WO2013111762A1 - バイオマスの糖化方法 - Google Patents
バイオマスの糖化方法 Download PDFInfo
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- WO2013111762A1 WO2013111762A1 PCT/JP2013/051247 JP2013051247W WO2013111762A1 WO 2013111762 A1 WO2013111762 A1 WO 2013111762A1 JP 2013051247 W JP2013051247 W JP 2013051247W WO 2013111762 A1 WO2013111762 A1 WO 2013111762A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K13/00—Sugars not otherwise provided for in this class
- C13K13/002—Xylose
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K13/00—Sugars not otherwise provided for in this class
- C13K13/007—Separation of sugars provided for in subclass C13K
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a biomass saccharification method, and more particularly to a method of saccharifying lignocellulosic biomass with an enzyme.
- the technology for saccharifying lignocellulosic biomass and obtaining monosaccharides as fermentation raw materials is a very important technology from the viewpoint of non-edible biomass resources and energy that do not compete with food.
- Methods for saccharifying lignocellulosic biomass are broadly classified into acid saccharification methods in which hydrolysis is performed using an acid such as sulfuric acid, and enzyme saccharification methods in which hydrolysis is performed using an enzyme.
- the acid saccharification method has an advantage that the reaction rate is high, but has a problem that an acid-resistant reactor is required and a step of neutralizing and recovering the acid after use is necessary.
- the enzymatic saccharification method has an advantage that the utility, equipment cost, or reaction selectivity is high as compared with the acid saccharification method because the decomposition reaction proceeds under relatively mild reaction conditions.
- the alkali pretreatment method pretreats biomass using an alkaline compound such as NaOH (Patent Documents 1 to 3), and breaks down the structure of biomass by effectively decomposing lignin and the like. It enhances the action.
- Patent Documents 1 to 3 an alkaline compound such as NaOH
- it is superior in that it can be pretreated under relatively mild conditions and can be applied to biomass having a high lignin content that is not easily saccharified.
- Another advantage is that the alkali metal corrosivity is low.
- the subject of the alkali pretreatment method is the cost of the alkali used, and it is required to achieve a high sugar yield with a smaller amount of alkali.
- reduction of water usage in pretreatment, further relaxation of processing conditions, and shortening of time are also required.
- the degradation product of biomass produced by alkali pretreatment may cause fermentation inhibition, and is generally removed by washing with water and then subjected to enzymatic saccharification. It is practically very important to obtain a saccharified solution that does not cause fermentation inhibition while reducing this washing water.
- Non-Patent Documents 4 to 6, Non-Patent Documents 1 and 2 an effective measure for reducing enzyme costs is the recovery and reuse of saccharifying enzymes, and various methods are known (Patent Documents 4 to 6, Non-Patent Documents 1 and 2).
- saccharifying enzymes such as cellulase exhibit high adsorptivity to carbohydrates and lignin, they are adsorbed to biomass residues that are difficult to decompose after the saccharification reaction. This adsorption phenomenon to the residue makes it difficult to recover and reuse the enzyme.
- the difference in the pretreatment method results in the enzyme adsorption, but there has been a demand for the development of a pretreatment method that can reduce the adsorption of the enzyme to the residue more effectively and at a low cost.
- the present invention can be applied to lignocellulosic biomass having a high lignin content, reduces the amount of alkali and water used in the pretreatment process, improves the sugar yield in the saccharification process, reduces the reaction time, and reduces the enzyme to the biomass residue. It is an object of the present invention to provide a method for saccharification of lignocellulosic biomass that can reduce the amount of adsorption and improve the enzyme recovery rate. It is another object of the present invention to provide a saccharification method capable of obtaining a saccharified solution having excellent fermentation characteristics while reducing the load of removing decomposition products generated in the pretreatment step.
- the present invention relates to the following inventions in order to solve the above problems.
- the solid-liquid ratio calculated by the following formula (I) in the pretreatment step is 2 to 20 for the mixture before solid-liquid separation, and 1 to 6 for the mixture after solid-liquid separation.
- Formula (I): Solid-liquid ratio total mass of all liquid components in the mixture / solid mass of lignocellulosic biomass in the mixture [3]
- the heat treatment is performed at 100 to 200 ° C. [1] Or the saccharification method as described in [2].
- the saccharification step is performed in the presence of a pretreated decomposition product of the solubilized lignocellulosic biomass produced in the pretreatment step, according to any one of the above [1] to [3] Saccharification method.
- a pretreated decomposition product of the solubilized lignocellulosic biomass produced in the pretreatment step according to any one of the above [1] to [3] Saccharification method.
- the saccharification method according to any one of [1] to [4], wherein the residual rate of the pretreated decomposition product calculated by the following formula (II) is 2 to 20% by mass.
- Residual rate of pretreated degradation product solid content mass of remaining pretreatment degradation product / solid content mass of lignocellulosic biomass
- the proportion of C5 sugar in the saccharified solution obtained in the saccharification step is based on the total sugar components
- the present invention can be applied to lignocellulosic biomass having a high lignin content, reducing the amount of alkali and water used in the pretreatment process, improving the sugar yield in the saccharification process, reducing the reaction time, and after enzymatic saccharification It is possible to provide a method for saccharification of lignocellulosic biomass that can reduce the amount of enzyme adsorbed and improve the enzyme recovery rate. Furthermore, in the present invention, the amount of enzyme used can be reduced and the enzyme cost can be greatly reduced by performing the saccharification step by recycling the collected enzyme. Moreover, in this invention, it is also possible to obtain the saccharified liquid excellent in the fermentation characteristic, reducing the load of the decomposition product produced
- FIG. 1 shows the measurement results of sugar yield and enzyme recovery of Examples 1 and 2 and Comparative Example 1.
- the present invention provides a method for saccharification of lignocellulosic biomass, that is, a method for producing saccharides (glucose, xylose, arabinose, etc.) obtained by saccharifying lignocellulosic biomass.
- the saccharification method of the present invention includes (1) a pretreatment step of preparing a mixture obtained by impregnating lignocellulosic biomass with an alkaline aqueous solution, then performing solid-liquid separation to remove a portion of the alkaline aqueous solution, and performing a heat treatment; (2) What is necessary is just to include the saccharification process which decomposes
- the raw material of the saccharification method of the present invention is not particularly limited as long as it contains lignocellulosic biomass, and the lignolin content may be high.
- Lignocellulosic biomass (hereinafter simply referred to as biomass) is mainly composed of cellulose, hemicellulose, and lignin, and includes woody plants, herbaceous plants, processed products thereof, wastes thereof, and the like. Specifically, for example, wood, thinned wood, residual lumber, building waste, bark, fruit bunches, fruit shells, foliage, straw, bagasse, waste paper and the like can be mentioned.
- wood such as oil palm, date palm, sago palm, coconut palm (stem, foliage, empty fruit bunch, fruit fiber), sugar cane (bagasse, foliage), corn (cob, foliage), eucalyptus, poplar, cedar, etc. (Bark, xylem), rice straw, straw, switchgrass, napiergrass, Eliansus, Miscanthus, Susuki. More preferred are empty fruit bunches of palm, sugarcane bagasse, corn cob, rice straw, straw, eucalyptus and cedar, and more preferred are empty fruit bunches of oil palm. Oil palm empty fruit bunches are biomass discharged in the palm oil extraction process and are abundant in Southeast Asia.
- the empty fruit bunch of oil palm has a high lignin content and is obtained in a high water content state, so its use is limited.
- the method of the present invention is particularly effective for such biomass feedstock.
- the size, shape, and the like of the biomass are not particularly limited, but those made into a powder, chip, or strip by cutting, pulverization, or the like, or those made into fibers by defibration are preferable.
- the size of the biomass raw material is preferably about 0.1 to 30 cm on average, more preferably about 1 to 10 cm as the length of the longest side. By setting the size within this range, high enzyme saccharification, solid-liquid separation, transportability and the like can be obtained.
- the moisture content (moisture content) of the biomass is not particularly limited, but the moisture content is preferably 0 to 90%, more preferably 30 to 90%, still more preferably 40 to 80%, and particularly preferably. 50 to 80%.
- the lignin content is, for example, 10% or more, preferably 20% or more, based on the solid content (absolute dryness).
- “%” means mass%, and represents mass% unless otherwise specified.
- a pretreatment step is performed.
- the pretreatment step after preparing a mixture obtained by impregnating biomass with an alkaline aqueous solution, solid-liquid separation is performed to remove a part of the alkaline aqueous solution, and heat treatment is performed.
- a high pretreatment effect can be obtained even with a smaller amount of alkali and water. That is, in the saccharification step, the enzyme contacts the cellulose and hemicellulose easily, the efficiency of the enzyme reaction is improved, and the sugar yield is improved. Moreover, enzyme adsorption to the biomass residue after the saccharification reaction is reduced, and enzyme recovery from the residue is facilitated.
- the technical feature of the pretreatment method of the present invention is that when the alkali is impregnated into the biomass, it is performed at a relatively high solid-liquid ratio (ratio of biomass solid to liquid), and solid-liquid separation is performed to reduce the solid-liquid ratio. The subsequent heat treatment is performed at a low solid-liquid ratio.
- the alkali can be impregnated into the biomass at high speed and uniformly, and the biomass can be efficiently applied to the biomass.
- the present invention can also be suitably applied to biomass having a high water content or lignin content.
- an alkaline aqueous solution is first prepared.
- the alkaline compound used in the alkaline aqueous solution is selected from the group consisting of hydroxides, oxides, sulfides, carbonates and bicarbonates of at least one metal selected from the group consisting of sodium, calcium, potassium and magnesium. At least one compound or the like can be used. Ammonia can also be used. Sodium hydroxide, sodium sulfide, sodium carbonate, calcium hydroxide, potassium hydroxide and potassium carbonate are preferred, and sodium hydroxide, calcium hydroxide and potassium hydroxide are more preferred.
- One alkali compound may be used, or a mixture of plural kinds may be used.
- alkali compounds are dissolved in water and used as an alkaline aqueous solution.
- the alkali compound concentration in the aqueous alkali solution is preferably from 0.1 to 30%, more preferably from 0.5 to 20%, particularly preferably from 1 to 10%.
- the pH of the alkaline aqueous solution is preferably pH 11 to 15, more preferably pH 12 to 14.5, and particularly preferably pH 12.5 to 14.
- anthraquinones such as anthraquinone and sulfonated anthraquinone may be added to the alkaline aqueous solution.
- the amount of anthraquinones added is not particularly limited as long as it does not interfere with the effects of the present invention.
- a step of bringing the alkaline aqueous solution into contact with biomass and impregnating the biomass with an alkaline compound is performed (impregnation step). Specifically, first, a mixture in which biomass and an aqueous alkali solution are mixed is prepared, treated under various conditions, and impregnated with the aqueous alkali solution. In the impregnation step, it is important to impregnate the alkali quickly and uniformly into the biomass. Therefore, it is preferable to prepare the mixture at a relatively high solid-liquid ratio.
- the solid-liquid ratio of the mixture in the impregnation step is preferably 1 to 30, and more preferably 1 to 20 2-20 are more preferable, and 2-10 are particularly preferable.
- total liquid components refers to the alkaline aqueous solution to be contacted, the moisture contained in the raw material biomass, and all other liquids Means the sum of the liquid components in the combined mixture.
- the “solid mass of biomass” means the solid mass of raw material biomass that does not contain liquid components such as moisture.
- the alkaline aqueous solution that has come into contact with the biomass is absorbed and impregnated inside the biomass, but if the alkaline aqueous solution (or all liquid components) exceeds the maximum (saturated) water content of the biomass, the biomass cannot fully absorb the alkaline aqueous solution.
- the aqueous alkali solution also exists in the voids (the space between the biomasses).
- the impregnation step is preferably performed under such conditions that the alkaline aqueous solution is also present in the voids.
- the concentration of the aqueous alkali solution in contact with the biomass changes when mixed with moisture contained in the biomass, but the alkali concentration in the mixture is in the range of 0.1 to 30% as the alkali compound concentration with respect to the total liquid components.
- 0.5 to 20% is more preferable, and 1 to 10% is particularly preferable.
- the pH of all liquid components in the mixture is preferably pH 11 to 15, more preferably pH 12 to 14.5, and particularly preferably pH 12.5 to 14.
