JP5728817B2 - Method for producing xylose sugar solution - Google Patents

Description

  The present invention relates to a method for producing a xylose sugar solution from xylan-containing biomass.

  In recent years, saccharides are produced from xylan-containing biomass, and fermentative production processes of chemicals such as ethanol using this saccharide raw material have been actively studied. As a method for producing such sugar, a method for producing a sugar solution by acid hydrolysis of concentrated biomass using concentrated sulfuric acid (Patent Documents 1 and 2), after hydrolyzing biomass with dilute sulfuric acid, cellulase and the like A method for producing a sugar solution by enzyme treatment is disclosed (Non-patent Document 1). Moreover, as a method not using an acid, a method of hydrolyzing biomass using subcritical water at about 250 to 500 ° C. to produce a sugar solution (Patent Document 3), and after treating the biomass with subcritical water, further enzyme A method for producing a sugar solution by treatment (Patent Document 4), and a method for producing a sugar solution by hydrolyzing biomass with pressurized hot water at 240 to 280 ° C. and further carrying out an enzyme treatment (Patent Document 5). ) Is disclosed. The monosaccharide obtained by such a technique contains not only xylose but also glucose. In addition, such monosaccharides are included, but low-molecular components such as acetic acid, formic acid, HMF, furfural, and vanillin, which are called fermentation inhibitors, are included (Non-Patent Document 2 or 3). In the case of producing a chemical such as ethanol by fermentation using such a sugar solution containing a large amount of fermentation-inhibiting components as a carbon source, it is a problem to cause inhibition of microbial growth and a decrease in the yield of the product. Such a fermentation inhibitor is known to have an adverse effect on growth or fermentation by microorganisms that use xylose as a carbon source as a sugar component (Non-Patent Document 4).

  As a method for removing such a fermentation inhibitor from a sugar solution, a method for removing the fermentation inhibitor using a nanofiltration membrane (Patent Document 6) is disclosed. Specifically, by filtering a sugar solution containing a fermentation inhibitor obtained from biomass through a nanofiltration membrane, the fermentation inhibitor is removed from the permeate side, and the fermentation inhibitor is removed as a non-permeate. It is disclosed that a sugar solution can be obtained. However, there is room for improvement in the removal rate of the fermentation inhibitor hydroxymethylfurfural (hereinafter also referred to as HMF) in this technology.

  In addition, as a method for solving the fermentation inhibition by the fermentation inhibitor contained in the biomass-derived sugar liquid, a microorganism having impurity resistance and ethanol fermentation ability was obtained from xylan-containing biomass in the fermentation production of ethanol. A method is disclosed in which secondary fermentation is performed by adding a microorganism having xylose assimilation and ethanol fermentation ability after adding to sugar solution and performing primary fermentation (Patent Document 7). Specifically, fermentation inhibitors such as HMF contained in sugar solution obtained from xylan-containing biomass in primary fermentation are taken into the body during primary fermentation by S. cerevisiae. It is said that ethanol is efficiently produced without inhibiting fermentation by an inhibitory substance. However, in the removal of the fermentation inhibitor using the aforementioned microorganism (yeast), there is still a problem that the effect of removing organic acids such as formic acid, acetic acid and levulinic acid is small.

Japanese National Patent Publication No. 11-506934 JP 2005-229821 A Japanese Patent Laid-Open No. 2003-212888 JP 2001-95597 A Japanese Patent No. 3041380 WO2009 / 110374 JP 2005-270056 A

A. Aden et al. “LignocellulosicBiomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover” NREL Technical Report (2002) S. Larson et al., “The generation of fermentation inhibitors during dilute acid hydrolysis of softwood” Enzyme Microb. Technol. 24.151-159. (1999) HB. Klinkle et al., “Inhibition of ethanol producing yeast and bacteria by degradation products produced during pre-treatment of biomass” Appl. Microbiol. Biotechnol. 66. 10-26 (2004) J.P. Delgenes et al., “Effects of lignocellulose degradation products on ethanol fermentation of glucose and xyloseby Saccharomyces cerevisiase, Zymomonas mobilis, Pichia stipitis, and Candida shehatae” Enzyme Microbial. Technol. 19. 220-225. (1996) M. Alfred et al., “Effect of pH, time and temperature of overliming on detoxification of dilute-acid hydrolyzates for fermentation by Saccaromyces cerevisiase” Process Biochemistry, 38,515-522 (2002)

  As described above, there is a technology for producing xylose sugar liquid from xylan-containing biomass and using it as a fermentation raw material for microorganisms. However, as to a method for efficiently removing fermentation inhibitory substances contained in xylose sugar liquid, there is still a technology. Many challenges remained. In this invention, when manufacturing a xylose sugar liquid from a xylan containing biomass, it makes it a subject to manufacture efficiently a xylose sugar liquid with very few fermentation inhibitors.

The present invention has the following configurations [1] to [ 4 ].

[1] A method for producing a xylose sugar solution from xylan-containing biomass,
(1) Hydrothermally treating xylan-containing biomass to obtain a hydrothermally treated product,
(2) adding a saccharifying enzyme to the hydrothermally treated product, further adding a microorganism selected from the group of Saccharomyces and Candida as a microorganism that does not assimilate xylose, and obtaining a saccharification and fermentation broth;
(3) The saccharification and fermentation broth was subjected to solid-liquid separation using at least a microfiltration membrane, and the obtained filtrate was filtered through a nanofiltration membrane and / or a reverse osmosis membrane, and concentrated and purified from a non-permeate. A method for producing a xylose sugar solution comprising the step of obtaining a xylose solution.

  [2] Production of xylose sugar solution according to [1], wherein the hydrothermally treated product of step (1) is a hydrothermal treatment liquid and / or hydrothermal treatment biomass obtained by treating xylan-containing biomass with high-pressure hot water at 180 to 300 ° C. Method.

[ 3 ] The method for producing a xylose sugar solution according to [1] or [2] , wherein the saccharification and fermentation solution in step (3) is adjusted to a pH range of 1 to 5.

[ 4 ] The method for producing a xylose sugar solution according to any one of [1] to [ 3 ], wherein the average pore size of the microfiltration membrane in step (3) is 0.4 μm or less.

  According to the present invention, a xylose sugar solution can be selectively obtained from a xylan-containing biomass, and further, fermentation inhibitory substances such as HMF, furfural, and vanillin are removed from the obtained xylose sugar solution. Can be used as a carbon source for microorganisms that assimilate xylose.

FIG. 1 is a schematic diagram showing the procedure in the method for producing a xylose sugar solution of the present invention. FIG. 2 is a diagram showing the relationship between membrane flux and transmembrane pressure when a microfiltration membrane is used in the solid-liquid separation of the present invention. FIG. 3 is a diagram showing an example in which a chemical (ethanol) is produced by culturing a microorganism using the xylose sugar solution obtained by the xylose production method of the present invention as a carbon source.

  Xylan-containing biomass is herbaceous biomass such as bagasse, switchgrass, napiergrass, eliansus, corn stover, beet pulp, cottonseed husk, palm empty shell bunch, rice straw, straw, bamboo, straw, etc., or birch, beech In the present invention, these are used as a raw material for the xylose sugar solution. Specifically, xylan-containing biomass contains hemicellulose components xylan, cellulose, aromatic polymer lignin, etc. In the present invention, xylose sugar solution is obtained by hydrolyzing xylan-containing biomass. obtain.

  In the method for producing xylose of the present invention, a hydrothermally treated product is obtained by first hydrothermally treating the xylan-containing biomass as the step (1) using the xylan-containing biomass as a raw material. Next, a hydrolysis reaction of xylan-containing biomass is performed by adding a saccharifying enzyme to the hydrothermally treated product obtained in step (1). Next, a microorganism that does not assimilate xylose is added, and glucose is grown as a carbon source. In step (2), a saccharification and fermentation broth can be obtained by the action of microorganisms that do not assimilate saccharifying enzymes and xylose. Next, in step (3), the obtained saccharification and fermentation broth is subjected to solid-liquid separation using at least a microfiltration membrane to obtain a filtrate from which xylan-containing biomass undegraded organisms and microorganisms that do not assimilate xylose are removed. The obtained filtrate is filtered through a nanofiltration membrane and / or a reverse osmosis membrane to obtain a xylose sugar solution obtained by concentrating and purifying a xylose-containing sugar solution from a non-permeate.

  First, the step (1) will be described.

  In the step (1), there is no particular limitation on the pretreatment step for hydrothermally treating the xylan-containing biomass, but in order to improve the efficiency of hydrothermal treatment described later, pre-cutting, pulverization, etc. to an appropriate particle size / size, etc. It is preferable to perform the process.

