Enzymatic hydrolysis of biomasses having a high dry matter (dm) content

Procedure for liquefaction and saccharification of polysaccharide containing biomass.

AF

214493

Field of the Invention

The present invention relates to a process for liquefaction and saccharification of biomass containing polysaccharides, having a high dry matter content and preferably having fibers and particles with a large average size. In addition, the present invention relates to the additional use of said processed biomass, for example for subsequent fermentation in bio-ethanol, special carbohydrates for nutrients and foods, as well as carbon filler material for processing for plastics and chemicals.

Background of the Invention

Numerous industrial and agricultural procedures, for example, municipal operations, nutrient processing and food and forest engineering, generate biomass, wastes and by-products containing polymeric sugars, for example in the form of starch, cellulose and hemicellulose. Agribusiness and chemical industries, as well as public organizations have a great interest in the development of procedures to convert such biomass into higher value materials. Therefore, by way of example, said biomass could potentially be converted into bioethanol, biogas or chemicals, using microorganisms and / or hydrolytic enzymes. However, most of the currently known procedures have not achieved a large-scale commercial practice due to their high cost of production, their high demand for energy and therefore due to the inherent uncertainty of economic feasibility.

In addition to being important as food and nutrients, biomass carbohydrates can be used as fillers for a large number of industrial processes. A well-known product in the form of polymers is paper, where cellulose is the main component. However, when processed in the form of oligomers and monomers, carbohydrates constitute an important loading material for a variety of industrial processes. As will be described in detail, they are necessary for a variety of microbial processes, but they can also be used as a loading material for, for example, enzymatic processing for obtaining special carbohydrates, for food and nutrients, for example trehalose. In addition, oligomers and monomers can substitute petrochemicals to process plastics and organic chemicals. In addition, carbohydrates can be used as hydrogen carriers in catalytic hydrogenation.

Therefore, it is clear that if an economical and abundant source of carbohydrates can be achieved for industrial processes, this could have substantial economic potential.

Starch is the storage carbohydrate most found in plants and occurs in the form of granules, which differ markedly in size and physical characteristics from species to species. Starch granules are generally quite resistant to the penetration of both water and hydrolytic enzymes, due to the formation of hydrogen bonds within the same molecule and with other adjoining molecules. However, these inter- and intra-hydrogen bonds can weaken as the temperature of the suspension rises. When an aqueous suspension of starch is heated, hydrogen bonds weaken, water is absorbed, and starch granules swell. This process is commonly called gelatinization, because the solution formed has a gelatinous, highly viscous consistency. Chemically, starch is a natural glucose polymer, which is generally insoluble, but dispersible in water at room temperature and is constituted by a repeating unit similar to that of cellulose and is linked together by glucosidic bonds a-1,4 and a -1.6, as opposed to the β-1,4 glucosidic bonds for cellulose. The units form a linear chain component, called amylose, or a branched chain component, called amylopectin. Most plant seeds, grains and tubers contain approximately 20-25% amylose. But some, such as pea starch, have 60% amylose and certain species of corn have 80% amylose. Waxy grain varieties, such as rice, have a low amylose content.

Apart from starch, the three main constituents in plant biomass are cellulose, hemicellulose and lignin, which are commonly referred to by the generic term, lignocellulose. Biomass that contain polysaccharides as a generic term include both starch and lignocellulosic biomass.

Cellulose, hemicellulose and lignin are present in different amounts in different plants and in different parts of the plant and are closely associated to form the structural skeleton of the plant.

Cellulose is a homopollsaccharide composed entirely of D-glucose linked together with p-1,4-glucosidic bonds and with a degree of polymerization of up to 10,000. The linear structure of cellulose allows the formation of both intra- and intramolecular hydrogen bonds, which results in the aggregation of cellulose chains in micro fibrils. The regions with high order micro fibrils are called crystalline and with less ordered regions they are called amorphous. The micro fibrils are assembled into fibrils, which then form the cellulose fibers. The partially crystalline cellulose structure together with the microfibrillar arrangement provides cellulose with high tensile strength, makes cellulose insoluble in most solvents, and is partially responsible for cellulose resistance against microbial degradation, is say enzymatic hydrolysis.