- the concentration and amount of the alkaline aqueous solution to be contacted are preferably set as appropriate in consideration of the amount of water contained in the biomass, the required alkali concentration, and the like.
- the treatment temperature in the impregnation step is preferably 20 to 100 ° C., more preferably 20 to 70 ° C., and particularly preferably 20 to 50 ° C.
- the impregnation step may be performed under normal pressure, but may be performed under reduced pressure conditions or pressurized conditions. By performing these pressure operations, the alkali impregnation rate can be increased. In the case of a pressurizing condition, it is preferably performed at 0.01 to 2 MPaG (gauge pressure), more preferably 0.05 to 0.5 MPaG.
- the impregnation time is preferably 0.1 to 10 hours, more preferably 0.1 to 3 hours, and particularly preferably 0.1 to 1 hour.
- the impregnation step can be carried out either batchwise or continuously.
- the impregnation rate may be increased by performing mixing, stirring, liquid circulation, or the like.
- the “partial alkaline aqueous solution” means an alkaline aqueous solution that can be removed by solid-liquid separation, and is a part of the alkaline aqueous solution in the mixture prepared in the impregnation step.
- the alkaline aqueous solution is considered to exist inside and outside (voids) of the biomass.
- Some alkaline aqueous solutions that are removed by solid-liquid separation are mainly alkaline aqueous solutions that exist in the voids of the biomass, but they also vary depending on the solid-liquid separation conditions, and include the alkaline aqueous solution that exists inside the biomass. May be.
- the purpose of the solid-liquid separation is to mainly remove the alkaline aqueous solution present in the voids of the biomass and lower the solid-liquid ratio. By reducing the solid-liquid ratio, the reaction field can be limited, and the action efficiency of alkali on solid biomass can be dramatically increased.
- alkali-impregnated biomass For solid-liquid separation, filtration, centrifugation, centrifugal filtration, cyclone, filter press, screw press, decanter and the like can be used.
- the mixture obtained after solid-liquid separation (hereinafter referred to as alkali-impregnated biomass) is subjected to the next heat treatment step.
- a part of the alkaline aqueous solution removed by solid-liquid separation is preferably reused in the impregnation step.
- Some alkaline aqueous solutions removed by solid-liquid separation contain a trace amount of biomass-derived components, and can be expected to improve the alkali impregnation rate and reduce alkali. You may use it, after supplementing the reduced alkali compound or the aqueous alkali solution as needed.
- the amount of alkali compound (alkaline impregnation amount) present in all the liquid components is preferably 0.1 to 30% with respect to the biomass solid mass, and preferably 1 to 20%. % Is more preferable, and 2 to 15% is particularly preferable.
- the heat treatment temperature is preferably 20 to 250 ° C, more preferably 100 to 200 ° C, and particularly preferably 150 to 200 ° C.
- the heat treatment time is preferably 0.1 to 100 hours, more preferably 0.1 to 24 hours, and particularly preferably 0.1 to 1 hour. By performing the heat treatment in this temperature and time range, the sugar yield and the enzyme recovery rate are improved.
- the atmosphere in the gas phase during the heat treatment is not particularly limited, such as oxygen gas, nitrogen gas, oxygen / nitrogen mixed gas, and air.
- the oxygen concentration is preferably 1 to 100% by volume, more preferably 10 to 95% by volume, and particularly preferably 15 to 80% by volume.
- One of the preferred forms is a method using air that can be used at low cost. Further, when heat treatment is performed in the presence of oxygen, oxygen is consumed with time. Therefore, it is preferable to perform the heat treatment while adding oxygen (while maintaining the oxygen concentration).
- the pressure (gauge pressure) at the time of heat processing is not specifically limited, 5 MPaG or less is preferable, 3 MPaG or less is more preferable, and 1 MPaG or less is especially preferable.
- the alkaline aqueous solution in the voids is removed, the surface area of the biomass is increased, and the gas in the gas phase can be efficiently taken in. Therefore, pretreatment in the presence of oxygen is a preferred method of implementing the present invention.
- Biomass mainly lignin
- Biomass is decomposed and solubilized by the heat treatment to produce a compound having a phenolic hydroxyl group or a carboxyl group.
- the pH of all liquid components in the mixture after the heat treatment is preferably pH 6 to 14, more preferably pH 7 to 13, and particularly preferably pH 8 to 12. By setting it as this pH range, the load of the following removal process can be reduced, utilizing an impregnated alkali efficiently.
- pH of all the liquid components after heat processing can be estimated by the above-mentioned method.
- the heat treatment conditions such as temperature, gas phase atmosphere, and pressure may be changed during the heat treatment.
- the heat treatment without supplying (restricting) oxygen is performed first, and the heat treatment supplying oxygen is performed later.
- the heat treatment conditions in each stage of this method are the same as the above conditions, and can be carried out in combination.
- the biomass after the heat treatment may be subjected to the next saccharification step as it is, but a step of removing a part of the solubilized biomass decomposition product (hereinafter referred to as pretreatment decomposition product) generated in the pretreatment step (removal step) ) Is preferably used for the saccharification step (the biomass after the removal step is hereinafter referred to as pretreated biomass).
- the pretreatment decomposition product is a soluble solid produced by decomposition of biomass by alkali, and is a decomposition product mainly composed of various components such as decomposed lignin and organic acid (such as acetic acid). Also contains an alkaline component.
- washing of biomass with a washing solvent such as water, solid-liquid separation by pressing, centrifugation, or the like can be raised, and a method of washing with water is preferable.
- a washing solvent such as water, solid-liquid separation by pressing, centrifugation, or the like
- organic solvents such as alcohol and ketone, or acids for pH adjustment may be added.
- the amount of the washing solvent containing water is preferably 0.1 to 100 times, more preferably 0.5 to 30 times the mass of the alkali-impregnated biomass after the heat treatment. It is particularly preferable that the amount be ⁇ 10 times.
- a washing solvent is added to the biomass after heat treatment to elute the pretreated decomposition product, and then solid-liquid separation is performed to separate the pretreated biomass and the washing solution (containing the pretreatment decomposition product).
- the washing operation can be performed under the same conditions as in the impregnation step.
- the washing operation may be performed once or a plurality of times.
- a batch, semi-batch, or continuous method can be used, but a semi-batch method or a continuous method is preferred to increase efficiency.
- cleaning since the structure of biomass may become firm when dried, it is preferable to use for the next saccharification process with a water-containing state, without drying.
- the method of removing by solid-liquid separation such as compression or centrifugation is advantageous in that the amount of water used can be reduced.
- a part of the pretreated decomposition product can be removed. You may combine washing
- the pretreated decomposition product is present in a high concentration in the saccharified solution, it may adversely affect the fermentation. Therefore, it is preferable to remove a part in the removing step.
- the present inventors have found that the pretreated degradation product reduces nonspecific adsorption of the enzyme to biomass.
- the saccharification step and / or the enzyme recovery step in the presence of the pretreatment degradation product, merits such as an increase in sugar yield, a reduction in the amount of enzyme, and an increase in the enzyme recovery rate can be obtained. Was revealed.
- the residual rate of the pretreated decomposition product in the pretreated biomass is preferably 1 to 30%, more preferably 2 to 20%, and particularly preferably 5 to 20%.
- the solid content mass of the remaining pretreatment decomposition product is obtained by sampling a part of the pretreatment biomass and thoroughly washing it (removing the pretreatment decomposition product sufficiently), and the amount of solid content in the resulting cleaning liquid (i.e. This can be determined by measuring the amount of processed decomposition product.
- the solid content mass of lignocellulosic biomass is the solid content mass of the pretreatment biomass which does not contain a pretreatment decomposition product, and is the solid content mass of the pretreatment biomass after fully washing
- the pretreated biomass obtained in the pretreatment step is decomposed with an enzyme to obtain a saccharified solution. That is, a mixture (hereinafter referred to as a reaction mixture) in which an enzyme, water and a pH adjuster as necessary are added to pretreated biomass is prepared, and a saccharification reaction is performed.
- a reaction mixture in which an enzyme, water and a pH adjuster as necessary are added to pretreated biomass is prepared, and a saccharification reaction is performed.
- a reaction mixture in which an enzyme, water and a pH adjuster as necessary are added to pretreated biomass is prepared, and a saccharification reaction is performed.
- a pH adjusting agent may be added during the removing step and the pH adjustment may be performed first. It is preferable to add the enzyme after adjusting the pH.
- the enzyme to be used may be an enzyme that can hydrolyze cellulose into a monosaccharide (glucose) or an enzyme that can hydrolyze hemicellulose into a monosaccharide (xylose, mannose, arabinose, etc.).
- Such an enzyme is generally called cellulase or hemicellulase, and is composed of a plurality of enzymes.
- the enzyme used for the saccharification method of this invention should just contain cellulase or hemicellulase, in order to improve saccharification efficiency, it is preferable to use what contains both.
- the pretreatment method of the present invention is also characterized in that since the alkali pretreatment proceeds efficiently, the hemicellulose is hardly solubilized and the hemicellulose content in the pretreated biomass is high. Therefore, a method of simultaneously decomposing cellulose and hemicellulose using an enzyme including cellulase and hemicellulase is a preferred embodiment. At the same time, enzymatic degradation can provide merits such as shortening the reaction time or increasing the sugar concentration. In a pretreatment method in which hemicellulose is solubilized (such as acid treatment, hydrothermal treatment, or alkali treatment under severe conditions), it is necessary to decompose cellulose and hemicellulose separately.
- the cellulase preferably contains cellobiohydrolase, ⁇ -glucanase and ⁇ -glucosidase.
- the hemicellulase preferably contains xylanase and ⁇ -xylosidase.
- Other hemicellulases include acetyl xylan esterase, ⁇ -arabinofuranosidase, mannanase, ⁇ -galactosidase, xyloglucanase, pectinase, pectinase and the like. Further, it may contain other enzymes involved in plant cell wall degradation, such as ferulic acid esterase, coumaric acid esterase, and protease. Whether or not these enzymes are contained can be confirmed by examining the enzyme activity using the substrate of each enzyme.
- the origin of the enzyme is not particularly limited, but includes the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Humicola, the genus Bacils, and the like.
- An enzyme derived from genus Trichoderma, Acremonium, and Aspergillus is preferable, and an enzyme derived from Trichoderma is more preferable.
- enzymes are commercially available and can be suitably used in the production method of the present invention.
- Commercially available enzyme preparations include Novozymes' Celic series (C-Tech, H-Tech, etc.), Novozymes 188, Cellcrust, Pulpzyme, Genencor's Accelase series (Trio, Duet, etc.), Multifect series Meiji Seika's Mecellase, Yakult Onozuka, Amano Enzyme's Cellulase (A, T), and the like.
- the celic series and the accelerator series are preferred.
- These enzyme preparations contain cellobiohydrolase, ⁇ -glucanase, ⁇ -glucosidase, xylanase, ⁇ -xylosidase, and may be used alone or in combination in consideration of the composition of the raw material biomass and the enzyme activity contained. it can. It is preferable to use a combination of an enzyme preparation having a high cellulase activity and an enzyme preparation having a high hemicellulase activity, for example, using a combination of Celic C-Tech series (cellulase is the main component) and Celic H-Tech series (contains hemicellulase is the main component). Is preferred.
- enzyme adsorbed on the undecomposed residue of the biomass by alkali treatment.
- an enzyme having high alkali stability and thermal stability it is possible to use an enzyme having high alkali stability and thermal stability.
- Enzymes may be modified chemically or genetically engineered (protein engineering). The modification can increase the enzyme stability, reduce the adsorptivity to the residue, and increase the enzyme recovery efficiency, so that it can be suitably used in the present invention.
- the amount of the enzyme used is not particularly limited, but is preferably 0.01 to 10%, more preferably 0. 0% as the solid mass (protein mass) of the enzyme active ingredient with respect to the solid mass of the pretreated biomass. Add 05-5%.
- the amount of water added is not particularly limited, and it is not necessary to add water if the pretreated biomass contains a sufficient amount of water. Preferably, it is added in an amount of 0 to 20 times, more preferably 0 to 10 times the solid content mass of the pretreated biomass. Further, the solid content concentration of the pretreated biomass in the reaction mixture is preferably 1 to 50%, more preferably 3 to 30%, and particularly preferably 5 to 25%.
- an acid or an alkali can be appropriately selected and used.
