ADDITION OF RAW SUGAR JUICE

Field of the invention

The present inventions relates to biotechnology, in particular to methods pertaining to the production of bioproducts - such as alcohols, fermentable sugars and/or fermentation products in general - from plant biomass, including plant primary sugars, such as fructose and sucrose, as well as from monomeric sugars derived from cellulose and other polysaccharides, such as glucose and xylan. In particular, the current invention concerns a method and products related to the production of bioproducts such as alcohol from biomass, said method comprising the integrated conversion of primary and secondary lignocellulosic sugars.

Background of the invention

Sugar cane and sweet sorghum processing to produce ethanol is typically conducted in a similar manner: Fresh canes are pressed to produce a sugar- rich juice, also called "raw juice", which is typically concentrated and effectively sterilized by evaporative processes, then directly fermented to ethanol.

Sorghum further comprises starch-rich seeds, which are typically subject to hydrolysis using amylase and glucoamylase enzymes for instance to produce a fermentable solution. Residual lignocellulosic materials in both cases such as bagasse, straw and leaves have been used typically as a fuel for steam (heat and power) generation. In recent years, there was considerable interest for the integrated processing schemes, whereby whole-crop sugar cane and sweet sorghum can be utilized in ethanol production. Integration requires conversion not only of primary sugars such as sucrose, glucose and fructose in pressed juice, through so- called "first generation (1 G)" processes, but also conversion of cellulosic sugars, and eventually also hemicellulosic sugars, through "second generation (2G)" technologies. Lignocellulosic 2G sugars are typically obtained through a process whereby bagasse, straw and/or leaves are first pretreated and then subject to enzymatic hydrolysis using a cellulase based enzyme preparation. Because of limitations of its physical structure, lignocellulosic biomass cannot be effectively converted to fermentable sugars by enzymatic hydrolysis without some pretreatment process. A wide variety of different pretreatment schemes have been reported, each offering different advantages and disadvantages. For review see Agbor et al. (201 1 ); Girio et al. (2010) ; Alvira et al. (2010) ;

Taherzadeh and Karimi (2008). From a sustainability perspective, hydrothermal pretreatments are especially attractive. These processes utilize pressurized steam/liquid hot water at temperatures in the order of 1 60 - 230°C to gently melt hydrophobic lignin that is intricately associated with cellulose strands, to solubilize a major part of the hemicellulose, rich in five carbon (C5) sugars, and to disrupt cellulose strands so as to improve accessibility to productive enzyme bindings. Hydrothermal pretreatments can be conveniently integrated with existing coal- and biomass-combustion electrical power generation plants to efficiently utilize turbine steam and power production capacity. WO2015/120859 concerns methods and products related to the production of alcohol from sugar cane or sweet sorghum with integration of 1 st and 2nd generation (1 G/2G) biorefining, thus comprising the integrated conversion of primary and secondary soft lignocellulosic biomass. A number of schemes and process arrangements have been reported for optimizing ethanol production from sugar cane by integration of 1 G and 2G processes. See e.g. (for sugar cane) Dias et al. (2013) ; Palacios-Bereche et al. (2013); Macrelli et al. (2012) ; Dias et al. (201 2); Dias et al. (201 1 ); Walter et al. (2010) and (for sorghum) Kim et al. (201 2); Ceclan et al. (2012). In all such integrated processing schemes reported to date, overall conservation of process steam is considered to be a critical factor. Steam is typically used to power processes such as evaporative concentrators, ethanol distillation systems, hydrothermal pretreatment systems, and other systems. The more process steam can be conserved, the more lignocellulosic material can be utilized for ethanol production, rather than for steam generation.

Two recent studies considered a variety of different process configurations in optimizing integrated 1 G/2G ethanol production from sugar cane, Dias et al. (2013) and Palacios-Bereche et al. (2013). One process variable considered in detail was the conditions under which enzymatic hydrolysis of pretreated bagasse was conducted. It is well known in the art that higher monomeric sugar yields can be obtained at any given enzyme dose where enzymatic hydrolysis is conducted at low total solids concentration. To the extent that the concentration of pretreated biomass total solids in the hydrolysis slurry is greater than about 5% by weight, the greater the biomass concentration, the lower will be the conversion yield at any given enzyme dose. Both Dias et al. (2013) and Palacios-Bereche et al. (2013) concluded that, despite higher yields at the enzymatic hydrolysis stage, low solids hydrolysis was disadvantageous overall because a greater quantity of steam is required to concentrate the resulting cellulosic sugar stream prior to fermentation, compared with hydrolysis at higher solids content, which results in higher sugar concentrations and reduced requirement for evaporative concentration. In alternative process scenarios, where the lignocellulosic hydrolysate is directly fermented without evaporative concentration, and without blending in to the 1 G sugar process stream, higher sugar concentrations in the hydrolysate are advantageous because this, in turn, results in higher ethanol concentration in the eventual fermentation broth. Higher ethanol concentrations in fermentation broth leads to lower steam consumption in distillation (ethanol recovery). Lower steam consumption directly correlates with higher ethanol production, where lignocellulosic bagasse is alternatively used to produce steam or lignocellulosic sugars for fermentation.

Summary of the invention

The current invention concerns methods and products related to the integration of bioproducts-production, such as 2G bioethanol production with e.g. sugar production, fruit- or vegetable juice production, and/or 1 G bioethanol production.

Surprisingly and unexpectedly, the inventors have realized that a carbohydrate comprising aqueous fraction can be added to pretreated lignocellulosic biomass prior to enzymatic hydrolysis without negative impact on the hydrolysis yields.

Thus, in a first aspect, the current invention concerns a method or process for the production of a bioproduct, comprising the steps of: (a) Pretreating a lignocellulosic biomass;

(b) Providing a raw juice comprising carbohydrates such as sucrose; said raw juice optionally comprising undissolved lignocellulosic plant material ;

(c) Diluting the pretreated biomass from step (a) with some quantity of the raw juice from step (b);

(d) Hydrolysing the diluted biomass from step (c) by enzymatic hydrolysis using a cellulase and/or xylanase comprising enzyme preparation.

In particular, the first aspect relates to methods/processes, wherein the lignocellulosic biomass and/or the raw juice is not from sugar cane or sweet sorghum ; and/or wherein the lignocellulosic biomass and the raw juice are from a different plant. Thus, the lignocellulosic biomass can e.g. be derived from sugar cane, while the raw juice is derived from sweet sorghum, and vice versa. According to some embodiments, when the raw juice is from sugar cane or sweet sorghum, the lignocellulosic biomass is from another plant, such as derived from sugar beet. Likewise, when the lignocellulosic biomass is from sugar cane or sweet sorghum, the raw juice is from another plant, such as derive from sugar beet. While in some embodiments, the bioproduct is an alcohol, such as EtOH, in some embodiments the bioproduct is not EtOH, and can e.g. be one or more of primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof. In some embodiments, the bioproduct is a fermentation product, and the above method comprises a fermentation step (e), wherein the diluted and hydrolysed biomass from step (d) is fermented to provide a fermentation product, such as e.g. EtOH. In some embodiments, the raw juice provided in step (b) is a liquid stream from sugar production, fruit juice production or 1 ^st generation bioethanol production.

A second aspect pertains to one or more bioproducts, including fermentation product(s), hydrolysates, dissolved solids, mixtures or dissolved solids and raw juice, as well as intermediary products, said product(s) being obtained or obtainable by a method or process according to the first aspect of the invention. Such bioproducts may include fuel or fuel additives for generation of power, heat and/or steam. In some embodiments, the bioproduct is an alcohol, such as EtOH.