  As conditions for the hydrothermal treatment in the step (1), after adding water so that the xylan-containing biomass is 0.1 to 50% by weight, high-pressure hot water of 100 to 400 ° C., preferably 180 to 300 ° C. is used. Process for seconds to 60 minutes. By treating under such temperature conditions, hydrolysis of xylan, cellulose, lignin, etc., which are components in the xylan-containing biomass, occurs. The number of processes is not particularly limited, and the process may be performed once or more. When the process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions. The hydrothermal treatment in step (1) is a pretreatment for improving the saccharification efficiency by the saccharifying enzyme performed in step (2) described later, and is not a treatment for completely hydrolyzing xylan or cellulose.

  The hydrothermally treated product of xylan-containing biomass is composed of a hydrothermally treated solid and hydrothermally treated water. The hydrothermally treated solid mainly includes cellulose, cellooligosaccharide, xylan, xylooligosaccharide, lignin and the like that could not be sufficiently decomposed by hydrothermal treatment. On the other hand, hydrothermally treated water contains monosaccharides, glucose, xylose, water-soluble cellooligosaccharides, and water-soluble xylooligosaccharides. The ratio or solid content of hydrothermally treated solid and hydrothermally treated water in the hydrothermally treated product of step (1) is not particularly limited. Alternatively, the hydrothermally treated product may be solid-liquid separated and separated into hydrothermally treated water and hydrothermally treated solid, and each component may be used as a hydrothermally treated product in the step (2) described later. As an example of a technique for solid-separating the hydrothermally treated product, a technique using a hydrothermal decomposition apparatus such as JP 2010-29862 can be exemplified and preferably used.

  Next, the step (2) will be described.

  The saccharifying enzyme in the step (2) refers to an enzyme agent containing an enzyme component capable of decomposing xylan or cellulose of a hydrothermally treated product, that is, an enzyme component called cellulase or hemicellulase. More specifically, it refers to an enzyme agent containing enzyme components such as cellobiohydrase, endoglucanase, exoglucanase, β-glucosidase, xylanase, and xylosidase.

  The enzyme component contained in the saccharifying enzyme will be described. Cellobiohydrase is a general term for cellulases characterized by hydrolysis from the terminal portion of cellulose. The enzyme group belonging to cellobiohydrase is represented by EC number: EC3.2.1.91. Have been described. Endoglucanase is a general term for cellulases characterized by hydrolysis from the central part of the cellulose molecular chain. EC numbers: EC 3.2.1.4, EC 3.2.1.6, EC 3.2.1 .39, EC 3.2.1.73, an enzyme group belonging to endoglucanase is described. Exoglucanase is a general term for cellulases characterized by hydrolysis from the end of a cellulose molecular chain, and is assigned to the exoglucanase as EC numbers: EC3.2.1.74 and EC3.2.1.58. Enzyme groups are described. β-glucosidase is a general term for cellulases characterized by acting on cellooligosaccharide or cellobiose, and an enzyme group belonging to β-glucosidase is described as EC number: EC 3.2.1.21. Xylanase is a general term for cellulases characterized by acting on hemicellulose or in particular xylan, and an enzyme group belonging to xylanase is described as EC number: EC3.2.1.8. Xylosidase is a general term for cellulases characterized by acting on xylo-oligosaccharides, and an enzyme group belonging to xylosidase is described as EC number: EC 3.2.1.37.

  The composition ratio of the above-mentioned enzyme component in the saccharifying enzyme in step (2) is not particularly limited, but hydrolysis of the hydrothermally treated product by the saccharifying enzyme is made efficient by the concerted or complementary action of the above-mentioned enzyme component. Since it is known, an enzyme agent comprising a plurality of enzyme components is preferable.

  The saccharifying enzyme in step (2) is preferably a saccharifying enzyme derived from the genus Trichoderma. Trichoderma microorganisms are known to secrete and produce a plurality of the above-described enzyme components in a large amount in the culture solution during the culturing process, and a large amount of saccharifying enzyme can be obtained at low cost from such a culture solution.

  The microorganisms of the genus Trichoderma are not particularly limited, but Trichoderma reesei is preferable, and specifically, Trichoderma reesei QM9414 (Trichoderma reesei QM9414), Trichoderma reesei QM9123 (TrichodermaR9 -30 (Trichoderma reeseiRut C-30), Trichoderma reesei PC3-7 (Trichoderma reesei PC3-7), Trichoderma reesei CL-847 (Trichoderma reeseiCL-847), Trichoderma reesei MCG77 (Trichorema reesei MCG77 (Trichoderma reesei) Examples thereof include derma reesei MCG80 (Trichoderma reeseiMCG80) and Trichoderma viride QM9123 (Trichoderma violet 9123). Further, it may be a microorganism derived from the aforementioned genus Trichoderma, which has been subjected to a mutation treatment with a mutation agent or ultraviolet irradiation to improve cellulase productivity.

  The saccharifying enzyme in the step (2) can be isolated from the culture supernatant obtained by culturing the microorganism for an arbitrary period in a medium adjusted so that the aforementioned Trichoderma microorganisms produce cellulase. The medium component to be used is not particularly limited, but a medium to which cellulose or cellulose-containing biomass is added can be generally used to promote cellulase production. A culture supernatant obtained by removing such a culture solution as a crude enzyme product or removing Trichoderma cells is preferably used. Further, a heterologous or homologous β-glucosidase, xylanase, or xylosidase may be added to the saccharifying enzyme derived from the genus Trichoderma. In addition, a heterologous or homologous β-glucosidase, xylanase, or xylosidase gene is introduced into a Trichoderma microorganism, and the Trichoderma microorganism that has been genetically modified to be produced in the culture medium is cultured. The enzyme obtained by separation may be used as a saccharifying enzyme.

  By adding the aforementioned saccharifying enzyme to the hydrothermally treated product, hydrolysis of cellulose, cellooligosaccharide, xylan, and xylooligosaccharide can be performed. By this hydrolysis, a solution containing glucose and xylose can be obtained. The glucose and xylose concentrations obtained are not particularly limited, but are generally about 5 g / L to 100 g / L, respectively. Glucose obtained here is consumed as a carbon source for the growth or proliferation of “microorganisms that do not assimilate xylose” described later.

  The amount of saccharifying enzyme added is not limited as long as sufficient hydrolysis efficiency of the hydrothermally treated product is obtained. Moreover, after adding a saccharifying enzyme, it can hydrolyze by hold | maintaining 1 minute-240 hours on optimal reaction temperature and pH conditions of a saccharifying enzyme. When the aforementioned Trichoderma saccharifying enzyme is used as the saccharifying enzyme, the reaction temperature is preferably in the range of 40 to 60 ° C. and the pH is preferably in the range of 3.0 to 7.5.

  The “microorganism that does not assimilate xylose” in the step (2) refers to a microorganism that does not assimilate xylose, which is a pentose, as a carbon source, or has an extremely slow assimilation rate. Usually, assimilation of glucose, which is a hexasaccharide, as a carbon source is a universal feature of microorganisms, on the other hand, there are microbial species that originally have the ability to assimilate xylose as a carbon source. Therefore, “microorganisms that do not assimilate xylose” referred to herein refers to all microorganisms excluding microorganisms having the following characteristics 1) and 2).

  Characteristic 1) A microorganism with a high ability to assimilate xylose as a carbon source has developed a metabolic pathway for xylose, takes xylose into a cell as a carbon source, and specifically metabolizes xylose via the pentose phosphate pathway. . Such microorganisms include Pichia, in particular Pichia stipitis, Candida, in particular Candida shehatae, Candida intermedia, Pachisoren, Pachisolen, Pachisolen, Pachisolen, Pachisolen, Pachisolen, Pachisole. Thermus, especially Thermus thermophilus, Escherichia, especially Escherichia coli, Bacillus, especially Bacillus subtilis, Rhodospodium, especially Rhodospodium Rhodosporidium torroi des), the genus Lipomyces, in particular Lipomyces starkey, and the genus Breltanomyces, in particular Brentanomyces cransenii. In addition to the microorganisms exemplified here, microorganisms that can utilize xylose as a carbon source are included in this category.

  Characteristic 2) A microorganism imparted with xylose assimilation by a gene recombination technique refers to a microorganism that can assimilate xylose by gene recombination technique, particularly by introducing or enhancing a xylose metabolism gene. Examples of the xylose metabolic gene include enzymes such as xylose isomerase, xylose reductase, xylitol dehydrogenase, and xylulose kinase. Examples of microorganisms imparted with xylose assimilation by such genetic recombination techniques include Japanese Patent No. 4124270 and Japanese Translation of PCT International Publication No. 2010-504756. In addition to the microorganisms exemplified here, microorganisms that can utilize xylose as a carbon source are included in this category.

