JP2014039492A - Method of producing ethanol from lignocellulose-containing biomass - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of efficiently producing ethanol from a lignocellulose raw material.SOLUTION: In a method of producing ethanol from a lignocellulose-based raw material where pre-treated lignocellulose-based raw material suspension, which is a raw material suitable for enzymatic saccharification reaction, is supplied to two culture tanks (a primary culture tank and a secondary culture tank) connected in series, and parallel saccharifying fermentation is performed in the two culture tanks by using cellulose-saccharifying enzyme and Issatchenkia orientalis as yeast, parallel saccharifying fermentation is performed within a ratio between a solution capacity in the primary culture tank and a solution capacity in the secondary culture tank of 7:3 to 3:7, and the parallel saccharifying fermentation is performed in a state where the temperature of raw material suspension in the secondary culture tank is higher than the temperature of raw material suspension in the primary culture tank by 1°C or more.

Description

  The present invention relates to a method for efficiently producing ethanol from biomass containing lignocellulose.

Attempts to produce ethanol from biomass resources such as bagasse, rice straw, and wood chips, which are renewable resources, and to use them as energy and chemical raw materials are underway in Japan and overseas.
As a method for producing monosaccharides and small saccharides as fermentation substrates from polysaccharides in plant biomass, there is an enzyme saccharification method in which hydrolysis is performed using an enzyme or a microorganism that produces the enzyme. By enzymatic decomposition, cellulose and hemicellulose contained in the biomass are decomposed to produce hexoses such as glucose, galactose and mannose, and pentoses such as xylose and arabinose.

It is possible to produce ethanol by fermentation with microorganisms such as yeast using saccharides (hexose sugar, pentose sugar) produced by enzymatic decomposition as raw materials. In the case of producing ethanol, parallel saccharification and fermentation in which saccharification by enzyme and fermentation by yeast are carried out in the same culture tank is known. Parallel saccharification and fermentation has the advantage that the reaction time can be shortened compared to the method in which saccharification and fermentation are performed in separate culture vessels. However, since the enzyme reaction and yeast growth are carried out in the same reaction vessel, the enzyme reaction and yeast There is a problem that the reaction cannot be carried out at the optimum temperature suitable for the growth of the rice. In general, Saccharomyces cerevisiae is used for ethanol production. When Saccharomyces cerevisiae is used as a yeast in concurrent saccharification and fermentation, Saccharomyces cerevisiae can grow at about 30 ° C., so it is necessary to carry out concurrent saccharification and fermentation at about 30 ° C., which reduces the reaction of saccharifying enzymes (saccharifying enzymes) This reaction is generally suitable at 40 to 50 ° C.). In recent years, attempts have been made to create thermotolerant yeasts in order to overcome the above problems, and as an example, Isatchenkia orientalis that can grow at around 40 ° C. has been reported. When ethanol production is performed, the growth conditions of yeast or the ethanol production efficiency by yeast vary depending on the type of yeast. Therefore, it is necessary to optimize conditions suitable for ethanol production in parallel saccharification and fermentation (or fermentation process).

In a method for producing ethanol by concurrent saccharification and fermentation of cellulosic biomass, a method for performing concurrent saccharification and fermentation (simultaneous saccharification and fermentation) by reducing the reaction temperature stepwise or continuously from the initial temperature has been reported (Patent Literature). 1). However, since the growth conditions of yeast or the ethanol production efficiency by yeast vary depending on the type of yeast, it is necessary to optimize conditions suitable for the ethanol production of the yeast when a specific yeast is used.

JP 2010-246422 A

  The subject of this invention is providing the method of manufacturing ethanol efficiently in the method of manufacturing ethanol using Isachenchia orientalis as yeast from biomass containing lignocellulose by parallel saccharification and fermentation.

