JP7652067B2 - Method for producing sugar solution - Google Patents

Description

The present invention relates to a method for producing a sugar liquid from cellulose-containing biomass that can be used as a fermentation raw material, etc.

Fermentation production processes for chemicals using carbohydrates as raw materials are used to produce various industrial raw materials. Currently, carbohydrates derived from edible raw materials such as sugar cane, starch, and sugar beet are used industrially as fermentation raw materials. However, due to the rising prices of edible raw materials due to the future increase in the world population and the ethical aspect of competing with food, it is necessary to develop a process to more efficiently produce sugar liquid from renewable non-edible resources, i.e., cellulose-containing biomass, or to efficiently convert the obtained sugar liquid into industrial raw materials as a fermentation raw material.

The carbohydrates in cellulose-containing biomass feedstocks are embedded in cell walls with complex structures. Therefore, to ensure that the enzymes act efficiently, it is preferable to subject the biomass feedstocks to an alkaline treatment prior to enzymatic hydrolysis.

For example, it has been disclosed that in order to improve the rate of enzymatic hydrolysis of cellulose , a specific pretreatment is applied to cellulose-containing biomass, in which a cellulose-containing material is brought into contact with an alkaline aqueous solution and the alkaline filtrate is repeatedly used, thereby producing a sugar liquid containing sugars at high purity (Patent Document 1).

It has also been disclosed that a plant biomass raw material containing cellulose is treated with a treating agent containing ammonia and then immersed in water at 40 to 100°C to dissolve polysaccharides into the water, thereby obtaining a raw material for enzymatic saccharification to be subjected to an enzymatic saccharification step. According to the present invention, enzymatic saccharification can be carried out efficiently, and therefore the method is used in a sugar production method with improved sugar production efficiency (Patent Document 2).

However, there is a quality issue in that fermentation inhibitors such as organic acids and aromatic compounds that are produced during the alkaline treatment are contaminated into the sugar liquid, and there is still a demand for a method for producing cellulose sugar liquid with reduced fermentation inhibitors.

WO2017/170552 WO2012/096236

The present invention aims to provide a method for reducing fermentation inhibitors, such as organic acids and aromatic compounds, that are produced by alkaline treatment of cellulose-containing biomass when producing sugar liquid from cellulose-containing biomass.

The inventors have now discovered that a sugar liquid with reduced fermentation inhibitors can be efficiently obtained by crushing cellulose-containing biomass to a specific degree of crushing, contacting it with an alkaline aqueous medium, adding water, subjecting the pretreated biomass to which water has been added to solid-liquid separation, and hydrolyzing the pretreated biomass.

The present invention is based on such findings and comprises the following [1] to [10].
[1] A method for producing a sugar liquid using cellulose-containing biomass as a raw material, comprising: (1) pulverizing the cellulose-containing biomass so that the weight ratio of the cellulose-containing biomass that does not pass through a sieve having an opening of 1 mm is 50% or less by dry weight;
Step (2): contacting the pulverized biomass obtained in step (1) with an alkaline aqueous medium to obtain a pretreated biomass;
step (3): adding water to the pretreated biomass obtained in step (2) and performing solid-liquid separation to obtain a cellulose-containing solid fraction; and step (4): hydrolyzing the cellulose-containing solid fraction obtained in step (3) to obtain a sugar liquid.
A method comprising:
[2] The method for producing a sugar liquid according to [1], wherein the step (2) is a step of passing an alkaline medium through the pulverized biomass obtained in the step (1) to obtain a pretreated biomass.
[3] The method for producing a sugar liquid according to [1] or [2], wherein the step (2) is a step of supplying the pulverized biomass obtained in the step (1) and an alkaline medium to a filter and passing the pulverized biomass and the alkaline medium through the filter.
[4] The method for producing a sugar liquid according to any one of [1] to [3], wherein the alkaline aqueous medium in the step (2) is an aqueous medium containing sodium hydroxide and/or potassium hydroxide.
[5] The method for producing a sugar liquid according to any one of [1] to [4], wherein the solid-liquid separation in the step (3) is pressing.
[6] The method for producing a sugar liquid according to any one of [1] to [5], wherein the cellulose-containing biomass is bagasse.
[7] Step (5): filtering the sugar liquid obtained in step (4) through a nanofiltration membrane or a reverse osmosis membrane, recovering the sugar liquid as a non-permeated liquid, and reusing the permeated liquid as water to be added to the pretreated biomass in step (3);
The method for producing a sugar liquid according to any one of [1] to [6], further comprising:
[8] A cellulose-containing solid content comprising a pulverized cellulose-containing biomass and an alkaline aqueous medium, the weight ratio of which not passing through a sieve with a mesh size of 1 mm being 50% or less by dry weight, and a moisture content of 50% by weight or more but less than 70% by weight.
[9] The cellulose-containing solid content according to [8], wherein the cellulose-containing biomass is bagasse.
[10] A pulverized cellulose-containing biomass having a weight ratio that does not pass through a sieve with 1 mm openings of 40 to 50% by dry weight.

According to the present invention, a high-purity sugar liquid can be obtained in which the fermentation inhibitors derived from the alkaline treatment of cellulose-containing biomass are reduced.