In some embodiments, a further product is provided, said further product comprising 0.1 -99.9% weight/weight or volume/volume one or more of the bio- or fermentationproduct according to the second aspect of the present invention. Short description of the drawings

Figure 1 : Glucan conversion as function of time for the six shake flasks.

Conditions: 170 h hydrolysis at 50 °C with 0.1 6 mL (10.4 FPU) AcTrio/g glucan, 12 % TS (total (suspended) solids), pH 4.7 - 5.2 adjusted with Ca(OH) ₂ . Figure 2: Average glucan conversion after 145 h over % of added 1 G sugar juice (raw juice). Conditions: 145 h hydrolysis at 50 °C with 0.1 6 mL (10.4 FPU) AcTrio/g glucan, 12 % TS (total (suspended) solids), pH 4.7 - 5.2 adjusted with Ca(OH) ₂ .

Figure 3: Increase in glucose concentration in pre-treated wheat straw fiber fractions after 20 h hydrolysis. Conditions: 50 °C with 0.075 g CTec3/g glucan, 10 % TS, pH 4.7-5.2 adjusted with NaOH.

Figure 4: Increase in xylose concentration in pre-treated wheat straw fiber fractions after 20 h hydrolysis. Conditions: 50 °C with 0,075 g CTec3/g glucan, 10 % TS, pH 4.7-5.2 adjusted with NaOH.

Detailed description of the invention

Definitions

The term "dry matter (D %)" as used herein refers to total solids (dissolved and undissolved) expressed as weight %, unless indicated otherwise.

"Autohydrolysis" refers to a pretreatment process wherein it is believed that acetic acid liberated by hemicellulose hydrolysis during pretreatment further catalyzes hemicellulose hydrolysis. This may apply to any hydrothermal pretreatment of lignocellulosic biomass, usually conducted at pH between 3.5 and 9.0.

In the context of the present invention, the term "bioproduct" is meant to comprise one or more of: alcohol, EtOH, sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combinations thereof.

"Plant" refers mainly multicellular eukaryotes of the kingdom Plantae, in particular to plant families or species. Expressions like "lignocellulosic biomass and/or raw juice being from a different plant" are meant to be interpreted as not only being from a different individual plant, but lignocellulosic biomass and/or raw juice being derived from different plant taxons, such as different plant families or species, e.g. sugar beets, sugar cane, or sweet sorghum. In another context, "plant" may also refer to a production facility, such as an electrical power generation plant. In the context of the present invention, the term "raw juice" is meant not to be limited to raw juice from sugar production, such as from sugar cane, where fresh canes are pressed to produce a sugar rich juice, and the residual lignocellulosic material is called "bagasse". Thus, the term "raw juice" is to be interpreted broader, comprising in its broadest definition any fermentable sugar comprising aqueous fluid, wherein said fermentable sugar is directly or indirectly derived from plant biomass. This includes any liquid/pumpable streams from e.g. sugar production from e.g. sugar beet, sugar cane, or sweet sorghum; fruit or vegetable juice production; or 1 G ethanol production. The "raw juice" according to the present invention may comprise insoluble components, such as fibres.

As used herein the term "whole slurry" refers to an enzymatic hydrolysis reaction mixture in which the ratio by weight of undissolved to dissolved solids at the start of enzymatic hydrolysis is less than 2.2:1 .

In the context of the present invention, the term "cellulase" is meant to comprise enzyme compositions that hydrolyse cellulose (beta-1 , 4-D-glucan linkages) and/or derivatives thereof. Cellulases include the classification of exo- cellobiohydrolases (CBH), endoglucanases (EG) and beta-glucosidases (BG) (EC3.2.1 91 , EC3.2.1 .4 and EC3.2.1 .21 ). Examples of cellulases include cellulases from e.g. Penicillium, Trichoderma, Humicola, Fusarium,

Thermomonospora, Cellulomonas, Clostridium and Aspergillus. Suitable cellulases are commercially available and known in the art. Commercial cellulase preparations may comprise one or more further enzymatic activities. Furthermore, "cellulase" can also be used interchangeably with "cell-wall modifying enzyme", referring to any enzyme capable of hydrolysing or modifying the complex matrix polysaccharides of the plant cell wall, such as any enzyme that will have activity in the "cell wall solubilisation assay" as e.g. described in W0101 1 5754, which is herewith included by reference. Included within this definition of "cell-wall modifying enzyme" are cellulases, such as

cellobiohydrolase I and cellobiohydrolase II, endo-glucanases and beta- glucosidases, xyloglucanases and hemicellulolytic enzymes, such as xylanases. Commercially available cellulase preparation(s) suitable in the present context are often optimized for lignocellulosic biomass conversion and may comprise a mixture of enzyme activities that is sufficient to provide enzymatic hydrolysis of pretreated lignocellulosic biomass, often comprising endocellulase

(endoglucanase), exocellulase (exoglucanase), endoxylanase, xylosidase and B-glucosidase activities. The term "optimized for lignocellulosic biomass conversion" refers to a product development process in which enzyme mixtures have been selected and/or modified for the specific purpose of improving hydrolysis yields and/or reducing enzyme consumption in hydrolysis of pretreated lignocellulosic biomass to fermentable sugars. In the context of the present invention, the term "glucan" is meant to comprise cellulose as well as other gluco-oligomers and other gluco- polymers. Such oligo- or polysaccharides consist of glucose monomers, linked by glycosidic bonds. "Hydrothermal pretreatment" commonly refers to the use of water, either as hot liquid, vapor steam or pressurized steam comprising high temperature liquid or steam or both, to "cook" biomass, at temperatures of 120 degrees centigrade or higher, either with or without addition of acids or other chemicals. "Solid/liquid separation" -related terms refer to an active mechanical process, whereby liquid is separated from solid by application of force through pressing, centrifugal or other force, whereby "solid" and "liquid" fractions are provided. The separated liquid is collectively referred to as "liquid fraction." The residual fraction comprising considerable insoluble solid content is referred to as "solid fraction." A "solid fraction" will have a dry matter content and will typically also comprise some residual of "liquid fraction."

The term "lignocellulosic biomass" is meant to comprise any biomass obtained, obtainable or derived from a lignin comprising plant, such as annual or a perennial plants, such as one or more of: cereal, wheat, wheat straw, rice, rice straw, corn, corn fiber, corn cobs, corn stover, hardwood bulk, softwood bulk, sugar cane, sweet sorghum, bagasse, nut shells, empty fruit bunches, grass, straw, cotton seed hairs, barley, rye, oats, sorghum, brewer's spent grains, palm waste material, wood, soft lignocellulosic biomass, algae, and any combination thereof.

In the context of the present invention, the term "soft lignocellulosic biomass" indicates non-wood biomass. The terms "about", "around", "approximately", or "~" indicate e.g. the measuring uncertainty commonly experienced in the art, which can be in the order of magnitude of e.g. +/- 1 , 2, 5, 10, 20, or even 50 percent (%), usually +/- 10%.

The term "comprising" is to be interpreted as specifying the presence of the stated parts, steps, features, or components, but does not exclude the presence of one or more additional parts, steps, features, or components. For example, a composition comprising a chemical compound may thus comprise additional chemical compounds. Further definitions may be found elsewhere in this document.