  Among such “microorganisms that do not assimilate xylose”, microorganisms that can be preferably used are microorganisms belonging to eukaryotic microorganisms. Eukaryotic microorganisms are called alias fungi, and are further classified into Ascomycota, Zygomycota, Basidiomycota, and Chytridymycota. Such eukaryotic microorganisms have a relatively high resistance to low molecular weight compounds such as acetic acid and furfural contained in hydrothermally treated products as compared to prokaryotic microorganisms, and can be preferably used in the present invention.

  Among eukaryotic microorganisms, the genus Saccharomyces or Candida can be preferably used. Saccharomyces spp. Or Candida spp. 1) Relatively high resistance to low molecular weight compounds, and excellent growth using glucose as a carbon source even in hydrothermally treated products 2) Aromatic aldehydes or furanaldehydes such as HMF, furfural, vanillin From the point of being excellent in decomposition or adsorption, it can be preferably used in the present invention. Of the genus Saccharomyces or Candida, Saccharomyces cerevisiae or Candida glabrata is more preferable.

  Such a microorganism that does not assimilate xylose can grow using a monosaccharide such as glucose as a carbon source without assimilating xylose, but may be a microorganism that produces a fermentation product during the growth process. Although it does not specifically limit as a fermentation product, The substance currently mass-produced in fermentation industry, such as alcohol, an organic acid, an amino acid, and a nucleic acid, can be mentioned as a specific example. Many Saccharomyces cerevisiae or Candida glabrata exhibit ethanol fermentability, can grow using glucose as a carbon source, and can preferably produce ethanol as its fermentation product.

  In step (2), a microorganism that does not assimilate the saccharifying enzyme and xylose is added to the hydrothermally treated product to produce a saccharified fermentation broth. At this time, saccharification and fermentation may be performed simultaneously by simultaneously introducing a saccharification enzyme and a microorganism that does not assimilate xylose, or a microorganism that does not assimilate xylose after adding a saccharification enzyme and saccharifying the hydrothermally treated product. May be added and fermented. The reaction time, temperature, and amount of saccharifying enzyme added in saccharification and fermentation may be appropriately set in consideration of the characteristics of the enzyme or microorganism used. However, in the saccharification and fermentation of step (2), 1) glucose is consumed by the action of microorganisms that do not assimilate xylose, and the ratio of xylose components in the sugar solution is increased. 2) Impurities such as HMF, furfural, and vanillin are removed. Since the purpose is to decompose or adsorb, the reaction time of saccharification and fermentation is such that the concentration of glucose and fermentation inhibitors such as HMF, furfural and vanillin in the resulting saccharification and fermentation solution is sufficiently reduced to the desired amount. It is preferable to set and process the reaction time. Such reaction time varies depending on the input amount of microorganisms and the content of HMF and the like contained in the hydrothermally treated product, but can be set in the range of 1 to 150 hours.

  Depending on the nature of the microorganism that does not assimilate xylose, the saccharification and fermentation broth obtained in step (2) may contain a fermentation product metabolically converted from glucose. If an ethanol-fermenting microorganism is used, ethanol produced from glucose is contained in the saccharification and fermentation broth. Alternatively, when a lactic acid fermentable microorganism is used, lactic acid produced from glucose is contained in the saccharification and fermentation solution. The amount of fermentation product contained in such a saccharification and fermentation broth depends on the amount of glucose produced from xylan-containing biomass. The fermentation product in the saccharification and fermentation broth in this step (2) may be removed as a nanofiltration membrane permeate by filtering through a nanofiltration membrane and / or a reverse osmosis membrane in step (3) described later. Alternatively, this permeate may be recovered.

  Next, the step (3) will be described.

  In step (3), the saccharification and fermentation broth is subjected to solid-liquid separation using at least a microfiltration membrane. By this solid-liquid separation, undecomposed solid matter is removed by the saccharification reaction of the hydrothermally treated product in step (2). The undegraded solid material here includes lignin components, cellulose components, xylan components, etc. that could not be sufficiently decomposed by saccharifying enzymes, and the microorganisms introduced in step (2) are also undegraded by solid-liquid separation. Included in solids. On the other hand, xylose is contained in the filtrate obtained by solid-liquid separation using a microfiltration membrane.

  The microfiltration membrane used in the step (3) is called a membrane filtration, and is a separation membrane that can separate and remove particles of about 0.01 to 10 μm from a fine particle suspension using a pressure difference as a driving force. . In other words, the surface of the microfiltration membrane has pores in the range of 0.01 to 10 μm, and fine particle components exceeding the pores can be separated and removed to the membrane side.

  In the step (3), it is preferable to use a microfiltration membrane having an average pore diameter of 0.4 μm or less. By using a microfiltration membrane with an average pore size of 0.4 μm or less, it has the effect of removing HMF and furfural, which are fermentation inhibitors contained in saccharification and fermentation broth, and has less impurities and xylose sugar A liquid can be produced. Such an average pore diameter may adopt the nominal diameter presented by each separation membrane manufacturer or may be actually measured. As a method for measuring the average pore size of the microfiltration membrane, there is a bubble point method. The bubble point method measures the minimum pressure at which bubbles can be observed on the membrane surface by applying air pressure from the membrane secondary side, and calculates the average pore diameter from the relationship between the surface tension and pressure of the liquid used. It is a technique. Specifically, it is possible to measure in accordance with ASTM F316-03 (Bubble Point Method), for example, using a penetrating pore distribution / gas permeability analyzer manufactured by Nippon Bell Co., Ltd. Can do.

  Examples of the material of the microfiltration membrane include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, ceramic, polypropylene, polycarbonate, and polytetrafluoroethylene (Teflon (registered trademark)). Although not particularly limited, the material of the microfiltration membrane used in the step (3) is a precision made of polyvinylidene fluoride in terms of antifouling property, chemical resistance, strength, filterability among the above materials. A filtration membrane is preferred.

  Examples of the method of microfiltration using a microfiltration membrane include pressure filtration, vacuum filtration, crossflow filtration, and centrifugal filtration. Crossflow filtration with less membrane clogging is preferred. Moreover, although filtration operation is divided roughly into constant pressure filtration, constant flow filtration, and non-constant pressure non-constant flow filtration, it is not specifically limited.

  Solid-liquid separation in step (3) may be performed at least using a microfiltration membrane, but depending on the case, other solid-liquid separation methods may be combined in addition to microfiltration. It is not something. Specific examples of other solid-liquid separation methods include press filtration and centrifugation.

  Moreover, after performing solid-liquid separation of a process (3), it is also one of the preferable aspects that the filtrate obtained is filtered through an ultrafiltration membrane. As an effect of the ultrafiltration membrane treatment, 1) the saccharification enzyme used in step (2) can be recovered and reused, so that the economy is improved. 2) the nanofiltration membrane and / or reverse osmosis in step (3) The clogging of the membrane in the membrane is further reduced, and 3) the Maillard reaction with the amino acid or protein of the xylose sugar solution can be suppressed. The ultrafiltration membrane to be used is not particularly limited, but is preferably a synthetic polymer membrane such as polyethersulfone, and is preferably an ultrafiltration membrane having a fractional molecular weight of 1000 to 200000.

  Next, the filtration component obtained by the above-described solid-liquid separation will be described with respect to filtration through a nanofiltration membrane and / or a reverse osmosis membrane.

  In the step (3), “filtering through a nanofiltration membrane and / or reverse osmosis membrane” means filtering the filtrate obtained by solid-liquid separation of a saccharification and fermentation broth through a nanofiltration membrane and / or a reverse osmosis membrane. By this operation, dissolved sugar, particularly xylose sugar solution, can be blocked or filtered to the non-permeate side, and the fermentation inhibitor can be permeated as a permeate or a filtrate.

  The fermentation inhibitor herein is a compound that is produced by hydrolysis of cellulose-containing biomass, and is a substance that acts inhibitory as described above in the fermentation process using the sugar solution obtained by the production method of the present invention as a raw material. In particular, it is broadly classified into organic acids, furan compounds, and phenol compounds produced in the acid treatment step of cellulose-containing biomass.

  Specific examples of the organic acid include acetic acid, formic acid, levulinic acid and the like. Examples of the furan compounds include HMF and furfural. Such an organic acid or furan compound is a product of decomposition of monosaccharides such as glucose or xylose.

  In addition, examples of phenolic compounds include vanillin, acetovanillin, vanillic acid, syringic acid, gallic acid, coniferyl aldehyde, dihydroconiphenyl alcohol, hydroquinone, catechol, acetogicon, homovanillic acid, 4-hydroxybenzoic acid, 4-hydroxybenzoic acid Specific examples include hydroxy-3-methoxyphenyl derivatives (Hibbert's ketones), and these compounds are derived from lignin or lignin precursors.