FIG. 1 is a process flow diagram showing an example of a method for producing a sugar liquid of the present invention.

The present invention is a method for producing a sugar liquid using cellulose-containing biomass as a raw material, and is characterized by including step (1): pulverizing the cellulose-containing biomass so that the proportion of the material that does not pass through a sieve with 1 mm mesh size is 50% or less by dry weight, step (2): contacting the pulverized biomass with an alkaline aqueous medium to obtain a pretreated biomass, step (3): adding water to the pretreated biomass to separate it into solid and liquid, and isolating the cellulose-containing solids, and step (4): hydrolyzing the cellulose-containing solids. An example of a process flow diagram of the present invention is shown in Figure 1, but the flow of the present invention is not limited thereto.

In the treatment of wastewater containing a powder that has been turned into a slurry, it is known that the higher the degree of grinding of the powder, the more difficult it is to dehydrate it (see Table 2 in J. Soc. Powder Technol., Japan, 38, 177-183 (2001)). However, in the present invention, when the pretreated biomass obtained by treating cellulose-containing biomass pulverized to a certain degree with alkali is hydrated and subjected to solid-liquid separation, the moisture content after solid-liquid separation is unexpectedly reduced compared to the case where such grinding is not performed, and this reduces the fermentation inhibitors derived from the pretreated biomass that are carried over during the subsequent hydrolysis step, making it possible to obtain a high-purity sugar liquid.

The steps of the present invention are explained below.

Cellulose-containing biomass refers to biological resources that contain at least cellulose. Suitable examples of cellulose-containing biomass include herbaceous biomass such as bagasse, switchgrass, napier grass, erianthus, corn stover, straw (rice straw, wheat straw), and empty fruit bunches of oil palm, as well as woody biomass such as trees, wood chips, and waste building materials, as well as biomass derived from aquatic environments such as algae and seaweed, and grain husk biomass such as the residue after starch extraction from corn husks, wheat husks, soybean husks, rice husks, and cassava meal. Herbaceous biomass such as bagasse, rice straw, and empty fruit bunches of oil palm are preferred.

The moisture content of the cellulose-containing biomass is not particularly limited, but preferred ranges are, for example, about 3% or more, about 3% to about 60%, about 5% or more, about 5% to about 60%, about 5% to about 55%, and about 5% to about 50%. The moisture content of the cellulose-containing biomass is measured by a method described in detail in the Examples.

In step (1), the cellulose-containing biomass is pulverized to a specific degree of pulverization to obtain pulverized biomass. The pulverization method is not particularly limited, and can be carried out using a machine commonly used for coarse pulverization of various materials, such as a ball mill, a vibration mill, a cutter mill, a hammer mill, a Willey mill, or a jet mill. This mechanical pulverization may be either dry or wet, but is preferably dry pulverization.

The degree of pulverization of the pulverized biomass obtained in step (1) is pulverized until the weight ratio (dry weight %) that does not pass through a sieve with a mesh size of 1 mm is about 50% or less by dry weight, preferably about 40% to about 50%, more preferably about 40% to about 45% or about 45% to about 50%, and the moisture content after solid-liquid separation is surprisingly reduced compared to when such pulverization is not performed, thereby reducing the fermentation inhibitors derived from the pretreated biomass that are brought in during the subsequent hydrolysis. The degree of pulverization is evaluated by drying the pulverized biomass to a moisture content of 20% or less and then passing the pulverized biomass through a sieve with a mesh size of 1 mm. In this specification, if the weight ratio (dry weight %) of the pulverized biomass that does not pass through a sieve with a mesh size of 1 mm is low, it may be expressed as a high degree of pulverization. The sieving method and conditions are in accordance with ISO 2591-1.

In step (2), the pulverized biomass obtained in step (1) is contacted with an alkaline aqueous medium to obtain a pretreated biomass. The pretreated biomass referred to here refers to a solid content of cellulose-containing biomass that has been subjected to an alkaline treatment so that the cellulose-containing biomass is efficiently hydrolyzed in the subsequent step (4).

The method for contacting the pulverized biomass obtained in step (1) with the alkaline aqueous medium is not particularly limited as long as it is an alkaline treatment method for cellulose-containing biomass well known to those skilled in the art, but preferably, the alkaline treatment method described in WO2017/170552 is used. This method is a method in which an alkaline aqueous medium is passed through a cellulose-containing biomass to obtain a pretreated biomass and an alkaline treatment liquid rich in coumaric acid and ferulic acid, and in the present invention, it is sufficient to pass an alkaline aqueous medium through the pulverized biomass.

An example of a method for passing an alkaline aqueous medium through pulverized biomass is a method in which the pulverized biomass and the alkaline aqueous medium are supplied to a filter and the pulverized biomass and the alkaline medium are passed through the filter in accordance with the method described in WO2017/170552. The pulverized biomass and the alkaline aqueous medium may be mixed in advance and supplied to the filter, or the two may be supplied separately to the filter. However, it is preferable to add the alkaline aqueous medium onto the pulverized biomass that has already been supplied to the filter.

