NL2010305C2

NL2010305C2 - Process for the treatment of lignocellulosic biomass.

Info

Publication number: NL2010305C2
Application number: NL2010305A
Authority: NL
Inventors: Adrianus Theodorus Smit; Wouter Johannes Joseph Huijgen
Original assignee: Stichting Energie
Priority date: 2013-02-15
Filing date: 2013-02-15
Publication date: 2014-08-18

Description

Process for the treatment of lignocellulosic biomass

[0001] The present invention relates to an advanced process for the fractionation of lignocellulosic biomass using organosolv.

Background

[0002] Biomass, especially lignocellulosic biomass is a valuable resource for the production of (bio)fuels, chemicals, performance products and energy. Lignocellulose is the most abundant renewable biomass available on land, and therefore relatively cheap. It comprises mainly cellulose, hemicellulose and lignin. Many research efforts have been devoted to the development of processes for the cost-effective conversion of biomass, especially lignocellulosic biomass, to valuable compounds. An example thereof is the conversion of cellulose to glucose, which in turn may serve e g. as a precursor for ’second-generation’ bioethanol (by fermentation of glucose), and is thus suitable for the preparation of biofuels.

[0003] The main structural components of biomass are cellulose, hemicelluloses and lignin. The two main types of hemicelluloses are xylans (xylose (C5) backbones, sometimes substituted with arabinose or glucuronic acid side groups), which are predominant in hardwood and grasses, and (gluco)mannans (glucose-mannose (C6), ratio about 1:3, backbones sometimes substituted with galactose side groups), which are predominant in softwood. Minor hemicellulose types include xyloglucans, arabino-galactans, and mixed P-l,3-glucans. Hemicelluloses may be chemically linked to lignin. Table 1 below gives approximate compositions of the structural components of some biomass types.

[0004] Table 1: Compositions of the structural components of some biomass types (in wt% based on dry weight)

[0005] The so-called organosolv process can be used to treat biomass (pretreatment), in order to make cellulose polymers better accessible for hydrolytic enzymes converting cellulose to glucose, or for pulping or fractionating of the biomass. Without pretreatment, the cellulose within lignocellulose biomass is poorly accessible for the hydrolytic enzymes, as it is shielded by other biomass components such as lignin and hemicelluloses. Organosolv involves high-temperature treatment of the biomass with a (water-miscible) organic solvent and optionally an (acidic) catalyst. During organosolv, the lignocellulose biomass is fractionated into a cellulose-enriched solid product stream (pulp) and a liquid product stream (liquor) comprising lignin and hemicellulose derivatives.

[0006] The hemicelluloses present in the lignocellulosic biomass are relatively unstable and break down during organosolv, especially as a result of the elevated temperatures employed. Hemicellulose is first hydrolysed into sugar monomers (C5 and/or Ce sugars), which may subsequently dehydrate to furans such as furfural, and react further to other compounds (including condensation products with lignin (“pseudo-lignin”)). Most of these latter compounds are less valuable than hemicellulose itself or the products directly obtained from it such as monomeric sugars and furfural. These degradation products may pollute the cellulose stream and/or the lignin stream, which are produced by the organosolv process, thereby reducing the efficiency of further treatment of these streams to produce valuable end-products, such as ethanol. In addition, the potentially valuable compounds that can be derived from the hemicellulose (e.g. monomeric sugars and furfural) are lost, thus reducing the effectiveness of the conversion of biomass into valuable components.

[0007] Especially the cellulose-enriched product stream obtained from the organosolv process comprises many impurities. Although organosolv treatment separates large parts of lignin and hemicellulose from the cellulose pulp, the cellulosic pulp typically still comprises significant amounts of lignin, as well as pseudo-lignins which may be formed during pretreatment by reaction of lignin with e.g. proteins, other extractives and/or furfural. These impurities significantly hamper the enzymatic hydrolysis of cellulose to glucose, which is to date still not applied on a commercial scale, since it cannot compete yet with glucose produced from first generation biomass sources (starch, sucrose etc.), in view of the high costs of the pretreatment step and required amounts of enzyme. Alternatives to enzymatic hydrolysis of cellulose, e.g. concentrated acid treatment, are undesirable for environmental reasons, corrosion of equipment and associated costs, and because they yield more by-products by sugar degradation reactions. Hence, one of the challenges of current research is to find means to enhance the efficiency of enzymatic hydrolysis of cellulose, in order to allow application on an industrial scale.

