98% or 99% or 99.5% (wt.) or even higher. Other separation steps, such as filtration, centrifugation, extraction, adsorption, etc. can also be a part of some recovery processes for alcohol as a product or bio fuel, or other bio fuels or chemical products.

[00262] In one embodiment, a process can be scaled to produce commercially useful bio fuels. In another embodiment, biocatalyst is used to produce an alcohol, e.g., ethanol, butanol, propanol, methanol, or a fuel such as hydrocarbons hydrogen, TAG, and hydroxy compounds. In another embodiment, biocatalyst is used to produce a carbonyl compound such as an aldehyde or ketone {e.g. acetone, formaldehyde, 1-propanal, etc.), an organic acid, a derivative of an organic acid such as an ester {e.g. wax ester, glyceride, etc.), 1, 2-propanediol, 1, 3 -propanediol, lactic acid, formic acid, acetic acid, succinic acid, pyruvic acid, or an enzyme such as a cellulase, polysaccharase, lipases, protease, ligninase, and hemicellulase.

[00263] TAG biosynthesis is widely distributed in nature and the occurrence of TAG as reserve compounds is widespread among plants, animals, yeast and fungi. In contrast, however, TAGs have not been regarded as common storage compounds in bacteria. Biosynthesis and accumulation of TAGs have been described only for a few bacteria belonging to the

actinomycetes group, such as genera of Streptomyces, Nocardia, Rhodococcus, Mycobacterium, Dietzia and Gordonia, and, to a minor extent, also in a few other bacteria, such as Acinetobacter baylyi and Alcanivorax borkumensis. Since the mid-1990's, TAG production in hydrocarbon- degrading strains of those genera has been frequently reported. TAGs are stored in spherical lipid bodies as intracellular inclusions, with the amounts depending on the respective species, cultural conditions and growth phase. Commonly, the important factor for the production of TAGs is the amount of nitrogen that is supplied to the culture medium. The excess carbon, which is available to the culture after nitrogen exhaustion, continues to be assimilated by the cells and, by virtue of oleaginous bacteria possessing the requisite enzymes, is converted directly into lipid. The compositions and structures of bacterial TAG molecules vary considerably depending on the bacterium and on the cultural conditions, especially the carbon sources. See, Brigham CJ, et al. (2011) J Microbial Biochem Technol S3:002.

[00264] In one embodiment, useful biochemicals can be produced from non-food plant biomass, with a steam or hot-water extraction technique that is carried out by contacting a charge of non-food plant pretreated biomass material such as corn stover or sorghum with water and/or acid (with or without additional process enhancing compounds or materials), in a pressurized vessel at an elevated temperature up to about 160 -220° C. and at a pH below about 7.0, to yield an aqueous (extract solution) mixture of useful sugars including long-chain saccharides (sugars), acetic acid, and lignin, while leaving the structural (cellulose and lignin) portion of the lignocellulosic material largely intact. In combination, these potential inhibitory chemicals especially sugar degradation products are low, and the plant derived nutrients that are naturally occurring lignocellulosic-based components are also recovered that are beneficial to a C5and C6 fermenting organism. Toward this objective, the aqueous extract is concentrated (by

centrifugation, filtration, solvent extraction, flocculation, evaporation), by producing a concentrated sugar stream, apart from the other hemicellulose (C5 rich) and cellulosic derived sugars (C6 rich) which are channeled into a fermentable stream.

[00265] In another embodiment, following enzyme/acid hydrolysis, additional chemical compounds that are released are recovered with the sugar stream resulting in a short-chain sugar solution containing xylose, mannose, arabinose, rhamnose, galactose, and glucose (5 and 6- carbon sugars). The sugar stream, now significantly rich in C5 and C6 substances can be converted by microbial fermentation or chemical catalysis into such products as triacylglycerol or TAG and further refined to produce stream rich in JP8 or jet fuels . If C5 sugar percentage correction has not been performed, it can be performed before fermentation to satisfy desired combination of C5 and C6 sugars for the fermentation organism and corresponding end product.

[00266] Bio fuel plant and process of producing bio fuel:

[00267] Large Scale Fuel and Chemical Production from Biomass

[00268] Generally, there are several basic approaches to producing fuels and chemical end-products from biomass on a large scale utilizing of microbial cells. In the one method, one first pretreats and hydrolyzes a biomass material that includes high molecular weight

carbohydrates to lower molecular weight carbohydrates, and then ferments the lower molecular weight carbohydrates utilizing of microbial cells to produce fuel or other products. In the second method, one treats the biomass material itself using mechanical, chemical and/or enzymatic methods. In all methods, depending on the type of biomass and its physical manifestation, one of the processes can comprise a milling of the carbonaceous material, via wet or dry milling, to reduce the material in size and increase the surface to volume ratio (physical modification).

[00269] In one embodiment, hydrolysis can be accomplished using acids, e.g., Bronsted acids {e.g., sulfuric or hydrochloric acid), bases, e.g., sodium hydroxide, hydrothermal processes, ammonia fiber explosion processes ("AFEX"), lime processes, enzymes, or combination of these. Hydrogen, and other end products of the fermentation can be captured and purified if desired, or disposed of, e.g., by burning. For example, the hydrogen gas can be flared, or used as an energy source in the process, e.g., to drive a steam boiler, e.g., by burning. Hydrolysis and/or steam treatment of the biomass can, e.g., increase porosity and/or surface area of the biomass, often leaving the cellulosic materials more exposed to the biocatalyst cells, which can increase fermentation rate and yield. Removal of lignin can, e.g., provide a combustible fuel for driving a boiler, and can also, e.g., increase porosity and/or surface area of the biomass, often increasing fermentation rate and yield. Generally, in any of the these embodiments, the initial concentration of the carbohydrates in the medium is greater than 20 mM, e.g., greater than 30 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, or even greater than 500 mM.

