WO2025079414A1 - Method and apparatus for producing target substance - Google Patents

Abstract

The present disclosure provides a method and an apparatus with which a target substance can be produced more efficiently from a lignocellulose-based material. The target substance is produced by: (a) allowing saccharification and a microbial reaction to proceed in parallel in a reaction solution containing a pretreated lignocellulose-based material, at least one saccharifying enzyme, and a microorganism, thereby producing at least one target substance; (b) subjecting at least a portion of the reaction solution to a solid-liquid separation treatment during or after the saccharification and the microbial reaction in step (a), thereby obtaining a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and a reaction residue; and (c) circulating at least a portion of the at least one saccharifying enzyme contained in the fraction (X) obtained in step (b) back to the reaction solution in step (a).

Description

Method and apparatus for producing target material

The present disclosure relates to a method and apparatus for producing a target substance.
This application claims priority based on Japanese Patent Application No. "Patent Application No. 2023-174873" submitted to the Japan Patent Office on October 9, 2023, and the contents of the Japanese patent application are incorporated herein for all purposes. It constitutes a part of this specification.

In recent years, from the perspective of effective measures against global warming caused by greenhouse gas emissions and realizing sustainable industries, biofuels such as bioethanol and biomass-derived chemicals, including biomass plastics, have been attracting attention as alternatives to petrochemical products. Currently, edible biomass such as sugarcane and corn is generally used as the raw material for such biomass-derived chemicals.

However, since edible biomass is originally derived from crops produced for food and feed use, it is a limited resource, and there is a risk that it will become difficult to procure a stable supply of raw materials if economic circumstances such as price hikes occur. Therefore, in recent years, attempts have been made to develop manufacturing processes for biomass-derived chemical products using non-edible biomass derived from various herbaceous plants and woody plants as a raw material. Non-edible biomass is a material that contains a large amount of lignocellulose, which is the main component of plant cell walls and gives strength to the plant body. Lignocellulose is composed of cellulose, hemicellulose, and lignin, and non-edible biomass is subjected to pretreatment to reduce lignin, which cannot be used as a substrate for microbial reactions. Next, cellulose and hemicellulose are converted into C6 and C5 monosaccharides such as glucose by saccharification treatment using saccharification enzymes, and various chemical products are produced by microbial fermentation using these monosaccharides as substrates.

For example, Patent Document 1 describes a method for producing ethanol, which includes a pretreatment step in which a fine bark slurry with a pH of 4 to 7 is prepared from bark raw material by treatment with an alkaline solution containing calcium hydroxide and mechanical treatment, a parallel saccharification and fermentation treatment step in which the fine bark slurry is treated by a parallel saccharification and fermentation method, an ethanol and enzyme recovery step in which the produced ethanol is recovered from the fermentation liquid and the enzyme-containing liquid is returned to the parallel saccharification and fermentation treatment step, and a calcium hydroxide recovery step in which the calcium content contained in the fermentation residue fraction separated from the fermentation liquid is recovered in the form of calcium hydroxide and circulated as calcium hydroxide for the alkaline solution. Patent Document 1 suggests that this method is a method for avoiding the inhibition of saccharification and fermentation reactions caused by the accumulation of alkaline ions and counter ions by using calcium hydroxide as the alkaline compound in the pretreatment by alkaline treatment.

Furthermore, Patent Document 2 describes a method for producing ethanol, which includes a sieving process in which a lignocellulosic raw material suspension made into a raw material is sieved using a 60-600 mesh filter to separate fibers from the lignocellulosic raw material (raw material suspension), and an enzymatic saccharification process in which the fibers separated in the sieving process are saccharified. As described above, the method described in Patent Document 2 improves the sugar yield in the saccharification reaction and the ethanol yield in ethanol fermentation by providing a sieving process in which fibers are separated using a filter having a mesh size within a specified range, but in reality, it also employs a chemical treatment process using alkaline chemicals or the like prior to the enzymatic saccharification process.

Furthermore, Patent Document 3 describes a method for producing ethanol, which includes a chemical treatment step of chemically treating lignocellulosic raw materials, and a saccharification and fermentation step of saccharifying the chemically treated raw material suspension with an enzyme and fermenting it with a fermenting microorganism, and is characterized in that the ratio of the weight of solids after the chemical treatment to the weight of solids before the chemical treatment in the chemical treatment step is controlled within a predetermined range. Patent Document 3 suggests that, as described above, by controlling the ratio of the weight of solids after the chemical treatment to the weight of solids before the chemical treatment in the chemical treatment step within a predetermined range, it is possible to reduce the amount of undecomposed residue discharged after saccharification, and to maintain high ethanol productivity for a long period of time.

Furthermore, Patent Document 4 describes a method for producing a chemical product, which includes filtering a culture solution of a microorganism with a separation membrane, retaining or refluxing the unfiltrate in the culture solution, adding a fermentation raw material to the culture solution, and recovering a product in the filtrate, wherein the microorganism is a microorganism that is subject to catabolite repression, and the fermentation raw material includes hexose and pentose. According to Patent Document 4, in a fermentation process using a microorganism that is subject to catabolite repression, when a mixed sugar of pentose and hexose is used as a substrate, assimilation of pentose is suppressed, but it is suggested that by adopting a configuration for continuous culture using a separation membrane, consumption of pentose by the microorganism is improved, and the yield of the chemical product is improved.

Furthermore, Patent Document 5 describes a method for producing a compound derived from lignocellulosic biomass, which includes a stirring step in which water is added to the fermentation residue obtained after fermenting a saccharification liquid derived from lignocellulosic biomass and the fermentation residue solution is stirred while maintaining a predetermined temperature, and a circulation step in which the fermentation residue solution produced in the stirring step is mixed with pretreated lignocellulosic biomass and saccharification enzymes in a saccharification step, and reused for saccharification and circulated. The method described in Patent Document 4 is recognized as employing the above stirring and circulation steps in order to reuse the fermentation residue obtained after fermenting a saccharification liquid derived from lignocellulosic biomass in the saccharification reaction in the system, since the saccharification enzymes that still retain enzymatic activity are adsorbed to the fermentation residue.

JP 2011-152067 A JP 2013-240320 A JP 2015-167484 A WO2013/105651 Patent Publication No. 2018-64515

The object of the present invention is to provide a method and apparatus for more efficiently producing target substances from lignocellulosic materials.

According to aspects of the present invention, the following is provided:

[1] (a) carrying out saccharification and a microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism, thereby producing at least one target substance;
(b) subjecting at least a portion of the reaction liquid to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue;
(c) circulating at least a portion of the at least one saccharifying enzyme contained in fraction (X) obtained in step (b) to the reaction solution in step (a);
A method for producing a target substance, comprising:

[2] (d) circulating at least a portion of the microorganisms and reaction residues contained in the fraction (Y) obtained in the step (b) to the reaction solution in the step (a);
The method according to [1], further comprising:

[3] (a) carrying out saccharification and a microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism, thereby producing at least one target substance;
(b) subjecting at least a portion of the reaction liquid to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue;
(d) circulating at least a portion of the microorganisms and reaction residues contained in the fraction (Y) obtained in the step (b) to the reaction solution in the step (a);
A method for producing a target substance, comprising:

[4] The method according to any one of [1] to [3], in which in step (a), at least a portion of the reaction liquid is continuously transferred through a plurality of reaction systems connected in series, and saccharification and microbial reaction are continuously carried out in each of the plurality of reaction systems, and in step (b), at least a portion of the reaction liquid present in the reaction system located at the very end of the plurality of reaction systems is subjected to the solid-liquid separation treatment.

[5] The method according to any one of [1] to [4], in step (c), fraction (X) obtained in step (b) is subjected to a concentration treatment to concentrate the at least one saccharifying enzyme, and the concentrate of the at least one saccharifying enzyme obtained by the concentration treatment is circulated to the reaction solution in step (a).

[6] In step (b), at least a part of the reaction solution is subjected to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and the at least one target substance, and a fraction (Y) containing the microorganism and reaction residue;
The method according to any one of [1] to [5], wherein in step (c), fraction (X) obtained in step (b) is subjected to a saccharifying enzyme/target substance separation treatment to obtain a fraction (X1) containing the at least one saccharifying enzyme and a fraction (X2) containing the at least one target substance, and fraction (X1) is circulated to the reaction solution in step (a).

[7] The method according to any one of [1] to [6], further comprising purifying the at least one target substance from at least a portion of fraction (X) and/or fraction (X2) obtained in step (c).

[8] The method according to any one of [1] to [7], wherein at least one of the following (i) and (ii) is obtained as the at least one target substance:
(i) fraction (X), a concentrate of fraction (X), or a material extracted and/or purified from fraction (X);
(ii) Fraction (Y), a concentrate or treatment of fraction (Y), or a material extracted and/or purified from fraction (Y).

[9] The method according to any one of [1] to [8], wherein in step (b), a solid-liquid separation treatment is performed on at least a part of the reaction liquid using a filter.
Here, the filter is preferably a ceramic filter.

[10] The method according to any one of [1] to [9], in step (b), a solid-liquid separation process is performed on at least a portion of the reaction liquid using a cross-flow filter.

[11] The method according to any one of [1] to [10], wherein in step (b), solid-liquid separation of at least a portion of the reaction solution is carried out using a microfiltration filter having a pore size of 50 nm to 2.0 μm.

[12] The method according to any one of [1] to [11], in which in step (b), a solid-liquid separation process is carried out using an ultrafiltration filter having a molecular weight cutoff of 3100 or more.

[13] The method according to any one of [1] to [12], wherein the kappa number of the pretreated lignocellulosic material is 15 or less.

[14] The method according to any one of [1] to [13], wherein the at least one saccharifying enzyme includes cellulase.

[15] The method according to any one of [1] to [14], wherein the microorganism is a microorganism capable of assimilating pentose.

[16] The method according to any one of [1] to [15], wherein the microorganism is an alcohol-fermenting yeast.

[17] The method according to any one of [1] to [16], wherein the microorganism is an ethanol-fermenting yeast.

[18] The method according to any one of [1] to [17], wherein in step (a), the saccharification and microbial reaction are carried out at a temperature in the range of 30°C to 40°C.

[19] The method according to any one of [1] to [18], wherein in step (a), the saccharification and microbial reaction are carried out at a pH in the range of 4 to 5.5.

[20] (A) a saccharification/microbial reaction unit that performs saccharification and microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism;
(B) a solid-liquid separation unit that separates at least a portion of the reaction solution into a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and the reaction residue;
(C) a saccharification enzyme circulation unit that circulates at least a portion of the at least one saccharification enzyme contained in the fraction (X) obtained in the solid-liquid separation unit to the saccharification and microbial reaction proceeding in parallel in the saccharification/microbial reaction unit;
An apparatus comprising:

[21] (D) The apparatus described in [20] further comprises a microorganism/reaction residue circulation unit that circulates at least a portion of the microorganisms and reaction residue contained in fraction (Y) obtained in the solid-liquid separation unit to the reaction liquid in the saccharification/microbial reaction unit.

[22] (A) a saccharification/microbial reaction unit that performs saccharification and microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism;
(B) a solid-liquid separation unit that separates at least a portion of the reaction solution into a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and the reaction residue;
(D) a microorganism/reaction residue circulation unit that circulates at least a portion of the microorganisms and reaction residue contained in the fraction (Y) obtained in the solid-liquid separation unit to the reaction liquid in the saccharification/microbial reaction unit; and
An apparatus comprising:

[23] The saccharification/microbial reaction unit comprises a plurality of reaction vessels or reaction compartments connected in series,
At least a part of the reaction solution is continuously moved through the plurality of reaction vessels or reaction compartments, and saccharification and microbial reaction are continuously performed in each of the plurality of reaction vessels or reaction compartments,
The apparatus according to any one of [20] to [22], wherein at least a portion of the reaction liquid present in the reaction tank or reaction compartment located at the rearmost position among the plurality of reaction tanks or reaction compartments is supplied to the solid-liquid separation unit.

[24] The solid-liquid separation unit is provided with a filter that separates at least a portion of the reaction liquid into a fraction (X) and a fraction (Y). The production apparatus according to any one of [20] to [23].
Here, the filter is preferably a ceramic filter.

[25] The saccharifying enzyme circulation unit includes a concentration treatment device that concentrates the at least one saccharifying enzyme in the fraction (X) obtained in the solid-liquid separation unit,
The concentrate of the at least one saccharification enzyme obtained by the concentration treatment device is configured to be circulated to the reaction liquid in the saccharification/microbial reaction unit.
The device according to any one of [20] to [24].

[26] The apparatus described in any one of [20] to [25], wherein the solid-liquid separation unit is configured to separate at least a portion of the reaction liquid in the saccharification/microbial reaction unit into a fraction (X) containing the at least one saccharification enzyme and at least one target substance, and a fraction (Y) containing the microorganism and reaction residue.

[27] The saccharification enzyme circulation unit is configured to separate the fraction (X) obtained in the solid-liquid separation unit into a fraction (X1) containing the at least one saccharification enzyme and a fraction (X2) containing the at least one target substance, and to circulate the fraction (X1) to the reaction liquid in the saccharification/microbial reaction unit.
The device according to any one of [20] to [26].

[28] The device according to any one of [20] to [27], further comprising a target substance extraction/purification unit for extracting or purifying the at least one target substance from at least a portion of fraction (X) and/or fraction (X2).

[29] The apparatus according to any one of [20] to [28], further comprising a target substance extraction/purification unit, the target substance extraction/purification unit including at least one distillation apparatus for distilling or fractionating at least a portion of fraction (X) and/or fraction (X2) to obtain an extract or purified product of at least one volatile organic compound as the at least one target substance.

[30] The apparatus described in any one of [20] to [29], further comprising a pretreatment unit that performs pretreatment on the raw lignocellulosic material to reduce the lignin content, and obtains the pretreated lignocellulosic material.

According to an embodiment of the present invention, the saccharification reaction of lignocellulosic materials such as biomass materials and the microbial reaction capable of producing a target substance are carried out in parallel, thereby reducing the labor hours and number of parts required for substance production, which results in reduced production costs and shorter production lead times. In addition, according to an embodiment of the present invention, the saccharification enzymes and microorganisms used in the saccharification/microbial reaction are circulated and reused in the target substance production process, eliminating the wasteful consumption of resources and materials, further reducing production costs, and enabling more efficient substance production.

1 is a schematic diagram showing an embodiment of an apparatus according to the present invention; FIG. 2 is a schematic diagram showing another embodiment of the device according to the present invention. FIG. 2 is a schematic diagram showing another embodiment of the device according to the present invention.

Methods
According to an aspect of the present invention, there is provided a method for producing a target substance, comprising at least the following steps (a), (b) and (c), or steps (a), (b) and (d):

(a) causing saccharification and a microbial reaction to proceed in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism, thereby producing at least one target substance;
(b) subjecting at least a portion of the reaction liquid to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue;
(c) circulating at least a portion of the at least one saccharifying enzyme contained in fraction (X) obtained in step (b) to the reaction solution in step (a);
(d) circulating at least a portion of the microorganisms and reaction residue contained in fraction (Y) obtained in step (b) to the reaction solution in step (a).

The above-mentioned methods according to the present invention will be described in detail below by showing examples of various embodiments. However, each method according to the present invention is not limited to the embodiments shown below.