Hemicellulose is a complex heterogeneous polysaccharide composed of a variety of monomeric residues: D-glucose, D-galactose, D-mannose, Dxilose, L-arabinose, D-glucuronic acid, and 4-O-methyl-D-glucuronic acid . Hemicellulose has a degree of polymerization below 200, has secondary chains and may be acetylated. In softwoods, such as spruce, pine and spruce, galactoglucomannan and arabino-4-O-methyl-glucuronoxylan are the main hemicellulose fractions. In hardwoods, such as birch, aspen, white poplar or oak, 4-O-acetyl-glucuronoxylan and glucomannan are the main constituents of hemicellulose. Pastures such as rice, wheat, oats and grass have hemicellulose composed mainly of glucuronoarabinoxylan.

Lignin is a complex network formed by the polymerization of phenyl propane units and constitutes the most abundant non-polysaccharide fraction of lignocellulose. The three monomers in lignin are p-coumaryl alcohol, conferl alcohol and synaptic alcohol, and very frequently they are linked through aryl glyceryl-aryl ether bonds. Lignin is linked to hemicellulose and incorporates carbohydrates, thus offering protection against microbial and chemical degradation.

As stated earlier, processed biomass could potentially be converted into bioethanol or chemicals, through the use of microorganisms and / or hydrolytic enzymes, or the carbohydrates of processed biomass could be used as a loading material for a variety of industrial processes, for example in enzymatic procedures for obtaining special carbohydrates for food and nutrients or as substitutes for petrochemicals in the production of plastics and organic chemicals. In addition, the processing of biomass carbohydrates, in accordance with the present invention, can be combined with the separation and fractionation of non-carbohydrate components. A particularly preferred use of a process according to the present invention is an Integrated part of a process for the production of blo-ethanol.

The production of blo-ethanol from biomass containing polysaccharides can be divided into three stages: 1) pre-treatment, 2) hydrolysis of polysaccharides in fermentable carbohydrates 3) and fermentation of carbohydrates.

Pretreatment is required when subsequent hydrolysis (for example enzymatic hydrolysis) of polysaccharides requires the decomposition of a structure, which on the other hand is protective (for example lignin) of plant materials. Several pre-treatment techniques are known. For cereals and grains, this pre-treatment can be in the form of a simple dry grinding so that the surfaces become accessible, but thermal and / or chemical processes are also needed for lgnocellulosic biomass. A biomass containing polysaccharides consisting, for example, of refined starch, does not require such pre-treatment methods before the enzymatic process. The pretreatment processes may be based on acid hydrolysis, steam explosion, oxidation, alkali or ethanol extraction, etc. A common feature of pre-treatment techniques is that, in combination with the action of possible added reagents, they take advantage of the softening and loosening of plant materials produced at temperatures above 100 ° C.

Following the pre-treatment, the next step in the use of biomass containing polysaccharides for the production of bio-ethanol or other biochemicals, is the hydrolysis of the starch, cellulose or hemicellulose released into fermentable sugars. If done enzymatically, a large number of different enzymes are required, with different modes of action. Enzymes can be added externally or the microorganisms that develop in the biomass can provide them.

Cellulose is hydrolyzed into glucose by carbohydrolytic cellulases. The prevailing understanding of the cellulolytic system divides cellulases into three classes; exo-1,4-pD-glucanases or cellobiohydrolases (CBH) (EC 3.2.1.91), which dissociate the cellobiose units from the ends of the cellulose chains; endo-1,4-β-D-glucanases (EG) (EC 3..2.1.4), which hydrolyse the random β-1,4glucosidic bonds in the cellulose chain; 1,3-β-D-glucosidase (EC 3.2.2.21) that hydrolyze the cellobiose to glucose and also dissociate glucose units from celooligosaccharides.

The different sugars in hemicellulose are released by hemicellulases. The hemicellulite system is more complex than the cellulotic system due to the heterologous nature of the hemicellulose. The system involves among others, the sendo-1,4-β-D-xylanases (EC 3.2.1.8), which hydrolyse the internal bonds in the xylan chain; 1,4-β-D-xylosidases (EC 3.2.1.37), which attack xylooligosaccharides from the non-reducing end and release xylose; endo1,4β-D-mannanases (EC 3.2.1.78), which dissociate the internal bonds; 1,4-β-ϋmannosidases mannanases (EC 3.2.1.25), which dissociate mannooligosaccharides from mannose. Secondary groups are removed by a variety of enzymes; α-D-galactosidases (EC 3.2.1.22), α-L-arabinofuranosidases (EC 3.2.1.55), α-Dglucuronidases (EC 3.2.1.139), cinnamoyl esterases (EC 3.2.2.-), acetyl xilan esterases (EC 3.1.1.6) and feruloyl esterases (EC 3.1.1.73).