As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have two tanks in which lignocellulosic raw material suspensions that have been pretreated as raw materials suitable for enzymatic saccharification are connected in series. From the lignocellulosic raw material, which is supplied to a culture tank (primary culture tank and secondary culture tank) and subjected to parallel saccharification and fermentation using cellulose saccharifying enzyme and yeast Isachenchia orientalis in the two tanks In the ethanol production method, the saccharification and fermentation is performed in a ratio of the solution volume in the primary culture tank and the solution volume in the secondary culture tank in the range of 7: 3 to 3: 7, and the raw material suspension in the primary culture tank It has been found that ethanol can be produced efficiently by carrying out parallel saccharification and fermentation at a temperature of 1 ° C. or more higher than the temperature of the raw material suspension in the secondary culture tank. Completed the invention.

(1) Supplying to the two culture tanks (primary culture tank and secondary culture tank) which connected in series the lignocellulosic raw material suspension in which the pretreatment made into the raw material suitable for enzyme saccharification reaction is performed, In the method for producing ethanol from lignocellulosic raw materials in which saccharification and fermentation is performed using cellulose saccharifying enzyme and Isatchenkia orientalis as yeast in the two tanks, the solution volume and the secondary volume in the primary culture tank The parallel saccharification and fermentation is performed in a ratio of the solution volume in the culture tank of 7: 3 to 3: 7, and the temperature of the raw material suspension in the secondary culture tank is determined from the temperature of the raw material suspension in the primary culture tank. A method for producing ethanol from a lignocellulosic material, characterized in that parallel saccharification and fermentation is performed at a temperature of 1 ° C or higher.

(2) Parallel saccharification and fermentation are performed in a temperature range of 36 to 38 ° C. of the raw material suspension in the primary culture tank and a temperature of the raw material suspension in the secondary culture tank of 39 to 43 ° C. The method for producing ethanol from the lignocellulosic raw material according to (1).

(3) The lignocellulosic raw material that has been pretreated as a raw material suitable for the enzymatic saccharification reaction is selected from chemical treatment, pressurized hot water treatment, and mechanical treatment for the lignocellulose raw material. The method for producing ethanol from a lignocellulosic raw material according to (1) or (2), which comprises a lignocellulose-containing biomass that has been subjected to pretreatment including one or more treatments.

(4) The method for producing ethanol from a lignocellulosic material according to any one of (1) to (3), wherein the lignocellulosic material is a forest land residue.

According to the present invention, a lignocellulosic raw material suspension, which has been pretreated as a raw material suitable for enzymatic saccharification reaction, is supplied to two culture tanks (primary culture tank and secondary culture tank) connected in series. In the method for producing ethanol from a lignocellulosic raw material in which saccharification and fermentation is performed using cellulose saccharifying enzyme and yeast as Isachenchia orientalis in the two culture tanks, the solution volume in the primary culture tank and The ratio of the solution volume in the secondary culture tank is subjected to parallel saccharification and fermentation in the range of 7: 3 to 3: 7, and the temperature of the raw material suspension in the secondary culture tank is determined from the temperature of the raw material suspension in the primary culture tank. It is possible to efficiently produce ethanol by carrying out parallel saccharification and fermentation at a temperature higher by 1 ° C. or higher.

It is a figure which shows an example of the apparatus for implementing the continuous production method of the saccharide | sugar from the lignocellulose raw material of this invention, or ethanol.

  Hereinafter, the present invention will be described in more detail.

<Lignocellulose raw material>
As a lignocellulosic raw material used as a raw material in the method of the present invention, as a woody system, chips or bark of papermaking trees, forest land residual materials, thinned wood, etc., sprouts generated from stumps of woody plants, sawmills, etc. Sawdust or sawdust that is generated, pruned branches and leaves of street trees, building waste, etc., and herbaceous agricultural waste such as kenaf, rice straw, straw, corn cob, bagasse, industrial crop residues such as oil crops and rubber, and Examples include wastes (for example, EFB: Empty Fruit Bunch), herbaceous energy crops Eliansus, Miscanthus, and Napiergrass.
Further, as biomass, food waste such as paper derived from wood, waste paper, pulp, pulp sludge, sludge, sewage sludge, and the like can be used as raw materials. These biomasses can be used alone or in combination. The biomass can be used as a dry solid, a solid containing water, or a slurry.