[Step (a)]
(Pretreated lignocellulosic material)
In the present invention, the term "lignocellulosic material" refers to a material that contains lignocellulose, which mainly contains polysaccharides cellulose and hemicellulose, and polymeric compound lignin, and in which a high-dimensional structure is formed by these structural units. Specifically, lignocellulose is a component contained in the cell walls of plants, etc. The term "lignocellulosic material" includes various biomass materials, and examples of such materials include various parts (e.g., leaves, stems, branches, trunks, roots) of plants including herbaceous plants (e.g., rice, wheat, crops, weeds) and woody plants (e.g., coniferous trees, broadleaf trees), construction waste, felling and pruning waste, plant bodies and plant waste after weeding and crop harvesting (e.g., rice straw, wheat straw, corn stover, crop stems and leaves, weed waste, discarded harvested material, sugarcane bagasse, and other agricultural and forestry plant waste), wood chips, bark, wood fiber, pulp, paper including waste paper, algae, and microalgae. In the present invention, the "lignocellulosic material" may be a single type of lignocellulosic material or a mixture of multiple types of lignocellulosic materials. Although the term "lignocellulosic material" literally means a material that contains lignocellulose, it does not exclude the inclusion of components other than lignocellulose.
In certain embodiments, the "lignocellulosic material" may be woody biomass.

In the present invention, the meaning of "pretreated" in "pretreated lignocellulosic material" is "lignocellulosic material" that has been subjected to any pretreatment in order to facilitate saccharification by at least one saccharification enzyme in step (a). Various methods of pretreatment are known, such as pulping treatment in the papermaking industry and pretreatment techniques for saccharifying biomass materials, and one of these known methods may be used, or a combination of multiple types may be used. However, it should be noted that in the present invention, "pretreatment" is not limited to known methods.

The following are examples of various pretreatment techniques:

Pulverization using a mill or other pulverizing tool/machine; digestion using high-temperature and high-pressure steam; steaming/explosion method; supercritical water/subcritical water treatment; acid treatment using inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, and phosphoric acid; alkali chemicals [e.g., NaOH, KOH, Ca(OH _{) 2} , _Na2CO3 , _NaHCO3 , _Na2SO3 , ammonia, _sodium sulfide ( _Na2 S), sodium polysulfide] and optionally various auxiliaries (for example, quinone compounds such as anthraquinone, sodium sulfate, sodium hydrogen sulfide); alkaline digestion treatment combining the above-mentioned alkaline treatment and digestion treatment (for example, alkaline sulfite-quinone digestion); digestion treatment using alcohols or organic solvents such as methanol, ethanol, propanol, phenol, etc.; digestion treatment using organic acids such as acetic acid; bleaching treatment by exposure to oxygen or ozone (optionally, alkaline chemicals may be added to the treatment medium); bleaching treatment using various bleaching agents such as chlorine dioxide and hydrogen peroxide.

Depending on the type and properties of the lignocellulosic material used as the raw material, one of the above-mentioned methods may be selected and used, or a combination of multiple methods may be used to obtain the pretreated lignocellulosic material to be subjected to step (a). However, the pretreatments that can be used to obtain the pretreated lignocellulosic material are not limited to the above-mentioned examples, and any pretreated lignocellulosic material obtained using any method will suffice.

　In the lignocellulosic material that has been pretreated as described above (i.e., "pretreated lignocellulosic material"), the lignin present in the lignocellulosic material is decomposed or reduced to a certain extent, or the three-dimensional structure formed by the bonds between lignin and cellulose and cellulose is destroyed, so that in step (a), the cellulose and hemicellulose, which are substrates for the saccharification enzyme, are easily decomposed by the saccharification enzyme in the reaction liquid to produce hexoses (e.g., glucose, mannose, arabinose, galacturonic acid, etc.), pentoses (e.g., xylose), and oligosaccharides, which are used as substrates for microbial reactions (e.g., metabolic reactions including fermentation by microorganisms and biosynthesis of specific substances) or microbial growth, and the microbial reactions and/or microbial growth proceed.

In some embodiments, the pretreated lignocellulosic material to be subjected to step (a) may be a lignocellulosic material that has been subjected to at least one of the following treatments: alkaline heating treatment, alkaline oxygen treatment, and bleaching treatment.

(Alkaline heat treatment)
The lignocellulosic material as a raw material is heat _- treated for a predetermined treatment time in an alkaline aqueous solution containing water as a main solvent, at least one alkaline agent (i) (e.g., at least one selected from the group consisting of NaOH, KOH, _Ca (OH) ₂ , _Na2CO3 , _NaHCO3 , _Na2SO3 , and ammonia), and at least one sulfide (ii) [e.g., at least one of sodium _{sulfide (Na2S) and sodium polysulfide (Na2Sn , n} = 2 to 5)]. The heat treatment can be performed using any heat treatment device such as an autoclave. In addition, the lignocellulosic material to be subjected to this treatment includes not only untreated lignocellulosic material, but also lignocellulosic material that has been previously subjected to any treatment, including mechanical crushing treatment or the alkaline oxygen treatment described below.

Here, the concentration of the at least one alkaline agent (i) in the alkaline aqueous solution is, for example, about 0.1 to about 70% by mass, about 1 to about 70% by mass, about 2 to about 50% by mass, preferably about 2 to about 30% by mass, and more preferably about 2 to about 10% by mass. In terms of the ratio between the amount of lignocellulosic material charged and the amount of the at least one alkaline agent (i) used, the amount of the at least one alkaline agent (i) used per about 100 parts by mass of lignocellulosic material (dry mass) can be, for example, about 1 to about 20 parts by mass, preferably about 3 to about 15 parts by mass, more preferably about 5 to about 15 parts by mass, and even more preferably about 8 to about 15 parts by mass.

Furthermore, the concentration of the at least one sulfide (ii) in the alkaline aqueous solution is, for example, about 0.1 to about 70% by mass, about 1 to about 70% by mass, about 2 to about 50% by mass, preferably about 2 to about 30% by mass, and more preferably about 2 to about 10% by mass. In terms of the ratio between the amount of lignocellulosic material charged and the amount of the at least one sulfide (ii) used, the amount of the at least one sulfide (ii) used per about 100 parts by mass of lignocellulosic material (dry mass) can be, for example, about 1 to about 20 parts by mass, preferably about 2 to about 15 parts by mass, more preferably about 3 to about 15 parts by mass, even more preferably about 4 to about 15 parts by mass, and particularly preferably about 5 to about 10 parts by mass.

Furthermore, the amount of lignocellulosic material added to the alkaline aqueous solution is not particularly limited, so long as a "pretreated lignocellulosic material" capable of undergoing the saccharification reaction in step (a) can be obtained. For example, the amount of lignocellulosic material added (dry mass) relative to about 100 parts by mass of the alkaline aqueous solution is, for example, about 10 to about 100 parts by mass, about 20 to about 90 parts by mass, preferably about 30 to about 80 parts by mass, more preferably about 40 to about 70 parts by mass, and even more preferably about 40 to about 60 parts by mass.

Furthermore, the heating temperature is, for example, about 80 to about 250°C, preferably about 120 to about 240°C, more preferably about 130 to about 230°C, even more preferably about 140 to about 220°C, particularly preferably about 150 to about 210°C, and most preferably about 160 to about 200°C. In addition, in certain embodiments, the heating rate in the heat treatment may be controlled, and the heat treatment can be performed at a heating temperature of, for example, about 10 to about 30°C/min, preferably about 15 to about 25°C/min.

Furthermore, the heat treatment time may be appropriately determined taking into consideration the concentration of the above-mentioned agent, the heating temperature, the desired properties of the pretreated lignocellulosic material to be obtained, etc., and is not particularly limited, but is, for example, about 10 to about 180 minutes, preferably about 20 to about 120 minutes, and more preferably about 30 to about 90 minutes.

The pretreated lignocellulosic material obtained by the various embodiments employing any combination of the ranges of the above-mentioned drug concentration, heating temperature, and heat treatment time can be preferably used because the lignin in the lignocellulosic material is substantially decomposed or reduced, and the excessive dissolution or over-decomposition of cellulose and hemicellulose, which serve as substrates for the saccharification reaction, into the solution is suppressed.

In some embodiments, the solid content of the lignocellulosic material that has been subjected to the above-mentioned alkaline heat treatment may be optionally washed with a solvent such as water, and then the fibers may be loosened using a pulp disaggregator or the like to form a fibrous sample (pulp-like sample). Furthermore, in certain embodiments, the above-mentioned fibrous sample (pulp-like sample) may be dehydrated so that the moisture content is about 40 to about 60%, more preferably about 45 to about 55%, for example about 50%, after removing unreacted lignocellulosic material remaining in the sample as necessary using a specified screen device or sieve.

(Alkaline oxygen treatment)
In an alkaline aqueous solution containing water as a main solvent and at least one of the above-mentioned alkaline agents (i), the lignocellulosic material is heat-treated for a predetermined treatment time under a flow of oxygen gas and a predetermined pressurized condition. The heat and pressure treatment can be performed using any heat and pressure treatment device such as an autoclave. In addition, the lignocellulosic material to be subjected to this treatment includes not only untreated lignocellulosic materials (e.g., biomass materials) but also lignocellulosic materials that have been subjected to at least one of mechanical crushing treatment, the above-mentioned alkaline heating treatment, and any other pretreatment.

Here, the concentration of the at least one alkaline agent (i) in the alkaline aqueous solution is, for example, about 0.1 to about 15% by mass, about 0.2 to about 12% by mass, about 0.25 to about 10% by mass, preferably about 0.3 to about 8.5% by mass, and more preferably about 0.3 to about 8.0% by mass. In terms of the ratio between the amount of lignocellulosic material charged and the amount of the at least one alkaline agent (i) used, the range of the amount of the at least one alkaline agent (i) used per about 100 parts by mass of lignocellulosic material (dry mass) can be, for example, about 0.5 to about 10 parts by mass, preferably about 0.8 to about 8 parts by mass, more preferably about 1 to about 6 parts by mass, and even more preferably about 1.5 to about 5 parts by mass.

Furthermore, the amount of lignocellulosic material to be charged is not particularly limited as long as it is possible to obtain pretreated lignocellulosic material that can undergo saccharification in step (a), but the amount of lignocellulosic material to be charged (dry weight) can be set within the range of, for example, about 10 to about 100 parts by weight, about 10 to about 60 parts by weight, preferably about 10 to about 50 parts by weight, more preferably about 15 to about 40 parts by weight, and even more preferably about 20 to about 30 parts by weight, relative to about 100 parts by weight of the alkaline aqueous solution.

Furthermore, the pressure conditions are, for example, about 0.25 to about 1 MPa, preferably about 0.3 to about 0.9 MPa, and more preferably about 0.4 to about 0.8 MPa.

Furthermore, the heating temperature is, for example, about 80 to about 200°C, preferably about 90 to about 150°C, more preferably about 90 to about 130°C, even more preferably about 95 to about 120°C, particularly preferably about 98 to about 110°C, and most preferably about 100°C.

Furthermore, the heat treatment time may be appropriately determined taking into consideration the concentration of the above-mentioned agent, the heating temperature, and the desired properties of the pretreated lignocellulosic material to be obtained, and is not particularly limited. However, when the above-mentioned ranges of agent concentration and heating temperature are adopted, it is, for example, about 10 to about 180 minutes, preferably about 20 to about 120 minutes, more preferably about 30 to about 90 minutes, and even more preferably about 40 to about 70 minutes.

The pretreated lignocellulosic material obtained by the various embodiments employing any combination of the ranges of the above-mentioned drug concentration, heating temperature, and heat treatment time can be preferably used because the lignin in the lignocellulosic material is decomposed or reduced to a considerable extent, while the excessive dissolution or over-decomposition of cellulose and hemicellulose, which serve as substrates for saccharification, into the solution is suppressed.

In some embodiments, the solid content of the lignocellulosic material that has been subjected to the alkaline oxygen treatment may be optionally washed with a solvent such as water, and then suspended in a predetermined amount of a solvent such as water to form a fibrous sample (pulp-like sample), which may then be dehydrated to a moisture content of about 40 to about 60%, more preferably about 45 to about 55%, for example about 50%.

(Bleaching treatment)
The lignocellulosic material is bleached with an arbitrary bleaching agent (bleaching solution) at a predetermined treatment temperature and treatment time. The lignocellulosic material to be subjected to this bleaching treatment includes not only untreated lignocellulosic material (e.g., biomass material) but also lignocellulosic material that has been previously subjected to at least one of the above-mentioned alkali heating treatment, alkali oxygen treatment, and arbitrary pretreatment. The temperature for the bleaching treatment is not particularly limited, but is, for example, about 20 to about 90°C, about 30 to about 90°C, preferably about 40 to about 80°C, more preferably about 50 to about 80°C, even more preferably about 60 to about 80°C, and most preferably about 65 to about 75°C. In addition, the treatment time is not particularly limited, but is, for example, about 10 to about 180 minutes, preferably about 20 to about 120 minutes, more preferably about 30 to about 90 minutes, and even more preferably about 40 to about 70 minutes.
A more specific example of the bleaching treatment will be described below.

There are no particular limitations on the type of bleaching agent that can be used as long as it produces a pretreated lignocellulosic material suitable for the saccharification and microbial reaction carried out in step (a). Specifically, hypochlorite bleaching [e.g., a bleaching solution containing at least one selected from the group consisting of hypochlorites including NaClO, KClO, CaCl.H ₂ O, and Ca(ClO) ₂ ]; bleaching using peroxides such as hydrogen peroxide (H ₂ O ₂ ) and sodium peroxide (Na ₂ O ₂ ) (peroxide bleaching); hydrosulfite bleaching (e.g., a bleaching solution containing Na ₂ S ₂ O ₄ , Na ₂ S ₂ O ₅ , or Na ₂ CO, or a mixture of Na ₂ O ₂ and/or ZnS ₂ O ₄ and sodium tripolyphosphate); a bleaching solution containing NaBH ₄ (sodium borohydride) and a sulfite such as NaHSO ₃ ; chlorine dioxide (ClO ₂ ) bleaching; peracetic acid (C ₂ H ₄ O _{3 ).} ) Bleaching, etc. are examples.

In some embodiments, for example, an aqueous bleaching solution containing water as a main solvent and an optional bleaching component may be used. In this case, the upper limit of the concentration of the bleaching component is not particularly limited, but may be, for example, about 0.05% by weight or more, about 0.1% by weight or more, about 0.12% by weight or more, about 0.13% by weight or more, about 0.14% by weight or more, about 0.15% by weight or more, about 0.16% by weight or more, about 0.17% by weight or more, about 0.18% by weight or more, about 0.19% by weight or more, about 0.2% by weight or more, about 0.21% by weight or more, about 0.22% by weight or more, about 0.23% by weight or more, or about 0.24% by weight or more, and the lower limit of the concentration of the bleaching component is about 6% by weight, about 5% by weight or less, about 4% by weight or less, about 3% by weight or less, or about 2% by weight or less. Furthermore, in certain embodiments, the concentration of the bleaching component may be a range of values that does not contradict any one of the upper limits described above and any one of the lower limits described above, and it should be understood that embodiments that use such ranges are expressly set forth in this specification.

Furthermore, the ratio between the amount of lignocellulosic material to be subjected to the bleaching treatment and the amount of bleaching agent (bleaching component) used can be set appropriately taking into consideration the type and properties of each bleaching agent, and is not limited. For example, the amount of bleaching agent (bleaching component) used for about 100 parts by mass of lignocellulosic material (dry mass) can be, for example, about 0.05 to about 5 parts by mass, preferably about 0.08 to about 4 parts by mass, more preferably about 0.09 to about 3 parts by mass, and even more preferably about 0.10 to about 3 parts by mass.