[00270] Biomass processing plant and process of producing products from biomass

[00271] In one aspect, a fuel or chemical plant that includes a pretreatment unit to prepare biomass for improved exposure and biopolymer separation, a hydrolysis unit configured to hydrolyze a biomass material that includes a high molecular weight carbohydrate, and one or more product recovery system(s) to isolate a product or products and associated by-products and co-products is provided. In another aspect, methods of purifying lower molecular weight carbohydrate from solid byproducts and/or toxic impurities are provided.

[00272] In another aspect, methods of making a product or products that include combining biocatalyst cells of a microorganism and a biomass feed in a medium wherein the biomass feed contains lower molecular weight carbohydrates and unseparated solids and/or other liquids from pretreatment and hydrolysis, and fermenting the biomass material under conditions and for a time sufficient to produce a biofuel, chemical product or fermentive end-products, e.g. ethanol, propanol, hydrogen, succinic acid, lignin, terpenoids, and the like as described above, is provided.

[00273] In another aspect, products made by any of the processes described herein are also provided herein.

[00274] Figure 1 is an example of a method for producing chemical products from biomass by first treating biomass with an acid at elevated temperature and pressure in a hydrolysis unit. The biomass may first be heated by addition of hot water or steam. The biomass may be acidified by bubbling gaseous sulfur dioxide through the biomass that is suspended in water, or by adding a strong acid, e.g., sulfuric, hydrochloric, or nitric acid with or without preheating/presteaming/water addition. During the acidification, the pH is maintained at a low level, e.g., below about 5. The temperature and pressure may be elevated after acid addition. In addition to the acid already in the acidification unit, optionally, a metal salt such as ferrous sulfate, ferric sulfate, ferric chloride, aluminum sulfate, aluminum chloride, magnesium sulfate, or mixtures of these can be added to aid in the acid hydrolysis of the biomass. The acid- impregnated biomass is fed into the hydrolysis section of the pretreatment unit. Steam is injected into the hydrolysis portion of the pretreatment unit to directly contact and heat the biomass to the desired temperature. The temperature of the biomass after steam addition is, e.g., from about 130° C to 220° C. The acid hydro lysate is then discharged into the flash tank portion of the pretreatment unit, and is held in the tank for a period of time to further hydrolyze the biomass, e.g., into oligosaccharides and monomeric sugars. Other methods can also be used to further break down biomass. Alternatively, the biomass can be subject to discharge through a pressure lock for any high-pressure pretreatment process. Hydro lysate is then discharged from the pretreatment reactor, with or without the addition of water, e.g. , at solids concentrations from about 10% to about 60%.

[00275] After pretreatment, the biomass may be dewatered and/or washed with a quantity of water, e.g. by squeezing or by centrifugation, or by filtration using, e.g. a countercurrent extractor, wash press, filter press, pressure filter, a screw conveyor extractor, or a vacuum belt extractor to remove acidified fluid. Wash fluids can be collected to concentrate the C5 saccharides in the wash stream. The acidified fluid, with or without further treatment, e.g.

addition of alkali (e.g. lime) and or ammonia (e.g. ammonium phosphate), can be re-used, e.g., in the acidification portion of the pretreatment unit, or added to the fermentation, or collected for other use/treatment. Products may be derived from treatment of the acidified fluid, e.g., gypsum or ammonium phosphate. Enzymes or a mixture of enzymes can be added during pretreatment to hydrolyze, e.g. endoglucanases, exoglucanases, cellobiohydrolases (CBH), beta-glucosidases, glycoside hydrolases, glycosyltransferases, alphyamylases, chitinases, pectinases, lyases, and esterases active against components of cellulose, hemicelluloses, pectin, and starch, in the hydrolysis of high molecular weight components.

[00276] A fermentor, attached or at a separate site, can be fed with hydrolyzed biomass, any liquid fraction from biomass pretreatment, an active seed culture of a biocatalyst, such as a yeast, if desired a co-fermenting microbe, e.g., another yeast or E. coli, and, if required, nutrients to promote growth of the biocatalyst or other microbes. Alternatively, the pretreated biomass or liquid fraction can be split into multiple fermenters, each containing a different strain of a biocatalyst and/or other microbes, and each operating under specific physical conditions.

Fermentation is allowed to proceed for a period of time, e.g., from about 1 to about 150 hours, while maintaining a temperature of, e.g., from about 25° C to about 50° C. Gas produced during the fermentation is swept from fermentor and is discharged, collected, or flared with or without additional processing, e.g. hydrogen gas may be collected and used as a power source or purified as a co-product.

[00277] In another aspect, methods of making a fuel or fuels that include combining one or more biocatalyst and a lignocellulosic material (and/or other biomass material) in a medium, adding a lignin fraction from pretreatment, and fermenting the lignocellulosic material under conditions and for a time sufficient to produce a fuel or fuels, e.g., ethanol, propanol and/or hydrogen or another chemical compound is provided herein.