- the alkali treatment is performed as the pretreatment step, the pretreated biomass is alkaline, so the acid is adjusted to a pH suitable for saccharification.
- a sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, an acetic acid, a citric acid, a succinic acid, a carbon dioxide etc. are mentioned, Preferably they are a sulfuric acid, hydrochloric acid, an acetic acid, and a carbon dioxide.
- the reaction conditions in the saccharification step are not particularly limited as long as hydrolysis by an enzyme proceeds.
- the reaction temperature is usually 20 to 80 ° C., preferably 30 to 60 ° C., more preferably 40 to 55 ° C.
- the reaction time is usually 1 to 300 hours, preferably 10 to 150 hours, more preferably 20 to 100 hours.
- the reaction pH may be set according to the optimum pH of the enzyme, but is usually pH 3 to 7, preferably pH 4 to 6, more preferably pH 4.5 to 5.5.
- the pH adjusting agent may be added or a buffer component may be added. Specifically, various organic acids can be used as the buffer component, and acetic acid, citric acid, and succinic acid are preferable.
- the saccharification step may be performed in the presence of various compounds.
- such compounds include proteins, surfactants, lignin degradation products, etc., but these compounds have an effect of reducing nonspecific adsorption of enzymes to biomass. Benefits such as improved recovery and reduced enzyme usage are obtained.
- it is a lignin degradation product, More preferably, it is a pretreatment degradation product produced
- the method for allowing the pretreatment decomposition product to exist in the saccharification step may be performed by using the pretreated biomass prepared as described above (with the pretreatment decomposition product remaining), but the pretreatment decomposition product is added separately. May be.
- the concentration of the pretreated decomposition product is preferably 1 to 30%, more preferably 2 to 20%, and particularly preferably 5 to 20% with respect to the solid mass of the pretreated biomass.
- the method for the saccharification reaction is not particularly limited, and the saccharification reaction may be performed while stirring or liquid circulation of the reaction mixture or in a stationary state. In order to accelerate the saccharification reaction, stirring or liquid circulation is preferable.
- a reaction mixture containing a saccharified solution is obtained.
- This reaction mixture is a mixture of a saccharified solution (a liquid containing a soluble low-molecular saccharide produced by hydrolysis of biomass and a free enzyme) and a residue (an undegraded biomass and a solid containing an adsorbing enzyme).
- the saccharified solution may be used in the form of a reaction mixture (as a mixture with the residue) or may be used after being separated from the residue by a method such as solid-liquid separation (in this case, enzyme recovery described later). Considered part of the process). It is one of the preferred embodiments of the present invention that the saccharifying enzyme is adsorbed on the residue, and the enzyme adsorbed on the residue is reused.
- the amount of enzyme used can be reduced.
- a method of reusing the adsorbing enzyme (a) a method of reusing undegraded biomass containing the adsorbing enzyme for the reaction as it is, and (b) a method of desorbing and recovering the adsorbing enzyme from the residue and reusing it, etc. Can be given.
- a specific method of (a) for example, a method in which a saccharified solution and undecomposed biomass (adsorbed enzyme) are separated during or after the saccharification reaction, and the undegraded biomass is used for the next saccharification reaction. can give.
- the saccharification reaction (separation from undegraded biomass) and the addition of fresh biomass raw materials are performed sequentially or continuously, and the saccharification reaction is performed using the adsorbed enzyme continuously.
- the method is a preferred method. A specific method of (b) will be described later.
- an enzyme recovery step for recovering an enzyme after the saccharification step is completed.
- enzyme recovery enzyme adsorption to biomass becomes a problem, but according to the pretreatment method of the present invention, enzyme recovery can be reduced and enzyme recovery can be performed more efficiently.
- the reaction mixture obtained in the saccharification step is subjected to solid-liquid separation and separated into a saccharification solution and a residue.
- the solid-liquid separation method is not particularly limited, and for example, filtration, centrifugation, centrifugal filtration, cyclone, filter press, screw press, decanter and the like can be used.
- Free enzyme non-adsorbed enzyme
- the adsorbed enzyme can be recovered from the residue.
- the recovered adsorbed enzyme may be reused as it is, but the enzyme may be recovered by desorption from the residue.
- the method of recovering the enzyme from the residue is not particularly limited, but the method of washing the residue using water, the method of recovering the enzyme using acid, the method of recovering the enzyme using alkali (alkali treatment), etc. Is mentioned.
- an alkali treatment is included. Since the adsorbed enzyme is desorbed by alkali treatment, a high enzyme recovery rate can be realized.
- the method for the alkali treatment is not particularly limited as long as it is a method in which an alkali is added to act on the residue.
- the alkali treatment may be performed before or after solid-liquid separation of the reaction mixture.
- (A) a step of adding an alkali to the reaction mixture before solid-liquid separation to perform an alkali treatment
- a process including a combination of both In any of the methods, it is important to adjust the pH of the alkali treatment liquid (the residue and added alkali, the whole treatment liquid containing the added water, or the whole reaction mixture) to a predetermined range.
- the adsorbed / desorbed state of the enzyme and the residue changes depending on the pH of the treatment solution, and the desorbing enzyme increases as the pH is increased.
- the enzyme is deactivated by alkali at high pH.
- the pH of the alkali treatment solution after the alkali addition is preferably pH 6 to 11, more preferably pH 7 to 10, and particularly preferably pH 7.5 to 9.5.
- the same alkali compounds as used in the pretreatment step can be used, and preferred compounds are also the same.
- the alkali compound is preferably added as an aqueous solution, and the concentration and amount of the alkali compound to be added may be those that achieve the above pH range. However, if the concentration is too low, the amount of addition increases and the recovered enzyme concentration also decreases. To do.
- the alkali concentration of the aqueous alkali solution to be added is preferably 0.01 to 10%, more preferably 0.1 to 5%.
- an additive may be added to promote desorption of the enzyme.
- additives include proteins, surfactants, lignin degradation products, and the like, preferably lignin degradation products.
- the amount of the pretreatment decomposition product to be added is 1 to 30%, more preferably 2 to 20%, particularly preferably 5 to 20%, based on the solid content mass of the residue.
- stirring, heating, or the like may be performed to promote enzyme desorption from the residue.
- the temperature for the alkali treatment is preferably 5 to 60 ° C, more preferably 10 to 40 ° C.
- the treatment time is preferably 0.1 to 10 hours, more preferably 0.1 to 1 hour.
- the solid-liquid separation method in this case is not particularly limited, and for example, filtration, centrifugation, centrifugal filtration, cyclone, filter press, decanter, and the like can be used.
- the steps from alkali treatment to solid-liquid separation may be performed batchwise or continuously.
- the step (A) is preferably carried out batchwise.
- the step (B) may be a batch type or a continuous type, but a continuous type is preferred.
- the alkali treatment (and recovery of the alkali treatment liquid) may be performed only once or a plurality of times.
- the pH of the alkali treatment solution (enzyme recovery solution) at the end of the gradual increase in pH is preferably pH 6 to 11, more preferably pH 7 to 10, and pH 7.5 It is particularly preferable that the ratio is ⁇ 9.5.
- Saccharifying enzyme is composed of a plurality of enzymes, and it has been confirmed that the adsorption / desorption characteristics of each enzyme are different.
- the method of performing the alkali treatment step (B) so that the pH of the alkali treatment solution gradually increases is that saccharification of a plurality of enzyme mixtures (for example, a mixture of a plurality of cellulases and a plurality of hemicellulases) having different pH and stability that are easy to desorb. Even when used as an enzyme, it is very useful in that a high enzyme recovery rate can be realized.
- the pH of the enzyme recovery solution obtained in the enzyme recovery step is preferably pH 3 to 7, and more preferably pH 4 to 6.
- the obtained enzyme recovery solution can be reused in the saccharification step. If necessary, the enzyme recovery solution is concentrated by a method such as ultrafiltration and reused. Further, since the saccharified solution obtained by solid-liquid separation of the reaction mixture also contains free enzyme (and saccharide), it is preferable to separate the enzyme and saccharide by a method such as ultrafiltration and reuse the enzyme. Further, the enzyme can be easily reused by utilizing the adsorption phenomenon to the biomass raw material. That is, a saccharified solution or an enzyme recovery solution is brought into contact with a fresh biomass raw material. Since only the enzyme is adsorbed to the biomass material, the saccharide and the enzyme (the state adsorbed to the biomass material) can be separated by solid-liquid separation.
- the enzyme recovery liquid containing saccharides may be used for fermentation as it is.
- a part of fresh enzyme may be added.
- the enzyme to be added may be the same as the enzyme composition used for the first time.
- ⁇ -glucosidase is likely to be adsorbed to the reaction residue, and the recovery rate may be lower than that of other enzymes. In such a case, it is preferable to add an enzyme solution containing a large amount of ⁇ -glucosidase.
- the products obtained in the present invention are low-molecular sugars, pretreated decomposition products, and undecomposed residues.
- the obtained saccharide include monosaccharides, disaccharides, and oligosaccharides. Specifically, glucose, mannose, galactose, xylose, arabinose, glucuronic acid, galacturonic acid, cellobiose, xylobiose, cellooligosaccharide, xylooligosaccharide, etc. is there.
- Disaccharides and oligosaccharides may be used after being saccharified using an enzyme or the like.
- the use of the obtained saccharide is not particularly limited, it can be suitably used for fermentation raw materials, chemical raw materials, feed, fertilizers and the like.
- ethanol 1-butanol, isobutanol, 2-propanol, lactic acid, succinic acid, acetic acid, 3-hydroxypropionic acid, pyruvic acid, citric acid, acrylic acid, itaconic acid, fumaric acid, various It can be suitably used for fermentative production of chemicals such as amino acids, isoprene and 1,3-propanediol.
- the pretreatment decomposition product generated in the pretreatment process is a decomposition product of lignin generated by alkaline decomposition of biomass, and can be used as an additive in a saccharification process or as a chemical product of a lignin decomposition product. is there.
- the cleaning liquid or the like obtained in the removing step also contains an alkali used in the pretreatment step, and the alkali may be recovered and reused.
- methods generally known in the pulp manufacturing process can be used (black liquor concentration, combustion, ash dissolution, causticization).
- the residue can be used as biomass fuel for steam and power production.
- the apparatus used in each step is not particularly limited, but the reactor used in the pretreatment step and the saccharification step may be, for example, a batch type, continuous type, or semi-continuous type device.
- a screw feeder type continuous reaction apparatus it is preferable to use a screw feeder type continuous reaction apparatus. In this case, at the entrance of the screw feeder, it is possible to simultaneously raise the pressure while charging a reactor while partially removing the alkaline aqueous solution by solid-liquid separation.
- the saccharification step a form in which the raw material biomass is filled in the reactor and the saccharification reaction is performed by circulating the saccharified solution while performing solid-liquid separation is preferable. It is also possible to perform the pretreatment step, removal step, saccharification step, and enzyme recovery step in one reactor (one-pot reaction). A filter press, screw press, centrifugal separation, centrifugal filtration, cyclone, decanter, etc. can be used as the solid-liquid separation device.
- the same apparatus (reactor) as in the saccharification step can be used, but preferably an apparatus capable of continuously adding an aqueous alkaline solution and extracting the enzyme recovery solution. You may use the apparatus of a saccharification process as it is.
- the pretreatment process of the present invention can be performed using a continuous digester known as a Kamiya type.
- a pretreatment step with oxygen addition using an oxygen bleaching tower.
- the saccharification method of the present invention Since the recovery rate of the enzyme recovered using the saccharification method of the present invention (the enzyme activity of the recovered enzyme relative to the enzyme activity of the enzyme used in the saccharification step) is very high, the recovered enzyme is effectively reused. be able to.
- the saccharification method of the present invention when the enzyme recovered from the residue and the enzyme recovered from the saccharified solution are combined, the enzyme activity is at least 50% or more based on the amount of enzyme used in the saccharification step, and 70% or more depending on the conditions. Can be recovered. Therefore, the saccharification method of the present invention is a very useful technique in that the amount of enzyme used can be reduced and the enzyme cost can be greatly reduced.
- the sugar yield of the lignocellulosic biomass obtained in the present invention is not particularly limited, but as the glucose yield (%), a value calculated according to the following formula is preferably 65% or more, and 75% or more. Is more preferable, and 85% or more is particularly preferable.