The bleaching process may be carried out by selecting one type of bleaching method (bleaching agent), or by combining multiple types as long as this does not cause technical problems. For example, examples of bleaching processes using a combination of multiple bleaching methods (bleaching agents) include a combination of hydrogen peroxide bleaching/hydrosulfite bleaching, and a combination of hypochlorous acid bleaching/hydrosulfite bleaching. In addition, a multi-stage method may be used in which bleaching processes using one or multiple bleaching methods are carried out multiple times in sequence.

In some embodiments, at least one bleaching treatment of _chlorine dioxide bleaching and peroxide bleaching (e.g. _, bleaching using _H2O2 or _Na2O2 ) may be performed once or multiple times. Furthermore, in a specific embodiment, the lignocellulosic material that has been subjected to the above-mentioned alkali heating treatment and alkali oxygen treatment may be subjected to chlorine dioxide bleaching and peroxide bleaching once or more, preferably twice or more, more preferably three or more times in any order in multiple stages. Furthermore, as a particularly preferred embodiment, the lignocellulosic material that has been subjected to the above-mentioned alkali heating treatment and alkali oxygen treatment may be subjected to at least three bleaching treatments, for example, in the order of chlorine dioxide bleaching, peroxide bleaching, and chlorine dioxide bleaching; or in the order of peroxide bleaching, chlorine dioxide bleaching, and peroxide bleaching.

Furthermore, in a specific embodiment, the bleaching solution containing chlorine dioxide contains water as a main solvent and chlorine dioxide as a bleaching component, and the concentration of chlorine dioxide in the bleaching solution can be any of the upper or lower limits or numerical ranges of the concentrations of the bleaching components described above.

Furthermore, in a specific embodiment, the bleaching solution containing peroxide contains water as a main solvent and _H2O2 or _Na2O2 and optionally NaOH as bleaching components, and the concentration of _H2O2 or _Na2O2 and the concentration of NaOH in the bleaching solution can be any of the upper or _lower limits or numerical ranges of the bleaching component concentrations described above. In addition, the ratio of the amount _of lignocellulosic material charged (dry mass) to the _{amount of H2O2} or _Na2O2 and NaOH used can be any of the ranges of the _{amount of} bleaching agent (bleaching component) used per about 100 parts by mass of lignocellulosic material (dry mass).

In some embodiments, the solid content of the lignocellulosic material that has been subjected to the above-mentioned bleaching treatment may be optionally washed with a solvent such as water, and then suspended in a predetermined amount of a solvent such as water to form a fibrous sample (pulp-like sample), which may then be dehydrated so that its moisture content is about 40 to about 60%, more preferably about 45 to about 55%, for example about 50%. In addition, when the bleaching treatment is performed multiple times in multiple stages, the above-mentioned optional washing and dehydration treatments may be performed immediately after each bleaching treatment step.

The "pretreated lignocellulosic material" used in step (a) includes lignocellulosic materials obtained by various pretreatments according to the above-mentioned embodiments, but is not limited to these, and also includes lignocellulosic materials that have been subjected to pretreatments that reduce or decompose lignin to a certain degree.

As a preferred embodiment, the pretreated lignocellulosic material to be subjected to step (a) may have an upper limit of kappa number of about 15.0 or less, preferably about 14.5 or less, more preferably about 14.4 or less, even more preferably about 14.3 or less, and particularly preferably about 14.2 or less. In a specific embodiment, the pretreated lignocellulosic material has a kappa number of about 12.0 or less. Since the lower the kappa number, the more preferable it is, the lower limit is not particularly limited, and may be, for example, about 1.0 or more, about 2.0 or more, or about 3.0 or more. As a specific embodiment, the pretreated lignocellulosic material may be subjected to step (a) whose kappa number falls within a numerical range that combines one of the lower limit values and one of the upper limit values.

The kappa number of the pretreated lignocellulosic material may be measured by various known methods. For example, the kappa number can be measured according to JIS P8211 "Test method for pulp kappa number" used in the examples described below.

In addition, the method of the present invention may further include, although not essential, (p) pretreating the raw lignocellulosic material prior to step (a) to obtain a pretreated lignocellulosic material, and examples of embodiments of step (p) are as described above.

(Saccharification and microbial reactions proceed in parallel)
The reaction liquid in step (a) contains at least one saccharification enzyme and a pretreated lignocellulosic material that serves as a substrate for the at least one saccharification enzyme, and therefore, as described above, a saccharification reaction proceeds in which the cellulose and hemicellulose contained in the pretreated lignocellulosic material are saccharified by the catalytic action of the saccharification enzyme, resulting in the production of carbohydrates (various hexoses, various pentoses, oligosaccharides, etc.) that serve as substrates for microbial reactions and microbial growth. Furthermore, since the reaction liquid also contains microorganisms, when carbohydrates are produced by the saccharification reaction, the microorganisms use the carbohydrates as substrates, and the microbial reaction and/or microbial growth also proceeds. Furthermore, since at least one saccharification enzyme, the microorganisms, and the pretreated lignocellulosic material that is a substrate for the saccharification enzyme are contained in the same reaction system, the saccharification reaction and the microbial reaction and/or microbial growth proceed in parallel.

In the present invention, the term "microbial reaction" is a concept that includes not only the metabolic reaction of microorganisms that occurs in a reaction liquid using carbohydrates generated by the saccharification of pretreated lignocellulosic material as a substrate, but also microbial growth that may occur as a result of the metabolic reaction. However, a "microbial reaction" does not necessarily have to involve microbial growth, and includes, for example, metabolic reactions of microorganisms that occur under non-growth conditions of microorganisms without substantial growth of microorganisms, and biosynthetic reactions of specific substances (for example, metabolic reactions that proceed under reducing conditions such as a complete or incomplete reductive TCA cycle, and biosynthetic reactions of specific substances).

(Saccharification enzyme)
In the present invention, the saccharifying enzyme that can be used is not particularly limited as long as it has the activity to decompose the cellulose and/or hemicellulose contained in the pretreated lignocellulosic material and produce carbohydrates (various hexoses, various pentoses, oligosaccharides, etc.) that serve as substrates for microbial reactions and microbial growth. Specifically, the saccharifying enzyme that can be used is one that has cellulase activity and/or hemicellulase activity.

In some embodiments, the saccharification enzyme may be a cellulase having at least one of the following enzyme activities: endoglucanase activity (EC 3.2.1.4) that cleaves from the inside of the cellulose molecule, and exoglucanase (cellobiohydrolase) activity (e.g., reducing end decomposition type: EC 3.2.1.176, non-reducing end decomposition type EC 3.2.1.91) that decomposes the reducing end or non-reducing end of cellulose to release cellobiose, etc.

In some embodiments, the saccharification enzyme may be a hemicellulase that breaks down hemicellulose into monosaccharides or oligosaccharides such as xylose. Typical examples of hemicellulose include mannan, β-1,4-glucan, xylan, and xyloglucan, and in certain embodiments, a hemicellulase that breaks down these hemicelluloses may be used. In more specific embodiments, at least one hemicellulase that exhibits at least one enzyme activity selected from the group consisting of xylanase, xylosidase, mannanase, pectinase, galactosidase, glucuronidase, and arabinofuranosidase may be used.

In addition, it is preferable to use an enzyme mixture that combines multiple cellulases and hemicellulases as the saccharification enzyme in order to exert sufficient enzyme activity.

The origin of cellulase and hemicellulase is not limited, and for example, cellulase and hemicellulase derived from microorganisms such as filamentous fungi, basidiomycetes, and bacteria can be used. They may be wild-type enzymes or genetically modified enzymes with enhanced specific functions. Examples of microorganisms that possess cellulases and hemicellulases include filamentous fungi, basidiomycetes, and bacteria such as those of the genera Trichoderma, Acremonium, Aspergillus, Bacillus, Pseudomonas, Penicillium, Aeromonus, Irpex, Sporotrichum, and Humicola. One type of cellulase and/or hemicellulase derived from these microorganisms may be used, or a combination of multiple types may be used.

Furthermore, various commercially available saccharifying enzymes may also be used. For example, product names include Novozym (registered trademark) 613, Novozym (registered trademark) 476, Celluzyme (registered trademark), Celluclast (registered trademark), Carezyme (registered trademark), FiberCare (registered trademark), Cellic (registered trademark) CTec/CTec2/CTec3, and the like (Novozymes); Optimase CX, Multifect (registered trademark) A40, Pergalase (registered trademark), Optimase (registered trademark), Accellerase ^™ 1000, Accellerase ^™ 1500, Accellerase ^™ TRIO, and the like (DuPont, Danisco); cellulase Examples of such products include Onozuka (registered trademark) and Maceroteam (registered trademark) (Yakult Pharmaceutical Co., Ltd.); Spartec (trademark) CEL100, Pyrolase (registered trademark) Cellulase, Pyrolase (registered trademark) 200 cellulase, Pyrolase (registered trademark) HT Cellulase (BASF Corporation); Acremo Cellulase KM, Cellulase TP5-KYOWA (Kyowa Kasei Co., Ltd.); GODO-TCL, Besselex (Godo Shusei Co., Ltd.); and Cellulizer ACE (Nagase ChemteX Corporation).

(Microorganisms)
In the present invention, the type of microorganism is not particularly limited and may be appropriately selected according to the type of microorganism suitable for producing a desired target substance or the species of microorganism to be grown. In addition, in the present invention, the microorganism may be a prokaryote or a eukaryote, and may be a wild type, a mutant, or a genetically modified organism.

In some embodiments, the microorganism is at least one selected from the group consisting of archaea, bacteria, cyanobacteria, microalgae, and fungi. Furthermore, in some embodiments, the microorganism is a microorganism capable of producing a specific substance. In addition, in certain embodiments, the microorganism is a microorganism that produces at least one substance selected from the group consisting of volatile substances (more specifically, volatile organic compounds, more specifically, alcohols including alkanols, e.g., methanol, ethanol, propanol, butanol); organic acids including acetic acid, lactic acid, butyric acid, and various amino acids; nucleic acids; and vitamins. Furthermore, in a preferred embodiment, the microorganism is an alcohol-producing microorganism (more preferably, alcohol-fermenting yeast, particularly preferably, ethanol-fermenting yeast). Examples of alcohol-producing microorganisms include Saccharomyces cerevisiae and other Saccharomyces genus, Schizosaccharomyces genus, Schizosaccharomyces pombe and other Schizosaccharomyces genus, Pichia stipitis and Pichia kudriavzevii (e.g., RZ8-1) and other Pichia genus, Pachysolen tannophilus and other Pachysolen genus, Kluyveromyces lactis and Kluyveromyces marxianus, ces marxianus (e.g., DMKU3-1042) and other members of the genus Kluyveromyces, Candida shehatae, Candida shehatae, Candida glabrata NF (e.g., Examples include yeasts belonging to the genus Candida, such as Candida spp. (RI3163), and some bacteria, such as Clostridium thermocellum, Clostridium thermohydrosulfuricum, Thermoanaerobacter ethanolicus, and Zymomonas mobilis.

Furthermore, in certain embodiments, a microorganism having pentose assimilation ability (e.g., a microorganism having xylose and/or arabinose assimilation ability) may be used as the microorganism. More specifically, a microorganism having xylose and/or arabinose assimilation ability may be, for example, an ethanol-fermenting genetically modified yeast to which xylose and/or arabinose assimilation ability has been imparted. Ethanol-fermenting yeast to which xylose and/or arabinose assimilation ability has been imparted by the introduction of various heterologous genes is also known, and such yeasts may be used as the microorganism in the present invention [for example, Appl. Biochem. Biotechnol., 105-108:277-286 (2003); Appl. Microbiol. Biotechnol., 73:1039-1046 (2007); J. Biosci. Bioeng. , 106:306-309 (2008). ;Appl. Microbiol. Biotechnol. , 82, 1037-1047 (2009); Appl. Environ. Microbiol. , 69, 4144-4150 (2003); Appl. Environ. Microbiol. , 73, 4881-4891 (2007); Microb. Cell Fact. , 8, 40 (2009); Appl. Environ. Microbiol. , 75, 907-914 (2009)].

Furthermore, in another embodiment, the microorganism may be a microorganism having cellobiose assimilation ability. For example, ethanol-fermenting yeasts that have been imparted with cellobiose assimilation ability by the introduction of various heterologous genes are known, and these may be used as the microorganism in the present invention [for example, (Science, 330, 84-86 (2010); Proc. Natl. Acad. Sci. U.S.A., 108, 504-509 (2011)). Furthermore, in another embodiment, a thermotolerant yeast capable of fermentation at temperatures above 37°C, around 40-50°C, may be used (e.g., J. Biosci. Bioeng. 2010; 110: 176-179; Braz. J. Microbiol. 49(2): 378-391 (2018); World J Microbiol Biotechnol. 1992; 8: 259-263).

As mentioned above, known microorganisms may be used as the microorganisms, but it should be noted that the "microorganisms" in the present invention are not limited to known ones.

(Reaction solution)
In the present invention, the "reaction liquid" in step (a) contains, in addition to the above-mentioned "pretreated lignocellulosic material" (a substrate for the saccharification enzyme that produces carbohydrates in the microbial reaction), the saccharification enzyme, and the microorganism, any additional components may be contained, although these are not essential, from the viewpoint of desired expression of the saccharification enzyme activity and the microbial reaction.

Furthermore, in some embodiments, the reaction solution may contain at least one of the following (P) to (T) in addition to water as the main solvent:
(P) at least one selected from the group consisting of molasses (e.g., from sugar cane, sugar beet, corn), malt extract, and whey;
(Q) at least one selected from the group consisting of corn steep liquor, yeast extract, soybean meal, and peptone (protein hydrolysate);
(R) at least one nitrogen source [e.g., inorganic nitrogen sources including ammonium salts (e.g., ammonium sulfate, ammonium carbonate, ammonium bicarbonate, ammonium phosphate), sodium nitrate, potassium nitrate, and ammonia; organic nitrogen sources including urea];
(S) at least one _inorganic buffer ₍ e.g., _K2HPO4 , _KH2PO4 , _CaCO3 );
(T) Antifoaming agent.

Furthermore, in certain embodiments, the reaction solution contains the above (P) and/or (Q) and (R).
Here, the contents of components (P) and/or (Q) in the reaction liquid may be appropriately determined depending on the properties of the saccharifying enzyme and microorganism used, and the desired saccharification and microbial reaction, and are not particularly limited, but are, for example, about 0.5 to about 60 parts by mass, about 0.5 to about 50 parts by mass, about 0.5 to about 40 parts by mass, preferably about 1.0 to about 40 parts by mass, about 1.0 to about 30 parts by mass, about 1.0 to about 20 parts by mass, more preferably about 1.5 to about 20 parts by mass, and even more preferably about 2.0 to about 10 parts by mass, relative to about 100 parts by mass of water as the main solvent. In addition, the content of the above-mentioned (R) in the reaction liquid is not limited, similar to the content of the components (P) and/or (Q), but is, for example, about 0.1 to about 5 parts by mass, about 0.1 to about 4 parts by mass, preferably about 0.5 to about 3 parts by mass, about 0.5 to about 4 parts by mass, more preferably about 1.0 to about 4 parts by mass, or about 1.0 to about 3 parts by mass, relative to about 100 parts by mass of water as the main solvent.
The content of the component (T) may be appropriately set within a range that does not inhibit saccharification and microbial reaction in the reaction liquid and can exhibit the desired defoaming effect.