  The filtrate obtained by solid-liquid separation using the microfiltration membrane in step (3) contains at least one of the substances as a fermentation inhibitor, and actually contains a plurality of kinds. These fermentation-inhibiting substances can be detected and quantified by a general analytical technique such as thin phase chromatography, gas chromatography, and high performance liquid chromatography.

  The nanofiltration membrane used in the step (3) is also called a nanofilter (nanofiltration membrane, NF membrane), and “a membrane that transmits monovalent ions and blocks divalent ions” It is a generally defined membrane. It is a membrane that is considered to have a minute gap of about several nanometers, and is mainly used to block minute particles, molecules, ions, salts, and the like in water. The reverse osmosis membrane used in the step (3) is also called a RO membrane, and is a membrane generally defined as “a membrane having a desalting function including monovalent ions”. It is a membrane that is thought to have ultrafine pores of about several nanometers, and is mainly used for removing ionic components such as seawater desalination and ultrapure water production.

  As a method of evaluating the performance of the nanofiltration membrane and reverse osmosis membrane used in step (3), by calculating the permeability (%) of the target compound (fermentation inhibitor, monosaccharide, etc.) contained in the sugar liquid Can be evaluated. The calculation method of the transmittance (%) is shown in Formula 1.

  Transmittance (%) = (concentration of target compound on transmission side / target compound concentration of non-permeate) × 100 (Equation 1).

  The concentration of the target compound in Formula 1 is not limited as long as it is an analytical technique that can be measured with high accuracy and reproducibility, but high-performance liquid chromatography, gas chromatography, or the like can be preferably used. When the target compound is a monosaccharide, the nanofiltration membrane and / or reverse osmosis membrane used in the present invention preferably has a lower permeability, whereas when the target compound is a fermentation inhibitor, the permeability is low. High is preferred.

  The pH of the sugar solution used for the nanofiltration membrane and / or the reverse osmosis membrane is not particularly limited, but is preferably in the range of 1 to 5. When the pH is less than 1, the membrane is denatured when used for a long period of time and the membrane performance such as flux and transmittance is remarkably lowered. When the pH is greater than 5, the removal rate of organic acids such as acetic acid, formic acid and levulinic acid is remarkably increased. This is because it may decrease. Since nanofiltration membranes and / or reverse osmosis membranes are charged on the membrane surface, substances that are ionized in the solution are easier to remove or block than non-ionized substances. When the content of the organic acid contained in is high, or when a high removal effect is required, the removal efficiency can be drastically improved by adjusting the sugar solution to a pH within the above range. Moreover, there exists a fouling suppression effect of a film | membrane as an effect which adjusts the pH of a sugar liquid to 1-5, and filters it through a nanofiltration membrane and / or a reverse osmosis membrane. In general, the initial flux value decreases as the pH decreases, but the long-term stability of the membrane can be maintained under the previous pH conditions, particularly only for sugar solutions derived from xylan-containing biomass.

In particular, in a reverse osmosis membrane, it is more preferable to adjust the pH of the sugar solution to 1 to 3. As with nanofiltration membranes, if the pH is less than 1, the membrane will be denatured when used for a long period of time, and the membrane performance such as flux and permeability will decrease significantly. This is because there is a case where the sufficient value cannot be obtained. This is because the reverse osmosis membrane has a smaller ionic radius, which is the effective radius of the organic acid, if the ionic-derived charge of the elution component is not further suppressed for reasons such as the pore size being smaller than that of the nanofiltration membrane. It is presumed that the removal performance equivalent to that cannot be maintained. If a reverse osmosis membrane of a low pressure / ultra-low pressure type that can reduce the operating pressure among reverse osmosis membranes is used, a reverse osmosis membrane that is not a low-pressure / ultra-low pressure type even if the adjusted pH of the raw water is greater than 3. It has the same organic acid removal rate, and the effect of reducing the amount of acid used for pH adjustment and the amount of alkali used for pH adjustment in the subsequent fermentation process can be obtained. Since the removal rate of organic acid is improved as compared with a reverse osmosis membrane which is not an ultra-low pressure type, it is preferably used in the present invention. Here, the low pressure / ultra-low pressure type reverse osmosis membrane is a permeation flow rate (m ³ / m ² / day) of sodium chloride (500 mg / L) per unit membrane area at a filtration pressure of 0.75 MPa and pH 6.5. Refers to a reverse osmosis membrane of 0.4 or more. As a method for evaluating the permeation flow rate (membrane permeation flux or membrane flux) per unit area of the membrane, the permeate amount, the time when the permeate amount was sampled, and the membrane area can be measured and calculated by Equation 2. it can.

Membrane permeation flux (m ³ / m ² / day) = permeate amount / membrane area / water sampling time (formula 2).

  The acid or alkali used for adjusting the pH of the sugar solution is not particularly limited. The acid is preferably hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, more preferably sulfuric acid, nitric acid, phosphoric acid from the viewpoint of preventing inhibition during fermentation, more preferably sulfuric acid from the viewpoint of economy. The alkali is preferably ammonia, sodium hydroxide, calcium hydroxide and an aqueous solution containing them from the viewpoint of economy, more preferably ammonia, sodium which is a monovalent ion from the viewpoint of membrane fouling, and more preferably inhibition during fermentation. Ammonia from the viewpoint of hardly occurring.

  The step of adjusting the pH of the sugar solution may be performed before the nanofiltration membrane and / or the reverse osmosis membrane treatment. When an enzyme is used for hydrolysis of cellulose-containing biomass, the pH may be adjusted to 5 or less during the hydrolysis reaction. In addition, when using a process that recycles an enzyme using an ultrafiltration membrane, the pH of the filtrate after the ultrafiltration membrane treatment should be adjusted because the enzyme tends to be deactivated when the pH is lowered to 4 or less. Is preferred.

  The temperature of the sugar solution used for the nanofiltration membrane and / or the reverse osmosis membrane in the present invention is not particularly limited, but can be appropriately set for the purpose of enhancing the fermentation inhibitor removal ability during filtration of the membrane to be used. Specifically, when filtering with a nanofiltration membrane, if the temperature of the sugar solution is 40 to 80 ° C., the ability to remove the fermentation-inhibiting substance of the nanofiltration membrane is increased, and therefore it is preferably set. Although the removal ability increases from the range of 40 ° C or higher when the temperature of the sugar solution is filtered through the nanofiltration membrane, if the temperature is higher than 80 ° C, the nanofiltration membrane may be denatured and the membrane characteristics may be lost. It is.

  Moreover, when filtering with a reverse osmosis membrane, if the temperature of a sugar solution is 1-15 degreeC, since the fermentation inhibitor removal ability of a reverse osmosis membrane will increase, it sets preferably. If the temperature of the sugar solution when filtering through a reverse osmosis membrane is lower than 1 ° C., it causes a failure of the apparatus due to freezing in the piping. In the temperature control, when the temperature is high, the membrane expands and a larger molecular weight is removed, and the removal amount tends to be improved. When the temperature is low, the membrane shrinks and the pore size of the membrane becomes small. This is because the loss of sugar to the filtrate side may be reduced.

  As a material for the nanofiltration membrane used in the present invention, a polymer material such as cellulose acetate polymer, polyamide, polyester, polyimide, vinyl polymer can be used. It is not limited to this, and a film including a plurality of film materials may be used. In addition, the membrane structure has a dense layer on at least one side of the membrane, and on the asymmetric membrane having fine pores gradually increasing from the dense layer to the inside of the membrane or the other side, or on the dense layer of the asymmetric membrane. Either a composite film having a very thin functional layer formed of another material may be used. As the composite membrane, for example, a composite membrane described in JP-A-62-201606 in which a nanofilter composed of a polyamide functional layer is formed on a support membrane made of polysulfone as a membrane material can be used.

  Among these, a composite film having a high-pressure resistance, high water permeability, and high solute removal performance and having an excellent potential and using a polyamide as a functional layer is preferable. In order to maintain durability against operating pressure, high water permeability, and blocking performance, a structure in which polyamide is used as a functional layer and is held by a support made of a porous membrane or nonwoven fabric is suitable. As the polyamide semipermeable membrane, a composite semipermeable membrane having a functional layer of a crosslinked polyamide obtained by polycondensation reaction of a polyfunctional amine and a polyfunctional acid halide on a support is suitable.

  In the nanofiltration membrane having a polyamide as a functional layer, preferable carboxylic acid components of monomers constituting the polyamide include, for example, trimesic acid, benzophenone tetracarboxylic acid, trimellitic acid, pyrometic acid, isophthalic acid, terephthalic acid, naphthalene Aromatic carboxylic acids such as dicarboxylic acid, diphenyl carboxylic acid, pyridine carboxylic acid and the like can be mentioned, but considering solubility in a film forming solvent, trimesic acid, isophthalic acid, terephthalic acid, and a mixture thereof are more preferable.