- Glucose yield% theoretical glucose yield obtained from the amount of produced glucose / raw biomass (based on solid content)
- the sugar yield of the lignocellulosic biomass obtained in the present invention is the following C5 sugar yield (%): The value calculated according to the formula is preferably 80% or more.
- C5 sugar means xylose, arabinose, xylobiose and the like.
- C5 sugar yield% total amount of C5 sugar produced / total yield of C5 sugar obtained from raw material biomass (solid content basis) Furthermore, as the sugar yield of the lignocellulosic biomass obtained in the present invention, glucose and C5 sugar are The total yield of sugars to be contained is preferably 70% or more, more preferably 75% or more, and particularly preferably 80% or more.
- the proportion of C5 sugar is preferably 20 to 50%, more preferably 25 to 45%, more preferably 30 to 45% with respect to the total sugar components. It is particularly preferred.
- the C5 sugar is as described above, and the total sugar component refers to all sugar components including C5 sugar and C6 sugar (such as glucose).
- C5 sugar is easier to decompose than C6 sugar and it is difficult to obtain a high yield
- the method of the present invention is characterized by a high yield of C5 sugar, and the ratio of C5 and the like is increased to the above range. Can do.
- the total saccharide concentration is preferably 5 to 20%, more preferably 7 to 15%.
- the total sugar concentration is the concentration of the above-mentioned total sugar component in the saccharified solution.
- the present invention also includes an invention of a saccharified solution characterized in that the pretreated decomposition product produced in the pretreatment step is contained at a constant concentration.
- concentration of the pretreated decomposition product in the saccharified solution is preferably 1 to 30%, more preferably 2 to 20%, more preferably 5 to 20%, based on the total sugar components in the saccharified solution. It is particularly preferred that By setting the content concentration of the pretreated decomposition product in the above range, it is not affected by fermentation inhibition, and merits such as an increase in fermentation products and an improvement in fermentation rate can be obtained.
- the load in the pretreatment degradation product removal process is reduced, and there are effects such as reduction of washing water, improvement of reaction efficiency in the saccharification process, and reduction of enzyme usage. This is advantageous because it is obtained.
- a method for including the pretreated decomposition product in the saccharified solution a method of preparing and saccharifying pretreated biomass in which the pretreated decomposition product remains, or a method of adding the pretreated decomposition product in the saccharification step, or obtaining And a method of adding to the obtained saccharified solution.
- the saccharified solution is analyzed (chromatographic method), and the lignin degradation products (phenolic polymer or monomolecular compound) contained in the saccharified solution are quantified.
- the method can be used.
- the concentration of the pretreated degradation product relative to the total sugar component can be known.
- the enzyme recovery rate in the present invention is not particularly limited, but the cellobiohydrolase (CBH) recovery rate is preferably 40% or more, more preferably 55% or more, and particularly preferably 60% or more. .
- the recovery rate of ⁇ -glucosidase (GLD) is preferably 10% or more, preferably 30% or more, and preferably 50% or more.
- the recovery rate of ⁇ -xylosidase (XLD) is preferably 30% or more, more preferably 40% or more, and particularly preferably 50% or more.
- the carboxymethyl cellulase (CMC) recovery rate is preferably 40% or more, more preferably 45% or more, and particularly preferably 50% or more.
- the recovery rate of xylanase (XYN) is preferably 40% or more, more preferably 45% or more, and particularly preferably 50% or more. A combination of these enzyme recovery rates is particularly preferred.
- CBH means cellobiohydrolase
- GLD means ⁇ -glucosidase
- XLD means ⁇ -xylosidase
- CMC means carboxymethyl cellulase ( ⁇ -glucanase)
- XYN means Means xylanase.
- [Analysis method] The following method was used for measuring enzyme activity. As a specific measuring method, the method disclosed in Japanese Patent Application Laid-Open No. 2012-223113 was used.
- CBH activity a colorimetric method using p-nitrophenyl- ⁇ -D-cellobioside as a substrate.
- GLD activity a colorimetric method using p-nitrophenyl- ⁇ -D-glucopyranoside as a substrate.
- XLD activity a colorimetric method using p-nitrophenyl- ⁇ -D-xylopyranoside as a substrate.
- CMC activity a colorimetric method using carboxymethylcellulose as a substrate.
- a DNS (dinitrosalicylic acid) method was used to measure the amount of reducing sugar.
- XYN activity a colorimetric method using soluble xylan as a substrate. The DNS method was used to measure the amount of reducing sugar.
- C5 sugar is xylose, arabinose, xylobiose Means.
- the theoretical yield of sugars obtained from the empty fruit bunch (EFB) of oil palm used as a raw material is as follows: glucose is 42%, C5 sugar total is 26% (xylose 25%, arabinose 1%), and sugar total is 68% (Mass yield based on EFB solids).
- Example 1 (1) Pretreatment step In a 100 ml glass reactor, 5.5 g of fibrous EFB (moisture content: 8.9%, solid content: 5.0 g) was added, and 4.0% NaOH aqueous solution as an alkaline aqueous solution. 50.0 g was added to prepare a mixture, which was sufficiently impregnated in EFB (still at room temperature for 15 minutes under reduced pressure). The solid-liquid ratio of this mixture is 10.1 (total liquid components 0.5 + 50.0 g ⁇ 5.0 g EFB solid content).
- EFB containing an alkaline aqueous solution alkali-impregnated EFB
- EFB containing an alkaline aqueous solution alkali-impregnated EFB
- the mass of the alkali-impregnated EFB was 16.9 g
- the solid-liquid ratio after the solid-liquid separation was 2.4 (total liquid components 11.9 g ⁇ EFB solid content 5.0 g).
- a part of the alkaline aqueous solution recovered by solid-liquid separation was about 38 g. Since the recovered aqueous alkali solution can be recycled, it is considered that the substantial amount of alkali used is 9.5%.
- the obtained alkali-impregnated EFB was put into a 100 ml pressure-resistant reactor equipped with a thermometer and a pressure gauge, and the inside of the reactor was replaced with nitrogen gas, and then sealed. The reactor was put into an oil bath and heat-treated at 100 ° C. (reactor internal temperature) for 1 hour.
- Pretreatment decomposition product removal process cleaning process
- a washing operation with water 50.0 g of pure water was added to 16.9 g of EFB after the heat treatment, and the mixture was stirred and mixed for 10 minutes to elute the solubilized biomass decomposition product (pretreatment decomposition product) generated in the pretreatment step.
- Solid-liquid separation was performed by filtration to obtain 12.8 g of pretreated EFB (water wet body) and about 53 g of filtrate (referred to as pretreatment liquid, alkaline aqueous solution containing about 3% of pretreated decomposition product, pH 12.7).
- pretreatment liquid alkaline aqueous solution containing about 3% of pretreated decomposition product, pH 12.7
- the reaction mixture was prepared as follows in a 50 ml glass reactor. 12.8 g of pretreated EFB, 1.6 mg of tetracycline hydrochloride, 1.2 mg of cycloheximide, 20 ml of 0.1M acetate buffer (pH 5.5), 0.30 g of enzyme solution (1: 1 mixture of enzyme A and enzyme B) are reacted. After adding to the vessel and adjusting the pH to 5.5 with 10% aqueous acetic acid, the total mass was adjusted to 40.0 g with water. Subsequently, the reactor was sealed, and saccharification reaction was carried out at 45 ° C. for 72 hours while shaking with a constant temperature shaker.
- the yield of glucose was 81% (theoretical yield of glucose) and the yield of C5 sugar was 83% (compared to the C5 sugar total theoretical yield), the total sugar yield was 82% (theoretical total sugar yield), and high sugar yields were obtained for both glucose and C5 sugar.
- the alkaline aqueous solution was sufficiently infiltrated into the EFB (still at room temperature for 15 minutes under reduced pressure), and the inside of the reactor was replaced with nitrogen gas and sealed without performing solid-liquid separation. Further, a heat treatment and a cleaning step were performed in the same manner as in Example 1 to obtain a pre-processed EFB. However, in the washing step, the same washing operation was performed after first filtering to remove the liquid. Subsequently, the saccharification step and the enzyme recovery step (including water washing) were performed in the same manner as in Example 1, and the sugar yield and the enzyme recovery rate were measured. The results shown in Table 1 and FIG. 1 were obtained.
- Example 2 The pretreatment was performed in the same manner as in Example 1 except that the heat treatment in the pretreatment step was performed by setting the gas phase portion at 80 volume% oxygen / 20 volume% nitrogen instead of the nitrogen atmosphere.
- the pressure at that time was 0.2 MPaG (gauge pressure) as a total pressure at 100 ° C., and the pressure drop due to oxygen consumption was maintained by adding oxygen gas.
- the washing step, the saccharification step, and the enzyme recovery step were performed in the same manner as in Example 1. The results shown in Table 1 and FIG. 1 were obtained.
- Example 3 As shown in Table 1 below, EFB saccharification experiments were conducted in the same manner as in Example 1 or 2 under various conditions. The results are shown in Table 1 below.
- Example 4 the pressure was raised to 1.0 MPaG with 80% by volume oxygen / 20% by volume nitrogen at room temperature, and then the temperature was raised. Heat treatment was performed without compensating for the pressure drop due to oxygen consumption.
- Example 8 Until the preparation of the alkali-impregnated EFB, the experiment was carried out in the same manner as in Example 5 (impregnation with 3% NaOH), and the experiment was conducted by changing the heat treatment method as follows. That is, the obtained alkali-impregnated EFB was placed in a 100 ml plastic beaker, and the beaker was further placed in a 2 L plastic container and covered (covered with a cloth soaked with 100 ml water on the bottom of the 2 L container and subjected to heat treatment). The container was filled with water vapor). A small hole was made in the lid to prevent the internal pressure from increasing. The reaction vessel thus prepared was placed in an oven at 80 ° C. and left to stand for 12 hours for heat treatment (the atmosphere in the gas phase was air and oxygen was sufficiently present. The pressure was atmospheric pressure. ). The process after the heat treatment was performed in the same manner as in Example 1. The results are shown in Table 1.
- Example 9 As shown in Table 1, EFB saccharification experiments were conducted in the same manner as in Example 8 with the conditions changed. The results are shown in Table 1.
- Example 11 Preparation of alkali-impregnated EFB and heat treatment were performed in the same manner as in Example 5. First, heat treatment was performed at 100 ° C. for 1 hour under nitrogen (under oxygen limitation). Subsequently, the EFB after the heat treatment was subjected to a heat treatment at 80 ° C. for 6 hours in the same manner as in Example 8 (air atmosphere, atmospheric pressure), and a heat treatment was performed under an oxygen supply. The process after the heat treatment was performed in the same manner as in Example 1. The results are shown in Table 1.
- Example 8 An alkaline aqueous solution was impregnated in EFB.
- the subsequent steps were the same as in Example 8.
- the results are shown in Table 2.
- the NaOH concentration of all liquid components in the alkali-impregnated EFB was calculated to be 3.7%, and the alkali impregnation amount was calculated to be 8.5%.
- Example 13 A saccharification experiment of EFB (water wet body) was performed in the same manner as in Example 12 except that the impregnation conditions of the alkaline aqueous solution were changed.
- the alkali impregnation conditions were set to 70 ° C. for 30 minutes at atmospheric pressure (without air pressurization). The results are shown in Table 2.
- Example 14 The experiment was performed under the same pretreatment and saccharification conditions as in Example 1 except that the residue treatment conditions in the enzyme recovery step were changed as follows.
- the residue treatment conditions in the enzyme recovery step were changed as follows.
- the primary treatment of the residue first, 15 g of water was added to the residue and mixed with stirring, and then a small amount of 1% NaOH aqueous solution was further added to adjust the pH of the treatment solution to 8.0. After gently stirring and mixing for 30 minutes, the enzyme was desorbed under alkaline conditions, and the primary treatment liquid was recovered by filtration.
- the secondary treatment was performed with water in the same manner as in Example 1, and the secondary treatment liquid was recovered. When the total enzyme recovery rate in the recovered liquid was measured, the results shown in Table 3 were obtained.
- Example 15 As shown in Table 3, the EFB saccharification experiment was conducted in the same manner as in Example 14 by changing the conditions of the primary treatment and the secondary treatment of the residue in the enzyme recovery step. The results are shown in Table 3.