[00278] In another aspect, the products made by any of the processes described herein is provided. EXAMPLES

[00279] The following examples serve to illustrate certain embodiments and aspects and are not to be construed as limiting the scope thereof.

[00280] Two Stage Pretreatment Experiments

[00281] A two-stage pretreatment process was performed to show that enzyme dosing can be reduced when carrying out an enzymatic hydrolysis following optimal stage autohydrolysis. The first stage of pretreatment was performed in a Biogasol's CarboFrac system (Biogasol ApS, Denmark). The pretreated corncob biomass material was then washed twice with equal volumes of warm water before being soaked under various acidic conditions for about 12 hours. After the soaking, the biomass was pretreated in Sweetwater's 10L reactor at various time intervals for the second-stage. For each condition tested with sulfurous acid in the 10L processor, lOOg of material were weighed out into 250mL flasks. The enzyme dosing variables tested were 1.0%, 0.50% and 0.25% loading based on total dry solids loaded into the 10L reactor. Each flask was placed on a shaker at 150 rpm at 50°C for a total of 72 hours. Samples were taken at 24-hour intervals. To compare and determine maximum carbohydrate yields, one-stage pretreated corncobs were washed twice to remove C5 soluble sugars, and then hydro lyzed as a 10%> solids solution with a normal (DOE -NREL recommended) dose of 5% on solids of commercial enzyme. The enzyme used throughout the experiment was Novozymes CTec3 (Novozymes A/S, Denmark). Carbohydrate concentration was determined through HPLC analysis on a Bio- Rad Carbohydrate column.

[00282] Table 1 indicates the initial amounts of carbohydrate and inhibitors with stage one process samples and in stage two samples. These samples were taken prior to enzyme addition. Results indicate free sugars or carbohydrate levels recorded prior to enzyme hydrolysis as a baseline. The results in Table 2 show the final carbohydrate reading after a 72-hour hydrolysis at the conditions indicated. To get the total sum of sugars as carbohydrates obtained from enzymatic hydrolysis alone, concentrations in Table 1 were subtracted from those in Table 2.

[00283] Table 1.

Glucose Xylose Arabinose Formic Acetic HMF Furfural

Acid Acid

One-stage 5.12 7.69 0.88 0.22 1.09 0.22 0

Two-stage

2%* -5min 2.8 9.35 1.01 0.25 1.01 0.11 0.24

3% - 5min 3.47 9.85 1.13 0.33 1.25 0.14 0.32 2% - 3.48 7.1 1.14 0.57 1.35 0.26 0.87 lOmin

3% - 2.73 5.24 0.78 0.42 0.95 0.15 0.64 lOmin

2% -15min 3.06 5.24 0.8 0.48 0.99 0.21 0.64

3% - 3.03 5.4 0.72 0.47 0.98 0.22 0.68 15min

[00284] * 10 % solids [00285] Table 2. The sum of C5 and C6 sugars for 5% solids with high enzyme loading

[00286] Final recordings for each condition tested. (Control numbers were not subtracted from these final readings.)

[00287] Table 3.

A. One-stage pretreatment, single acid treatment, final (g/L) washed material solids at 10% solids.*

Enzyme Glucose Xylose Arabinose Formic Acetic HMF Furfural loading Acid Acid

No 0.61 6.84 0.55 0.78

enzyme

0.25"-» 4^ι) KLV) 0 52 0 94

0.50% 42.53 11.67 0.65 0.97

i .oo". ;, 53.94 1 2.S7 0 ⁷6 1 02

*The maximum sum of C5 and C6 sugars for 10 % solids with high enzyme loading is 76.7 g L

Two-Stage Sulfurous Pretreatment

2% Sulphurous acid- 5 min pretreatment - final monomeric sugars (g/L) 8.5% solids

Glucose Xylose Arabinose Formic Acetic HMF Furfural

Acid Acid

No 1.82 8.09 0.87 ^~024 0.91 0.05 0.09 enzyme

[00288] Maximum carbohydrate yields obtained from 5% enzyme dosing are shown below in Table 4 after the 48-hour hydrolysis, which both indicate nearly 100% conversion. Conversion efficiency for each trial was calculated using final concentrations (Table 2) divided by maximum concentration based on percent solids present, and is shown in Table 5.

[00289] Table 4. Maximum yields of washed corncobs (only pretreated once) in g/L.

Table 5. Conversion efficiency of cellulose and hemicellulose based on estimated solids hydrolyzed.

[00291] Direct comparisons of the maximum carbohydrate yields obtained from a one- stage pretreatment versus those obtained from a two-stage pretreatment are shown in Figure 5 and Table 6. Two-stage pretreatment concentrations were derived from the pretreatment using 3% acid and run for 15 minutes. One-stage pretreatment data was derived from a 10% solid solution and two-stage pretreatment data was derived from about a 4.7% solution with percent conversion based on solids loaded taken into account to make them comparable.

[00293] The conversion efficiency was calculated based on the initial percent solids in each flask individually. Those percent solids are in Table 5, and in Table 3.

[00294] Overall, the highest carbohydrate yields were seen out of the two-stage pretreated material at 3% acid for 15 minutes. Figure 5 represents results from both a one-stage

pretreatment and a two-stage pretreatment. The final carbohydrate yields were compared at each enzyme dosing. The two-stage carbohydrate yields exceeded the one-stage yields at all enzyme- dosing levels. The difference was not as significant at the 0.25%> enzyme dosing level, but it is clearly seen at 0.50%> and 1.0% enzyme dosing levels. It is also significant that the 0.50% dosing and 1.0% dosing yielded nearly the same amount of carbohydrates overall in a one-stage process indicating that maximum carbohydrate conversion was reached. An enzyme dosing reduced down to 0.50% loading based on solids is sufficient for a two-stage pretreated material, which showed about a 99% increase in final carbohydrate conversion than a one-stage pretreatment alone.

