methods to produce a sugar liquid and to produce a chemical

专利摘要:
METHODS FOR PRODUCING A SUGAR LIQUID AND METHOD FOR PRODUCING A CHEMICAL This is the provision of a method for producing a sugar liquid, where the method comprises Steps (1) to (3): Step (1): one step of adding a filamentous fungus-derived cellulase to a cellulose pretreated product to obtain a hydrolyzate; Step (2): a step of adding residual molasses to said hydrolyzate to obtain a mixed sugar liquid; and Step (3): a step of subjecting said mixed sugar liquid to liquid-solid separation and filtering the obtained solution component through an ultrafiltration membrane to recover the filamentous fungus-derived cellulase as a non-permeate and to obtain a sugar liquid as a permeate. By the present invention, the recovery of filamentous fungus-derived cellulase enzyme from a cellulose hydrolyzate is improved so that the amount of cellulase used in the process to produce a sugar liquid can be reduced. Additionally, in the present invention, by adding residual molasses to the cellulose hydrolyzate to prepare a mixed sugar liquid, the sugar components can be recovered not only from cellulose, but also from (...).
公开号:BR112013022233B1
申请号:R112013022233-6
申请日:2012-03-02
公开日:2021-04-20
发明作者:Hiroyuki Kurihara；Katsushige Yamada；Yuki Yamamoto
申请人:Toray Industries, Inc.；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to a method for producing a sugar liquid from biomass. BACKGROUND OF THE INVENTION
[002] In recent years, methods for producing a sugar liquid by pre-treating a cellulose-containing biomass with an acid, hot water, alkali or the like and then adding cellulase to it to perform hydrolysis have been extensively studied. However, these methods that use cellulase have a disadvantage that since a large amount of cellulase is used and cellulase is expensive, the cost to produce a sugar liquid is high.
[003] As methods to solve the problem, methods in which the cellulase used for cellulose hydrolysis is recovered and reused have been proposed. Disclosed examples of such methods include a method in which continuous solid and liquid separation is performed with a spin filter and the obtained sugar liquid is filtered through an ultrafiltration membrane to recover cellulase (Patent Document 1), a method in that a surfactant is fed at the enzymatic saccharification stage to suppress cellulase adsorption and thereby enhance the recovery efficiency (Patent Document 2) and a method in which the residue produced by enzymatic saccharification is subjected to electrical treatment to recover the component of cellulase (Patent Document 3), but these methods have fundamentally failed to solve the problem. PRIOR ART DOCUMENTS Patent Documents Patent Document 1 in JP 2006-87319 A Patent Document 2 in JP 63-87994 A Patent Document 3 in JP 2008-206484 A BRIEF DESCRIPTION OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION
[004] The present invention aims, as described above, to reduce the amount of cellulase used in the hydrolysis of cellulose. MEANS TO SOLVE PROBLEMS
[005] The present inventors have intensively studied in order to solve the problem described above and, as a result, have focused attention on adding residual molasses to cellulose hydrolyzate. As a result, the present inventors have found that this improves the amount of cellulase recovered from the cellulose hydrolyzate, thereby completing the present invention.
[006] That is, the present invention has the constitutions [1] to [7] below.
[007] [1] A method for producing a sugar liquid, the method comprising Steps (1) to (3) below: Step (1): a step of adding a filamentous fungus-derived cellulase to a product cellulose pretreated to obtain a hydrolyzate; Step (2): a step of adding residual molasses to the hydrolyzate to obtain a mixed sugar liquid; and Step (3): a step in which the mixed sugar liquid undergoes separation between solid and liquid and filters the obtained solution component through an ultrafiltration membrane to recover the filamentous fungus-derived cellulase as a non-permeate and to get a sugar liquid as a permeate.
[008] [2] The method for producing a sugar liquid according to [1], wherein the filamentous fungus-derived cellulase of Step (1) is Trichoderma-derived cellulase.
[009] [3] The method for producing a sugar liquid according to [1] or [2], wherein the pretreated cellulose product of Step (1) is one or more of the products selected from the group consisting of products obtained by hydrothermal treatment, dilute sulfuric acid treatment or alkali treatment.
[010] [4] The method for producing a sugar liquid according to any one of [1] to [3], wherein, in Step (2), the residual molasses is added to the hydrolyzate to prepare a sugar liquid blend whose sugar concentration is within the range of 50 to 200 g/L.
[011] [5] The method for producing a sugar liquid according to any one of [1] to [4], wherein Step (2) comprises a process of incubating the mixed sugar liquid at a temperature within the range from 40 to 60 °C.
[012] [6] The method for producing a sugar liquid according to any one of [1] to [5], the method comprising the step of filtering the sugar liquid of Step (3) through a membrane of nanofiltration and/or reverse osmosis membrane to remove fermentation inhibitors as a permeate and to obtain a sugar concentrate as a non-permeate.
[013] [7] A method for producing a chemical, the method comprising culturing a fermentation of a microorganism that has an ability to produce a chemical with the use of, as a fermentation feedstock , a sugar liquid obtained by the method for producing a sugar liquid according to any one of [1] to [6]. EFFECT OF THE INVENTION
[014] Through the present invention, the enzymatic recovery of cellulase derived from filamentous fungus from a cellulose hydrolyzate is improved, so that the amount of cellulase used in the process to produce a sugar liquid can be reduced. Additionally, in the present invention, by adding residual molasses to the cellulose hydrolyzate to prepare a mixed sugar liquid, the sugar components can be recovered not only from the cellulose, but also from the residual molasses. BRIEF DESCRIPTION OF THE DRAWINGS
[015] Figure 1 is a schematic flowchart showing the steps of the present invention.
[016] Figure 2 is a schematic diagram of an apparatus to carry out the present invention.
[017] Figure 3 is a schematic diagram of an apparatus for producing a concentrated sugar liquid.
[018] Figure 4 is a schematic diagram that shows the production of a chemical with the use of a sugar liquid and/or concentrated sugar liquid as a fermentation feedstock.
[019] Figure 5 is a schematic diagram showing the constitution of an apparatus in a case where a liquid sugar manufacturing plant comprising the apparatus of Figure 2 is built next to an existing sugar manufacturing plant. BEST WAY TO PERFORM THE INVENTION
[020] The embodiments to carry out the present invention are described below in detail for each Step. STEP 1
[021] The cellulose pretreated product in Step (1) means a biomass that contains cellulose that has been pretreated by hydrolysis. Specific examples of cellulose-containing biomass include herbaceous biomasses such as bagasse, fast-growing grass, Bermuda grass, Erianthus, corn husk, rice straw and wheat straw; wood biomass such as trees and waste building materials; and biomass derived from the aquatic environment such as algae and seaweed. Such biomasses contain, in addition to cellulose and hemicellulose (hereafter referred to as “cellulose” as a general term for cellulose and hemicellulose), lignin as aromatic macromolecules and the like. In particular, in the present invention, pretreatment of a cellulose-containing biomass is performed in order to improve the efficiency of hydrolysis of the biomass by cellulase derived from filamentous fungus and the product obtained as a result is called cellulose pretreated product.
[022] Examples of pretreatment include acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, hydrothermal treatment, subcritical water treatment, spray treatment, evaporation treatment and drying treatment. In the present invention, the pretreatment is preferably hydrothermal treatment, dilute sulfuric acid treatment or alkali treatment since alkali treatment, hydrothermal treatment and dilute sulfuric acid treatment show better enzymatic saccharification efficiencies and require lower amounts of enzyme compared to other methods.
[023] In the case of hydrothermal treatment, water is added so that the concentration of biomass containing cellulose is 0.1 to 50% by weight, and the resulting mixture is treated at a temperature of 100 to 400 °C for 1 second to 60 minutes. By treatment under such temperature condition, a pretreated cellulose product which can be easily hydrolyzed by cellulase can be obtained. The number of treatment times is not restricted and the treatment can be performed 1 or more times. 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 subsequent treatments.
[024] 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 can 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 can be set within the range of 1 second to 60 minutes. The number of treatment times is not restricted and the treatment can be performed 1 or more times. 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 subsequent treatments. Since the hydrolyzate obtained by treating dilute sulfuric acid contains an acid, neutralization is necessary in order to further carry out the hydrolysis reaction with cellulase or in order to use the hydrolyzate as a fermentation raw material.