- Example 15 the pH of the primary treatment was changed to 9.0.
- Example 16 the primary treatment was carried out at pH 8.0, and in the secondary treatment, NaOH was further added to raise the pH of the treatment solution to 9.0 stepwise to recover the enzyme.
- Example 17 an experiment was performed by adding 10% of the pretreatment liquid obtained in the cleaning step to the treatment liquid during the primary treatment. The results are shown in Table 3.
- Example 18 The same pretreatment and saccharification conditions as in Example 2 were performed except that the residue treatment conditions in the enzyme recovery step were changed.
- the same procedure as in Example 14 was performed under the conditions shown in Table 3. The results are shown in Table 3.
- Example 20 After the EFB saccharification step was performed in the same manner as in Example 2, the enzyme recovery step was performed in the same manner as in Example 16 (residual alkali treatment was pH 8.0 ⁇ pH 9.0) to obtain a recovered solution It was. The total amount of the recovered solution obtained was subjected to ultrafiltration (using Kurabo Industries, Centricut U-10, molecular weight cut off 10,000, membrane material polysulfone) and concentrated to about 10 g to obtain a recovered enzyme solution. Using this recovered enzyme solution, the EFB saccharification experiment was performed again (enzyme recycling reaction). That is, pretreatment EFB was prepared in the same manner as in Example 2, and a reaction mixture was prepared as follows.
- Pretreatment EFB water wet body
- tetracycline hydrochloride 1.6 mg, cycloheximide 1.2 mg, 0.1 M acetate buffer (pH 5.5) 10 ml
- recovered enzyme solution about 10 g
- fresh enzyme solution (1 of enzyme A and enzyme B) : 1 mixture
- 0.06 g was mixed, adjusted to pH 5.5 with 10% aqueous acetic acid, and the total mass was adjusted to 40.0 g with water.
- the initial 20% (fresh) enzyme solution is replenished as a loss. This was saccharified under the same conditions as in Example 2.
- the glucose yield was 89%
- the C5 saccharide yield was 81%
- the total saccharide yield was 86% (the saccharide yield is a yield considering the amount of saccharide brought from the recovered enzyme solution).
- the saccharide yield is a yield considering the amount of saccharide brought from the recovered enzyme solution).
- a sugar yield equivalent to that of the first time was obtained.
- Example 21 An EFB saccharification experiment was conducted under the same conditions as in Example 8 except that the alkaline aqueous solution in the pretreatment step was changed. As the alkaline aqueous solution, 5% KOH was used. The results are shown in Table 1.
- Example 22 EFB saccharification experiment was conducted in the same manner as in Example 1. However, at the time of preparing the reaction mixture in the saccharification step, 10.0 g of the pretreatment liquid obtained in the washing step is added (the amount of the acetate buffer is reduced accordingly, a total of 40.0 g, pH 5.5), and the saccharification reaction is performed. It was. Further, the enzyme recovery step was performed in the same manner as in Example 1. As for the sugar yield, the glucose yield was 83%, the C5 sugar yield was 85%, and the total sugar yield was 84%. The enzyme recovery rate was 71% for CBH, 32% for GLD, and 65% for XLD, and a higher sugar yield and enzyme recovery rate than Example 1 were obtained.
- Example 23 A rice straw saccharification experiment was conducted in the same manner as in Example 5 except that the biomass raw material was changed to rice straw. That is, 5.7 g of rice straw (produced in Nagano Prefecture, water content 11.6%, solid content 5.0 g) was used as a raw material, impregnated with 3% NaOH (solid-liquid ratio 10), and solid-liquid separation was performed. . The mass of the alkali-impregnated rice straw was 23.8 g, and the solid-liquid ratio after solid-liquid separation was 3.8. Further, the steps after the heat treatment were performed in the same manner as in Example 5. However, in the saccharification process, the reaction time was changed to 20 hours.
- the mass yield of raw rice straw was 32% for glucose, 13% for C5 sugar, and 45% for total sugar. If the theoretical yield of rice sugar was 70%, the theoretical yield was 64%.
- the enzyme recovery rate was 95% for CBH and 90% for XLD, and a very high recovery rate was obtained.
- Example 24 An alkali-impregnated EFB was prepared in the same manner as in Example 1. However, the amount of 4% NaOH aqueous solution at the time of impregnation was 75 g (solid-liquid ratio before solid-liquid separation was 15.1). After the solid-liquid separation, the alkali-impregnated EFB was put in a pressure-resistant reactor, sealed in an air atmosphere, put in an oil bath, and maintained at 180 ° C. (reactor internal temperature) for 15 minutes to perform heat treatment ( Heat treatment in high temperature and short time). Then, the washing
- Example 25 to 37 Various conditions were changed as shown in Table 4, and experiments were conducted in the same manner as in Example 24. The results are also shown.
- experiments were performed by changing the alkaline aqueous solution and the heat treatment conditions in various ways.
- a mixed solution of NaOH and sodium carbonate (concentration: 1% each) was used as the alkaline aqueous solution.
- Example 32 a 4% aqueous ammonia solution was used.
- Example 33 a 0.5% slurry of calcium oxide was used as the alkaline aqueous solution.
- a pressure reduction treatment was performed at the time of impregnation, and the reactor was sealed and subjected to a treatment of holding at 50 ° C.
- Example 34 the solid-liquid ratio before solid-liquid separation was lowered.
- Example 35 the solid-liquid ratio after solid-liquid separation was lowered (after solid-liquid separation, the solid-liquid ratio was lowered by absorbing the liquid using a filter paper).
- Example 36 reuse experiment of alkaline aqueous solution was conducted. That is, the filtrate obtained by solid-liquid separation (filtration) after alkali impregnation in Example 24 was reused as an aqueous alkaline solution. However, at this time, the insufficient NaOH and water were replenished to obtain an alkaline aqueous solution similar to that in Example 24.
- Example 37 the heat treatment was performed by setting the gas phase portion during the heat treatment to 50 volume% oxygen / 50 volume% nitrogen instead of the air atmosphere, and further setting the initial pressure during preparation to 0.6 MPaG.
- Comparative Examples 2 to 4 In Comparative Example 2, pretreatment was performed by a method that does not perform solid-liquid separation as in Comparative Example 1. The heat treatment conditions were the same as in Example 24, 180 ° C. and 15 minutes. In Comparative Examples 3 and 4, experiments were performed in the same manner as in Example 24 using pure water instead of the alkaline aqueous solution. Experimental conditions and results are shown in Table 4.
- Example 38 An experiment was conducted to reuse the enzyme adsorbed on the raw material. That is, the experiment was conducted up to the saccharification step under the same conditions as in Example 25. However, the saccharification reaction was stopped at 24 hours. The resulting reaction mixture was filtered and separated into a saccharified solution (about 30 g) and a wet undecomposed raw material (about 10 g). Subsequently, this undecomposed raw material and a separately prepared pretreated EFB (same conditions as in Example 25) were mixed, and the reaction mixture (40.0 g) was re-prepared (however, the enzyme was not 0.30 g but 1 The saccharification reaction was resumed at 45 ° C.
- the sugar yield of the whole experiment (vs. theoretical yield, twice as much raw material basis) was 90% for glucose, 89% for C5 sugar, and 90% for total sugar.
- Example 39 The EFB pretreatment step was performed in the same manner as in Example 25. In the next washing step, the same water washing operation as in Example 1 was repeated four times. That is, 50.0 g of pure water was added to the EFB after the heat treatment, and the mixture was stirred and mixed for 10 minutes to elute the pretreated decomposition product. Solid-liquid separation was performed by filtration to obtain an EFB solid content and a water-washed filtrate (pretreatment liquid 1). This water washing operation was further repeated three times to obtain a water washing filtrate (pretreatment liquids 2 to 4) and a sufficiently washed pretreatment EFB. The water in the pretreatment liquids 1 to 4 was evaporated and the solid content (pretreatment decomposition product amount) was measured.
- the pretreatment liquid 1 contains 1.54 g
- the pretreatment liquid 2 contains 0.16 g
- the pretreatment liquid 3 contains 0.03 g
- the pretreatment liquid 4 contains ⁇ 0.01 g of solids. 0.73 g.
- the washing method in this way, it is possible to prepare pretreatment raw materials containing pretreatment decomposition products at various concentrations, and further saccharifying them to prepare saccharified solutions containing pretreatment decomposition products at various concentrations It is also possible to do.
- Example 1 (and an example under equivalent implementation conditions), it was found that the residual rate of the pretreatment decomposition product in the pretreatment EFB was about 6%.
- the saccharification reaction was carried out in the presence of about 6% of a pretreatment decomposition product.
- the concentration of the pretreated decomposition product in the saccharified solution was considered to be about 7% with respect to the total sugar components.
- Example 40 The effects of pretreatment degradation products on fermentation were investigated.
- An EFB saccharification experiment was conducted in the same manner as in Example 25 except that the scale was 20 times (raw material EFB 100 g).
- the water washing operation was performed three times in the same manner as in Example 39 (pretreatment decomposition residual ratio ⁇ 0.3%).
- the saccharification step tetracycline hydrochloride and cycloheximide were not added, and saccharification was performed by increasing the solid content concentration of the pretreated EFB (the amount of the reaction mixture was reduced to 540 g by reducing water).
- the reaction mixture after the reaction was subjected to solid-liquid separation to obtain a saccharified solution A.
- saccharified solution A The total sugar concentration of saccharified solution A was 11.0%, and the C5 sugar ratio was 38%.
- This saccharified solution and the filtrate obtained in the first washing operation (pretreatment liquid A, containing 3.3% of the pretreated decomposition product based on the solid content) were mixed at the ratio shown in Table 5, and the pretreatment decomposition product was mixed.
- Saccharified solutions B to D containing various concentrations (ratio) were prepared. These are prepared by simulating saccharified liquids obtained under various washing conditions, and saccharified liquid C has a composition equivalent to that of the saccharified liquid obtained in one washing operation (see Example 39). Subsequently, butanol fermentation was performed using the saccharified liquids A to D.
- the medium was adjusted to a sugar concentration of 40 g / L (total sugar), a medium component (TYA medium) was added, and the pH was adjusted to 6-7.
- a control experiment an experiment using the glucose solution of the reagent instead of the EFB saccharified solution (control 1) and an experiment using the reagent glucose + xylose (mass ratio 6: 4) solution (control 2) were performed.
- the strain was Clostridium saccharoper butylacetonicum (ATCC 27021 strain), an ATCC strain, and fermented under static conditions at 30 ° C. for 48 hours after preculture. The results are shown in Table 5.
- the bacterial cell concentration (OD660), produced butanol concentration, and butanol mass yield (consumed sugar) at 33 hours and 48 hours of fermentation were shown.
- the numerical value is an average value of two experiments.
- Example 1 in which solid-liquid separation was performed after alkali impregnation, the total sugar yield was 13% higher even with a smaller amount of alkali compared to Comparative Example 1 in which solid-liquid separation was not performed. It was. Further, a high enzyme recovery rate was obtained in Example 1, but the enzyme recovery rate of Comparative Example 1 was very low, and it was found that the present invention is superior in enzyme recovery. Moreover, the solid-liquid ratio in the pretreatment of Example 1 is 2.4, which is about one-fourth the amount of water used as compared with 10 in Comparative Example 1, and it was found that water can be reduced.
- Example 2 it was confirmed that the addition of oxygen in the pretreatment further improved the sugar yield and the enzyme recovery rate.
- Example 3 it was found that a high sugar yield and enzyme recovery rate could be obtained even with a low concentration of oxygen.
- Example 4 it was found that a higher sugar yield and enzyme recovery rate could be obtained by changing the pressure and heat treatment conditions. In particular, the GLD recovery rate was improved.
- Examples 5 and 6 are experiments in which the concentration of impregnated alkali was lowered (with a small amount of alkali), but higher sugar yield and enzyme recovery were obtained compared to Comparative Example 1, and the amount of alkali used It was found that it is possible to reduce In Example 7, high sugar yield and enzyme recovery were obtained even under high temperature and short time heat treatment conditions.
- Example 8 it was found that high sugar yield and enzyme recovery were obtained even under normal pressure, low oxygen concentration air atmosphere, and low temperature and long time heat treatment conditions.