The most important enzymes used in starch hydrolysis are alpha-amylases (1,4-α-d-glucan glucanhydrolases, (EC 3.2.1.1). These are endo-active hydrolases that dissociate the 1,4- bonds. α-d-glucosides and may exceed, but not hydrolyze, the 1,6-alpha-D-glucosidic branching points, but also exo-active glycoamylases such as beta-amylase (EC 3.2.1.2) and pululanase ( EC 3.2.1.41) can be used for starch hydrolysis. The result of starch hydrolysis is mainly glucose, maltose, maltotirose, α-dextrin and varied amounts of oligosaccharides. When a starch-based hydrolyzate is used for fermentation, it may be advantageous to add proteolytic enzymes. Such enzymes can prevent the flocculation of the microorganism and can generate amino acids available to the microorganism.

In combination with the pretreatment and enzymatic hydrolysis of lignocellulosic biomass, it has been found that the use of oxidative enzymes can also have a positive effect on general hydrolysis, as well as on the viability of the microorganisms used for, for example, fermentation subsequent. The reason for this effect is the oxidative cross-linking of lignins and other phenolic inhibitors caused by oxidative enzymes. Typically, laccase (EC 1.10.3.2) or peroxidase (EC. 1.11.1.7) is used either externally or by incorporation of a laccase gene into the applied microorganism.

The enzymatic hydrolysis of biomass has been previously described. However, in the case of lignocellulosic biomass, only the material consisting of fibers and particles with an average size below 25.4 mm (1 inch) and which also has a relatively low dry matter content, that is to say Below 20% (w / w), they have been successfully hydrolyzed by this method.

US4409329 describes the hydrolysis of the solid cellulose material to sugar, where the cellulose is hydrolyzed to simple sugars by treating a 3-20% granular suspension (w / w) of solid nutrients containing 30-80% (w / p) cellulose, with an enzymatic cellulase complex. The filler material containing solid cellulose had an average particle size of from 0.0254 mm - 25.4 mm (0.01 to 1 inch) in diameter. For mixing, perforated rotor blades were used.

US 2002117167 A describes the enzymatic hydrolysis of hemicellulose to biomass material, which comprises solubilizing at least a portion of hemicellulose and hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. The biomass used is preferably an aqueous suspension of crude or pretreated material. The biomass material can be any cellulosic material that includes hemicellulose. The procedure is described as an especially effective procedure with grain fibers such as corn, rice, wheat, oats or barley.

US 2004005674A describes a process for enzymatic hydrolysis of lignocellulose. The degradation of lignocellulose to sugars comprises contacting the lignocellulose with at least one auxiliary enzyme and with at least one cellulase. The lignocellulosic material was crushed (the average fiber size of the material was not specified) and had a low dry matter content (0.2 g of crushed forage material in 10 ml of enzymatic solution).

Compendium of the invention

The present invention relates to a process for liquefaction and saccharification of biomass containing polysaccharides, which has a relatively high dry matter content, preferably above 20%, and which preferably consists of relatively large fibers and particles where at least 20% (w / w) of the biomass is within 26-70 mm. In addition, the process is particularly suitable for liquefaction and saccharification of biomass containing polysaccharide consisting mainly of starch, refined starch, cellulose, hemicellulose and lignin, for example grains or wheat straw. In the case of lignocellulosic biomass, these are preferably pre-treated, subjecting them to temperatures between 110-250 ° C for 1-60 minutes in a manner that ensures the accessibility of cellulose to enzymes and at the same time ensures a limited content. of fermentation inhibitors in pre-treated biomass. The present invention combines enzymatic hydrolysis based on the combination of hydrolytic enzymes that include a carbohydrolytic enzyme and an oxidative enzyme with a type of mixture that is based on the principle of gravity that ensures the application of mechanical forces, mainly shear and shear forces. tearing, for biomass. Preferred types of mixers are for example free fall mixers, such as drum mixers, rotating mixers, or similar mixing devices.