The pH of the alkaline filtrate may be within the same range as that of the alkaline aqueous medium, and the preferred pH range is, for example, 7 to 12, 8 to 12, more preferably 9 to 12, and even more preferably 10 to 12. The pH of the alkaline filtrate tends to decrease as the reaction proceeds. This is because the soluble lignin component acts as a neutralizer as the alkaline reaction proceeds, and the progress of the reaction can be measured by the degree of decrease. In particular, the pH range of the pulverized biomass after contacting the pulverized biomass with the alkaline aqueous medium (after the reaction) can be appropriately adjusted depending on the initial alkali concentration, etc., and is preferably, for example, 7 to 12.5, 8 to 12.5, more preferably 8 to 12, and even more preferably 8 to 11. Measuring whether the pH of the alkaline filtrate is within the above range is an effective means of evaluating whether the reaction has progressed to a level sufficient for the subsequent hydrolysis step.

In addition, it is preferable that the temperature of the alkaline aqueous medium in contact with the pulverized biomass is substantially maintained. When the filter is equipped with a filtration section that passes the alkaline aqueous medium through the pulverized biomass, it is preferable that the temperature of the alkaline filtrate filtered through the filtration section is also substantially maintained. The temperature of the alkaline aqueous medium and the alkaline filtrate can be maintained by installing a known heat-retaining device or heating device in the filter.

The filtrate coming out of the filtration section after contacting with the alkaline aqueous medium may be circulated and brought into contact with the pulverized biomass again. The number of times this is repeated is not particularly limited, but suitable numbers are, for example, at least 2 times, 2 to 20,000 times, 2 to 10,000 times, 2 to 1,000 times, 3 to 10,000 times, 3 to 1,000 times, or 3 to 100 times.

Examples of alkaline aqueous media include alkaline aqueous solutions such as ammonia, ammonia water, and aqueous media containing hydroxides, but are preferably aqueous media containing sodium hydroxide and/or potassium hydroxide, more preferably aqueous media containing sodium hydroxide, and even more preferably aqueous solutions of sodium hydroxide and/or potassium hydroxide.

The alkali concentration of the alkaline aqueous medium can be calculated from the content of the alkaline substance (alkali solids such as hydroxides) in the alkali-containing medium. The upper limit of the alkali concentration of the alkaline aqueous medium is not particularly limited, but is preferably about 3, 2, 1.5, 1, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2% by weight, and the lower limit is preferably about 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5% by weight. The preferred range of the alkali concentration is, for example, about 0.05% by weight to about 0.3% by weight, about 0.1% by weight to about 3% by weight, about 0.1% by weight to about 2% by weight, more preferably about 0.1% by weight to about 2% by weight, about 0.25% by weight to about 1.5% by weight, about 0.25% by weight to about 1.5% by weight, and even more preferably about 0.25% by weight to about 1.0% by weight.

The lower limit of the pH of the alkaline aqueous medium is not particularly limited as long as it is alkaline, but is 7 or more, preferably 8 or more, more preferably 9 or more, and even more preferably 10 or more. The upper limit of the pH is not particularly limited as long as it is less than pH 14, but can be set to pH 13.5 or less from the viewpoint of reducing the amount of alkali used. The preferred pH range is, for example, 7 to 13.5, 8 to 13.5, more preferably 9 to 13.5, and even more preferably 10 to 13.5.

The upper limit of the temperature of the alkaline aqueous medium is not particularly limited, but is preferably about 110, 100, 95, 90, 80, 75, 70 ° C, and the lower limit is preferably about 35, 40, 50, 60, 65 ° C. In addition, the preferred temperature range of the alkaline aqueous medium is, for example, about 35 ° C to about 100 ° C, about 40 ° C to about 100 ° C, about 50 ° C to about 100 ° C, about 60 ° C to about 100 ° C, about 65 ° C to about 100 ° C, about 80 ° C to about 100 ° C, more preferred temperature ranges are about 60 ° C to about 100 ° C, about 65 ° C to about 100 ° C, about 80 ° C to about 100 ° C, and even more preferred temperature ranges are about 65 ° C to about 100 ° C or about 80 ° C to about 100 ° C.

The weight ratio of the alkaline aqueous medium to the pulverized biomass (dry weight) is not particularly limited, but preferred ranges are, for example, 100:1 to 2:1, 90:1 to 3:1, 50:1 to 5:1, 30:1 to 5:1, 25:1 to 7:1, 25:1 to 5:1, and 20:1 to 5:1.

The ratio of the alkali-containing aqueous medium to the pulverized biomass (dry weight) can be set using the amount of alkali used (also called the amount of alkali reaction) calculated by the calculation method of the amount of alkali reaction described in the Examples as an index. The preferred range of the amount of alkali used is, for example, from about 20 mg/g to about 300 mg/g, from about 30 mg/g to about 200 mg/g, from about 40 mg/g to about 200 mg/g, from about 45 mg/g to about 180 mg/g, from about 45 mg/g to about 150 mg/g, from about 50 mg/g to about 120 mg/g, and from about 60 mg/g to about 120 mg/g. The more preferred range of the amount of alkali used is from about 45 mg/g to about 150 mg/g, from about 50 mg/g to about 120 mg/g, and from about 60 mg/g to about 120 mg/g.