- Example 11 high sugar yield and enzyme recovery rate were obtained even in a shorter time by performing the heat treatment in two stages (no oxygen supply + oxygen supply).
- Example 21 good results were obtained even when an alkali other than NaOH was used.
- Examples 12 and 13 are experiments conducted using raw materials of water wet bodies (high water content) and changing various alkali impregnation conditions. It was found that high sugar yield and enzyme recovery were obtained.
- Table 3 shows the results of enzyme recovery from the residue under various conditions.
- the enzyme recovery rate was improved by adding an alkali in the enzyme recovery step, and it was found that the recovery rate was higher at a higher pH.
- a higher enzyme recovery rate was obtained by performing the alkali treatment in a manner of gradually increasing the pH.
- Example 17 it was found that a higher enzyme recovery rate can be obtained by adding the pretreated degradation product and performing enzyme recovery as compared with Example 14 under the same pH conditions.
- Examples 18 and 19 it was found that a higher enzyme recovery rate can be obtained by performing pretreatment in the presence of oxygen and further adding an alkali to perform enzyme recovery. In particular, the GLD recovery rate was improved.
- Example 20 was an experiment in which the recovered enzyme was reused. Since the results equivalent to the first reaction were obtained, it was found that the recovered enzyme could be reused. In Example 22, it was found that the sugar yield and the enzyme recovery rate were improved by adding the pretreatment decomposition product and performing the saccharification reaction. In addition, Example 23 was an experiment using rice straw which is herbaceous biomass as a raw material, and it was found that high sugar yield and enzyme recovery rate could be obtained even if rice straw was used.
- Example 24 to 37 it was found that the sugar yield was improved by performing the heat treatment in the pretreatment step at a high temperature (around 150 ° C. to 200 ° C.) for a short time. The sugar concentration and C5 sugar ratio were also shown.
- Example 36 it was found that the alkaline aqueous solution recovered by solid-liquid separation can be reused. Compared with Comparative Examples 2 to 4, the effect of improving the sugar yield by performing solid-liquid separation and using an alkali was also confirmed in a high temperature region.
- Example 38 a sequential saccharification reaction was performed in which the enzyme adsorbed on the biomass was reused, and the effect of reducing the enzyme and the effect of improving the sugar productivity were shown.
- Example 39 the removal process of the pretreatment decomposition product was examined, and the relationship between the cleaning method and the residual rate of the pretreatment decomposition product was clarified.
- Example 40 a fermentation experiment of a saccharified solution was performed, and high fermentation results were obtained. Furthermore, it was found that when the pretreated decomposition product was present in the saccharified solution, an improvement in the fermentation product concentration or an improvement in the yield of sugar was observed.
- alkali acts on biomass efficiently, it is possible to achieve a high sugar yield even with a smaller amount of alkali and water, and in addition, a high enzyme recovery rate. It can also be achieved. Further, by performing pretreatment in the presence of oxygen, a higher sugar yield and enzyme recovery rate can be obtained. In the method of the present invention, it is also possible to obtain a saccharified solution having excellent fermentation characteristics while reducing the load for removing the pretreated decomposition product.
- the present invention is useful as a method for saccharification of lignocellulosic biomass in order to obtain saccharides as fermentation raw materials.
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Abstract
Description
[1](1)リグノセルロース系バイオマスにアルカリ水溶液を含浸させた混合物を調製した後、固液分離を行って一部のアルカリ水溶液を除去し、熱処理を行う前処理工程、及び(2)前処理工程で得られたリグノセルロース系バイオマスを酵素で分解して糖化液を得る糖化工程を含むことを特徴とするリグノセルロース系バイオマスの糖化方法。
[2]前処理工程において、下記式(I)で算出される固液比が、固液分離前の混合物は2~20であり、固液分離後の混合物は1~6であることを特徴とする前記[1]に記載の糖化方法。
式(I):
固液比=混合物中の全液体成分の総質量/混合物中のリグノセルロース系バイオマスの固形分質量
[3]前処理工程において、熱処理を100~200℃で行うことを特徴とする前記[1]又は[2]に記載の糖化方法。
[4]糖化工程が、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物の存在下で行われることを特徴とする前記[1]~[3]のいずれかに記載の糖化方法。
[5]前処理工程と糖化工程の間に、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物の一部を除去する除去工程を含み、除去工程後のリグノセルロース系バイオマスにおいて、下記式(II)で算出される前処理分解物の残存率が2~20質量%であることを特徴とする前記[1]~[4]のいずれかに記載の糖化方法。
式(II):
前処理分解物の残存率=残存する前処理分解物の固形分質量/リグノセルロース系バイオマスの固形分質量
[6]糖化工程で得られる糖化液中のC5糖の割合が、全糖成分に対して20~50質量%であることを特徴とする前記[1]~[5]のいずれかに記載の糖化方法。
[7]糖化工程で得られる糖化液の全糖濃度が5~20質量%であることを特徴とする前記[1]~[6]のいずれかに記載の糖化方法。
[8]糖化工程で得られる未分解のリグノセルロース系バイオマスに吸着した酵素を再利用することを特徴とする前記[1]~[7]のいずれかに記載の糖化方法。
[9]前処理工程において、酸素を添加して熱処理を行うことを特徴とする前記[1]~[8]のいずれかに記載の糖化方法。
[10]糖化工程に続いて、糖化工程終了後に酵素を回収する酵素回収工程を含むことを特徴とする前記[1]~[9]のいずれかに記載の糖化方法。
[11]酵素回収工程において、未分解のリグノセルロース系バイオマスに吸着した酵素をアルカリ処理により脱着させ回収する工程を含むことを特徴とする前記[10]に記載の糖化方法。
[12]含水率が30~90%のリグノセルロース系バイオマスを前処理工程に供することを特徴とする前記[1]~[11]のいずれかに記載の糖化方法。
[13]糖化工程で得られる糖化液であって、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物が、糖化液中の全糖成分に対して、2~20質量%含まれることを特徴とする糖化液。
固液比=混合物中の全液体成分の総質量/混合物中のバイオマスの固形分質量
ここで「全液体成分」とは、接触させるアルカリ水溶液、および原料バイオマスが含む水分や、その他の液体全てを合わせた混合物中の液体成分の合計を意味する。また、「バイオマスの固形分質量」とは、水分等の液体成分を含まない原料バイオマスの固形分質量を意味する。含浸工程の混合物(すなわち、固液分離前の混合物)の固液比を前記範囲にすることで、高いアルカリ含浸速度と均一性を達成することができる。
アルカリ含浸バイオマス(すなわち、固液分離後の混合物)における固液比(=混合物中の全液体成分の総質量/混合物中のリグノセルロース系バイオマスの固形分質量)は、好ましくは1~10であり、より好ましくは1~6であり、特に好ましくは1~4である。また固液分離後の混合物において、全液体成分中に存在するアルカリ化合物量(アルカリ含浸量)としては、バイオマス固形分質量に対して、0.1~30%であることが好ましく、1~20%であることがより好ましく、2~15%であることが特に好ましい。
熱処理時の気相部の雰囲気は、酸素ガス、窒素ガス、酸素/窒素混合ガス、空気等、特に限定されない。酸素存在下でアルカリ前処理を行うことで、糖化工程におけるバイオマスへの酵素吸着が低減し、糖収率向上や酵素回収率向上が期待できる。具体的には、酸素濃度が1~100体積%であることが好ましく、10~95体積%であることがより好ましく、15~80体積%であることが特に好ましい。好ましい形態の一つとしては、安価に利用できる空気を使用する方法である。また、酸素存在下で熱処理を行う場合、酸素は時間とともに消費されるため、酸素を添加しながら(酸素濃度を維持しながら)熱処理を行うことが好ましい。
熱処理によってバイオマス(主にはリグニン)は分解、可溶化され、フェノール性水酸基やカルボキシル基を有する化合物を生成する。この分解反応によってアルカリ成分は消費(中和)されるため、バイオマスのpHは低下する。熱処理後の混合物中の全液体成分のpHとしては、pH6~14が好ましく、pH7~13がより好ましく、pH8~12が特に好ましい。このpH範囲とすることで、含浸アルカリを効率的に利用しつつ、次の除去工程の負荷を低減することができる。なお、熱処理後の全液体成分のpHは、上述の方法で見積もることができる。