Description of the invention

The production of concentrated sugar solutions is beneficial in relation to subsequent fermentation or other microbial processes due to the improved volumetric productivity and the reduced cost of downstream processing. In the case of bio-ethanol production, the energy requirements for distillation are significantly reduced if the fermentation broth contains more than 4% ethanol) Galbe Y Zacchi, 2002). This requires a sugar concentration above 8%, which with most types of lignocellulosic biomass corresponds to an initial dry matter content of 20%. In other words, it is desirable to subject the biomass containing polysaccharides with high dry mass content, preferably above 20%, for enzymatic hydrolysis in order to subsequently produce fermentation broths containing bio-ethanol, suitable for distillation. of ethanol

The processes of the present invention provide a degree of enzymatic hydrolysis of typically 30-50%. However, under optimal conditions, even a higher degree of enzymatic hydrolysis can be obtained. The liquefied and saccharified biomass will consequently contain relatively large amounts of glucose, xylose, cellobiose, non-degraded cellulose lignin and hemicellulose and even active enzymes appropriate for further processing, i.e. fermentation processes (ethanol, lactic acid, etc.). Liquefied biomass will also be appropriate for gasification, hydrogenation, organic synthesis, or biogas and nutrient production.

If the biomass containing polysaccharide is Ignocl-3, the pretreatment must ensure that the structure of the lignocellulosic content will be more accessible to enzymes, and at the same time the concentrations of harmful inhibitory by-products such as acetic acid, furfural and hydroxymethyl furfural will remain substantially low. There are several strategies to achieve this, which imply, all subject the lignocellulosic material at temperatures between 110-250 ° C for 1-60 minutes for example:

• Extraction of hot water • Hydrolysis of dilute acid, in multiple stages, which removes dissolved material before the inhibitory substances are formed • Hydrolysis of diluted acid under conditions of relatively low intensity • Wet alkaline oxidation • Vapor explosion • Almost any pre-treatment with subsequent detoxification

Biomass containing polysaccharides according to the present invention include any material containing polymeric sugars, for example, in the form of starch, as well as in the form of refined starch, cellulose and hemicellulose. Biomass having a dry matter content above 20% are preferred.

Relevant types of biomass for enzymatic hydrolysis and mixing, according to the present invention, may include biomass derived from agricultural crops such as for example:

• Starch, for example grains containing starch and refined starch • corn fodder • Bagasse • ​​Straw, for example of rice, wheat, rye, oats, barley, rye, rapeseed sorghum • Softwoods for example Pinus sylvestris, Pinus radiata • Woods hard, Salix spp. Eucalyptus spp.

• Tubers, for example beet and potato • Cereals of, for example, rice, wheat, rye, oats, barley, rye, rapeseed, sorghum and corn.

• Paper waste, biogas processing fiber fractions, fertilizer, palm oil processing waste, municipal solid waste or the like with a similar dry matter content.

If the biomass containing polysaccharides are llnocellulosic, the material can be cut into pieces, where 20% (w / w) of the biomass is preferably within a range of 26-70 mm, before pretreatment. The pre-treated material preferably has a dry matter content above 20% before entering the mixing device. In addition to releasing the carbohydrates from the biomass, the pre-treatment procedure sterilizes and partially dissolves the biomass and at the same time cleanses the fraction of lignin from potassium chloride.

The mixture obtained in a process according to the present invention meets at least four objectives.

In the first place, it ensures a close contact between the enzymes used and the biomass (substrate) that contains polysaccharide, since this will be insoluble or only very slightly soluble in most cases.

Secondly, the mechanical work done on the material during mixing helps to tear fibers and particles of the biomass, larger, and therefore help to increase the surface area of ​​the material. This will increase the accessibility of, for example, cellulose and hemicellulose to the enzymes used. To further increase the mechanical work on the material, steel balls or similar means that collide with the material could be added to the drum.

Third, the mixing of the material prevents the local accumulation of a high concentration of cellobiose, which - as is well known to those skilled in the art - could inhibit, for example, cellulase enzymes, especially cellobiohydrolases.