Summary of the invention

[0008] The invention relates to an advanced organosolv process of lignocellulosic biomass, resulting in a cellulose-enriched product (pulp) containing less impurities and a lignin-enriched product containing a higher content of native lignin. Surprisingly, the inventors have found that performing an extraction with an organic solvent prior to organosolv fractionation results in a better performance of the organosolv step itself. Moreover, using pre-extracted biomass according to the invention allows a more efficient enzymatic hydrolysis of cellulose and thus a reduction of the required enzyme load and associated costs. Furthermore, the composition of the products obtained from the organosolv process according to the invention is less influenced by the compositional differences of various types of biomass or various batches of the same type of biomass. These variations are suppressed by removal of (large parts of) the extractives by organic solvent (and optionally aqueous) extraction.

Preferred embodiments 1. A process for fractionating lignocellulosic biomass, comprising the steps of: (a) extracting the biomass with an extracting liquid comprising a first organic solvent at a temperature below 100 °C; (b) treating the extracted biomass with a treatment liquid comprising a second organic solvent and optionally an acid at a temperature above 100 °C.

2. The process according to embodiment 1, wherein the first organic solvent is a water-miscible solvent selected from lower alcohols, ethers and ketones.

3. The process according to embodiment 2, wherein the first organic solvent comprises ethanol.

4. The process according to any one of embodiments 1-3, wherein the extracting liquid comprises between 0 and 30 vol.% of water.

5. The process according to any one of embodiments 1-4, wherein extraction step (a) is performed at a temperature between 30 °C and 80 °C.

6. The process according to any one of embodiments 1-5, wherein extraction step (a) is preceded by a pre-extraction step in which the biomass is extracted with water at a temperature below 100 °C, preferably between 20 °C and 60 °C.

7. The process according to any one of embodiments 1-6, wherein treatment step (b) is performed at a temperature between 120 °C and 170 °C.

8. The process according to any one of embodiments 1-7, wherein the first organic solvent is the same as the second organic solvent, preferably both comprise ethanol.

9. The process according to any one of embodiments 1-8, wherein the lignocellulosic biomass is selected from herbaceous biomass, softwood and hardwood and combinations thereof, preferably the lignocellulosic biomass comprises herbaceous biomass.

10. A process for fractionation of herbaceous biomass, comprising the steps of: (a) extracting the biomass with an extracting liquid comprising a first organic solvent, preferably ethanol, at a temperature below 100 °C; (b) treating the extracted biomass with a treatment liquid comprising a second organic solvent at a temperature between 120 °C and 170 °C, and at a pH between 1 and 5.

11. The process according to any one of embodiments 1-10, further comprising a step of: (c) subjecting a cellulose-enriched product stream resulting from step (b) to enzymatic hydrolysis.

12. The process according to embodiments 6 and 11, wherein step (c) is preformed in the presence of an aqueous extract obtained in the pre-extraction step.

13. A composition obtainable by the process of any one of embodiments 1-10, comprising, based on total dry weight: (a) at least 65 wt% cellulose; (b) at most 12 wt% lignin; (c) at most 6 wt% ash; and (d) at most 8 wt% hemicellulose.

14. A process for producing glucose, comprising bringing the composition according to embodiment 13 in contact with at least hydrolytic enzyme, preferably a cellulase.

Detailed description

[0009] The invention relates to a process for fractionating lignocellulosic biomass into a cellulose-enriched product stream (pulp) and a lignin-enriched product stream (liquor), comprising extracting the biomass and subsequently subjecting the extracted biomass to an organosolv step. Without being bound by a theory, it is believed that preextraction of the biomass using an organic solvent removes biomass components (extractives) which hamper the organosolv process and/or the subsequent enzymatic cellulose hydrolysis, but leaves the valuable structural components (cellulose, hemicellulose and lignin) intact.

[0010] Thus, the invention relates to a biomass fractionation process, comprising: (a) extracting the biomass with an extracting liquid comprising a first organic solvent at a temperature below 100 °C; (b) treating the extracted biomass with a treatment liquid comprising a second organic solvent and optionally an acid at a temperature above 100 °C.