The time for which the pulverized biomass is contacted with the alkaline aqueous medium is not particularly limited, but the contact time may be, for example, from about 20 minutes to about 72 hours, from about 20 minutes to about 48 hours, from about 20 minutes to about 24 hours, from about 30 minutes to about 48 hours, from about 30 minutes to about 24 hours, from about 30 minutes to about 12 hours, from about 30 minutes to about 6 hours, or from about 30 minutes to about 3 hours.

The pretreated biomass obtained by contacting the pulverized biomass with the alkaline aqueous medium is preferably separated and recovered by a method known to those skilled in the art and supplied to the subsequent step (3). When a filter is used to contact the pulverized biomass with the alkaline aqueous medium, the solids remaining in the filter can be recovered as the pretreated biomass.

The moisture content of the pretreated biomass supplied to step (3) is not particularly limited, but preferred ranges are, for example, from about 50% by weight to about 99% by weight, from about 60% by weight to about 99% by weight, from about 70% by weight to about 99% by weight, from about 80% by weight to about 99% by weight, or from about 80% by weight to about 95% by weight, more preferably from about 80% by weight to about 99% by weight, and even more preferably from about 80% by weight to about 95% by weight. The moisture content of the pretreated biomass is measured by a method described in detail in the Examples.

In step (3), water is added to the obtained pretreated biomass, and solid-liquid separation is performed in order to separate fermentation inhibitors from the pretreated biomass. This step reduces fermentation inhibitors that are generated by the alkali treatment of the cellulose-containing biomass contained in the pretreated biomass and are thought to be derived from lignin in the cellulose-containing biomass. Specific examples of fermentation inhibitors include organic acids such as formic acid, acetic acid, citric acid, and lactic acid, aromatic compounds such as ferulic acid, coumaric acid, vanillin, vanillic acid, acetovanillin, 4-hydroxybenzoic acid, syringic acid, and gallic acid, and furan compounds such as HMF and furfural.

The amount of water added to the pretreated biomass is the weight ratio of the pretreated biomass (dry weight) to the water added, and is not particularly limited; however, preferred ranges are, for example, 1:1 to 1:100, 1:1 to 1:50, 1:1 to 1:30, 1:1 to 1:20, 1:1 to 1:10, 1:1 to 1:5, and 1:1 to 1:3.

The pH of the water added to the pretreated biomass is not particularly limited, but preferred ranges are, for example, 3 to 9, 3 to 8, 3 to 7, 4 to 9, 4 to 8, 4 to 7, 5 to 9, 5 to 8, and 5 to 7.

The temperature of the water added to the pretreated biomass is not particularly limited, but preferred ranges are, for example, 10 to 60°C, 10 to 50°C, 10 to 40°C, 20 to 60°C, 20 to 50°C, 20 to 40°C, 30 to 60°C, 30 to 50°C, and 30 to 40°C.

Water can be added to the pretreated biomass by mixing the pretreated biomass with the water to be added in a reaction tank, or the reaction tank may be equipped with an agitator. The shape of the reaction tank is not important as long as it allows contact between the pretreated biomass and the water to be added. The pretreated biomass can be transported to the reaction tank by a conveyor or by natural fall. As long as contact between the pretreated biomass and water can be established, water can be added during transport by a conveyor or the like, even if it is not in a reaction tank.

As the apparatus used for solid-liquid separation, centrifugation, squeezing, etc. can be applied, with squeezing being preferred. As the apparatus for squeezing, screw press, belt press, filter press, etc. can be used, with the screw press being preferred.

From the viewpoint of reducing fermentation inhibitors derived from the pretreated biomass, the moisture content of the cellulose-containing solids after solid-liquid separation is preferably from about 50% by weight to about 85% by weight, more preferably from about 50% by weight to less than about 70% by weight. The moisture content of the cellulose-containing solids is measured by the moisture content measurement method described in the Examples.

The content of the alkaline aqueous medium in the cellulose-containing solids is preferably from about 5 mg/g biomass to about 40 mg/g biomass, more preferably from about 7 mg/g biomass to about 35 mg/g biomass, and even more preferably from about 10 mg/g biomass to about 30 mg/g biomass. By reducing the content of the alkaline aqueous medium in the cellulose-containing solids to the above range, it is possible to reduce fermentation inhibitors derived from the pretreated biomass that are carried over during the subsequent hydrolysis.

In step (4), the cellulose -containing solid content is hydrolyzed to obtain a sugar solution. In the hydrolysis step, known hydrolysis methods such as acid hydrolysis, alkali hydrolysis, and enzymatic hydrolysis can be applied, but it is preferable to hydrolyze the cellulose- containing solid content with an enzyme in an aqueous medium. The sugar solution obtained by such an enzymatic hydrolysis step can be obtained as an aqueous solution containing oligosaccharides and/or monosaccharides, such as glucose, xylose, arabinose, galactose, xylobiose, and cellobiose.