Fourth, an important characteristic of cellulase enzymes is the influence of cellulose binding domains (CBD) on enzyme performance. CBDs are functional parts of cellulose degrading enzymes. CBDs allow adhesion of the water-soluble enzyme on an insoluble substrate surface (cellulose). The close relationship between the enzyme and the cellulose provided by the CBD improves the catalytic speed and the stability of the enzyme. To hydrolyze cellulose, the enzyme must change the position of the CBD on the cellulose chain. It is believed that the mechanical action, that is the mixture, is important for the movement of the CBD and therefore for the enzymatic action of the enzymes along the cellulose chain.

Furthermore, it should be noted that enzymatic biomass hydrolysis has traditionally been carried out in agitated tank reactors equipped with impellers (for example Rushton turbine or Intemig impeller) mounted on a centrally positioned drive shaft, similar to those that are They use in the fermentation industry. Due to this equipment, high viscosity solutions, of very sticky or very dry dry material, cannot be agitated efficiently, since they result in very poor areas or without any mixing. In addition, the agitation of such solutions requires a very large energy input, which is detrimental to the economy of the procedure. When operating with biomass containing polysaccharides, the upper possible limit has been previously restricted to approximately 20%. The gravity-based mixing principle according to the present invention overcomes this problem and can be used for biomass containing polysaccharide with a dry matter content of up to 80%, preferably 20-50%. The gravity mixing principle according to the present invention can easily be carried out in ascending scale and can be applied to all kinds of biomass, in addition to refined starch, which contains up to more than 80% cellulose.

Unlike conventional stirred tank reactors, traditionally used for enzymatic hydrolysis, a gravity-based mixing principle, that is, a drum mixer, a mixer with a rotating shaft that lifts the biomass or a similar mixing device, which uses a principle of free fall, allow to obtain at the same time, an efficient mixture, even with small energy revenues and high dry matter contents and they also perform mechanical processing / degradation through gravity forces, including shear and tear forces between the material and the drum, as well as the forces resulting from the impact between the material that falls and the bottom of the drum and at the same time positively affect the influence of cellulose adhesion domains (CBD) on the yield of the enzyme.

Although the processing of non-miscible materials, of plants, such as, for example, biomass containing polysaccharides with a relatively high dry matter content and a high average fiber and particle size, is known from fermentation or state bioreactors solid, where rotating mixers are used for mixing (Giovanozzi et al. 2002), this principle has not been previously implemented in a liquefaction / saccharification process or in a bio-ethane fermentation process.

The present invention provides a process for processing biomass with a relatively high dry matter content, for example a dry matter content of between 20-80%, preferably between 20-50%. In addition, the process according to the present invention ensures efficient liquefaction and saccharification that allows the direct use of the product in, for example, fermenters.

Enzymes capable of converting starch, cellulose and hemicellulose or parts thereof into glucose, xylose and cellobiose are added to the biomass naturally or in the form of microbial organisms that give rise to the accumulation of said enzymes. The pH and temperature of the biomass are adjusted with reference to the optimum pH and the optimum temperature of the enzymes applied.

Depending on the enzyme load, the biomass will be liquefied and saccharified to a liquid without any or with only a few remaining fibers and large particles within 3-24 hours. The addition of a glucose metabolizing microorganism at any time during hydrolysis and liquefaction can improve the degree of enzymatic hydrolysis as long as the enzyme inhibitor products are removed in this way.

Detailed description of the invention

A process according to the present invention can be carried out using the following preferred technical parameters.

• Dry matter content: 20-80%, preferably 25-70%, more preferably 25-60%, even more preferably 25-50% or 25-40%, and more preferably 25-35%.

• Distribution of fiber and particle sizes of lignocellulosic biomass: 0-150 mm, preferably 5-125 mm, more preferably

10-100 mm, even more preferably 15-90 mm or 20-80 mm and more preferably 26-70 mm. The preferred distribution of fiber and particle sizes is defined as at least 20% (w / w) of the lignocellulosic biomass that is within the preferred Interval.

If the biomass containing polysaccharide is lignocellulosic, it must be pretreated, for example with extraction of hot water. If a hydro thermal pretreatment is chosen, the following technical data are preferred:

• Pretreatment temperature: 110-250 ° C, preferably 120-240 ° C, more preferably 130-230 ° C, more preferably 140-220 ° C, more preferably 150-210 ° C, more preferably 160-200 ° C, even more preferably 170-200 ° C or more preferably 180-200 ° C.