[0011] Biomass suitable for the process according to the invention includes ligno-cellulosic biomass, such as softwood, hardwood, and herbaceous biomass, including grasses and straws, and can be supplied in the form of forestry residues, agricultural residues, yard waste, animal and human waste (e.g. biodegradable municipal waste). Such biomass comprises in general 20 to 80 wt.% carbohydrates (based on dry matter), which are valuable starting materials for production of fuels and chemicals in a biorefinery. Lignocellulosic biomass (so-called second generation biomass) is cheaper than starch-containing biomass (first generation biomass) and does not compete with (human) dietary needs. Preferably, herbaceous biomass in the form of agricultural residues and/or biodegradable municipal waste is used in the process according to the invention, more preferably, the herbaceous biomass is selected from straw, leaves, (fresh or dried) grasses and combinations thereof, most preferably straw (e.g. rice straw, barley straw, wheat straw).

Extraction and pre-extraction

[0012] Extraction comprises at least one extraction step, wherein the biomass is extracted with an extraction liquid comprising a first organic solvent (referred to as organic extraction). In one embodiment, extraction contains only one extraction step, wherein the biomass is extracted with an extraction liquid comprising a first organic solvent, optionally as a mixture with water. Extraction may also involve multiple extraction steps, wherein the extraction liquid in each individual extraction step comprises water or a first organic solvent or a mixture thereof. Thus, extraction may contain at least one, at least two, at least three, or at least four separate extraction steps.

Preferably, extraction contains at least two extraction steps, more preferably it contains two extraction steps. It is preferred that each individual extraction step is performed using a different extraction liquid.

[0013] Conveniently, at least one extraction step is performed using water as extraction liquid and one or more other extraction steps using a first organic solvent and/or mixtures of water and a first organic solvent as extraction liquid. The extraction liquid preferably comprises at least 20 wt% of the first organic solvent (i.e. between 20 wt% and 100 wt%), more preferably at least 50 wt%, even more preferably at least 70 wt%, most preferably at least 90 wt%. The extraction liquid preferably comprises at most 50 wt% water (i.e. between 0 wt% and 50 wt%), more preferably at most 30 wt%, even more preferably at most 10 wt%, most preferably at most 5 wt%. Whenever it is mentioned that the extraction liquid is a certain solvent, it is meant that the extraction liquid comprises at least 90 wt% of that solvent, preferably at least 95 wt%.

[0014] In case the extraction comprises two or more extraction steps, it is preferred that one extraction step is performed using water as extraction liquid (i.e. containing less than 20 % of organic solvent), and the other extraction step is performed using at least 20%, preferably at least 50%, more preferably at least 70 % of a first organic solvent, preferably ethanol, as extraction liquid. In a most preferred embodiment, extraction contains two extraction steps, a first extraction step, referred to below as pre-extraction, wherein the biomass is extracted with water as extraction liquid, and a second extraction step, wherein the water-extracted biomass is extracted with a first water-miscible organic solvent, preferably ethanol, as extraction liquid.

[0015] Suitable first organic solvents to be used according to the invention include, but are not limited to, lower alcohols and diols, ethers, ketones, amides, lower alkanes, carboxylic acids and CO2 (sc). Herein, “lower” means containing 1-6 carbon atoms (Ci-Có), especially C1-C4. The first organic solvent is preferably water-miscible or capable of dissolving at least 10 wt% of water. Examples of suitable first organic solvents include methanol, ethanol, propanol, isopropanol, butanol and its isomers, ethylene glycol, propylene glycol, methoxyethanol, dimethoxyethane, diethylene glycol, dioxane, acetone, methyl ethyl ketone, tetrahydrofuran, dimethyl formamide, dimethyl acetamide, N-methylpyrrolidone etc. Further polar (co)solvents can be used as well, although these are slightly less preferred, for example acetonitrile, formic acid, acetic acid, methyl acetate, ethyl acetate, non-apolar haloalkanes such as dichloromethane, and CO2 (sc). Apolar solvents, such as hydrocarbons, e.g. pentane, cyclopentane, hexane, toluene or mixtures thereof, such as petroleum ether, can be also used as (co)solvents, or as solvents in an optional post-extraction step. In the context of the present invention, mixtures of miscible organic solvents are also encompassed in the term “organic solvent”. Preferably, the first organic solvent is selected from methanol, ethanol, propanol, butanol and acetone. Conveniently, at least one extraction step of the extraction is performed using an extraction liquid comprising a first organic solvent, which is the same organic solvent to be used during the subsequent organosolv step (referred to as the second organic solvent, see further below). Preferably, the extraction liquid comprises ethanol as first organic solvent, most preferably the extraction liquid is ethanol.