[025] The alkali treatment is a method in which an alkali selected from sodium hydroxide, calcium hydroxide and ammonia is allowed to act on a biomass that contains cellulose. As the alkali to be used in the alkali treatment, ammonia can be used especially preferentially. Ammonia treatment can be carried out by methods described in documents in JP 2008-161125 A and JP 2008-535664 A. For example, ammonia is added at a concentration within the range of 0.1 to 15% by weight for a biomass which contains cellulose and the treatment is carried out at 4 to 200 °C, preferably 90 to 150 °C. The ammonia to be added can be in a liquid or gas state. Additionally, the form of ammonia to be added can be pure ammonia or aqueous ammonia. The number of treatment times is not restricted and the treatment can be performed 1 or more times. 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 subsequent treatments. The treated product obtained by ammonia treatment needs to be subjected to ammonia neutralization or ammonia removal in order to further carry out the enzymatic hydrolysis reaction. Ammonia neutralization can be carried out after removal of the solid component from the hydrolyzate by separation of solid and liquid or in a state in which the solid component is contained. The acid reagent to be used for neutralization is not restricted. For ammonia removal, the ammonia-treated product can be kept under pressure to allow the ammonia to evaporate in the gaseous state. The removed ammonia can be recovered and reused.
[026] In Step (1), the pretreated cellulose product described above is subjected to hydrolysis with cellulase to obtain a hydrolyzate. Cellulose hydrolysis means decreasing the molecular weight of cellulose. Additionally, in cellulose hydrolysis, hemicellulose components such as xylan, mannan and arabinan are hydrolyzed at the same time. Examples of monosaccharide components contained in the hydrolyzate include glucose, xylose, mannose and galactose and the main monosaccharide component is glucose, which is a hydrolyzate of cellulose. Additionally, in cases where hydrolysis is insufficient, disaccharides such as cellobiose and xylobiose; cello-oligosaccharides; xylooligosaccharides; and the like; are contained.
[027] In Step (1), the pretreated cellulose product is hydrolyzed with a cellulase derived from filamentous fungus. Examples of filamentous fungus derived cellulase include those derived from Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor, Talaromyces, Phanerochaete, white rot fungi and brown rot fungi. In the present invention, among such cellulases derived from filamentous fungus, cellulase derived from Trichoderma, which has high cellulose degrading activity, is used preferentially.
[028] Cellulase derived from Trichoderma is an enzymatic composition comprising cellulase derived from a microorganism that belongs to the genus Trichoderma as a main component. The microorganism belonging to the genus Trichodermanão is restricted and is preferably Trichoderma reesei. Specific examples of such a microorganism include Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei Rut C-30, Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80, and Trichoderma viride. Cellulase can also be derived from a mutant strain originated from the Trichoderma microorganism described above, which mutant strain was prepared by mutagenesis using a mutagenic agent, UV irradiation or the like to enhance cellulase productivity .
[029] Trichoderma-derived cellulase used in the present invention is an enzymatic composition comprising a plurality of enzymatic components such as cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, xylanase and xylosidase, whose enzymatic composition has an activity to hydrolyze cellulose to cause saccharification. In the degradation of cellulose, cellulase derived from Trichoderma has a coordinated or complementary effect by the plurality of enzymatic components and enables a more efficient hydrolysis of the cellulose through it. The cellulase used in the present invention especially preferably comprises cellobiohydrolase and xylanase derived from Trichoderma.
[030] Cellobiohydrolase is a general term for cellulases that hydrolyze cellulose from the terminal portions. The group of enzymes belonging to cellobiohydrolase is described with the EC number: EC3.2.1.91.
[031] Endoglucanase is a general term for cellulases that hydrolyze cellulose molecular chains from their core portions. The group of enzymes belonging to endoglucanase is described with EC numbers: EC3.2.1.4, EC3.2.1.6, EC3.2.1.39 and EC3.2.1.73.
[032] Exoglucanase is a general term for cellulases that hydrolyze cellulose molecular chains from their termini. The group of enzymes belonging to exoglucanase is described with EC numbers: EC3.2.1.74 and EC3.2.1.58.
[033] β-glucosidase is a general term for cellulases that act on cello-oligosaccharides or cellobiose. The group of enzymes belonging to β-glucosidase is described with the EC number: EC3.2.1.21.
[034] Xylanase is a general term for cellulases that act on hemicellulose or especially xylan. The group of enzymes belonging to the xylanase is described with the EC number: EC3.2.1.8.
[035] Xylosidase is a general term for cellulases that act on xylo-oligosaccharides. The group of enzymes belonging to xylosidase is described with the EC number: EC3.2.1.37.
[036] As the cellulase derived from Trichoderma, a crude enzyme product is preferably used. The raw enzyme product is derived from a culture supernatant obtained by culturing a Trichoderma microorganism for an arbitrary period in a medium prepared so that the microorganism produces cellulase. The medium components to be used are not restricted and a medium supplemented with cellulose in order to promote cellulase production can be used in general. Like the raw enzyme product, the culture liquid can be used as it is or a culture supernatant processed only by removing Trichoderma cells is preferably used.
[037] The weight ratios of the enzymatic components in the raw enzymatic product are not restricted. For example, a culture liquid derived from Trichoderma reese contains 50 to 95% by weight of cellobiohydrolase and also contains as other components endoglucanase, β-glucosidase and the like. While microorganisms belonging to Trichoderma produce strong cellulase components inside the culture liquid, the β-glucosidase activity in the culture liquid is low since β-glucosidase is retained in cells or cell surfaces. Therefore, β-glucosidase from a different species or the same species can be added to the raw enzyme product. As β-glucosidase from a different species, β-glucosidase derived from Aspergillus can be used with preference. Examples of the β-glucosidase derived from Aspergillus include Novozyme 188, which is commercially available from Novozyme. The method of adding β-glucosidase from a different species or the same species to the raw enzyme product can also be a method in which a gene is introduced into a microorganism that belongs to Trichoderma to carry out genetic recombination of the microorganism in a way that β-glucosidase is produced inside the culture liquid and the micro-organism belonging to Trichoderma is then cultivated, followed by isolation of the culture liquid.
[038] The reaction temperature for hydrolysis with cellulase derived from filamentous fungus is preferably within the range of 15 to 100 °C, more preferably within the range of 40 to 60 °C, most preferably 50°C . The pH for the hydrolysis reaction is preferably within the range of pH 3 to 9, more preferably within the range of pH 4 to 5.5, most preferably pH 5. In order to adjust the pH, an acid or alkali can be added so that a desired pH is reached. Additionally, as required, a tampon can be used.
[039] Additionally, in the hydrolysis of a cellulose pretreated product, stirring/mixing is preferably performed in order to promote contact between the cellulose pretreated product and the filamentous fungal cellulase and to achieve a concentration of uniform sugar in the hydrolyzate. The solid concentration of the cellulose pretreated product is more preferably within the range of 1 to 25% by weight. In particular, in Step (1), the solid concentration of the cellulose pretreated product is preferably defined within the range of as low as 2 to 10% by weight. This aims to ensure a sufficient amount of liquid to dilute residual molasses in Step (2) at a later stage. Another advantage of setting the solid concentration within the range of as low as 2 to 10% by weight is the improvement in the hydrolysis efficiency of the cellulose pretreated product. This is due to the property of cellulase derived from a filamentous fungus that the enzymatic reaction is inhibited by sugar products such as glucose and cellobiose, which are products produced by hydrolysis.
[040] The concentration of sugar in the hydrolyzate obtained in Step (1) of the present invention is not limited and is preferably within the range of 10 to 100 g/L, more preferably within the range of 20 to 80 g/ L in terms of monosaccharide concentration. This is because a sugar concentration within this range allows adjustment of the sugar concentration to the most appropriate value by mixing it with residual molasses at a later stage. STEP 2
[041] In the present invention, the residual molasses is added to the hydrolyzate obtained in Step (1) described above. Residual molasses (Molasess) means a by-product produced in the sugar manufacturing process from a juice from a sugar crop such as sugar cane, sugar beet, Beta vulgaris, beet or grape, or from raw sugar prepared by a crystallization of such juice from a sugar crop. Residual molasses is a solution that contains sugar components that remained after the sugar crystallization step in the sugar manufacturing process. In general, the sugar crystallization step is normally carried out a plurality of times so that a first sugar is produced as a crystalline component obtained by the first crystallization, a second sugar is produced as a crystalline component obtained by crystallizing the residual liquid of the first sugar (first molasses), a third sugar is produced by crystallizing the residual liquid from the second sugar (second molasses) and the step is repeated further. The final molasses obtained as a residual liquid is called residual molasses. The sugar components contained in the residual molasses are mainly sucrose, glucose and fructose and certain amounts of other sugar components such as xylose and galactose can also be contained therein. As the number of times of crystallization increases, concentrations of components other than sugar components derived from the sugar crop increase in the residual molasses. Therefore, it is known that residual molasses also contains a large amount of fermentation inhibitors. Examples of fermentation inhibitors contained in residual molasses include acetic acid, formic acid, hydroxymethylfurfural, furfural, vanillin, acetovanilone, guaiacol and various inorganic substances (ions). However, the components and amounts of sugars and fermentation inhibitors contained in the residual molasses are not limited.
