WO2009034235A1

WO2009034235A1 - Method of processing a carbohydrate raw-material

Info

Publication number: WO2009034235A1
Application number: PCT/FI2008/050510
Authority: WO
Inventors: Petro Lahtinen; Anne Kallioinen; Matti Siika-Aho; Anna SUURNÄKKI; Tarja Tamminen; Jaana Uusitalo; Timo Repo; Markku Leskelä; Niklas Von Weymarn
Original assignee: Valtion Teknillinen Tutkimuskeskus
Priority date: 2007-09-14
Filing date: 2008-09-15
Publication date: 2009-03-19
Also published as: FI20075644A0; FI120878B; FI20075644A

Abstract

Method of processing a carbohydrate raw-material. The method comprises subjecting the raw-material to a treatment carried out with an alkaline agent in an aqueous medium in the presence of O2 for degrading at least a part of the raw-material in order to produce an aqueous suspension comprising solids and dissolved components, recovering at least a part of the aqueous suspension, and using at least a part of the recovered suspension for non-fibrous applications. According to the invention, the carbohydrate raw-material is subjected to the first treatment in the presence of a transition metal catalyst formed by a copper ion and a nitrogen donor ligand. The present invention provides efficient conversion of any lignocellulosic material (wood, straw, bagasse, etc.) to hydrolysable form. The present invention also provides separation of lignin from lignocellulosic materials and dissolution of the lignin thus separated into the water phase.

Description

Method of processing a carbohydrate raw-material

The present invention relates to processing of carbohydrate raw-materials. In particular the present invention concerns a method according to the preamble of claim 1 for treating carbohydrates in an alkaline aqueous phase in the presence of a catalyst for producing an aqueous solution or dispersion which can be used as such or as a starting material for obtaining materials for non-fibrous applications.

Conversion of biomass to value-added products, especially to different forms of energy has received growing attention as a mean of replacing energy and other end-products derived from fossil raw materials. For instance, of the conventional bio fuels for transport (bioethanol, bio-ETBE, pure vegetable oil, biodiesels and biomethane), bioethanol has a long proven history and in some cases also environmental advantages compared to fossil fuels.

Bioethanol can be produced from a variety of renewable raw materials. Traditionally, it has been produced from starch or sugar-containing agricultural crops, but in the future the focus will be directed towards different raw materials rich in lignocellulose. Such materials include agricultural and forestry residues, side streams from food and forest industries, municipal and industry wastes, as well as energy crops. Besides widening the raw material basis for bioethanol production and perhaps the economics of production, the "second generation" raw materials (lignocellulosics) and conversion technologies required to convert these into ethanol are expected to decrease the carbon dioxide footprint of bioethanol production.

Converting biomass into ethanol requires biotechnology. In a sequence of catalytic reactions micro-organisms (typically yeast) convert sugars (preferably glucose or sucrose) into ethanol and by-products such carbon dioxide. In conventional bioethanol production the sugars are obtained by simply pressing out the sucrose-rich juice from e.g. crushed sugar cane and sugar beet. Alternatively, starch is hydro lysed in the presence of cheap enzymes and moderate heat increase to a glucose-rich solution. The structure of lignocellulosic materials is, however, much more complex, and releasing the sugars requires much harsher processing. Enzymes are typically chosen to perform the main hydrolysis of available polysaccharides in lignocellulose. However, for the enzymes to work efficiently the lignocellulosic material must first be pre-treated. Methods for breaking down or modifying the native structure of lignocellulose are thus called pre -treatment methods. Several such methods are known in the art. These include steam explosion, with or without chemicals, such as sulphuric acid, ammonia, etc., hot water treatment, mild acid hydrolysis, CaO treatment, wet oxidation, organic solvent treatment, ammonium treatment, etc.

The main difficulties associated with existing techniques are incomplete decomposition/modifying of the lignocellulosic material and the formation of toxic compounds during pre -treatment, which makes the further processing less feasible and efficient. In particular, the crude hemicellulose or cellulose filtrate from a conventional pretreatment contains usually various degradation products of lignocellulose. These may be lignin and sugar decomposition products, including furfural, hydroxymethyl furfural and formic and acetic acid. Most of these components are toxic to enzymes and microorganisms slowing the subsequent hydrolysis and fermentation process.

It is an aim of the present invention to eliminate at least a part of the above problems of the art and to provide a novel method for processing carbohydrate raw-material obtainable from various sources so as to provide, for example, an improved starting material for ethanol production.

It is another aim of the present invention to provide a method of producing novel liquid compositions containing dissolved carbohydrates and lignin.