The inventors discovered that cellulase enzyme preparations are

comparatively uninhibited in an environment comprising a high percentage of raw juice, such as raw juice from sugar cane or sweet sorghum. It is also believed that the same is true for other raw juices, such as sugar beet or fruit juices. As a consequence, enzymatic hydrolysis using these enzyme preparations can be advantageously conducted at lower solid content where the hydrolysis mixture is supplemented with raw juice, instead of fresh water or recycled process water. The resulting hydrolysate comprises higher sugar concentration, combining other sugars than 2G sugars, such as 1 G sugars with 2G sugars, and thereby permits a combined ethanol fermentation that will reach levels of ethanol in weight % that are advantageously high in terms of distillation (ethanol recovery) costs. It is believed that, mutatis mutandis, similar advantages of e.g. reduced water needs and/or reduced product

purification/recovery/processing costs and can be obtained for other bioproducts obtained or obtainable according to the present invention, thus not limited to ethanol or other alcohols, and also not limited to bioproducts requiring a fermentation step for their provision. Table 1 shows an accounting of expected final ethanol concentration in fermentation of hydrolysate, where sugar cane bagasse has been subject to hydrothermal pretreatment and hydrolysed at various different levels of dry matter (total solids) % to equivalent conversion. Shown are values of expected ethanol in weight % where the hydrolysate is dilute using a mixture comprising 90% water, 1 0% cane juice, or 70% water and 30% cane juice, or 50% water and 50% cane juice. Also shown are expected ratios of enzyme consumption at the various levels of dry matter, in the absence of cane juice

supplementation. Table 1. Final ethanol concentration and relative enzyme consumption as a function of DM% and % cane juice supplementation.

Hydrolysis Relative enzyme Final ethanol wt %

DM% dose w/out juice 0% juice 10% juice 30% juice 50% juice

18 1 .000 4.63 4.91 5.47 6.03

17 0.970 4.37 4.65 5.21 5.77

1 6 0.936 4.1 1 4.39 4.95 5.51

15 0.903 3.85 4.13 4.69 5.25

14 0.873 3.60 3.88 4.44 5.00

13 0.837 3.34 3.62 4.18 4.74

12 0.81 2 3.08 3.36 3.92 4.48

As shown, by using a mixture of cane juice and water as diluent in hydrolysis of pretreated bagasse, equivalent final ethanol concentrations in the fermentation broth can be achieved using a substantially reduced enzyme dose. Even where cane juice imparts some inhibition of enzyme activity, it can nevertheless be advantageous to supplement hydrolysis with cane juice diluent. For example, using a commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and provided by GENENCOR Tm under the tradename ACCELLERASE TRIO Tm, supplementation of diluent in hydrolysis at 12% DM is expected to impart a loss of glucan conversion of approximately 4% in absolute yield terms, relative to hydrolysis with pure water as diluent. Yet this 4% loss in glucan conversion is readily compensated for by an approximately 1 6-19% savings in enzyme dose, where the hydrolysis can be run at 12% DM compared with 18% DM which would normally be required to reach final ethanol yields of 4.63 weight %, in the absence of cane juice supplementation. It is well known in the art that distillation costs are exponentially increased at ethanol concentrations beneath 4.0 weight % and fall still sharply between 4.0% and 5.0%.

The precise amount of raw juice supplementation to be used is a variable to be optimized, in light of the degree of inhibition experienced in a raw juice environment by each given cellulase enzyme preparation, as well as the sugar/carbohydrate of the raw juice added, including economical concerns. In particular, this invention is seen to be relevant, when balancing the increased costs for extracting the remaining percentages of sugars, and/or fruit juice in sugar or fruit juice production versus the increased bioproduct yields, such as EtOH in 2G bioethanol production obtained according to the present invention. This provides a hitherto unprecedented flexibilities and alternatives.

First aspect

In a first aspect, the current invention concerns a method for the production of a bioproduct, said method comprising the steps of:

(a) Pretreating a lignocellulosic biomass, such as soft lignocellulosic biomass;

(b) Providing a raw juice comprising carbohydrates such as sucrose; said raw juice optionally comprising undissolved lignocellulosic plant material ;

(c) Diluting the pretreated biomass from step (a) with some quantity of the raw juice from step (b); (d) Hydrolysing the diluted biomass from step (c) by enzymatic hydrolysis using a cellulase and/or xylanase comprising enzyme preparation.

In some embodiments, the lignocellulosic biomass is soft lignocellulosic biomass. In other embodiments, the lignocellulosic biomass is not soft lignocellulosic biomass, such as wood.

In some embodiments, the raw juice comprises undissolved components or constituents, such as fibers and/or lignocellulosic plant materials. In some embodiments, the raw juice does not comprise undissolved components or constituents.

In some embodiments, the bioproduct is a fermentation product, and the above method comprises a fermentation step (e), wherein the diluted and hydrolysed biomass from step (d) is fermented to provide a fermentation product, such as an alcohol, e.g. EtOH.

In some embodiment, wherein the aqueous liquid phase of the hydrolysis mixture in step (d) comprises at least 5 g/L sucrose derived from the added raw juice, such as at least 1 0 g/L or more; such as 1 5 g/L or more; such as 20 g/L or more; such as, 25 g/L or more; such as 30 g/L or more; such as 40 g/L or more; such as 50 g/L or more; such as 60 g/L or more sucrose derived from the added raw juice.

In some embodiments, the initial dissolved sucrose from the added raw juice is between 5 and 60 g/L, and/or around 5, 1 0, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, or more than 120 g/L. g/L. In some embodiments, the initial dissolved sucrose from the added raw juice is in the range of 1 -120, 2.5-100, or 5-60 g/L.

In some embodiments, a pretreated biomass and/or a fiber fraction is

hydrolysed under conditions where initial dissolved sucrose from the added raw juice is between 5 and 60 g/L by weight, and/or around 5, 10, 1 5, 20, 25, 30, 40, 50, or 60 g/L.

In some embodiments, the pretreated biomass and/or fiber fraction is hydrolysed under conditions where initial dissolved sucrose from the added raw juice is between 5 and 60 g/L, 10 and 60 g/L, 15 and 60 g/L, 20 and 60 g/L, 25 and 60 g/L, 30 and 60 g/L, 40 and 60 g/L, or 50 and 60 g/L.

In some embodiments, the ratio of added raw juice to pretreated biomass in weight/weight (w/w) or volume/volume (v/v) in step (c) and/or (d) is in the range of 0.01 to 4, 0.1 to 3, or 0.5 to 2; and/or in the range of 0.01 -0.1 ; 0.1 -0.25; 0.25- 0.5; 0.5-1 .0; 1 .0-1 .5; 1 .5-2.0; 2.0-2.5; 2.5-3.0; 3.0-3.5; 3.5-4.0.

