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Manufacturing Method For Sugar Solution And Device For Same

Abstract: A method for cellulolysis which uses a cellulase from a filamentous fungus as a saccharifying enzyme and in which: cellulose undergoes primary hydrolysis by adding a saccharifying enzyme thereto after which the primary hydrolysis product is separated into a primary sugar solution and solid material by solid liquid separation; water is added to the solid material and a secondary hydrolysis is carried out after which the secondary hydrolysis product is separated into a secondary sugar solution and residue by solid liquid separation; and the primary sugar solution and/or the secondary sugar solution is/are passed through an ultrafiltration membrane after which the saccharifying enzyme is collected from the film side and the sugar solution is collected from the permeating side. Thus provided is a method for reducing the amount of an enzyme such as cellulase used in a method for manufacturing a sugar solution from a pretreated cellulose material.

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Patent Information

Application #
Filing Date
08 October 2012
Publication Number
51/2015
Publication Type
INA
Invention Field
BIO-CHEMISTRY
Status
Email
Parent Application
Patent Number
Legal Status
Grant Date
2019-06-28
Renewal Date

Applicants

TORAY INDUSTRIES INC.
1 1 Nihonbashi Muromachi 2 chome Chuo ku Tokyo 1038666

Inventors

1. KURIHARA Hiroyuki
c/o Basic Research Center Toray Industries Inc. 10 1 Tebiro 6 chome Kamakura shi Kanagawa 2488555
2. MINAMINO Atsushi
c/o Basic Research Center Toray Industries Inc. 10 1 Tebiro 6 chome Kamakura shi Kanagawa 2488555
3. YAMAMOTO Yuki
c/o Basic Research Center Toray Industries Inc. 10 1 Tebiro 6 chome Kamakura shi Kanagawa 2488555
4. YAMADA Katsushige
c/o Basic Research Center Toray Industries Inc. 10 1 Tebiro 6 chome Kamakura shi Kanagawa 2488555