The invention is based on the idea of subjecting the raw-material, optionally after homogenization and dispersing in liquid, to a treatment carried out in an alkaline aqueous medium in the presence of a catalyst and dioxygen. Examples of such catalysts include transition metal catalysts comprising copper coordinated with at least one aliphatic or aromatic nitrogen donor ligand. Such catalysts are capable of enhancing a breaking up of the structure of the lignocellulosic or cellulosic material. Partial or even total hydrolysis of the material can be achieved without the generation of toxic by-products which accompanies the performance of traditional pre-treatment processes. It is known in the art that copper complexes with 2,2'-bipyridine and 1,10-phenanthroline ligands are useful catalysts in the oxidation of veratryl alcohol. In this respect, reference is made to an article by H. Korpi et al. ("An efficient method to investigate metal-ligand combinations for oxygen bleaching", Applied Catalysis A. General 268 (2004) 199-206). These complexes have also been used as delignification catalysts in pulp bleaching experiments where they were able to decrease the kappa number of softwood pulp and increasing brightness. The publication is silent about the use of the described procedure which is intended as a pre-treatment for bleaching in a another process involving a far- reaching hydrolysis of a biomass for subsequent non- fiber applications..

WO 94/03646 discloses methods for the pre-treatment of lignocellulose-containing biomass comprising the addition of calcium hydroxide and water to the biomass and subjecting the mixture thus formed to relatively high temperatures for a period of time sufficient to render the biomass amenable to digestion. A pre-treatment of wheat straw using combined wet oxidation and alkaline hydrolysis is described by Bjerre et al. (Bjerre, Olesen, Fernqvist, Plόger & Skammelsen Schmidt, "Pretreatment of Wheat Straw Using Combined Wet Oxidation and Alkaline Hydrolysis Resulting in Convertible Cellulose and Hemicelllose", Biotechnology and Biengineering, Vol. 49 (1996), p. 568-577). Neither of the references contain any indication of the potential use of any particular metal catalysts in hydrolysis of biomass.

According to the present invention, the raw material is treated in alkaline slurry in the presence of a catalyst of the above-identified kind so as to provide a suspension of solids and dissolved components. The suspension, or at least a part thereof, is recovered and subjected to further processing or to separation of its components or a combination thereof.

More specifically, the method according to the present invention is mainly characterized by what is stated in the characterizing part of claim 1.

Considerable advantages are obtained by the present invention. Thus, the present invention offers a method for efficiently modifying the lignocellulosic material with reduced or - in practice - no formation of toxic compounds and thereby lessening the problems related to current pre-treatment techniques. Also, the carbohydrates obtained with the method described in the present invention are more susceptible to hydrolysis making the further processing of plant material more feasible than with known methods.

The present invention provides efficient conversion of any lignocellulosic material (wood, straw, bagasse, etc.) to hydrolysable form. The present invention also provides separation of lignin from lignocellulosic materials and dissolution of the lignin thus separated into the water phase.

Next, the invention will be examined more closely with the aid of a detailed description and with reference to the attached drawings and a number of non-limiting working examples.

Figure 1 is a bar chart showing the enzymatic hydrolysis of solid fraction by cellulases;

Figure 2 shows in graphical form the hydro lysability of alkaline catalytic treated and steam exploded spruce by cellulases;

Figure 3 shows the hydrolability of alkaline catalytic treated barley straw by cellulases;

Figure 4 is a (CE) electropherogram which indicates the acid composition of the alkaline filtrates of the three treatments described in Example 10, using semiquantitative quantitation by comparison to the standard mixtures; Figure 5 is a similar electropherogram indicating the acid composition of the alkaline filtrate of the standard treatment described in Example 10 at various dilution ratios; and

Figure 6 is a schematic presentation of the different stages and products obtained from a process based on the present catalytic lignocellulose pre-treament.

For the purpose of the present invention, the term "fibrous applications" stands for applications, where a carbohydrate raw material is converted to products that are based on a fibrous (self-supporting) network. The term "non-fibrous applications" is used for designating all other applications.

As discussed above, the present invention is based on the finding that lignocellulosic material is decomposed/modified in alkaline water solutions under 0₂-containing atmosphere when certain transition metal complexes (described later on) are present. Similar catalytic methods have been earlier used in the manufacture of pulp, but there is no suggestion in the art that these catalytic methods could be used as a pre-treatment in the manufacturing of other products than paper pulp.

According to a preferred embodiment, the raw-material is composed of a carbohydrate material, e.g. a material comprising cellulose, hemicellulose, lignin or combinations thereof. Optionally, the raw-material can be present in fibrous state, such as in the case of cellulosic and lignocellulosic materials.

The raw-material can be derived from biomass, including e.g. wood, pulp, agricultural and industrial sidestreams, forestry residues and thinnings; agricultural residues, such as straw, bagasse, olive thinnings; energy crops, such as reed canary grass, willow, switchgrass, Miscanthus; peat; bagasse and sea biomass. Also municipal and industrial wastes can be used, particularly organic, solid or liquid wastes, and they can be selected from refuse derived fuel (RDF); wastes from sawmills, plywood, furniture and other mechanical forestry wastes; and waste slurries (including industrial and municipal wastes).

According to a preferred embodiment, the raw-materials of the present method include wood, pulp, recycled fibres, straw, agricultural, municipal and industrial wastes and similar compositions which contain carbohydrates.

The raw-material, usually a solid material containing some moisture, is typically homogenized, optionally in water, and slurried. The slurry obtained preferably has a consistency of about 0.1 to 75 %, in particular about 1 to 15 % by weight, calculated from the weight of the water or aqueous solution or suspension.