[042] The residual molasses used in Step (2) is preferably the molasses obtained after multiple crystallizations. More specifically, residual molasses is a molasses that remained after repeating the crystallization preferably not less than 2 times, more preferably not less than 3 times. Additionally, the sugar concentration in the residual molasses is preferably not less than 200 g/L, more preferably not less than 500 g/L. In cases where the concentration of sugar component in the residual molasses is less than 200 g/L, the recovery of cellulase derived from filamentous fungus does not increase, which is not preferable. On the other hand, although the upper limit of the sugar component concentration in the residual molasses used in Step (2) is not limited, the upper limit of the sugar component concentration in the residual molasses obtained by a normal sugar manufacturing process is considered be 800 g/L. The sugar concentration in the residual molasses can be measured using a known measurement method such as HPLC. Additionally, the residual molasses preferably contains K+ ions in addition to the sugars described above sugars. The concentration of K+ ions in the residual molasses preferably used in the present invention is not less than 1 g/L, more preferably not less than 5 g/L, most preferably not less than 10 g/L.
[043] The residual molasses is added to the hydrolyzate from Step (1) to prepare a mixed sugar liquid. The sugar concentration in the blended sugar liquid is preferably not greater than 200 g/L, more preferably not greater than 150 g/L since, in cases where the sugar concentration in the blended sugar liquid it is too high, the viscosity is too high, so the flow in subsequent ultrafiltration membrane treatment can be low. On the other hand, in cases where the sugar concentration in the mixed sugar liquid is less than 40 g/L, the concentration of the finally obtained sugar liquid can be low, so that the sugar concentration in the mixed sugar liquid is preferably not less than 40 g/L, more preferably not less than 50 g/L. That is, the residual molasses is added so that the sugar concentration in the mixed sugar liquid is preferably within the range of 40 to 200 g/l, more preferably within the range of 50 to 150 g/l. Although the mixed sugar liquid can be incubated at room temperature (25°C), the liquid is preferably incubated at a temperature within the range of 40 to 60°C, more preferably incubated at a temperature of about 50°C. Through this, the amount of enzyme that can be recovered with an ultrafiltration membrane at a later stage increases, which is preferable.
[044] Some types of microorganisms to be used for fermentation have low efficacy of using sucrose, which is a main sugar in the residual molasses, as a carbon source. Therefore, in cases where residual molasses is used as a fermentation raw material in the production of a chemical with the use of such a microorganism, it is preferable to hydrolyze the sucrose contained in the residual molasses to glucose and fructose in advance. Residual molasses hydrolysis treatment can also be heat treatment under acidic or alkaline conditions. Additionally, an enzymatic treatment with invertase, sucrase and/or the like can be carried out. Invertase is also called β-fructofuranosidase and means an enzyme that hydrolyzes sucrose to glucose and fructose. Sucrase also means an enzyme that hydrolyzes sucrose to glucose and fructose. The invertase used in the present invention is not limited and commercially available yeast-derived invertase from Biocon (Japan) Ltd. or Mitsubishi-Kagaku Foods Corporation can be purchased and used. The treatment conditions for invertase can be those used normally for its effective action. Sucrase to be used is also not limited and sucrase commercially available from Wako Pure Chemical Industries, Ltd. or Mitsubishi-Kagaku Foods Corporation can be purchased and used. Treatment conditions for sucrase can be those used normally for its effective action. The invertase or sucrase treatment can be carried out by adding invertase or sucrase in advance to the residual molasses alone or can be carried out after preparing the mixed sugar liquid by adding the residual molasses to the hydrolyzate of Step (1). Since, as described above, incubating the mixed sugar liquid at a temperature within the range of 40 to 60 °C increases the amount of recovered enzyme, the sucrose hydrolysis reaction can also be performed by adding invertase or sucrase to it. process. STEP 3
[045] In Step (3), the mixed sugar liquid obtained in Step (2) is subjected to separation between solid and liquid and the solution component is recovered. The separation between solid and liquid can be carried out by a known solid and liquid separation method such as: centrifugation with the use of a screw decanter or the like; filtration including suction/pressure filtration; or membrane filtration which includes microfiltration. Such separation between solid and liquid can also be accomplished by a combination of more than one method and is not restricted as long as solids can be effectively removed therefrom. However, in view of the scale suppression of an ultrafiltration membrane at a later stage, the solution component after separation between solid and liquid is preferably free of solid as much as possible and, more specifically, it is preferable to carry out the first separation of solid and liquid by centrifugation or by filtration using a filter press or the like, followed by further subjecting the obtained solution component to membrane filtration through a microfiltration membrane in order to completely remove solids. A microfiltration membrane is also called a membrane filtration and is a separation membrane that can separate and remove particles that have a size of about 0.01 to 10 µm from a particulate suspension using a difference of pressure as a driving force. A microfiltration membrane has pores that have a size within the range of 0.01 to 10 µm on its surface and particulate components larger than the pores can be separated/removed towards the membrane side. Examples of a microfiltration membrane material include, without limitation, cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, ceramics, polypropylene, polycarbonate, and polytetrafluoroethylene (Teflon (trademark)). The membrane is preferably a polyvinylidene fluoride microfiltration membrane in view of contamination resistance, chemical resistance, strength, filtration performance and the like.
[046] Subsequently, the solution component is subjected to ultrafiltration membrane treatment. An ultrafiltration membrane generally means a separation membrane that has a pore size within the range of 1.5 nanometers to 250 nanometers and can block water-soluble macromolecules that have molecular weights within the range of 1,000 to 200,000 as a not permeated. The molecular weight cut-off of the ultrafiltration membrane is not limited as long as cellulase derived from filamentous fungus can be recovered and the molecular weight cut-off is preferably 1,000 to 100,000 Da, more preferably 10,000 to 30,000 Da. of the ultrafiltration membrane that can be used include polyether sulfone (PES), polyvinylidene fluoride (PVDF) and regenerated cellulose and, since cellulose is degraded by cellulase derived from filamentous fungus, the material of the ultrafiltration membrane is preferably a synthetic polymer such as such as PES or PVDF. Preferred examples of the shape of the ultrafiltration membrane include a tubular type, a spiral element and a flat membrane. Examples of the method of filtration through the ultrafiltration membrane include cross-flow filtration and conventional filtration and, in view of fouling and flow, cross-flow filtration is preferred.
[047] By filtering the solution component through the ultrafiltration membrane, a sugar liquid can be obtained as a permeate. The sugar liquid obtained is a liquid produced by almost complete removal of solids that were originally contained in the mixed sugar liquid by separation of solid and liquid. On the other hand, by filtration through the ultrafiltration membrane, colored substances and water-soluble macromolecules in the mixed sugar liquid are removed on the non-permeate side, whose water-soluble macromolecules contain the filamentous fungus-derived cellulase component used in Step ( 1). The filamentous fungus-derived cellulase component to be recovered is not limited and all or a part of the filamentous fungus-derived cellulase component used in the hydrolysis can be recovered as the non-permeate. Since the non-permeate also contains sugar components derived from the mixed sugar liquid, an operation of adding water to the non-permeate and further filtering the resultant through an ultrafiltration membrane can be repeated to recover such sugar components.
[048] Step (3) has an effect to remarkably increase the amount of filamentous fungus-derived cellulase enzyme contained in the recovered enzyme compared to conventional techniques and, among the filamentous fungus-derived cellulase components, cellobiohydrolase and xylanase are retrieved especially at high efficiency. By reusing the recovered filamentous fungus-derived cellulase for hydrolysis of the cellulose pretreated product, the amount of the filamentous fungus-derived cellulase to be used can be reduced. The recovered filamentous fungus-derived cellulase can be reused alone for hydrolysis or it can be reused after being mixed with freshly filamentous fungus-derived cellulase. Additionally, in some cases, the recovered filamentous fungus-derived cellulase can be used effectively in a use other than cellulose hydrolysis. SUGAR CONCENTRATION STAGE
[049] Through filtration, as in the method described in WO 2010/067785, the sugar liquid obtained in Step (3) through a nanofiltration membrane and/or reverse osmosis membrane, a concentrated sugar liquid that contains components Concentrated sugar can be obtained as a non-permeate.
[050] A nanofiltration membrane is also called a nanofilter (nanofiltration membrane, NF membrane) and in general defined as a “membrane that allows the permeation of monovalent ions, but blocks divalent ions”. The membrane is considered to have fine voids that are about several nanometers in size and primarily used to block fine particles, molecules, ions, salts and the like in water.
[051] A reverse osmosis membrane is also called an RO membrane and is generally defined as a “membrane that has a desalting function also for monovalent ions”. The membrane is considered to have ultra-fine voids that have sizes from about several angstroms to several nanometers, and is mainly used for removal of ionic components such as seawater desalination and ultrapure water production.
[052] Examples of nanofiltration membrane material or reverse osmosis membrane material that can 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 limited to a membrane made of one of the materials and can be a membrane comprising a plurality of the membrane materials.