[0016] For the optional pre-extraction step and other optional aqueous extraction steps during the one or more individual extraction steps of step (a) of the process according to the invention, it was found that (non-demineralised) tap water or filtered, relatively clean water, can be conveniently used as extraction liquid, while demineralised water is also suitable, with or preferably without added organic solvents or other additives. Aqueous (pre-)extraction steps may be performed using an aqueous extracting liquid, in particular water. The aqueous liquid may contain agents assisting in the dissolution of extractives, such as acids, bases, salts and surfactants. The pH may be from slightly alkaline to acidic, e.g. between 2 and 10, preferably between 4 and 8. If desired minor amounts of an organic solvent (e.g. as described above for the first organic solvent, alcohols, polyols, ethers and the like) may be added to the aqueous extracting liquid. However, the level of organic solvents is preferably kept low, e.g. below 20 wt%, more preferably below 10 wt%, most preferably below 2 wt%.

[0017] The biomass subjected to the extraction step (a) may be fresh or dried biomass, optionally after removal of large impurities such as stones and pieces of metal, and optionally chopped or milled to pieces for ease of handling (e.g. pieces of 0.01 to 50 cm, in particular 0.1-10 cm in length or diameter, depending on the type of biomass).

[0018] Each individual extraction step of the extraction may be performed using any extraction technique known in the art. Conveniently, extraction is performed by washing the biomass with the solvent, or by soaking the biomass in the solvent. In this embodiment, the biomass preferably soaks at least 1 minute in the solvent, more preferably between 5 minutes and 600 minutes, most preferably between 10 minutes and 120 minutes. The extraction may also be performed stage-wise, in a counter-current mode. In such a staged mode, relatively clean solvent is used for a second or later stage of the extraction and the extract of the second or later stage is used as an extracting liquid for the preceding (or first) stage. In this way the residual amount of extractives in the biomass is minimised while keeping the amount of extracting liquid relatively low. Counter-current extraction allows a reduction in the total amount of extraction solvent.

[0019] Each extraction step of the extraction can be performed with water and/or first organic solvent as extracting liquid, wherein the extracting liquid has a temperature between its melting temperature and its boiling temperature (or higher if pressurised), i.e. is in liquid form. Preferred extracting temperatures are from 10 to 100 °C. For aqueous extraction steps, the extraction temperature is more preferably from 15 to 75°C, most preferably from 20 to 60°C, and for organic extraction steps more preferably from 15 to 80°C, most preferably from 30 to 75°C. For extraction steps using a mixture of water and organic solvent, the skilled person will appreciate how to manipulate the temperatures for optimal results. For extraction step (a), i.e. using a first organic solvent, the amount of extracting liquid is preferably between 0.5 and 20 L of liquid per kg of biomass. For single stage extraction, the preferred amount is between 1 and 12 L, most preferred between 1.5 and 6 L of solvent per kg of biomass. For the optional aqueous pre-extraction step, the amount of extracting liquid (water) is preferably between 1 and 20 L of liquid per kg of biomass. For single stage extraction, the preferred amount is between 2 and 12 L, most preferred between 3 and 10 L of solvent per kg of biomass. For counter-current extraction, the preferred amount of extraction solvent is between 1 and 6 L, especially between 1.5 and 4 L solvent per kg biomass. The biomass weight is understood herein as the dry weight, without adherent water.

[0020] The mixture of biomass and extraction liquid may be filtered after each extraction step of the extraction, using a filter having small enough pores to retain the chopped and washed or soaked biomass, and large enough pores to allow the extract comprising extractives to pass. Typically, the pores of such a filter are between 10 pm and 10 mm in diameter, preferably between 100 pm and 1 mm. The retentate comprising biomass is used for further treatment by organosolv as described below.