  Preferred amine components of the monomers constituting the polyamide include m-phenylenediamine, p-phenylenediamine, benzidine, methylenebisdianiline, 4,4′-diaminobiphenyl ether, dianisidine, 3,3 ′, 4- Triaminobiphenyl ether, 3,3 ′, 4,4′-tetraaminobiphenyl ether, 3,3′-dioxybenzidine, 1,8-naphthalenediamine, m (p) -monomethylphenylenediamine, 3,3′- Monomethylamino-4,4′-diaminobiphenyl ether, 4, N, N ′-(4-aminobenzoyl) -p (m) -phenylenediamine-2,2′-bis (4-aminophenylbenzimidazole), 2 , 2′-bis (4-aminophenylbenzoxazole), 2,2′-bis (4-aminophenyl) Secondary diamines such as primary diamines having an aromatic ring such as nilbenzothiazole), piperazine, piperidine or derivatives thereof, among which nanofiltration membranes having a functional layer of a crosslinked polyamide containing piperazine or piperidine as a monomer are It is preferably used because it has heat resistance and chemical resistance in addition to pressure resistance and durability, and a nanofiltration membrane having a cross-linked piperazine polyamide as a functional layer is more preferably used. Specific examples of the crosslinked piperazine polyamide-based nanofiltration membrane include UTC60, a crosslinked piperazine polyamide-based nanofiltration membrane manufactured by Toray Industries, Inc.

  The nanofiltration membrane is generally used as a spiral membrane module, but the nanofiltration membrane used in the present invention is also preferably used as a spiral membrane module. Specific examples of preferred nanofiltration membrane modules include, for example, GEsepa, a nanofiltration membrane manufactured by GE Osmonics, which is a cellulose acetate-based nanofiltration membrane, NF99 or NF99HF, a nanofiltration membrane manufactured by Alfa Laval, whose functional layer is polyamide. NF-45, NF-90, NF-200, NF-270, or NF-400, a nanofiltration membrane manufactured by Filmtec Co., Ltd., having a cross-linked piperazine polyamide as a functional layer, and a main component of cross-linked piperazine polyamide The nanofiltration membrane module SU-210, SU-220, SU-600, or SU-610 manufactured by Toray Industries, Inc., which includes a functional layer of polyamide containing the constituents shown, including UTC60 manufactured by Toray Industries, Inc. is more preferable. Alfa Laval nano filter with polyamide as functional layer NF99 or NF99HF of membrane, NF-45, NF-90, NF-200 or NF-400 of Nanotec membrane manufactured by Filmtec Co., Ltd. with functional layer of cross-linked piperazine polyamide, or polyamide based on cross-linked piperazine polyamide It is a nanofiltration membrane module SU-210, SU-220, SU-600 or SU-610 manufactured by Toray Industries, Ltd., including UTC 60 made by Toray Industries, Inc. More preferably, a functional layer made of polyamide based on crosslinked piperazine polyamide It is the nano filtration membrane module SU-210, SU-220, SU-600 or SU-610 made by the company including UTC60 made by Toray Industries, Inc.

  The filtration with the nanofiltration membrane in the step (2) may apply pressure, and the filtration pressure is preferably in the range of 0.1 to 8 MPa. If the filtration pressure is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane may be damaged. Moreover, if the filtration pressure is in the range of 0.5 to 7 MPa, the membrane permeation flux is high, so that the sugar solution can be efficiently permeated, and the possibility of affecting the membrane damage is low. More preferably, the range of 1 to 6 MPa is particularly preferable.

  As a material of the reverse osmosis membrane used in the present invention, a composite membrane using a cellulose acetate-based polymer as a functional layer (hereinafter, also referred to as a cellulose acetate-based reverse osmosis membrane) or a composite membrane using a polyamide as a functional layer (hereinafter referred to as a cellulose acetate-based reverse osmosis membrane) And a polyamide-based reverse osmosis membrane). Here, as the cellulose acetate-based polymer, organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and the like, or a mixture thereof and those using mixed esters can be mentioned. It is done. The polyamide includes a linear polymer or a crosslinked polymer having an aliphatic and / or aromatic diamine as a monomer.

  Specific examples of the reverse osmosis membrane used in the present invention include, for example, ultra-low pressure type SUL-G10, SUL-G20, low pressure type SU-710, SU-, which are polyamide-based reverse osmosis membrane modules manufactured by Toray Industries, Inc. 720, SU-720F, SU-710L, SU-720L, SU-720LF, SU-720R, SU-710P, SU-720P, high-pressure type SU-810, SU-820 including UTC80 as a reverse osmosis membrane, SU-820L, SU-820FA, Cellulose acetate reverse osmosis membrane SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100 , SC-8200, NTR-759HR, NTR-729HF, NTR manufactured by Nitto Denko Corporation 70SWC, ES10-D, ES20-D, ES20-U, ES15-D, ES15-U, LF10-D, Alfa Laval RO98pHt, RO99, HR98PP, CE4040C-30D, GE GE Sepa, Filmtec BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30-4040, SW30HRLE-4040, KOCH TFC-HR, TFC-ULP, TRISEP ACM-1, ACM-2, ACM-4, etc. It is done.

  In the present invention, it is preferable to filter through a reverse osmosis membrane. In general, when a nanofiltration membrane is used, the permeability of xylose increases, and the amount that can be recovered as a non-permeate may decrease. However, with a reverse osmosis membrane, xylose is not lost as a non-permeate. Can be recovered. Therefore, when a xylose sugar solution is obtained by combining a nanofiltration membrane and a reverse osmosis membrane, the sugar solution is first filtered through a nanofiltration membrane, and the resulting filtrate is further filtered through a reverse osmosis membrane, whereby a nanofiltration membrane and a reverse membrane are obtained. Xylol sugar solution can be recovered without loss from the non-permeated solution of the osmotic membrane.

  The xylose sugar solution obtained by the above-mentioned step (3) can be used as an industrial raw material as it is, but may be further purified by a method such as activated carbon treatment, ion exchange membrane treatment, crystallization, and the like.

  The xylose sugar solution obtained by the present invention preferably has a xylose concentration of 50 to 200 g / L. If the xylose concentration is too low, it is difficult to use as an industrial raw material, and concentrating the xylose sugar solution to a range exceeding 200 g / L leads to the nanofiltration membrane and / or reverse osmosis membrane in step (3). In the process to filter, since a high pressure is required, it is not economically preferable.

  In addition, the concentration of HMF or furfural as a fermentation inhibitor contained in the xylose sugar solution is preferably in the range of 0 to 1000 ppm when the xylose sugar solution is used as a carbon source for the microorganisms described later.

  Examples of the use of xylose sugar solution as an industrial raw material include uses such as food additives, preservatives, and fragrances. Further, the xylose sugar solution of the present invention can be converted into xylitol by further hydrogenation reaction, and can be used as a sweetener such as gum. Moreover, a chemical can be produced by culturing microorganisms using xylose sugar solution as a carbon source.

  Hereinafter, a method for producing a chemical product by culturing a microorganism using a xylose sugar solution as a carbon source will be described.

  The xylose sugar solution obtained by the present invention may appropriately contain a nitrogen source, inorganic salts, and, if necessary, organic micronutrients such as amino acids and vitamins as necessary. In some cases, in addition to xylose, as a carbon source, saccharides such as glucose, sucrose, fructose, galactose, and lactose, starch saccharified solution containing these saccharides, sweet potato molasses, sugar beet molasses, high test molasses, acetic acid, etc. You may add acids, alcohols, such as ethanol, glycerol, etc., and use as a fermentation raw material. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, Corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added.

  Microorganisms for culturing microorganisms using xylose sugar solution as a carbon source are limited to microorganisms that can use xylose as a carbon source. This is because glucose is almost consumed by the action of the microorganism in the step (2). Therefore, only microorganisms that can use xylose as a carbon source can be used.

  Specific examples of microorganisms that can use xylose as a carbon source are roughly classified into two types as described above. That is, a microorganism that can assimilate xylose as a carbon source, and a microorganism that adds xylose assimilation by a genetic recombination technique.