In the slurry, the carbohydrate material is modified by degrading it at least partially in order to produce an aqueous suspension comprising solids and dissolved components. At least a part (e.g. at least about 5 wt-%, preferably 20 to 100 wt-%) of the aqueous suspension is recovered, and at least a part (e.g. at least about 10 wt-%, preferably 20 to 100 wt-%) of the recovered suspension is used for non- fibrous applications. A suitable pre-treatment solution capable of decomposing lignocellulosic material can be formed by mixing a salt or an oxide of a copper with a nitrogen containing organic compound in an aqueous solution/dispersion.

The solution or dispersion is alkaline, which in practice means that it, optionally in addition to the catalyst, contains an alkaline agent in the aqueous liquid phase.

The pH of the solution is initially at least 8, preferably at least 12. The temperature is typically about 50 to 150 ⁰C. The treatment can be carried out in air or in oxygen gas or in air enriched with oxygen. The pressure can be reduced, but is typically ambient (normal) pressure or excess pressure up to about 50 bar (abs.); the partial pressure of oxygen is preferably about 1 to 20 bar (abs.). Treatment times may range from 0.1 to 24 hours, preferably from about 10 minutes to about 20 hours.

The alkaline agent of the solution is selected from the group of alkali metal and earth alkaline metal hydroxides and carbonates and bicarbonates.

The transition metal catalyst comprises a catalyst containing (a) nitrogeneous ligand(s). The catalyst is of a kind capable of enhancing a breaking up of the structure of the lignocellulosic or cellulosic material. The transition metal catalyst preferably comprises copper coordinated with at least one nitrogen donor ligand. The catalyst may comprise a copper ion coordinated with one or more nitrogen donor ligands. The copper ion is preferably coordinated with at least a part of the nitrogen atoms of a nitrogen containing organic compound having a structure wherein at least two nitrogen atoms are spaced apart by a carbon chain of 1 to 4 carbon atoms, preferably 2 carbon atoms.

According to one embodiment, the nitrogen containing organic compound has a general structure selected from

wherein each substituent R¹ to R⁶ independently stands for hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or hydroxyl group which optionally is substituted or two of said substituents together with an intermediate nitrogen atom form a alicyclic or aromatic cyclic group comprising 1 to 3 rings, each group having 5 to 7 ring members and optionally containing at least one further hetero atom selected from N, O and S, m and n independently stand for 0 or 1 , and ^₁₁ designates a single or double bond.

The substituents can be selected from hydroxy, oxo, carboxy, carbonyl, sulfonyl, sulfoxyl, amido, nitro and/or amino groups.

Groups of the indicated kind may increase water- solubility of the ligands. It is also possible to provide substituents which increase solubility in non-polar media. Examples of such substituents are formed by hydrocarbyl radicals such as aliphatic and aromatic groups, in particular linear or branched alkyl, alkenyl and alkynyl groups and aryl groups.

As a specific example of the present ligands, it should be mentioned that the nitrogen containing organic compound can be a heterocyclic compound comprising two or more, fused or ring-assembled nitrogenous rings. The nitrogenous rings can be selected from rings derived from pyrrol, imidazol, pyrazol, pyridine, pyrazine, pyrimidine, pyridazine, indol, isoindol, indazol, purine, chinolin, and chinoxaline.

According to a particularly preferred embodiment, the nitrogen containing organic compound contains the structure of N-C-C-N (phenanthroline, bipyridine, cf. Formulas A to B), and the treatment is carried out in an alkaline solution (Na₂COs, NaOH, K2CO3, NaHCθ₃, KOH, etc.) under an oxygen-containing atmosphere.

Formulas A and B

One particularly preferred embodiment comprises copper phenantroline [Cu(II) 1.10- phenanthrolinate] . The concentration of the catalyst is about 0.00001 to 10 %, in particular about 0.0001 to 0.01 %, calculated from the weight of the liquid composition including the raw material and the liquid phase.

During the treatment, about 5 to 85 % by weight of the raw-material is dissolved and at least a part thereof is present in the aqueous phase of the aqueous suspension. Typically, material amounting to at least about 5 %, in particular about 10 to 85 %, for example 20 to 85 %, of the weight of the original raw-material is recovered in the form of the aqueous suspension.

The suspension obtained from the treatment contains solids and dissolved components; cellulose being present as a polymer, while most of the hemicellulose is partly or fully hydrolyzed/degraded. Lignin is mostly dissolved; pectins intact, and in addition there are small-molecular degradation products (e.g. acids).

The recovered material is used for producing carbohydrates selected from the group of monosaccharides, disaccharides, oligosaccharides and polysaccharides, alcoholic compounds, lignin, chemicals and nanoparticles and nanowhiskers.

For this purpose, the recovered portion, i.e. either the solid matter, the liquid portion or a mixture of solids and liquid, is subjected to a further treatment step selected from the group of mechanical treatments, chemical treatments and enzymatic treatments and combinations thereof, in particular it is subjected from the group of hydrolysis, fermentation and oxidation and combinations thereof optionally in further combination with preceding or subsequent mechanical treatments.