• Pre-treatment time: 1-60 min, preferably 2-55 min, more preferably 3-50 min, more preferably 4-45 min, more preferably 5-40 min, more preferably 5-35 min, more preferably 5- 30 min, more preferably 5-25 min, more preferably 5-20 min and more preferably 5-15 min.

• Dry matter content after pre-treatment of at least 20 p / p%.

Enzymatic treatment of biomass containing polysaccharide in a gravity mixer:

If a vessel based on the concept of a free fall mixture is used, in the form of a reactor with a horizontally placed agitator shaft, which elevates the biomass, or other similar mixing device, the following technical data are preferred:

• Rotation speed: 0-30 rpm, preferably 0-20 rpm, more preferably 0-25 rpm even more preferably 0-10 rpm and more preferably 0-5 rpm, • Rotation with periodically alternated direction of rotation.

• Rotation at pre-defined intervals.

The optimum rotation speed will depend on the volume of the container, the preferred rotation speed can therefore be relatively high when a procedure is carried out in a relatively small container, while it can be relatively low when the procedure is carried out in a relatively large container.

• Enzymes for lignocellulosic biomass:

- Cellobiasa (for example Novozym 188)

- Cellulase (for example Celluclast 1.5 FG L) • Enzyme loading in Filter Paper Units (FPU) / g DM. 1 FPU is equal to the amount of enzyme needed to hydrolyze 1 pmol / min of glycosidic bonds on Whatmann # 1 filter paper, under specified conditions well known to one skilled in the art. However, the enzymatic activity could in principle be provided in any conceivable form, including through the addition of microorganisms that give rise to the desired enzymatic activity: corresponding to 0.001-15 FPU / g of dry matter, preferably 0.01 - 10 FPU / g of dry matter, more preferably 0.1-8 FPU / g of dry matter, more preferably 1-7 FPU / g of dry matter and more preferably less than 6 FPU / g • Enzymes for starch-containing biomass :

- Enzymes in the starch processing: alpha-amylases and glucoamylases • Treatment time for enzymatic hydrolysis: 0-72 hours, preferably 1-60 hours, more preferably 2-48 hours, and more preferably 3-24 hours, such as 4 -24 hours, such as 6-24 hours, such as 8-24 hours, such as 10-24 hours, such as 12-24 hours, such as 1824 hours or 22 hours.

• Temperature for enzymatic hydrolysis. Adjusted with reference to the optimum temperatures of the enzymatic activities applied: 0-105 ° C, preferably 10-100 ° C, more preferably 15-90 ° C, more preferably 20-80 ° C, more preferably 25-70 ° C and more preferably 30-70 ° C such as 40-45 ° C or room temperature.

• pH of the biomass. Adjusted with reference to the optimum pH of the enzymatic activities applied: 3-12, such as 5-10, such as 6-9, such as 7-8 and preferably 4-11 • Enzymatic treatment can be carried out as batch, batch of feeding or continuous procedure.

Example 1: Enzymatic laboratory hydrolysis

Pre-treated, pressed wheat straw with an average size of approximately 40 mm (water withdrawal at 180-200 ° C for 5-10 min., Water flow ratio and dry matter of 5: 1) that corresponds at 25 g of dry weight (= 67.0 g of pre-treated straw) it was placed inside a plastic bag, 0.75 ml of Novozym, 188.375 mL of Celluclast 1.5 FG L and 11.9 mL of buffer of 50 mM sodium citrate, pH 5.9, mixed and sprayed on the straw. This resulted in a final dry matter content of 30%. The enzyme load corresponded to 10 Units of Filter Paper (FPU) / g DM.

The mixer consisted of a drum (1.0 m in length and 0.78 m in diameter) with 5 internal ribs along the long axis to ensure proper mixing of the material. The drum rotated along the horizontal axis with a speed of 26 rpm. Mixing / hydrolysis of the material was carried out for 824 hours at room temperature. This resulted in a thick paste without any large remaining fiber. A control bag, with the same enzymatic load but without mixing, showed no signs of straw degradation.