Specification

DESCRIPTION MANUFACTURING METHOD FOR SUGAR SOLUTION AND DEVICE FOR SAME TECHNICAL FIELD [0001] The present invention relates to a method for producing a sugar liquid from cellulose, and an apparatus for the method. BACKGROUND ART [0002] Processes of fermentation production of chemical products using sugars as raw materials have been used for producing various industrial materials. At present, as the sugars to be used as fermentation feedstocks, those derived from food materials such as sugar cane, starch and sugar beet are industrially used. However, in view of the fact that rise in the prices of food materials due to future increase in the world population is expected, or in an ethical view of the fact that sugars as industrial materials may compete with sugars for food, a process for efficiently producing a sugar liquid from a renewable nonfood resource, that is, a cellulose-containing biomass, or a process for using an obtained sugar liquid as a fermentation feedstock to efficiently convert the sugar liquid to an industrial material needs to be constructed in the future. [0003] Examples of disclosed methods for producing a sugar liquid from a cellulose-containing biomass include methods for producing sugar liquids by acid hydrolysis of cellulose and hemicellulose using concentrated sulfuric acid (Patent Documents 1 and 2) and a method wherein a cellulose-containing biomass is subjected to hydrolysis treatment using dilute sulfuric acid and then enzymatically treated with cellulase or the like to produce a sugar liquid (Non-patent Document 1). Further, examples of disclosed methods using no acid include a method wherein a cellulose-containing biomass is hydrolyzed using subcritical water at about 250°C to 500°C to produce a sugar liquid (Patent Document 3), a method wherein a cellulose-containing biomass is subjected to subcritical water treatment and then enzymatically treated to produce a sugar liquid (Patent Document 4), and a method wherein a cellulose-containing biomass is subjected to hydrolysis treatment with pressurized hot water at 240°C to 280°C and then enzymatically treated to produce a sugar liquid (Patent Document 5). [0004] In recent years, methods of hydrolysis of a biomass which use less energy and cause less environmental load but produce sugar at high yields have been extensively studied. However, such methods using enzymes have a drawback in that the costs of enzymes are high. [0005] For solving these technical problems, methods by recovering and reusing the enzymes used in the hydrolysis have been proposed. Examples of such methods disclosed include a method wherein continuous solid-liquid separation is carried out with a spin filter and the obtained sugar liquid is filtered through an ultrafiltration membrane to recover the enzymes (Patent Document 6), a method wherein a surfactant is fed at the stage of enzymatic saccharification, to suppress enzyme adsorption and thereby enhance the recovery efficiency (Patent Document 7), a method wherein the residue produced by enzymatic saccharification is subjected to electric treatment to recover the enzyme component (Patent Document 8) and a method wherein the residue produced by enzymatic saccharification is fed again to another batch of biomass and the enzymes is thereby reused (Patent Document 9). PRIOR ART DOCUMENTS Patent Documents [0006] Patent Document 1: Japanese Translated PCT Patent Application Laid-open No. 11-506934 Patent Document 2: JP 2005-229821 A Patent Document 3: JP 2003-212888 A Patent Document 4: JP 2001-95597 A Patent Document 5: JP 3041380 B Patent Document 6: JP 2006-87319 A Patent Document 7: JP 63-87994 A Patent Document 8: JP 2008-206484 A Patent Document 9: JP 55-144885 A Non-patent Documents [0007] Non-patent Document 1: A. Aden et al. "Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover" NREL Technical Report (2002) SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION [0008] Methods for producing sugar liquids by recovering/reusing enzyme have been developed as described above, but the effects of these methods have been insufficient in view of reduction in the amount of the enzyme used. Therefore, the present invention aims to develop a process wherein the effect of reducing the amount of enzyme is higher than those in the conventional methods. MEANS FOR SOLVING THE PROBLEMS [0009] The present inventors intensively studied to solve the above problems, and, as a result, a method of hydrolysis of cellulose using a filamentous fungus-derived cellulase as a carbohydrase was invented, which method comprises: the step of adding the carbohydrase to cellulose to perform primary hydrolysis and then subjecting the primary hydrolysate to solid-liquid separation into a primary sugar liquid and solids; the step of adding water to the solids and performing secondary hydrolysis, followed by subjecting the secondary hydrolysate to solid-liquid separation into a secondary sugar liquid and a residue; and the step of filtering the primary sugar liquid and/or secondary sugar liquid through an ultrafiltration membrane and recovering the carbohydrase from the feed side and recovering a sugar solution from the permeate side. [0010] That is, the present invention has the constituents (1) to (13) below.(1) A method for producing a sugar liquid by using as a carbohydrase afilamentous fungus-derived cellulase to hydrolyze cellulose, the method comprising: the step of adding said carbohydrase to cellulose to perform primary hydrolysis and then subjecting the primary hydrolysate to solid-liquid separation into a primary sugar liquid and solids; the step of adding water to the solids and performing secondary hydrolysis, followed by subjecting the secondary hydrolysate to solid-liquid separation into a secondary sugar liquid and a residue; and the step of filtering the primary sugar liquid and/or secondary sugar liquid through an ultrafiltration membrane, and recovering the carbohydrase from the feed side and recovering a sugar liquid from the permeate side. (2) The method for producing a sugar liquid according to (1), wherein the filamentous fungus-derived cellulase is Trichoderma-derived cellulase. (3) The method for producing a sugar liquid according to (1) or (2), wherein the cellulose is derived from a processed product prepared by ammonia treatment, hydrothermal treatment or dilute sulfuric acid treatment of biomass. (4) The method for producing a sugar liquid according to any of (1) to (3), wherein the secondary hydrolysis is hydrolysis in the presence of one or more selected from the group consisting of inorganic salts (excluding calcium salts), hydrophilic organic solvents, amino acids and nonionic surfactants, and sugar liquids comprising these substances. (5) The method for producing a sugar liquid according to (4), wherein the inorganic salt(s) (excluding calcium salts) is/are one or more selected from the group consisting of sodium salts, potassium salts, magnesium salts, sulfuric acid salts, ammonium salts, hydrochloric acid salts, phosphoric acid salts, acetic acid salts and nitric acid salts. (6) The method for producing a sugar liquid according to (5), wherein the inorganic salt(s) (excluding calcium salts) is/are one or more selected from the group consisting of sodium chloride, sodium acetate, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, ammonium chloride, dipotassium hydrogen phosphate, ammonium sulfate, magnesium chloride and magnesium sulfate. (7) The method for producing a sugar liquid according to (4), wherein the hydrophilic organic solvent(s) is/are one or more selected from the group consisting of methanol, ethanol, 1-propanol, isopropanol, N,N-dimethylformamide, butanol, acetone, acetonitrile, ethylene glycol and glycerin. (8) The method for producing a sugar liquid according to (4), wherein the amino acid(s) is/are one or more selected from the group consisting of arginine, cysteine, glutamic acid, histidine and lysine. (9) The method for producing a sugar liquid according to any of (1) to (8), wherein the solid-liquid separation of a primary hydrolysate and/or secondary hydrolysate is press filtration. (10) The method for producing a sugar liquid according to any of (1) to (9), the method comprising the step of filtering the sugar liquid through a reverse osmosis membrane and/or nanofiltration membrane to concentrate the sugar liquid. (11) An apparatus for the method for producing a sugar liquid according to any of (1) to (10), the apparatus comprising as constituents a stirring tank for primary hydrolysis; solid-liquid separation device; secondary hydrolysis tank or press filtration device for secondary hydrolysis; solid-liquid separation device(s) for the primary hydrolysate and/or secondary hydrolysate; and ultrafiltration membrane device for separating the carbohydrase and the sugar liquid from the primary sugar liquid and/or the secondary sugar liquid. (12) An apparatus for the method for producing a sugar liquid according to any of (1) to (10), the apparatus comprising as constituents a reaction vessel for primary hydrolysis; press filtration device having a warm-water supply tank; circulation line for circulating the filtrate from the press filtration device to the warm-water supply tank; and ultrafiltration membrane device for separating the carbohydrase and the sugar liquid from the primary sugar liquid and/or the secondary sugar liquid. (13) The apparatus according to (11) or (12), comprising as a constituent a sugar liquid concentrating device equipped with a reverse osmosis membrane and/or a nanofiltration membrane for concentrating the sugar liquid obtained with the ultrafiltration membrane device. EFFECT OF THE INVENTION [0011] In the present invention, primary hydrolysis is followed by solid-liquid separation, and the residual enzyme components contained in the obtained solids are used to perform secondary hydrolysis. This produces 1) an effect to increase the sugar yield and 2) an effect to increase the recovered amount of enzyme. Therefore, the present invention is economically advantageous over conventional techniques. BRIEF DESCRIPTION OF THE DRAWINGS [0012] Fig. 1 is a schematic diagram showing an embodiment of the method of the present invention for producing a sugar liquid. Fig. 2 is a schematic diagram showing an embodiment of the apparatus for carrying out the method of the present invention for producing a sugar liquid. Fig. 3 is a schematic diagram showing an embodiment of the apparatus for carrying out the method of the present invention for producing a sugar liquid. Fig. 4 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method of the present invention for producing a sugar liquid is carried out in a press filtration chamber. Fig. 5 is a schematic diagram showing an embodiment of the apparatus for carrying out the method of the present invention for producing a sugar liquid. Fig. 6 is a schematic diagram showing an embodiment of the apparatus for carrying out the method of the present invention for producing a sugar liquid. Fig. 7 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method of the present invention for producing a sugar liquid is carried out in a press filtration chamber. Fig. 8 is a schematic diagram showing an embodiment of the apparatus for carrying out the method of the present invention for producing a sugar liquid. Fig. 9 is a diagram showing the results of analysis of an enzyme contained in the secondary sugar liquid obtained by the method of the present invention for producing a sugar liquid. Fig. 10 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method of the present invention for producing a sugar liquid is carried out in a secondary hydrolysis tank which is separate from the tank for the primary hydrolysis. Fig. 11 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method of the present invention for producing a sugar liquid is carried out in a secondary hydrolysis tank which is separate from the tank for the primary hydrolysis. Fig. 12 is a schematic diagram showing an embodiment in which the secondary hydrolysis in the method of the present invention for producing a sugar liquid is carried out in a secondary hydrolysis tank which is separate from the tank for the primary hydrolysis. Fig. 13 is a schematic diagram showing an embodiment in which the primary hydrolysis and the secondary hydrolysis in the method of the present invention for producing a sugar liquid are carried out in the same tank. Fig. 14 is a schematic diagram showing an embodiment in which a microfiltration membrane device is placed upstream of an ultrafiltration membrane device. Fig. 15 is a schematic diagram showing an embodiment in which cross-flow filtration is performed using a microfiltration membrane module. Fig. 16 is a schematic diagram showing an embodiment in which dead-end filtration is performed using a microfiltration membrane module. BEST MODE FOR CARRYING OUT THE INVENTION [0013] Large amounts of celluloses are contained in herbaceous biomasses such as bagasse, switchgrass, napier grass, Erianthus, corn stover, rice straw and wheat straw; and woody biomasses such as trees and waste building materials. These cellulose-containing biomasses can be preferably used as raw materials in the present invention. [0014] Cellulose-containing biomass contains, in addition to cellulose and hemicellulose (hereinafter referred to as "cellulose" as a general term for cellulose and hemicellulose), lignin and the like which are aromatic macromolecules. Therefore, in cases where cellulose derived from a biomass is used as a raw material for a sugar liquid in the method of the present invention for producing a sugar liquid, the efficiency of enzymatic hydrolysis can be enhanced by pretreatment. Examples of the method of pretreatment of a cellulose-containing biomass include acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkaline treatment, caustic soda treatment, ammonia treatment, hydrothermal treatment, subcritical water treatment, pulverization treatment and steaming treatment. In the present invention, the method of pretreatment is preferably ammonia treatment, hydrothermal treatment or dilute sulfuric acid treatment. [0015] The ammonia treatment is carried out according to JP 2008-161125 A and JP 2008-535664 A. For example, ammonia is added to the biomass at a concentration within the range of 0.1 to 15% by weight, and the treatment is carried out at 4 to 200°C, preferably 90 to 150°C. The ammonia to be added may be in the state of either liquid or gas. Further, the form of the ammonia to be added may be either pure ammonia or aqueous ammonia. The number of times of the treatment is not restricted, and 1 or more times of the treatment may be carried out. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from those for the second and later treatments. The treated product obtained by the ammonia treatment needs to be subjected to neutralization of ammonia or removal of ammonia in order to further carry out enzymatic hydrolysis reaction. The neutralization of ammonia may be carried out either after removal of the solids from the hydrolysate by solid-liquid separation or in the state in which the solids are contained. The acid reagent to be used for the neutralization is not restricted. The ammonia can be removed by maintaining the ammonia-treated product under reduced pressure to allow evaporation of the ammonia into the state of gas. The removed ammonia may be recovered and reused. [0016] In the case of dilute sulfuric acid treatment, the concentration of sulfuric acid is preferably 0.1 to 15% by weight, more preferably 0.5 to 5% by weight. The reaction temperature may be set within the range of 100 to 300°C, and is preferably set within the range of 120 to 250°C. The reaction time may be set within the range of 1 second to 60 minutes. The number of times of the treatment is not restricted, and 1 or more times of the treatment may be carried out. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from those for the second and later treatments. Since the hydrolysate obtained by the dilute sulfuric acid treatment contains acid, neutralization is necessary in order to further carry out hydrolysis reaction with cellulase or in order to use the hydrolysate as a fermentation feedstock. [0017] In the case of hydrothermal treatment, water is added such that the cellulose-containing biomass is contained at 0.1 to 50% by weight, and the treatment is then carried out at a temperature of 100 to 400°C for 1 second to 60 minutes. By performing the treatment under such temperature conditions, hydrolysis of cellulose occurs. The number of times of the treatment is not restricted, and 1 or more times of the treatment may be carried out. In particular, in cases where the treatment is carried out 2 or more times, the conditions for the first treatment may be different from those for the second and later treatments. [0018] The cellulase used in the present invention is filamentous fungus-derived cellulase. Examples of the filamentous fungus include microorganisms such as Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor and Talaromyces. Since these microorganisms secrete cellulase into the culture medium, the culture medium may be used as it is as unpurified filamentous fungus-derived cellulase, or the culture medium may be purified and formulated to be used as a mixture containing filamentous fungus-derived cellulase. In cases where the filamentous fungus-derived cellulase is used as a purified and formulated product, a substance(s) other than the enzyme, such as a protease inhibitor, dispersant, solubilizer and/or stabilizer may be added to prepare the cellulase formulation. [0019] The filamentous fungus-derived cellulase used in the present invention is preferably cellulase produced by Trichoderma (hereinafter referred to as Trichoderma-derived cellulase). In the present invention, the Trichoderma-derived cellulase is preferably cellulase derived from Trichoderma reesei, and specific examples of preferred Trichoderma microorganisms from which the cellulase is to be derived include Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RutC-30, Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80 and Trichoderma viride QM9123 (Trichoderma viride 9123). The cellulase may also be derived from a mutant strain originated from the above-described Trichoderma microorganism, which mutant strain was prepared by mutagenesis using a mutagen, UV irradiation or the like to enhance the cellulase productivity. [0020] Filamentous fungus-derived cellulase is an enzyme composition comprising a plurality of enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, |3-glucosidase, xylanase and xylosidase, which enzyme composition has an activity to hydrolyze and saccharify cellulose. Since filamentous fungus-derived cellulase comprises such a plurality of enzyme components and allows, in cellulose degradation, efficient hydrolysis of cellulose due to their concerted effect or complementary effect, filamentous fungus-derived cellulase is preferably used in the present invention. [0021] Cellobiohydrolase is a general term for cellulases that hydrolyze cellulose from the terminal portions. The group of enzymes belonging to cellobiohydrolase are described as EC number: EC 3.2.1.91. [0022] Endoglucanase is a general term for cellulases that hydrolyze cellulose molecular chains from their central portions. The group of enzymes belonging to endoglucanase are described as EC number: EC 3.2.1.4. [0023] Exoglucanase is a general term for cellulases that hydrolyze cellulose molecular chains from their terminal portions. The group of enzymes belonging to exoglucanase are described as EC number: EC 3.2.1.74. [0024] (3-glucosidase is a general term for cellulases that acts on cellooligosaccharides or cellobiose. The group of enzymes belonging to (3-glucosidase are described as EC number: EC 3.2.1.21. [0025] Xylanase is a general term for cellulases that acts on hemicellulose or especially xylan. The group of enzymes belonging to xylanase are described as EC number: EC 3.2.1.8. [0026] Xylosidase is a general term for cellulases that acts on xylooligosaccharides. The group of enzymes belonging to xylosidase are described as EC number: EC 3.2.1.37. [0027] Such filamentous fungus-derived cellulase components can be separated by a known method such as gel filtration, ion exchange or two-dimensional electrophoresis, and the separated components can be subjected to amino acid sequence analysis (N-terminal analysis, C-terminal analysis or mass spectrometry), followed by comparison of the sequences with a database. [0028] The enzyme activity of filamentous fungus-derived cellulase can be evaluated based on its hydrolytic activities on polysaccharides, such as the Avicel-degrading activity, carboxymethyl cellulose (CMC)-degrading activity, cellobiose-degrading activity, xylan-degrading activity and mannan-degrading activity. The main cellulase components involved in the Avicel-degrading activity are cellobiohydrolase and exoglucanase, which degrade cellulose from its terminal portions. The main cellulase components involved in the xylan-degrading activity are xylanase and xylosidase. The main cellulase component involved in the cellobiose-degrading activity is P-glucosidase. The main cellulase components involved in the CMC-degrading activity are cellobiohydrolase, exoglucanase and endoglucanase. The term "main" herein is used to mean that the component(s) is/are involved in the degradation to the highest extent(s) although other enzyme components are also involved in the degradation. [0029] As the filamentous fungus-derived cellulase, a crude enzyme product is preferably used. The crude enzyme product is derived from the culture supernatant obtained after culturing a microorganism belonging to a genus of filamentous fungus for an arbitrary period in a medium prepared such that the microorganism produces cellulase. The medium components to be used are not restricted, and a medium supplemented with cellulose in order to promote production of cellulase may be generally used. As a crude enzyme product, the culture liquid may be used as it is, or the culture supernatant processed only by removal of the filamentous fungus may be preferably used. [0030] The weight ratios of enzyme components in the crude enzyme product are not restricted, and, for example, culture liquid derived from Trichoderma reesei contains 50 to 95% by weight cellobiohydrolase, and also contains as other components endoglucanase, |3-glucosidase and the like. Microorganisms belonging to Trichoderma produce strong cellulase components into the culture liquid, while the P-glucosidase activity in the culture liquid is low since P-glucosidase is retained in the cells or on the cell surfaces. Therefore, P-glucosidase from a different species or from the same species may be added to the crude enzyme product. As the P-glucosidase from a different species, P-glucosidase derived from Aspergillus may be preferably used. Examples of the P-glucosidase derived from Aspergillus include Novozyme 188, which is commercially available from Novozyme. The method of addition of P-glucosidase from a different species or from the same species to the crude enzyme product may be a method wherein a gene is introduced to a microorganism belonging to Trichoderma to perform genetic recombination of the microorganism such that P-glucosidase is produced into the culture liquid, and the microorganism belonging to Trichoderma is then cultured, followed by isolating the culture liquid. [0031] In the present invention, hydrolysis of cellulose with the filamentous fungus-derived cellulase is carried out in two steps, that is, primary hydrolysis and secondary hydrolysis. The steps are described below in order. [0032] The primary hydrolysis in the present invention means that carbohydrase is added to cellulose that has not been brought into contact with carbohydrase, to perform hydrolysis. The enzyme used for the primary hydrolysis may be either the later-mentioned fresh enzyme or recovered enzyme, and, in view of reduction in the amount of the enzyme used, especially in the amount of fresh enzyme used, it is preferred to use a mixture of both the recovered enzyme and fresh enzyme. [0033] The reaction temperature during the primary hydrolysis is preferably within the range of 40 to 60°C, and, especially in cases where Trichoderma-derived cellulase is used, the reaction temperature is more preferably within the range of 45 to 55°C. [0034] The reaction time of the primary hydrolysis is preferably within the range of 2 hours to 200 hours. In cases where the reaction time is less than 2 hours, sugar yield is insufficient, which is not preferred. On the other hand, in cases where the reaction time is more than 200 hours, the enzyme activity decreases, which is not preferred since, in the later-mentioned secondary hydrolysis, sugar yield is insufficient and the enzyme cannot be recovered. [0035] The pH during the primary hydrolysis is preferably within the range of 4.0 to 5.5. In cases where Trichoderma-derived cellulase is used as the filamentous fungus-derived cellulase, the optimum reaction pH is 5.0, but, especially in the case of primary hydrolysis, the pH changes during the hydrolysis. Therefore, it is preferred to perform the hydrolysis while maintaining a constant pH using an acid or alkali. [0036] The primary hydrolysate contains a primary sugar liquid and solids, and the solids contain polysaccharide components such as undegraded cellulose and hemicellulose, and components that cannot be originally degraded with carbohydrase, such as lignin. Further, a relatively large amount of filamentous fungus-derived cellulase is adhered to the solids. Therefore, in the present invention, in order to perform the later-mentioned secondary hydrolysis using the polysaccharide components and filamentous fungus-derived cellulase contained in the solids obtained by the primary hydrolysate, the obtained solids are recovered by solid-liquid separation. Examples of the method of solid-liquid separation include centrifugation and press filtration, and, in the present invention, recovering the solids by press filtration is preferred. [0037] A reason why press filtration is preferred for the solid-liquid separation is that 1) high yield of sugar liquid can be achieved. The present invention aims to achieve improvement of the sugar recovery and the enzyme recovery as compared to those in the conventional techniques. Therefore, the method of