In particular, the recovered material can also be subjected to a second treatment for increasing the yield or concentration of carbohydrates, chemicals, lignin, or nanoparticles or nanofϊbres. By extended hydrolysis of the polysaccharides and oligosaccharides, the yield of monosaccharides is increased. The monosaccharides, such as xylose and glucose and galactose, are capable of being used for production of ethanol by fermentation. Various combinations of treatment and fermentation can be employed. One preferred embodiment comprises conversion of cellulose (and hemicellulose) to ethanol involving either separate hydrolysis and fermentation (SHF) or simultaneous saccharifϊcation and fermentation (SSF). The latter alternative can be carried out as simultaneous saccharifϊcation and hemicellulose fermentation (SSHF), which is also referred to as simultaneous saccharifϊcation and co-fermentation (SSCF).

Generally, these embodiments can be applied to the production of any chemicals that can be prepared by fermentation from monosaccharides, such as ethanol, lactic acid, sugar acids, acetic acid and similar.

For producing ethanol, the fermentation step is carried out in the presence of a fermenting micro-organism, capable of fermenting major lignocellulose-derived carbohydrates (sugars), such as hexoses and pentoses. Typically, the fermenting organism is capable of producing ethanol from the major lignocellulose derived sugars at temperature of 30-70

⁰C.

Examples of suitable organisms are the following:

Yeasts: Saccharomyces cerevisiae, including genetically modified strains, such as VTT strain B-03339, Pichia stipitis, Candida shehatae, Hansenula polymorpha, Pachysolen tannophilus, Brettanomyces naardenensis, Pichia segobiensis, P .guillermondii, P. naganishii, Candida tenuis, C. albicans, C. tropicalis, C. maltosa, C. torresii, Metschnikowia bicuspidate, M. zobelii, Sporopachydermia quercuum, Wingea robertsii,

Bacteria: Zymomonas mobilis, Escherichia coli (genetically modified organisms (GMO) strain/strains), Klebsiella oxytoca (GMO strain)

Fungi: Fusarium oxysporum, Candida millerii, C. tropicalis, C. parapsilosis, Petromyces albertensis, Debaromyces hansenii, Cellulomonas cellulans, Corynebacterium sp., Serratia marcescens.

According to another embodiment, illustrate by the right-hand flow-sheet of Figure 6, the suspension of solids and dissolved components obtained from treatment, is taken to a liquid separation step, from which a liquid phase containing dissolved polymers (lignin, sugar oligomers, pectins, etc.) and other dissolved molecules (degradation products) are obtained. These can be recovered as such and/or upgraded.

Thus, at least a part (generally 10 - 100 % by weight) of the liquid portion can also be recovered and subjected to further treatment in order to precipitate at least some of the dissolved carbohydrate material or lignin.

The solid phase remaining after liquid separation consists mostly of cellulose. This portion of the raw-material can be used for cellulose hydrolysis to yield sugar monomers or as a starting material for the recovery of nanoparticles and other nanostructures. According to one option, the solid phase is treated as explained above as a fermentation substrate.

Based on the above, the present invention also comprises a method in which a cellulosic or lignocellulosic raw material is process with the objective of modifying it, or a part of it, into a material, which has non-fibrous applications, as well as applications based on the formed nanoparticles or nanostructures of the solid fraction, comprising the steps of

- optionally separating some fraction of the raw material and excluding it from further processing; - optionally bringing the raw material into a more homogenised form;

- subjecting the raw material to a treatment in the presence of a catalyst formed by a nitrogen donor ligand and copper ion;

- optionally separating the material into different fractions;

- recovering the material or at least a part of it; - optionally subjecting the modified material, or some part of it, to further upgrading; and

- using the recovered material in various applications.

The following examples illustrate the invention. Example 1

In one embodiment of the invention spruce chips (5 % d.w./w) are suspended in a solution of 130 mg/L CuSO₄, 300 mg/L 1,10-phenanthroline and 20 g/L Na₂CO₃. The solution is mechanically stirred under 10 atm O₂ pressure and kept at 100 ⁰C for 20 hours. The reaction yields a suspension with a solid and solution fractions which are suitable for further processing according to examples below.

Example 2

In another embodiment of the invention spruce chips (5 % d.w./w) are suspended in a solution of 130 mg/L CuSO₄, 260 mg/L 1,1 '-bipyridine and 20 g/L Na₂CO₃. The solution is mechanically stirred under 10 atm O₂ pressure and kept at 100 ⁰C for 20 hours. The reaction yields a suspension with a solid and solution fractions which are suitable for further processing according to examples below.

Example 3

This example discloses ethanol production from catalytically pretreated softwood according to Example 1 by using enzymatic prehydrolysis before fermentation.