In some embodiments, the ratio of added raw juice to pretreated biomass in weight/weight (w/w) or volume/volume (v/v) in step (c) and/or (d) is at least 0.10:1 0 or more; such as 0.25:10 or more; such as 0.5 :1 0 or more; such as

0.75:1 0 or more; such as 1 :10 or more; such as 1 .5:10 or more; such as 2:10 or more; such as 2.5:10 or more; such as 3:10 or more; such as 3.5:10 or more; such as 4:1 0 or more; such as 4.5:1 0 or more; such as 5:10 or more; such as 5.5:10 or more; such as 6 :10 or more; such as 6.5:1 0 or more; such as 7:1 0 or more; such as 7.5:10 or more; such as 8:10 or more; such as 8.5:10 or more; such as 9:1 0 or more; such as 9.5:1 0 or more; such as 10:10 or more, such as such as 1 1 :10 or more, such as 12:1 0 or more, such as 1 3:10 or more, such as 14:1 0 or more, such as 1 5:10 or more, such as 1 6:1 0 or more, such as 1 7:10 or more, such as 1 8:10 or more, such as 19:1 0 or more, such as 20:10 or more, such as 21 :10 or more, such as 22:1 0 or more, such as 23:10 or more, such as 24:1 0 or more, such as 25:10 or more, such as 26:1 0 or more, such as 27:10 or more, such as 28:10 or more, such as 29:1 0 or more, such as 30:10 or more, such as 31 :10 or more, such as 32:1 0 or more, such as 33:10 or more, such as 34:1 0 or more, such as 36:10 or more, such as 37:1 0 or more, such as 38:10 or more, such as 39:10 or more, such as 40:1 0 or more.

In some embodiments, the raw juice provided in step (b) is a liquid stream from sugar production, such as sugar production from sugar beet, sugar cane or sweet sorghum. "Liquid stream" is meant to comprise a pumpable, fluid, thus said liquid stream may comprise non-soluble components/precipitates. The raw juice is usually an aqueous liquid. According to some embodiments, said non- soluble components may be biomass, nutrients or other factors that contribute positively in the final yield of the provision of the bioproduct according to the invention. The non-soluble components may also be or comprise undissolved lignocellulosic plant material such as fibers. In some embodiments, the raw juice provided in step (b) is a liquid stream from fruit juice production. In some embodiments, the raw juice provided in step (b) is a liquid stream from a 1 ^st generation bioethanol production, such as a 1 ^st generation bioethanol production based on starch comprising plant material such as tubers including e.g. potatoes and cassava), cereals (e.g. maize, barley, wheat, sorghum, rye, oat), sugar cane, sweet sorghum and/or sugar beet.

The lignocellulosic biomass may be subjected to different forms of

pretreatment. Thus, according to one embodiment, the pretreatment in step (a) can e.g. be a hydrothermal pretreatment and/or a autohydrolysis pretreatment.

Different chemicals can be added during pretreatment. According to one embodiment, the pretreatment in step (a) may comprise: (i) addition of a base such as ammonia (NH ₄ OH) or NaOH ; and/or (ii) addition of an acid such as H2SO. . In a preferred embodiment, said pretreatment in step (a) does not comprise (i) addition of a base such as NH ₄ OH or NaOH ; and/or (ii) addition of an acid such as H2SO4. Regardless of addition of chemicals or not, such as H2S0 ₄ and/or NH ₄ OH or NaOH, the pretreatment according to the invention may, or may not comprise a processing step known as steam explosion.

In some embodiments, the pretreated biomass is subjected to at least one solid/liquid separation step to provide a fiber fraction and a liquid fraction ; and optionally washing the fiber fraction as to remove dissolved solids, such as conducting said washing by a series of pressing and dilution steps, or other washing steps known in the art.

In some embodiments, the pretreated biomass and/or the fiber fraction is hydrolysed under conditions where initial undissolved solids are between 1 0% and 25%, 1 0% and 20%, or around 15% (w/w). In some embodiments, the pretreated biomass and/or fiber fraction is hydrolysed under conditions where pH is maintained at or around pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0 or lower. In some embodiments, the pretreated biomass and/or fiber fraction is hydrolysed under conditions where pH is maintained at +/- 0.1 -0.25 pH units around pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. In some embodiments, the pretreated biomass and/or fiber fraction is hydrolysed under conditions where pH is maintained in the range of pH 7-4, 7-5, 7-6, 6-4, 6-5, 5- 4; and/or wherein the pH is maintained lower than pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0. In some embodiments, hydrolysis is conducted at a constant or approximately constant pH.

In some embodiments, the pretreated biomass and/or fiber fraction is hydrolysed using a cellulase preparation optimized for lignocellulosic biomass conversion, such as a commercially available cellulase preparation.

In one embodiment, the pretreated biomass and/or fiber fraction is hydrolysed using a cellulase preparation that is not inhibited more than 20% after 145 hours hydrolysis at an enzyme loading of at least 8 FPU/g DM under conditions appropriate for the tested enzyme preparation by added raw juice where sucrose derived from the added juice is at least 5 g/L.

In some embodiments the pretreated biomass and/or fiber fraction is hydrolysed using a cellulase preparation that is not inhibited more than 10, 15, 20, 25, 30, 35, 40, 45, or 50%, after 24, 48, 72, 96, 120, or 145 hours hydrolysis at an enzyme loading of at least 8 FPU/g DM under conditions appropriate for the tested enzyme preparation by added raw juice where sucrose derived from the added juice or raw juice is at least 5, 1 0, 15 or 20 g/L.

In some embodiments, the hydrolysate obtained after hydrolysis of pretreated biomass and/or fiber fraction is subject to at least one solid/liquid separation step to provide insoluble solids separated from dissolved solids, such as by using a filter press with internal wash, optionally comprising a further step such as a drying step.

In some embodiments, the insoluble solids provided according to the first aspect are suitable as a fuel and/or fuel additive, such as fuel and/or fuel additive for generation of power, heat and/or steam. This may require one or more conventional processing steps known in the art, such as pressing, drying, and/or pelleting. In some embodiments, the dissolved solids comprising cellulosic sugars and sugars derived from the raw juice are mixed with a further quantity of raw juice, optionally followed by a concentration step, such as evaporative concentration and/or reverse osmosis concentration. Other conventional concentration steps or procedures may be used as well.

In some embodiments, the further quantity of raw juice added can e.g. be in the range of around 1 , 2, 5, 10, 1 5, 20, 30, 40, or 50% by weight or volume; and/or at least 1 , 2, 5, 1 0, 1 5, 20, 30, 40, or 50% by weight or volume.

In some embodiments relating to the first aspect of the invention, a method is provided, wherein (i) the hydrolysate obtained according to any one of the preceding embodiments; (ii) the dissolved solids obtained according to any one of the preceding embodiments; (iii) the mixture of dissolved solids and raw juice obtained according to any one of the preceding embodiments; (iv) the concentrated solution provided through the concentration step according to any one of the preceding embodiments; and/or any combination of (i), (ii), (iii) and/or (iv) is subsequently fermented to provide one or more product(s), optionally after concentration, purification or any other step(s), such as unit operations known in the art.

In some embodiments, the hydrolysate obtained according to the first aspect of the invention is subsequently fermented to provide one or more product(s), optionally followed and/or preceded by a concentration, purification or any other processing step(s).

In some embodiments, the dissolved solids are subsequently fermented to provide one or more product(s), optionally followed and/or preceded by a concentration, purification or any other step(s).

In some embodiments, a mixture of dissolved solids and raw juice is

subsequently fermented to provide one or more product(s), optionally followed and/or preceded by a concentration, purification or any other step(s).

In some embodiments any combination of hydrolysate, dissolved solids, including mixture of dissolved solids and raw juice, as well as any concentrated solution subsequently fermented to provide one or more product(s), optionally followed and/or preceded by a concentration, purification or any other step(s). In some embodiment, the bioproduct is a fermentation product, such as one or more chemical, alcohol, ethanol and/or any combination thereof. In some embodiments, the bio- and/or fermentation product is one or more of: EtOH, primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof.