[053] As the nanofiltration membrane to be used in the present invention, a spirally wound membrane element is preferable. Specific examples of preferred nanofiltration membrane elements include a GE Sepa cellulose acetate nanofiltration membrane element, manufactured by GE Osmonics; NF99 and NF99HF nanofiltration membrane elements, manufactured by Alfa-Laval, which have functional polyamide layers; NF-45, NF-90, NF-200, NF-270, and NF-400 nanofiltration membrane elements, manufactured by FilmTec Corporation, which have functional layers of cross-linked polyamide piperazine; and SU-210, SU-220, SU-600, and SU-610 nanofiltration membrane elements, manufactured by Toray Industries, Inc., which comprises a UTC60 nanofiltration membrane, manufactured by the same manufacturer, which comprises a polyamide piperazine crosslinked with a main component. The nanofiltration membrane element is most 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 even more preferably SU-210, SU-220, SU-600 or SU-610.
[054] In terms of the material of the reverse osmosis membrane used in the present invention, examples of the membrane include a composite membrane comprising a cellulose acetate polymer as a functional layer (hereinafter referred to as cellulose acetate reverse osmosis membrane) and a composite membrane comprising a polyamide as a functional layer (hereafter called polyamide reverse osmosis membrane). Examples of the cellulose acetate polymer herein include polymers prepared with organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate and cellulose butyrate, which can be used alone, as a blend or as a blended ester. Examples of the polyamide include linear polymers and crosslinked polymers composed of aromatic and/or aliphatic diamine monomers.
[055] Specific examples of the reverse osmosis membrane used in the present invention include polyamide reverse osmosis membrane modules manufactured by TORAY INDUSTRIES, INC., SUL-G10 and SUL-G20, which are ultra-low pressure type modules, and 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, just like SU-810 , SU-820, SU-820L and SU-820FA, which are high pressure type modules that contain UTC80 as a reverse osmosis membrane; 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 LF10-D, manufactured by Nitto Denko Corporation; RO98pHt, RO99, HR98PP and CE4040C-30D, manufactured by Alfa-Laval; GE Sepa, manufactured by GE; BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040 and SW30HRLE-4040, manufactured by FilmTec Corporation; TFC-HR and TFC-ULP, manufactured by KOCH; and ACM-1, ACM-2 and ACM-4, manufactured by TRISEP.
[056] The sugar liquid concentration using a nanofiltration membrane and/or a reverse osmosis membrane has an advantage that the sugar concentration in the sugar liquid can be increased and fermentation inhibitors can be removed as a permeate . The term "fermentation inhibitors" herein means components other than sugars that inhibit fermentation in the fermentation stage at a later stage, and specific examples of fermentation inhibitors include aromatic compounds, furan compounds, organic acids and inorganic salts monovalents. Representative examples of such aromatic compounds and furan compounds include furfural, hydroxymethylfurfural, vanillin, vanillic acid, syringic acid, coniferyl aldehyde, coumaric acid and ferulic acid. Examples of organic acids and inorganic salts include acetic acid, formic acid, potassium and sodium. The sugar concentration in the concentrated sugar liquid can be adjusted arbitrarily within the range of 50 g/L to 400 g/L depending on the treatment conditions for the nanofiltration membrane and/or the reverse osmosis membrane, and can be arbitrarily adjusted depending on the use of concentrated sugar liquid and/or the like. In cases where more complete removal of fermentation inhibitors is required, water can be added to the sugar liquid or concentrated sugar liquid, followed by concentration of the resultant through a nanofiltration membrane and/or a reverse osmosis membrane to a desired sugar concentration. Through this, fermentation inhibitors can be removed as a permeate.
[057] The use of a nanofiltration membrane is more preferred as it has a superior effect of removing fermentation inhibitors than a reverse osmosis membrane. The use of a nanofiltration membrane or the use of a reverse osmosis membrane can be selected in consideration of the concentration of fermentation inhibitors contained in the mixed sugar liquid, or how fermentation at a later stage is influenced by the fermentation inhibitors.
[058] The concentrated sugar liquid described above can be further concentrated through the use of a vacuum evaporator, multi-effect evaporator, freeze dryer, spray dryer, hot air dryer and/or the like. SUGAR LIQUID / CONCENTRATED LIQUID SUGAR
[059] The sugar liquids and/or concentrated sugar liquids obtained by the present invention can be used for uses such as food, sweeteners, feed and fermentation raw materials. PROCESS FOR THE PRODUCTION OF CHEMICAL PRODUCT
[060] Using the sugar liquid and/or concentrated sugar liquid obtained by the present invention as a fermentation raw material to cultivate the microorganisms that have the ability to produce chemicals, various chemical substances can be produced. “The cultivation of microorganisms using the sugar liquid and/or concentrated sugar liquid as a fermentation raw material” in this document means that the sugar components or amino sources contained in the sugar liquid are used as nutrients for the microorganisms, to cause it, and to allow the continuation of the cultivation of the microorganisms. Specific examples of chemicals include alcohols, organic acids, amino acids and nucleic acids, which are mass-produced substances in the fermentation industry. Such chemicals are produced and accumulated inside and outside the living body using sugar components contained in the sugar liquid as carbon sources to be metabolized. Specific examples of chemicals that can be produced by microorganisms include alcohols such as ethanol, 1,3-propanediol, 1,4-propanediol and glycerol; organic acids such as acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid and citric acid; nucleosides such as inosine and guanosine; nucleotides such as inosinic acid and guanylic acid; and amine compounds such as cadaverine. Furthermore, the sugar liquid of the present invention can be applied to the production of enzymes, antibiotics, recombinant proteins and the like. The microorganism used to produce such a chemical is not limited as long as the microorganism has the ability to efficiently produce the chemical of interest, and examples of the microorganism that can be used include microorganisms such as E. coli, yeasts, filamentous fungi and Basidiomycetes.
[061] In cases where the sugar liquid and/or concentrated sugar liquid of the present invention is/are used as the fermentation feedstock for the production of a chemical, a source(s) ) nitrogen and/or inorganic salt(s) can be added thereto if necessary, and an organic micronutrient(s) such as an amino acid(s) and/ or vitamin(s) can be added to it if necessary. Furthermore, in some cases, the sugar liquid and/or concentrated sugar liquid may be supplemented with xylose and/or another/other carbon source(s) to prepare the fermentation feedstock, and examples of the(s) carbon source(s) include sugars such as glucose, sucrose, fructose, galactose and lactose; saccharified starch liquids containing these sugars; sweet potato molasses; sugar beet molasses; and high-quality molasses (high-test); and additionally, organic acids such as acetic acid; alcohols such as ethanol; and glycerin. Examples of nitrogen source(s) that can be used include ammonia gas; aqueous ammonia; ammonium salts; urea; nitrates; and other secondary used organic nitrogen sources such as oil tarts, soy hydrolysates, casein digests, other amino acids, vitamins, maize, yeast or yeast extracts, meat extracts, peptides such as peptone, and cells of various fermentation microorganisms and their hydrolysates. Examples of the inorganic salt(s) which may be added as appropriate include phosphates, magnesium salts, calcium salts, iron salts and manganese salts.
[062] Examples of the method for culturing the microorganism include known fermentation culture methods such as batch culture, fed batch culture and continuous culture. In particular, since solids are completely removed from the sugar liquid and/or concentrated sugar liquid of the present invention with the use of an ultrafiltration membrane and/or the like, it is possible to separate and collect the microorganism used for fermentation through a method such as centrifugation or membrane separation in order to reuse the microorganism. In such separation/collection and reuse of the microorganism, the microorganism may be separated/collected continuously while sugar liquid and/or fresh concentrated sugar liquid is/are added during the culture, or the microorganism can be sorted/collected upon completion of the crop to be reused for the next batch crop. CONSTITUTION OF A SUGAR LIQUID MANUFACTURING APPLIANCE
[063] The method for producing a sugar liquid of the present invention is described below with a focus on the apparatus used for this, with reference to the schematic drawings.
[064] Figure 1 is a schematic flow diagram showing the steps of the present invention. The details of it are as described above.