Spruce chips (4.9 % d.w./w) were catalytically pretreated according to Example 1. The prehydrolysis was done directly in the pre-treatment solution by adding commercial enzyme preparations and adjusting the temperature to 45 ⁰C and pH to 5.0. The enzyme preparations used were Celluclast 1,5 FG (10 FPU/g d.w.) and Novozym 188 (β- glucosidase dosage 100 nkat/g d.w.). After 4 hours the prehydrolysate was tempered to 30 ⁰C and inoculated with yeast strain (VTT-B-03339) to the concentration of 1 g/1 (d.w.) suspended before inoculation with nutrients in 10 vol-% (of the prehydrolysate) of YNB (Yeast Nitrogen Base). The fermentation was carried out at 30 ⁰C in waterlock flasks using slow agitation (100 rpm).

The fermentation using enzymatic pre-hydrolysis after alkaline catalytic treatment resulted in 118 hours an ethanol concentration of 8.8 g/L As a result, ethanol can be produced from alkaline catalytically pretreated softwood by using enzymatic prehydrolysis before fermentation.

Example 4

Ethanol production from catalytically pretreated softwood is disclosed by using simultaneous saccharification and fermentation (SSF).

Spruce chips (4.9 % d.w./w) were suspended in a solution of 240 mg/L CuSO4*5H₂O, 350 mg/L 1,10-phenanthroline and 10 g/L NaOH. The solution was mechanically stirred under 10 atm O₂ pressure and kept at 100 ⁰C for 20 hours. The simultaneous saccharification and fermentation was done directly in the pre-treatment solution by adding commercial enzyme preparations and adjusting the temperature to 45 ⁰C and pH to 5.0. The commercial enzyme preparations used were Celluclast 1,5 FG (10 FPU/g d.w.) and Novozym 188 (β- glucosidase dosage 100 nkat/g d.w.). The yeast (strain VTT-B-03339) to the concentration of 1 g/1 (d.w.) was inoculated immediately after enzyme addition. It was suspended before inoculation with nutrients in 10 vol-% (of the prehydrolysate) of YNB (Yeast Nitrogen Base). The hydrolysis and fermentation was carried out at 30 ⁰C in waterlock flasks using slow agitation (100 rpm).

Simultaneous saccharification and fermentation (SSF) after alkaline catalytic treatment resulted in 117 hours an ethanol concentration of 6.6 g/1.

Example 5

Spruce chips (4.9 % d.w./w) were suspended in a solution of 240 mg/L CuSO4*5H₂O, 350 mg/L 1,10-phenanthroline and 10 g/L NaOH. The solution was mechanically stirred under 10 atm O₂ pressure and kept at 100 ⁰C for 20 hours.

Alkaline catalytically treated material was filtered and the solid fibrous fraction was washed with water. The carbohydrate composition of washed solid fraction was analysed after acid hydrolysis to monosaccharides by HPLC (PuIs et al., 1985; Tenkanen and Siika- aho, 2000). Results show that after alkaline catalytic treatment a solid fraction having high glucose content and thus high cellulose content was obtained (Table 1). Table 1 Carbohydrate composition (% of dry weight) of alkaline catalytically treated washed fibres after acid hydrolysis by HPLC.

Example 6

A washed, solid fraction obtained by alkaline catalytic treatment was hydrolysed enzymatically to monosaccharides (from Example 5). Solid fraction was suspended into 50 mM sodium acetate buffer pH 5 into 10 mg/ml concentration. Enzymatic reaction was started by adding commercial cellulase Celluclast 1,5L FP 10 FPU/g dry weight and commercial β-glucosidase Novozym 188 100 nkat/g dry weight. Suspensions were incubated at 45 ⁰C with magnetic stirring for 24 hours.

The release of reducing sugars was analysed during the hydrolysis reaction and the results are indicated in Figure 1. The amounts of reducing sugars were determined by an analysis method based on reduction of 2-hydroxy-3,5-dinitrobenzoic acid (Bernfeld, 1955). As will appear from the Figure 1, 90 % of the carbohydrates in alkaline treated solid fraction were hydrolysed enzymatically in 24 hours. Results show that alkaline catalytic treatment produced solid fraction with high hydrolysability.

Example 7

It was an aim of this experiment to compare the hydrolysability of alkaline catalytically treated material with steam explosion treated spruce. Steam explosion is the state-of-the-art technology for pretreatment of lignocellulosic material for ethanol production.

Alkaline catalytically treated material from Example 1 was filtered and washed with water. After catalytic treatment, 53 % of treated wood remained as fibrous solid material. The carbohydrate composition of washed solid fraction was analysed after acid hydrolysis to monosaccharides by HPLC (PuIs et al, 1985; Tenkanen and Siika-aho, 2000) (Table 2)

Table 2. Carbohydrate composition (% of dry weight) of alkaline catalytically treated washed fibres after acid hydrolysis by HPLC

Washed, solid fractions obtained by alkaline catalytic treatment or steam explosion were hydrolysed enzymatically to monosaccharides. Steam explosion was carried out according to the method of Ohgren et al. (2006) by impregnating with 2.5 % SO₂, and steam pretreating at 210 ⁰C for 5 min. Solid fractions were suspended into 50 mM sodium acetate buffer pH 5 into 10 mg/ml concentration. Enzymatic reaction was started by adding Celluclast 1,5L FP 5 FPU/g and Novozym 188 in an amount 50 nkat/g dry weight. Suspensions were incubated at 45 ⁰C with magnetic stirring for 48 hours. The release of reducing sugars was analysed during the hydrolysis reactions (Bernfeld, 1955).