  Examples of microorganisms that can essentially assimilate xylose as a carbon source include Pichia, in particular Pichia stipitis, Candida, in particular Candida shehatae, Candida intermedia, Pachisoren Genus, Pachisolen tanophilus, Thermus, especially Thermus thermophilus, Escherichia, especially Escherichia coli, Bacillus silis, especially Bacillus b. Genus, especially Rhodospodium toluroides (Rhod sporidium toruloides), Lipomyces genus, especially Lipomyces Starkey (Lipomyces starkey), Brel Tano My genus, in particular, and the like can be exemplified breath Tano My Seth Kuranzeni (Brentanomyces clansenii). Among these microorganisms, Escherichia coli (E. coli) or Pichia stipitis is preferable, and Pichia stipitis is more preferable.

  Moreover, as an example of a microorganism to which xylose assimilation is added by a gene recombination technique, a microorganism into which a xylose metabolism gene is introduced can be exemplified. Examples of the xylose metabolic gene include enzymes such as xylose isomerase, xylose reductase, xylitol dehydrogenase, and xylulose kinase. Examples of microorganisms imparted with xylose assimilation by such genetic recombination techniques include Japanese Patent No. 4124270 and Japanese Translation of PCT International Publication No. 2010-504756. Such microorganisms are not particularly limited as long as they can efficiently assimilate using xylose as a carbon source and have the ability to produce a desired fermentation product.

  The culture of microorganisms is usually performed within the optimum pH and optimum temperature range of the microorganisms to be cultured. The pH of the culture solution is usually adjusted to a predetermined value within a pH range of 4 to 8 with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, or the like. If it is necessary to increase the oxygen supply rate, means such as adding oxygen to the air to keep the oxygen concentration at 21% or higher, pressurizing the culture, increasing the stirring rate, or increasing the aeration rate can be used.

  When culturing a microorganism using a xylose sugar solution as a carbon source, a fermentation culture method known to those skilled in the art can be employed, but from the viewpoint of productivity, the continuous culture method disclosed in WO2007 / 097260 is preferably employed.

  Specific examples of the chemical product obtained by the above-described method for producing a chemical product include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, nucleic acids, and diamines. For example, alcohols include ethanol, 1-propanol, isopropanol, butanol, isobutanol, 1,3-propanediol, 1,4-butanediol, glycerol, and the like, and organic acids include acetic acid, lactic acid, pyruvic acid, and succinic acid. Examples of malic acid, itaconic acid, citric acid, and nucleic acids include nucleosides such as inosine and guanosine, nucleotides such as inosinic acid and guanylic acid, and diamine compounds such as cadaverine. The present invention can also be applied to the production of substances such as enzymes, antibiotics, and recombinant proteins. The chemical preferably produced in the present invention is alcohol.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.

(Reference Example 1) Preparation of saccharifying enzyme (Trichoderma-derived saccharifying enzyme)
The saccharifying enzyme was prepared by the following method.

[Pre-culture]
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14 (w / vol), potassium dihydrogen phosphate 0.2 % (W / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol), zinc chloride 0.02% (w / vol) , Iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol), manganese chloride tetrahydrate 0.0008% ( w / vol), boric acid 0.0006% (w / vol), hexamolybdenum hexamolybdate tetrahydrate 0.0026% (w / vol) and added to distilled water, 100 mL is a triangle with 500 mL baffle Stick to flask, 121 Autoclaved at 15 ° C for 15 minutes. After standing to cool, PE-M and Tween 80, each autoclaved at 121 ° C. for 15 minutes, were added 0.01% (w / vol). Trichoderma reesei PC3-7 was inoculated to this preculture medium at 1 × 10 ⁵ cells / mL, and cultured at 28 ° C. for 72 hours with shaking at 180 rpm to prepare a preculture (shaking apparatus: TAITEC). BIO-SHAKER BR-40LF manufactured by the company).

[Main culture]
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), cellulose (Avicel) 10% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14% ( w / vol), potassium dihydrogen phosphate 0.2% (w / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol), Zinc chloride 0.02% (w / vol), iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol), Manganese chloride tetrahydrate distilled to 0.0008% (w / vol), boric acid 0.0006% (w / vol), hexamolybdenum hexamolybdate tetrahydrate 0.0026% (w / vol) Add to water and add 2.5L to 5L stirring jar ( Imposition to BLE Co. DPC-2A) containers and autoclaved for 15 minutes at 121 ° C.. After standing to cool, 0.1% each of PE-M and Tween 80 which were autoclaved at 121 ° C. for 15 minutes was added, and Trichoderma reesei PC3-7 pre-cultured in a liquid medium in advance by the above-described method was added. 250 mL was inoculated. Then, it culture | cultivated by 28 degreeC, 87 hours, 300 rpm, and aeration volume 1vvm, and after centrifugation, the supernatant liquid was membrane-filtered (Millipore Corporation Stericup-GV material: PVDF). 1/100 amount of β-glucosidase (Novozyme 188) as a protein weight ratio was added to the culture solution adjusted under the aforementioned conditions, and this was used as a saccharifying enzyme in the following examples.

Reference Example 2 Measurement of Sugar Concentration The glucose and xylose concentrations of the present invention were quantified by comparison with a standard under the HPLC conditions shown below. The hydrothermally treated product, saccharified solution, saccharified fermentation solution, and xylose sugar solution were centrifuged at 3500 G for 10 minutes, and the supernatant components were subjected to the following analysis.
Column: Luna NH ₂ (Phenomenex)
Mobile phase: Milli Q: Acetonitrile = 25: 75 (flow rate 0.6 mL / min)
Reaction solution: None Detection method: RI (differential refractive index)
Temperature: 30 ° C.

(Reference Example 3) Analysis of Fermentation Inhibitory Substances (HMF, Furfural, Vanillin, Acetic Acid, Formic Acid) Among fermentation inhibitory substances, HMF, furfural, and vanillin were quantified by comparison with a standard under the following HPLC conditions. The hydrothermally treated product, saccharified solution, saccharified fermentation solution, and xylose sugar solution were centrifuged at 3500 G for 10 minutes, and the supernatant components were subjected to the following analysis.
Column: Synergi HideRP 4.6 mm × 250 mm (Phenomenex)
Mobile phase: 0.1% acetonitrile _H 3 PO ₄ (flow rate 1.0 mL / min)
Detection method: UV (283 nm)
Temperature: 40 ° C.

Acetic acid and formic acid were quantified by comparison with a standard under the following HPLC conditions. The hydrothermally treated product, saccharified solution, saccharified fermentation solution, and xylose sugar solution were centrifuged at 3500 G for 10 minutes, and the supernatant components were subjected to the following analysis.
Column: Series mobile phase of Shim-Pack and Shim-Pack SCR101H (manufactured by Shimadzu Corporation): 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electric conductivity temperature: 45 ° C.

(Reference Example 4) Analysis of ethanol The ethanol accumulation concentration was measured by gas chromatography. Shimadzu GC-2010 Capillary GC TC-1 (GL science) 15 meter L. * 0.53 mm I.D. D. , Df1.5 μm was detected and calculated by a hydrogen salt ionization detector and evaluated.

Example 1
Step (1): Step of obtaining hydrothermally treated product from xylan-containing biomass Rice straw was used as xylan-containing biomass. The xylan-containing biomass was shredded with a cutter mill so as to be about 100 μm. Then, water was added so that it might become 15% of solid content weight of a xylan containing biomass. This xylan-containing biomass mixed solution was autoclaved (manufactured by Nitto Koatsu) at 200 ° C. for 15 minutes. After the treatment, the solid-liquid product was lightly settled, and then the supernatant component was used as a hydrothermally treated product. The hydrothermally treated product had a solid concentration of about 5%. The component composition contained in the hydrothermally treated product of Example 1 was analyzed according to the methods of Reference Examples 2 to 4. The analysis results are shown in Table 1.

Step (2): A step of adding a saccharifying enzyme to a hydrothermally treated product and further adding a microorganism that does not assimilate xylose to obtain a saccharified fermentation broth Saccharification adjusted in Reference Example 1 to the hydrothermally processed product adjusted in Step (1) Enzyme was added and saccharification was performed. The saccharification conditions are as follows.
[Saccharification conditions]
Hydrothermally treated product: 2 L (prepared in Example 1)
pH: 5.0
Saccharification enzyme: addition reaction temperature and time so that the protein concentration becomes 1 mg / mL: 50 ° C., 6 hours.

  Table 1 shows the results of analyzing the component composition of the saccharified solution obtained under the above conditions according to Reference Examples 2 to 4. It was confirmed that the glucose and xylose components were significantly increased by saccharification of the hydrothermally treated product.