  Examples of the woody lignocellulosic raw material include Eucalyptus genus plants, Salix genus plants, Poplar genus plants, Acacia genus plants, and Cryptomeria genus plants. Plants, genus Acacia and willow genus are preferable because they can be easily collected in large quantities as raw materials. Among the lignocellulosic raw materials derived from woody plants, forest land remnants (including bark and leaves) and bark are preferable. For example, bark of tree species such as Eucalyptus genus or Acacia genus commonly used for papermaking raw materials can be obtained in large quantities stably from lumber mills and chip factories for papermaking raw materials. Preferably used.

<Mechanical processing>
In the present invention, the lignocellulose raw material can be subjected to mechanical treatment. Examples of the mechanical treatment include any mechanical means such as cutting, cutting, crushing, and grinding, and making lignocellulose easy to be saccharified in the next chemical treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a cutting device, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader, a ball mill etc. can be used.

As a pre-process or post-process of the mechanical treatment, a foreign matter removing step by washing or the like for removing foreign matter (foreign matter other than lignocellulose such as stone, dust, metal, plastic) can be introduced.
Examples of the method for washing the raw material include a method of removing water from the foreign material mixed with the raw material by spraying water on the raw material, or a method of removing the foreign material by immersing the raw material in water and sedimenting the foreign material. Moreover, the method of isolate | separating a foreign material from a raw material using apparatuses, such as a metal trap, is mentioned.
If foreign materials are included in the raw materials, there is a possibility of causing damage to equipment parts used in mechanical processing such as refiner discs (plates), and causing problems in the manufacturing process such as clogging of piping. Therefore, it is desirable to introduce a foreign substance removing step.

<Chemical treatment>
Next, the mechanically treated lignocellulose raw material is then chemically treated. As the chemical treatment, one or more alkali chemicals selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate, or one or more selected from sodium sulfite and the above alkaline chemicals are used. This is a pretreatment including a chemical treatment of immersing in a solution containing an alkaline chemical. Further, chemical treatment with an oxidizing agent such as ozone or chlorine dioxide is also possible.
The chemical treatment is preferably performed as a post-treatment of the pretreatment in combination with the mechanical treatment.

  The amount of chemicals used in the chemical treatment can be arbitrarily adjusted depending on the situation, but in order to reduce chemical costs and to suppress the yield reduction due to cellulose elution / overdecomposition, It is desirable that it is 50 mass parts or less with respect to 100 mass parts of absolutely dry cellulosic raw materials. The immersion time and the treatment temperature of the chemical in the chemical treatment can be arbitrarily set depending on the raw materials and chemicals to be used, but a treatment time of 20 to 90 minutes and a treatment temperature of 80 to 200 ° C. are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing time is preferably 70 minutes or less and the processing temperature is preferably 180 ° C. or less.

  As chemical treatment, 10 to 50% by mass of sodium sulfite and 0.1 to 5% by mass of alkali as a pH adjuster can be added to the lignocellulose raw material (dry weight). When sodium sulfite is added alone to the lignocellulose in the above-mentioned addition amount and heat-treated, an organic acid such as acetic acid is generated during hydrolysis, so that the pH is lowered and the hydrolyzed solution becomes acidic. When hydrolysis is continued under acidic conditions, the problem arises that xylose produced by hydrolysis is converted to furfural. Since furfural is an inhibitor of ethanol fermentation, it is desirable not to produce it as much as possible. Moreover, since the yield of xylose which is a fermentation substrate falls, ethanol production efficiency falls as a result. In the present invention, by adding sodium sulfite and an alkali as a pH adjuster to the lignocellulose raw material in the above-described amount and heat-treating, the pH during hydrolysis is maintained from neutral to weakly alkaline. Production and reduction in xylose yield can be suppressed. Moreover, since the pH of the aqueous solution containing lignocellulose after heat treatment (after hydrolysis) is 4.0 to 7.0 (neutral to weakly alkaline), the waste liquid or hydrolyzate after the hydrolysis treatment is neutralized. There is a merit that the amount of chemicals used for the reduction can be reduced.