[065] Figure 2 is a schematic diagram of an apparatus to carry out the present invention. A pretreated cellulose product and a cellulase derived from filamentous fungi are fed to a hydrolysis reaction tank (2), where the hydrolysis is carried out. To efficiently carry out the hydrolysis, the hydrolysis tank (2) preferably comprises an incubator (1) and a mixer (3). Upon completion of the hydrolysis, the residual molasses is fed to the hydrolysis reaction tank (2). The residual molasses to be fed may be a preliminary diluted one in consideration of ease of handling. After adding residual molasses to the hydrolyzate, the resulting mixture is preferably mixed using the mixer (3). In order to increase the recovery efficiency of cellulase derived from filamentous fungi, the incubator (1) can be used to keep the temperature of the hydrolysis tank within the range of 40 to 60 °C. Thereafter, the mixed sugar liquid prepared in the hydrolysis tank (2) is fed to a solid-liquid separator (5) with the use of liquid transfer means such as a liquid sending pump (4). As the solid-liquid separator (5), a known solid-liquid separator such as a centrifuge, a filter press, a screw press, a rotary drum filter or a belt filter can be used. The separation between solid and liquid is preferably carried out through filtration using a separating membrane. The solution component obtained using the solid-liquid separator (5) can be further filtered through a microfiltration membrane device (6). Consequently, in the process of separation between solid and liquid, the solution component obtained through the solid-liquid separator (5) and the microfiltration membrane device (6) is collected in a solution collection tank (7). The solution collection tank (7) is connected by means of an ultrafiltration membrane pump (8) to an ultrafiltration membrane (9), where the above solution component is separated into cellulase derived from filamentous fungi and a liquid of sugar. The ultrafiltration membrane (9) is preferably one processed in the form of a module such as a spiral module. Cellulase derived from filamentous fungi separated through the ultrafiltration membrane (9) is collected as a non-permeate via the solution collection tank (7). On the other hand, the permeate from the ultrafiltration membrane (9) can be collected as a sugar liquid, and can be used for the production of a chemical or the like.
[066] Figure 3 is a schematic diagram of an apparatus for additional concentration of liquid sugar of the present invention. The sugar liquid of the present invention is held in a sugar liquid collection tank (10), which is connected by means of a high pressure pump (11) to a nanofiltration membrane and/or reverse osmosis membrane (12 ). The sugar component of the present invention is collected as a non-permeate from the nanofiltration membrane and/or the reverse osmosis membrane (12), and collected as a concentrated sugar liquid in the sugar liquid collection tank (10). As a permeate of the nanofiltration membrane and/or the reverse osmosis membrane (12), fermentation inhibitors are removed along with excess water.
[067] Figure 4 is a schematic diagram showing an apparatus for producing a chemical with the use of the sugar liquid and/or the concentrated sugar liquid of the present invention. The sugar liquid and/or concentrated sugar liquid of the present invention is fed to a fermenter (14) comprising an agitator (15) and an incubator (13). A microorganism is fed to and cultivated in the fermentor to produce a chemical, while the microorganism can be separated through a microorganism separation device (16) from the culture liquid comprising the chemical by means of completion of the culture or during the culture process. The microorganism separated by the microorganism separation device (16) is collected in the fermentor (14).
[068] Figure 5 is a schematic diagram showing the constitution of an apparatus in a case where a liquid sugar factory comprising the liquid sugar manufacturing apparatus of the present invention is built close to a liquid sugar manufacturing plant. existing sugar. The diagram shows an achievement where the existing sugar manufacturing plant uses sugar cane as a raw sugar material. The sugar manufacturing plant comprises a picadeira (slicing step) (17) for cutting sugar cane, a juicer (squeezing step) (18) for squeezing sugar cane to obtain cane juice. of sugar, a juice tank (19) to store the sugar cane juice, an effect evaporator (multi-effect evaporator) (20) to concentrate the sugar cane juice (concentration step), a crystallizer (21) for crystallizing the sugar contained in the concentrate (crystallization step) and a separating device (22) for separating the sugar from crystallized raw material. The residual molasses to be used in the present invention is discharged from the crystallizer (21) or from the separation device (22). The sugarcane bagasse discharged from the juicer (18) is transported through a conveyor (23) such as a conveyor to the sugar liquid factory. Sugarcane bagasse is sprayed through a sprayer (24) at the stage before the pre-treatment step. As a pulverizer, a crusher mill, cutter mill, hammer mill or the like can be used, or a combination of a plurality of these can be used. The pulverized sugarcane bagasse is pretreated in a heater (25) which has at least one heating function (pretreatment step). In the pretreatment, an acid, alkali, dilute sulfuric acid, ammonia, caustic soda or the like may be added as described above. The pre-treated cellulose product is subjected to hydrolysis with cellulase derived from filamentous fungi using the device in Figure 2 described above (Step 1). Thereafter, the residual molasses (molasses) discharged from the sugar manufacturing plant is added to the hydrolyzate from Step (1). The residual molasses is connected to the apparatus of Figure 2 via a transport line (26). The apparatus of Figure 3 for sugar concentration can be added next to the apparatus of Figure 2. Furthermore, as described above, the sugar liquid of the present invention can be used for food, feed, fermentation raw materials and the like. EXAMPLES
[069] The present invention is described below more specifically by way of the Examples. However, the present invention is not limited thereto. REFERENCE EXAMPLE 1
[070] Preparation of Pretreated Cellulose Product (Hydrothermal Treatment).
[071] As a biomass containing cellulose, sugarcane bagasse was used. The cellulose-containing biomass was immersed in water and subjected to treatment using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) with stirring at 180 °C for 20 minutes. After the treatment, the separation between solid and liquid was carried out by centrifugation (3,000 G) to separate the pre-treated cellulose product from the solution component. The obtained cellulose pretreated product was used in the Examples below. REFERENCE EXAMPLE 2
[072] Sugar Concentration Measurement
[073] The concentrations of sucrose, 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 REFERENCE EXAMPLE 3
[074] Analysis of Fermentation Inhibitors
[075] Aromatic compounds and furan compounds were quantified under the HPLC conditions described below based on comparison with standard samples. Each analysis sample was centrifuged at 3,500 G for 10 minutes and the supernatant component obtained was submitted for analysis below. Column: Synergi HidroRP 4.6 mm x 250 mm (manufactured by Phenomenex) Mobile phase: acetonitrile-0.1% H3PO4 (flow rate, 1.0 ml/minute) Detection method: UV (283 nm) Temperature: 40 °C
[076] Acetic acid and formic acid were quantified under the HPLC conditions described below based on comparison with standard samples. Each analysis sample was centrifuged at 3,500 G for 10 minutes and the supernatant component obtained was submitted for analysis below. Column: Shim-Pack and Shim-Pack SCR101H (manufactured by Shimadzu Corporation) which are arranged in a linear fashion Mobile phase: 5 mM p-toluenesulfonic acid (flow rate, 0.8 ml/minute) Reaction solution: 5 mM of p-toluenesulfonic acid, 20 mM Bis-Tris, 0.1 mM EDTA-2Na (flow rate, 0.8 ml/minute) Detection method: Electrical conductivity Temperature: 45 °C REFERENCE EXAMPLE 4
[077] Preparation of Cellulase derived from Trichoderma
[078] Trichoderma-derived cellulase was prepared by the following method. PRE-CULTURE
[079] A mixture of 5% (weight/volume) of maize, 2% (weight/volume) of glucose, 0.37% (weight/volume) of ammonium tartrate, 0.14 (weight/volume) of sulfate of ammonium, 0.2% (weight/volume) of potassium dihydrogen phosphate, 0.03% (weight/volume) of calcium chloride dihydrate, 0.03% (weight/volume) of hepta- magnesium sulfate hydrate, 0.02% (weight/volume) zinc chloride, 0.01% (weight/volume) iron(III) chloride hexahydrate, 0.004% (weight/volume) penta - copper (II) sultate hydrate, 0.0008% (weight/volume) of manganese chloride tetrahydrate, 0.0006% (weight/volume) of boric acid and 0.0026% (weight/volume) of hexa-ammonium heptamolybdate tetrahydrate in distilled water was prepared, and 100 ml of this mixture was placed in a 500 ml Erlenmeyer flask with protrusions on the bottom, then sterilized by autoclave 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 to it at 0.01% (weight/volume), each one. To these pre-culture media, Trichoderma reesei PC3-7 was inoculated at 1 x 105 cells/ml, and the cells were cultured at 28 °C for 72 hours with stirring at 180 rpm, to perform pre-culture (shaker: BIO-SHAKER BR-40LF, manufactured by TAITEC CORPORATION). MAIN CULTURE
[080] A mixture of 5% (weight/volume) of maize, 2% (weight/volume) of glucose, 10% (weight/volume) of cellulose (Avicel), 0.37% (weight/volume) of tartrate of ammonium, 0.14% (weight/volume) of ammonium sulfate, 0.2% (weight/volume) of potassium dihydrogen phosphate, 0.03% (weight/volume) of chloride dihydrate calcium, 0.03% (weight/volume) of magnesium sulfate heptahydrate, 0.02% (weight/volume) of zinc chloride, 0.01% (weight/volume) of magnesium chloride hexahydrate iron (III), 0.004% (weight/volume) of copper (II) sulfate pentahydrate, 0.0008% (weight/volume) of manganese chloride tetrahydrate, 0.0006% (weight/volume) ) of boric acid and 0.0026% (weight/volume) of hexaammonium heptamolybdate tetrahydrate in distilled water was prepared and 2.5 l of this mixture was placed in a 5 l stir jar (manufactured by ABLE , DPC-2A), then being sterilized through an autoclave 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 to it by 0.1% each. To the resulting mixture, 250 ml of the preculture of Trichoderma reesei PC3-7 prepared preliminary with a liquid medium by the method described above was inoculated. Then, cells were cultured at 28 °C for 87 hours at 300 rpm at an aeration rate of 1 vvm. After centrifugation, the supernatant was subjected to membrane filtration (Stericup-GV, manufactured by Millipore, material: PVDF). To the culture liquid prepared under the conditions described above, β-glucosidase (Novozyme 188) was added at a protein weight ratio of 1/100 and the resulting mixture was used as the cellulase derived from Trichoderma in the Examples below. REFERENCE EXAMPLE 5
[081] Method for Measuring the Amount of Recovered Filamentous Fungi-Derived Cellulase.