Generally, hydrolysis, in particular enzymatic hydrolysis can be performed in different ways, and many different methods are known in the art. According to one embodiment, hydrolysis is performed as whole slurry. Thus, in some embodiments, the hydrolysis in step (d) is either performed as whole slurry, or a solid/liquid separation step is performed prior to hydrolysis so as to provide a fiber fraction and a liquid fraction. In a further embodiment, said fiber fraction is separately subject to enzymatic hydrolysis.

In an embodiment according to the first aspect of the invention, the raw juice is not derived from sugar cane and/or sweet sorghum. In another embodiment, the lignocellulosic biomass is not derived from sugar cane and/or sweet sorghum. In a further embodiment, when the raw juice and/or the lignocellulosic biomass is not derived from sugar cane and/or sweet sorghum, the fermentation product is not EtOH.

Thus, in some embodiments of the first aspect of the invention, (i) the raw juice is not derived from sugar cane or sweet sorghum; (ii) the lignocellulosic biomass is not derived from sugar cane or sweet sorghum, and/or (iii) the fermentation product is not EtOH, including any combination of (i), (ii) and/or (iii).

Thus, in some embodiment of the invention, the lignocellulosic biomass is not from sugar cane or sweet sorghum. According to another embodiment, the raw juice is not from sugar cane or sweet sorghum.

Furthermore, in some embodiments, the bio- and/or fermentation product is not an alcohol, such as EtOH.

The flexibility of the current invention allows for a wide range of lignocellulosic biomasses and/or raw juices to be processed. Thus, in one embodiment, the lignocellulosic biomass and the raw juice are or are derived from a different plant.

In some embodiments, the lignocellulosic biomass is derived from sugar cane, and the raw juice is derived from sweet sorghum or from another plant than sugar cane, such as e.g. sugar beet, a fruit plant, etc. In some embodiments, the lignocellulosic biomass is derived from sweet sorghum, and the raw juice is derived from sugar cane or from another plant than sugarcane, such as e.g. sugar beet, a fruit plant, etc.

Commonly, fermentation, especially fermentation in the field of ethanol production is performed using yeast, often Saccharomyces, such as

Saccharomyces cerevisiae. Alternatives are known in the art, especially when aiming at provision of other fermentation products than ethanol.

Second aspect

The second aspect of the current invention relates, in the broadest sense, to any products obtained, or being obtainable according to any one of the preceding embodiments of the first aspect of the invention, including any combination thereof. This may include one or more bioproducts, including fermentation product(s), hydrolysates, dissolved solids, mixtures or dissolved solids and raw juice, as well as intermediary products, said product(s) being obtained or obtainable by a process according to the first aspect of the invention, including fuel or fuel additives for generation of power, heat and/or steam. In some embodiments, the bioproduct is EtOH.

In one embodiment according to the second aspect, a bioproduct is provided, such as a fermentation product, provided according to any one of the methods of the preceding embodiments. In a preferred embodiment, the bioproduct is a bio- alcohol, such as bioethanol.

In some embodiments, the bio- and/or fermentation product is one or more of: alcohol, EtOH, primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof.

Further embodiments relate to one or more products comprising or consisting essentially of the hydrolysate, the dissolved solids, the mixture of dissolved solids and raw juice, and any concentrate (s) and/or solid fraction(s) provided as described herein, such as in the first aspect. This includes also any combination of any hydrolysate, dissolved solids, mixtures of dissolved solids and raw juice, and any concentrate(s) and/or solid fraction(s).

Further product-related embodiments pertain to a fuel or fuel additive, such as fuel and/or fuel additive for generation of power, heat and/or steam. These can be provided from the insoluble solids separated from dissolved solids, such as by using a filter press with internal wash, optionally comprising a further drying step, such as described herein. Such fuel or fuel additives are believed to be very suitable for power, heat and/or steam generation. In some embodiments, the fuel is provided as solid fuel, such as in the form of pellets.

Some embodiments relate to further products according to the second aspect of the invention, said further products comprising 0.1 -99.9% weight/weight or volume/volume of any product according to the present invention.

In the following section, further embodiments are presented in the form of numbered embodiments. 1 . A method for the production of a bioproduct, said method comprising the steps of :

(a) Pretreating a lignocellulosic biomass, such as soft lignocellulosic biomass;

(b) Providing a raw juice comprising carbohydrates such as sucrose; said raw juice optionally comprising undissolved lignocellulosic plant material;

(c) Diluting the pretreated biomass from step (a) with some quantity of the raw juice from step (b); and

(d) Hydrolysing the diluted biomass from step (c) by enzymatic hydrolysis using a cellulase and/or xylanase comprising enzyme preparation; wherein the lignocellulosic biomass and/or the raw juice is not from sugar cane or sweet sorghum.

2. A method for the production of a bioproduct, said method comprising the steps of : (a) Pretreating a lignocellulosic biomass, such as soft lignocellulosic biomass;

(b) Providing a raw juice comprising carbohydrates such as sucrose; said raw juice optionally comprising undissolved lignocellulosic plant material;

(c) Diluting the pretreated biomass from step (a) with some quantity of the raw juice from step (b); and

(d) Hydrolysing the diluted biomass from step (c) by enzymatic hydrolysis using a cellulase and/or xylanase comprising enzyme preparation; wherein the lignocellulosic biomass and the raw juice are from a different plant.

3. A method according to embodiment 1 or 2, further comprising step of

fermenting the hydrolysed and diluted biomass from step (d) to provide a fermentation product.

4. A method according to any one of the preceding embodiments, wherein the aqueous liquid phase of the hydrolysis mixture in step (d) comprises at least

5 g/L sucrose derived from the added raw juice, such as at least 1 0 g/L or more; such as 15 g/L or more; such as 20 g/L or more; such as, 25 g/L or more; such as 30 g/L or more; such as 40 g/L or more; such as 50 g/L or more; such as 60 g/L or more sucrose derived from the added raw juice. 5. A method according to any one of the preceding embodiments, wherein the initial dissolved sucrose from the added raw juice is between 1 -1 20, 2.5-1 00, or 5-60 g/L; and/or around 5, 10, 1 5, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 1 20, or more than 120 g/L.

6. A method according to any one of the preceding embodiments, wherein the ratio of added raw juice to pretreated biomass in weight/weight (w/w) or volume/volume (v/v) in step (c) and/or (d) is in the range of 0.01 to 4, 0.1 to 3, or 0.5 to 2; and/or 0.01 -0.1 ; 0.1 -0.25; 0.25-0.5; 0.5-1 .0; 1 .0-1 .5; 1 .5-2.0; 2.0-2.5; 2.5-3.0; 3.0-3.5; 3.5-4.0.

