WO2009005389A1

WO2009005389A1 - The method of pre-treatment of the cellulose-containing biomass to produce ware-soluble carbohydrates

Info

Publication number: WO2009005389A1
Application number: PCT/RU2007/000364
Authority: WO
Inventors: Anatoly Aleksandrovich Politov; Olga Viktorovna Bershak; Oleg Ivanovich Lomovsky; Kirill Georgievich Korolev
Original assignee: 'arter Technology Limited'; ARTER Tech Ltd
Current assignee: 'arter Technology Limited'; ARTER Tech Ltd
Priority date: 2007-07-04
Filing date: 2007-07-04
Publication date: 2009-01-08
Anticipated expiration: 2010-01-04

Abstract

The present invention relates to the method of preparation of the cellulose- containing biomass to the next enzymatic hydrolysis of the cellulose and conversion of the polysaccharides in the raw stock into water-soluble carbohydrates. The effectiveness of hydrolytic conversion of the cellulose and accompanying polysaccharides into water- soluble carbohydrates can be promoted by combining the pre-treatment of the raw stock with cellulosolytic solutions, drying and subsequent mechanical grinding of the raw stock. The treatment of the original raw stock with cellulosolytic compositions is conducted at moderate temperatures between 20 and 50 °C and low hydromodulus 1 - 8. The operation implies partial hydrolysis of the cellulose and other polysaccharides. Then the raw stock is dried and ground in mills with free or constrained impacting. This preparation of the plant raw stock upsets the crystalline cellulose structure and expands the specific surface. Unlike the grinding conducted under the same conditions but without enzymatic pre-treatment and drying, the yield of the fine fraction is enlarged considerably. The product obtained according to the present method is suitable for further enzymatic hydrolysis into soluble carbohydrates and microbiological processing of the latter into alcohols.

Description

The Method of Pre-Treatment of the Cellulose-Containing Biomass to Produce Ware-Soluble Carbohydrates Field of the Invention

The present invention relates to the processes of pre-treatment of the cellulose- containing biomass and other accompanying polysaccharides and represents the method of intensification of subsequent enzymatic hydrolysis to convert high- molecular carbohydrates in the raw stock into water-soluble carbohydrates. A high enzymatic hydrolysis efficiency of cellulose is achieved by combining pre- treatment of the raw stock with the solutions of cellulosolytic enzymatic preparations, drying and subsequent mechanical grinding of the raw stock . The original raw stock is treated with cellulosolytic preparations at moderate temperatures between 20 and 50 °C and low duty of water 1-8. Then the raw stock is dried and ground in mills with free or constrained impact. Such preparation of the plant raw stock destroys the cellulose crystalline structure and expands the specific surface. Compared with grinding under the same conditions, introduction of enzymatic pre-treatment and drying boosts significantly the yield of the fine fraction.

Background Techniques below are known that enable to extend the effectiveness significantly the enzymatic hydrolysis of the biomass containing cellulose and accompanying polysaccharides.

Most researchers emphasize a pronounced relation between the crystallinity degree of cellulose and the rate of its hydrolysis. The more the cellulose becomes amorphous the higher is the rate of hydrolysis; in a number of cases the concentration rise of the amorphous phase from 20 to 80 percent leads to five or six fold acceleration. The crystalline cellulose biomass can be rendered amorphous by mechanical treatment of the raw stock in devices of various designs, such as extruders, ball, vibrocentrifugal, jet, roll mills etc.

Enzymatic hydrolysis of the cellulose-containing biomass evolves in a heterogeneous medium; hence, the effect of the phase interface on the hydrolysis rate is considerable. Therefore, when treating the cellulose-containing raw stock, all effort is made to achieve low average size of particles and ensure maximum expansion of the phase interface surface. Most of the known approaches to the intensification of enzymatic hydrolysis of polysaccharides relate to the mechanical treatment of the raw stock. In a number of cases, the pre-treatment combines chemical and mechanical effects.