前処理分解物の残存率=残存する前処理分解物の固形分質量/リグノセルロース系バイオマスの固形分質量
上記残存率の範囲にすることで、前処理分解物が糖化工程、酵素回収工程、または発酵においてポジティブな効果を示し、糖収率の向上、酵素回収率の向上、発酵生成物の増加等のメリットを得ることができる。また比較的高い前処理分解物の残存率でも許容されるため、除去工程での負荷が低下し、洗浄水削減等のメリットも得られる。
(a)の具体的方法としては、例えば、糖化反応の途中、または終了後に糖化液と未分解バイオマス(酵素を吸着)を分離し、次の糖化反応にその未分解バイオマスを使用する方法等があげられる。糖化反応の途中に、糖化液の回収(未分解バイオマスとの分離)と、フレッシュなバイオマス原料の追加を逐次的、もしくは連続的に行い、吸着酵素を継続的に利用しながら、糖化反応を行う方法は好ましい方法である。(b)の具体的方法は後述する。
酵素回収工程では、上述のように、糖化工程で得られた反応混合物を固液分離し、糖化液と残渣とに分離する。固液分離方法は特に限定されず、例えば、ろ過、遠心分離、遠心ろ過、サイクロン、フィルタープレス、スクリュープレス、デカンター等を使用することができる。糖化液からは遊離の酵素(非吸着酵素)を回収することができる。残渣からは吸着酵素を回収することができる。上述のように、回収した吸着酵素はそのまま再利用してもよいが、残渣から脱着させて酵素を回収してもよい。
アルカリ化合物は水溶液として添加することが好ましく、添加するアルカリ化合物の濃度、量は、上記pH範囲を達成するものであればよいが、濃度が低すぎると添加量が多くなり、回収酵素濃度も低下する。添加するアルカリ水溶液のアルカリ濃度は、0.01~10%であることが好ましく、0.1~5%であることがより好ましい。
アルカリ処理の際、残渣からの酵素脱着を促進させるために攪拌、加熱等を行ってもよい。アルカリ処理の温度は、5~60℃が好ましく、10~40℃がより好ましい。処理時間は、0.1~10時間が好ましく、0.1~1時間がより好ましい。
アルカリ処理から固液分離までの工程は、回分式で行ってもよく、連続式で行ってもよい。上記工程(A)は回分式で行うのが好ましい。上記工程(B)は回分式でも連続式でもよいが、連続式の方が好ましい。
グルコース収率%=生成グルコース量/原料バイオマス(固形分基準)から得られるグルコース理論収量
また、本発明で得られるリグノセルロース系バイオマスの糖収率としては、C5糖収率(%)として、下記式に従って算出された値が80%以上のものが好ましい。C5糖とは、キシロース、アラビノース、キシロビオース等を意味する。
C5糖収率%=生成C5糖合計量/原料バイオマス(固形分基準)から得られるC5糖合計理論収量
さらに、本発明で得られるリグノセルロース系バイオマスの糖収率としては、グルコースとC5糖を含む糖の合計収率が、70%以上のものが好ましく、75%以上のものがより好ましく、80%以上が特に好ましい。
(1)バイオマス
原料のリグノセルロース系バイオマスとして、パーム油を生産する際に排出されるアブラヤシの空果房(以下「EFB」という。)を原料に用いた(産地インドネシア)。EFBの形状としては、パーム油工場にてシュレッダー処理が施された繊維状のEFBを用いた。
(2)糖化酵素
ノボザイムズ社の酵素液セリック シーテック2(商品名、以下「酵素A」という。)及びセリック エイチテック2(商品名、以下「酵素B」という。)を所定の割合で混合して使用した。酵素Aは主にセルラーゼ(CBH、GLD、CMC)活性、酵素Bは主にヘミセルラーゼ(XLD、XYN)活性を有している。
酵素活性の測定には以下の方法を用いた。具体的な測定方法は、特開2012-223113号公報に開示されている方法を用いた。
CBH活性:p-ニトロフェニル-β-D-セロビオシドを基質とした比色法。
GLD活性:p-ニトロフェニル-β-D-グルコピラノシドを基質とした比色法。
XLD活性:p-ニトロフェニル-β-D-キシロピラノシドを基質とした比色法。
CMC活性:カルボキシメチルセルロースを基質とした比色法。還元糖量の測定にはDNS(ジニトロサリチル酸)法を用いた。
XYN活性:可溶性キシランを基質とした比色法。還元糖量の測定はDNS法を用いた。
グルコース収率%=生成グルコース量/原料バイオマス(未処理、固形分基準)から得られるグルコース理論収量
C5糖収率%=生成C5糖合計量/原料バイオマス(未処理、固形分基準)から得られるC5糖合計理論収量
糖合計収率%=(生成グルコース量+C5糖合計量)/原料バイオマス(未処理、固形分基準)から得られる糖類合計理論収量
ここでC5糖とは、キシロース、アラビノース、キシロビオースを意味する。なお、原料として用いたアブラヤシの空果房(EFB)から得られる糖類の理論収率は、グルコースが42%、C5糖合計は26%(キシロース25%、アラビノース1%)、糖合計では68%であった(EFB固形分基準の質量収率)。
酵素回収率は以下の式に従って算出した。
酵素回収率%=回収液(反応液、洗浄液)中の酵素活性/糖化反応仕込時の酵素活性
(1)前処理工程
100mlのガラス製反応器に、繊維状EFBを5.5g(含水率8.9%、固形分として5.0g)を入れ、更にアルカリ水溶液として4.0%のNaOH水溶液を50.0g添加して混合物を調製し、十分にEFB中に含浸させた(減圧下、室温で15分静置)。なお、この混合物の固液比は、10.1である(全液体成分0.5+50.0g÷EFB固形分5.0g)。続いて、ろ過によりアルカリ水溶液を含んだEFB(アルカリ含浸EFB)と、一部のアルカリ水溶液とを固液分離し、それぞれ回収した。アルカリ含浸EFBの質量は16.9gであり、固液分離後の固液比は2.4であった(全液体成分11.9g÷EFB固形分5.0g)。また、アルカリ含浸EFB中に含まれるNaOHの固形分質量は、全液体成分質量より0.48gと見積もられた(=11.9g×0.04、対原料EFBでのアルカリ含浸量としては9.5%)。固液分離にて回収された一部のアルカリ水溶液は約38gであった。回収されたアルカリ水溶液はリサイクル使用可能なため、実質的なアルカリ使用量は9.5%であると考えられる。続いて、得られたアルカリ含浸EFBを、温度計と圧力計を備えた100mlの耐圧反応器に入れ、窒素ガスで反応器内を置換した後、密閉した。反応器をオイルバスに投入し、100℃(反応器内部温度)で1時間、熱処理をした。
続いて、水による洗浄操作を行った。熱処理後のEFB16.9gに50.0gの純水を添加して10分間攪拌混合し、前処理工程にて生成する可溶化されたバイオマス分解物(前処理分解物)を溶出させた。ろ過により固液分離を行い、前処理EFB12.8g(水ウェット体)とろ液約53g(前処理液と称する。約3%の前処理分解物を含むアルカリ性水溶液、pH12.7)を得た。前処理EFBは乾燥せずにそのまま次の糖化工程へ供した。
50mlのガラス製反応器内で以下のように反応混合物を調製した。
前処理EFB12.8g、テトラサイクリン塩酸塩1.6mg、シクロヘキシミド1.2mg、0.1M酢酸バッファー(pH5.5)20ml、酵素液(酵素Aと酵素Bの1:1混合液)0.30gを反応器に添加し、10%酢酸水でpH5.5に調整後、総質量を40.0gに水で調整した。続いて反応器を密閉し、恒温振とう機で振とうしながら、45℃で72時間、糖化反応を行った。糖化液(未分解原料を除去した反応液)を一部サンプリングし、生成した糖類をHPLCで分析した結果、グルコース収率は81%(対グルコース理論収量)、C5糖収率は83%(対C5糖合計理論収量)、糖合計収率は82%(対糖合計理論収量)であり、グルコース、C5糖共に高い糖収率が得られた。
糖化工程後の反応混合物をろ過し、糖化液(糖類と遊離の酵素を含む)約35gと未分解残渣(ウェット)約5gとに固液分離した。残渣に残存している酵素を回収するために、残渣に水15gを加え、30分間ゆるやかに攪拌混合した後、ろ過を行い、ろ液を回収した(1次処理液、pH5.5)。更に、同様の水洗操作を行い、2次処理液を得た。糖化液、1次処理液、及び2次処理液を合わせて回収液(約65g)とした。
回収液中の総酵素回収率を求めたところ、CBHは64%、GLDは10%、XLDは61%、CMCは55%、XYNは51%であった。CBH、XLD、CMC、XYNは比較的高い酵素回収率が得られた。GLDは吸着力が極めて強く回収が困難であることが知られているが、10%の回収率が得られた。以上の実験条件及び実験結果を、表1及び図1にまとめた。ここで、アルカリ使用量は、アルカリ含浸EFB中のアルカリ含浸量である(固形分基準)。
実施例1と同等量のNaOHを用いて、固液分離を行わない以下の前処理方法でEFBの糖化実験を行った。
すなわち、温度計と圧力計を備えた100mlの耐圧反応器に、実施例1と同様に繊維状EFBを5.5g入れ、アルカリ水溶液として1.0%のNaOH水溶液を50.0g添加した(固液比10.1)。このアルカリ水溶液はNaOHを0.50g(=対原料EFBの使用量としては10%)を含み、実施例1と同等のNaOH使用量である。
続いて、アルカリ水溶液を十分にEFB中に浸透させた後(減圧下、室温で15分静置)、固液分離を行わずに、そのまま窒素ガスで反応器内を置換し密閉した。更に実施例1と同様に熱処理、洗浄工程を行い、前処理EFBを得た。ただし洗浄工程では、まずろ過して液体を除去した後、同様の洗浄操作を行った。
続いて実施例1と全く同様に糖化工程及び酵素回収工程(水洗含む)を行い、糖収率及び酵素回収率の測定を行ったところ、下記表1及び図1に示した結果となった。
前処理工程における熱処理の際の気相部を窒素雰囲気では無く、80体積%酸素/20体積%窒素として熱処理を行った点以外は、実施例1と同様に前処理を行った。その際の圧力は100℃における全圧で0.2MPaG(ゲージ圧)とし、酸素消費による圧力低下分は酸素ガスを追加して圧力を維持した。前処理の後、実施例1と同様に洗浄工程、糖化工程、及び酵素回収工程を行ったところ、下記表1及び図1に示した結果となった。
下記表1に示したように種々の条件を変えて、実施例1又は2と同様にEFBの糖化実験を行った。結果を下記表1に示す。なお実施例4では、室温において80体積%酸素/20体積%窒素で1.0MPaGに加圧してから昇温し、酸素消費による圧力低下分は補わずに熱処理を行った。
アルカリ含浸EFBの調製までは実施例5と同様に行い(3%NaOH含浸)、熱処理方法を以下のように変更して実験を行った。すなわち、得られたアルカリ含浸EFBを100mlのプラスチックビーカーに入れ、そのビーカーを更に2Lのプラスチック容器に入れて蓋をした(2L容器の底には100mlの水を含ませた布を敷き、熱処理中に容器内が水蒸気で満たされるようにした)。また蓋には小さな穴を開け、内圧が上がらないようにした。このように調製した反応容器を80℃のオーブンに入れ、12時間静置して熱処理を行った(気相部の雰囲気は空気であり、酸素は十分に存在する条件。圧力は大気圧である)。熱処理後の工程は実施例1と同様に行った。結果を表1に示す。
表1に示したように条件を変えて、実施例8と同様にEFBの糖化実験を行った。結果を表1に示す。
アルカリ含浸EFBの調製、及び熱処理までは実施例5と同様に行い、まず、窒素下(酸素制限下)での100℃、1時間の熱処理を行った。続いて、熱処理後のEFBを実施例8と同様の方法で80℃、6時間の熱処理を行い(空気雰囲気、大気圧)、酸素供給下での熱処理を行った。熱処理後の工程は実施例1と同様に行った。結果を表1に示す。
原料として水で膨潤させたEFB(水ウェットEFB)を用いて糖化実験を行った。すなわち、100mlの耐圧容器に、水ウェットEFBを14.6g(含水率65.8%、固形分として5.0g)入れ、更にアルカリ水溶液として6.0%のNaOH水溶液を15.0g添加した(固液比=4.9、全液体成分は9.6g+15.0g=24.6g、EFB固形分は5.0g)。更に攪拌棒で軽く混合した後、密閉し、容器を空気で0.2MPaGに加圧した。これを40℃で1時間保持し、アルカリ水溶液をEFB中に含浸させた。以降の工程は実施例8と同様に実験を行った。結果を表2に示す。なお、アルカリ含浸EFB中の全液体成分のNaOH濃度は3.7%、アルカリ含浸量は8.5%と計算された。
アルカリ水溶液の含浸条件を変えた他は実施例12と同様にEFB(水ウェット体)の糖化実験を行った。アルカリ含浸条件は大気圧(空気加圧なし)で70℃、30分間とした。結果を表2に示す。
酵素回収工程における残渣の処理条件を以下のように変更した以外は実施例1と同様の前処理、糖化条件で実験を行った。残渣の1次処理の際には、まず、水15gを残渣に加えて攪拌混合した後、更に1%NaOH水溶液を微量添加し、処理液のpHを8.0に調整した。30分間緩やかに攪拌混合し、アルカリ条件での酵素の脱着処理を行った後、ろ過により1次処理液を回収した。2次処理は実施例1と同様に水で行い、2次処理液を回収した。回収液中の総酵素回収率を測定したところ、表3に示した結果となった。
表3に示したように、酵素回収工程における残渣の1次処理、及び2次処理の条件を変えて、実施例14と同様にEFBの糖化実験を行った。結果を表3に示す。なお実施例15では1次処理のpHを9.0に変更した。実施例16では1次処理をpH8.0で行い、2次処理ではNaOHを更に添加して処理液のpHを9.0に段階的に上げて酵素回収を行った。実施例17では、1次処理の際に、洗浄工程で得られる前処理液を処理液中に10%添加して実験を行った。結果を表3に示す。
酵素回収工程における残渣の処理条件を変更した以外は実施例2と同様の前処理、糖化条件で行った。残渣の1次処理の際には、表3に示した条件で、実施例14と同様に行った。結果を表3に示す。
実施例2と同様の方法でEFBの糖化工程まで行った後、実施例16と同様の方法で酵素回収工程を実施し(残渣のアルカリ処理はpH8.0→pH9.0)、回収液を得た。得られた回収液全量を限外ろ過(クラボウ社、セントリカットU-10使用、分画分子量1万、膜材質ポリサルホン)にかけ、約10gまで濃縮して回収酵素液とした。この回収酵素液を用いて、EFBの糖化実験を再度行った(酵素リサイクル反応)。すなわち、実施例2と同様に前処理EFBを調製し、以下のように反応混合物を調製した。