The results are indicated in Figure 2.

As apparent, alkaline treated spruce was hydrolysed by enzymes more efficiently than steam exploded spruce: both hydrolysis rate and hydrolysis level at the end of reaction was higher. Example 8

Alkaline catalytically treated materials prepared according to Example 1 were filtered and the solid fractions were washed with water. The composition of solid fraction was analysed by HPLC after acid hydrolysis to monosaccharides (PuIs et al., 1985; Tenkanen and Siika- aho, 2000)

Table 3. Monosaccharide compositions of solid fractions (% of d.w.)

The filtrate was analysed by HPLC as such after hydrolysis by enzyme mixture (Buchert et al., 1993; Tenkanen et al, 1997; Tenkanen and Siika-aho, 2000). By this method no monosaccharides were detected from the filtrates.

By acid hydrolysis the oligosaccharides present in filtrate could be analysed by HPLC (Table 4) Table 4. Solubilization of sugars in alkaline catalytic treatments analysed by acid hydrolysis and HPLC (% of original sugars).

Example 9

An aim of this experiment was to find out the hydro lysability of alkaline catalytically treated barley straw.

Alkaline catalytically treated barley straw was filtered and washed with water. After catalytic treatment, 38 % of treated wood remained as fibrous solid material. The carbohydrate composition of washed solid fraction is presented in example 8.

A washed, solid fraction of barley straw obtained by alkaline catalytic treatment was hydrolysed enzymatically to monosaccharides. Solid fractions were suspended into 50 mM sodium acetate buffer pH 5 into 2 mg/ml concentration. Enzymatic reaction was started by adding Celluclast 1.5L FP 5 FPU/g and Novozym 188 in an amount of 50 nkat/g dry weight. Suspensions were incubated at 45 ⁰C with magnetic stirring for 72 hours. The release of reducing sugars was analysed during the hydrolysis reactions (Bernfeld, 1955). The results are presented in Figure 3.

As a result, carbohydrates in the alkaline treated barley straw were hydrolysed totally to monosaccharides in 72 hours enzymatic hydrolysis. Therefore, alkaline catalytic treatment is an efficient way to improve the hydro lysability of barley straw as well as spruce. Example 10

An aim of this experiment was to recover lignin from the dissolved fraction after alkaline catalytic treatment of Spruce chips (Standard sample was produced with the method described in Example 1 , Standard 5 h sample similarly with the exception that reaction time was 5 hours, Standard ¹A cat. sample similarly with the exception that 65 mg/L CuSO₄, 150 mg/L 1,10-phenanthroline was used as catalyst).

Filtered solutions from alkaline catalytic treatments of Spruce were precipitated by acidification with hydrochloric acid to pH 2.5. The precipitates were collected by centrifugation and washed repeatedly with water, pre-adjusted to pH 2.5 and freeze-dried.

Three chip treatment conditions were applied. The yields of the precipitates are given in Table 5.

Table 5. Yield of precipitated lignin

*46 g dry wood/1, 0.25 M NaOH, 48.6 mg CuSO₄*5H₂O, 70 mg phenanthroline 100⁰C, 20 h, 10 atm O₂

**46 g dry wood/1, 0.25 M NaOH, 48.6 mg CuSO₄*5H₂O, 70 mg phenanthroline 100⁰C, 5 h, 10 arm O₂

***46 g dry wood/1, 0.25 M NaOH, 24.3 mg CuSO₄*5H₂O, 35 mg phenanthroline 100⁰C,

2O h, 10 atm O₂

Example 11

An aim of this experiment was to recover pectic substances from the dissolved fraction after alkaline catalytic treatment of Spruce chips.

Lignin is oxidised and degraded under the alkaline catalytic treatment conditions, releasing originally lignin-bound polysaccharides into the solution (Laine and Tamminen 2002). Analysis of the filtrates in Example 8 showed that treatment of the filtrate with a mixture of hydro lysing enzymes acting on the main wood polysaccharides could only release minor amounts of monosaccharides, whereas non-selective acid hydrolysis released monosaccharides derived from pectic substances.

The pectic substances are water-soluble and thus remain in the filtrate after lignin precipitation. They can potentially be recovered by ultrafiltration.

Example 12

An aim of this experiment was to recover small-molecular organic acids from the dissolved fraction after alkaline catalytic treatment of Spruce chips.

Lignin and to some extent carbohydrates are degraded under the alkaline catalytic treatment conditions into small-molecular organic acids. After precipitation of lignin and ultrafiltration of pectic substances by ultrafiltration, small-molecular organic acids remain in the solution and can be recovered by distillation or selective complexing. Acid composition of the filtrates is seen in the capillary electrophoresis (CE) electropherogram in Figure 4.

Acetic and glycolic acids co-elute and would require separate analysis for complete analysis. Dilution improves the separation to some extent, showing that both components are present, whereas analysis at higher concentration reveals the presence of minor amounts of malonic and succinic acids (Figure 5).