In the present invention, the "reaction liquid" can be said to be essentially a reaction medium that does not impede the sufficient expression of saccharifying enzyme activity and is suitable for the survival of microorganisms or microbial reactions, and therefore may be formed by adding pretreated lignocellulosic material, saccharifying enzymes, and microorganisms to any microbial culture medium that can fulfill the meaning of such a reaction medium.

In addition, although not essential, the above-mentioned reaction liquid (saccharification/microbial reaction medium) is preferably subjected to a heat treatment using an autoclave or the like, or a sterilization and/or bacterial growth prevention treatment by adding an acid or the like, in order to prevent contamination and proliferation of unwanted bacteria prior to the addition of saccharification enzymes and microorganisms to the reaction liquid prior to carrying out saccharification and microbial reactions. Furthermore, in certain embodiments, the pretreated lignocellulosic material that serves as the substrate for the saccharification reaction may also be subjected to a similar sterilization and/or bacterial growth prevention treatment in advance.

In saccharification and microbial reaction, the pH of the reaction solution may be appropriately determined taking into consideration the optimum pH of the saccharification enzyme used and the characteristics of the microbial reaction, and is not particularly limited, but it is generally preferable to prepare the reaction solution in advance within the range of about 3.0 to about 10.0, about 3.5 to about 8.0, preferably about 3.5 to about 6.0, more preferably about 3.5 to about 5.8, even more preferably about 3.8 to about 5.7, and particularly preferably about 3.8 to about 5.6. In a more preferred embodiment, the pH of the reaction solution may be maintained within these ranges during saccharification and microbial reaction.
As the pH adjuster, one or more of alkalies such as NaOH, KOH, and ammonia, and acids such as hydrochloric acid, acetic acid, lactic acid, and citric acid can be used. Specifically, the pH of the reaction liquid can be adjusted by adding the pH adjuster to the reaction liquid in the form of an aqueous solution adjusted to a predetermined concentration.

The temperature of the saccharification and microbial reaction may be appropriately determined taking into consideration the optimum temperature of the saccharification enzyme and the optimum temperature for the microbial reaction, and is not particularly limited as long as it does not interfere with the saccharification and microbial reaction. In consideration of the growth temperatures of various microorganisms (e.g., psychrophilic bacteria, mesophilic bacteria, thermophilic bacteria, and hyperthermophilic bacteria), the temperatures of the saccharification and microbial reaction are generally about 10 to about 30°C, about 25 to about 35°C, about 30 to about 38°C, about 35 to about 45°C, about 40 to about 55°C, about 55 to about 68°C, or about 85 to about 100°C. In consideration of the ease of control of the saccharification and microbial reaction, it is preferable to select and use the saccharification enzyme and microorganism suitable for each temperature range as the temperature of the saccharification and microbial reaction. Furthermore, in certain embodiments, yeast (e.g., alcohol- or ethanol-fermenting yeast) is used as the microorganism, the temperature of the saccharification and microbial reaction is maintained in the range of about 28 to about 42°C, about 35 to about 53°C, or about 37 to about 52°C, and a saccharification enzyme with an optimum temperature within these temperature ranges may be used.

Furthermore, the saccharification and microbial reaction may be carried out in a semi-batch or batch manner, but is preferably carried out in a continuous manner. In addition, a microorganism that realizes a microbial reaction that proceeds under anaerobic or reducing conditions, or microaerobic or aerobic conditions, may be used to carry out the microbial reaction under any of the above conditions that are suitable for the microorganism. For example, when carrying out aerobic fermentation using alcohol-fermenting yeast or the like, the reaction system may be aerated at a predetermined aeration rate and saccharification and microbial reaction (i.e., alcohol fermentation) may be carried out while stirring at a predetermined stirring speed.

The method of preparing the reaction solution containing each of the reaction solution components, and the timing and method of adding the pretreated lignocellulosic material, saccharification enzyme, and microorganism to the previously prepared reaction medium may be selected as appropriate within the scope of not causing technical contradictions or hindrances, and are not particularly limited. For example, prior to or during the saccharification and microbial reaction, the following (s) to (u) may be added simultaneously to the previously prepared reaction medium, or in any order (for example, in the order of (s), (t), and (u)), or two of the following (s) to (u) may be added simultaneously and the remaining one before or after the simultaneous addition of the two.
(s) a predetermined amount of at least one saccharification enzyme;
(t) a predetermined amount of a preculture of a microorganism pre-grown in a predetermined medium;
(u) a predetermined amount of pretreated lignocellulosic material.

Furthermore, in some embodiments, at least one of the above elements (s) to (u) may be added to the reaction solution one or more times during the progress of saccharification and the microbial reaction. Furthermore, in certain embodiments, all of the above elements (s) to (u) may be added once each when the saccharification and the microbial reaction are started, and then the above element (u) may be added one or more times during the progress of the saccharification and the microbial reaction, so that the saccharification and the microbial reaction are continuously progressed by the active saccharification enzymes (element (s)) and the microorganisms (t) with the pretreated lignocellulosic material (element (u)) added continuously. According to such embodiments, more efficient production of target substances and/or proliferation of microorganisms can be achieved.

As described below, the method of the present invention employs a configuration in which at least a portion of at least one saccharification enzyme that has escaped from the saccharification and microbial reaction system and at least a portion of the microorganisms and reaction residue are recovered as fraction (X) and fraction (Y), respectively, in the latter steps (b) and (c), and are circulated to step (a) for reuse. Therefore, the embodiment in which element (u) is added one or more times during the progress of saccharification and microbial reaction as described above is particularly preferable because it allows the pretreated lignocellulosic material to be efficiently used as a substrate.

In a specific embodiment, while the saccharification and microbial reaction are proceeding, element (u) may be added to the reaction solution at a certain time period (e.g., a predetermined time period falling within a range of about 15 minutes to about 360 minutes, about 15 minutes to about 300 minutes, about 15 minutes to about 240 minutes, about 15 minutes to about 120 minutes, or about 15 minutes to about 60 minutes), in which case, the addition of element (u) to the reaction solution may be performed manually or automatically using a predetermined control device or control system.

As described below, when the saccharification and microbial reaction in step (a) are carried out in a plurality of saccharification and microbial reaction systems (hereinafter, sometimes simply referred to as "reaction systems") connected in series, one or more additional additions of at least one of the above (s) to (u) may be added to at least one reaction system (reaction tank) selected from the plurality of reaction systems (reaction tanks). The reaction system (reaction tank) to which the additional additions are made is not particularly limited. However, considering that in the plurality of reaction systems (reaction tanks) connected in series, at least a portion of the reaction liquid is transferred from upstream to downstream while the saccharification and microbial reaction proceed in parallel, as a specific embodiment, a form in which at least one of the above (s) to (u) is additionally added one or more times to the reaction system (reaction tank) located at the most upstream position among the plurality of reaction systems (reaction tanks) can be preferably adopted from the viewpoint of realizing an efficient process.

The amount of each of the above elements (s) to (t) added to the reaction medium or reaction liquid at a given timing may be appropriately set from the viewpoint of realizing an efficient process, and is not particularly limited. In addition, the amount of at least one saccharification enzyme [element (s)] used depends on conditions such as the enzyme activity units of various saccharification enzymes, but generally can be set in the range of, for example, about 0.005 to about 1000 parts by mass, about 0.01 to about 900 parts by mass, preferably about 0.5 to about 600 parts by mass, more preferably about 1 to about 500 parts by mass, about 5 to about 200 parts by mass, or about 5 to about 150 parts by mass, and most preferably about 5 to about 100 parts by mass, relative to 100 parts by mass of the pretreated lignocellulosic material [element (u)] to be saccharified in the reaction system. Furthermore, in certain embodiments, the amount of the predetermined amount of the microbial preculture [element (t)] added may be set in the range of, for example, about 0.01 to about 100 parts by mass, preferably about 0.5 to about 80 parts by mass, more preferably about 1 to about 60 parts by mass, or about 1 to about 50 parts by mass, per 100 parts by mass of the pretreated lignocellulosic material [element (u)].

Novozymes, which manufactures the above-mentioned Cellic (registered trademark) CTec3 and other products, has proposed an evaluation method using an enzyme activity unit called "BHU(2)HS" (roughly, an enzyme activity unit based on the hydrolysis activity for cellulose in crushed corn stover) as an index for evaluating the performance of a multiple saccharification enzyme mix suitable for the saccharification reaction of biomass materials and estimating the required enzyme activity unit (Novozymes technical information "BHU(2)HS, Biomass hydrolysis activity by FCD"). Therefore, the required amount of "at least one saccharification enzyme" used in the present invention may be appropriately determined using this enzyme activity unit as an index, or using a fluorescent in vitro enzyme assay system applied to measuring the enzyme activity unit (Methods Enzymol. 2012, 510, pp. 19-36.). For example, the saccharification enzyme mix Cellic (registered trademark) CTec3 HS provided by Novozymes has an enzyme activity of about 1000 to 2000 BHU (2) HS/g according to the above enzyme activity unit, and in a specific embodiment, at least one saccharification enzyme [element (s)] exhibiting an enzyme activity equivalent to about 1000 to 2000 BHU (2) HS/g may be prepared in advance and added in a predetermined amount (g) belonging to each of the above numerical ranges to 100 parts by mass of the pretreated lignocellulosic material [element (u)], or an amount of "at least one saccharification enzyme" [element (s)] equivalent to the amount of enzyme activity units (BHU (2) HS) equivalent to the total amount (g) of the predetermined amount may be added.

Furthermore, in another embodiment, the at least one saccharification enzyme [element (s)] may be added in an amount ranging from about 5.0 to about 2,000,000, about 10 to about 1,800,000, about 500 to about 1,200,000, about 1,000 to about 200,000, about 5,000 to about 400,000, or about 5,000 to about 300,000, and most preferably about 5,000 to about 200,000, in enzyme activity units by BHU(2)HS per 100 parts by mass of the pretreated lignocellulosic material [element (u)].

Further, as a preferred embodiment, in step (a), a plurality of "reaction systems" connected in series may be provided, and a predetermined amount of the reaction solution may be transferred from one reaction system to another in the plurality of reaction systems, and saccharification and microbial reaction may be performed in each reaction system. Here, as a more specific embodiment, a configuration may be adopted in which the main solvent water (e.g., sterilized water) or reaction solvent (culture medium) is supplied to each reaction system at a predetermined rate (e.g., about 1 to 20 mL/min), the reaction solution volume of each reaction system is controlled to a preset value (e.g., a predetermined value in the range of about 1 L to about 10,000 L), and the reaction solution exceeding the volume value is transferred to the next reaction system according to the volume, thereby uniformly controlling the reaction volume and reaction conditions in each reaction system. The plurality of reaction systems may be composed of a plurality of reaction tanks or reactors. In addition, the plurality of reaction systems may be realized by a plurality of reaction compartments or reaction tanks formed by a plurality of partitions in the same reaction device, and further, may be realized by connecting a plurality of such reaction devices.
Furthermore, in certain embodiments, the number of reaction systems connected in series is two or three.

In some embodiments, as described above, in step (a), a plurality of reaction systems connected in series are provided, and a predetermined amount of reaction liquid is transferred sequentially from one reaction system to another in the plurality of reaction systems, and saccharification and microbial reactions are performed in each reaction system. In step (b), at least a portion of the reaction liquid present in the reaction system located at the rear end of the plurality of reaction systems may be subjected to a solid-liquid separation process described below. Furthermore, in some embodiments, in step (a), a portion of the reaction liquid may be circulated from at least one reaction system other than the first reaction system among the plurality of reaction systems connected in series to at least one reaction system located in front of the at least one reaction system. Furthermore, in a specific embodiment, in step (a), a portion of the reaction liquid may be circulated from the rear reaction system to the front reaction system in two adjacent reaction systems among the plurality of reaction systems connected in series. Furthermore, in a specific embodiment, in step (a), when a portion of the reaction liquid is transferred from the front reaction system to the rear reaction system in two adjacent reaction systems among a plurality of reaction systems connected in series, at least a portion of the "part of the reaction liquid" sent from the front reaction system to the rear reaction system may be circulated to the front reaction system.

In step (a), by employing the various embodiments as described above, a saccharification reaction and a microbial reaction (e.g., biosynthesis of a target substance and/or microbial growth) can be achieved.
However, step (a) of the method according to the present invention is not limited to the above-mentioned embodiment.

In the method according to the present invention, the type of at least one target substance produced in step (a) is not particularly limited. Specifically, the target substance is a substance produced by a microbial reaction and a substance derived from the substance (e.g., a substance produced from a substance produced directly by a microorganism through a predetermined decomposition reaction or chemical reaction), and examples thereof include various volatile substances (more specifically, volatile organic compounds, more specifically, alkanols, e.g., alcohols including methanol, ethanol, propanol, and butanol), various organic acids, proteins, peptides, carbohydrates (monosaccharides, oligosaccharides, polysaccharides), nucleic acids, vitamins, and polyphenol compounds including flavonoids, as described above. Note that the at least one target substance may be a substance accumulated in the cells or bacterial body of a microorganism, and/or may be a substance secreted into the reaction medium (culture medium).

[Step (b)]
In step (b), at least a portion of the reaction liquid that has been subjected to step (a) is subjected to solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue. In other words, step (b) is a step of obtaining at least a portion of the reaction liquid in which saccharification and microbial reaction have progressed in step (a), for example, during or after the saccharification and microbial reaction in step (a), fractionating the reaction liquid into the fraction (X) and fraction (Y) by a solid-liquid separation technique, and obtaining these fractions.

In some embodiments of the method according to the present invention, the at least one target substance may be contained in fraction (X) and/or (Y) fractionated in step (b). Furthermore, in a specific embodiment, in step (b), at least a portion of the reaction liquid that has been subjected to step (a) is subjected to a solid-liquid separation process to obtain fraction (X) containing the at least one saccharifying enzyme and the at least one target substance, and fraction (Y) containing the microorganism and reaction residue.

(Solid-liquid separation process)
For the solid-liquid separation treatment, any solid-liquid separation technique can be used as long as it can fractionate the reaction liquid into the above-mentioned fraction (X) and fraction (Y). For example, solid-liquid separation techniques using filtration using a (vibrating) screen, a rotary drum screen, a belt screen, a multi-plate wave filter, or other various filters, centrifugal separation, vacuum dehydration, pressurized dehydration, a belt press, a screw press, a roller press, etc. can be used.

In some embodiments, the solid-liquid separation process may utilize a filter (e.g., a ceramic filter) capable of fractionating the filtrate into a fraction (X) containing at least one saccharification enzyme and a filtrate fraction (Y) containing microorganisms and other reaction residues (filtrate fraction containing insoluble solid residues).

The filtration method using a filter can be used without any particular limitations, for example, the dead-end (total filtration) method or the cross-flow (circulating filtration) method can be used.