[00295] Table 4 shows maximum carbohydrate yields possible at 98%> carbohydrates out of the corncob material. The highest one-stage pretreatment recovered only 70% total

carbohydrates. The two-stage pretreatment showed recovery of 94% total carbohydrates with 3% acid pretreatment for 15 minutes in the second stage. The two-stage pretreatment showed a 35% increase in total conversion over the one-stage pretreatment, and also showed a possibility for enzyme reduction to 0.50% loading.

[00296] Similar to Figure 5, Figure 6 results were extrapolated out to 5.00% loading using the one-stage pretreated material. These results demonstrate the conversion of carbohydrates peaks at about 1.00% loading for a one-stage washed pretreated material. For a two-stage pretreated material, conversion peaks sooner at 0.50%, demonstrating enzyme loading can be severely reduced to achieve the same results as standard loading. Loading 0.50%> enzyme showed an average 94 % conversion of total carbohydrates available, and a 99% increase in carbohydrate conversion in two-stage compared to a one-stage pretreatment process.

[00297] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A two stage method of producing sugars from a biomass comprising:

a) adding the biomass to a first liquid at a hydration temperature to produce a hydrated biomass;

b) mechanical size reduction of the hydrated biomass to produce a mixture of size reduced solid particles;

c) heating the mixture of size reduced solid particles at a first hydrolysis temperature for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction;

d) heating the first solid fraction in an acidic medium comprising an acid at a second hydrolysis temperature for a second hydrolysis time to produce a mixture; and

e) hydrolyzing the mixture with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solid fraction.

2. The method of claim 1, wherein the first liquid is water.

3. The method of claim 1 or 2, wherein the C5 sugars of the first liquid fraction comprise soluble polysaccharides.

4. The method of claim 3, further comprising hydrolyzing the first liquid fraction with one or more hemicellulase enzymes.

5. The method of claim 1, wherein the first liquid comprises from about 0.01% to about 10% of an acid.

6. The method of claim 1, wherein the first liquid comprises from about 0.01% to about 5% of an acid.

7. The method of claim 1, wherein the first liquid comprises from about 0.01% to about 1% of an acid.

8. The method of claim 1, wherein the first liquid comprises from about 0.1% to about 0.5% of an acid.

9. The method of claim 1, wherein the first liquid comprises from about 0.1% to about 0.3% of an acid.

10. The method of claim 5, 6, 7, 8, or 9, wherein the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof.

11. The method of claim 5, 6, 7, 8, or 9, wherein the first liquid is derived from S0₂ gas.

12. The method of claim 5, 6, 7, 8, or 9, wherein the first liquid is derived from

H2SO4 gas.

13. The method of claim 5, 6, 7, 8, or 9, wherein the first liquid has a pH of from about 1.5 to about 3.5.

14. The method of claim 1, wherein the hydration temperature is from about 20 °C to about 110 °C.

15. The method of claim 1, wherein the hydration temperature is from about 35 °C to about 70 °C.

16. The method of claim 1, wherein the hydration temperature is from about 45 °C to about 55°C.

17. The method of claim 1, wherein the hydration temperature is about 50°C.

18. The method of claim 1, wherein the hydrated biomass comprises about 2% to about 12% solids (w/v).

19. The method of claim 1, wherein the hydrated biomass comprises about 5-6% solids (w/v).

20. The method of claim 1, wherein the hydrated biomass comprises about 10% to about 30%) solids (w/v).

21. The method of claim 18, 19, or 20, wherein the hydrated biomass is dewatered to about 30-32%) solids (w/v) prior to heating at the first hydrolysis temperature.

22. The method of claim 1, wherein at least 50% of the solid particles in the mixture of size reduced solid particles are less than 10 mm in a dimension.

23. The method of claim 1, wherein at least 50% of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension.

24. The method of claim 1, wherein at least 50% of the solid particles in the mixture of size reduced solid particles are less than 5 mm in a dimension.

25. The method of claim 1, wherein at least 50% of the solid particles in the mixture of size reduced solid particles are less than 2.5 mm in a dimension.

26. The method of claim 1, wherein at least 50% of the solid particles in the mixture of size reduced solid particles are less than 1.5 mm in a dimension.

27. The method of claim 1, wherein at least 50% the solid particles in the mixture of size reduced solid particles are from about 0.1 mm to about 1 mm in a dimension.

28. The method of claim 1, wherein all of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension.

29. The method of claim 1, wherein all of the solid particles in the mixture of size reduced solid particles are less than 1 mm in a dimension.

30. The method of claim 22, 23, 24, 25, 26, 27, 28, or 29, wherein the dimension is diameter or width.

31. The method of claim 1 , wherein the first hydrolysis temperature is about 125 °C to about 200 °C.

32. The method of claim 1, wherein the first hydrolysis temperature is about 150 °C to about 170 °C.

33. The method of claim 1, wherein heating the hydrated biomass is performed at a pressure of from about 100 psig to about 175 psig.