[0021] During extraction, the total dry weight of the biomass may reduce, as water-soluble and/or organic solvent-soluble components will be washed away. These extractives may include salts, proteins, fatty acids, triglycerides, waxes, terpenes and resin acids. As the skilled person will appreciate, hydrophilic components (e.g. salts, water-soluble proteins) will predominantly be washed away during aqueous extraction, i.e. in the pre-extraction step, while lipophilic components are predominantly extracted during extraction with an organic solvent. The composition and concentration of washable components is highly dependent on the type of biomass. For example, annual fibres such as straw contain relatively large amounts of fatty acids and/or waxy materials, which may be extracted using organic solvent, and (soft)wood may contain significant amounts of terpenes and resin acids.

Organosolv

[0022] The extracted biomass is subsequently fed to an organosolv process step, which separates the lignocellulose biomass into a cellulose-enriched product stream (also referred to as ‘cellulose pulp’ or just ‘pulp’) and a lignin-enriched product stream (liquor). Any organosolv process step as known in the art may be employed in the process according to the invention. Generally, organosolv is performed at a temperature above 100 °C, such as between 100 °C and 280 °C, preferably between 125 °C and 250 °C, more preferably between 150 °C and 240 °C, most preferably between 170 °C and 220 °C, and/or at a pH below 6.0, preferably between 2.0 and 5.0. Typically, organosolv is performed either autocatalytically or acid-catalysed. Autocatalytic organosolv is performed by exposing lignocellulose biomass, which is suspended in a (water-miscible) second organic solvent optionally combined with water, to elevated temperatures, typically above 190 °C. During acid-catalysed organosolv, the temperature may be somewhat lower, typically above 170 °C, and some acid is added to the suspension of the biomass, the second (water-miscible) organic solvent and optionally water.

[0023] The process according to the invention is especially useful for low temperature organosolv processes, i.e. organosolv performed at a temperature below 170 °C, such as between 100 °C and 170 °C, preferably between 120 °C and 165 °C, most preferably between 130 °C and 160 °C. In specific embodiments the organosolv process is performed at temperature below 140°C, e.g. between 120 and 140°C. Low temperature organosolv is especially desirable for two reasons. First of all, it reduces the costs of the pretreatment step, and secondly it reduces degradation of the hemicellulose. As discussed above, hemicellulose degradation during pretreatment significantly reduces the effectiveness of the conversion of biomass into valuable components. However, at those reduced temperatures, non-structural organic components present in biomass may negatively affect the organosolv process. During high temperature organosolv (i.e. above 170 °C), the non-structural components of the biomass, when not extracted prior to organosolv, decompose or react with structural components. As these non-structural organic components or extractives are largely removed using the extraction step according to the invention, the organosolv process operates more smoothly, even at reduced temperatures. This is especially true for the low temperature organosolv process of annual fibres such as straw, which comprise significant amounts of apolar extractives such as fatty acids and waxy materials. At the temperatures employed during low temperature organosolv, these apolar extractives form aggregates during the organosolv process, thereby hindering the fractionation. It was observed that low temperature organosolv of wheat straw was less effective without prior extraction with an organic solvent.

[0024] Suitable second organic solvents for the organosolv step are known in the art and include lower alcohols, and ethers, such as methanol, ethanol, (iso)propanol, butanol, ethylene glycol, methoxyethanol, dimethoxyethane, dioxane. Preferred second organic solvents include ethanol. In a preferred embodiment, the solvent used during the organosolv step is the same solvent as used as first organic solvent in the extraction step, as discussed above.

[0025] Organosolv yields a cellulose pulp and a separate lignin-containing liquor. The cellulose pulp obtained by the process according to the invention contains a higher content of cellulose (i.e. the cellulose pulp has a higher purity), compared to cellulose pulp obtained by prior art organosolv processes. For example, the lignin content of the cellulose pulp is reduced, when compared to prior art cellulose pulp. Hence, the invention also relates to the products obtainable by the process according to the invention. Typically, the cellulose pulp according to the invention is a composition comprising, based on total dry weight: (a) at least 65 wt% cellulose, preferably at least 70 wt% cellulose, more preferably at least 75 wt% cellulose; (b) at most 12 wt% lignin, preferably at most 8 wt% lignin, more preferably at most 6 wt% lignin; (c) preferably at most 6 wt% ash, more preferably at most 4.5 wt% ash; and (d) preferably at most 8 wt% hemicelluloses (preferably xylans: polysaccharides predominantly having a backbone of β-D-xylose units with various side units such as arabinose, glucuronic acid and acetate; or mannans: polysaccharides having a backbone of mannose an glucose units, with possible galactose side units), more preferably at most 5 wt% xylan or mannan, most preferably at most 4 wt% xylan or mannan.