Figure 5 shows the acid compositions of the alkaline filtrate of the standard treatments described in Example 10 with varying dilution ratio.

The total content of acids in the standard sample after removal of lignin was analysed by potentiometric titration to be 0.18 M. By using 40 g/mol as a typical weight of acid, this corresponds to 7.3 g/1 and 15.7 weight-% on wood. Example 13

Alkaline catalytic treatment as pre -treatment for manufacturing of nanoparticles

Alkaline catalytically treated material from wood and/or straw was filtered and the solid fibrous fraction was washed with ion exchanged water. Washed, solid fraction obtained by alkaline catalytic treatment was further processed to nanoparticles.

Nanoparticles are also formed during the alkaline catalytic treatment and they can be detected from the filtrate.

Example 14

A summary of the various method steps are given in the form of a schematic presentation of two slightly different embodiments in Figures 6a and 6b. The first embodiment (Fig. 6a) describes the different stages and products obtained during processing of carbohydrates to sugar monomer and the second (Fig. 6b) describes processing of cellulose to monomers, including an intermittent liquid separation step. The catalytic pre-treatment method described in Examples 1 and 2 are referred to in the figures with reference numerals 3 and 13, respectively.

In the embodiment of Figure 6a, carbohydrate raw materials of the above discussed kind, usually solid materials with some moisture, are first conducted to a homogenization step 1 , where the materials may be dispersed in liquid, e.g. water. After that, the slurry thus obtained is conducted to a catalytic pre-treatment 3, where the slurry is mixed with catalyst and, if necessary, with a base in order to adjust the pH of the slurry. High pressure and temperature are applied. As a result a suspension of solids and dissolved components are obtained. Most of the cellulose, if any, from the feed is still present as polymer, while most of the hemicelluloses have been partly or fully hydro lyzed/degraded. Lignin is mostly dissolved, while pectins are intact. The solids and dissolved polymer portion contains typically also some small molecular degradation products, such as acids.

The suspension is conducted to further treatment, which in the drawing is illustrated with an enzymatic treatment, e.g. enzymatic hydrolysis. As discussed above, the further treatment can, however, comprise a number of different treatment steps, including not only chemical processing but also mechanical and physical treatment. After any hydrolysis, most of the cellulose and hemicellulose is hydro lyzed to monomers, while pectins still are intact. Naturally, the effluent contains small molecular degradation products (acids), and lignin in suspended or dissolved form.

In the second embodiment depicted in Figure 6b, the raw material (e.g. cellulose) is homogenized 11, if desired, and slurried. The slurry is subjected to a catalytic treatment 12 similar to the one in Fig. 6a. The suspension obtained contains both solids and dissolved components and it is conducted to a liquid separation step 14, where the liquid phase is separated from the solid matter. The solid phase, which mostly consists of cellulose can then be subjected to further treatment as discussed in connection with the process of Fig. 6a. As a result, most of the cellulose will be present in the hydrolysis effluent in the form of monomers.

The liquid phase can be separately processed to separate and recover dissolved polymers (lignin, sugar oligomers, pectins, etc.) and other dissolved molecules including various degradation products.

References

Bernfeld, P (1955) Amylases, a and b. In Colowick SP and Kaplan NO (eds) Methods of enzymology, VoI 1, Academic press, NY, pp 149-158.

Buchert, J, Siika-aho, M, Pere, J, Valkeajarvi, A and Viikari L (1993) Quantitative determination of wood derived soluble oligosaccharides by HPLC. Biotechnol. Techn. 7, 785-790.

PuIs, J., Poutanen, K., Kόrner, H. -U., and Viikari, L. (1985) Biotechnical utilization of wood carbohydrates after steaming pretreatment. Appl. Microbiol. Biotechnol. 22, 416- 423.

Tenkanen, M. and Siika-aho, M. (2000) An α-glucuronidase of Schitzophyllum commune acting on polymeric xylan. J. Biotechnol., 78:2, 149-161.

Tenkanen, M., Makkonen, M., Perttula, M., Viikari, L., and Teleman, A. (1997) Action of Tricoderma reesei mannanase on galactoglucomannan in pine kraft pulp. J. Biotechnol. 55, 191-204.

Laine, C, Tamminen, T. Origin of carbohydrates dissolved during oxygen delignification of birch and pine kraft pulp, Nordic Pulp and Paper Research Journal 17:2 (2002) 168-171.

Ohgren, K., Rudolf, A., Galbe, M., Zacchi, G. (2006) Fuel ethanol production from steam- pretreated corn stover using SSF at higher dry matter content. Biomass & Bioenergy 30, 863-869.

Claims

1. Method of processing a carbohydrate raw-material, comprising the steps of

- subjecting the raw-material to a treatment carried out with an alkaline agent in an aqueous medium in the presence OfO₂ for degrading at least a part of the raw- material in order to produce an aqueous suspension comprising solids and dissolved components,

- recovering at least a part of the aqueous suspension, and

- using at least a part of the recovered suspension for non-fibrous applications, wherein the carbohydrate raw-material is subjected to the first treatment in the presence of a transition metal catalyst formed by a copper ion and a nitrogen donor ligand.