34. The method of claim 1, wherein the first hydrolysis time is from about 1 minute to about 120 minutes.

35. The method of claim 1, wherein the first hydrolysis time is from about 5 minutes to about 60 minutes.

36. The method of claim 1, wherein the first hydrolysis time is from about 20 minutes to about 40 minutes.

37. The method of claim 1, wherein the first hydrolysis time is from about 5 minutes to about 15 minutes.

38. The method of claim 1, wherein the first hydrolysis time is less than about 20 minutes.

39. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 120 °C.

40. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 130 °C.

41. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 140 °C.

42. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 150 °C.

43. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 160 °C.

44. The method of claim 1, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 170 °C.

45. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 120 °C.

46. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 130 °C.

47. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 140 °C.

48. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 150 °C.

49. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 160 °C.

50. The method of claim 1, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 170 °C.

51. The method of claim 1 , wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 180 °C.

52. The method of claim 1, wherein the first liquid fraction further comprises low levels of an inhibitor compound.

53. The method of claim 52, wherein the inhibitor compound is furfural,

hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

54. The method of claim 1, further comprising separating the first liquid fraction and the first solid fraction.

55. The method of claim 1, further comprising concentrating the first liquid fraction.

56. The method of claim 1, wherein the acidic medium is an acidic solution.

57. The method of claim 1, wherein the acidic medium comprises water.

58. The method of claim 1, wherein the second hydrolysis temperature is from about 175 °C to about 275 °C.

59. The method of claim 1, wherein the second hydrolysis temperature is from about 190 °C to about 240 °C.

60. The method of claim 1, wherein the second hydrolysis time is from about 1 minute to about 120 minutes.

61. The method of claim 1, wherein the second hydrolysis time is from about 1 minute to about 60 minutes.

62. The method of claim 1, wherein the second hydrolysis time is from about 5 minutes to about 15 minutes.

63. The method of claim 1, wherein the second hydrolysis time is at least about 5 minutes.

64. The method of claim 1, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 190°C.

65. The method of claim 1, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 200°C.

66. The method of claim 1, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 210°C.

67. The method of claim 1, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 220°C.

68. The method of claim 1, wherein the acidic medium comprises from about 0.1% to about 10% of the acid.

69. The method of claim 1, wherein the acidic medium comprises from about 0.1% to about 5%> of the acid.

70. The method of claim 1, wherein the acidic medium comprises from about 1% to about 3%) of the acid.

71. The method of claim 1, 68, 69 or 70, wherein the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof.

72. The method of claim 1, 68, 69 or 70, wherein the acidic medium is derived from S0₂ gas.

73. The method of claim 1, 68, 69 or 70, wherein the acidic medium is derived from H₂SC"4 gas.

74. The method of claim 1, wherein the second liquid fraction further comprises low levels of an inhibitor compound.

75. The method of claim 74, wherein the inhibitor compound is furfural,

hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

76. The method of claim 1, wherein the one or more cellulase enzymes are at from about 0.1%) to about 20%> based on total dry solids.

77. The method of claim 1, wherein the one or more cellulase enzymes are at from about 0.1%) to about 10%> based on total dry solids.

78. The method of claim 1, wherein the one or more cellulase enzymes are at from about 0.1%) to about 5%> based on total dry solids.

79. The method of claim 1, wherein the one or more cellulase enzymes are at from about 0.25%) to about 1%> based on total dry solids.

80. The method of claim 1, wherein the one or more cellulase enzymes are at about 0.5%) based on total dry solids.

81. The method of claim 1 , further comprising separating the second liquid fraction from the second solid fraction.

82. The method of claim 1 or 81, further comprising concentrating the second liquid fraction.

83. The method of claim 1 or 81, wherein the first liquid fraction is combined with the second liquid fraction.

84. The method of claim 1, wherein the C5 sugars comprise xylose, arabinose, or a combination thereof.

85. The method of claim 1, wherein the C6 sugars comprise glucose.

86. The method of claim 1, wherein the biomass comprises cellulose, hemicellulose, or lignocellulose.

87. The method of claim 1 or 86, wherein the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

88. The method of claim 1 or 2, further comprising removing starch from the biomass prior to heating the hydrated biomass at the first hydrolysis temperature.

89. The method of claim 88, wherein removing starch from the biomass comprises heating the hydrated biomass at greater than 100 °C.

90. The method of claim 88, wherein the starch is hydro lyzed by one or more enzymes to produce glucose monomers.

91. The method of claim 90, wherein the one or more enzymes comprise a-amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof.

92. The method of claim 89, wherein the starch is hydrolyzed by one or more enzymes to produce glucose monomers.

93. The method of claim 92, wherein the one or more enzymes comprise a-amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof.