Preferably, the composition is a cellulose pulp obtained from herbaceous biomass, including grasses and straws, more preferably from straw. The cellulose pulp according to the invention is ideally suited to be used in enzymatic hydrolysis, as described further below.

[0026] Typically, the lignin liquor according to the invention comprises a higher content of native lignin, when compared to prior art lignin liquors. Without being bound to any theory, it is envisioned that fewer reactions take place between lignin and e.g. hemicellulose derivatives, degradation products thereof, or other reactive components present in the biomass such as proteins and other non-structural components, during the organosolv process according to the invention. These reactions are reduced in the process according to the invention by virtue of the prior extraction, which removes large parts of the non-structural components as extractives. This is especially true in case the process according to the invention is combined with low temperature organosolv, as at such reduced temperatures the hemicellulose or its monomeric derivatives (e.g. xylose, arabinose, mannose) is less prone to degrade, and thus less reactive towards lignin.

Enzymatic hydrolysis

[0027] In a preferred embodiment, the cellulose-enriched product stream, which is obtained as a product from the organosolv step, is subjected to enzymatic hydrolysis. Enzymatic hydrolysis of cellulose to glucose is accomplished by an enzyme or combination of enzymes, capable of hydrolysing cellulose, referred to as hydrolytic enzymes, preferably cellulases. Hydrolysis of cellulose is also known as cellulolysis. The activity of cellulase enzymes is typically measured in FPU (filter paper unit); see Ghose, T. K. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59, 257-268. The process according to the invention may be performed using any cellulase enzyme. Suitable cellulase enzymes are endocellulases (breaking cellulose at inner positions), exocellulases (cellobiohydrolases, breaking cellulose at more external positions to produce cellobiose or cellutetraose), beta-glucosidases (cellobiases, cleaving the exocellulase products into glucose units). Other cellulase enzymes, such as oxidative cellulases and cellulose phosphorylases, are less preferred. Preferably a combination of cellulase enzymes is used, in particular a combination of endo-cellulase, exo-cellulase and β-glucosidase. Also, hemicellulases (e.g. xylanases, arabinases, mannanases, etc.) may be present to decompose any residual hemicellulose remaining after the organosolv step.

[0028] In the process according to the invention, the enzymatic hydrolysis of cellulose may be performed in any manner known in the art. Thus, the cellulose-enriched product stream or cellulose-rich pulp is brought in contact with an enzyme capable of hydrolysing cellulose, preferably a mixture of cellulases. The resulting hydrolysate is rich in glucose, which may be further processed, optionally after separation of solid residues, such as fermented to produce e.g. ethanol or other alcohols or chemically treated to produce e.g. 5-hydroxymethyl-furfural and other furans, or the glucose may be used as such, as known in the art.

[0029] The invention also relates to a process for the enzymatic hydrolysis of cellulose by bringing the cellulose pulp according to the invention, as described above, in contact with one ore more cellulase enzymes. Using the process according to the invention, a reduced load of hydrolytic enzymes is needed to achieve the same glucose yield, when compared to prior art processes for the enzymatic hydrolysis of cellulose, which reduces the costs of the enzymatic production of glucose from cellulose.

[0030] In case an aqueous pre-extraction step (i.e. the extracting liquid comprises below 20 wt% organic solvent) is part of the prior extraction, the process according to the invention is readily combined with the use of an aqueous extract, in particular a protein-containing aqueous extract, which is obtained by aqueous pre-extraction as described above, during enzymatic hydrolysis of the cellulose pulp. The presence of such an aqueous extract, optionally after (partial) concentration by methods known in the art, results in significant enhancement of the enzyme activity during enzymatic hydrolysis of cellulose. As such, the enzyme loading during enzymatic hydrolysis step can be significantly reduced, without negatively affecting the yield of glucose. Surprisingly, the protein-rich aqueous biomass extract, suitable for enhancing the activity of the hydrolysing enzyme, may efficiently be prepared by extracting the biomass with water and subsequently filtering the mixture. No further purification steps are necessary for achieving a significant increase in enzyme activity during enzymatic hydrolysis. Other components, which may be co-extracted from the biomass during aqueous extraction, do not inhibit the hydrolytic enzyme (cellulase) during enzymatic hydrolysis of cellulose, or this inhibition is more than compensated by the activity raise caused by the presence of the aqueous extract. Protein from the biomass itself constitutes a relatively cheap and easily accessible protein source.