The separation ability of the filter can be appropriately determined taking into consideration the size of the microbial species used in the microbial reaction (i.e., the microorganisms separated as fraction (Y)) and the molecular weight of the target substance to be separated and extracted, but in terms of the pore size of the filter that separates the insoluble solid residue containing the microorganisms and reaction residue from the reaction liquid, it is generally possible to use a precision filtration filter or a filter with a similar separation ability (e.g., a ceramic filter), and the pore size is, for example, about 10 nm to about 15 μm, preferably about 10 nm to about 20 μm. 14 μm, more preferably about 10 nm to about 13 μm, even more preferably about 10 nm to about 12 μm. When fungi such as yeast or filamentous fungi are used as the microorganisms, the diameter is, for example, about 10 nm to about 12 μm, preferably about 10 nm to about 5 μm, more preferably about 10 nm to about 2 μm, even more preferably about 50 nm to about 1.8 μm, even more preferably about 50 nm to about 1.5 μm, about 80 nm to about 1.2 μm, particularly preferably about 100 nm to about 0.6 μm, and most preferably about 100 nm to about 0.5 μm.

Furthermore, in certain embodiments, the reaction liquid may be subjected to solid-liquid separation treatment using an ultrafiltration filter having a molecular weight cutoff of, for example, a lower limit of about 3000 or more, about 3100 or more, about 3200 or more, about 3300 or more, about 3400 or more, about 3500 or more, about 3600 or more, about 3700 or more, about 3800 or more, about 3900 or more, about 4000 or more, or about 5000 or more, preferably about 6000 or more, more preferably about 7000 or more, even more preferably about 8000 or more, particularly preferably about 9000 or more or about 9500 or more, and most preferably about 9600 or more, about 9700 or more, about 9800 or more, about 9900 or more, or about 10,000 or more. If a solid-liquid separation process of a reaction solution is performed using an ultrafiltration filter having such a molecular weight cutoff range, the solid-liquid separation can be performed into a filtrate as a fraction (X) containing at least one saccharification enzyme (e.g., various cellulases/hemicellulases) and a material that does not pass through the filter (filtrate) as a fraction (Y) containing the microorganisms and reaction residue. Note that since the upper limit of the molecular weight cutoff of an ultrafiltration filter is generally said to be 300,000, a suitable ultrafiltration filter having an upper limit of about 300,000 or less can be selected, for example, taking into consideration the molecular weight of the target substance to be separated/extracted.

In some embodiments, the solid-liquid separation process of the reaction liquid is performed using an ultrafiltration filter having an upper limit of the molecular weight cutoff of, for example, about 200,000 or less, about 100,000 or less, about 90,000 or less, about 80,000 or less, about 70,000 or less, about 60,000 or less, about 40,000 or less, about 30,000 or less, about 25,000 or less, about 24,000 or less, about 23,000 or less, about 22,000 or less, about 21,000 or less, or about 20,000 or less. In certain embodiments, the solid-liquid separation process of the reaction liquid may be performed using an ultrafiltration filter having a molecular weight cutoff that falls within a numerical range that is a combination of any one of the lower limit values of the molecular weight cutoff described above and any one of the upper limit values of the molecular weight cutoff described above without any contradiction.

In a more specific embodiment, a filter device including one or more microfiltration or ultrafiltration filters as described above, having a columnar shape and one or more flow paths in the longitudinal direction (e.g., a ceramic filter, in other words, the reaction liquid passes through one or more flow paths in the longitudinal direction of the columnar shape), may be used, and the reaction liquid may be separated into solid and liquid by a cross-flow (circulation filtration) method, with the inflow (circulation) linear velocity of the reaction liquid set within a predetermined range (value), for example, about 0.5 m/sec to about 7 m/sec, preferably about 1 m/sec to about 5 m/sec, more preferably about 1.5 m/sec to about 4.5 m/sec, and particularly preferably about 2 m/sec to about 4 m/sec, and the membrane pressure set within a predetermined range (value), for example, about 0.05 to about 0.5 MPa, preferably about 0.09 to about 0.3 MPa.

Here, the longitudinal length of the filter may be appropriately set in consideration of the volume and properties of the reaction liquid to be treated, and is not particularly limited, but may be selected from, for example, about 10 cm to about 1 m, about 15 cm to about 50 cm, and preferably about 20 cm to about 30 cm. In addition, in the columnar filter, the diameter of the flow path through which the reaction liquid can pass may be selected from a range that does not cause clogging, and is not particularly limited, but a columnar filter having a flow path diameter in the millimeter scale range may be used. In some embodiments, a columnar filter may be used whose flow path diameter is, for example, about 1 mm or more, about 1.5 mm or more, or about 2.0 mm or more as a lower limit. Furthermore, a columnar filter whose flow path diameter is about 10 mm or less, about 9 mm or less, about 8 mm or less, about 7 mm or less, about 6 mm or less, or about 5 mm or less as an upper limit may be used. Furthermore, in certain embodiments, a columnar filter may be used whose flow path diameter is within a range that is a compatible combination of any one of the above lower limit values and any one of the above upper limit values.

The material of the filter is not particularly limited, but a ceramic filter, for example, alumina or titania, can be used.

Needless to say, commercially available filters can be used for the solid-liquid separation process, for example, the ceramic membrane filter "Cefilt" series, preferably the MF membrane, which is a precision filtration filter, or the UF membrane, which is an ultrafiltration filter (NGK Insulators, Ltd.).

[Step (c)]
As described above, in step (c), at least a portion of the at least one saccharifying enzyme contained in fraction (X) obtained by solid-liquid separation in step (b) is circulated to the reaction solution in step (a). By circulating the saccharifying enzyme to the reaction solution in this manner, the saccharifying enzyme contained in fraction (X) is reused in the saccharification and microbial reaction in step (a), enabling efficient saccharification and microbial reaction.

In step (c), fraction (X) obtained in step (b) may be directly recycled to step (a), or fraction (X) may be subjected to a concentration treatment to concentrate at least one saccharifying enzyme, and the concentrate of at least one saccharifying enzyme obtained by the concentration treatment may be recycled to the reaction solution in step (a). There are no particular limitations on the concentration treatment, as long as it can obtain a concentrated fraction containing a saccharifying enzyme that can exhibit a certain enzymatic activity. For example, concentration treatments can be performed using various distillations (e.g., simple distillation, rectification, fractional distillation, reduced pressure distillation, flash distillation, molecular distillation, pressurized distillation, steam distillation, extractive distillation), ultrafiltration, nanofiltration, precipitation (salting out) using salts such as ammonium sulfate, affinity chromatography, and ion exchange chromatography, and one or more of these may be used.

When the microbial reaction is a reaction that produces at least one volatile substance or volatile organic compound (alcohols, other organic solvents, etc.) as a target substance, it is preferable to use distillation as the concentration treatment. This is because the distillation technique makes it possible to obtain an extract fraction containing a volatile compound as a target substance and a concentrated fraction (water-soluble fraction) containing at least one saccharification enzyme at low cost and easily, and to circulate the concentrated fraction to the saccharification and microbial reaction in step (a). In an embodiment in which a crude extract fraction containing a volatile substance or volatile organic compound and a concentrated fraction containing at least one saccharification enzyme are obtained by a crude extraction treatment such as reduced pressure distillation, and the concentrated fraction is circulated to the reaction system in step (a) to produce a target substance, as a more specific embodiment, the crude extract fraction containing a volatile substance or volatile organic compound may be further subjected to a concentration and purification treatment such as precision distillation to obtain a high-purity volatile substance or volatile organic compound as a target substance.

Furthermore, in some embodiments, in step (b), at least a portion of the reaction liquid that has been subjected to step (a) is subjected to a solid-liquid separation process to obtain a fraction (X) containing the at least one saccharifying enzyme and the at least one target substance, and a fraction (Y) containing the microorganism and reaction residue; in step (c), fraction (X) obtained in step (b) is subjected to a separation process of at least one saccharifying enzyme/target substance to obtain a fraction (X1) containing at least one saccharifying enzyme and a fraction (X2) containing at least one target substance, and fraction (X1) may be circulated to the reaction liquid in step (a).

In addition, in certain embodiments, the method may further include extracting or purifying the at least one target substance from at least a portion of fraction (X) and/or fraction (X2) obtained in step (c).

[Step (d)]
In step (b), fraction (Y) containing microorganisms and reaction residues is obtained by solid-liquid separation, so fraction (Y) is typically a fraction containing microorganisms and reaction residues and having a solid, semi-solid or slurry form. Therefore, the method according to the present invention may include step (d) as described above.

In step (d), an embodiment may be adopted in which at least a portion of fraction (Y) obtained in step (b) is directly circulated to the reaction liquid in step (a). However, step (d) is not limited to this embodiment, and the fraction (Y) obtained in step (b) may be subjected to various treatments such as concentration treatment, addition of a microbial preculture or any component (e.g., medium, medium components, water, etc.), moisture adjustment treatment, etc., and the treated product obtained thereby (i.e., a treated product containing the microorganisms and reaction residue contained in fraction (Y)) may be circulated to the reaction liquid in step (a). In other words, in step (d), it is sufficient that at least a portion of the microorganisms and reaction residue contained in fraction (Y) obtained in step (b) is circulated to the saccharification and microbial reaction (microbial growth) in step (a) and reused in the saccharification and microbial reaction (microbial growth), and the specific form is not important.

As a specific embodiment, in step (b), as described above, a filter device including one or more microfiltration filters or ultrafiltration filters having a columnar shape and one or more flow paths in the longitudinal direction is used, and solid-liquid separation is performed by a cross-flow (circulation filtration) method. In step (d), at least a portion of the filter refractory fraction (Y) that passes through the flow path without permeating the one or more filter materials (i.e., a solid, semi-solid, or slurry-like fraction that passes through the flow path without permeating the filter material) may be circulated to the reaction liquid in step (a). When such an embodiment is adopted, the one or more flow paths in the filter device through which the reaction liquid or the solid, semi-solid, or slurry-like fraction contained therein (microorganisms and reaction residues) passes may be connected to the reaction system (reaction tank) in step (a) via piping or tubes, and the microorganisms and reaction residues may be circulated to the reaction system in step (a) in step (d).

[Obtaining target substances]
In the method for producing a target substance according to the present invention, in step (a), a microbial reaction is carried out using the carbohydrate produced by saccharification of a pretreated lignocellulosic material as a substrate to produce at least one target substance, and the target substance is obtained.

Here, the scope of the target substance can be understood by literally interpreting the meaning of the wording of step (a), and includes various substances as described above. In addition, the target substance is not limited to a highly-purified substance that has been highly extracted and refined, but may be a mixture of multiple types of substances. Furthermore, the target substance may be a microbial bacterial body (cell), an organelle, a cellular part, a secretion secreted into a culture medium by a microbial reaction, or a reaction liquid containing the same.

In some embodiments, the target substance is at least one of the following (i) and (ii):
(i) fraction (X), a concentrate of fraction (X), or a material extracted and/or purified from fraction (X);
(ii) Fraction (Y), a concentrate or treatment of fraction (Y), or a material extracted and/or purified from fraction (Y).

In addition, various types of processing for obtaining a "processed product" include, for example, heat processing, freeze-drying, moisture adjustment processing, homogenization processing, granulation processing, solidification processing, formulation processing, pulverization processing, etc., and the processed product of fraction (Y) can be a processed product obtained by subjecting fraction (Y) to one type of processing selected from these, or a combination of multiple types of processing in any order.

When concentrating, extracting, and purifying a target substance, various techniques may be used, such as the above-mentioned various distillations, various organic solvent extractions, precipitation methods using various alcohols or organic solvents, salting out using ammonium sulfate, filtration, ultrafiltration, ion exchange chromatography, affinity chromatography, etc., and one of these may be used, or a combination of two or more may be used.

The uses of the target substance produced in the present invention are not limited in any way, but examples include pharmaceutical uses, industrial uses, fuel uses, and cosmetic uses. In addition, the target substance produced in the present invention may be a substance that is actually used for various purposes, or it may be an intermediate raw material used in the production of a final product.

<Apparatus>
According to another aspect of the present invention, there is provided an apparatus comprising at least the following (A), (B) and (C), or (A), (B) and (D).
(A) a saccharification/microbial reaction unit that performs saccharification and microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism;
(B) a solid-liquid separation unit for separating at least a part of the reaction solution into a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue;
(C) a saccharification enzyme circulation unit for circulating at least a portion of the at least one saccharification enzyme contained in the fraction (X) obtained in the solid-liquid separation unit to the saccharification and microbial reaction proceeding in parallel in the saccharification/microbial reaction unit;
(D) The above-mentioned device is a microorganism/reaction residue circulation unit that circulates at least a portion of the microorganisms and reaction residue contained in fraction (Y) obtained in the solid-liquid separation unit to the reaction liquid in the saccharification/microbial reaction unit.
Needless to say, the device may include all of the above (A) to (D).

In some embodiments, the apparatus is for use in producing at least one target substance, and more specifically, for use in carrying out the method of the present invention described above.

Below, a specific embodiment of the device according to the present invention will be shown and described in detail.

[Embodiment (I)]
FIG. 1A is a schematic diagram of an exemplary apparatus according to the present invention.

The apparatus 10 shown in FIG. 1A mainly comprises one reaction device (reaction tank) 2 constituting a saccharification/microbial reaction unit, a solid-liquid separation device 3 constituting a solid-liquid separation unit and a microorganism/reaction residue circulation unit, and a saccharification enzyme/target substance separation device 4 constituting a saccharification enzyme circulation unit and a target substance extraction/purification unit.
As shown in Fig. 1A, each device or unit such as the reaction device (reaction tank) 2 is connected through each pipe, and some of the pipes realize the function of each unit, and each pipe can be considered as an element constituting each unit. In addition, a liquid delivery means (not shown) such as a liquid delivery pump can be provided on each pipe as necessary.
The configuration of the device 10 will now be described in detail.

In detail, in FIG. 1A, as indicated by the arrows L, M, E, and C, pretreated lignocellulosic material L, microorganisms M (e.g., preculture), saccharification enzymes E, and reaction medium (culture medium) C are respectively introduced into the reaction tank of the reaction device 2 to form a reaction liquid. Then, in the reaction liquid formed inside the reaction tank of the reaction device 2, saccharification and microbial reaction proceed in parallel as described above. In this regard, in addition to the reaction tank, the saccharification/microbial reaction unit may include any elements (not shown), such as mechanisms or devices for measuring various parameters of the reaction liquid (e.g., pH, temperature, dissolved oxygen/dissolved carbon dioxide concentration, redox potential, microbial density), a stirring mechanism or stirring device for the reaction liquid, and various mechanisms or devices for controlling various parameters of the reaction liquid to certain values or ranges.

Next, the inside of the reaction tank of the reaction device 2 is connected to the solid-liquid separation device 3 via piping 6, and at least a portion of the reaction liquid in which the saccharification and microbial reaction have progressed is transferred from the inside of the reaction tank of the reaction device 2 to the solid-liquid separation device 3 via piping 6 using a liquid delivery means (not shown), such as a pump.

The solid-liquid separation device 3 is a solid-liquid separation means or device (e.g., a filter or a filter device equipped with the same) based on the various solid-liquid separation methods as described above, and the reaction liquid transferred from the reaction tank of the reaction device 2 to the solid-liquid separation device 3 is separated into a solid-liquid fraction (X) containing at least one saccharification enzyme and a fraction (Y) containing microorganisms and reaction residues by the solid-liquid separation device 3. Note that in this embodiment, it is assumed that the fraction (X) is a fraction that contains the target substance T in addition to at least one saccharification enzyme.