To obtain a fine-dispersed product from the plant cellulose-containing raw stock, it is mechanically ground in various mills, then the obtained product is dried and screened. For instance, the method of producing plant flour (Emil H. BaIz,

Andrew W. Kassay, 1944, US 2362528, B27L 11/06) includes grinding of the raw stock in a drum impact mill, separation of fine particles and repeated grinding in a rotary mill of the remaining raw stock. The method advanced by N. S. Enikopolov, etc. implies that the raw stock is wetted before grinding, and then it is ground in a screw mixer heated to 120 ⁰C under pressure up to 50 MPa (SU 4260749, 1988,

D21B 1/06).

The drawbacks of the above methods of producing fine-dispersed products from the plant cellulose-containing raw stock are wasteful energy consumption for grinding and drying of the raw stock and poor grinding effectiveness. The latter is due to the fibrous structure of the plant raw stock, presence of accompanying cellulose components, such as lignins , hemicellulose that make natural cellulose- containing materials especially strong. Hence, various methods were advance how to produce fine-dispersed products from the plant raw stock, including chemical pre-treatment of the raw stock followed by its mechanical treatment.

Chemical treatment enables to modify the raw stock in the manner that preparation of the raw stock for enzymatic hydrolysis by mechanical treatment would be smoother. These methods comprise acidic hydrolysis of the cellulose and hemicellulose the plant raw stock contains, alkalinic treatment to eliminate lignins and treatment with different solvents.

In particular, a method was advanced how to obtain fine-dispersed products based on the treatment of wood or any other cellulose-containing raw stock with a urea solution followed by drying and grinding in a ball mill (Andrew W. Kassay, et al., 1942, US 2364721, C08L 97/02). To destroy the crystalline cellulose structure and to increase the specific surface of the raw stock in order to intensify the subsequent enzymatic hydrolysis of polysaccharides, it was proposed to treat cellulose-containing waste in two-screw extruders at an elevated temperature (up to 200 °C). As the raw stock, rice corn, cotton lint, guza-paya, etc., can be used (RU 2223327, 2004, C13K 1/02). G.R. Huber et al. proposed to treat the cellulose-containing raw stock in extruders at elevated temperatures and pressure (G.R. Huber, et al., 1992, US 5114488, B30B 11/22, C13K 1/00). Pre-treatment of the cellulose-containing raw stock can be performed in extruders in the presence of alkaline solutions (T. Inoi, et al., 1987, US 4642287, C12P 19/00, C13K 1/000) that enables to achieve high effectiveness of the subsequent enzymatic hydrolysis of polysaccharides in the raw stock.

A method is advanced of mechanical treatment of the lignocellulose raw stock in the presence of water, solutions of alkalis, acids, and enzymes. The method is based on the treatment of pulp in microcavitation devices so that intensive shear strain would destroy the structure of fibers in the lignocellulose biomass boosting the substrate responsivity to subsequent enzymatic hydrolysis (E.D. Stuart, et al., 1996, US 5498766, C08H 5/04, C13K 1/00).

The drawbacks of the above methods of pre-treatment of the cellulose- containing biomass are application of chemical agents restricting use of obtained intermediate products, highly cost of elimination of solvents, acids, etc.

Close technical solutions are methods of treatment of the cellulose-containing biomass when the raw stock is pre-treated with solutions of acids. By the method advanced by D.L. Brink (US 5536325, 1996, B02C 13/18, C13K 1/00), easily hydrolysable polysaccharide in the raw stock (primarily the hemicellulose and amorphous cellulose are subjected to hydrolysis at a temperature 160 °C and higher under the effect of nitric acid. Then the hydrolysate is separated from the dry residue, the latter is subjected to mechanical grounding in impact mills. The obtained ground product possesses higher reactivity in acidic hydrolysis. The fine- dispersed product can also be produced by pre-steaming of the raw stock, its treatment with sulfuric acid solution and following grinding (Z.I. Grebenkina, et al., 1982, SU 1518123). The drawbacks of these methods are use of solution of acids that acids saturate the raw stock, therefore acid resistant metallic grinding, drying and transporting equipment is needed. Summary of the Invention

The technical task is to develop a cheaper, energy saving and environmentally friendly method of pre-treatment of the cellulose-containing raw stock and obtain a fine-dispersed substrate as an intermediate product that would better react in the process of enzymatic hydrolysis of polysaccharides it contains.