7. A method according to any one of the preceding embodiments, wherein the ratio of added raw juice to pretreated biomass in weight/weight (w/w) or volume/volume (v/v) in step (c) and/or (d) is at least 0.10:10 or more; such as 0.25:10 or more; such as 0.5:10 or more; such as 0.75:1 0 or more; such as 1 :10 or more; such as 1 .5:10 or more; such as 2:10 or more; such as 2.5:10 or more; such as 3 :10 or more; such as 3.5:10 or more; such as 4:10 or more; such as 4.5:1 0 or more; such as 5:1 0 or more; such as 5.5:10 or more; such as 6:1 0 or more; such as 6.5:1 0 or more; such as 7:10 or more; such as 7.5:1 0 or more; such as 8:1 0 or more; such as 8.5:1 0 or more; such as 9:10 or more; such as 9.5:10 or more; such as 1 0:10 or more, such as such as 1 1 :10 or more, such as 12:10 or more, such as 13:1 0 or more, such as 14:10 or more, such as 1 5:10 or more, such as 1 6:10 or more, such as 17:10 or more, such as 18:10 or more, such as 19:10 or more, such as 20:10 or more, such as 21 :10 or more, such as 22:10 or more, such as 23:10 or more, such as 24:10 or more, such as 25:10 or more, such as 26:10 or more, such as 27:10 or more, such as 28:10 or more, such as 29:10 or more, such as 30:10 or more, such as 31 :10 or more, such as 32:10 or more, such as 33:10 or more, such as 34:10 or more, such as 35:10 or more, such as 36:10 or more, such as 37:10 or more, such as 38:10 or more, such as 39:10 or more, such as 40:10 or more.

A method according to any one of the preceding embodiments, wherein the raw juice provided in step (b) is a liquid stream from sugar production, such as sugar production from sugar beet, sugar cane or sweet sorghum.

A method according to any one of the preceding embodiments, wherein the raw juice provided in step (b) is a liquid stream from fruit juice production. A method according to any one of the preceding embodiments, wherein the raw juice provided in step (b) is a liquid stream from a 1 ^st generation bioethanol production, such as a 1 ^st generation bioethanol production based on starch comprising plant material such as tubers including e.g. potatoes and cassava), cereals (e.g. maize, barley, wheat, sorghum, rye, oat), sugar cane, sweet sorghum and/or sugar beet.

A method according to any one of the preceding embodiments, wherein the pretreatment in step (a) is hydrothermal and/or autohydrolysis pretreatment. A method according to any one of the preceding embodiments, wherein the pretreatment in step (a) comprises one or more of:

- addition of a base such as NH ₄ OH or NaOH ;

- (ii) addition of an acid such as H2S0 ₄ ; and/or

- (iii) steam explosion.

A method according to any one of the preceding embodiments, wherein the pretreatment in step (a) does not comprises one or more of:

- (iv) addition of a base such as NaOH or NH ₄ OH;

- (v) addition of an acid such as H2SO ₄ ; and/or - (vi) steam explosion.

14. A method according to any one of the preceding embodiments, wherein the pretreated biomass is subject to at least one solid/liquid separation step to provide a fiber fraction and a liquid fraction; and optionally washing the fiber fraction as to remove dissolved solids, such as conducting said washing by a series of pressing and dilution steps.

15. A method according to any one of the preceding embodiments, wherein the pretreated biomass and/or the fiber fraction according to embodiment 14 is hydrolysed under conditions where initial undissolved solids are between 10% and 25%, 1 0% and 20%, or around 1 5% (w/w).

1 6. The method according to any one of the preceding embodiments, wherein the pretreated biomass and/or fiber fraction is hydrolysed under conditions where pH is maintained at or around pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5 or 4.0 or lower.

17. A method according to any one of the preceding embodiments, wherein the pretreated biomass and/or fiber fraction is hydrolysed using a cellulase preparation optimized for lignocellulosic biomass conversion, such as a commercially available cellulase preparation.

18. A method according to any one of the preceding embodiments, wherein the pretreated biomass and/or fiber fraction is hydrolysed using a cellulase preparation that is not inhibited more than 20% after 145 hours hydrolysis at an enzyme loading of at least 8 FPU/g DM under conditions appropriate for the tested enzyme preparation by added raw juice where sucrose derived from the added juice is at least 5 g/L.

19. A method according to any one of the preceding embodiments, wherein the hydrolysate obtained after hydrolysis of pretreated biomass and/or fiber fraction is subject to at least one solid/liquid separation step to provide insoluble solids separated from dissolved solids, such as by using a filter press with internal wash, optionally comprising a further step such as a drying step.

20. A method according to embodiment 19, wherein insoluble solids are suitable as a fuel and/or fuel additive, such as fuel and/or fuel additive for generation of power, heat and/or steam.

21 . A method according to any one of the preceding embodiments, wherein dissolved solids comprising cellulosic sugars and sugars derived from the raw juice are mixed with a further quantity of raw juice, optionally followed by a concentration step, such as evaporative concentration and/or reverse osmosis concentration.

The method according to any one of the preceding embodiments, wherein the further quantity of raw juice added is around 1 , 2, 5, 10, 15, 20, 30, 40, or 50% by weight or volume.

A method according to any one of the preceding embodiments, wherein (i) the hydrolysate obtained according to any one of the preceding

embodiments; (ii) the dissolved solids obtained according to any one of the preceding embodiments; (iii) the mixture of dissolved solids and raw juice obtained according to any one of the preceding embodiments; (iv) the concentrated solution provided through the concentration step according to any one of the preceding embodiments; and/or any combination of (i), (ii), (iii) and/or (iv) is subsequently fermented to provide one or more product(s), optionally after concentration, purification or any other step(s).

A method according to any one of the preceding embodiments, wherein the fermentation product is one or more chemical, alcohol, ethanol or any combination thereof.

The method according to any one of the preceding embodiments, wherein the bio- and/or fermentation product is one or more of: EtOH, primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof.

A method according to any one of the preceding embodiments, wherein the hydrolysis is either performed as whole slurry, or wherein a solid/liquid separation step is performed prior to hydrolysis so as to provide a fiber fraction and a liquid fraction, wherein the fiber fraction is separately subject to enzymatic hydrolysis.

The method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is derived from sugar cane, and the raw juice is derived from sweet sorghum, or another plant than sugar cane.

The method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is derived from sweet sorghum, and the raw juice is derived from sugar cane, or another plant than sweet sorghum.

A method according to any one of the preceding embodiments, wherein the raw juice is not derived from sugar cane or sweet sorghum. 30. A method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is not derived from sugar cane or sweet sorghum.

31 . The method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is derived from sugar cane, and the raw juice is derived from sweet sorghum.

32. The method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is derived from sweet sorghum, and the raw juice is derived from sugar cane.

33. The method according to any one of the preceding embodiments, wherein the lignocellulosic biomass is a soft lignocellulosic biomass.

34. The method according to any one of the preceding embodiments, wherein the raw juice comprises undissolved lignocellulosic plant material.

35. A method according to any one of the preceding embodiments, wherein the bioproduct is not EtOH.

36. A method according to any one of the preceding embodiments, wherein the fermentation product is one or more of: alcohol, EtOH, primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof.

37. A bioproduct, such as a fermentation product, provided according to any one of the preceding embodiments.

38. The bioproduct according to embodiment 37, said product being one or more of: alcohol, EtOH, primary sugar, glucose, fructose, xylan, sucrose, monomeric sugar derived from cellulose or other polysaccharides, including any combination thereof.

39. A product comprising or consisting essentially of (i) the hydrolysate

provided according to any one of the preceding embodiments; (ii) the dissolved solids provided according to any one of the preceding

embodiments; (iii) the mixture of dissolved solids and raw juice provided according to any one of the preceding embodiments; (iv) the concentrated solution provided through the concentration step according to any one of the preceding embodiments; and/or or any combination of (i), (ii), (iii) and/or (iv).

40. A fuel or fuel additive, such as fuel and/or fuel additive for generation of power, heat and/or steam provided according to any one of the preceding embodiments. 41 . A further product, comprising 0.1 -99.9% weight/weight or volume/volume one or more of the product according to any one of embodiments 37-40.