The enzyme to be used is not particularly limited as long as it is a cellulose or hemicellulose hydrolase, but may be, for example, a relatively inexpensive commercially available product. Examples of cellulase enzyme preparations that can be used include "Acremonium Cellulase" (Meiji Seika Pharma Co., Ltd.), which is an enzyme derived from the genus Acremonium, "Accelrace Duet" (Danisco Japan Co., Ltd.), and "Celluclast" 1.5L (Novozymes Co., Ltd.), which are enzymes derived from the genus Trichoderma. Examples of hemicellulase enzyme preparations that can be used include "Optimash BG" (Genencor Co., Ltd.). The origin of the enzyme is not particularly limited, but more preferably it is an enzyme derived from a filamentous fungus. Enzymes derived from filamentous fungi are rich in decomposing enzymes for polysaccharides derived from cellulose-containing biomass, such as cellulase, hemicellulase, and β-glucosidase, and are advantageous in carrying out a hydrolysis reaction on biomass after alkali treatment.

The enzymes used may be used alone or in combination, taking into consideration the properties of the enzyme preparation, the composition of the desired product, etc. The amount of the suitable enzyme is also not particularly limited and can be determined appropriately. The amount of such an enzyme can be, for example, 0.01 g to 1 g per 1 g of raw material substrate, and preferably 0.001 g to 0.1 g. The temperature, pH, and time of the enzymatic hydrolysis can be appropriately set depending on the properties and combination of the enzymes. For example, the suitable range is the temperature from 30 to 60°C, more preferably from 35 to 50°C. The pH is, for example, from pH 3 to 8, preferably from pH 4 to 7. The reaction time is, for example, from 1 hour to 48 hours, preferably from 6 to 24 hours.

The concentration of glucose, xylose or xylobiose in the sugar solution obtained in step (4) is not particularly limited, and can be appropriately set by adjusting the reaction conditions of each step. Suitable glucose concentrations are, for example, about 5 g/L to about 1000 g/L, about 5 g/L to about 700 g/L, about 5 g/L to about 550 g/L, or about 10 g/L to about 550 g/L. Suitable xylose concentrations are, for example, about 1 g/L to about 100 g/L, about 1 g/L to about 50 g/L, or about 1 g/L to about 10 g/L. Suitable xylobiose concentrations are, for example, about 1 g/L to about 100 g/L, about 1 g/L to about 50 g/L, about 1 g/L to about 20 g/L, or about 1 g/L to about 15 g/L.

The sugar liquid produced in step (4) can be used as is for various industrial applications, but may be subjected to post-processing if desired, such as membrane processing, centrifugation, concentration, drying, etc.

Preferred membrane treatments for the sugar liquid produced in step (4) include nanofiltration membrane or reverse osmosis membrane treatments.

Nanofiltration membranes, also known as nanofilters (nanofiltration membranes, NF membranes), are generally defined as membranes that allow monovalent ions to pass through and block divalent ions. These membranes are thought to have tiny pores of about a few nanometers in size, and are primarily used to block tiny particles, molecules, ions, salts, etc. in water.

Reverse osmosis membranes, also known as RO membranes, are generally defined as "membranes with desalination capabilities, including for monovalent ions." They are thought to have ultra-small pores of a few angstroms to a few nanometers, and are primarily used to remove ionic components in seawater desalination and ultrapure water production.

The nanofiltration membrane or reverse osmosis membrane treatment is a treatment in which the sugar liquid produced in step (4) is filtered through a nanofiltration membrane or reverse osmosis membrane to block or filter out dissolved sugars, particularly monosaccharides such as glucose and xylose, and oligosaccharides such as xylobiose and cellobiose, on the non-permeated side of the membrane, and allow fermentation inhibitors remaining in the sugar liquid to permeate as a permeate, and can be carried out by the method described in WO2010/067785.

The sugar liquid recovered from the non-permeated side of the nanofiltration membrane or reverse osmosis membrane has further reduced fermentation inhibitors compared to before the membrane treatment, and the fermentation performance can be improved compared to before the membrane treatment. Furthermore, the permeate recovered from the permeated side of the nanofiltration membrane or reverse osmosis membrane can be used as water to be added to the pretreated biomass in the above step (3) (step (5)). This step can reduce the amount of water used in all steps, and can have the unexpected effect of further removing aromatic compounds, which are fermentation inhibitors, by the solid-liquid separation in step (3).

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Furthermore, unless otherwise indicated, the units and measurement methods described in this specification are in accordance with the Japanese Industrial Standards (JIS).

The various analytical methods used in this example are as follows:

[Method of measuring sugar concentration]
The sugar concentration in the saccharified solution obtained in each of the Examples and Comparative Examples was quantified by comparison with a standard sample under the high performance liquid chromatography (HPLC) conditions shown below.
Column: Luna _NH2 (Phenomenex)
Mobile phase: ultrapure water; acetonitrile = 25:75
Flow rate: 0.6mL/min
Reaction solution: None Detection method: RI (differential refractive index)
Temperature: 30°C.