94. The method of claim 90, wherein the glucose monomers are combined with the second liquid fraction.

95. The method of claim 92, wherein the glucose monomers are combined with the second liquid fraction.

96. The method of claim 1, wherein the yield of C5 or C6 sugars is at least about 80% of a theoretical maximum.

97. The method of claim 1, wherein the yield of C5 sugars or C6 sugars is at least about 90% of a theoretical maximum.

98. A composition comprising the C5 sugars produced by the method of claim 1.

99. A composition comprising the C6 sugars produced by the method of claim 1.

100. A composition comprising the C5 sugars and the C6 sugars produced by the method of claim 1.

101. A system for two stage production of sugars from a biomass comprising:

a) a slurry mixer containing a first liquid at a hydration temperature;

b) a rotary feeder that adds the biomass to the first liquid;

c) a dewatering chamber that removes liquid from the biomass; d) a cutter pump that reduces the particle size of the biomass and pumps the biomass from the slurry mixer to the dewatering chamber;

e) a microreactor that further reduces the particle size of the biomass to produce a mixture of size reduced solid particles;

f) a hemicellulose reactor where the mixture of size reduced solid particles is heated at a first hydrolysis temperature and a first hydrolysis pressure for a first hydrolysis time to produce a first liquid fraction comprising C5 sugars, and a first solid fraction;

g) a first flash tank for reducing temperature and pressure of the first liquid fraction and the first solid fraction;

h) a first separator to separate the first liquid fraction from the first solid fraction;

i) a retention module mixer that mixes the first solids fraction with an acidic medium comprising an acid, and heats the first solids fraction at a second hydrolysis temperature for a second hydrolysis time to produce a mixture; and j) a first enzyme reactor, wherein the mixture is hydrolyzed with one or more cellulase enzymes to produce a second liquid fraction comprising C6 sugars, and a second solids fraction.

102. The system of claim 101, further comprising a second flash tank for reducing temperature and pressure of the mixture.

103. The system of claim 101, further comprising a second separator to separate the second liquid fraction from the second solid fraction.

104. The system of claim 101, wherein the first liquid is water.

105. The system of claim 101 or 104, wherein the C5 sugars of the first liquid fraction comprise soluble polysaccharides.

106. The system of claim 105, further comprising a second enzyme reactor, wherein the first liquid fraction is hydrolyzed with one or more hemicellulase enzymes.

107. The system of claim 101, wherein the first liquid comprises from about 0.01% to about 10% of an acid.

108. The system of claim 101, wherein the first liquid comprises from about 0.01% to about 5% of an acid.

109. The system of claim 101, wherein the first liquid comprises from about 0.01% to about 1% of an acid.

110. The system of claim 101, wherein the first liquid comprises from about 0.1% to about 0.5% of an acid.

111. The system of claim 101, wherein the first liquid comprises from about 0.1 % to about 0.3% of an acid.

112. The system of claim 107, 108, 109, 110, or 111, wherein the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof.

113. The system of claim 107, 108, 109, 110, or 111, wherein the first liquid is derived from S0₂ gas.

114. The system of claim 107, 108, 109, 110, or 111, wherein the first liquid is derived from H₂SO₄ gas.

115. The system of claim 107, 108, 109, 110, or 111, wherein the first liquid has a pH of from about 1.5 to about 3.5.

116. The system of claim 101, wherein the hydration temperature is from about 20 °C to about 110 °C.

117. The system of claim 101, wherein the hydration temperature is from about 35 °C to about 70 °C.

118. The system of claim 101, wherein the hydration temperature is from about 45 °C to about 55°C.

119. The system of claim 101, wherein the hydration temperature is about 50°C.

120. The system of claim 101, wherein the biomass is added to the first liquid at about 2% to about 12% solids (w/v).

121. The system of claim 101, wherein the biomass is added to the first liquid at about 5-6% solids (w/v).

122. The system of claim 101, wherein the dewatering chamber comprises one or more screw-type rotors.

123. The system of claim 101, wherein the biomass is dewatered in the dewatering chamber to about 30-32%> solids (w/v).

124. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are less than 10 mm in a dimension.

125. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension.

126. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are less than 5 mm in a dimension.

127. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are less than 2.5 mm in a dimension.

128. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are less than 1.5 mm in a dimension.

129. The system of claim 101, wherein at least 50% the solid particles in the mixture of size reduced solid particles are from about 0.1 mm to about 1 mm in a dimension.

130. The system of claim 101, wherein all of the solid particles in the mixture of size reduced solid particles are less than 7.5 mm in a dimension.

131. The system of claim 101, wherein all of the solid particles in the mixture of size reduced solid particles are less than 1 mm in a dimension.

132. The system of claim 124, 125, 126, 127, 128, 129, 130, or 131, wherein the dimension is diameter or width.

133. The system of claim 101, wherein the hemicellulose reactor is a double-jacketed, screw type retention module.

134. The system of claim 101, wherein the first hydrolysis temperature is about 125 °C to about 200 °C.

135. The system of claim 101, wherein the first hydrolysis temperature is about 150 °C to about 170 °C.

136. The system of claim 101, wherein first hydrolysis pressure is from about 100 psig to about 175 psig.

137. The system of claim 101, wherein the first hydrolysis time is from about 1 minute to about 120 minutes.

138. The system of claim 101, wherein the first hydrolysis time is from about 5 minutes to about 60 minutes.

139. The system of claim 101, wherein the first hydrolysis time is from about 20 minutes to about 40 minutes.

140. The system of claim 101, wherein the first hydrolysis time is from about 5 minutes to about 15 minutes.

141. The system of claim 101, wherein the first hydrolysis time is less than about 20 minutes.

142. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 120 °C.

143. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 130 °C.

144. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 140 °C.

145. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 150 °C.

146. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 160 °C.

147. The system of claim 101, wherein the first hydrolysis time is about 5 minutes and the first hydrolysis temperature is about 170 °C.

148. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 120 °C.

149. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 130 °C.

150. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 140 °C.

151. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 150 °C.

152. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 160 °C.

153. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 170 °C.

154. The system of claim 101, wherein the first hydrolysis time is about 15 minutes and the first hydrolysis temperature is about 180 °C.