  Next, a microorganism that does not assimilate xylose was added to the saccharified solution for fermentation. As a microorganism that does not assimilate xylose, Saccharomyces cerevisiae OC-2 (wine yeast) or Candida grabrata (NBRC 103857) was used. The above two types of microorganisms that do not assimilate xylose are precultured at 25 ° C. for 1 day in YPD medium (2% glucose, 1% yeast extract (Bacto Yeast Extract / BD), 2% polypeptone (Nippon Pharmaceutical)). Went. Next, the obtained culture solution was added to 1% (20 mL) with respect to the hydrothermally treated product after saccharification. After adding the microorganism, it was incubated at 25 ° C. for 2 days. What was obtained by this operation was used as a saccharification and fermentation broth. In particular, the saccharification and fermentation broth prepared by adding Saccharomyces cerevisiae OC-2 (wine yeast) was referred to as “saccharification and fermentation broth 1”, and the saccharification and fermentation broth prepared by adding Candida grabrata (NBRC103857) was referred to as “saccharification and fermentation broth 2”. Table 1 shows the results of analyzing the component composition of the saccharification and fermentation broth 1 and saccharification and fermentation broth 2 (both pH 5.0) according to Reference Examples 2 to 4.

  Fermentation by addition of microorganisms that do not assimilate xylose causes 1) selective consumption of glucose and production of ethanol as a fermentation product, and 2) removal (consumption) of HMF, furfural, and vanillin. It could be confirmed. Further, by comparing the components of the saccharified fermentation broth 1 and the saccharified fermented broth 2, it was found that Candida grabrata was slightly better at removing HMF, furfural, and vanillin than Saccharomyces cerevisiae OC-2 (wine yeast).

Step (3): The saccharification and fermentation broth is subjected to solid-liquid separation with at least a microfiltration membrane, the obtained filtrate is filtered through a nanofiltration membrane and / or a reverse osmosis membrane, and concentrated and purified from the membrane fraction. First, solid-liquid separation using a microfiltration membrane was performed for the saccharification and fermentation broth 1 and saccharification and fermentation broth 2 obtained in step (2). The microfiltration membrane used was a polysulfone microfiltration membrane (GE) having an average pore size of 0.4 μm. In a small flat membrane filtration device (SEpa (registered trademark) CF II Med / High Foulant System manufactured by GE), a microfiltration membrane was installed, and the saccharification and fermentation solution of Example 2 under the conditions of 0.1 m ³ / m ² / day 1. The cross-flow filtration of the saccharification fermentation liquid 2 was performed, and the microfiltration membrane filtrate 1 and the microfiltration membrane filtrate 2 were obtained as a membrane permeate, respectively. The results of analyzing the microfiltration membrane filtrate according to Reference Examples 2 to 4 are shown in Table 2.

  Next, 1 L of the solution component obtained by solid-liquid separation using a microfiltration membrane was filtered through a nanofiltration membrane or a reverse osmosis membrane. Cross-linked piperazine polyamide-based nanofiltration membrane “UTC60” (manufactured by Toray Industries, Inc.) as a nanofiltration membrane, and a cross-linked wholly aromatic polyamide-based reverse osmosis membrane “SUL-G” (manufactured by Toray Industries, Inc.) as a reverse osmosis membrane, Each was set in a small flat membrane filtration device (GE-made Sepa (registered trademark) CF II Med / High Foulant System), filtered at a raw water temperature of 25 ° C. and a high-pressure pump pressure of 3 MPa, and 0.8 L of permeate Got. Table 2 shows the component composition of the xylose solution obtained as the non-permeate liquid (membrane fraction) of the nanofiltration membrane, and Table 3 shows the component composition of the xylose solution obtained as the non-permeate liquid (membrane fraction) of the reverse osmosis membrane. Show. In addition, what processed the saccharification fermentation liquid 1 was made into the xylose sugar liquid 1, and what processed the saccharification fermentation liquid 2 was made into the xylose sugar liquid 2.

  Compared to saccharification and fermentation broth 1 and saccharification and fermentation broth 2 (Table 1), HMF and furfural are better in microfiltration membrane filtrate 1 and microfiltration membrane filtrate 2 (Table 2), which are solid-liquid separated by a microfiltration membrane. , Vanillin was found to decrease. It was also found that xylose can be concentrated by filtering through a nanofiltration membrane or reverse osmosis membrane, while concentration of acetic acid, formic acid, etc. can be kept low. That is, it was found that the xylose sugar solution can be concentrated while greatly reducing the concentration of acetic acid and formic acid with respect to the xylose concentration. Further, when xylose concentration in the nanofiltration membrane (Table 2) and xylose concentration in the reverse osmosis membrane (Table 3) were compared, it was found that the reverse osmosis membrane can concentrate with less loss of xylose.

(Example 2) Effect of pH adjustment of saccharification and fermentation broth on nanofiltration membrane or reverse osmosis membrane filtration In Example 3, nanofiltration after saccharification and fermentation broth having a pH of 5.0 was separated using a microfiltration membrane. It is filtered through a membrane or reverse osmosis membrane. Therefore, the pH during the nanofiltration membrane or reverse osmosis membrane treatment is 5.0. In this example, the pH of the saccharification and fermentation broth 1 prepared in step (2) of Example 1 was adjusted to pH 1, pH 2.4, pH 3.2, pH 4.0, pH 5.0, pH 6.0, pH 7.0 in advance. Prepared. The pH was adjusted using a 0.1N NaOH aqueous solution or a 0.1N dilute sulfuric acid solution. The pH-adjusted saccharification and fermentation broth was subjected to solid-liquid separation using the microfiltration membrane described in Step (2) of Example 1, and then filtered through a nanofiltration membrane or a reverse osmosis membrane, and the resulting xylose sugar solution was obtained. The component comparison of was carried out. The results are shown in Table 4 (nanofiltration membrane treatment) and Table 5 (reverse osmosis membrane treatment).

  In both cases of nanofiltration membranes and reverse osmosis membranes, it was found that organic acid components, particularly acetic acid, are concentrated when the pH is higher than pH 5.0. On the other hand, in the range of pH 1.0 to 5.0, it was found that the organic acid, particularly acetic acid, can be efficiently removed as a permeate, so that the concentration of acetic acid contained in the resulting xylose sugar solution can be kept low. This is because the permeation of the nanofiltration membrane and the reverse osmosis membrane is increased by increasing the non-dissociated acetic acid ratio by adjusting the pH of the sugar solution supplied to the nanofiltration membrane or the reverse osmosis membrane to a range of 1 to 5. It was inferred that the performance was improved.

(Example 3) Effect of average pore size of microfiltration membrane in solid-liquid separation In order to confirm the effect of average pore size of microfiltration membrane used for solid-liquid separation in step (3), various average pore sizes were selected. The components contained in the filtrate obtained by solid-liquid separation of the saccharification and fermentation broth were compared using the microfiltration membrane. As the separation membrane, an isopore manufactured by Millipore (average pore diameter: 0.1 μm, 0.2 μm, 0.4 μm, 0.6 μm, 0.8 μm, 1.2 μm) was used. The separation membrane is a microfiltration membrane having a very uniform pore size distribution because the pores are formed by electron beams with respect to polycarbonate. The aforementioned microfiltration membrane was set in a stirring cell (Model 8050), and microfiltration of the saccharification and fermentation broth 1 or saccharification and fermentation broth 2 obtained in step (2) of Example 1 was performed. In the microfiltration, a silicon tube having an inner diameter of 2 mm and an outer diameter of 4 mm was connected to a stirring cell, and filtration was performed at a constant flow rate. The results of analyzing the obtained filtrates by the methods described in Reference Examples 2 to 4 are shown in Table 6 (microfiltration membrane filtrate of saccharification and fermentation broth 1) and Table 7 (microfiltration membrane filtrate of saccharification and fermentation broth 2). .

  It has been found that both the saccharification and fermentation liquid 1 and the saccharification and fermentation liquid 2 can suppress the concentration of HMF and furfural in the filtrate to a low level by solid-liquid separation using a microfiltration membrane having an average pore size of 0.4 μm or less. The microorganism that does not assimilate xylose used in the saccharification and fermentation broth 1 and saccharification and fermentation broth 2 is a yeast of the genus Saccharomyces or Candida, and the cell size is said to be around 10 μm. On the other hand, the average pore size of the microfiltration membrane used in this example is 1.2 μm at most, and it is possible to sufficiently separate the microorganisms into solid and liquid even when any microfiltration membrane is used. Although considered, in this example, it was found that the HMF and furfural concentrations in the filtrate increase and decrease with an average pore diameter of 0.4 μm as a boundary. This phenomenon is a boundary between whether or not the fragments derived from microorganisms retaining HMF and furfural produced by solid-liquid separation of microorganisms that have taken up HMF and furfural into cells are separated by a microfiltration membrane. Was estimated to have an average pore diameter of 0.4 μm.