2. The method according to claim 1, wherein the carbohydrate raw-material is selected from the group of carbohydrate-containing biomasses, consisting of wood, agricultural and industrial sidestreams, energy crops such as willow, reed canary grass, switchgrass and Miscanthus, as well as bagasse, sea biomass and municipal waste.

3. The method according to claim 1 or 2, wherein the raw-material is treated at a consistency of about 0.1 to 75 % (w/w), in particular about 1 to 15 % by dry weight.

4. The method according to any of claims 1 to 3, wherein about 5 to 90 % by weight of the raw-material is dissolved and at least a part thereof is recovered in the form of the aqueous phase of the aqueous suspension.

5. The method according to any of claims 1 to 4, wherein the recovered material is used for producing carbohydrates selected from the group of monosaccharides, disaccharides, oligosaccharides and polysaccharides or chemicals selected from group alcohols, lignin and extractives.

6. The method according to any of claims 1 to 4, wherein the recovered material is used for producing nanoparticles, nanowhiskers or other nano-scale structures composed of substances selected from group carbohydrates, lignin and extractives.

7. The method according to any of claims 1 to 6, wherein the recovered material is subjected to a further treatment step selected from the group of fractionation, mechanical treatments, chemical treatments and enzymatic treatments and combinations thereof,

8. The method according to any of claims 1 to 7, wherein the recovered material is subjected to a further treatment step by hydrolysis, fermentation or oxidation or a combination thereof.

9. The method according to any of claims 1 to 8, comprising subjecting the recovered material to a second treatment for increasing the yield of carbohydrates, chemicals, lignin, nanoparticles, nanowhiskers, nanofϊbrils or nanofibres.

10. The method according to any of claims 1 to 9, comprising recovering at least a part of the solid portion of the modified material.

11. The method according to claim 10, wherein the solid portion is subjected to simultaneous saccharification and fermentation for producing a fermentation product selected from the group of ethanol, lactic acid, sugar acids, acetic acid and similar chemicals that can be prepared by fermentation from monosaccharides.

12. The method according to claim 10, wherein the solid portion is subjected to separate saccharification and fermentation for producing a fermentation product selected from the group of chemicals that can be prepared by fermatation from monosaccharides, in particular ethanol, lactic acid and sugar acids.

13. The method according to claim 11 or 12, wherein at least a part of the liquid portion is recovered and subjected to saccharification and fermentation optionally together with the recovered part of the solid portion.

14. The method according to any of claims 1 to 13, comprising recovering at least a part of the liquid portion and subjecting it to further treatment in order to recover at least some of the dissolved carbohydrate material or lignin.

15. The method according to any of the preceding claims, wherein the raw-material is treated with the alkaline agent in aqueous liquid phase.

16. The method according to any of the preceding claims, wherein the raw-material is treated with the alkaline agent at a pH of at least 8, preferably at least 12.

17. The method according to any of the preceding claims, wherein the raw-material is treated with the alkaline agent at a temperature of 50 to 150 ⁰C.

18. The method according to any of the preceding claims, wherein the alkaline agent is selected from the group of alkali metal and earth alkaline metal hydroxides and carbonates and bicarbonates.

19. The method according to any of the preceding claims, wherein the transition metal catalyst comprises a homogeneous catalyst.

20. The method according to any of the preceding claims, wherein the transition metal catalyst is capable of enhancing a breaking up of the structure of the lignocellulosic or cellulosic material.

21. The method according to any of the preceding claims, wherein the transition metal catalyst comprises a transition metal ion coordinated with an aliphatic or aromatic amine.

22. The method according to any of the preceding claims, wherein the transition metal catalyst comprises a copper ion coordinated with nitrogen atoms of a nitrogen containing organic compound having a structure wherein at least two nitrogen atoms are spaced apart by a carbon chain of 1 to 4 carbon atoms, preferably 2 carbon atoms.

23. The method according to claim 21 or 22, wherein the nitrogen containing organic compound is a heterocyclic compound comprising two or more, fused or ring-assembled nitrogenous rings.

24. The method according to claim 23, wherein the nitrogenous rings are selected from rings derived from pyrrol, imidazol, pyrazol, pyridine, pyrazine, pyrimidine, pyridazine, indol, isoindol, indazol, purine, chinolin, and chinoxaline.

25. The method according to claim 23 or 24, wherein the catalyst is copper phenantroline [Cu(II) 1.10-phenanthrolinate].

26. The method according to claim 21 or 22 wherein aliphatic or aromatic amine c has a general structure selected from

R¹ (R²WN_[CR³R⁴]_M._^N^(R⁵)_nR⁶

wherein each substituent R¹ to R⁶ independently stands for hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or hydroxyl group, which optionally is substituted, or two of said substituents together with an intermediate nitrogen atom form a alicyclic or aromatic cyclic group comprising 1 to 3 rings, each group having 5 to 7 ring members and optionally containing at least one further hetero atom selected from N, O and S, m and n independently stand for 0 or 1, and

^ designates a single or double bond.