前処理EFB(水ウェット体)、テトラサイクリン塩酸塩1.6mg、シクロヘキシミド1.2mg、0.1M酢酸バッファー(pH5.5)10ml、回収酵素液約10g、フレッシュ酵素液(酵素Aと酵素Bの1:1混合液)0.06gを混合し、10%酢酸水でpH5.5に調整後、総質量を40.0gに水で調整した。なお、初回の20%分の(フレッシュ)酵素液を損失分として補充している。
これを実施例2と同様の条件で糖化した。生成した糖類をHPLCで分析した結果、グルコース収率は89%、C5糖収率は81%、糖合計収率は86%(糖収率は回収酵素液から持ち込まれる糖量を考慮した収率)であり、初回と同等の糖収率が得られた。
前処理工程におけるアルカリ水溶液を変えた点以外は、実施例8と同様の条件でEFB糖化実験を行った。アルカリ水溶液としては5%KOHを使用した。結果を表1に示す。
実施例1と同様にEFBの糖化実験を行った。ただし、糖化工程での反応混合物調製の際に、洗浄工程で得られた前処理液10.0gを添加し(その分酢酸バッファーを減量、合計40.0g、pH5.5)、糖化反応を行った。更に実施例1と同様に酵素回収工程を行った。糖収率はグルコース収率が83%、C5糖収率が85%、糖合計収率は84%であった。酵素回収率はCBHが71%、GLDが32%、XLDが65%であり、実施例1よりも高い糖収率、酵素回収率が得られた。
バイオマス原料を稲わらに変更した点以外は、実施例5と同様の方法で稲わらの糖化実験を行った。すなわち、原料として稲わら5.7g(長野県産、含水率11.6%、固形分として5.0g)を用い、3%NaOHを含浸させ(固液比10)、固液分離を行った。アルカリ含浸稲わらの質量は23.8gであり、固液分離後の固液比は3.8であった。更に実施例5と同様に熱処理以降の工程を行った。ただし、糖化工程では反応時間を20時間に変更した。糖収率は、対原料稲わら(未処理、固形分基準)の質量収率として、グルコース収率が32%、C5糖収率は13%、糖合計収率は45%であった。稲わらの糖類理論収率を70%とすると、対理論収率は64%であった。酵素回収率はCBHが95%、XLDが90%であり、非常に高い回収率が得られた。
実施例1と同様にアルカリ含浸EFBを調製した。ただしここでは含浸時の4%NaOH水溶液の量を75gとした(固液分離前の固液比15.1)。固液分離後、アルカリ含浸EFBを耐圧反応器に入れて空気雰囲気のまま密閉し、オイルバス中に投入して180℃(反応器内部温度)で15分間温度を維持し、熱処理を行った(高温短時間での熱処理)。その後、実施例1と同様に洗浄工程及び糖化工程を実施した。結果を表4に示す。ここでは糖化液の組成として、糖合計濃度(グルコース+C5糖濃度)、及びC5糖割合(糖合計に対するC5糖の質量割合)も合わせて示した。
表4に示したように種々の条件を変更し、実施例24と同様に実験を行った。結果を合わせて示す。実施例25~30では、アルカリ水溶液、及び熱処理条件を種々変えて実験を行った。実施例31では、アルカリ水溶液としてNaOHと炭酸ナトリウムの混合液(濃度は各1%)を用いた。実施例32では、4%アンモニア水溶液を用いた。
実施例33では、アルカリ水溶液として酸化カルシウムの0.5%スラリーを用いた。含浸時には減圧処理を行い、さらに反応器を密閉してローテーターにて転回混合しながら50℃で1時間保持する処理を行った後、固液分離を行い、アルカリ含浸EFBを得た。
実施例34では、固液分離前の固液比を低下させた。実施例35では、固液分離後の固液比を低下させた(固液分離後に、ろ紙を用いて液体分を吸水することで固液比を低下させた)。実施例36では、アルカリ水溶液の再利用実験を行った。すなわち、実施例24のアルカリ含浸後の固液分離(ろ過)で得られたろ液をアルカリ水溶液として再利用した。ただしこの際、不足するNaOH、及び水は、新たに補充して実施例24と同様のアルカリ水溶液とした。
実施例37では、熱処理時の気相部を空気雰囲気ではなく、50体積%酸素/50体積%窒素として、さらに仕込み時の初期圧を0.6MPaGとして、熱処理を行った。
比較例2では、比較例1と同様に固液分離を行わない方法で前処理を行った。熱処理条件は実施例24と同じ180℃、15分で行った。比較例3、及び4では、アルカリ水溶液の代わりに純水を用いて、実施例24と同様の方法で実験を行った。実験条件及び結果を表4に合わせて示す。
未分解原料への吸着酵素を再利用する実験を行った。すなわち、実施例25と同様の条件で糖化工程まで実験を行った。ただし糖化反応は、途中24時間の時点で反応を停止した。得られた反応混合物をろ過し、糖化液(約30g)とウェットの未分解原料(約10g)に分離した。続いてこの未分解原料と、別途調製した前処理EFB(実施例25と同条件)とを混合し、反応混合物(40.0g)を再調製して(ただし酵素は0.30gではなく、1/3量の0.10gを添加)、45℃で糖化反応を再開した。反応をさらに72時間行った時点で終了し、糖化液を分析した。実験全体の糖収率は(対理論収量、2倍量の原料基準)、グルコースが90%、C5糖が89%、糖合計では90%であった。
実施例25と同様にEFBの前処理工程を行った。次の洗浄工程では、実施例1と同じ水洗操作を4回繰り返した。すなわち、熱処理後のEFBに50.0gの純水を添加して10分間攪拌混合し、前処理分解物を溶出させた。ろ過により固液分離を行い、EFB固形分と水洗ろ液(前処理液1)を得た。この水洗操作をさらに3回繰り返し、水洗ろ液(前処理液2~4)と、十分に洗浄された前処理EFBを得た。
前処理液1~4の水分を蒸発させ固形分量(前処理分解物量)を測定した。その結果、前処理液1が1.54g、前処理液2が0.16g、前処理液3が0.03g、前処理液4が<0.01gの固形分を含んでおり、合計では1.73gであった。この結果より、水洗各段階での前処理分解物の残存量は、水洗操作1回で0.19g(=1.73-1.54)、2回で0.03g(=0.19-0.16)、3回で<0.01gと考えられた。また、4回水洗を繰り返した後の前処理EFBを乾燥させ、固形分量を測定したところ、3.2gであった。したがって、水洗各段階での前処理分解物の残存率(=残存する前処理分解物の固形分量/前処理EFBの固形分量)としては、水洗操作1回(洗浄水50g)で5.9%、2回(同100g)で0.9%、3回(同150g)で<0.3%と見積もられた。このように洗浄方法を変えることで、種々濃度で前処理分解物を含む前処理原料を調製することができ、さらにそれらを糖化することで、種々濃度で前処理分解物を含む糖化液を調製することも可能である。なお、実施例1(及び同等の実施条件の実施例)においては、前処理EFB中の前処理分解物の残存率は、約6%であったことがわかった。また糖化工程では、約6%の前処理分解物の存在下にて糖化反応を行っていたと考えられた。また糖化液中の前処理分解物の含有濃度は、全糖成分に対して、約7%であると考えられた。
前処理分解物の発酵への影響を検討した。実施例25と同様の方法で、ただしスケールをすべて20倍(原料EFB100g)としてEFBの糖化実験を行った。洗浄工程では、実施例39と同様に水洗操作を3回行った(前処理分解物の残存率<0.3%)。また糖化工程では、テトラサイクリン塩酸塩、及びシクロヘキシミドを添加せず、かつ、前処理EFBの固形分濃度を高めて糖化した(水を減量して反応混合物量を540gとした)。反応後の反応混合物を固液分離し、糖化液Aを得た。糖化液Aの糖合計濃度は11.0%であり、C5糖割合は38%であった。この糖化液と、水洗操作1回目で得たろ液(前処理液A、前処理分解物を固形分基準で3.3%含有)を表5に示した割合で混合し、前処理分解物を種々の濃度(割合)で含む糖化液B~Dを調製した。これらは種々の洗浄条件から得られる糖化液を模擬的に調製したものであり、糖化液Cが、水洗操作1回(実施例39を参照)の時に得られる糖化液と同等の組成である。
続いて、糖化液A~Dを用いてブタノール発酵を実施した。培地は、糖濃度40g/L(糖合計)に調整し、培地成分(TYA培地)を添加し、pHを6~7に調整して用いた。また対照実験として、EFB糖化液の代わりに試薬のグルコース液を用いた実験(対照1)、及び試薬のグルコース+キシロース(質量比6:4)液を用いた実験(対照2)を実施した。菌株はATCC株のクロストリジウムサッカロパーブチルアセトニカム(ATCC27021株)を用い、前培養の後、30℃で48時間、静置条件で発酵を行った。結果を表5に示す。発酵33時間と48時間での菌体濃度(OD660)、生成ブタノール濃度、及びブタノール質量収率(対消費糖)を示した。数値は2回の実験の平均値である。
実施例38では、バイオマスに吸着した酵素を再利用する逐次的な糖化反応を行い、酵素の削減効果、及び糖生産性の向上効果が示された。実施例39では、前処理分解物の除去工程の検討を行い、洗浄方法と、前処理分解物の残存率の関係を明らかにした。実施例40では、糖化液の発酵実験を行い、高い発酵成績が得られた。さらには、前処理分解物が糖化液中に存在した場合、発酵生成物濃度の向上、あるいは対糖収率の向上が認められることが分かった。
Claims (13)
- (1)リグノセルロース系バイオマスにアルカリ水溶液を含浸させた混合物を調製した後、固液分離を行って一部のアルカリ水溶液を除去し、熱処理を行う前処理工程、及び(2)前処理工程で得られたリグノセルロース系バイオマスを酵素で分解して糖化液を得る糖化工程を含むことを特徴とするリグノセルロース系バイオマスの糖化方法。
- 前処理工程において、下記(I)で算出される固液比が、固液分離前の混合物は2~20であり、固液分離後の混合物は1~6であることを特徴とする請求項1に記載の糖化方法。
式(I):
固液比=混合物中の全液体成分の総質量/混合物中のリグノセルロース系バイオマスの固形分質量 - 前処理工程において、熱処理を100~200℃で行うことを特徴とする請求項1又は2に記載の糖化方法。
- 糖化工程が、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物の存在下で行われることを特徴とする請求項1~3のいずれかに記載の糖化方法。
- 前処理工程と糖化工程の間に、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物の一部を除去する除去工程を含み、除去工程後のリグノセルロース系バイオマスにおいて、下記式(II)で算出される前処理分解物の残存率が2~20質量%であることを特徴とする請求項1~4のいずれかに記載の糖化方法。
式(II):
前処理分解物の残存率=残存する前処理分解物の固形分質量/リグノセルロース系バイオマスの固形分質量 - 糖化工程で得られる糖化液中のC5糖の割合が、全糖成分に対して20~50質量%であることを特徴とする請求項1~5のいずれかに記載の糖化方法。
- 糖化工程で得られる糖化液の全糖濃度が5~20質量%であることを特徴とする請求項1~6のいずれかに記載の糖化方法。
- 糖化工程で得られる未分解のリグノセルロース系バイオマスに吸着した酵素を再利用することを特徴とする請求項1~7のいずれかに記載の糖化方法。
- 前処理工程において、酸素を添加して熱処理を行うことを特徴とする請求項1~8のいずれかに記載の糖化方法。
- 糖化工程に続いて、糖化工程終了後に酵素を回収する酵素回収工程を含むことを特徴とする請求項1~9のいずれかに記載の糖化方法。
- 酵素回収工程において、未分解のリグノセスロース系バイオマスに吸着した酵素をアルカリ処理により脱着させ回収する工程を含むことを特徴とする請求項10に記載の糖化方法。
- 含水率が30~90%のリグノセルロース系バイオマスを前処理工程に供することを特徴とする請求項1~11のいずれかに記載の糖化方法。
- 糖化工程で得られる糖化液であって、前処理工程で生成する可溶化されたリグノセルロース系バイオマスの前処理分解物が、糖化液中の全糖成分に対して、2~20質量%含まれることを特徴とする糖化液。
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JP2015159755A (ja) * | 2014-02-27 | 2015-09-07 | 王子ホールディングス株式会社 | リグノセルロース含有バイオマスからのエタノール製造方法 |
JP5857149B1 (ja) * | 2015-07-16 | 2016-02-10 | 新日鉄住金エンジニアリング株式会社 | イネ科植物の茎葉由来発酵生成物の製造方法及び製造装置 |
WO2018001366A1 (zh) * | 2016-07-01 | 2018-01-04 | 青岛鑫垚地农业科技股份有限公司 | 有机-无机聚合保水肥料及其制备方法 |
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ITTO20111219A1 (it) * | 2011-12-28 | 2013-06-29 | Beta Renewables Spa | Procedimento migliorato di pre-impregnazione per la conversione di biomassa |
GB201315475D0 (en) | 2013-08-30 | 2013-10-16 | Green Biologics Ltd | Solvent production |
CN111218490A (zh) * | 2018-11-27 | 2020-06-02 | 南京理工大学 | 利用氨和助剂对木质纤维素进行预处理的方法 |
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WO2018001366A1 (zh) * | 2016-07-01 | 2018-01-04 | 青岛鑫垚地农业科技股份有限公司 | 有机-无机聚合保水肥料及其制备方法 |
US10544065B2 (en) | 2016-07-01 | 2020-01-28 | Qingdao Xinyaodi Agricultural Technology Joint-Stock Co., Ltd. | Organic-inorganic polymeric water-retaining fertilizer and preparation method of the same |
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