Fraction (Y) containing microorganisms and reaction residues is then circulated from solid-liquid separation device 3 to the reaction tank of reaction device 2 via pipe 7, and reused in the saccharification/microbial reaction taking place inside the reaction tank. Meanwhile, fraction (X) containing at least one saccharification enzyme and target substance T is transferred from solid-liquid separation device 3 to saccharification enzyme/target substance separation device 4 via pipe 8, where it is separated into fraction (X1) containing at least one saccharification enzyme and fraction (X2) containing target substance T. Then, fraction (X1) containing at least one saccharification enzyme separated by a liquid delivery means (not shown), such as a pump, is circulated from saccharification enzyme/target substance separation device 4 via pipe 9 to the inside of the reaction tank of reaction device 2, and reused in the saccharification reaction taking place inside the reaction tank.

On the other hand, the fraction (X2) containing the target substance T separated in the saccharification enzyme/target substance separation device 4 may be optionally collected in a collection tank (not shown) via piping 11, or transferred to a purification device (not shown) for further purification of the target substance.

[Embodiment (II)]
Fig. 1B is a schematic diagram showing another example of a typical apparatus according to the present invention. The apparatus 100 shown in Fig. 1B has a different configuration from the apparatus 10 shown in Fig. 1A in that it is provided with two reaction apparatuses (reaction tanks), a first reaction apparatus (first reaction tank) 2a and a second reaction apparatus (second reaction tank) 2b, which are connected in series via a pipe 6a.

In other words, the device 100 shown in FIG. 1B mainly comprises a first reaction device 2a and a second reaction device 2b that constitute a saccharification/microbial reaction unit, a solid-liquid separation device 3 that constitutes a solid-liquid separation unit and a microbial/reaction residue circulation unit, and a saccharification enzyme/target substance separation device 4 that constitutes a saccharification enzyme circulation unit and a target substance extraction/purification unit.

In detail, in FIG. 1B, as indicated by the arrows L, M, E, and C, pretreated lignocellulosic material L, microorganisms M (e.g., preculture), saccharification enzymes E, and reaction medium (culture medium) C are introduced into the reaction tank of the first reaction device 2a to form a reaction liquid. Then, in the reaction liquid formed inside the reaction tank of the first reaction device 2a, saccharification and microbial reactions proceed in parallel as described above. The inside of the reaction tank of the first reaction device 2a is connected to the inside of the reaction tank of the second reaction device 2b via piping 6a, and a predetermined amount of the reaction liquid in the reaction tank of the first reaction device 2a is transferred into the reaction tank of the second reaction device 2b via piping 6a by any liquid delivery means (not shown), such as a pump, and saccharification and microbial reactions proceed in parallel in the reaction liquid in the second reaction device 2b.

The inside of the reaction tank of the second reaction device 2b is connected to the solid-liquid separation device 3 via piping 6b, and at least a portion of the reaction liquid in which the saccharification and microbial reaction have progressed is transferred from the inside of the reaction tank of the second reaction device 2b to the solid-liquid separation device 3 via piping 6b by a liquid delivery means (not shown), such as a pump.

The solid-liquid separator 3 is a solid-liquid separator based on the various solid-liquid separation methods as described above, and the reaction liquid transferred from the reaction tank of the second reaction device 2b to the solid-liquid separator 3 is separated into a fraction (X) containing at least one saccharification enzyme and a fraction (Y) containing microorganisms and reaction residues by the solid-liquid separator 3. In this embodiment, it is assumed that the fraction (X) is a fraction that contains the target substance T in addition to at least one saccharification enzyme.

Then, from the solid-liquid separation device 3, the fraction (Y) containing the microorganisms and the reaction residue is circulated through the pipe 7 into the reaction tank of the first reaction device 2a and the reaction tank of the second reaction device 2b, and is reused for the saccharification/microbial reaction proceeding inside each of these reaction tanks. Meanwhile, the fraction (X) containing at least one saccharification enzyme and the target substance T is transferred from the solid-liquid separation device 3 to the saccharification enzyme/target substance separation device 4 through the pipe 8, and is separated into a fraction (X1) containing at least one saccharification enzyme and a fraction (X2) containing the target substance T in the saccharification enzyme/target substance separation device 4. Then, the separated fraction (X1) containing at least one saccharification enzyme is circulated through the pipe 9 from the saccharification enzyme/target substance separation device 4 to the reaction tanks of the first reaction device 2a and the second reaction device 2b, and is reused for the saccharification reaction proceeding inside each of these reaction tanks.
Other matters are similar to those in the above-mentioned embodiment (I).

[Embodiment (III)]
The apparatus 200 shown in FIG. 2 is a further embodiment of an apparatus having two reactors (reaction tanks), a first reactor (second reaction tank) and a second reactor (second reaction tank), which are connected in series, like the apparatus 100 shown in FIG. 1B.

As shown in FIG. 2, the apparatus 200 is equipped with a screw press 15 into which pretreated lignocellulosic material (not shown) is fed, and the pretreated lignocellulosic material is dehydrated by the screw press 15 to adjust the moisture content of the pretreated lignocellulosic material to a predetermined range.
As indicated by an arrow D, the waste liquid (drainage water) discharged from the screw press 15 is collected or discarded.

When saccharification and microbial reactions are carried out in the reaction tank of the first reaction device 2a, a reaction medium in which the saccharification and microbial reactions can proceed may be prepared in advance inside the reaction tank. The reaction medium may be prepared manually inside the reaction tank of the first reaction device 2a, but the reaction medium may be automatically prepared inside the reaction tank by providing the apparatus 200 with a reaction medium preparation unit including a device or mechanism that prepares a reaction medium according to the type and properties of the target microorganism or microbial reaction and supplies the reaction medium into the reaction tank of the first reaction device 2a (not shown in FIG. 2).

Meanwhile, the pretreated lignocellulosic material with the adjusted moisture content is transferred from the screw press 15 to the inside of the reaction tank of the first reaction device 2a via the pipe 16a. In addition, the device 200 further includes a pre-culture device 12 for pre-culture of microorganisms and a saccharification enzyme storage tank 13, and the pre-culture of the microorganisms pre-cultured and grown in the pre-culture device 12 is supplied from the pre-culture device 12 to the inside of the reaction tank of the first reaction device 2a via the pipe 16b, and at least one saccharification enzyme stored in the saccharification enzyme storage tank 13 is also supplied from the saccharification enzyme storage tank 13 to the inside of the reaction tank of the first reaction device 2a via the pipe 16c.

As described above, inside the reaction tank of the first reaction device 2a, a reaction liquid for saccharification and microbial reaction is formed from the pretreated lignocellulosic material, reaction medium, microbial preculture, and saccharification enzymes, and the saccharification and microbial reaction proceed in parallel in the reaction liquid.

The apparatus 200 further includes a sterile water storage tank 14 that stores sterile water and supplies it to the inside of the reaction tank of the first reaction device 2a. A predetermined amount of the reaction liquid present in the reaction tank of the first reaction device 2a is transferred to the reaction tank of the second reaction device 2b. For example, sterile water is supplied from the sterile water storage tank 14 to the inside of the reaction tank of the first reaction device 2a via piping 16d, and the amount of reaction liquid present in the reaction tank of the first reaction device 2a is controlled to be constant.

Furthermore, similarly to the apparatus 100, in the apparatus 200, the inside of the reaction tank of the first reaction apparatus 2a is connected to the reaction tank of the second reaction apparatus 2b via the pipe 6a and the pump 18a, and a predetermined amount of the reaction liquid in the reaction tank of the first reaction apparatus 2a is transferred via the pipe 6a to the inside of the reaction tank of the second reaction apparatus 2b, where saccharification and microbial reaction further proceed in parallel in the reaction liquid in the reaction tank.
In the apparatus 200 shown in FIG. 2, the pipe 6a connecting the insides of the first and second reaction tanks 2a and 2b has a branch point midway downstream of the pump 18a, and in addition to the branch pipe connecting to the inside of the reaction tank of the second reaction tank 2b, a branch pipe connecting to the inside of the reaction tank of the first reaction tank 2a is provided, and a part of the reaction liquid transferred from the inside of the reaction tank of the first reaction tank 2a to the inside of the reaction tank of the second reaction tank 2b through the branch pipe is circulated inside the reaction tank of the first reaction tank 2a.

Next, the reaction tank of the second reaction device 2b is connected to the solid-liquid separation device 3 via piping 6b and pump 18b, and at least a portion of the reaction liquid in which the saccharification and microbial reaction have progressed is transferred from inside the reaction tank of the second reaction device 2b to the solid-liquid separation device 3 via piping 6b.

As described above, the solid-liquid separation device 3 is a solid-liquid separation device based on various solid-liquid separation techniques, and the reaction liquid transferred from inside the reaction tank of the second reaction device 2 to the solid-liquid separation device 3 is separated by the solid-liquid separation device 3 into a fraction (X) containing at least one saccharification enzyme and the target substance T, and a fraction (Y) containing microorganisms and reaction residues.
Also in the apparatus 200, the fraction (Y) containing microorganisms and reaction residues from the solid-liquid separation apparatus 3 is circulated through the pipe 7 into each of the reaction tanks of the first reaction apparatus 2a and the second reaction apparatus 2b, and is reused for the saccharification/microbial reaction proceeding inside each of these reaction tanks.

2, in the apparatus 200, the pipe 7 branches at a branch point into a branch pipe that communicates with the inside of the reaction tank of the second reaction apparatus 2b and a branch pipe that communicates with the pipe 16d that communicates between the sterile water storage tank 14 and the inside of the reaction tank of the first reaction apparatus 2a, and a flow control valve 22 is provided in the middle of the branch pipe that communicates with the pipe 16d. In the pipe 7, the section from the solid-liquid separation apparatus 3 to the branch point and the section of the branch pipe that communicates with the inside of the reaction tank of the second reaction apparatus 2b are upstream sections of the pipe 7 and are always in an open state, so that most of the fraction (Y) sent from the solid-liquid separation apparatus 3 to the pipe 7 is circulated inside the reaction tank of the second reaction apparatus 2b. On the other hand, in the pipe 7, the downstream section of the branch pipe communicating with the pipe 16d is provided with the flow control valve 22 as described above, and by controlling the opening and closing of the flow control valve 22, a part (predetermined amount) of the fraction (Y) is transferred to the pipe 16d and supplied to the inside of the reaction tank of the first reaction device 2a via the pipe 16d. When a part (predetermined amount) of the fraction (Y) is transferred to the pipe 16d by controlling the opening and closing of the flow control valve 22, sterile water may be sent from the sterile water storage tank 14, and a part of the fraction (Y) transferred to the pipe 16d may be pushed in the direction of the first reaction device 2a by the flow of the sterile water and supplied to the inside of the reaction tank of the first reaction device 2a.

On the other hand, the "fraction (X) containing at least one saccharification enzyme and target substance T" (i.e., filtrate) generated in the solid-liquid separation device 3 is transferred from the solid-liquid separation device 3 to the filtrate recovery tank 17 via pipe 8a and temporarily stored therein. Furthermore, the fraction (X) stored in the filtrate recovery tank 17 is transferred to the crude extraction device 4a corresponding to the saccharification enzyme/target substance separation device via pipe 8b in response to the drive of pump 18c provided on pipe 8b located downstream. Then, in the crude extraction device 4a, the transferred fraction (X) is separated into a fraction (X1) containing at least one saccharification enzyme and a fraction (X2) containing at least one target substance T. Then, the fraction (X1) containing at least one saccharification enzyme is transferred from the crude extraction device 4a to pipe 16d via pipe 9 connected to pipe 16d, and then circulated inside the reaction tank of the first reaction device 2a via pipe 16d. When fraction (X1) is transferred to pipe 16d, sterile water may be sent from sterile water storage tank 14, and fraction (X) transferred to pipe 16d may be pushed in the direction of first reaction device 2a by the flow of the sterile water and supplied to the inside of the reaction tank of first reaction device 2a.

Meanwhile, the fraction (X2) produced in the crude extraction device 4a is transferred via pipe 11a to a purification device 4b that purifies at least one target substance T, and purification of the at least one target substance T is carried out in the purification device 4b to produce a product containing the at least one target substance T at a high purity. The product produced in the purification device 4b is transferred from the purification device 4b to a target substance recovery tank 19 via pipe 11b and recovered. Note that such devices or components are elements that may constitute a target substance extraction/purification unit that may be included in the device of the present invention. In addition, as shown by arrow D in FIG. 2, the waste liquid (wastewater) produced in the purification device 4b is recovered or discarded.

In addition, in a specific embodiment of the present invention, a substance containing alcohols such as ethanol and other volatile substances such as organic solvents can be produced as the target substance T through saccharification and microbial reaction, and in the apparatus 200, the crude extraction apparatus 4a and the purification apparatus 4b may be, in order, a crude distillation apparatus (e.g., a crude distillation column) and a rectification apparatus (e.g., a rectification column), which are distillation apparatuses based on the principles of distillation or fractionation.

In addition, it goes without saying that the device produces multiple types of target substances T and may include one or more crude extraction devices and/or purification devices, and may further include multiple target substance recovery tanks in which the multiple purified target substances T are respectively recovered.

All other details are the same as those in the above-mentioned embodiment (I) or (II).

[General Apparatus According to the Invention]
In some embodiments, the saccharification/microbial reaction unit in the apparatus may include a plurality of reaction devices or reaction tanks, the plurality of reaction devices or reaction tanks being connected in series, and the reaction liquid may be continuously moved inside the reaction devices or reaction tanks, and the saccharification and microbial reaction may proceed in parallel inside the reaction devices or reaction tanks.Further, in a specific embodiment, the saccharification/microbial reaction unit in the apparatus may include a plurality of reaction lines in which two or more reaction devices or reaction tanks are connected in series, and the reaction liquid in each reaction line may be continuously moved inside two or more reaction devices or reaction tanks, and the saccharification and microbial reaction may proceed in parallel inside the reaction devices or reaction tanks.

Furthermore, the type of saccharification/microbial reaction unit or reaction device or reaction tank can be used without any particular restrictions as long as it is capable of proceeding with saccharification and microbial reaction in parallel, and various types can be used depending on the purpose. For example, stirring type, bubble column type, fluidized bed type, packed bed type, etc. can be mentioned. Furthermore, in a specific embodiment, a type can be adopted in which saccharification enzymes and microorganisms are immobilized on a polymer carrier or membrane, etc., and saccharification of pretreated lignocellulosic material and microbial reaction using the carbohydrates produced by the saccharification as a substrate are carried out.

In certain embodiments, the saccharification/microbial reaction unit may further include a mechanism or device for adjusting and controlling various reaction conditions for the saccharification and microbial reaction carried out in the reaction device or reaction tank [e.g., at least one selected from the group consisting of pH in the reaction liquid, temperature, dissolved oxygen concentration, dissolved carbon dioxide concentration, redox potential, microbial cell mass (cell density), aeration rate, stirring speed, selection of various gas inflows and/or inflow rates, presence or absence of light irradiation and the amount of light irradiation, and addition and addition amounts of various reaction media or their components].

In some embodiments, the solid-liquid separation unit includes a filter member that separates the reaction liquid into the above-mentioned fraction (X) and fraction (Y). Furthermore, in certain embodiments, the saccharification enzyme circulation unit may include a concentration treatment device that concentrates at least one saccharification enzyme in fraction (X) obtained in the solid-liquid separation unit. Needless to say, embodiments of the present invention may also include those in which the solid-liquid separation device also has the function of a concentration treatment device.

In some embodiments, the system further includes a target substance extraction/purification unit that extracts and/or purifies at least one target substance from at least a portion of the liquid fraction (X) obtained in the solid-liquid separation unit. In the examples of embodiments (I) to (III) (FIGS. 1A and 1B and FIG. 2), the unit is composed of a saccharification enzyme/target substance separation device 4, or a crude extraction device 4a and a purification device 4b.