The technical result of the present invention is the method of pre-treatment of the cellulose-containing biomass that enables to increase the effectiveness of the subsequent enzymatic hydrolysis and conversion of the polysaccharides in the raw stock into water-soluble carbohydrates. The needed process effect is achieved due to higher effectiveness of mechanical treatment of the raw stock, namely by production of an intermediate product with finer particles.

This result ensures production of the fine-dispersed intermediate product by the method that obviates pre-treatment with acids that can reduce the yield of the target products (partial degradation of carbohydrates). It prevents appearance of toxic substances that may exclude further use of the intermediate product for the subsequent enzymatic hydrolysis into soluble carbohydrates and their microbiological processing into alcohols. The formulated task is solved because by the claimed method the polysaccharides in the raw stock are subjected to partial enzymatic hydrolysis, then the raw stock is dried and undergoes mechanical treatment resulting in the fine-dispersed product .

The following cellulose-containing biomass is applicable to pre-treatment by this method:

3.1. Al kinds of plants can serve as the raw stock, namely, algae, grass, bush, and trees. All parts of these plants can be used that contain cellulose.

3.2. The raw stock can also include wastes of agriculture, food industries, and woodworking. 3.3. The fine-dispersed product in the present context implies powder with sizes

500 μm and smaller obtained from the raw stock listed in item 3.1 above.

It is preferable to treat with a solution of the enzymes possessing hydrolytic activity towards polysaccharides and do it at the ratio between the solid substrate and the solution (S: L) between 1 : 1 and 1 :8 at a temperature not higher than 60 °C. The sense of the invention is that the plant raw stock containing cellulose is subjected to treatment with a complex of enzymes prior to grinding.

The enzymes catalyze the cellulose breaking the bonds of the polymeric chain and partial dissolution of the amorphous cellulose portions. In its turn, it destroys the supramolecular cellulose structure. The degree of enzymatic hydrolysis can amount to less than 1-2 percent of the total cellulose mass, but it is enough to upset the integrity of the cellulose skeleton and to reduce noticeably its strength.

After the enzymatic treatment, the weaker plant raw stock than the original raw stock disintegrates more effectively. Hence, the fine-dispersed intermediate product the cellulose contains with the upset supramolecular structure and a high specific structure can be produced by a milder mechanical treatment compared with the raw stock untreated with enzymes.

Unlike the selective effect of enzymes, the treatment of the cellulose-containing biomass with alkalis, acids and other chemical agents produces the hydrolysis of polysaccharides without any accumulation of toxic products in the material after treatment. The chemical treatment inhibits further use of biochemical

(biotechnologϊcal) of the intermediate product as a source of carbohydrates.

Brief description of the drawings Fig. 1 shows the granulometric composition (in %) of the intermediate product resulting from mechanical grinding of saw dust.

Fig. 2 shows the grain-size distribution (in %)of the intermediate product resulting from grinding by the present method, example 1.

Fig. 3 shows the grain-size distribution (% by mass) of the intermediate product resulting from grinding of the raw stock (the corn straw) in the disintegrator, example 2.

Fig. 4 shows the grain-size distribution (in %) of the intermediate product according to example 2.

Fig. 5 shows the grain-size distribution (% by mass)of the intermediate product resulting from mechanical grinding of wheat straw.

Fig. 6 shows the grain-size distribution (% by mass)of the intermediate product resulting from grinding of the raw stock by the present method, example 3. Fig. 7 shows the grain-size distribution (% by mass)of the intermediate product resulting from mechanical grinding of wood sawdust without pre-treatment with the solution of enzymes .

Fig. 8. shows the grain-size distribution (% by mass)of the intermediate product resulting from grinding of the raw stock by the present method, example 4. Fig. 9 shows the grain-size distribution (% by mass)of the intermediate product resulting from mechanical grinding of the corn straw without pre-treatment with the solution of enzymes .

Fig. 10 shows the grain-size distribution (% by mass)of the intermediate product resulting from grinding of the raw stock by the present method, example 5.

Fig. 1 1 shows the sequence of deformation during grinding. Above (A) - constrained impact, below (B) - free impact.

Preferred embodiment Detailed Description of the Invention The proposed method is embodied in the following manner.