[082] The amount of cellulase derived from filamentous fungi that can be recovered in Step (3) was quantified by measuring 3 types of degradation activities (hereinafter referred to as activity values): 1) degrading activity of crystalline cellulose; 2) cellobiose degrading activity; and 3) xylan degrading activity.
[083] 1) Degrading Activity of Crystalline Cellulose To an enzyme liquid (prepared under predetermined conditions), an Avicel microcrystalline cellulose (Microcrystalline Cellulose, manufactured by Merch) was added in 1 g/L and sodium acetate buffer (pH 5 ,0) was added to 100 mM, then 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 conditions described above. Thereafter, the tube was subjected to centrifugation, and the concentration of glucose 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 glucose produced (g/L) was used as the same as the activity level of the degrading activity of crystalline cellulose, and used for comparing the amount of enzyme recovered.
[084] 2) Cellobiose Degrading Activity For 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, then 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 conditions described above. Thereafter, the tube was subjected to centrifugation and the concentration of glucose in the supernatant component was measured. Measurement of glucose concentration was performed according to the method described in Reference Example 2. The concentration of glucose produced (g/L) was used as the same as the activity level of cellobiose degrading activity and used for comparison of the amount of enzyme recovered.
[085] 3) Xylane Degrading Activity For 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 in 100 mM, then 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 conditions described above. Thereafter, the tube was subjected to centrifugation and the concentration of xylose 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 xylose produced (g/L) was used as it is as the activity level of the xylose degrading activity and used for comparison of the amount of enzyme recovered. REFERENCE EXAMPLE 6
[086] Measurement of Inorganic Ion Concentration The concentrations of cations and anions contained in the sugar liquid were quantified under the HPLC conditions shown below based on comparison with standard samples. 1) Cation Analysis Column: Ion Pac AS22 (manufactured by DIONEX) Mobile phase: 4.5 mM Na2CO3/1.4 mM NaHCO3 (flow rate, 1.0 ml/minute) Reaction solution: None detection: Electrical conductivity (through the use of a suppressor) Temperature: 30 °C 2) Anion analysis Column: Ion Pac CS12A (manufactured by DIONEX) Mobile phase: 20 mM methanesulfonic acid (flow rate, 1.0 ml/ minute) Reaction solution: None Detection method: Electrical conductivity (through the use of a suppressor) Temperature: 30 °C REFERENCE EXAMPLE 7
[087] Residual Molasses Component Analysis As a residual molasses, "residual molasses (Molasses Agri)" (manufactured by Organic Land Co,. Ltd.) was used. The raw material of the residual molasses was raw sugar derived from sugar cane. The results of the analysis of sugar components, organic acids, furan/aromatic compounds and inorganic ions in the residual molasses are shown in Tables 1 to 4. The total concentration of each group of components is shown in Table 5. The component analysis was performed in accordance with Reference Example 2, Reference Example 3, and Reference Example 6. TABLE 1
TABLE 2
TABLE 3
TABLE 4
TABLE 5
TOTAL CONCENTRATION OF EACH COMPONENT GROUP REFERENCE EXAMPLE 8
[088] Ethanol Concentration Analysis The accumulated ethanol concentration was quantified by gas chromatography. Its evaluation was carried out through detection and calculation with a hydrogen salt ionization detector using Shimadzu GC-2010 Capillary GC TC-1 (GL Science) 15 meters per l. x 0.53 mm I.D., df 1.5 µm. COMPARATIVE EXAMPLE 1
[089] Production of Liquid Sugar without Addition of Residual Molasses to the Hydrolysate STEP 1):
[090] To the cellulose pretreated product (0.5 g) prepared in Reference Example 1, distilled water was added and 0.5 ml of the cellulase derived from Trichoderma prepared in Reference Example 4 was added, followed by the addition additional distilled water to a total weight of 10 g. Thereafter, dilute sulfuric acid or dilute caustic soda was added to the resulting composition so that the pH of the composition was within the range of 4.5 to 5.3. After pH adjustment, the composition was transferred to a side-arm test tube (manufactured by Tokyo Rikakikai Co., Ltd., Φ30 NS14/23, CPS-1000 compact mechanical stirrer, conversion adaptor, feed inlet with a three-way stopcock, MG-2200 incubator), and incubated and shaken at 50 °C for 24 hours to obtain a hydrolyzate. STEP(2):
[091] Step (2) was not performed in this Comparative Example. STEP (3):
[092] The hydrolyzate obtained in Step (1) was subjected to separation between solid and liquid through centrifugation (3,000 G, 10 minutes), and thus separated into the solution component (6 ml) and solids. The solution component was further filtered using a Millex HV filter unit (33 mm; made of PVDF; pore size, 0.45 µm). The sugar concentrations (glucose and xylose concentrations) in the obtained solution component were measured according to the method described in Reference Example 2. The measured sugar concentrations are shown in Table 6. The obtained solution component was filtered through an ultrafiltration membrane which has a molecular weight cut-off of 10,000 (VIVASPIN 20, manufactured by Sartorius stedim biotech, material: PES) and centrifuged at 4,500 G until the membrane fraction is reduced to 1 ml. To the membrane fraction, 10 ml of distilled water was added, and the resulting mixture was centrifuged again at 4,500 G until the membrane fraction was reduced to 1 ml. This operation was performed once more, and the recovered enzyme liquid was collected from the membrane fraction. The amount of recovered enzyme was quantified by measuring each activity value in accordance with Reference Example 5. The activity value measured in the present Comparative Example 1 was defined as "1 (reference)", and used for comparison with the quantities of enzyme recovered in Comparative Example 2 and Example 1 (Table 7) described below. COMPARATIVE EXAMPLE 2
[093] Production of Liquid Sugar by Addition of Liquid Sugar Reagent. STEP 1)
[094] Step (1) was performed using the same procedure as Step (1) of Comparative Example 1. STEP (2)
[095] A reagent sugar liquid, which is not residual molasses, was added to the hydrolyzate from Step (1). The reactant sugar liquid was prepared so that the sugar concentrations were the same as those in the residual molasses described in Reference Example 7. That is, the reactant sugar liquid was prepared by completely dissolving 148 g of glucose, 163 g of fructose and 371 g of sucrose in 1 liter of RO water. To the hydrolyzate of Step (1), 0.5 ml of the liquid sugar reagent thus obtained was added. Thereafter, the resulting mixture was stirred at room temperature (25°C) for about 5 minutes to prepare a mixed sugar liquid as a uniform liquid. STEP (3)
[096] The mixed sugar liquid obtained in Step (2) was used to effect the separation between solid and liquid and the ultrafiltration membrane treatment through the same procedure as in Step (3) of Comparative Example 1. The sugar concentrations obtained are shown in Table 6. Each measured activity value was divided by the activity value from Comparative Example 1. The obtained value is shown in Table 7 as the amount of enzyme recovered from Comparative Example 2. EXAMPLE 1
[097] Method for Preparation of Liquid Sugar by Addition of Residual Molasses to Cellulose Hydrolysate. STEP 1)
[098] Step (1) was performed using the same procedure as Step (1) of Comparative Example 1. STEP (2)
[099] To the hydrolyzate (10 ml) obtained in Step (1), 0.5 g of residual molasses (Reference Example 7; sugar concentration, 689 g/L) was added. Thereafter, the resulting mixture was stirred at room temperature (25°C) for about 5 minutes to prepare a mixed sugar liquid as a uniform liquid. STEP (3)
[100] With the use of the mixed sugar liquid obtained in Step (2), the separation between solid and liquid and the ultrafiltration membrane treatment were carried out by the same procedure as in Step (3) of Comparative Example 1. The concentrations of sugars obtained are shown in Table 6. Each measured activity value was divided by the activity value from Comparative Example 1. The obtained value is shown in Table 7 as the amount of enzyme recovered from Example 1.