[Method for measuring the concentration of furan and aromatic compounds]
The concentrations of furan compounds (HMF, furfural) and phenolic compounds (vanillin, etc.) contained in the sugar solution were analyzed by HPLC under the conditions shown below and quantified by comparison with standards.
Column: Synergi HydroRP 4.6 mm x 250 mm (Phenomenex)
Mobile phase: acetonitrile-0.1% H ₃ PO ₄ (flow rate 1.0 mL/min) Detection method: UV (283 nm)
Temperature: 40°C.

[Method of measuring organic acid concentration]
The organic acids (acetic acid, formic acid) contained in the sugar solution were analyzed by HPLC under the conditions shown below and quantified by comparison with standards.
Column: Shim-Pack SPR-H and Shim-Pack SCR101H (Shimadzu Corporation) in series Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL/min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM bistris, 0.1 mM EDTA.2Na (flow rate 0.8 mL/min)
Detection method: electrical conductivity Temperature: 45°C.

[Method for measuring moisture content]
The moisture content of the cellulose-containing biomass, pretreated biomass, and cellulose-containing solid used in the following experiments was measured. The moisture content (wt%; hereinafter, simply indicated as %) was measured using an infrared moisture meter (FD-720, Kett Electric Laboratory) by holding the sample at a temperature of 120°C and measuring the moisture content, which is the value obtained from the difference between the stable value after evaporation and the initial value.

For reference, the moisture contents of various cellulose-containing biomass measured by this method are shown in Table 1. Bagasse, rice straw, and oil palm empty fruit bunches are classified as herbaceous biomass.

[Calculation method for alkali reaction amount]
Regarding the method for calculating the alkali reaction amount, for example, when a (g) of cellulose-containing biomass raw material a (g) having a moisture content x (%) is added and reacted with an aqueous sodium hydroxide solution b (g) of y (%), the alkali reaction amount (unit: mg/g-dry biomass) was calculated using the following formula 1.

Amount of alkali reaction = y x b x 1000/{(100-x) x a}... (Equation 1).

Comparative Reference Example 1: Relationship between the degree of crushing of bagasse during hydrothermal treatment and the moisture content of the cellulose-containing solid content In step (1), bagasse with a moisture content of 50% was crushed using a cutter mill (Nara Machinery Works, Ltd., Varionyx BRX-400). The crushing conditions were set to a screen hole diameter of 30 mm, and crushing was performed while supplying at a rotation speed of 600 rpm and a supply rate of 1000 kg/hr. The crushed biomass was dried to a moisture content of 20% in order to measure the dry weight ratio, and passed through a sieve with 1 mm openings under the conditions of ISO 2591-1. The weight ratio that did not pass through was measured and found to be 45%.

In step (2), the crushed bagasse (moisture content: 50%) was subjected to hydrothermal treatment at high pressure at 180°C for 10 minutes with a solid content of 5%. The obtained pretreated bagasse was filtered through a stainless steel sieve with 3 mm openings (opening rate: 30%), and the solids remaining on the top surface of the sieve (pretreated biomass) were pressed against the surface of the sieve by hand and squeezed. The moisture content of the obtained pretreated biomass was 90%.

In step (3), water was added to the pretreated biomass in an amount 1.6 times the solid content (dry weight), and the pretreated biomass to which water had been added was subjected to solid-liquid separation using a small laboratory screw press (HX100, manufactured by Fukoku Kogyo Co., Ltd., frequency 10 Hz). The moisture content of the cellulose-containing solid content after solid-liquid separation was measured and found to be 75%. The results are shown in Table 2.

Comparative Reference Example 2: Relationship between bagasse crushing degree during hydrothermal treatment and moisture content of cellulose-containing solid content The crushing conditions were the same as those of Comparative Reference Example 1, except that the screen hole diameter of the cutter mill was 40 mm. In addition, the crushed biomass was dried to a moisture content of 20% in order to measure the dry weight ratio, and sieved through a sieve with 1 mm openings under the conditions of ISO 2591-1. The weight ratio that did not pass through was measured and found to be 50%. The moisture content of the cellulose-containing solid content after solid-liquid separation was 73%. The results are shown in Table 2.

Comparative Reference Example 3: Relationship between bagasse crushing degree during hydrothermal treatment and moisture content of cellulose-containing solid content The crushing conditions were the same as those of Comparative Reference Example 1, except that the screen hole diameter of the cutter mill was 50 mm. In addition, the crushed biomass was dried to a moisture content of 20% in order to measure the dry weight ratio, and sieved through a sieve with 1 mm openings under the conditions of ISO 2591-1. The weight ratio that did not pass through was measured and found to be 55%. The moisture content of the cellulose-containing solid content after solid-liquid separation was 70%. The results are shown in Table 2.

Comparative Reference Example 4: Relationship between bagasse pulverization degree during alkali treatment and moisture content of cellulose-containing solids In step (1), pulverization was performed under the same pulverization conditions as in Comparative Example 3, except that the screen hole diameter of the cutter mill was 50 mm. The pulverized biomass was dried to a moisture content of 20% in order to measure the dry weight ratio, and passed through a sieve with 1 mm openings under the conditions of ISO 2591-1. The weight ratio that did not pass through was measured and found to be 55%.