The cellulose-containing raw stock is mixed with the solution of the enzymes in their concentration up to 1 % by mass at the ratio between the solid substrate and the solution (S: L) between 1 :1 and 1 :10; the mixture is kept during 1-24 hours at a temperature not higher than 60 ⁰C . The enzymes induce partial hydrolysis of the polysaccharides in the raw stock. The enzymatic complexes containing endo- and exodepolymerases hydrolytically active towards the cellulose , hemicellulose and starch, can serve for the hydrolysis . After treatment with the solution of the enzymes, the raw stock is dried at a temperature not higher than 100⁰C and subject to mechanical grounding . The pre-treatment of the raw stock with the solution of enzymes enables:

• to conduct partial enzymatic hydrolysis of inner glycoside links in the polysaccharide molecules upsetting the cellulose supramolecular structure and integrity of plant fibers expending the effectiveness of subsequent mechanical treatment; • to avoid the energy consuming stage and use of acids .

The published patents show no such methods of preparation of the cellulose- containing biomass for the following enzymatic hydrolysis to produce water- soluble carbohydrates. Hence, the claimed method satisfies the criterion of novelty.

The claimed technical solution has the following distinctive features: • the raw stock is subjected to the partial enzymatic hydrolysis prior to mechanical treatment of ;

• treatment with the solution of enzymes lasts 1-24 hours ; • If it is necessary to intensify the process of hydrolysis, the mixture of the raw stock and the solution of enzymes is treated with ultrasound.

The combination of the substantial distinctive features is unknown at the current state-of-the-art, still it enables to resolve the formulated task and to conclude that the claimed technical solution satisfies the criterion of the invention level.

The plant raw stock of different origins includes the main components, such as cellulose , hemicellulose , lignins , pectins, starchy substances, and. minor components, such as fats, plant protein, low-molecular organic compounds, inorganic compounds. Cellulose is the main component of cellular membranes of higher plants.

Together with its accompanying substances, it acts as a skeleton enduring the most of the mechanical loading.

Cellulose has a complex supramolecular structure resulting from intermolecular interactions between molecules. The weakest cellulose link is the primary fibril in which groups of parallel-arranged macromolecules are cross-linked by multiple hydrogen bonds. The cellulose macromolecules in the primary fibrils form highly ordered crystalline zones alternating with inhomogeneous, less ordered amorphous zones. The crystalline zones of the primary fibril zones extend for 15 run, their cross section is 3-7 nm. The hydrogen bonds of microfibrils of the cellulose primary fibrils cross-link and form main links of the structure of cellulose fibers. According to the currently generally accepted Freigh-Wissling model, occluded water, lignin, and hemicellulose between the primary fibrils play a substantial role in formation of microfibrils. This specific setup of the cellulose morphological structure ensures its stability when exposed to considerable mechanical loads. Cellulose is also quite stable to acids , enzymes, and microorganisms .

Hemicellulose is a polysaccharide that plant tissues contain together with cellulose and lignin in the plant cellular membranes. It is a branched polymer of different structures, the main monomeric components of the hemicellulose being xylose , arabinose, galactose, glucose, mannose and uronic acid .

Hemicellulose differs from cellulose by better solubility in alkaline solutions and reactivity in the processes of acidic and enzymatic hydrolysis. As a rule, the degree of polymerization of hemicellulose is inferior to that of cellulose. The monosaccharide remnants are usually linked by β-l,4-bonds, they have the lateral bonds of a different type. The hemicellulose main component is xylose (50-70 % of monomeric links), the main hemicellulose class is xylanes . Lignin is an amorphous cross-linked phenolic polymer that only vascular plants have and that can amount to 30 % of their mass.

Compared with the crystalline cellulose, hemicellulose and amorphous lignocellulose material undergo much easier the catalytic hydrolysis producing water-soluble carbohydrates . Hydrolysis of polysaccharides can be conducted using two main types of catalysts: acids and enzymes . The solid residue after hydrolysis with sulfuric acid contains lignins and unhydrolyzed cellulose .