[0031] As a result of the reduced enzyme requirement, this preferred embodiment of the present invention meets the need for reducing the costs of enzymatic hydrolysis of cellulose. Without being bound to a theory, the inventors assume that proteins from the aqueous extract are adsorbed to the lignin present in the cellulosic substrate. As such, the amount of hydrolytic enzyme inactivated by adsorption onto liberated lignin decreases. Thus, a cellulose-rich pulp, originating from pretreated biomass is contacted simultaneously with (i) an enzyme capable of hydrolysing cellulose, preferably a (mixture of) cellulase(s), and with (ii) a protein-rich aqueous extract originating from aqueous extraction of biomass. In an especially preferred embodiment, the biomass is pretreated according to the present invention, i.e. subjected to extraction and subsequent organosolv as described above, and the protein-rich aqueous biomass extract used during the enzymatic hydrolysis of biomass originates from aqueous extraction of the same biomass, prior to organosolv. The protein-rich extract may be the directly obtained extract or a concentrate thereof.

Examples

[0032] The following examples are intended to illustrate the invention, not to limit the scope.

Example 1: (Pre-)extraction + low temperature organosolv

[0033] A first batch (batch 1) of wheat straw was pre-extracted using 10 L water per kg biomass. A second batch (batch 2) of wheat straw was first pre-extracted using the same conditions as for the first batch, and subsequently extracted with 10 L ethanol (containing about 4 wt% water) per kg biomass. Both pre-extraction and extraction were performed using a Soxhlet extractor. Both batches 1 and 2 of wheat straw were subjected to organosolv, as well as a batch of wheat straw, which has not undergone (pre-)extraction (control). Organosolv conditions employed: 140 °C; 120 min; solvent = ethanol/water (60/40 w/w); liquid/solid ratio = 10 L/kg; 50 mM H2SO4 for the (pre-)extracted wheat straw; 60 mM H2SO4 for the control experiment. The increased H2SO4 concentration for the control experiment is to counteract the higher acid-neutralisation capacity of the mineral part of the original biomass, which is lowered during pre-extraction by (partial) removal of the mineral part.

[0034] During organosolv of the control experiment, formation of balls of fatty acids and/or waxy material was observed, which hindered the fractionation of straw into the lignin-enriched liquor and the cellulose-enriched pulp, giving i.a. rise to a lower cellulose concentration in the pulp. These balls were not observed during organosolv of the second batch of extracted wheat straw.

[0035] The negative effects of the extractives present in biomass on organosolv are reflected in the pulp yield and fractionation degrees obtained in the organosolv process, as shown in table 2, and in the compositions of the cellulose pulp obtained during organosolv, as shown in table 3. Compositions are determined using the method described in W.J.J. Huijgen, A.T. Smit, P.J. de Wild, H. den Uil, BioResource Technology, 2012, 114, 389-398.

[0036] Table 2: Pulp yield andfractionation degrees

* Based on dry weight of the fresh biomass before (pre-)extraction.

** Degree of xylan hydrolysis, based on amount of xylan present in the fresh biomass. *** Reduction in lignin content in pulp compared to fresh biomass.

[0037] Table 3: Cellulose pulp composition (in wt% based on dry weight)

[0038] Pulp yields are higher for the control experiment and the water pre-extracted batch 1, which are indicative of a less effective fractionation (i.e. more impurities are present in the cellulose pulp). This is confirmed in the degree of delignification and in the compositions of the pulp, as shown in table 3. Ethanol extraction prior to organosolv results in a cellulose pulp comprising significantly increased amount of cellulose (based on measured glucose monomeric units) and significantly decreased amounts of lignin and xylan, when compared with cellulose pulp obtained by organosolv of biomass pre-extracted with water or not (pre-)extracted at all. Water preextraction leads to a significant reduction in ash content of the cellulose pulp. In addition, water pre-extraction and ethanol extraction give rise to more hydrolysis of the hemicellulose (xylan) during organosolv. Reduced amounts of lignin imply a more effective fractionation or delignification of the biomass (separation of lignin from cellulose).