In step (2), 5.0 kg of the obtained crushed bagasse (water content 50%) was put into a multifunctional extractor (manufactured by Izumi Food Machinery Co., Ltd.), 45 kg of a sodium hydroxide solution of a predetermined concentration (initial temperature: 90°C, pH about 13) was added from the spray ball at the top of the tank of the multifunctional extractor, and the liquid obtained by gravity filtration from the filter net installed in the tank (alkaline filtrate) was added again from the spray ball. The reaction was carried out for a predetermined time while monitoring the temperature by providing a heating mechanism between the filter net and the upper spray ball. During the reaction, the alkaline filtrate was adjusted so that it did not drop below 90°C. In addition, the stirring blade attached to the multifunctional extractor was not used, and the bagasse and cellulose-containing solids were placed on the filter net, and no operation was performed to shape them or turn them into a slurry using a stirring blade or the like. The alkaline filtrate was kept circulating for a predetermined reaction time. The obtained sample was further filtered through a stainless steel sieve with 3 mm openings (opening ratio: 30%), and the solid matter remaining on the top surface of the sieve (pretreated biomass) was pressed against the surface of the sieve by hand and squeezed. The moisture content of the obtained pretreated biomass was 90%.

In step (3), water was added to the pretreated biomass in an amount 1.6 times the solid content (dry weight), and the pretreated biomass was subjected to solid-liquid separation using a small laboratory screw press. The moisture content of the cellulose-containing solid content after solid-liquid separation was measured and found to be 75%. The results are shown in Table 2.

Reference Example 1: Relationship between bagasse crushing degree during alkali treatment and moisture content of cellulose-containing solids The crushing conditions were the same as those of Comparative Reference Example 4, except that the screen hole diameter of the cutter mill was 40 mm. In addition, the crushed biomass was dried to a moisture content of 20% in order to measure the dry weight ratio, and sieved through a sieve with 1 mm openings under the conditions of ISO 2591-1. The weight ratio that did not pass through was measured and found to be 50%. The moisture content of the cellulose-containing solids after solid-liquid separation was 64%. The results are shown in Table 2.

Reference Example 2: Relationship between bagasse crushing degree during alkali treatment and moisture content of cellulose-containing solids The crushing conditions were the same as those of Comparative Reference Example 4, except that the screen hole diameter of the cutter mill was 30 mm. In addition, the crushed biomass was dried to a moisture content of 20% in order to measure the dry weight percentage, and sieved through a sieve with 1 mm openings under the conditions of ISO 2591-1, and the weight percentage was measured to be 45%. In addition, the moisture content of the cellulose-containing solids after solid-liquid separation was 60%. The results are shown in Table 2.

As can be seen from Table 2 , when the pretreatment is a hydrothermal treatment, as the proportion of the pulverized biomass that does not pass through a 1 mm sieve (dry weight %) decreases (the degree of pulverization increases), the moisture content (%) of the cellulose-containing solids after solid-liquid separation increases, and this result is consistent with information known to those skilled in the art that the higher the degree of pulverization of the powder in the treatment of wastewater in which the powder is slurried, the more difficult it is to dehydrate it (see Table 2 in J. Soc. Powder Technol., Japan, 38, 177-183 (2001)). On the other hand, when the pretreatment is an alkali treatment, as the proportion of the pulverized biomass that does not pass through a 1 mm sieve (dry weight %) decreases (the degree of pulverization increases), the moisture content (%) of the cellulose-containing solids after solid-liquid separation decreases, contrary to information known to those skilled in the art, and it has been found that by increasing the degree of pulverization of the pulverized biomass, it is possible to reduce fermentation inhibitors derived from the pretreatment that are mixed in when producing a sugar solution by hydrolysis in the subsequent stage.

Comparative Example 1: Example of production of sugar liquid from bagasse (steps (1) and (3) not performed)
The same pulverization and pretreatment conditions as those of Comparative Reference Example 4 were carried out to obtain a pretreated biomass. The obtained pretreated biomass was not subjected to the addition of water and solid-liquid separation in step (3), but was added with pure water and 35% hydrochloric acid in step (4) so that the solid concentration on a dry basis was 5% and the pH was 5.0 to prepare a slurry containing a cellulose-containing solid content. 5 mL of "Accelrace Duet", an enzyme manufactured by Danisco Japan Co., Ltd., was added to 500 mL of the obtained slurry, and the slurry temperature was maintained at 50 ° C. and constantly stirred to perform a reaction for 8 hours. The sugar, acetic acid, and aromatic compound concentrations of the obtained reaction solution were measured, and the weights of acetic acid and coumaric acid per 100 g of the obtained glucose were calculated. The results are shown in Table 3.

Comparative Example 2: Example of production of sugar liquid from bagasse (step (1) not performed)
The same pulverization conditions, pretreatment conditions, water addition and solid-liquid separation conditions were carried out as in Comparative Example 4. Using the cellulose-containing solid fraction after solid-liquid separation, the same hydrolysis operation as in Comparative Example 1 was carried out as step (4). The concentrations of sugar, acetic acid and aromatic compounds in the resulting reaction solution were measured, and the weights of acetic acid and coumaric acid per 100 g of the resulting glucose were calculated. The results are shown in Table 3.