solid-liquid separation is preferably one with which larger amounts of sugar liquid components can be recovered at once. The recovery of sugar liquid components by the solid-liquid separation can be improved especially by increasing the amount of water to be added after the secondary hydrolysis. However, an increase in the amount of water to be added causes a decrease in the sugar concentration in the secondary sugar liquid, which is not preferred. Therefore, in view of suppressing the amount of water used, while achieving a high sugar recovery, the solid-liquid separation is preferably performed by press filtration. Another reason why press filtration is preferred is that 2) a clear filtrate can be obtained. In the present invention, the primary sugar liquid and/or secondary sugar liquid obtained by solid-liquid separation is/are filtered through an ultrafiltration membrane to recover enzyme components. The sugar liquid to be passed through the ultrafiltration membrane preferably contains only small amounts of solids and particulate components in view of prevention of membrane fouling, and, in the case of press filtration, the filtrate contains only small amounts of solids and particulate components and may therefore be preferably used in the present invention. [0038] The secondary hydrolysis in the present invention means that the second hydrolysis is preformed for the solids obtained by the solid-liquid separation of the primary hydrolysate, using only the filamentous fungus-derived cellulase adsorbed on the solids. That is, in the secondary hydrolysis, hydrolysis of solids is performed only with the adsorbed enzyme, without further addition of carbohydrase. [0039] In contrast to conventional techniques (wherein only primary hydrolysis is performed), the present invention is characterized in that secondary hydrolysis is performed without further addition of enzyme, to improve the sugar yield and/or the enzyme recovery rate. Production of sugar and/or recovery of enzyme can of course be done also in the conventional techniques, but, by performing the secondary hydrolysis of the present invention, more sugar and enzyme can be recovered. A major reason for this is prevention of enzyme inhibition by removal of the produced sugar. The hydrolysate after the primary hydrolysis contains a large amount of sugar components. By performing solid-liquid separation to remove the sugars (glucose, xylose and oligosaccharides) produced by the hydrolysis and further adding water, the concentration of the produced sugars contained as solution components can be decreased. By this, inhibition of the enzyme by the products can be prevented, and the secondary hydrolysis can be sufficiently carried out with only the enzyme adsorbed on the solids. Therefore, even with the same amount of enzyme used as that in a conventional technique, more sugar and/or enzyme can be recovered by performing the secondary hydrolysis of the present invention. [0040] The amount of water to be added in the present invention is not restricted, and the addition is preferably carried out such that the solid concentration before the secondary hydrolysis is between 1% by weight and 20% by weight. In cases where the solid concentration is higher than 20% by weight, and in cases where the solid concentration is lower than 1% by weight, the yield of sugar and/or the recovered amount of enzyme may be low, which is inefficient and not preferred. [0041] The reaction temperature during the secondary hydrolysis is preferably within the range of 40 to 60°C, and, especially in cases where Trichoderma-derxved cellulase is used, the reaction temperature is more preferably within the range of 40 to 55°C, still more preferably within the range of 50°C. [0042] The reaction time of the secondary hydrolysis is preferably within the range of 5 to 180 minutes. In cases where the reaction time is less than 5 minutes, the recovery efficiency of the adsorbed enzyme is low, while even in cases where the reaction is carried out for not less than 180 minutes, the recovery efficiency of the adsorbed enzyme does not increase, which is inefficient. [0043] The pH during the secondary hydrolysis is preferably within the range of 6.0 to 8.0. In cases where Trichoderma-derived cellulase is used as the filamentous fungus-derived cellulase, the optimum reaction pH is 5.0, and, especially in the case of primary hydrolysis, the reaction is preferably performed at a pH of 5.0. On the other hand, in the secondary hydrolysis, since the main purpose is recovery of the adsorbed enzyme, the reaction is preferably carried out at a pH within the range of 6.0 to 8.0, in which the recovery efficiency of the adsorbed enzyme is high. At a pH of less than 6.0, the amount of recovery of enzyme decreases, while with a pH higher than 8.0, carbohydrase is deactivated, which is not preferred. That is, at a pH within the range of 6.0 to 8.0, the degree of deactivation of carbohydrase is extremely low and the recovery efficiency of carbohydrase can be high. [0044] The secondary hydrolysate contains a secondary sugar liquid and solids, and, similarly to the case of the primary hydrolysis, these can be separated from each other by solid-liquid separation, preferably press filtration. [0045] In the secondary hydrolysis, one or more compounds selected from nonionic surfactants, amino acids, inorganic salts (excluding calcium salts) and hydrophilic organic solvents may be added. By adding such a compound(s), any one or more of the sugar yield, the amount of recovered enzyme and the activity of recovered enzyme can be increased. In particular, in cases where the activity of recovered enzyme is high, the amount of the fresh enzyme to be added upon the reuse of the recovered enzyme can be reduced, which is economically preferred. [0046] The secondary hydrolysis may be performed in the presence of a surfactant, and the surfactant is preferably a nonionic surfactant. This is because, in cases where a cationic surfactant, anionic surfactant or amphoteric surfactant is used, the surfactant promotes deactivation of carbohydrase and has an inhibitory action on the secondary hydrolysis reaction. Further, the activity of the recovered enzyme is also decreased, which is not preferred. On the other hand, with a nonionic surfactant, a high sugar yield efficiency and a high enzyme recovery efficiency can be obtained, so that a nonionic surfactant is preferably used. [0047] The nonionic surfactant is also called the non-ionic surfactant, and is a surfactant whose hydrophilic moiety is constituted by a nonelectrolyte. Specific examples of the notiionic surfactant include polyoxyethylene alkyl ethers, polyoxypropylene block copolymers, polyoxyethylene alkyl allyl ethers, polyoxyethylene fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene nonyl phenyl ethers, polyoxyethylene naphthyl ethers, polyoxyethylene octylphenyl ethers, polyoxyethylene alkyl amines, glycerin fatty acid esters and acetylene series polyoxyethylene oxides, and these may be used individually or as a mixture of two or more thereof. The nonionic surfactant is preferably a polyoxypropylene block copolymer. The molecular weight of the polyoxypropylene block copolymer is preferably within the range of 500 to 15000. [0048] The nonionic surfactant is preferably added at a concentration within the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05 by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is more than 5% by weight, deactivation of carbohydrase is promoted, which is economically disadvantageous and hence not preferred. [0049] The secondary hydrolysis may be carried out in the presence of an inorganic salt(s), and examples of the inorganic salt(s) which may be used include sodium salts, potassium salts, magnesium salts, sulfuric acid salts, ammonium salts, hydrochloric acid salts, phosphoric acid salts, acetic acid salts and nitric acid salts. Examples of more preferred inorganic salts include sodium chloride, sodium acetate, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium chloride, dipotassium hydrogen phosphate, ammonium sulfate, magnesium chloride and magnesium sulfate. Among these, sodium chloride, sodium sulfate and sodium hydrogen sulfate, which are sodium salts; and magnesium chloride and magnesium sulfate, which are magnesium salts; are most preferred. By addition of such an inorganic salt(s), the Avicel-degrading activity and the xylan-degrading activity in the recovered enzyme can be increased. [0050] Further, as an alternative to such inorganic salts, sea water may be used. Sea water is an aqueous inorganic salt solution which contains 2.6 to 2.7% sodium chloride, 0.3 to 0.4% magnesium chloride, 0.1 to 0.2% magnesium sulfate and about 0.07% potassium chloride and which occurs in nature in the largest amount. Therefore, sea water may be used as an aqueous inorganic salt solution in the secondary hydrolysis. The pH of sea water is mostly dependent on its salt composition, and generally within the range of 8.2 to 8.5. Sea water may be used in the secondary hydrolysis either without changing the pH or after adjusting the pH to an arbitrary value. It is preferred to adjust the pH to a value within the range of 5 to 8.3 in view of enhancement of the cellulase activity of the recovered enzyme. For the adjustment of the pH, a common acid such as sulfuric acid or hydrochloric acid may be used, and the acid is not restricted. [0051] Further, as an alternative to such an inorganic salt(s), ash prepared by subjecting cellulose-containing biomass, a pretreated product of cellulose-containing biomass, the saccharification residue obtained after hydrolysis of cellulose-containing biomass, or the like to boiler combustion may be used. Such ash contains a large amount of potassium salts, and an aqueous inorganic salt solution can be prepared by dissolving the salts in water. [0052] The inorganic salt(s) is/are preferably added at a concentration within the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05 by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is more than 5% by weight, deactivation of carbohydrase is promoted, which is economically disadvantageous and hence not preferred. In cases where sea water is used as the aqueous inorganic salt solution, the dilution rate of the sea water is preferably set within the range of 1/10 to 1. [0053] The secondary hydrolysis may be carried out in the presence of a hydrophilic organic solvent(s). The hydrophilic organic solvent in the present invention means one having a solubility of not less than 100 g/L in water at 20°C. On the other hand, an organic solvent having a solubility of less than 100 g/L under the above conditions is called a hydrophobic organic solvent. Examples of hydrophobic organic solvents include, but are not limited to, 1-butanol (74 g/L), 1-pentanol (27 g/L), 1-hexanol (5.8 g/L), ethyl acetate (83 g/L), hexane (trace amount) and chloroform (trace amount). Representative examples of the hydrophilic organic solvent in the present invention include methanol, ethanol, 1-propanol, isopropanol, dimethyl sulfoxide, N,N-dimethylformamide, acetone, acetonitrile, ethylene glycol and glycerin. By adding such a hydrophilic organic solvent, the Avicel-degrading activity of the recovered enzyme can be enhanced, which is preferred. [0054] The above hydrophilic organic solvent(s) is/are preferably added at a concentration within the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05 by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is more than 5% by weight, deactivation of carbohydrase is promoted, which is economically disadvantageous and hence not preferred. [0055] The secondary hydrolysis may be carried out in the presence of an amino acid(s), and examples of the amino acid(s) which may be used include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine, and derivatives thereof. Among these amino acids, alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan and valine, which have high solubility in water, are preferred. Arginine, cysteine, glutamic acid, histidine and lysine, with which recovered enzyme having high Avicel-degrading activity can be obtained, are most preferred. [0056] The above amino acid(s) is/are preferably added at a concentration within the range of 0.05 to 5% by weight. In cases where the concentration is less than 0.05 by weight, the recovery efficiency of carbohydrase is low, while in cases where the concentration is more than 5% by weight, deactivation of carbohydrase is promoted, which is economically disadvantageous and hence not preferred. [0057] In the present invention, the primary sugar liquid and/or secondary sugar liquid is/are filtered through an ultrafiltration membrane, and carbohydrase is separated/recovered from the feed side, and a sugar solution is recovered from the permeate side. The molecular weight cutoff of the ultrafiltration membrane used in the present invention is not restricted as long as it allows permeation of glucose (molecular weight, 180), which is a monosaccharide, and allows blocking of the enzyme. More specifically, the molecular weight cutoff may be within the range of 500 to 50000, and the ultrafiltration membrane has a molecular weight cutoff of preferably within the range of 5000 to 