The common biochemical catalyst are enzymes (cellulases , xylanases, etc.), that are produced, as a rule, in the form of a complex preparation by superfiltration of cultural fluids of definite microorganisms . The enzymes in the compositions of these complexes possess definite specialization: some of them effectively hydrolyze inner glycoside links with monosaccharose remnants remote from polysaccharide terminal groups (endopolymerases, endogluconases, endoenzymes ); others preferably split the external glycoside links at the ends of the polysaccharide chain (exodepolymerases, exogluconases, exoenzymes ); still others, glucosidases, hydrolyze glycoside links of di- and oligosaccharides.

The essence of the invention is that, under the effect of the enzymatic complex, the amorphous cellulose is hydrolyzed and it determines the strength of the crystalline cellulose and hemicellulose fibers ensuring adhesive bonding of the cellulose and lignin fibers.

Depending on what part of the cellulose and hemicellulose is hydrolyzed and disintegrate, several mechanisms of weakening of the wood can be assumed. Denser parts in the microfibrils of the crystalline cellulose alternate with amorphous less dense parts. Such system can be represented as a sequence of chained elastic and viscous elements. In case of mechanical deformation, the viscous elements restrain the elastic deformation . In case of enzymatic hydrolysis, the susupramolecular structure of the cellulose amorphous with higher reactivity is the first to fail. The remaining crystalline potion is intensively amorphized when the deformation is more extensive. This breaks microfibrils and weakens the particles.

The glycoside links in the crystalline cellulose can disrupt in case of the enzymatic hydrolysis facilitating the breaking of the microfibril fibers. Finally, the hydrolysis of hemicellulose breaks the link between cellulose and the lignin matrix. When the structure of such natural composite is upset, the strength of the particles in the raw stock weakens too.

In general, the selective disintegration of the cellulose and hemicellulose produces a material with new mechanical properties that enable to obtain the fine- dispersed intermediate product with a higher reactivity more effectively on the devices and mills of various types.

As enzymatic preparations, it is preferable to use mass-produced compositions produced by Sibbiopharm (Russia), Genencor International, Iogen Corporation, Novozyme, Primalco. The useful enzymatic preparations can be produced directly while producing the compositions of enzymes that are separated by superfiltration of the cultural fluids of Trichoderma viride (reesei) and/or Aspergillus awamori and/or Bacillus subtilis.

The mass-produced composition with the cellulase and xylonase activity of Genencor or Sibbiopharm (Russia) was used in the paper. Mechanical treatment and mechanochemical activation

The mechanical treatment of solids is a well-known process that leads to grinding and activation. Activation is intensification of reactivity of treated materials and substances that is due both to growth of the specific surface in grinding and formation of pinpointed and linear defects in the solid, occurrence of amorphization and formation of radicals. Changes in the physical-chemical properties of the solids and evolution of their chemical transformation during mechanical treatment are studied by mechanochemistry, so the chemical reactions under such conditions are termed mechanochemical reactions. Due to the process intricacy, the mechanical effect on dispersed solids and due to a large variety of the physical-chemical properties of solids, at present there is no general theory equally well describing the mechanochemical processes (Beyer, M.K., Clausen- Schaumann, H., Mechanochemistry: the Mechanical Activation of Covalent Bond, Chem. Rev., 2005, vol.105, no. 8, pp. 2921 - 2948). H. Rumpf (Chem. Ing. Techn., 1959, vol. 31, pp. 333) assumed that there two principally different types of mechanical actions applied to solids. With one type of the effect, the particles experience a constrained impact between two moving surfaces , while another type of particles interacts with just one moving surface. The diagram in Fig. 11 illustrates well the difference between these effects (Heinicke, G., Tribochemistry, 1984, Berlin: Akademie-Verlag). According to this diagram, the mills and activators can be divided into two types whether the material is subjected to a constrained or a free impact. Though both grinding and activation occur with these two types of effects, it is customary to assume that alongside with grinding the conditions appear in the mills with the constrained impact for mechanical activation, meanwhile, grinding occurs in the first place in the mills with the free impact.

When treating the plant raw stock in the constrained impact mills the processes of deformation and mixing take place that can lead to decomposition of the organic matter, including polysaccharides. Therefore, if the biomass containing biologically active substances is ground, it is essential to select a proper device for their mechanical treatment . Usually ball or hammer mills are selected for grinding wood. However, we have shown that, after enzymatic pre-treatment, both the mills with the free impact, vortex mills, and disintegrators yield also effectively the fϊne- dispersed intermediate product.