EXAMPLES

Example 1

Characterization of raw juice from sugar cane and sweet sorghum.

Juice from sugar cane and sweet sorghum were analyzed to determine their sugar composition.

Cane juice was extracted by pressing to provide juice, then irradiated using X- ray irradiation to eliminate contaminating microorganisms, then stored at 4° C until use. Typical concentration (s)/composition(s) of sugar cane juice are in the range of 13 to 1 6 Brix, with around the following sugar composition : total sugar = saccharose (97%) + fructose (1 .5%) + glucose (1 .5%). Composition of sugar cane juice soluble dry substance have also been published to be:

Juice constituent g/100 g

Sugars 75.0 - 94.0

Sucrose 70.0 - 90.0

Glucose 2.0 - 4.0

Fructose 2.0 - 4.0

Oligosaccharides 0.001 - 0.050

Salts 3.0 - 4.5

of inorganic acids 1 .5 - 4.5

of organic acids 1 .0 - 3.0

Organic acids 1 .5 - 5.5

Carboxylic acids 1 .1 - 3.0

Amino acids 0.5 - 2.5

Other organic non-sugars

Protein 0.5 - 0.6

Starch 0.001 - 0.18

Soluble polysaccharides 0.03 - 0.50

Waxes, fats, phosphatides 0.04 - 0.15

(Source: Sugar Technology, Beet and Cane Sugar Manufacture (1998) Van der Poel, P. W.; Schiweck, H.; Schwartz, T; see e.g. Table 2/21, pag. 153)

Example 2

Characterization of selected cellulase enzyme preparations.

A cellulase preparation can be obtained from Trichoderma reesei RUT-C30 raised on C5- rich liquid fraction from pretreated sorghum bagasse as carbon source, as described by Korpos et al. (2012). A cellulase preparation can be obtained from Penicillium echinulatum raised on pretreated sugar cane bagasse as carbon source, as described by Pereira et al. (2013).

A cellulase preparation can be obtained from Aspergillus sp. S4 B2 F raised on wheat bran as carbon source, as described by Soni et al. (2010)

A cellulase preparation from Trichoderma harzianum raised on pretreated sugar cane bagasse as carbon source, as described by Delabona et al. (2012).

A cellulase preparation from Bacillus subtilis KIBGE HAS raised on sugar cane bagasse as carbon source, as described by Bano et al. (2013).

A commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and sold by NOVOZYMES™ under the tradename CELLIC CTEC3™ can be obtained commercially.

A commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and sold by GENENCOR™ under the tradename

ACCELLERASE TRIO™ can be obtained commercially.

A commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and sold by DSM Tm can be obtained commercially.

A commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and sold by Dyadic Tm can be obtained commercially.

The cellulase activity of the cellulase preparations can be determined and expressed per unit volume or mass as "filter paper units" as determined by the method of Adney, B. and Baker, J., Laboratory Analytical Procedure #006, "Measurement of cellulase activity", August 12, 1996, the USA National Renewable Energy Laboratory (NREL), which is expressly incorporated by reference herein in entirety. It will be readily understood by those skilled in the art that FPU provides a measure of cellulase activity, but additional enzyme activities may be usefully included in an effective mixture of cellulytic enzymes, including but not limited to hemicellulase enzyme activities.

Further examples of cellulase preparations can e.g. be found herein , e.g. in the section "Definitions", as well as e.g. in WO101 1 5754. Example 3 Comparative performance of enzymatic activity of selected cellulase

preparations in a raw juice environment.

Any of the enzyme preparations mentioned in example 2 can be used for comparative performance measurements in the presence of various amounts of cane juice, as described in Example 4

Example 4

Optimization of raw juice supplementation for a given cellulase preparation.

For any given enzyme preparation, the raw juice supplementation which will be advantageous will be that where the final ethanol concentration in fermentation broth with added juice is equivalent to "base case" conditions, but at which the

DM % of hydrolysis is sufficiently lowered so as to provide better conversion at a given enzyme dose overall, notwithstanding some inhibition of conversion imposed by the added juice.

A commercially available cellulase preparation optimized for conversion of lignocellulosic biomass and sold by GENENCOR™ under the tradename

ACCELLERASE TRIO™ was examined.

Cellulase activity measurements in Filter Paper Units (FPU) were determined for ACCELLERASE TRIO™ by the method of Ghose (1987) and found to be 65 FPU/g enzyme preparation. An enzyme dose of 0.1 6 ml / g glucan (or 10.4 FPU / g glucan) was used in the experiments.

A set of six shake flasks was set up with double determination of the three conditions: 100 wt-% 1 G sugar juice,50 wt-% 1 G sugar juice and 0 % sugar juice (pure water) as reference. Shake flasks were incubated with agitation at 250 rpm and 50 °C.

Bagasse obtained after extraction of cane juice as described in example 1 was pretreated in the Inbicon 100 kg/h pilot plant with a feed flow of 50 kg TS/h, as described by Petersen et al. (2009). Before pretreatment, the fresh bagasse (SCB batch E) was soaked in water to achieve a dry matter content of 40 wt-% TSio5°c at ambient temperature without addition of any chemicals. Pretreatment conditions were 195° C, residence time 12minutes, log severity Ro 3.88. After leaving the pretreatment reactor the pretreated biomass slurry was pressed to a fiber fraction of approximately 55 % DM and a liquid fraction. An adjustment period of 3 h before steady state was kept and samples were taken. The pretreated material, fiber fraction as well as liquid fraction, was collected and analysed. The dry matter and composition of the samples were determined.

The pretreated bagasse fiber fraction obtained as described herein was used in shake flask experiments at a dry matter content 12 % without any additives other than AcTRIO and pH adjustment chemicals. The pH was adjusted with 20 % Ca(OH)2 to pH 5. Before enzyme addition the sugar content was measured by HPLC. When preparing the sample for the HPLC the solution was diluted with sulphuric acid, whereby the sucrose is split into glucose and fructose. To follow the hydrolysis, samples were measured on HPLC after 6, 24, 50, 72, 145 and 170 hours. From the measured glucose and xylose

concentrations the values measured before enzyme addition were subtracted to eliminate the contribution from 1 G sugar.

The glucan conversion over time was calculated based on the sugars from the fiber fractions, the sugar from 1 G juice having been subtracted. Figure 1 shows glucan conversion for the six shake flasks. The obtained glucan conversions after 170 h for the shake flasks without use of 1 G sugar juice (0 % OAI sugar juice), with 50 % 1 G sugar juice and with 100 % 1 G sugar juice were

determined to be approx. 73 %, 70 % and 65 %, respectively. "OAI sugar juice" indicates sugar cane raw juice.

Figure 2 shows the average glucan conversion after 145 h hydrolysis over the percentage of 1 G sugar juice added to hydrolysis. This relation can be described by a linear function and shows a decrease by 8 % conversion

(absolute) when going from 0 % sugar juice to 100 % sugar juice. It is assumed that a similar or slightly lower decrease would be obtained for higher dry matter contents.