(Comparative example 1) When performing centrifugation as solid-liquid separation in step (3) Saccharification and fermentation broth 1 and saccharification and fermentation broth 2 (each 1 L) obtained in step (2) of Example 1 are used at 2500 G for 10 minutes. Centrifuged. The obtained supernatant was analyzed by the method described in Reference Examples 2-4. For comparison, Table 8 also shows the analysis results of the microfiltration membrane (filtrate) 1 and the microfiltration membrane (filtrate) 2 obtained in step (3) of Example 1.

  In solid-liquid separation by centrifugation, it was found that HMF and furfural components are contained in a larger amount in the supernatant after solid-liquid separation than in the case of solid-liquid separation by a microfiltration membrane. In solid-liquid separation by centrifugation, it was speculated that the fine particle components including HMF and furfural were not sufficiently removed.

  Next, 0.8 L of the obtained supernatant component was filtered through a nanofiltration membrane or a reverse osmosis membrane by the method described in Step (3) of Example 1. However, from the point of time when about 0.1 L of permeate was filtered, it became difficult to filter and a concentrated solution could not be obtained. This is presumably due to the fact that a large amount of microbe components derived from microorganisms or biomass remain, and the clogging of the nanofiltration membrane or the reverse osmosis membrane progressed rapidly. That is, it was found that at least solid-liquid separation with a microfiltration membrane is necessary from the viewpoint of performing nanofiltration membrane treatment or reverse osmosis membrane treatment.

(Example 4) Influence of fermentation by microorganisms that do not assimilate xylose in step (2) on solid-liquid separation by microfiltration membrane in step (3) Fermentation by microorganisms that do not assimilate xylose in step (2) In order to confirm the influence on the solid-liquid separation by the microfiltration membrane in the step (3), the saccharified solution (before adding the microorganism) prepared in the step (2) of Example 1, the saccharified fermentation solution 1 and the saccharified fermentation solution 2 The effect on the microfiltration treatment of selenium was investigated. In the small flat membrane filtration apparatus provided with the same separation membrane as the microfiltration membrane described in the step (3) of Example 1, the membrane flux is changed from 0.05 m ³ / m ² / day to 0.4 m ³ / m ² / day. The transmembrane pressure difference (kPa) of the microfiltration membrane was measured while changing to. The result is shown in FIG.

  It was confirmed that the transmembrane pressure difference due to the change in membrane flux of the saccharification and fermentation broth 1 and saccharification and fermentation broth 2 had almost the same tendency. On the other hand, in the saccharified liquid (before the addition of microorganisms), it was found that the transmembrane pressure difference rapidly increased at a lower membrane flux than in the saccharified and fermented liquid 1 and the saccharified and fermented liquid 2. This indicates that the saccharification liquid (before adding the microorganism) has poor filterability compared to the saccharification fermentation liquid 1 and the saccharification fermentation liquid 2. In other words, it was found that the filterability of the microfiltration membrane in the step (3) is improved by adding and fermenting a microorganism that does not assimilate xylose in the step (2) of the present invention. This result shows the decomposition (or adsorption) of unknown substances that cause clogging of not only aromatic and furan compounds such as HMF, furfural and vanillin but also other microfiltration membranes by fermentation of microorganisms that do not assimilate xylose. ) Suggests the results.

(Comparative example 2) When a microorganism that does not assimilate xylose is not added in step (2) No microorganism that does not assimilate xylose is added to the saccharified solution (before adding the microorganism) obtained in step (2) of Example 1 The solid-liquid separation by the microfiltration membrane and the nanofiltration membrane treatment were performed according to the procedure described in the step (3) of Example 1. Table 9 shows components of the obtained xylose sugar solution (comparative example). Table 9 also shows the components of the saccharified solution in Step (2) of Example 1 (before addition of microorganisms) and the components of Xylose Sugar Solution 1 in Step (3) of Example 1 for component comparison.

  In the xylose sugar solution (Comparative Example 2), since no microorganism that does not assimilate xylose was added, glucose was directly concentrated by the nanofiltration membrane. Moreover, compared with the xylose sugar liquid 1, the content of HMF, furfural, and vanillin increased in the xylose sugar liquid (Comparative Example 2).

(Example 5) Production example of a chemical by culturing microorganisms using xylose sugar solution as a carbon source (ethanol fermentation by Pichia stepitis)
As the xylose sugar solution, the xylose sugar solution 1 obtained in the step (3) of Example 1 was used as the carbon source of the microorganism. For comparison, the xylose sugar solution obtained in Comparative Example 2 (Comparative Example 2) and the saccharified solution obtained in Step (2) of Example 1 were used as a carbon source for microorganisms.

  As the microorganism, Pichia stipitis NRRL Y-7124 strain was used.

  The medium and ethanol fermentation conditions were carried out under the following conditions.

[Pre-culture]
5% xylose, 1% yeast extract (Bacto Yeast Extract / BD) and 2% polypeptone (Nippon Pharmaceutical) were dissolved in 50 mL distilled water in a 200 mL Erlenmeyer flask, and then autoclaved at 120 ° C. for 20 minutes. The resulting medium was inoculated with Pichia stititis NRRL Y-7124 grown on potato dextrose agar plates. The Erlenmeyer flask was incubated at 28 ° C. for 2 days. The obtained preculture liquid was used for ethanol fermentation mentioned later.

[Ethanol fermentation]
The xylose sugar solution 1 and the xylose sugar solution (Comparative Example 2) were diluted with distilled water so that the xylose concentration was 50 g / L. The saccharified solution was used as it was without adjusting the sugar concentration. Furthermore, xylose sugar solution 1, xylose sugar solution (Comparative Example 2), and saccharified solution were added to 1% yeast extract (Bacto Yeast Extract / BD) and 2% polypeptone (Nippon Pharmaceutical) (all final concentrations). Then, high-pressure sterilization was performed at 120 ° C. for 20 minutes. 5 mL of the preculture was added to the obtained medium, and ethanol fermentation was performed at 28 ° C.

  Ethanol accumulated in the culture broth was analyzed according to the method described in Reference Example 4. The transition of ethanol produced 0 to 120 hours after ethanol fermentation is shown in FIG. It was found that the saccharified solution hardly produced ethanol as a fermentation product. This was presumed to be because 1) glucose and xylose had low sugar concentrations and carbon sources were insufficient, and 2) there were many fermentation inhibitors such as HMF, furfural, vanillin, and acetic acid. On the other hand, in xylose sugar solution 1 and xylose sugar solution (Comparative Example 2), production and accumulation of ethanol as a fermentation product were confirmed. However, it was found that the xylose sugar solution 1 produced more ethanol and produced more fermentation product than the xylose sugar solution (Comparative Example 2). As shown in the analysis values of Table 9, the xylose sugar solution 1 and the xylose sugar solution (Comparative Example 2) have the same xylose sugar concentration. Rather, the xylose sugar solution (Comparative Example 2) is added to xylose. Although the glucose component is also included and the carbon source is abundant, the xylose sugar solution 1 is superior in fermentation performance when the xylose sugar solution 1 is used as the carbon source. The xylose sugar solution 1 has HMF, furfural, vanillin, etc. This is probably because the weight of the fermentation inhibitory substance is extremely small.

  Therefore, in Example 5, it became clear that a chemical could be produced by culturing microorganisms using the xylose sugar solution obtained by the present invention as a carbon source. Further, it has been found that the xylose sugar solution obtained by the present invention is superior as a carbon source for microorganisms than the xylose sugar solution obtained by the prior art.

  The xylose sugar solution obtained by the present invention can be widely used as a fermentation raw material for microorganisms, and as an industrial raw material.

Claims (4)

A method for producing a xylose sugar solution from xylan-containing biomass,
(1) Hydrothermally treating xylan-containing biomass to obtain a hydrothermally treated product,
(2) adding a saccharifying enzyme to the hydrothermally treated product, further adding a microorganism selected from the group of Saccharomyces and Candida as a microorganism that does not assimilate xylose, and obtaining a saccharification and fermentation broth;
(3) The saccharification and fermentation broth was subjected to solid-liquid separation using at least a microfiltration membrane, and the obtained filtrate was filtered through a nanofiltration membrane and / or a reverse osmosis membrane, and concentrated and purified from a non-permeate. A method for producing a xylose sugar solution comprising the step of obtaining a xylose solution.   The method for producing a xylose sugar solution according to claim 1, wherein the hydrothermally treated product of step (1) is a hydrothermal treatment liquid and / or hydrothermally treated biomass obtained by treating xylan-containing biomass with high-pressure hot water at 180 to 300 ° C. The manufacturing method of the xylose sugar liquid of Claim 1 or 2 which adjusts the saccharification fermentation liquid of a process (3) to the range of pH 1-5. The method for producing a xylose sugar solution according to any one of claims 1 to 3 , wherein an average pore diameter of the microfiltration membrane in the step (3) is 0.4 µm or less.

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