Some of the resulting material after enzymatic hydrolysis for 24 hours (an amount corresponding to 29 g of dry matter) was diluted to 15% of dry matter in a blue cap bottle and yeast was added (baker's yeast, De Danske Spritfabrikker ) The bottle was closed with pneumatic locking and placed for 72 hours at 30 ° C with shaking at 500 rpm. The resulting liquid contained 33 g / L of ethanol, 10 g / L of xylose. No glucose was detected which indicates that the yeast is able to use all the glucose produced during hydrolysis. Assuming a yield of ethanol in glucose of 0.5 g of ethanol per g of glucose, this corresponded to a 70% conversion of the original cellulose.

Example 2. Enzymatic hydrolysis at pilot scale

Pre-treated, pressed wheat straw with an average size of approximately 40 mm (pre-treated by extracurrent water extraction at

180-200 ° C for 5-10 min., With a ratio of water flow and dry matter of 5: 1) corresponding to 7 kg DW (= 20 kg of pre-treated straw) was deposited in a conventional rotating mixer for cement, with a horizontal axis inclined to approximately 10 degrees. The mixer had 2 Internal ribs along the long axis to ensure the mixing of the material. A lid was mounted on the opening to prevent evaporation from the mixer. The mixer drum rotated along the horizontal axis with a speed of 29 rpm. 200-1150 mL of Celluclast 1.5 FG L and 40-225 mL of Novozym 188 were added to the straw. This resulted in a final dry matter content of 30%. The enzyme load corresponded to 3-15 FRU / g DM. The pH was adjusted to 4.8 to 5.0 by the addition of sodium carbonate.

The cement mixer was heated to 40-45 ° C by using a fan heater. Mixing / hydrolysis of the material was carried out for 22 hours. Depending on the enzyme load, this resulted in a more or less viscous liquid without any remaining large fibers. The pre-treated straw degraded to a paste in approximately 3-5 hours. After 5-24 hours of mixing, the paste became a viscous liquid. Control experiments with pre-treated wheat straw only or with pre-treated wheat straw at only 160 ° C, but using the same enzyme load, showed no sign of straw liquefaction.

Simultaneous saccharification and fermentation was carried out by adding yeast to the cement mixer, after 24 hours of hydrolysis at 40-45 ° C using an enzyme load of 10-15 FPU / g DM. The temperature was allowed to cool below 35 ° C and compressed yeast (baker's yeast, De Danske Spritfabrikker) was added to a concentration of approximately 1% (w / w) based on dry straw. The saccharification and fermentation was continued for 48 hours at 25 ° C.

The resulting material was centrifuged for 15 min. at 2500 rpm The supernatant was filtered through a 0.45 pm filter and analyzed to determine the sugars by HPLC. With an enzyme load of 15 FPU / g DM; The supernatant contained 70 g / L of glucose, 30 g / L of xllose after 24 hours of hydrolysis. This corresponded to a 50% hydrolysis of the cellulose and hemicellulose originally present in the straw. Simultaneous saccharification and fermentation, using an enzyme load of 10 FPU / g DM resulted in 42 g / L of ethanol and 30 g / L of xylose.

Example 3: Liquefaction, hydrolysis and fermentation

The hydrolysis reactor was designed to carry out experiments with liquefaction and solid hydrolysis concentrations above 20% DM (fig 1). The reactor consisted of a drum placed horizontally divided into 5 separate chambers, each 20 cm wide and 60 cm in diameter. A horizontal rotating shaft mounted with three blades in each chamber was used for mixing / stirring. A 1.1 kW motor was used for propulsion and the rotational speed was adjusted within the range of 2.5 and 16.5 rpm. The direction of rotation was programmed to change within a range of 2.5 and 16.5 rpm. The direction of rotation was programmed to change twice per minute between the clockwise direction and the opposite direction. A heating jacket filled with water, on the outside allowed the temperature control up to 80 ° C.

The chambers were filled with pressed pretreated wheat straw with an average size of approximately 40 mm (pretreated by countercurrent water extraction at 180-200 ° C for 5-10 min. With a water flow ratio and dry matter of 5: 1) and water to provide an initial DM content of 20 to 40%. Celluclast 1.5 FG L and Novozym 188 were added in a 5: 1 ratio to provide an enzyme load of 7 FPU per g DM. The liquefaction and hydrolysis were carried out at 50 ° C and at a pH of 4.8 to 5.0. The mixing speed was 6.6 rpm. Simultaneous saccharification and fermentation (SSF) experiments were carried out by lowering the temperature to 32 ° C after 8 h of liquefaction and hydrolysis and then 15 g of compressed baker's yeast (De Danske Spritfabrikker) per kg of initial DM was added.