155. The system of claim 101, wherein the first liquid fraction further comprises low levels of an inhibitor compound.

156. The system of claim 155, wherein the inhibitor compound is furfural, hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

157. The system of claim 101, wherein the acidic medium is an acidic solution.

158. The system of claim 101, wherein the acidic medium comprises water.

159. The system of claim 101, wherein the second hydrolysis temperature is from about 175 °C to about 275 °C.

160. The system of claim 101, wherein the second hydrolysis temperature is from about 190 °C to about 240 °C.

161. The system of claim 101, wherein the second hydrolysis time is from about 1 minute to about 120 minutes.

162. The system of claim 101, wherein the second hydrolysis time is from about 1 minute to about 60 minutes.

163. The system of claim 101, wherein the second hydrolysis time is from about 5 minutes to about 15 minutes.

164. The system of claim 101, wherein the second hydrolysis time is at least about 5 minutes.

165. The system of claim 101, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 190°C.

166. The system of claim 101, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 200°C.

167. The system of claim 101, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 210°C.

168. The system of claim 101, wherein the second hydrolysis time is at least about 5 minutes and the second hydrolysis temperature is about 220°C.

169. The system of claim 101, wherein the acidic medium comprises from about 0.1% to about 10 % of the acid.

170. The system of claim 101, wherein the acidic medium comprises from about 0.1% to about 5 % of the acid.

171. The system of claim 101, wherein the acidic medium comprises from about 1% to about 3%) of the acid.

172. The system of claim 101, 169, 170, or 171, wherein the acid is sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid or a combination thereof.

173. The system of claim 101, 169, 170, or 171, wherein the acidic medium is derived from SC"₂ gas.

174. The system of claim 101, 169, 170, or 171, wherein the acidic medium is derived from H2SO4 gas.

175. The system of claim 101, wherein the second liquid fraction further comprises low levels of an inhibitor compound.

176. The system of claim 175, wherein the inhibitor compound is furfural,

hydroxymethylfurfural (HMF), formic acid, acetic acid, or a combination thereof.

177. The system of claim 101, wherein the one or more cellulase enzymes are at from about 0.1% to about 20% based on total dry solids.

178. The system of claim 101, wherein the one or more cellulase enzymes are at from about 0.1%) to about 10%> based on total dry solids.

179. The system of claim 101, wherein the one or more cellulase enzymes are at from about 0.1%) to about 5%> based on total dry solids.

180. The system of claim 101, wherein the one or more cellulase enzymes are at from about 0.25%) to about 1%₀ based on total dry solids.

181. The system of claim 101, wherein the one or more cellulase enzymes are at about 0.5%) based on total dry solids.

182. The system of claim 101, wherein the C5 sugars comprise xylose, arabinose, or a combination thereof.

183. The system of claim 101, wherein the C6 sugars comprise glucose.

184. The system of claim 101, wherein the biomass comprises cellulose,

hemicellulose, or lignocellulose.

185. The system of claim 101 or 184, wherein the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

186. The system of claim 101 or 104, further comprising a microreactor for hydrolyzing starch with one or more enzymes to produce glucose monomers.

187. The system of claim 186, wherein the hydration temperature is greater than 100 °C to remove starch from the biomass.

188. The system of claim 186, wherein the one or more enzymes comprise a-amylase, β-amylase, glucoamylase, pullulinase, or a combination thereof.

189. The system of claim 186, further comprising a separator to remove the glucose monomers from the biomass.

190. A two stage method of producing sugars from a biomass comprising:

a) reducing the size of the biomass to smaller particles;

b) adding a 0.01-0.5 %> acid solution to the biomass to produce a slurry of 10- 30% w/v solids; c) treating the slurry for less than 20 minutes at 120-180°C to produce a first liquid fraction containing C5 sugars and a first solid fraction;

d) separating and neutralizing the first liquid fraction;

e) further treating the first solid fraction with a temperature greater than 190°C for greater than 5 minutes in a 0.5-10% acid solution to produce a mixture; f) neutralizing the mixture;

g) hydrolyzing the mixture with at least one cellulase enzymes to produce a second liquid fraction and a second solid fraction; and

h) separating the second liquid fraction from the second solid fraction.

191. The method of claim 190, wherein the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

192. The method of claim 190, wherein the smaller particles are less than 10 mm in a dimension.

193. The method of claim 190, wherein the smaller particles are less than 5 mm in a dimension.

194. The method of claim 190, wherein the smaller particles are less than 2 mm in a dimension.

195. The method of claim 190, wherein the smaller particles are less than 1 mm in a dimension.

196. The method of claim 190, wherein the smaller particles are less than 0.2 mm in a dimension.

197. The method of claim 190, wherein the smaller particles are uniform in size.

198. The method of claim 190, wherein the step c is carried out at a temperature of 120°C for 5 minutes.

199. The method of claim 190, wherein the step c is carried out at a temperature of 130°C for 5 minutes.

200. The method of claim 190, wherein the step c is carried out at a temperature of 140°C for 5 minutes.

201. The method of claim 190, wherein the step c is carried out at a temperature of 150°C for 5 minutes.

202. The method of claim 190, wherein the step c is carried out at a temperature of

170°C for 5 minutes.