In a specific embodiment, the apparatus according to the present invention further includes a pretreatment unit that performs a pretreatment on the lignocellulosic material as a raw material to reduce the lignin content, and obtains the pretreated lignocellulosic material. The specific contents of the pretreatment are as described in the method according to the present invention. It should be understood that an apparatus including a treatment unit that can perform at least one of the steps that can be included in the method according to the present invention or the various treatments that constitute them, as described above, are explicitly described in this specification as various embodiments.

It should be understood that the above-described embodiments, elements, and other invention-specific matters of the method and apparatus according to the present invention may be combined or substituted for each other in both cases, unless there is a particular contradiction, and that various embodiments resulting from the mutual adoption of such embodiments, elements, and other invention-specific matters are expressly set forth in this specification.

　Specific embodiments of the present invention have been described above in detail, but the present invention is not limited to the above-mentioned embodiments. Various modifications, alterations, and combinations of each configuration, element, and feature may be adopted without departing from the gist of the present invention.

In addition, the contents of the patent application from which this application claims priority, Japanese Patent Application No. 2023-174873, and the prior art documents referenced in this specification are incorporated herein by reference for all purposes and constitute a part of this specification.

In this application, unless otherwise specified, the terms "contain," "constitute," "include," "have," and "consist of" do not exclude the presence of elements other than those explicitly stated with these terms, and these terms are often used interchangeably.

Preparation of pretreated lignocellulosic material
(Alkaline heat treatment)
500g (absolute dry weight) of eucalyptus chips and a solution of 60g of NaOH and 39g of _Na2S dissolved in 1L of water were added to the inside of a jar of a horizontal rotary autoclave (manufactured by Kumagaya Riki Kogyo Co., Ltd.), mixed, heated to 175°C at a temperature increase rate of 20°C/min, and held for 45 minutes to dissolve the lignin in the eucalyptus chips. Thereafter, the mixture was removed from the autoclave and thoroughly washed with water, and then the fibers were loosened using a standard pulp disintegrator (manufactured by Kumagaya Riki Kogyo Co., Ltd.) to obtain a fibrous pulp sample. Next, unreacted chips were removed from the pulp sample using a test flat screen (manufactured by Kumagaya Riki Kogyo Co., Ltd.) equipped with a screen with a mesh size of 2mm, and further dehydrated to a moisture content of 50% using a centrifugal dehydrator (manufactured by Kumagaya Riki Kogyo Co., Ltd.).

(Alkaline oxygen treatment)
120 g of the pulp sample obtained by the above-mentioned alkali treatment was mixed with an alkali solution obtained by dissolving 2.4 g of NaOH in 0.48 L of water, and the resulting mixture was placed in the jar of an indirect heating autoclave (manufactured by Toyo Koatsu Co., Ltd.), compressed oxygen gas with a purity of 99.9% was injected, and an alkali oxygen treatment was performed for 60 minutes under conditions of a gauge pressure of 0.5 MPa and a heating temperature of 100° C. After the alkali oxygen treatment, the pulp sample was removed from the autoclave and suspended in 1.4 L of ion-exchanged water, and then dehydrated using a centrifugal dehydrator (manufactured by Kumagai Riki Kogyo Co., Ltd.) until the moisture content reached 50%.

(Bleaching treatment)
120 g of the pulp sample that had been subjected to the alkaline oxygen treatment as described above was placed in a plastic bag. A solution obtained by dissolving 1.2 g of chlorine dioxide in 0.48 L of water was further placed in the plastic bag, and the bag was immersed in a thermostatic water bath (manufactured by Tokyo Rikakikai Co., Ltd.) at a temperature of 70° C. for 60 minutes to perform chlorine dioxide bleaching. After the reaction was completed, the pulp sample was suspended in 1.4 L of ion-exchanged water, and then dehydrated using a centrifugal dehydrator (manufactured by Kumagaya Riki Kogyo Co., Ltd.) until the moisture content reached 50%.

Next, the pulp sample after chlorine dioxide bleaching was placed in a plastic bag, and a solution obtained by dissolving 0.6 g of NaOH and 0.12 g of hydrogen peroxide in 0.48 L of water was added and mixed. The plastic bag was then immersed in a thermostatic water bath (Tokyo Rikakikai Co., Ltd.) at 70°C for 60 minutes to perform hydrogen peroxide bleaching. After the reaction was completed, the pulp sample was suspended in 1.4 L of ion-exchanged water, and then dehydrated using a centrifugal dehydrator (Kumagaya Riki Kogyo Co., Ltd.) until the moisture content reached 50%.

Next, the pulp sample after hydrogen peroxide bleaching was placed in a plastic bag, and a solution obtained by dissolving 0.18 g of chlorine dioxide in 0.48 L of water was added and mixed. The plastic bag was then immersed in a thermostatic water bath (manufactured by Tokyo Rikakikai Co., Ltd.) at a temperature of 70° C. for 60 minutes to perform chlorine dioxide bleaching. After the reaction was completed, the pulp sample was suspended in 1.4 L of ion-exchanged water and then dehydrated using a centrifugal dehydrator (manufactured by Kumagai Riki Kogyo Co., Ltd.) until the moisture content reached 50%.
As described above, a pretreated pulp sample was obtained as "pretreated lignocellulosic material."

[Test Example 1]
In this test example, a saccharification/microbial reaction apparatus having a configuration roughly similar to that of the example apparatus shown in Figure 2 was constructed, and the pretreated pulp sample (pretreated lignocellulosic material) obtained as described above was used as the raw material. In addition, specific saccharification enzymes and ethanol-fermentation yeast were used as described below to carry out parallel saccharification and fermentation to produce so-called bioethanol.
The procedure is described in detail below.

<Saccharification, fermentation and solid-liquid separation>
(Medium preparation)
In a 15 L jar fermenter No. 1 (manufactured by Biot Co., Ltd.; corresponding to the first reaction tank 2a in FIG. 2; hereinafter sometimes referred to as "tank No. 1"), 120 g of corn steep liquor (hereinafter CSL, manufactured by Japan Corn Starch Co., Ltd.), 12 g of ammonium sulfate, and 4.8 L of water were placed, and heat sterilization was performed at 121° C. for 20 minutes.

(Culture preparation)
After cooling with aeration at 120 mL/min, the mixture was stirred at a stirring speed of 150 rpm while automatically adjusting the temperature at 35° C., and the pH was automatically adjusted using 1 M sodium hydroxide solution. After the pH value stabilized at 5, 180 g of cellulase [Novozymes Cellic (registered trademark) CTec3] (enzyme activity ≧1000 BHU (2) HS/g, enzyme activity unit "1000 BHU (2) HS/g": Novozymes technical information "BHU (2) HS, Biomass hydrolysis activity by FCD"] was added.

(Saccharification and fermentation)
First, an ethanol-fermenting yeast [ethanol-producing recombinant yeast (Saccharomyces cerevisiae)] that has been given the ability to utilize xylose through a specified genetic recombination operation was prepared as a microorganism, and this was pre-cultured in a specified medium to prepare a preculture.

After seeding 120 g of the above-mentioned ethanol fermentation yeast preculture into the reaction medium prepared in the No. 1 tank, 25 g of the above-mentioned pretreated pulp sample (moisture content: 50%) was added every 30 minutes, and the saccharification reaction and ethanol fermentation were allowed to proceed under conditions of an aeration rate of 120 mL/min, a temperature of 35°C, and an agitation speed of 150 rpm, while the pH of the reaction medium was automatically adjusted to 5.0.

Furthermore, in the saccharification/microbial reaction apparatus used in this test example, as shown in Figure 2, tank No. 1 is connected to another 15 L jar fermenter No. 2 (manufactured by Biot, which corresponds to the second reaction tank 2b in Figure 2; sometimes referred to as "tank No. 2") that had been previously sterilized and empty, via piping and a pump (reference numbers 6a and 18a in Figure 2, respectively). Immediately after 120 g of the above-mentioned ethanol-fermentation yeast preculture was inoculated into the reaction medium prepared in tank No. 1, the circulation pump (reference number 18a in Figure 2) was started at a flow rate of 1 L/min, and sterilized water was supplied to tank No. 1 at a rate of 4 mL/min from 24 hours after the start of sample introduction, so that the reaction medium corresponding to the portion where the liquid level of the reaction medium (saccharification fermentation liquid) exceeded the 6 L level was circulated between tank No. 1 and tank No. The mixture was transferred to tank No. 2 by momentarily opening a valve (not shown in FIG. 2) installed on a pipe (6a in FIG. 2) that connected tank No. 2. In tank No. 2, the saccharification reaction and ethanol fermentation were further carried out while the pH of the reaction medium was automatically adjusted to 5.0 under the conditions of an aeration rate of 120 mL/min, a temperature of 35° C., and an agitation speed of 150 rpm, just like tank No. 1.

In the saccharification/microbial reaction apparatus used in this test example, as shown in FIG. 2, pipe 6a branches downstream of pump 18a into a pipe connected to tank No. 2 and a pipe connected to tank No. 1. After the circulation pump (FIG. 2, 18a) is started, a predetermined amount of saccharification fermentation liquid discharged from tank No. 1 to pipe 6a is circulated to tank No. 1 via the pipe connected to tank No. 1.

(Solid-liquid separation)
48 hours after the start of sample introduction, when the reaction medium liquid level in the No. 2 tank reached 6 L, the solution in the No. 2 tank was filtered using a precision filtration filter "Cefilt MF" (manufactured by NGK Insulators, Ltd., made of ceramic) with a pore size of 0.1 um and a length of 25 cm, under conditions of a circulation linear velocity of 3 m/sec (= 9 L/min) and a membrane pressure of 0.1 MPa, and 1/48 (250 mL per hour) of the total liquid volume in 1 hour was supplied to a reduced pressure distillation tower described below.

On the other hand, most of the material that did not pass through the filter (microorganisms and reaction residues) was circulated to the reaction liquid inside tank No. 2, and the remainder was circulated to the reaction liquid in tank No. 1, in accordance with the configuration of pipes 7 and 16d shown in Figure 2.

Even after the filtration process using the above filter, the above pretreated pulp sample was continued to be added to Tank No. 1, and the pretreated pulp sample, sterilized water, and the concentrated cellulase solution obtained in the reduced pressure distillation process described below were added to Tank No. 1, so that the reaction medium liquid volume in Tank No. 1 was always controlled to a level of 6 L.

Furthermore, in tank No. 2, the volume of the reaction medium (saccharification fermentation liquid) is theoretically understood to decrease from time to time by the amount of liquid that passes through the filter, but this decrease is compensated for by sending liquid from tank No. 1, and the volume of the reaction liquid in tank No. 2 is controlled to be constantly maintained at 6 L.

<Distillation process>
(Reduced pressure distillation)
The filter-passing liquid, supplied at 250 mL per hour from the filter, was distilled under reduced pressure using a vacuum distillation apparatus (a flash evaporator manufactured by Tokyo Rikakikai Co., Ltd.) set at a reduced pressure of 60 hPa and a temperature of 40° C. to obtain an ethanol solution with a concentration of 15 vol % as a fraction containing ethanol, the fermentation product, and also obtain a concentrated liquid (remainder) in which active cellulase was concentrated, and this concentrated liquid was returned to the saccharification and fermentation liquid in tank No. 1 (i.e., the cellulase concentrated liquid was supplied to the saccharification and fermentation liquid in tank No. 1 via pipe 9 in FIG. 2).

(Precision distillation)
The 15 vol % ethanol solution obtained by the above-mentioned reduced pressure distillation was subjected to a concentration treatment using a bioethanol dehydration and purification apparatus (manufactured by Kiriyama Co., Ltd.) to produce ethanol with a purity of 95 vol % as the target substance.

[Test Examples 2 to 17]
Ethanol was produced as a target substance in the same manner as in Test Example 1, except that the conditions in Test Example 1 were changed as shown in the table below.

[Test Example 18]
In this test example, ethanol fermentation production was carried out by adopting a configuration in which three saccharification and fermentation tanks, tank No. 1, tank No. 2, and tank No. 3, were connected in series in the following manner in accordance with the procedure of Test Example 1. That is, this test example is an example of adopting a configuration in which another reaction device is provided between the first reaction tank 2a and the second reaction tank 2b, as will be described with reference to FIG.
For conditions not described below, the same conditions as those in Test Example 1 were used.

That is, in the saccharification and fermentation process in Test Example 1, the reaction medium liquid volume that exceeded the 4 L level in Tank No. 1 was transferred to Tank No. 2, and saccharification reaction and ethanol fermentation were allowed to proceed under the same conditions as Tank No. 1. Furthermore, the reaction medium liquid volume that exceeded the 4 L level in Tank No. 2 was transferred to another 15 L jar fermenter No. 3 (manufactured by Biot Co., Ltd.; hereinafter sometimes referred to as "Tank No. 3") that had been empty and sterilized in advance, and saccharification reaction and ethanol fermentation were allowed to proceed under the same conditions as Tank No. 1 and Tank No. 2. 48 hours after the start of pulp sample introduction, when the liquid level of the reaction medium in Tank No. 3 reached the 4 L level, Tank No. The reaction medium in the three tanks was filtered using a precision filtration filter "Cefilt MF" (made by NGK Insulators, Ltd., made of ceramic) with a pore size of 0.1 μm and a length of 25 cm, at a circulation linear velocity of 3 m/sec and a membrane pressure of 0.1 MPa.

In this test example, similar to test example 1, a branch pipe was provided in the middle of the pipe for transferring the reaction liquid from tank No. 1 to tank No. 2 to circulate a portion of the reaction liquid in tank No. 1, and a branch pipe was also provided in the middle of the pipe for transferring the reaction liquid from tank No. 2 to tank No. 3 to circulate a portion of the reaction liquid in tank No. 2, and the reaction liquid was circulated in tank No. 1 and tank No. 2, respectively.

Furthermore, the volume of each reaction medium in tanks No. 2 and 3 is theoretically understood to be reduced by the volume of liquid that passed through the microfiltration filter, but the volume of the reaction medium in each tank was controlled to maintain 4 L by the inflow of reaction liquid from the upstream tank.

As described in Test Example 1, in the reduced pressure distillation of the distillation process, the filtered liquid supplied from the filter at 250 mL per hour was distilled under reduced pressure using a reduced pressure distillation apparatus (Tokyo Rikakikai flash evaporator) set at a reduced pressure of 60 hPa and a temperature of 40°C, to obtain a 15 vol% ethanol solution as a fraction containing ethanol, the fermentation product, and also obtain a concentrated liquid (remainder) in which active cellulase was concentrated, and this concentrated liquid was returned to the saccharification fermentation liquid in Tank No. 1.

On the other hand, the 15 vol% ethanol solution obtained by the above reduced pressure distillation was subjected to precision distillation in the same manner as in Test Example 1 to produce ethanol as the target substance.

[Test Example 19]
In this test example, ethanol was produced by adopting the procedure of Test Example 1 with the following modifications.

In other words, in the saccharification and fermentation process, 37 g of pretreated pulp sample (moisture content 50%) was added to tank No. 1 every 30 minutes, and from 16 hours after the start of pulp sample addition, sterilized water was added to tank No. 1 at a rate of 5 mL/min, and the reaction medium in tank No. 1 where the liquid level exceeded the 6 L level was transferred to tank No. 2.