The mechanical treatment is conducted with the well known equipment. The mills preferable for the purposes of the invention should possess definite operating parameters, such as the impact intensity and duration of the operation. The examples are the following: • planetary ball mills or vibratory ball mills in which the intensity of mechanical treatment is characterized by the acceleration of balls. The optimum acceleration range of balls is 60-400 m/s². For comparison, the coefficient of acceleration of the balls in a common gravitation mill is about 10 m/s². The time in the zone of treatment is 0.5-2.0 minutes. The mass-produced vibration mills of the VCM and CEM series by Novits (Russia) or Tribochem (Germany) are applicable for the process;

• rotary mills in which grinding is effected by collision of particles with the vanes that have speed 6,000-12,000 r.p.m. the time in the zone of treatment is 0.2 - 2.0 minutes. For example, the disintegrators and mass-produced rotary mills of Titan (Saint-Petersburg, Russia) or Arter (Moscow) are applicable for the process;

• vortex or jet mills in which particles of the original material are accelerated by the air or gas stream to 10-120 m/s. The treatment of the material is de to collisions of particles against deflecting obstacles. The time in the zone of treatment is 0.2-2.0 minutes. The mills Vortex Mills of Hydan Technologies (USA) or Jet Micronizers of Sturtevent Inc. (USA) and vortex pneumatic mills of the VIT Ltd. (Novosibirsk, Russia) can be used. The mechanical treatment can be conducted within a broad range of intensity yielding close results. The duration and intensity of the mechanical treatment are selected to avoid the conditions when a significant amount of enzymes undergoes denaturation.

The lower limit of intensity and duration of the mechanical treatment with the said equipment types is determined by the moment the process of disintegration of the particles of the consumed enzymes begins in the raw stock. The upper limit is determined by the moment the degradation of the reacting components begins, primarily by reduction of the activity of the enzymes.

The lower and upper frequency limits of the ultrasound treatment are 17 and 44 kHz, respectively. They are determined by similar processes of the onset of disintegration of associations of particles and components.

Examples . The following examples explain the present. Example 1. The pre-treatment of wood sawdust.

Enzymatic treatment . Wood sawdust was treated with a 0.5 % solution of enzymes obtained from the cultural fluid of Trichoderma viride (reesei). The solution of the enzymes was prepared on the citrate buffer with pH 5. The S:L ratio was 1 :5. The substrate was exposed to the temperature 50 °C during 24 hours.

Then the raw stock was dried at the temperature 100 °C.

Mechanical treatment . Dried sawdust was ground in the Fritsch Pulverizette mill (Germany) during 2 minutes.

Table 1 shows the grain-size composition of the fine-dispersed product obtained according to this example. The table shows that the sizes of particles diminish, the obtained intermediate product contains 30 % by mass the particles smaller than 100 μm, meanwhile the original sawdust contains 4 % by mass of the particles smaller than 100 μm, meanwhile the ground particles account for 11 % if ground without pre-treatment with the solution of enzymes Fig. 1 shows the grain-size composition (in %) of the intermediate product resulting from mechanical grinding of sawdust.

Fig. 2 shows the grain-size composition (in %) of the intermediate product resulting from grinding, according to the present method. The grinding according to the present method yields the method yields the intermediate product with smaller particles .

Example 2. Pre-treatment of corn straw.

Enzymatic treatment . the corn straw was treated with the solution of a mixture of enzymes obtained from the cultural fluid of Trichoderma viride (reesei) and Bacillus subtilis in the ratio 4:1, respectively, (pH 4,7; 1 % of the enzymes to the substrate mass). The ratio of the substrate to the solution of enzymes is 1 :8. Hydrolysis was conducted at 50 °C during 20 hours . Then the raw stock was dried at the temperature 100 °C. After drying, the treated enzymes in the raw stock were ground in the laboratory disintegrator IA 28 (Disintegrator, Estonia).

Table 2 shows the grain-size composition of the intermediate product obtained according to the present example; it shows too the grain-size composition of the original raw stock and the intermediate product resulting from the mechanical grinding of the raw stock without the enzymatic pre-treatment .