203. The method of claim 190, wherein the step c is carried out at a temperature of 180°C for 5 minutes.

204. The method of claim 190, wherein step e is carried out at a temperature of 190°C for greater than 5 minutes.

205. The method of claim 190, wherein step e is carried out at a temperature of 200°C for greater than 5 minutes.

206. The method of claim 190, wherein step e is carried out at a temperature of 210°C for greater than 5 minutes.

207. The method of claim 190, wherein step e is carried out at a temperature of 220°C for greater than 5 minutes.

208. The method of claim 190, wherein the acid solution of step b is derived from S0₂ gas.

209. The method of claim 190, wherein the acid solution of step b is derived from H₂SC"4 gas.

210. The method of claim 190, wherein the acid solution for step e is derived from S0₂ gas.

211. The method of claim 190, wherein the acid solution for step e is derived from H₂SC"4 gas.

212. The method of claim 208 or 209, wherein the acid solution is 0.1-0.3% w/v.

213. The method of claim 210 or 211 , wherein the acid solution is 1-3% w/v.

214. The method of claim 190, wherein the acid solution in step b is selected from the group consisting of: sulfurous acid, sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid and combinations thereof.

215. The method of claim 190, wherein the acid solution in step e is selected from the group consisting of: sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid and combinations thereof.

216. The method of claim 190, wherein the first liquid fraction of (d) is combined with the second liquid fraction of (h).

217. A two stage method of producing sugars from a biomass comprising:

a) reducing the size of the biomass to smaller particles;

b) adding water to the biomass to produce a slurry of 10-30%) w/v solids;

c) treating the 10-30% biomass (w/v) with water for no more than 20 minutes at

120-180°C to produce a first liquid fraction containing C5 sugars and a first solid fraction; d) removing and concentrating the first liquid fraction;

e) hydrolyzing the first liquid fraction with at least one hemicellulase enzymes; f) treating the first solid fraction with a temperature greater than 190°C for greater than 5 minutes in a 0.5-10% acid solution to produce a mixture;

g) neutralizing the mixture;

h) hydrolyzing the mixture with cellulase enzymes to produce a second liquid fraction and a second solid fraction; and

i) separating the second liquid fraction from the second solid fraction.

218. The method of claim 217, wherein the biomass comprises corn, corn syrup, corn stover, corn cobs, molasses, silage, grass, straw, grain hulls, bagasse, distiller's grains, distiller's dried solubles, distiller's dried grains, condensed distiller's solubles, distiller's wet grains, distiller's dried grains with solubles, wood, bark, sawdust, paper, poplars, willows, switchgrass, alfalfa, prairie bluestem, algae, fruit peels, pits, sorghum, sweet sorghum, sugar cane, switch grass, rice, rice straw, rice hulls, wheat, wheat straw, barley, barley straw, bamboo, seeds, seed hulls, oats, oat hulls, food waste, municipal sewage waste, or a combination thereof.

219. The method of claim 217, wherein the smaller particles are less than 10 mm in a dimension.

220. The method of claim 217, wherein the smaller particles are less than 5 mm in a dimension.

221. The method of claim 217, wherein the smaller particles are less than 2 mm in a dimension.

222. The method of claim 217, wherein the smaller particles are less than 1 mm in a dimension.

223. The method of claim 217, wherein the smaller particles are less than 0.2 mm in a dimension.

224. The method of claim 217, wherein the smaller particles are uniform in size.

225. The method of claim 217, wherein the step c is carried out at a temperature of 120°C for 5 minutes.

226. The method of claim 217, wherein the step c is carried out at a temperature of 130°C for 5 minutes.

227. The method of claim 217, wherein the step c is carried out at a temperature of 140°C for 5 minutes.

228. The method of claim 217, wherein the step c is carried out at a temperature of 150°C for 5 minutes.

229. The method of claim 217, wherein the step c is carried out at a temperature of

170°C for 5 minutes.

230. The method of claim 217, wherein the step c is carried out at a temperature of 180°C for 5 minutes.

231. The method of claim 217, wherein step f is carried out at a temperature of 190°C for greater than 5 minutes.

232. The method of claim 217, wherein step f is carried out at a temperature of 200°C for greater than 5 minutes.

233. The method of claim 217, wherein step f is carried out at a temperature of 210°C for greater than 5 minutes.

234. The method of claim 217, wherein step f is carried out at a temperature of 220°C for greater than 5 minutes.

235. The method of claim 217, wherein the temperature for step c is 160°C for 15 minutes.

236. The method of claim 217, wherein the temperature for step c is 170°C for 15 minutes.

237. The method of claim 217, wherein the acid solution for step f is derived from S0₂ gas.

238. The method of claim 217, wherein the acid solution for step f is derived from H₂SC"4 gas.

239. The method of claim 237or 238, wherein the acid solution is 1-3% w/v.

240. The method of claim 217, wherein the acid solution in step f is selected from the group consisting of: sulfuric acid, formic acid, hydrochloric acid, acetic acid, carbonic acid, oxalic acid and combinations thereof.

241. The method of claim 217, wherein the first liquid fraction of (d) is combined with the second liquid fraction of (i).

242. The method of either of claims 190 or 217, wherein starch is removed from a biomass prior to step (c).

243. The method of claim 242, wherein the starch is hydro lyzed to glucose monomers by enzymatic digestion.

244. The method of claim 243, wherein the enzymatic digestion is carried out with enzymes selected from the group consisting of a-amylase, β-amylase, glucoamylase, pullulinase, and a combination thereof.

245. The method of claim 244, wherein the glucose monomers is combined with the second liquid fraction of step (i).