Other saccharification and fermentation conditions in tanks No. 1 and No. 2 were the same as in Test Example 1, with aeration of 120 mL/min, temperature of 35°C, stirring at 150 rpm, and automatic adjustment to pH 5.0.

32 hours after the start of the pulp sample addition, when the liquid level of the reaction medium in the No. 2 tank reached a level of 6 L, the reaction medium in the No. 2 tank was filtered through a ceramic filter (manufactured by NGK Insulators, Ltd.) with a pore size of 0.1 um and a length of 25 cm at a circulation linear velocity of 3 m/sec and a membrane pressure of 0.1 MPa, and 1/32 of the total liquid volume (373 mL per hour) of the membrane-passed liquid was supplied to the reduced pressure distillation tower in 1 hour. The other procedures and conditions were the same as those in Test Example 1.

[Test Example 20]
In this test example, ethanol was produced by adopting the procedure of Test Example 1 with the following modifications.
That is, in the saccharification and fermentation process, 17 g of a pretreated pulp sample (moisture content: 50%) was added to No. 1 tank every 30 minutes, and from 32 hours after the start of the pulp sample addition, sterilized water was added to No. 1 tank at a rate of 3 mL/min, and the reaction medium in No. 1 tank, where the liquid level exceeded the 6 L level, was transferred to No. 2 tank.
Other saccharification and fermentation (reaction culture) conditions in tanks No. 1 and No. 2 were the same as in Test Example 1, with aeration of 120 mL/min, temperature of 35° C., stirring at 150 rpm, and automatic adjustment to pH 5.0.

64 hours after the start of the pulp sample addition, when the liquid level of the reaction medium in the No. 2 tank reached a level of 6 L, the reaction medium in the No. 2 tank was filtered using a ceramic filter (manufactured by NGK Insulators, Ltd.) with a pore size of 0.1 um and a length of 25 cm, at a circulation linear velocity of 3 m/sec and a membrane pressure of 0.1 MPa, and 1/64 of the total liquid volume (168 mL per hour) of the membrane-passed liquid was supplied to the reduced pressure distillation tower in 1 hour. The other procedures and conditions were the same as those in Test Example 1.

[Test Example 21]
Ethanol production was carried out in the same manner as in Test Example 1, except that the temperature of the reduced pressure distillation in the distillation step was changed to 50°C.

[Test Example 22]
In this test example, ethanol was produced in the same manner as in Test Example 1, except that the following changes were made.
That is, in Test Example 1, the No. 2 tank was omitted, and saccharification and fermentation was carried out only in the No. 1 tank, thereby producing ethanol as a target substance. In detail, in the saccharification and fermentation process, 48 hours after the start of pulp sample introduction, when the liquid level of the reaction medium in the No. 1 tank reached a level of 12 L, the reaction medium in the No. 1 tank was filtered using a precision filtration filter (manufactured by NGK Insulators, Ltd., made of ceramic) with a pore size of 0.1 um and a length of 25 cm, under conditions of a circulation linear velocity of 3 m/sec and a membrane pressure of 0.1 MPa, and 1/48 (250 mL per hour) of the total liquid volume was supplied to the reduced pressure distillation tower in 1 hour. Note that even after the operation using the precision filtration filter was started, the pretreated sample was continuously introduced into the No. 1 tank, and the No. In addition to the pretreated pulp sample and sterilized water, the concentrated liquid obtained in the reduced pressure distillation process was added to tank No. 1, and the liquid level of the reaction medium in tank No. 1 was controlled to always be at a level of 12 L.

In this test example, as in test example 1, a branch pipe was provided in the middle of the pipe connecting tank No. 1 and the microfiltration filter, and a part of the reaction liquid transferred from tank No. 1 to the microfiltration filter was circulated to tank No. 1 via the branch pipe, and the concentrated liquid fraction containing active cellulase obtained by reduced pressure distillation in the distillation process was circulated to the saccharification fermentation liquid in tank No. 1.

Other procedures and conditions were the same as in Test Example 1.

<Various analyses and evaluations>
In the above-mentioned Test Examples 1 to 22, the samples were analyzed and evaluated as follows.

(Kappa number)
For the pretreated pulp samples obtained in each test example, the kappa number was measured according to JIS P8211 "Testing method for pulp kappa number".

(SS concentration)
At the point when 144 hours had elapsed from the start of the introduction of the pretreated pulp sample, 10 mL of the saccharification and fermentation liquid was collected from tank No. 2 in Test Examples 1 to 21, and 10 mL of the saccharification and fermentation liquid was collected from tank No. 1 in Test Example 22. The collected saccharification and fermentation liquid was suction filtered using a Buchner funnel equipped with a pre-weighed glass fiber filter paper (GS-25 manufactured by Advantec Co., Ltd.) and dried in a dryer at 105°C, the amount of SS (suspended solids) present in the saccharification and fermentation liquid was measured, and the SS concentration (vol%) was calculated.

(95 vol% ethanol yield)
In each test example, the amount of 95 vol. % ethanol obtained in the bioethanol dehydration and purification apparatus was measured using a measuring cylinder.
Table 2 below shows the results of the above analysis.

The results shown in Table 2 show that in all test examples 1 to 22, which adopted a configuration in which yeast and unreacted residues derived from the saccharification and fermentation liquid, as well as a specific fraction containing cellulase, were circulated and reused, a significant amount of ethanol was produced as the target substance.

Furthermore, in Test Example 22, in which the saccharification reaction was carried out in only one reaction tank, the 95 vol% ethanol yield was 314 mL, whereas in Test Examples 1 to 21, in which the saccharification reaction was carried out in two or more reaction tanks, the 95 vol% ethanol yield exceeded 1,200 mL, making it possible to produce ethanol at a significantly higher yield. Thus, it has become clear that a further improvement in ethanol yield can be expected by carrying out the saccharification reaction liquid in a configuration in which the reaction liquid is transferred between two or more reaction tanks connected in series in a specified manner, and by adopting a configuration in which the yeast and unreacted residue derived from the saccharification fermentation liquid, as well as a specified fraction containing cellulase, are circulated and reused for saccharification and fermentation.

In all of Test Examples 1 to 22, the pretreated pulp samples used had a kappa number of less than 15, and the SS concentration was relatively low. In other words, in addition to the above-mentioned structure of recycling and reusing the yeast, unreacted residue, and cellulase, the use of pretreated pulp samples with a relatively low kappa number (pretreated pulp samples with a kappa number of less than 15) was also thought to have contributed to the realization of a high ethanol yield. More specifically, it can be inferred that when pretreated lignocellulosic material with a relatively low kappa number is used, most of the cellulose and hemicellulose contained in the material is efficiently saccharified by the saccharification enzyme, and the resulting C5 and C6 monosaccharides are efficiently used in ethanol fermentation, leading to a high ethanol yield.

In addition, as a result of the above test example, it was found that when using eucalyptus chips to produce ethanol using the above-mentioned specified cellulase and yeast, it is preferable to adopt the following conditions in order to achieve a high ethanol yield.

(Pretreatment conditions)
In addition to the alkali treatment, an oxygen treatment is adopted, and the oxygen addition concentration in the oxygen treatment is set to 10 parts by mass or more per 100 parts by mass of the pretreated pulp sample (pretreated lignocellulosic material).
(Saccharification and fermentation conditions)
Reaction culture temperature: 30 to 40°C range Reaction medium pH: 4 to 5.5 range Aeration rate: 0.001 vvm or more (solid-liquid separator/filter conditions)
・Molecular weight cutoff: 10,000 or more ・Pore diameter: 0.5 um or less ・Circulation flow linear velocity: 2 to 4 m/sec
(Reduced pressure distillation conditions)
Reduced pressure distillation temperature: 40-50°C

As described above, Test Examples 1 to 22 demonstrated that the specific configuration of the present invention makes it possible to efficiently produce target substances through saccharification reactions and microbial reactions using lignocellulosic materials as raw materials.

The present invention has high industrial applicability in fields such as material production.

2. Reactor (reactor, fermenter)
2a First reaction apparatus (first reaction tank, first fermenter)
2b Second reaction apparatus (second reaction tank, second fermenter)
3. Solid-liquid separation device (filter device)
4 Saccharifying enzyme/target substance separation device 4a Crude extraction device (coarse particle device or crude distillation column, saccharifying enzyme/target substance separating device)
4b Purification apparatus (rectification apparatus or rectification column)
6, 6a, 6b, 7, 8, 8a, 8b, 9, 11, 11a, 11b Piping 12 Pre-culture device 13 Saccharification enzyme storage tank 14 Sterile water storage tank 15 Screw press 17 Filtrate recovery tank 16a, 16b, 16c, 16d Piping 18a, 18b, 18c Pump 19 Target substance recovery/storage tank 22 Flow control valve L Pretreated lignocellulosic material M Microorganism E Saccharification enzyme C Reaction medium (culture medium)
D Wastewater (wastewater)

Claims (30)

(a) causing saccharification and a microbial reaction to proceed in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism, thereby producing at least one target substance;
(b) subjecting at least a portion of the reaction liquid to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and reaction residue;
(c) circulating at least a portion of the at least one saccharifying enzyme contained in fraction (X) obtained in step (b) to the reaction solution in step (a);
A method for producing a target substance, comprising: (d) circulating at least a portion of the microorganisms and reaction residues contained in the fraction (Y) obtained in the step (b) to the reaction solution in the step (a);
The method of claim 1 further comprising: The method according to claim 1, wherein in step (a), at least a portion of the reaction liquid is continuously transferred through a plurality of reaction systems connected in series, and saccharification and microbial reaction are continuously carried out in each of the plurality of reaction systems, and in step (b), at least a portion of the reaction liquid present in the reaction system located at the end of the plurality of reaction systems is subjected to the solid-liquid separation treatment. The method according to claim 1, wherein in step (c), the fraction (X) obtained in step (b) is subjected to a concentration treatment to concentrate the at least one saccharifying enzyme, and the concentrate of the at least one saccharifying enzyme obtained by the concentration treatment is circulated to the reaction solution in step (a). In step (b), at least a portion of the reaction solution is subjected to a solid-liquid separation treatment to obtain a fraction (X) containing the at least one saccharifying enzyme and the at least one target substance and a fraction (Y) containing the microorganism and reaction residue;
The method according to claim 1, wherein in step (c), fraction (X) obtained in step (b) is subjected to a saccharifying enzyme/target substance separation treatment to obtain a fraction (X1) containing the at least one saccharifying enzyme and a fraction (X2) containing the at least one target substance, and fraction (X1) is circulated to the reaction solution in step (a). The method according to claim 4, further comprising purifying the at least one target substance from at least a portion of fraction (X) and/or fraction (X2) obtained in step (c). The method according to claim 1, wherein at least one of the following (i) and (ii) is obtained as the at least one target substance:
(i) fraction (X), a concentrate of fraction (X), or a material extracted and/or purified from fraction (X);
(ii) Fraction (Y), a concentrate or treatment of fraction (Y), or a material extracted and/or purified from fraction (Y). The method according to claim 1, wherein in step (b), a solid-liquid separation process is performed on at least a portion of the reaction liquid using a filter. The method of claim 8, wherein the filter is a ceramic filter. The method according to claim 1, wherein in step (b), a cross-flow filter is used to perform a solid-liquid separation process on at least a portion of the reaction liquid. The method according to claim 1, wherein in step (b), solid-liquid separation of at least a portion of the reaction liquid is performed using a microfiltration filter having a pore size of 50 nm to 2.0 μm. The method according to claim 1, wherein in step (b), solid-liquid separation is carried out using an ultrafiltration filter having a molecular weight cutoff of 3100 or more. The method of claim 1, wherein the pretreated lignocellulosic material has a kappa number of 15 or less. The method of claim 1, wherein the at least one saccharification enzyme comprises cellulase. The method according to claim 1, wherein the microorganism is a microorganism capable of assimilating pentose. The method according to claim 1, wherein the microorganism is an alcohol-fermenting yeast. The method according to claim 1, wherein the microorganism is an ethanol-fermenting yeast. The method according to claim 1, wherein in step (a), the saccharification and microbial reaction are carried out at a temperature in the range of 30°C to 40°C. The method according to claim 1, wherein in step (a), the saccharification and microbial reaction are carried out at a pH in the range of 4 to 5.5. (A) a saccharification/microbial reaction unit that performs saccharification and microbial reaction in parallel in a reaction liquid containing a pretreated lignocellulosic material, at least one saccharification enzyme, and a microorganism;
(B) a solid-liquid separation unit that separates at least a portion of the reaction solution into a fraction (X) containing the at least one saccharifying enzyme and a fraction (Y) containing the microorganism and the reaction residue;
(C) a saccharification enzyme circulation unit that circulates at least a portion of the at least one saccharification enzyme contained in the fraction (X) obtained in the solid-liquid separation unit to the saccharification and microbial reaction proceeding in parallel in the saccharification/microbial reaction unit;
An apparatus comprising: (D) The apparatus according to claim 20, further comprising a microorganism/reaction residue circulation unit that circulates at least a portion of the microorganisms and reaction residue contained in fraction (Y) obtained in the solid-liquid separation unit to the reaction liquid in the saccharification/microbial reaction unit. The saccharification/microbial reaction unit comprises a plurality of reaction vessels or reaction compartments connected in series,
At least a part of the reaction solution is continuously moved through the plurality of reaction vessels or reaction compartments, and saccharification and microbial reaction are continuously performed in each of the plurality of reaction vessels or reaction compartments,
At least a part of the reaction liquid present in the reaction tank or reaction compartment located at the rearmost position among the plurality of reaction tanks or reaction compartments is configured to be supplied to the solid-liquid separation unit.
21. The apparatus of claim 20. The manufacturing apparatus according to claim 20, wherein the solid-liquid separation unit is equipped with a filter that separates at least a portion of the reaction liquid into a solid-liquid fraction (X) and a fraction (Y). The device of claim 23, wherein the filter is a ceramic filter. the saccharifying enzyme circulation unit includes a concentration treatment device that concentrates the at least one saccharifying enzyme in the fraction (X) obtained in the solid-liquid separation unit,
The concentrate of the at least one saccharification enzyme obtained by the concentration treatment device is configured to be circulated to the reaction liquid in the saccharification/microbial reaction unit.
21. The apparatus of claim 20. The apparatus of claim 20, wherein the solid-liquid separation unit is configured to separate at least a portion of the reaction liquid in the saccharification/microbial reaction unit into a fraction (X) containing the at least one saccharification enzyme and at least one target substance, and a fraction (Y) containing the microorganism and reaction residue. the saccharification enzyme circulation unit is configured to separate the fraction (X) obtained in the solid-liquid separation unit into a fraction (X1) containing the at least one saccharification enzyme and a fraction (X2) containing the at least one target substance, and to circulate the fraction (X1) to the reaction liquid in the saccharification/microbial reaction unit.
27. The apparatus of claim 26. The device according to claim 26 or 27, further comprising a target substance extraction/purification unit for extracting or purifying the at least one target substance from at least a portion of fraction (X) and/or fraction (X2). Further comprising a target substance extraction/purification unit,
28. The apparatus according to claim 26 or 27, wherein the target substance extraction/purification unit comprises at least one distillation apparatus for distilling or fractionating at least a portion of fraction (X) and/or fraction (X2) to obtain an extract or purification product of at least one volatile organic compound as said at least one target substance. The apparatus of claim 20, further comprising a pretreatment unit for performing a pretreatment on the raw lignocellulosic material to reduce a lignin content, thereby obtaining the pretreated lignocellulosic material.

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