[101] Based on a comparison of Comparative Example 1, Comparative Example 2 and Example 1, the amount of enzyme recovered was greater in Example 1 than in Comparative Example 1, so it was suggested that residual molasses contains an amount increasing component of recovered enzyme. Additionally, since the amount of enzyme recovered from Reference Example 2 was almost the same as that of Reference Example 1, it was suggested that the sugar components contained in the residual molasses (sucrose, glucose and fructose) did not affect the amount of enzyme recovered, and what other component is involved in increased enzyme recovery. TABLE 6 SUGAR CONCENTRATION
EXAMPLE 2
[102] Relation between Amount of Residual Molasses Added and Amount of Recovered Enzyme
[103] Step (2) was carried out in the same manner as in Example 1 except that the residual molasses was added in an amount of 0.1 g (Example 1), 0.5 g, 1 g, 2 g or 5 g , and the amount of cellulase derived from filamentous fungus that could be recovered was compared. Each recovered enzyme activity value in each experimental group was divided by the activity value of Comparative Example 1. The obtained value is shown in Table 8 as the amount of recovered enzyme (relative value). As a result, it has been found that as the amount of residual molasses added increases, the amount of enzyme recovered, especially the amount of enzyme involved in the crystalline cellulose degradation activity, increases. TABLE 8 Relationship between the amount of residual molasses added and the amount of enzyme recovered (relative value)
COMPARATIVE EXAMPLE 3
[104] Relation between Amount of Reagent Sugar Added and Amount of Recovered Enzyme
[105] Step (2) was carried out in the same manner as in Comparative Example 2 except that the reactant sugar liquid was added in an amount of 0.1 g (Comparative Example 2), 0.5 g, 1 g, 2 g or 5 g, and the amount of cellulase derived from filamentous fungus that could be recovered was compared. As a result, it was found as shown in Table 9 that, unlike the result of Example 2, the amount of recovered enzyme does not increase in terms of any of the activities, even if the amount of added reactant sugar increases. That is, it has been found that a component other than the sugars contained in the residual molasses is involved in the increased amount of recovered enzyme. TABLE 9 Relationship between the amount of reagent sugar added and the amount of enzyme recovered

EXAMPLE 3
[106] Relationship between Incubation Temperature of Mixed Sugar Liquid and Amount of Recovered Enzyme
[107] Step (2) was carried out in the same manner as in Example 1 except that the incubation temperature after adding 0.5 g of residual molasses to prepare a mixed sugar liquid was set to 25 °C (Example 1) , 40°C, 50°C, 60°C or 70°C. The activity value of the recovered enzyme was measured to compare the amount of recovered enzyme among the experimental groups (Table 10). As a result, it has been found that incubation of the mixed sugar liquid within the temperature range of 40 to 60 °C further increases the amount of enzyme recovered. TABLE 10 Relationship between the incubation temperature of the mixed sugar liquid and the amount of enzyme recovered

EXAMPLE 4
[108] Step to Obtain Concentrated Sugar Liquid using Nanofiltration Membrane or Reverse Osmosis Membrane SUGAR LIQUID PREPARATION
[109] A sugar liquid (1 L) was prepared under the following conditions. STEP 1)
[110] 1.4 g of cellulase derived from Trichoderma was added to the cellulose pretreated product (400 g) obtained in the Reference Example, and distilled water was added further to a total weight of 8 kg. Additionally, the pH of the composition was adjusted with diluted caustic soda to a value within the range of 4.5 to 5.3. Although the liquid was incubated such that a liquid temperature of 45 to 50 °C was maintained, and although dilute sulfuric acid and/or dilute caustic soda was/have been added to the liquid such that the pH was maintained within the range of 4.5 to 5.3, the liquid was incubated for 24 hours to obtain 8 kg of a hydrolyzate. STEP (2)
[111] 0.4 kg of residual molasses was added to 8 kg of the hydrolyzate obtained in Step (1). After that, the resulting mixture was mixed for 5 minutes to obtain a sugar liquid mixed as a uniform liquid. STEP (3)
[112] The mixed sugar liquid obtained in Step (2) was subjected to solid-liquid separation and treatment by ultrafiltration membrane. For solid-liquid separation, a compact filter press apparatus (MO-4 filter press, manufactured by Yabuta Industries Co., Ltd.) was used. As a filter cloth, a polyester fabric (T2731C, manufactured by Yabuta Industries Co., Ltd.) was used. 8 l of the mixed sugar liquid was placed in a small tank. Under aeration with compressed air from the bottom, a liquid inlet was opened to feed the spray liquid slowly into a filtration chamber using an air pump (66053-3EB, manufactured by Taiyo International Corporation). Subsequently, a compression step was carried out by inflating a diaphragm attached to the filtration chamber. The compression pressure was slowly increased to 0.5 MPa, and the apparatus was then left to stand for about 30 minutes to recover the solution component as a filtrate. The total volume of solution component obtained was 6 l. The remaining liquid component has been lost because of the dead volume of the device. Subsequently, the solution component was filtered through a microfiltration membrane after solid-liquid separation. Microfiltration was performed using 1,000 ml Stericup HV (manufactured by Millipore; PVDF; mean pore size, 0.45 μm) to obtain 5 l of a filtrate. The obtained filtrate (solution component) was processed using a compact flat membrane filtration device (Sepa (registered trademark) CF II Med/High Foulant System, manufactured by GE) equipped with a flat ultrafiltration membrane having a molecular weight cutoff of 10,000 (PW series SEPA, manufactured by GE, functional surface material: polyether sulfone). Although the operating pressure was controlled in such a way that the flow rate on the feed side was constantly 2.5 l/minute and the membrane flow was constantly 0.1 m/D, 4 l of 5 l of the filtered above were filtered to obtain a sugar liquid. TREATMENT OF SUGAR LIQUID WITH THE USE OF NANOFILTRATION MEMBRANE OR REVERSE OSMOSIS MEMBRANE
[113] Using 1 l of the sugar liquid produced in Steps (1) to (3), concentration through a nanofiltration membrane or concentration through a reverse osmosis membrane was performed. As the nanofiltration membrane, “DESAL-5L” (manufactured by Desalination) was used. Like the reverse osmosis membrane, a fully crosslinked “UTC80” aromatic reverse osmosis membrane (manufactured by Toray Industries, Inc.) was used. Each membrane was mounted in a compact flat membrane filtration device ("Sepa" (registered trademark) CF II Med/High Foulant System, manufactured by GE), and filtration treatment was carried out at a raw liquid temperature of 25° C at a pressure of 3 MPa using a high pressure pump. By this treatment, 0.5 l of a nanofiltration membrane concentrate and 0.5 l of a permeate were obtained (double concentration). The concentrated sugar liquid obtained using the nanofiltration membrane is shown in Table 11, and the concentrated sugar liquid obtained using the reverse osmosis membrane is shown in Table 12. As shown in Table 11 and Table 12, it was found that, although concentration of sugar components is possible with either a nanofiltration membrane or a reverse osmosis membrane, concentration through a nanofiltration membrane has a greater effect of removing fermentation inhibitors such as HMF, furfural, acetic acid and potassium ions. TABLE 11 Concentrated sugar liquid prepared with a Nanofiltration membrane

TABLE 12 CONCENTRATED SUGAR LIQUID PREPARED WITH A REVERSE OSMOSIS MEMBRANE

EXAMPLE 5
[114] Ethanol Fermentation Test using Liquid Sugar as Fermentation Raw Material
[115] Using the sugar liquid obtained from the sugar liquid preparation process of Example 4 (performed by Steps (1) to (3)) and using a yeast (Saccharomyces cerevisiae OC-2: yeast from wine), an ethanol fermentation test was performed. For comparison, a mixed sugar liquid obtained by carrying out Steps (1) and (2) of the sugar liquid preparation process of Example 4 was also used as a fermentation raw material. Yeast was pre-cultured in YPD medium (2% glucose, 1% yeast extract (Bacto Yeast Extract, manufactured by BD), 2% polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.)) for 1 day at 25 °C. Subsequently, the obtained culture liquid was added to the sugar liquid or the mixed sugar liquid (sugar concentration, 57 g/L) in a concentration of 1% (20 ml). After the addition of the microorganism, incubation was carried out at 25 °C for 2 days. The culture liquid obtained by this operation was subjected to accumulated ethanol concentration analysis by the procedure of Reference Example 8. As shown in Table 13, it was found that direct use of a mixed sugar liquid prepared only by mixing a cellulose hydrolyzate with residual molasses results in low concentration of accumulated ethanol. This was assumed due to the action of substances that must be removed in Step (3) as fermentation inhibiting factors. TABLE 13 ETHANOL FERMENTATION TEST
EXAMPLE 6
[116] Ethanol Fermentation Test Using Concentrated Sugar Liquid as Fermentation Raw Material