50000, more preferably within the range of 10000 to 30000. Examples of the material which may be used for the functional membrane of the ultrafiltration membrane include polyether sulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfonated polyether sulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate and polytetrafluoroethylene. Since regenerated cellulose, cellulose and cellulose ester undergo degradation by cellulase, an ultrafiltration membrane using a synthetic polymer material such as PES or PVDF is preferably used. Examples of the method of filtration through an ultrafiltration membrane include dead-end filtration and cross-flow filtration, and the method is preferably cross-flow filtration in view of suppression of membrane fouling. Examples of the form of the ultrafiltration membrane which may be used as appropriate include the flat membrane, spiral-wound membrane, tubular membrane and hollow fiber membrane. Specific examples of the ultrafiltration membrane include Type G-5, Type G-10, Type G-20, Type G-50, Type PW and Type HWS UF, manufactured by DESAL; HFM-180, HFM-183, HFM-251, HFM-300, HFM-116, HFM-183, HFM-300, HFK-131, HFK-328, MPT-U20, MPS-U20P and MPS-U20S, manufactured by KOCH; SPE1, SPE3, SPE5, SPE10, SPE30, SPV5, SPV50 and SOW30, manufactured by Synder; products of Microza (registered trademark) UF series, manufactured by Asahi Kasei Corporation, having molecular weight cutoffs of 3000 to 100000; andNTR7410 and NTR7450, manufactured by Nitto Denko Corporation. [0058] In cases where a compound(s) such as a nonionic surfactant(s), inorganic salt(s), hydrophilic organic solvent(s), amino acid(s) and/or the like is/are added for the secondary hydrolysis, the secondary sugar liquid, of course, contains these compounds added. Such compounds may have inhibitory actions on the later fermentation step depending on their types and the amounts of addition. In such a case, only the recovered enzyme may be separated/recovered from the secondary sugar liquid using an ultrafiltration membrane and the sugar liquid containing inorganic salts obtained in the permeate side may be treated as a waste liquid. [0059] In the present invention, it is preferred to filter the primary sugar liquid and secondary sugar liquid through an ultrafiltration membrane and to further filter the sugar liquid obtained from the permeate side through a reverse osmosis membrane and/or nanofiltration membrane. In the present invention, the secondary sugar liquid is likely to have a lower sugar concentration compared to the primary sugar liquid because, for example, 1) since the secondary sugar liquid is produced by hydrolysis reaction using only carbohydrase adhered to the solids, the absolute amount of carbohydrase is smaller; and 2) the hydrolysis efficiency of lignocellulose which has remained as solids is low. Therefore, in cases where only the secondary sugar liquid, or a mixture of the secondary sugar liquid and the primary sugar liquid is used in the later fermentation step, the concentration of the fermentation product may become low due to low sugar concentration. However, by filtering the sugar liquid through a reverse osmosis membrane and/or nanofiltration membrane, the decrease in the sugar concentration in the sugar liquid can be prevented. The sugar concentration herein means the total amount of monosaccharide components, especially glucose and xylose. The concentration rate in such sugar concentration is not restricted as long as the concentration is carried out to achieve a concentration appropriate for the later fermentation step. The sugar concentration in the sugar solution before the concentration is not restricted, and is preferably 10 g/L to 100 g/L. The sugar concentration after the concentration is not restricted, and the sugar liquid can be generally preferably used in the later fermentation step in cases where the sugar concentration is 50 g/L to 200 g/L. [0060] Examples of the material of the nanofiltration membrane or reverse osmosis membrane which may be used in the present invention include polymer materials such as cellulose acetate polymers, polyamides, polyesters, polyimides, vinyl polymers and polysulfones. The membrane is not restricted to a membrane constituted by only one of the materials, and may be a membrane comprising a plurality of membrane materials. [0061] As the nanofiltration membrane to be used in the present invention, a spiral-wound membrane element is preferred. Specific examples of the preferred nanofiltration membrane element include a cellulose acetate nanofiltration membrane element GE Sepa, manufactured by GE Osmonics; nanofiltration membrane elements NF99 and NF99HF, manufactured by Alfa-Laval, which have polyamide functional layers; nanofiltration membrane elements NF-45, NF-90, NF-200, NF-270 and NF-400, manufactured by FilmTec Corporation, which have cross-linked piperazine polyamide functional layers; and nanofiltration membrane elements SU-210, SU-220, SU-600 and SU-610, manufactured by Toray Industries, Inc., comprising a nanofiltration membrane UTC60, manufactured by the same manufacturer, which comprises a cross-linked piperazine polyamide as a major component. The nanofiltration membrane element is more preferably NF99 or NF99HF; NF-45, NF-90, NF-200 or NF-400; or SU-210, SU-220, SU-600 or SU-610. The nanofiltration membrane element is still more preferably SU-210, SU-220, SU-600 or SU-610. [0062] As the reverse osmosis membrane to be used in the present invention, a spiral-wound membrane element is preferred as in the case of the nanofiltration membrane. Specific examples of the preferred reverse osmosis membrane element include polyamide reverse osmosis membrane modules manufactured by TORAY INDUSTRIES, INC. SU-710, SU-720, SU-720F, SU-710L, SU-720L, SU-720LF, SU-720R, SU-710P and SU-720P, which are low-pressure type modules, as well as SU-810, SU-820, SU-820L and SU-820FA containing UTC70 as a reverse osmosis membrane, which are high-pressure type modules; cellulose acetate reverse osmosis membranes manufactured by the same manufacturer SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100 and SC-8200; NTR-759HR, NTR-729HF, NTR-70SWC, ES10-D, ES20-D, ES20-U, ES15-D, ES15-U and LFIO-D, manufactured by Nitto Denko Corporation; R098pHt, R099, HR98PP and CE4040C-30D, manufactured by Alfa-Laval; GE Sepa, manufactured by GE; and BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040 and SW30HRLE-4040, manufactured by FilmTec Corporation. [0063] The apparatus for carrying out the above-described method of the present invention for producing a sugar liquid is described below. The apparatus for carrying out the method of the present invention for producing a sugar liquid needs to comprise as constituents at least: a stirring tank 2 for carrying out the primary hydrolysis; secondary hydrolysis tank 28 or press filtration device 8 for carrying out the secondary hydrolysis; solid-liquid separation device(s) (25, 30) for the primary hydrolysate and the secondary hydrolysate; and ultrafiltration membrane device (12, 33) for separating the carbohydrase and the sugar liquid from the primary sugar liquid and/or the secondary sugar liquid. For describing embodiments of such an apparatus, specific examples are shown in Fig. 2 to Fig. 8 and Fig. 10 to Fig. 16. The apparatuses in Fig. 2 to Fig. 8 and Fig. 10 to Fig. 16 were classified into Form 1 to Form 4 based on their characteristics. Form 1 is an apparatus form in which the secondary hydrolysis is carried out in a press filtration tank 8, and corresponds to Fig. 2 to Fig. 8. [0064] Form 1 is an embodiment wherein water is circulated into the press filtration chamber, and it is an apparatus form with which the secondary hydrolysis of the present invention can be carried out as long as the apparatus has a press filtration device 8 for solid-liquid separation. This has an advantage in that the constitution of the apparatus is simple and the cost for the apparatus can hence be suppressed. However, it has a drawback in that the primary sugar liquid and the secondary sugar liquid are contaminated with each other in the apparatus. [0065] Form 2 is an apparatus form comprising a secondary hydrolysis tank 28 for performing the secondary hydrolysis. Form 2 comprises a stirring tank 2 and a secondary hydrolysis tank 28 separately. Form 2 has an advantage in that solids can be resuspended in the secondary hydrolysis tank 28 and the efficiency of the secondary hydrolysis is high. Depending especially on the types and the concentrations of the compounds added for the secondary hydrolysis, the compounds may have inhibitory actions on the later fermentation of a sugar liquid. Therefore, in view of avoiding contamination of the primary sugar liquid and the secondary sugar liquid with each other, it is advantageous to have, as in Form 2, the secondary hydrolysis tank 28 dedicated to the secondary hydrolysis separately from the stirring tank 2 for carrying out the primary hydrolysis, and to further have a solid-liquid separation 30 dedicated to the secondary sugar liquid and an ultrafiltration membrane device 33 dedicated to the secondary sugar liquid. However, Form 2 has a drawback in that, as the total number of equipments including the secondary hydrolysis tank 28 increases, the equipment cost increases. [0066] Form 3, which is an apparatus form wherein the secondary hydrolysis is carried out in the stirring tank 2 in which the primary hydrolysis is also carried out, is shown in a figure. Form 3 is an apparatus form wherein the hydrolysate obtained in the stirring tank 2 is subjected to solid-liquid separation and then returned again to the stirring tank 2, followed by adding water thereto to perform the secondary hydrolysis. Form 3 has an advantage in that the number of equipments can be the smallest and the equipment cost can be reduced. However, Form 3 has a drawback in that the primary sugar liquid and the secondary sugar liquid are contaminated with each other in the apparatus. [0067] Examples of Form 4, which is an embodiment wherein a microfiltration membrane device 36 is placed between a solid-liquid separation device 25 and an ultrafiltration membrane device 12 are partially shown in figures. The placement of the microfiltration membrane device has an advantage in that insoluble microparticles that could not be sufficiently removed by the solid-liquid separation can be removed, and membrane clogging in the ultrafiltration membrane device 12 can be reduced in a later step. [0068] [Table 1] [0069] Form 1, which is an embodiment wherein the secondary hydrolysis is carried out in a press filtration device, is described below using the schematic diagrams shown in Fig. 2. to Fig. 8. [0070] Examples of the apparatus for carrying out the method of the present invention for producing a sugar liquid include an apparatus comprising as constituents a stirring tank for the primary hydrolysis 2, press filtration device 8 having a warm-water supply tank 6, circulation line 10 for circulating the filtrate from the press filtration device 8 to the warm-water supply tank 6, and ultrafiltration membrane device 12 for separating the carbohydrase and the sugar solution from the primary sugar liquid and/or the secondary sugar liquid. The apparatus for the method of the present invention for producing a sugar liquid is described below with reference to the examples of the apparatus shown in figures. [0071] Fig. 2 and Fig. 3 are schematic diagrams showing apparatuses each of which uses a press filtration device 8 having a warm-water inlet 15 and a hydrolysate inlet 14 shown in Fig. 4 separately. Fig. 5 and Fig. 6 are schematic diagrams showing apparatuses each of which uses a press filtration device 8 having a hydrolysate-inlet-cum-warm-water inlet 21 shown in Fig. 7. Fig. 2 and Fig. 5 are schematic diagrams showing apparatuses each of which has a stirring tank 2 and a warm-water supply tank 6 separately. On the other hand, Fig. 3 and Fig. 6 are schematic diagrams showing apparatuses each of which uses a stirring tank 2 also as a warm-water supply tank. [0072] The apparatus shown in Fig. 2 is described below in detail. The stirring tank 2 for carrying out the primary hydrolysis has an inlet 3 for supplying cellulose, stirring device 4 for stirring/mixing lignocellulose, and thermostat 1 for keeping the temperature of the stirring tank. The primary hydrolysate obtained in the stirring tank 2 is fed to the press filtration device 8 from a hydrolysate inlet 14. In the press filtration device 8, solid-liquid separation is carried out by compression with a compressor 9, and warm water is supplied from a warm-water supply tank 6 to the solids retained in the press filtration chamber through a warm-water inlet 15. The warm-water supply tank 6 has a water supply line 5, warm-water supply tank thermostat 7 for keeping the temperature of the warm water at a predetermined value, and a circulation line 10 for circulating the filtrate obtained by the press filtration. The primary sugar liquid and/or the secondary sugar liquid obtained by the press filtration is retained in a filtrate recovery tank 11, and filtered through an ultrafiltration membrane device 12. The recovered carbohydrase is recovered and/or reused through a carbohydrase