Fig. 3 shows the grain-size composition (% by mass) of the intermediate product resulting from grinding of the raw stock (the corn straw) in the disintegrator. Fig. 4 shows the grain-size composition (in %) of the intermediate product obtained according to the present example .

These data vividly demonstrate how the sizes of particles according to the present method diminish.

Example 3. Pre-treatment of wheat straw. Enzymatic treatment . the raw stock (the wheat straw) was treated with the solution of the mixture of enzymes obtained from the cultural fluid of Trichoderma viride (reesei) and Aspergillus awamori in the ratio 4:1, respectively, (pH 4,7; 1 % enzymes to the substrate mass). The ratio of the substrate to the solution of enzymes was 1 :5. Hydrolysis was conducted at 50 °C during 24 hours . Then the raw stock was dried at the temperature 100 °C.

Mechanical treatment of the raw stock . The raw stock treated with enzymes was ground in the vortex pneumatic mill MP-20, the VIT Ltd. (Novosibirsk, Russia).

Table 3 shows the grain-size composition of the intermediate product obtained according to the present example , the grain-size composition of the original raw stock and the intermediate product resulting from mechanical raw stock with out enzymatic pre-treatment . It shows vividly how the sizes of particles diminish after treatment according to the present method.

Fig. 5 shows the grain-size composition (% by mass) of the intermediate product resulting from the mechanical grinding of the wheat straw.

Fig. 6 shows the grain-size composition (% by mass) of the intermediate product resulting from the grinding of the raw stock according to the present method. The grinding yields the product with smaller particles . Example 4. Pre-treatment of wooden sawdust.

Enzymatic treatment . The wooden sawdust was treated with the solution of the enzymatic complex Cellolux of Sibbiopharm, Russia (pH 4.5; 0.5 % of the complex to the substrate mass) with the S:L proportion 1 :5. Hydrolysis was conducted at 50 °C during 24 hours . Then the raw stock was dried at the temperature 100 °C.

The mechanical treatment implied grinding of the raw stock treated with enzymes in a vortex pneumatic mill . Table 4 shows the grain-size composition of the intermediate product according to the present example , the grain-size composition of the original raw stock and the intermediate product resulting from the mechanical grinding of the raw stock without enzymatic pre-treatment. It shows vividly how the sizes of particles diminish after treatment according to the present method. Fig. 7 shows the grain-size composition (% by mass) of the intermediate product resulting from mechanical grinding of wooden sawdust without pre- treatment with the solution of enzymes . Fig. 8. shows the grain-size composition (% by mass) of the intermediate product resulting from grinding of the raw stock according to the present method. The grinding yields the product with smaller particles .

Example 5. Pre-treatment of corn straw. Enzymatic treatment . The raw stock was treated with the solution of the enzymatic complex Cellolux of Sibbiopharm, Russia (pH 4.7;

0.5 % of the complex to the substrate mass) with the S:L proportion 1 :5. Hydrolysis was conducted at 50 °C during 24 hours , the pulp was periodically exposed to ultrasound with the frequency 17-44 kHz. The treatment duration was 5-7 minutes with the periodicity 1-2 times per hour. Then the raw stock was dried at the temperature 100 ⁰C.

Mechanical treatment of the raw stock . The raw stock treated with enzymes was ground in the planetary mill APF-4 (Institute of Chemistry & Technology of the Siberian Branch of the Russian Academy of Science, Novosibirsk, Russia). The enzymatic hydrolysis together with pulp ultrasound treatment expands the specific surface of the raw stock, mainly because the number of macropores grows. A larger specific surface capable to sorb the enzymes (i.e. the number of macropores) is accompanied by a higher degree of hydrolysis of polysaccharides in the raw stock facilitating considerably the next process of grinding . Table 5 shows the grain-size composition of the intermediate product obtained according to the present example, the grain-size composition of the original raw stock and the intermediate product resulting from the mechanical grinding of the raw stock without enzymatic pre-treatment. It shows vividly how the sizes of particles diminish after treatment according to the present method. Fig. 9 shows the grain-size composition (% by mass) of the intermediate product resulting from mechanical grinding of the corn straw without pre-treatment with the solution of enzymes .