[0039] The cellulose-enriched pulp was subsequently subjected to enzymatic hydrolysis (conditions: 20 FPU per gram pulp of cellulase enzyme (Accelerase 1500, DuPont Industrial Biosciences); 1.50 g pulp per 50.0 mL water buffered at pH 4.8; time = 72 h). The progress of the enzymatic hydrolysis was monitored by determination of the glucose concentration at various intervals for 72 h. The figure summarizes the results obtained using the cellulose-enriched pulp, obtained by organosolv of batch 1, batch 2 and the control experiment. Water pre-extraction (batch 1) slightly increases the final glucose concentration, when compared to the control experiment, while ethanol extraction (batch 2) markedly increases the obtained final glucose concentration. Thus, employing organic extraction prior to organosolv increases the glucose concentration during enzymatic hydrolysis. Notably, in all experiments the final glucose concentration was already achieved after 24 h, which implies that the cellulose in the pulp obtained by the low temperature organosolv process of this example is readily accessible by the cellulase enzyme. Thus, the organosolv process is efficiently performed at reduced temperatures, such as at 140 °C or lower, preferably in combination with organic extraction prior to organosolv.

Claims

A method for fractionating lignocellulose biomass, wherein: (a) the biomass is extracted at a temperature below 100 ° C with an extraction liquid which contains at least 20% by weight of a first organic solvent selected from lower alcohols and diols, ethers, ketones, amides, lower alkanes, carboxylic acids and CO 2 (sc), wherein the amount of extraction fluid is between 0.5 and 20 L per kg of biomass; (b) subjecting the extracted biomass to an organosol step at a temperature between 120 ° C and 280 ° C with a treatment liquid comprising a second organic solvent selected from lower alcohols, ethers and ketones, water and optionally an acid.

The method of claim 1, wherein the first organic solvent is a water-soluble solvent selected from lower alcohols, ethers, and ketones.

The method of claim 2, wherein the first organic solvent comprises ethanol.

A method according to any one of claims 1-3, wherein the extraction liquid comprises between 0 and 30% by weight of water.

The method according to any of claims 1-4, wherein extraction step (a) is carried out at a temperature between 30 ° C and 80 ° C.

A method according to any one of claims 1-5, wherein extraction step (a) is preceded by a pre-extraction step in which the biomass is extracted with water at a temperature below 100 ° C, preferably between 20 ° C and 60 ° C.

A method according to any one of claims 1-6, wherein the treatment step (b) is carried out at a temperature between 125 ° C and 250 ° C.

A method according to any one of claims 1-6, wherein the treatment step (b) is carried out at a temperature between 120 ° C and 170 ° C.

The method of any one of claims 1-8, wherein the first organic solvent is the same as the second organic solvent, and preferably both comprise ethanol.

The method according to any of claims 1-9, wherein the lignocellulose biomass is selected from grassy biomass, softwood, hardwood and combinations thereof, preferably the lignocellulose biomass comprises grassy biomass.

11. A method for fractionating grassy biomass, wherein: (a) the biomass is extracted at a temperature below 100 ° C with an extraction liquid that contains at least 20% by weight of a first organic solvent selected from lower alcohols and diols, ethers, ketones , amides, lower alkanes, carboxylic acids and CO2 (sc), preferably ethanol, wherein the amount of extraction fluid is between 0.5 and 20 L per kg of biomass; (b) subjecting the extracted biomass to an organosolv step at a temperature between 120 ° C and 170 ° C, and at a pH between 1 and 5 with a treatment liquid containing a second organic solvent selected from lower alcohols, ethers and ketones, water and optionally an acid.

The method of any one of claims 1 to 11, further comprising: (c) subjecting the cellulose-enriched product stream resulting from step (b) to enzymatic hydrolysis.

Process according to claims 6 and 12, wherein step (c) is carried out in the presence of an aqueous extract obtained from the pre-extraction step.