recovery line. [0073] The apparatus shown in Fig. 3 is described below in detail. The stirring tank 2 for carrying out the primary hydrolysis has an inlet 3 for feeding cellulose, stirring device 4 for stirring/mixing cellulose, and thermostat 1 for keeping the temperature of the stirring tank. The primary hydrolysate obtained in the stirring tank 2 is fed to the press filtration device 8 from a hydrolysate inlet 14. In the press filtration device 8, solid-liquid separation is carried out by compression with a compressor 9, and warm water is supplied from a warm-water supply tank 6 to the solids retained in the press filtration chamber through a warm-water inlet 15. A hydrolysate inlet 14 and a warm-water inlet 15 are connected to the press filtration device 8, and the flow can be switched with a valve. The primary sugar liquid and/or the secondary sugar liquid obtained by the press filtration is retained in a filtrate recovery tank 11, and filtered through an ultrafiltration membrane device 12. The recovered carbohydrase is recovered and/or reused through a carbohydrase recovery line. [0074] The apparatus shown in Fig. 5 is described below in detail. The stirring tank 2 for carrying out the primary hydrolysis has an inlet 3 for feeding cellulose, stirring device 4 for stirring/mixing cellulose, and thermostat 1 for keeping the temperature of the stirring tank. The primary hydrolysate obtained in the stirring tank 2 is fed to the press filtration device 8 from a hydrolysate-inlet-cum-warm-water inlet 21. In the press filtration device 8, solid-liquid separation is carried out by compression with a compressor 9, and warm water is supplied from a warm-water supply tank 6 to the solids retained in the press filtration chamber through the hydrolysate-inlet-cum-warm-water inlet 21. The warm-water supply tank 6 has a water supply line 5, warm-water supply tank thermostat 7 for keeping the temperature of the warm water at a predetermined value, and a circulation line 10 for circulating the filtrate obtained by the press filtration. The primary sugar liquid and/or the secondary sugar liquid obtained by the press filtration is/are retained in a filtrate recovery tank 11, and filtered through an ultrafiltration membrane device 12. The recovered carbohydrase is recovered and/or reused through a carbohydrase recovery line. [0075] The apparatus shown in Fig. 6 is described below in detail. The stirring tank 2 for carrying out the primary hydrolysis has an inlet 3 for feeding cellulose, stirring device 4 for stirring/mixing cellulose, and thermostat 1 for keeping the temperature of the stirring tank. The primary hydrolysate obtained in the stirring tank 2 is fed to the press filtration device 8 from a hydrolysate-inlet-cum-warm-water inlet 21. In the press filtration device 8, solid-liquid separation is carried out by compression with a compressor 9, and warm water is supplied from the stirring tank 2 to the solids retained in the press filtration chamber through the hydrolysate-inlet-cum-warm-water inlet 21. The primary sugar liquid and/or the secondary sugar liquid obtained by the press filtration is/are retained in a filtrate recovery tank 11, and filtered through an ultrafiltration membrane device 12. The recovered carbohydrase is recovered and/or reused through a carbohydrase recovery line. [0076] In the above-described apparatuses, the secondary hydrolysis can be carried out by subjecting the primary hydrolysate to press filtration and feeding and/or circulating warm water at 40 to 60°C to the filtration chamber tank retaining the obtained solids. Since the solids after press filtration have low moisture content and low fluidity, performing the secondary hydrolysis in a separate stirring vessel or the like requires power to supply energy for re-dispersion of the solids. By feeding warm water prewarmed to a temperature within the range of 40 to 60°C in the warm- water supply tank 6 to the press filtration chamber, the activity of the enzyme components adsorbed to the solids can be increased, so that the secondary hydrolysis of the present invention can be carried out. In cases where the amount of the warm water fed is too large, the sugar concentration in the secondary sugar liquid is too low, which is not preferred. On the other hand, in cases where the amount of the warm water fed is too small, the reaction temperature in the filtration chamber cannot be sufficiently kept, which is not preferred. It should be noted that, by heating the once-fed water to 40 to 60°C and circulating the water again, the reaction temperature can be maintained, and the sugar concentration in the secondary sugar liquid can be increased. The length of time of the feeding and/or circulation of warm water is preferably within the range of 5 minutes to 180 minutes. In cases where the length of time is shorter than 5 minutes, the secondary hydrolysis cannot be sufficiently carried out, while in cases where the length of time is longer than 180 minutes, the sugar production rate tends to be saturated, which is not preferred from the viewpoint of energy. [0077] The press filtration device is shown in Fig. 4 and Fig. 7 as schematic diagrams. In the device shown in Fig. 4, the primary hydrolysate is fed from the hydrolysate inlet 14 into the press filtration chamber 20, and solid-liquid separation is carried out by compression with a pressing plate 19. Thereafter, warm water is fed through the warm-water inlet 15 to bring the warm water into contact with the solids (primary hydrolysate) 18, followed by being filtered through a filter cloth 17. The filtrate is further circulated through a thermostat, and fed again into the press filtration chamber through the warm-water inlet 15. By allowing such circulation, the secondary hydrolysis can be carried out in the press filtration chamber. Fig. 7 is a schematic diagram showing a method for supplying warm water through a hydrolysate-inlet-cum-warm-water inlet 21. That is, the primary hydrolysate is fed through the hydrolysate-inlet-cum-water inlet 21 into the press filtration chamber 20 and subjected to compression with a pressing plate 19, by which solid-liquid separation is carried out. Thereafter, warm water is fed through the hydrolysate-inlet-cum-warm-water inlet 21 to bring the warm water into contact with the solids (primary hydrolysate) 18, followed by being filtered through a filter cloth 17. The filtrate is further circulated through a thermostat, and fed again into the press filtration chamber through the hydrolysate-inlet-cum-warm-water inlet 21. By allowing such circulation, the secondary hydrolysis can be carried out in the press filtration chamber. The length of time of the feeding and/or circulation of warm water into the press filtration chamber is preferably within the range of 5 minutes to 180 minutes. In cases where the length of time is shorter than 5 minutes, the secondary hydrolysis cannot be sufficiently carried out, while in cases where the length of time is longer than 180 minutes, the sugar production rate tends to be saturated, which is not preferred from the viewpoint of energy. In cases where the amount of the warm water fed is too large, the sugar concentration in the secondary sugar liquid is too low, which is not preferred. On the other hand, in cases where the amount of the warm water fed is too small, the reaction temperature in the filtration chamber cannot be sufficiently kept, which is not preferred. In such cases, by heating the once-fed water to 40 to 60°C and circulating the water again, the reaction temperature can be maintained, and the sugar concentration in the secondary sugar liquid can be increased. [0078] Fig. 8 is a schematic diagram showing an apparatus wherein a sugar concentrating device having a reverse osmosis membrane and/or nanofiltration membrane for concentrating the sugar liquid is further attached to the apparatus shown in Fig. 2. More specifically, this apparatus comprises, in the filtrate side of the ultrafiltration membrane device 12, a sugar solution tank 22; nanofiltration membrane device and/or reverse osmosis membrane device 23 connected thereto via a pump; and a filtrate line 24. In cases where the nanofiltration membrane device and/or reverse osmosis membrane device is/are connected to the apparatus shown in Fig. 3, 5 or 6, the nanofiltration membrane device and/or reverse osmosis membrane device may be connected, in the same manner as in Fig. 8, downstream of the ultrafiltration membrane device 12, which is included in the apparatus shown in Fig. 3, 5 or 6 similarly to the apparatus shown in Fig. 2. [0079] Form 2, which is an embodiment wherein the secondary hydrolysis is carried out in a secondary hydrolysis tank, is described below with reference to Fig. 10 to Fig. 12. [0080] Fig. 10 is a diagram showing an example of an apparatus system having a stirring tank for the primary hydrolysis 2 and a secondary hydrolysis tank 28 separately. The solid-liquid separation device 25 is not restricted as long as it enables solid-liquid separation of the primary hydrolysate using a centrifuge, filter press, belt filter or the like. The solids obtained with the solid-liquid separation device 25 is transferred to the secondary hydrolysis tank 28 by a solid transfer means 26. The solid transfer means 26 is not restricted as long as it is suitable for the properties of the solids, and examples of the means include a belt conveyer and a screw pump. The secondary hydrolysis tank 28 at least comprises a thermostat 2 (secondary hydrolysis tank) 27 for carrying out the secondary hydrolysis. The secondary hydrolysis tank 28 may further comprise a stirring device 2 (secondary hydrolysis tank) 29 for mixing the solids by stirring. The secondary hydrolysis tank 28 further comprises a solid-liquid separation device 2 (secondary hydrolysis tank) 30 for carrying out solid-liquid separation of the secondary hydrolysate. The secondary sugar liquid separated through the solid-liquid separation device 2 (secondary hydrolysis tank) 30 is transferred to a secondary sugar liquid recovery tank 32. The secondary sugar liquid in the secondary sugar liquid recovery tank 32 is filtered through a secondary sugar liquid ultrafiltration membrane device 33 to recover the enzyme. [0081] Fig. 11 shows an embodiment of an apparatus system which has a stirring tank 2 for carrying out the primary hydrolysis and a secondary hydrolysis tank 28 separately, and a solid-liquid separation device 25 and a solid-liquid separation device 2 (secondary hydrolysate) 30 separately, while an ultrafiltration membrane device 12 is shared by the primary sugar liquid and the secondary sugar liquid. As in Fig. 10, the secondary hydrolysate obtained in the secondary hydrolysis tank 28 is subjected to solid-liquid separation in the solid-liquid separation device 2 (secondary hydrolysate) 30, and transferred a filtrate tank 11 through a secondary sugar liquid transfer line 34. The primary sugar liquid and the secondary sugar liquid recovered into the filtrate tank 11 are filtered through an ultrafiltration membrane 12 at once or sequentially, and the enzyme and the sugar are thereby separated. [0082] Fig. 12 shows an embodiment of an apparatus system which has a stirring tank 2 for carrying out the primary hydrolysis and a secondary hydrolysis tank 28 separately, while a solid-liquid separation device 25 and an ultrafiltration membrane device 12 are commonly used by the primary sugar liquid and the secondary sugar liquid. The secondary hydrolysate obtained in a secondary hydrolysis tank 28 is transferred to a solid-liquid separation device 25 through a secondary hydrolysate transfer line 35, and separated into the secondary sugar liquid and solids. The primary sugar liquid and the secondary sugar liquid separated in the solid-liquid separation device 25 is recovered into a filtrate recovery tank 11 and filtered through an ultrafiltration membrane 12 at once or sequentially, and the enzyme and the sugar are thereby separated. [0083] Form 3, which is an embodiment wherein the secondary hydrolysis is carried out in a tank which is also used for the primary hydrolysis, is described below with reference to Fig. 13. [0084] The apparatus shown in Fig. 13 is for an embodiment wherein the primary hydrolysate obtained in a stirring tank 2 for carrying out the primary hydrolysis is separated by a solid-liquid separation device 25, and the obtained solids are circulated to the primary hydrolysis tank through a secondary hydrolysate transfer line 35, followed by carrying out the secondary hydrolysis in the primary hydrolysis tank. The primary sugar liquid and the secondary sugar liquid separated in the solid-liquid separation device 25 are collected into a filtrate recovery tank 11, and filtered through an ultrafiltration membrane device 12 to separate the enzyme and the sugar. [0085] Apparatus forms wherein a microfiltration membrane device is placed upstream of an ultrafiltration membrane device are described below with reference to Fig. 14 to Fig. 16. Fig. 14 shows an embodiment (partial diagram) wherein a microfiltration membrane device 36 is placed upstream of an ultrafiltration membrane device 12. The microfiltration membrane device 36 is not restricted as long as it can remove insoluble