Fig. 10. shows the grain-size composition (% by mass) of the intermediate product resulting from grinding of the raw stock according to the present method. The grinding yields the product with smaller particles .

Example 6. Enzymatic hydrolysis of the fine-dispersed intermediate product according to the present method. A sample of the fine-dispersed intermediate product (4 grams) was placed in a 0.05-0.1 M acetate or citrate buffer pH = 4.5 (40 ml) containing 0.1-0.2 % formaldehyde as a preservative . The obtained mixture was hydrolyzed while stirring in a magnetic mixer in a glass reactor at a temperature 48 ± 1 ⁰C. After 10 hours, the initial average hydrolysis rate was measured. The concentration of the water-soluble carbohydrates was determined by high efficiency liquid chromatography. Table 6 shows the results.

The Table shows that pre-treatment combining partial enzymatic hydrolysis , drying and mechanical grinding accelerates the initial hydrolysis rate 50-70 % versus the usual mechanical grinding .

It is established that enzymatic pre-treatment boosts the effectiveness of amorphization of the crystalline cellulose . The crystallinity index determined according to Seagull was 65-70 % of the original raw stock 35-40 % of the samples ground without pre-treatment with enzymes, 15-25 % for the samples obtained according to examples 1 -4 with pre-treatment with the solution of enzymes, 10-14 % according to example 5. An extra advantage of the claimed approach is significant expansion of the specific surface. For instance, the specific surface of the original biomass was 0.9-1.2 m²/g, that of the samples after mechanical treatment without preliminary enzymatic hydrolysis was 0.9-1.6 m /g, that of the samples obtained by grinding of the biomass after contact with the solution of enzymes was 2.5-4.5 m²/g. the fine-dispersed intermediate product obtained according to example 5 demonstrated the maximum specific surface.

Table 1. Grain-size composition (% by mass) of the intermediate product according to example 1.

Table 2. Grain-size composition (% by mass) of the intermediate product according to example 2.

Table 3. Grain-size composition (% by mass) of the intermediate product according to example 3.

Table 4. Grain-size composition (% by mass) of the intermediate product according to example 4.

Table 5. Grain-size composition (% by mass) of the intermediate product according to example 5.

Table 6. The initial hydrolysis rate of the fine-dispersed intermediate product (in percent in respect to the intermediate product obtained by mechanical treatment without preliminar enz matic hydrol sis.

Thus, the invention led to a cheaper, more energy saving and environmentally friendly method of pre-treatment of the cellulose-containing raw stock yielding an intermediate product on the fine-dispersed substrate possessing high reactivity towards the process of enzymatic hydrolysis of polysaccharides it contains. Industrial Applications

The present invention is embodied with multipurpose equipment extensively employed by the industry.

Claims

1. A method of pre-treatment of the cellulose-containing biomass to produce ware-soluble carbohydrates that results from enzymatic pre-treatment of the plant cellulose-containing raw stock by drying the treated raw stock and mechanical grinding of the treated and dried raw stock in a mill to the particle size under 160 μm.

2. The method in claim 1 characterized in that the enzymatic treatment is conducted at the room temperature.

3. The method in claim 1 characterized in that the treatment of the raw stock is conducted at such ratio of the solid substrate to the solution at which the original biomass retains its looseness.

4. The method in claim 1 characterized in that the enzymatic composition is used as a powder or a cultural fluid obtained by cultivating microorganisms producers of enzymes capable to hydrolyze polysaccharides. 5. the method in claim 1 characterized in that the mechanical treatment is performed in ball planetary or vibratory mills with acceleration of the grinding balls from 60 to 400 m/s² during 0.

5-2.0 minutes, or in rotary mills with the speed of the grinding rotors 10-120 m/s during 0.2-2.0 minutes, or in pneumatic vortex mills with the gas flow rate 10-120 m/s during 0.2-2.0 minutes, or in designators with the speed of the blades 6,000-12,000 r.p.m.

6. The method in claim 1 characterized in that the enzymatic hydrolysis preceding grounding is conducted with periodic exposure of the pulp to ultrasound with the 17-44 kHz.