Comparative Example 3: Example of production of sugar liquid from bagasse (step (3) not performed)
The same operation as in Comparative Example 4 was carried out except that the milling conditions were a cutter mill screen hole diameter of 40 mm and solid-liquid separation was carried out without adding water to the pretreated biomass. Using the cellulose-containing solid fraction obtained after solid-liquid separation, a hydrolysis operation similar to that in Comparative Example 1 was carried out as step (4). The sugar, acetic acid, and aromatic compound concentrations of the obtained reaction solution were measured, and the weights of acetic acid and coumaric acid per 100 g of the obtained glucose were calculated. The results are shown in Table 3.

Example 1: Production of sugar liquid from bagasse (steps (1) to (4) carried out)
Using the cellulose-containing solid fraction after solid-liquid separation obtained in Reference Example 1, a hydrolysis operation was carried out as step (4) in the same manner as in Comparative Example 1. The concentrations of sugar, acetic acid, and aromatic compounds in the resulting reaction solution were measured, and the weights of acetic acid and coumaric acid per 100 g of the resulting glucose were calculated. The results are shown in Table 3.

Example 2: Example of production of sugar liquid from bagasse (steps (1) to (4) carried out)
Using the cellulose-containing solid fraction after solid-liquid separation obtained in Reference Example 2, a hydrolysis operation was carried out as step (4) in the same manner as in Comparative Example 1. The concentrations of sugar, acetic acid, and aromatic compounds in the resulting reaction liquid were measured, and the weights of acetic acid and coumaric acid per 100 g of the resulting glucose were calculated. The results are shown in Table 3.

Example 3: Production of sugar liquid from bagasse (steps (1) to (5) carried out)
The sugar solution after hydrolysis in step (4) obtained in Example 1 was subjected to solid-liquid separation by centrifugation (Saito Centrifuge Co., Ltd., 2,000 rpm), and then filtered using a microfiltration membrane (Millipore, PVDF membrane with a pore size of 0.05 μm). The sugar solution was then passed through a reverse osmosis membrane (Toray Industries, Inc., cross-linked wholly aromatic polyamide reverse osmosis membrane UTC80) to recover the sugar solution as a concentrated liquid, and water was recovered as a permeate so as to be concentrated to one-fourth the volume of the raw water.

Steps (1) to (4) were carried out again under the same conditions as in Example 1, and the water added in step (3) was the water recovered as the permeate of the reverse osmosis membrane (step (5)). The sugar, acetic acid, and aromatic compound concentrations of the sugar solution obtained in step (4) were measured, and the weights of acetic acid and coumaric acid per 100 g of the obtained glucose were calculated. The results are shown in Table 3.

As can be seen from Table 3, the weight of acetic acid and coumaric acid relative to glucose was relatively reduced in Examples 1 to 3 compared to Comparative Examples 1 to 3 in which none of steps (1) to (4) were performed. In particular, the comparison results of Comparative Example 2 and Examples 1 and 2 revealed that when the weight ratio (dry weight %) of powdered biomass that does not pass through a 1 mm mesh sieve exceeds 50%, the concentration of fermentation inhibitors in the sugar solution increases. Furthermore, the results of Examples 1 and 3 revealed that the concentration of coumaric acid in the sugar solution was further reduced by performing step (5).

Claims (7)

A method for producing a sugar liquid using cellulose-containing biomass as a raw material, comprising the steps of:
Step (1): A step of crushing a cellulose-containing biomass so that the weight ratio that does not pass through a sieve having an opening of 1 mm is 50% or less by dry weight;
Step (2): contacting the pulverized biomass obtained in step (1) with an alkaline aqueous medium to obtain a pretreated biomass;
step (3): adding water to the pretreated biomass obtained in step (2) and performing solid-liquid separation to obtain a cellulose-containing solid fraction having a moisture content of 50% by weight or more and less than 70% by weight; and step (4): hydrolyzing the cellulose-containing solid fraction obtained in step (3) to obtain a sugar liquid.
A method comprising: The method for producing sugar liquid according to claim 1, wherein the step (2) is a step of passing an alkaline medium through the pulverized biomass obtained in the step (1) to obtain a pretreated biomass. The method for producing sugar liquid according to claim 1 or 2, wherein the step (2) is a step of supplying the pulverized biomass and the alkaline medium obtained in the step (1) to a filter and passing the pulverized biomass and the alkaline medium through the filter. The method for producing a sugar solution according to any one of claims 1 to 3, wherein the alkaline aqueous medium in step (2) is an aqueous medium containing sodium hydroxide and/or potassium hydroxide. The method for producing sugar liquid according to any one of claims 1 to 4, wherein the solid-liquid separation in step (3) is pressing. The method for producing sugar liquid according to any one of claims 1 to 5, wherein the cellulose-containing biomass is bagasse. Step (5): filtering the sugar liquid obtained in step (4) through a nanofiltration membrane or a reverse osmosis membrane to recover the sugar liquid as a non-permeated liquid, and reusing the permeated liquid as water to be added to the pretreated biomass in step (3);
The method for producing a sugar liquid according to any one of claims 1 to 6, further comprising:

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