As shown, compared with a "base case" hydrolysis of pretreated bagasse as whole slurry at 18% DM, in light of Table 1 , and with the results presented in this example, use of raw juice, such as cane juice supplementation in enzymatic hydrolysis of separated fiber fraction using the enzyme preparation

ACCELLERASE TRIO™ could be particularly advantageous where about 30% of diluent used in hydrolysis is cane juice, with 70% water. Under these conditions, equivalent final ethanol concentrations can be achieved in fermentation broth, but hydrolysis can be conducted at 1 5% DM. A reduction in glucan conversion corresponding to 71 % vs 73% (71 /73 = 0.973) is expected. However, at the same time, an increased conversion yield is expected at equivalent enzyme dose, where the relative enzyme equivalence dose is only 0.903. Accordingly, the conversion yield at equivalent dose is expected to be in the order of 1 /0.903 = 1 .107. A total net gain in conversion yield where hydrolysis is conducted at 1 5% DM using diluent comprising 30% cane juice is expected at equivalent enzyme dose of (1 .1 07) ^* (0.973) = 1 .08, that is, approximately 5% absolute conversion increase at equivalent enzyme dose. It will be readily understood by one skilled in the art that a similar optimization procedure can be applied to any particular enzyme preparation, based on the results.

In this case, where diluent used to dilute fiber fraction was 30% cane juice, and where hydrolysis was conducted at 15% DM, the sucrose concentration in the hydrolysate at the start of enzymatic hydrolysis comprised at least 12 g/L, assuming a sucrose concentration of at least 70 g/L in the cane juice.

Example 5

Sugar inhibition analysis

A set of shake flasks was set up with double or single determination of the seven conditions:

1 ) Negative control (pre-treated wheat straw fiber fraction (ptwsff) without any additional chemicals and/or enzymes)

2) ptwsff + 55 g cane sugar/kg in shake flask before enzyme addition 3) ptwsff + 1 1 0 g cane sugar/kg in shake flask before enzyme addition

4) ptwsff + 55 g glucose sugar/kg in shake flask before enzyme addition

5) ptwsff + 1 1 0 g glucose sugar/kg in shake flask before enzyme addition

6) ptwsff + 55 g pure sucrose/kg in shake flask before enzyme addition

7) ptwsff + 1 1 0 g pure sucrose/kg in shake flask before enzyme addition Shake flasks were incubated with agitation at 250 rpm and 50 °C.

Wheat straw was pretreated in the Inbicon 100 kg/h pilot plant with a feed flow of 50 kg TS/h, as described by Petersen et al. (2009). Before pretreatment, wheat straw (Wheat straw batch PWS_F) was soaked in water to achieve a dry matter content of 40 wt-% TSios°c at ambient temperature without addition of any further chemicals. Pretreatment conditions were 189 °C, residence time 12 minutes. After leaving the pretreatment reactor, the pretreated biomass slurry was pressed to a fiber fraction of 49% dry matter (DM) and a liquid fraction. An adjustment period of 3 h was kept before reaching steady state, whereupon samples were taken. The pretreated material, fiber fraction as well as liquid fraction, were collected and analyzed. The dry matter and composition of the samples were determined.

The pretreated wheat straw fiber fraction was used in shake flask experiments at a dry matter content 10 %. The pH was adjusted with 8 % NaOH and maintained in the range of pH 4.7 to 5.2, as the pH decreased during hydrolysis presumably due to release of acetic acid. Before enzyme addition (CTec3; dosage 0.075 g CTec3/g glucan, 10 % TS, pH 4.7-5.2), the sugar content was measured by HPLC. The hydrolysates and controls were sampled and diluted 1 :10 before monomeric carbohydrate yield was quantified by HPLC with a Phenomenex Rezex H+ column (Kristensen et al. 2009). To follow the hydrolysis, samples were measured on HPLC after 20 hours. From the measured glucose and xylose concentrations the values measured before enzyme addition were subtracted to eliminate the contribution from additives. The glucose and xylose concentrations after 20 hours were calculated based on the sugars derived from fiber fraction, and the added sugars (cane sugar, glucose or sucrose) were subtracted. Glucose and xylose yields are presented in Figures 3 and 4, respectively. The data is also summarized in Table 2 below Table 2: Impact of carbohydrate addition on glucose and xylose yields.

References

Kristensen, J. B., C. Felby and H. Jorgensen (2009). "Determining Yields in High Solids Enzymatic Hydrolysis of Biomass." Appl. Biochem. Biotechnol. 156:557-562.

Selig M.J., Hsieh C.W., Thygesen L.G., Himmel M. E., Felby C, and Decker S. R. (2012) Considering water availability and the effect of solute concentration on high solids saccharification of lignocellulosic biomass. Biotechnol. Prog. 28(6):1478-1490.

Selig M.J., Thygesen L.G. and Felby C. (2014). Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose saccharification. Biotechnol. Biofuels 7(1 ):159.

Qing Q., Yang B. and Wyman C.E. (2010): Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Biores. Technol. 101 (24) :9624-9630.

Agbor, V., et al. "Biomass pretreatment: Fundamentals toward application", Biotechnology Advances (201 1 ) 29:675

Alvira, P., et al. "Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review", Bioresource Technology (201 0) 101 :4851

Girio, F., et al., "Hemicelluloses for fuel ethanol : A review," Bioresource

Technology (201 0), 1 01 :4775

Taherzadeh, M., et al. "Pretreatment of Lignocellulosic Wastes to Improve Ethanol and Biogas Production: A Review" International Journal Molecular Science (2008) 9:1 621

Ceclan R. E., Pop A. and Ceclan M., (2012), Studies concerning the integrated use of sweet sorghum for bioethanol production in Romania, Chemical Engineering Transactions, 29, 877-882

Korpos, M. et al., (2012) Processing sweet sorghum into bioethanol - an integrated approach PERIODICA POLYTECHNICA-CHEMICAL

ENGINEERING, 56(1 ):21

Dias, M., et al., (2013), Evaluation of process configurations for second generation integrated with first generation bioethanol production from sugarcane, Fuel Processing Technology 109:84.

Palacios-Bereche, R., et al., (2013) Exergetic analysis of the integrated first- and second- generation ethanol production from sugarcane,

http://dx.doi.ora/10.1 01 6/i.enerav.201 3.05.010

Macrelli, S., et al., (201 2) Techno-economic evaluation of 2d generation bioethanol production from sugar cane bagasse and leaves integrated with the sugar-based ethanol process, Biotechnology for Biofuel 5:22

Dias, M., et al., (2012) Improving second generation ethanol production through optimisation of first generation production process from sugarcane, Energy 43:246

Dias, M., et al., (201 1 ) Simulation of integrated first and second generation bioethanol production from sugarcane: comparison between different biomass pretreatment methods, J. Ind. Microbiol. Biotechnol. 38:955

Walter, A., and Ensinas, A., (2010) Combined production of second-generation biofuels and electricity from sugarcane residues, Energy 35:874

Kim, M., et al., (201 2) Utilization of whole sweet sorghum containing juice, leaves and bagasse for bio-ethanol production, Food Sci. Biotechnol.

21 (4):1 075

Pereira, B., et al., (2013) Cellulase on-site production from sugar cane bagasse using Penicillium echinulatum, Bioenerg. Res. 6:1052

Petersen, M., et al. (2009) Optimization of hydrothermal pretreatment of wheat straw for production of bioethanol at low water consumption without addition of chemicals, Biomass and Bioenergy 33:834.

Soni, S., et al., (2010) Bioconversion of sugarcane bagasse into second generation bioethanol after enzymatic hydrolysis with in-house produced cellulases from Aspergillus sp. S4B2F, BioResources 5(2) :741

Bano, S., et al., (2013) High production of cellulose degrading endo-1 ,4,B-D- glucanase using bagasse as a substrate from Bacillus subtilis KIBGE HAS, Carbohydrate Polymers 91 :300