Liquefaction and hydrolysis was possible with an initial DM content of up to 40% DM (fig. 2 and 3). With initial 40% DM it was possible to reach glucose concentrations of 80 g kg '¹ after 96 h. It was also possible to operate the procedure as SSF (fig 3), thus reducing the inhibition of product, of cellulases, caused by the accumulation of glucose. It was possible to ferment the hydrolysates with up to 40% of the initial DM content using normal baker's yeast. Under conditions not completely anaerobic, the ethanol yield was 80, 79, 76, 73 and 68% of what is theoretically obtainable at 20, 25, 30, 35 and 40% DM, respectively.

Example 4: Liquefaction, saccharification and fermentation of the complete agricultural product

Biomass containing lignocellulosic and starch can be processed simultaneously using gravity mixing and a mixture of cellulases, hemicellulases and amylases. Lignocellulosic biomass can be derived from agricultural crops consisting of for example wheat, fodder, straw, for example rice, wheat, rye, oats, barley, rye, rapeseed and sorghum, tubers for example, beet, potato, cereals for example rice, wheat, rye, barley rye oats, rapeseed, sorghum, wood consisting of softwood, for example Pinus sylvestris, Pinus radiata, hardwoods, for example Salix spp., Eucalyptus spp. municipal solid waste, paper waste and similar biomass.

For the experiments, the hydrolysis reactor described in the example was used

3. Wheat straw (primarily a source of lignocellulose) was pretreated using countercurrent water extraction at 180-200 ° C for 5-10 min., With a water flow and dry matter ratio of 5: 1. Wheat grains (mainly a source of starch) were crushed dry using a Kongskilde roller mill. Wheat grain and pretreated straw with an average size of approximately 4 ° mm, was mixed in a 1: 1 ratio on a dry basis. The DM was adjusted to between 30 and 40% by adding water. Celluclast 1.5 FG L and Novozym 188 were added in a 5: 1 ratio to provide an enzyme load of 7 FPU per g DM of straw. Starch hydrolysis was carried out using cold-crushed NS59933 enzyme (Novozymes A / S, Bagsvaerd, Denmark) in a load of 3.5 g per kg of wheat grain. The liquefaction and hydrolysis was carried out at 50 ° C and at a pH of 4.8 to 5.0. After 8 h. the temperature decreased to 34 ° C and 15 g of compressed baker's yeast (De Danske Spritfabrikker) per kg of initial DM was added. An experiment with straw with only 30% DM was carried out in parallel.

The mixing of straw with grains resulted in a rapid initial accumulation of glucose in the liquefaction and hydrolysis stage compared to the application of straw only (Figure 4). After 96 h of liquefaction and SSE, the concentration of ethanol was 41 g kg '¹ using only wheat straw as the only substrate (fig 4). In the experiment with straw and grain the concentration of ethanol reached 68 g kg¹.

Example 5: Low temperature liquefaction of starch or starch-containing materials.

A process according to the present invention can also be applied to process, at a low temperature, refined starch or starch-containing materials, (for example, beets, potatoes, cereals, for example, rice, wheat, rye, oats, barley, oatmeal, sorghum). According to example 4, pre-treatment of the grain with heat is not necessary for liquefaction and hydrolysis of starch. Dry grinding is used, on the other hand, generally for the pre-treatment of grains containing starch. Dry ground grains with a dry matter content of 20-60% are charged to the mixer by gravity. The cold-crushed NS50033 enzyme (Novozymes A / S, Bagsvaerd, Denmark) or alpha-amylases and glucoamylases are added simultaneously. Then, liquefaction and complete saccharification of starch is possible in a single stage procedure. The temperature and pH ranges during the enzymatic hydrolysis process are defined by enzymes and will be in the range of 25-60%, preferably 49-55% and a pH of 3-12, preferably a pH of 3-8, respectively .

The procedure can be combined with SSF.