microparticle components contained in the primary sugar liquid and/or the secondary sugar liquid obtained in the solid-liquid separation device 33 or the press filtration device 8, and examples of the microfiltration membrane device 36 include microfiltration membranes having average pore sizes within the range of 0.01 um to 1 um. The method of filtration in the microfiltration membrane device 36 may be either cross-flow filtration (Fig. 15) or dead-end filtration (Fig. 16). [0086] Fig. 15 shows an embodiment of the microfiltration membrane device 36 for performing the microfiltration by cross-flow filtration. The primary sugar liquid and/or the secondary sugar liquid may be stored in a microfiltration membrane raw liquid tank 37 and filtered through a microfiltration membrane 38 while being circulated by a pump. [0087] Fig. 16 shows an embodiment wherein the microfiltration is carried out by dead-end filtration. The primary sugar liquid and/or the secondary sugar liquid is/are stored in a microfiltration membrane raw liquid tank 37 and filtered through a microfiltration membrane 38. In the case of dead-end filtration, a compressed-air supply device 39 for performing air-bubble washing of the membrane surface may be provided as appropriate, and a reverse-washing pump 40 for reverse washing may be placed. The reverse washing may be carried out with the filtrate recovered into a microfiltrate recovery tank 41, or, in some cases, with a common membrane washing liquid or liquid agent. The microfiltration membrane 38 may be in the form of either a flat membrane or hollow fiber membrane. The hollow fiber membrane may be either an internal-pressure type membrane or an external-pressure type membrane. EXAMPLES [0088] The present invention is described below more specifically by way of Examples. However, the present invention is not restricted to these Examples. [0089] (Reference Example 1) Preparation of Pretreated Cellulose 1. Preparation of Pretreated Cellulose 1 (Dilute Sulfuric Acid Treatment) As the cellulose, rice straw was used. The cellulose was soaked in 1% aqueous sulfuric acid solution, and subjected to treatment using an autoclave (manufactured by Nitto Kdatsu Co., Ltd.) at 150°C for 30 minutes. After the treatment, solid-liquid separation was carried out to separate sulfuric acid-treated cellulose from the aqueous sulfuric acid solution (hereinafter referred to as "dilute-sulfuric-acid treatment liquid"). Subsequently, the sulfuric acid-treated cellulose was mixed with the dilute-sulfuric-acid treatment liquid with stirring such that the concentration of the solid contents is 10% by weight, and the pH was adjusted to about 5 with sodium hydroxide. The resulting mixture was used in the Examples below as the pretreated cellulose 1. [0090] 2. Preparation of Pretreated Cellulose 2 (Ammonia Treatment) As the cellulose, rice straw was used. The cellulose was fed into a compact reactor (manufactured by Taiatsu Techno Corporation, TVS-N2 30 ml), and cooled with liquid nitrogen. Into this reactor, ammonia gas was flown, and the sample was completely soaked in liquid ammonia. The lid of the reactor was closed, and the reactor was left to stand at room temperature for about 15 minutes. Subsequently, the reactor was processed in an oil bath at 150°C for 1 hour. Thereafter, the reactor was removed from the oil bath, and the ammonia gas was leaked in a fume hood, followed by vacuuming the inside of the reactor to 10 Pa with a vacuum pump, thereby drying the cellulose. The resultant was used in the Examples below as the pretreated cellulose 2. [0091] 3. Preparation of Pretreated Cellulose 3 (Hydrothermal Treatment) As the cellulose, rice straw was used. The cellulose was soaked in water, and subjected to treatment using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 180°C for 20 minutes with stirring. The treatment was carried out at a pressure of 10 MPa. After the treatment, solid-liquid separation was carried out by centrifugation (3000 G) to separate the processed biomass component from the solution component (hereinafter referred to as "hydrothermally treated liquid"). The processed biomass component was used in the Examples below as the pretreated cellulose 3. [0092] (Reference Example 2) Measurement of Sugar Concentration The concentrations of glucose and xylose contained in the sugar liquid were measured under the HPLC conditions described below based on comparison with standard samples. Column: Luna NH2 (manufactured by Phenomenex, Inc.) Mobile phase: MilliQ:acetonitrile = 25:75 (flow rate, 0.6 mL/minute) Reaction solution: None Detection method: RI (differential refractive index) Temperature: 30°C [0093] (Reference Example 3) Preparation of Trichoderma-derived Cellulase Trichoderma-derived cellulase was prepared by the following method. [0094] [Preculture] The mixture of 5% corn steep liquor (w/vol), 2% glucose (w/vol), 0.37% ammonium tartrate (w/vol), 0.14% (w/vol) ammonium sulfate, 0.2% (w/vol) potassium dihydrogen phosphate, 0.03% (w/vol) calcium chloride dihydrate, 0.03% (w/vol) magnesium sulfate heptahydrate, 0.02% (w/vol) zinc chloride, 0.01% (w/vol) iron (TTI) chloride hexahydrate, 0.004% (w/vol) copper (II) sulfate pentahydrate, 0.0008% (w/vol) manganese chloride tetrahydrate, 0.0006% (w/vol) boric acid and 0.0026% (w/vol) hexaammonium heptamolybdate tetrahydrate in distilled water was prepared, and 100 mL of this mixture was placed in a baffled 500-mL Erlenmeyer flask, followed by being sterilized by autoclaving at 121 °C for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each of which was sterilized by autoclaving at 121 °C for 15 minutes separately from the mixture, were added thereto at 0.01% (w/vol) each. To this preculture medium, Trichoderma reesei PC3-7 was inoculated at 1 x 105 cells/mL, and the cells were cultured at 28°C for 72 hours with shaking at 180 rpm, to perform preculture (shaker: BIO-SHAKER BR-40LF, manufactured by TAITEC CORPORATION). [0095] [Main Culture] The mixture of 5% corn steep liquor (w/vol), 2% glucose (w/vol), 10% (w/vol) cellulose (Avicel), 0.37% ammonium tartrate (w/vol), 0.14% (w/vol) ammonium sulfate, 0.2% (w/vol) potassium dihydrogen phosphate, 0.03% (w/vol) calcium chloride dihydrate, 0.03% (w/vol) magnesium sulfate heptahydrate, 0.02% (w/vol) zinc chloride, 0.01% (w/vol) iron (III) chloride hexahydrate, 0.004% (w/vol) copper (II) sulfate pentahydrate, 0.0008% (w/vol) manganese chloride tetrahydrate, 0.0006% (w/vol) boric acid and 0.0026% (w/vol) hexaammonium heptamolybdate tetrahydrate in distilled water was prepared, and 2.5 L of this mixture was placed in a 5-L stirring jar (manufactured by ABLE, DPC-2A), followed by being sterilized by autoclaving at 121 °C for 15 minutes. After allowing the mixture to cool, PE-M and Tween 80, each of which was sterilized by autoclaving at 121°C for 15 minutes separately from the mixture, were added thereto at 0.1% each. To the resulting mixture, 250 mL of preculture of Trichoderma reesei PC3-7 preliminarily prepared with a liquid medium by the method described above was inoculated. The cells were cultured at 28°C for 87 hours at 300 rpm at an aeration rate of 1 wm. After centrifugation, the supernatant was subjected to membrane filtration (Stericup-GV, manufactured by Millipore, material: PVDF). To the culture liquid prepared under the above-described conditions, p-glucosidase (Novozyme 188) was added at a protein weight ratio of 1/100, and the resulting mixture was used as Trichoderma- derived cellulase in the Examples below. [0096] (Reference Example 4) Method of Measurement of Cellulase Activity The cellulase activity was measured and evaluated by the following procedures in terms of four types of degradation activities: 1) Avicel-degrading activity; 2) carboxymethyl cellulose (CMC)-degrading activity; 3) cellobiose-degrading activity; and 4) xylan-degrading activity. [0097] 1) Avicel-degrading Activity To an enzyme liquid (prepared under predetermined conditions), Avicel (Cellulose Microcrystalline, manufactured by Merch) was added at 1 g/L and sodium acetate buffer (pH 5.0) was added at 100 mM, followed by allowing the resulting mixture to react at 50°C for 24 hours. This reaction liquid was prepared in a 1-mL tube, and the reaction was allowed to proceed with mixing by rotation under the above-described conditions. Thereafter, the tube was subjected to centrifugation, and the glucose concentration in the supernatant component was measured. The measurement of the glucose concentration was carried out according to the method described in Reference Example 2. The concentration of the produced glucose (g/L) was used as it is as the activity value of the Avicel-degrading activity. [0098] 2) CMC-degrading Activity To an enzyme liquid, carboxymethyl cellulose was added at 10 g/L and sodium acetate buffer (pH 5.0) was added at 100 mM, followed by allowing the resulting mixture to react at 50°C for 0.5 hour. This reaction liquid was prepared in a 1-mL tube, and the reaction was allowed to proceed with mixing by rotation under the above-described conditions. Thereafter, the tube was subjected to centrifugation, and the glucose concentration in the supernatant component was measured. The measurement of the glucose concentration was carried out according to the method described in Reference Example 2. The concentration of the produced glucose (g/L) was used as it is as the activity value of the CMC-degrading activity. [0099] 3) Cellobiose-degrading Activity To an enzyme liquid, cellobiose (Wako Pure Chemical Industries, Ltd.) was added at 500 mg/L and sodium acetate buffer (pH 5.0) was added at 100 mM, followed by allowing the resulting mixture to react at 50°C for 0.5 hour. This reaction liquid was prepared in a 1-mL tube, and the reaction was allowed to proceed with mixing by rotation under the above-described conditions. Thereafter, the tube was subjected to centrifugation, and the glucose concentration in the supernatant component was measured. The measurement of the glucose concentration was carried out according to the method described in Reference Example 2. The concentration of the produced glucose (g/L) was used as it is as the activity value of the cellobiose-degrading activity. [0100] 4) Xylan-degrading Activity To an enzyme liquid, xylan (Birch wood xylan, Wako Pure Chemical Industries, Ltd.) was added at 10 g/L and sodium acetate buffer (pH 5.0) was added at 100 mM, followed by allowing the resulting mixture to react at 50°C for 4 hours. This reaction liquid was prepared in a 1-mL tube, and the reaction was allowed to proceed with mixing by rotation under the above-described conditions. Thereafter, the tube was subjected to centrifugation, and the xylose concentration in the supernatant component was measured. The measurement of the xylose concentration was carried out according to the method described in Reference Example 2. The concentration of the produced xylose (g/L) was used as it is as the activity value of the xylose-degrading activity. [0101] (Example 1) Hydrolysis of Cellulose with Trichoderma-derwed Cellulase The results of the primary hydrolysis and the secondary hydrolysis in hydrolysis of cellulose using Trichoderma-derived cellulase are described in the Examples below. The method of the experiment was as follows. [0102] (Step 1: Primary Hydrolysis) To each of the pretreated celluloses 1 to 3 (1 g each), distilled water was added, and 10 mg of Trichoderma-deriwed cellulase was added, followed by adding distilled water such that the total weight is 10 g. Further, dilute sulfuric acid or dilute caustic soda was added thereto to adjust the pH of the composition to a value within the range of 4.5 to 5.3. The composition after the pH adjustment was transferred to a side-arm test tube (q>30 NS14/23, manufactured by Tokyo Rikakikai Co., Ltd.), and the composition was transferred to a side-arm reactor (cp30 NS14/23, manufactured by Tokyo Rikakikai Co., Ltd.), followed by performing hydrolysis at 50°C for 24 hours with incubation and stirring (compact mechanical stirrer CPS-1000, manufactured by Tokyo Rikakikai Co., Ltd., conversion adapter, feed inlet with a three-way stopcock, incubator MG-2200). The hydrolysate was subjected to solid-liquid separation by centrifugation (3000 G, 10 minutes), and, by this, the hydrolysate was separated into the primary sugar liquid (6 mL) and solids. The glucose and xylose concentrations in the obtained primary sugar liquid were measured by the methods described in Reference Example 2. The sugar yield (mg) in the primary sugar liquid was calculated according to the following equation. Table 2 shows a summary of the results. Sugar yield (mg) in the primary sugar liquid = {sugar concentration (g/L) after 24 hours of incubation - sugar concentration (g/L) after 0 hour of incubation} x 6 (mL) [0104] (Step 2: Secondary Hydrolysis) To the solids obtained in Step 1, distilled water was added such that the total weight is 10 g. Further, dilute sulfuric acid or dilute caustic soda was added thereto to adjust the pH of the composition to a value within the range of 4.5 to 5.3. The composition was transferred to a side-arm test tube (q>30 NS14/23, manufactured by Tokyo Rikakikai Co., Ltd.), and the composition was transferred to a side-arm reactor (