[117] The concentrated sugar liquid prepared using a nanofiltration membrane and the concentrated sugar liquid prepared using a reverse osmosis membrane in Example 4 were diluted twice with RO water, and used as fermentation media by the same procedure as in Example 5. As a result, as shown in Table 14, both concentrated sugar liquids show higher fermentation performance than the sugar liquid of Example 5, and it was found that a concentrated sugar liquid prepared with the use of a nanofiltration membrane is especially excellent as a fermentation medium. TABLE 14 ETHANOL FERMENTATION TEST
EXAMPLE 7
[118] Mixed Sugar Liquid Invertase Treatment 0.5 g residual molasses (Reference Example 7; sugar concentration, 689 g/L) was added to the hydrolyzate (10 ml) from Step (2) of Example 1. After that, the resulting mixture was stirred at room temperature (50 °C) for about 5 minutes to prepare a mixed sugar liquid as a uniform liquid. Subsequently, 1 g of yeast-derived invertase (Yeast Invertase Solution; Wako Pure Chemical Industries, Ltd.) was added to the mixed sugar liquid, and the resulting mixture was allowed to stand for an additional 1 hour. STEP (3)
[119] Using the mixed sugar liquid obtained in Step (2), solid-liquid separation and ultrafiltration membrane treatment were carried out by the same procedure as in Step (3) of Comparative Example 1. The sugar concentrations obtained are shown in Table 15. TABLE 15 SUGAR CONCENTRATIONS

[120] As shown in Table 15, it has been found that, because of sucrose hydrolysis, sugar and fructose sugar concentrations increase compared to their concentrations in Example 1. EXAMPLE 8
[121] Production of l-lactic acid using Invertase Treated Sugar Liquid
[122] The strain of Lactococcus lactis JCM7638 was inoculated into 5 ml of the sugar liquid from Example 7, and static culture was carried out for 24 hours at a temperature of 37 °C. The concentration of l-lactic acid in the culture liquid was analyzed under the following conditions. Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation) Mobile phase: 5 mM p-toluenesulfonic acid (flow rate, 0.8 ml/min.) Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bis-Tris, 0.1 mM EDTA-2Na (flow rate, 0.8 ml/min.) Detection method: Electrical conductivity Temperature: 45 °C
[123] As a result of the analysis, accumulation of 26 g/L of l-lactic acid was found, and it could be confirmed that lactic acid can be produced using the sugar liquid of the present invention. COMPARATIVE EXAMPLE 4
[124] Production of L-lactic Acid Using Reactant Sugar Liquid
[125] For comparison, glucose, xylose, sucrose, and fructose were mixed together in such a way that the sugar concentrations described in Table 15 were reached, to prepare 5 ml of a liquid sugar reagent. The strain of Lactococcus lactis JCM7638 was inoculated into the reagent sugar liquid, and static culture was carried out for 24 hours at a temperature of 37 °C. However, no growth can be observed. This was considered due to the absence, unlike the sugar liquid of Example 8, of amino acids, vitamins and the like for lactic acid bacteria growth in the reactant sugar liquid. REFERENCE EXAMPLE 9
[126] As the biomass containing cellulose, sugarcane bagasse was used. The cellulose-containing biomass was immersed in 1% aqueous sulfuric acid solution, and subjected to treatment using an autoclave (manufactured by Nitto Koatsu Co., Ltd.) at 150 °C for minutes. Thereafter, solid-liquid separation was carried out to separate the resultant into an aqueous sulfuric acid solution (hereinafter referred to as a liquid treated with dilute sulfuric acid) and a pre-treated cellulose product (diluted sulfuric acid). COMPARATIVE EXAMPLE 5
[127] Production of Liquid Sugar without Addition of Residual Molasses in Hydrolysate
[128] Using the cellulose pretreated product (diluted sulfuric acid) prepared in Reference Example 9, a liquid of recovered enzyme was collected by the same procedure as in Comparative Example 1. The amount of recovered enzyme was quantified by measuring. if each activity value according to Reference Example 5. The activity value measured in the present Comparative Example 5 was defined as "1 (reference)", and used for comparison with the amounts of enzyme recovered in Comparative Example 6 described later and Example 9 (Table 16). COMPARATIVE EXAMPLE 6
[129] Production of Liquid Sugar with Addition of Liquid Sugar Reagent in Hydrolysate STEP 1)
[130] Step (1) at least the procedure that in Step (1) of Comparative Example 1. STEP (2)
[131] Step (2) was performed by the same procedure as in Comparative Example 2. STEP (3)
[132] Using the mixed sugar liquid obtained in Step (2), solid-liquid separation and ultrafiltration membrane treatment were carried out by the same procedure as in Step (3) of Comparative Example 1. Each measured activity value was divided by the activity value of Comparative Example 5. The obtained value is shown in Table 16 as the amount of enzyme recovered from Comparative Example 6. EXAMPLE 9
[133] Method for Producing Liquid Sugar with Addition of Residual Molasses in Cellulose Hydrolysate STEP 1)
[134] Step (1) was performed by the same procedure as in Step (1) of Comparative Example 1. STEP (2)
[135] Step (2) was performed by the same procedure as in Example 1. STEP (3)
[136] Using the mixed sugar liquid obtained in Step (2), solid-liquid separation and ultrafiltration membrane treatment were carried out by the same procedure as in Step (3) of Comparative Example 1. Each measured activity value was divided by the activity value of Comparative Example 5. The obtained value is shown in Table 16 as the amount of enzyme recovered from Example 9.
[137] Based on a comparison of Comparative Example 5, Comparative Example 6 and Example 9, the amount of enzyme recovered was greater in Example 9 than in Comparative Example 5, so it was suggested that residual molasses contains an amount increasing component of recovered enzyme. Additionally, since the amount of enzyme recovered from Comparative Example 6 was almost the same as that of Comparative Example 5, it was suggested that the sugar components contained in the residual molasses (sucrose, glucose and fructose) did not affect the amount of recovered enzyme and that another component is involved in increased enzyme recovery. The present results indicate that residual molasses increases recovered enzyme activities, regardless of whether the cellulose was pretreated or not. TABLE 16 RECOVERED ENZYME QUANTITY 2 (RELATIVE VALUE)

INDUSTRIAL APPLICABILITY
[138] The present invention allows for production of a sugar liquid from biomass that contains cellulose, wherein sugar liquid can be used as a fermentation feedstock for fermentation production of various chemicals. SYMBOL DESCRIPTIONS 1 Incubator 2 Hydrolysis tank 3 Mixer 4 Liquid delivery pump 5 Liquid to solid separator 6 Microfiltration membrane device 7 Solution collection tank 8 Ultrafiltration membrane pump 9 Ultrafiltration membrane 10 Collection tank sugar liquid 11 High pressure pump 12 Nanofiltration membrane and/or reverse osmosis membrane 13 Incubator 14 Fermenter 15 Stirrer 16 Microorganism separation device 17 Chopper 18 Juicer 19 Juice tank 20 Effect evaporator 21 Crystallizer 22 Device separation 23 Conveyor 24 Sprayer 25 Heater 26 Conveyor line

权利要求:
Claims (5)
[0001]
1. METHOD FOR PRODUCING A SUGAR LIQUID, characterized in that it comprises Steps (1) to (3) below: Step (1): a step of adding a filamentous fungus-derived cellulase to a pre-treated cellulose product to obtain a hydrolyzed; Step (2): a step of adding 40 to 200 g/L of residual molasses to said hydrolyzate to obtain a mixed sugar liquid, and incubating said mixed sugar liquid at a temperature within the range of 40 to 60 °C ; and Step (3): a step of subjecting said mixed sugar liquid to solid-liquid separation and filtering the obtained solution component through an ultrafiltration membrane (9), to recover the filamentous fungus-derived cellulase as a non-permeate and to obtain a sugar liquid as a permeate.
[0002]
2. METHOD, according to claim 1, characterized in that said cellulase derived from filamentous fungus of Step (1) is cellulase derived from Trichoderma.
[0003]
3. METHOD according to any one of claims 1 to 2, characterized in that said pre-treated cellulose product of Step (1) is one or more products selected from the group consisting of products obtained by hydrothermal treatment, treatment by dilute sulfuric acid or alkali treatment.
[0004]
4. METHOD according to any one of claims 1 to 3, characterized in that it comprises the step of filtering said sugar liquid from Step (3) through a nanofiltration membrane and/or a reverse osmosis membrane (12) to remove fermentation inhibitors as a permeate and to obtain a sugar concentrate as a non-permeate.
[0005]
5. METHOD FOR PRODUCING A CHEMICAL, characterized in that it comprises producing a sugar liquid, as defined in any one of claims 1 to 4, and carrying out the fermentation culture of a microorganism that has an ability to produce a chemical using said sugar liquid as a fermentation raw material.

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法律状态:
2018-09-25| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-08-25| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-02-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-20| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/03/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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JP2011-046623|2011-03-03|

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JP2011046623|2011-03-03|

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PCT/JP2012/055323|WO2012118171A1|2011-03-03|2012-03-02|Method for producing sugar solution|

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