Documents

Orders

Section Controller Decision Date

Application Documents

# Name Date
1 8583-CHENP-2012 PCT 08-10-2012.pdf 2012-10-08
1 8583-CHENP-2012-RELEVANT DOCUMENTS [30-09-2023(online)].pdf 2023-09-30
2 8583-CHENP-2012 FORM-5 08-10-2012.pdf 2012-10-08
2 8583-CHENP-2012-RELEVANT DOCUMENTS [23-09-2022(online)].pdf 2022-09-23
3 8583-CHENP-2012-RELEVANT DOCUMENTS [11-08-2021(online)].pdf 2021-08-11
3 8583-CHENP-2012 FORM-3 08-10-2012.pdf 2012-10-08
4 8583-CHENP-2012-RELEVANT DOCUMENTS [03-03-2020(online)].pdf 2020-03-03
4 8583-CHENP-2012 FORM-2 08-10-2012.pdf 2012-10-08
5 8583-CHENP-2012-IntimationOfGrant28-06-2019.pdf 2019-06-28
5 8583-CHENP-2012 FORM-1 08-10-2012.pdf 2012-10-08
6 8583-CHENP-2012-PatentCertificate28-06-2019.pdf 2019-06-28
6 8583-CHENP-2012 ENGLISH TRANSLATION 08-10-2012.pdf 2012-10-08
7 Abstract_Granted 315129_28-06-2019.pdf 2019-06-28
7 8583-CHENP-2012 DRAWINGS 08-10-2012.pdf 2012-10-08
8 Claims_Granted 315129_28-06-2019.pdf 2019-06-28
8 8583-CHENP-2012 DESCRIPTION (COMPLETE) 08-10-2012.pdf 2012-10-08
9 8583-CHENP-2012 CORRESPONDENCE OTHERS 08-10-2012.pdf 2012-10-08
9 Description_Granted 315129_28-06-2019.pdf 2019-06-28
10 8583-CHENP-2012 CLAIMS 08-10-2012.pdf 2012-10-08
10 Drawings_Granted 315129_28-06-2019.pdf 2019-06-28
11 8583-CHENP-2012 ABSTRACT 08-10-2012.pdf 2012-10-08
11 Marked Up Claims_Granted 315129_28-06-2019.pdf 2019-06-28
12 8583-CHENP-2012-FORM 3 [22-05-2019(online)].pdf 2019-05-22
12 8583-CHENP-2012.pdf 2012-10-09
13 8583-CHENP-2012 FORM-3 04-04-2013.pdf 2013-04-04
13 Correspondence by Agent_Power Of Attorney_27-02-2019.pdf 2019-02-27
14 8583-CHENP-2012 CORRESPONDENCE OTHERS 04-04-2013.pdf 2013-04-04
14 8583-CHENP-2012-Annexure (Optional) [22-02-2019(online)].pdf 2019-02-22
15 8583-CHENP-2012-Written submissions and relevant documents (MANDATORY) [22-02-2019(online)].pdf 2019-02-22
15 8583-CHENP-2012_Marked-up Version.pdf 2014-03-12
16 8583-CHENP-2012-FORM 3 [09-01-2019(online)].pdf 2019-01-09
16 8583-CHENP-2012_Form 13.pdf 2014-03-12
17 8583-CHENP-2012-ExtendedHearingNoticeLetter_11Feb2019.pdf 2019-01-08
17 8583-CHENP-2012 Amended claims.pdf 2014-03-12
18 8583-CHENP-2012-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [07-01-2019(online)].pdf 2019-01-07
18 Form-18(Online).pdf 2014-03-17
19 8583-CHENP-2012 FORM-3 18-06-2015.pdf 2015-06-18
19 8583-CHENP-2012-HearingNoticeLetter.pdf 2018-12-11
20 8583-CHENP-2012 CORRESPONDENCE OTHERS 18-06-2015.pdf 2015-06-18
20 Correspondence by Agent_Power of Attorney, Assignment_20-06-2018.pdf 2018-06-20
21 8583-CHENP-2012-ABSTRACT [13-06-2018(online)].pdf 2018-06-13
21 8583-CHENP-2012-Form 3-231115.pdf 2016-06-09
22 8583-CHENP-2012-CLAIMS [13-06-2018(online)].pdf 2018-06-13
22 8583-CHENP-2012-Correspondence-231115.pdf 2016-06-09
23 8583-CHENP-2012-COMPLETE SPECIFICATION [13-06-2018(online)].pdf 2018-06-13
23 Form 3 [13-06-2016(online)].pdf 2016-06-13
24 8583-CHENP-2012-Correspondence-Change In Applicant Address-010116.pdf 2016-06-14
24 8583-CHENP-2012-DRAWING [13-06-2018(online)].pdf 2018-06-13
25 8583-CHENP-2012-FER_SER_REPLY [13-06-2018(online)].pdf 2018-06-13
25 Form 3 [24-11-2016(online)].pdf 2016-11-24
26 8583-CHENP-2012-FORM 3 [13-06-2018(online)].pdf 2018-06-13
26 8583-CHENP-2012-FORM 3 [27-11-2017(online)].pdf 2017-11-27
27 8583-CHENP-2012-FER.pdf 2018-02-23
27 8583-CHENP-2012-OTHERS [13-06-2018(online)].pdf 2018-06-13
28 8583-CHENP-2012-PETITION UNDER RULE 137 [13-06-2018(online)]-1.pdf 2018-06-13
28 8583-CHENP-2012-Proof of Right (MANDATORY) [13-06-2018(online)].pdf 2018-06-13
29 8583-CHENP-2012-PETITION UNDER RULE 137 [13-06-2018(online)].pdf 2018-06-13
30 8583-CHENP-2012-PETITION UNDER RULE 137 [13-06-2018(online)]-1.pdf 2018-06-13
30 8583-CHENP-2012-Proof of Right (MANDATORY) [13-06-2018(online)].pdf 2018-06-13
31 8583-CHENP-2012-FER.pdf 2018-02-23
31 8583-CHENP-2012-OTHERS [13-06-2018(online)].pdf 2018-06-13
32 8583-CHENP-2012-FORM 3 [13-06-2018(online)].pdf 2018-06-13
32 8583-CHENP-2012-FORM 3 [27-11-2017(online)].pdf 2017-11-27
33 8583-CHENP-2012-FER_SER_REPLY [13-06-2018(online)].pdf 2018-06-13
33 Form 3 [24-11-2016(online)].pdf 2016-11-24
34 8583-CHENP-2012-Correspondence-Change In Applicant Address-010116.pdf 2016-06-14
34 8583-CHENP-2012-DRAWING [13-06-2018(online)].pdf 2018-06-13
35 Form 3 [13-06-2016(online)].pdf 2016-06-13
35 8583-CHENP-2012-COMPLETE SPECIFICATION [13-06-2018(online)].pdf 2018-06-13
36 8583-CHENP-2012-CLAIMS [13-06-2018(online)].pdf 2018-06-13
36 8583-CHENP-2012-Correspondence-231115.pdf 2016-06-09
37 8583-CHENP-2012-ABSTRACT [13-06-2018(online)].pdf 2018-06-13
37 8583-CHENP-2012-Form 3-231115.pdf 2016-06-09
38 8583-CHENP-2012 CORRESPONDENCE OTHERS 18-06-2015.pdf 2015-06-18
38 Correspondence by Agent_Power of Attorney, Assignment_20-06-2018.pdf 2018-06-20
39 8583-CHENP-2012 FORM-3 18-06-2015.pdf 2015-06-18
39 8583-CHENP-2012-HearingNoticeLetter.pdf 2018-12-11
40 8583-CHENP-2012-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [07-01-2019(online)].pdf 2019-01-07
40 Form-18(Online).pdf 2014-03-17
41 8583-CHENP-2012 Amended claims.pdf 2014-03-12
41 8583-CHENP-2012-ExtendedHearingNoticeLetter_11Feb2019.pdf 2019-01-08
42 8583-CHENP-2012-FORM 3 [09-01-2019(online)].pdf 2019-01-09
42 8583-CHENP-2012_Form 13.pdf 2014-03-12
43 8583-CHENP-2012-Written submissions and relevant documents (MANDATORY) [22-02-2019(online)].pdf 2019-02-22
43 8583-CHENP-2012_Marked-up Version.pdf 2014-03-12
44 8583-CHENP-2012 CORRESPONDENCE OTHERS 04-04-2013.pdf 2013-04-04
44 8583-CHENP-2012-Annexure (Optional) [22-02-2019(online)].pdf 2019-02-22
45 8583-CHENP-2012 FORM-3 04-04-2013.pdf 2013-04-04
45 Correspondence by Agent_Power Of Attorney_27-02-2019.pdf 2019-02-27
46 8583-CHENP-2012-FORM 3 [22-05-2019(online)].pdf 2019-05-22
46 8583-CHENP-2012.pdf 2012-10-09
47 8583-CHENP-2012 ABSTRACT 08-10-2012.pdf 2012-10-08
47 Marked Up Claims_Granted 315129_28-06-2019.pdf 2019-06-28
48 8583-CHENP-2012 CLAIMS 08-10-2012.pdf 2012-10-08
48 Drawings_Granted 315129_28-06-2019.pdf 2019-06-28
49 Description_Granted 315129_28-06-2019.pdf 2019-06-28
49 8583-CHENP-2012 CORRESPONDENCE OTHERS 08-10-2012.pdf 2012-10-08
50 8583-CHENP-2012 DESCRIPTION (COMPLETE) 08-10-2012.pdf 2012-10-08
50 Claims_Granted 315129_28-06-2019.pdf 2019-06-28
51 8583-CHENP-2012 DRAWINGS 08-10-2012.pdf 2012-10-08
51 Abstract_Granted 315129_28-06-2019.pdf 2019-06-28
52 8583-CHENP-2012 ENGLISH TRANSLATION 08-10-2012.pdf 2012-10-08
52 8583-CHENP-2012-PatentCertificate28-06-2019.pdf 2019-06-28
53 8583-CHENP-2012-IntimationOfGrant28-06-2019.pdf 2019-06-28
53 8583-CHENP-2012 FORM-1 08-10-2012.pdf 2012-10-08
54 8583-CHENP-2012-RELEVANT DOCUMENTS [03-03-2020(online)].pdf 2020-03-03
54 8583-CHENP-2012 FORM-2 08-10-2012.pdf 2012-10-08
55 8583-CHENP-2012-RELEVANT DOCUMENTS [11-08-2021(online)].pdf 2021-08-11
55 8583-CHENP-2012 FORM-3 08-10-2012.pdf 2012-10-08
56 8583-CHENP-2012-RELEVANT DOCUMENTS [23-09-2022(online)].pdf 2022-09-23
56 8583-CHENP-2012 FORM-5 08-10-2012.pdf 2012-10-08
57 8583-CHENP-2012 PCT 08-10-2012.pdf 2012-10-08
57 8583-CHENP-2012-RELEVANT DOCUMENTS [30-09-2023(online)].pdf 2023-09-30

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1 8583_chenp_2012_09-02-2018.pdf

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