  Examples of the alkali used as the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate and the like, but are not particularly limited to these chemicals. The alkali used is preferably sodium hydroxide.

The heat treatment temperature when the heat treatment is performed by adding 10 to 50% by weight of sodium sulfite and 0.1 to 5% by weight of alkali as a pH adjuster to the lignocellulose raw material (dry weight) is 80 -200 degreeC is preferable and 120-180 degreeC is more preferable. Moreover, although heat processing time can be performed in 10 to 300 minutes, 30 to 120 minutes are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing temperature is preferably 180 ° C. or lower and the processing time is 120 minutes or shorter.

(Grinding treatment)
In the present invention, the lignocellulosic raw material obtained by the chemical treatment is preferably ground in a refiner disk (plate) clearance of 0.1 to 2.0 mm, preferably in the range of 0.1 to 1.0 mm. Further preferred. As a refiner to be used, a single disk refiner, a double disk refiner, or the like can be used, and any refiner that can set the clearance of the opposing disk within a range of 0.1 to 2.0 mm can be used without particular limitation. it can. If the disc clearance exceeds 2.0 mm, the sugar yield obtained by saccharification or concurrent saccharification and fermentation is added, which is not preferable. On the other hand, if the disc clearance is lower than 0.1 mm, the yield of the hydrolyzate (solid content) after grinding with a refiner is not preferable. Also, if the disc clearance is lower than 0.1 mm, the electricity consumption required for the operation of the refiner increases, which is not preferable.

  The lignocellulosic raw material that has been subjected to the above grinding treatment is subjected to solid-liquid separation into an aqueous solution and a solid content, and the solid content is used as a raw material for saccharification or concurrent saccharification and fermentation. As a method for solid-liquid separation, for example, an apparatus that can be separated into an aqueous solution and a solid content using a screw press or the like and can be used without limitation as long as it can be separated into an aqueous solution and a solid content.

It is preferable to perform sterilization treatment before saccharification or parallel saccharification fermentation using the raw material after the solid separation. When miscellaneous bacteria are mixed in the lignocellulosic biomass raw material, there is a problem that the miscellaneous bacteria consume sugar when the enzyme is saccharified and the yield of the product decreases.
The sterilization treatment may be a method in which the raw material is exposed to a pH at which bacteria are difficult to grow, such as an acid or an alkali. About the raw material after an acid and an alkali treatment, it is preferable to use as a raw material, after adjusting to neutrality vicinity or pH suitable for a saccharification and / or saccharification fermentation process. In addition, even when pasteurized at a high temperature, it is preferably used as a raw material after the temperature is lowered to room temperature or a temperature suitable for the saccharification and fermentation process. Thus, by feeding out the raw material after adjusting the temperature and pH, it is possible to prevent the enzyme from being exposed to the outside of the preferred pH and the preferred temperature and being deactivated.

  The lignocellulose raw material subjected to the pretreatment is supplied to the concurrent saccharification and fermentation step.

<Concurrent saccharification and fermentation process>
The lignocellulosic raw material that has been subjected to pretreatment suitable for enzymatic saccharification reaction is supplied to the parallel saccharification and fermentation process, mixed with an appropriate amount of water and enzyme to form a raw material suspension, and further yeast: Isatchenkia Orientalis (Issatchenkia) orientalis). The lignocellulosic raw material is saccharified by an enzyme, and the produced saccharide is fermented to ethanol by yeast.

  In the present invention, two culture tanks (a primary culture tank and a secondary culture tank) are connected in series in the parallel saccharification and fermentation step. If the said culture tank is a culture tank which can perform parallel saccharification fermentation, the capacity | capacitance, shape, and material of a culture tank will not be restrict | limited in particular. The ratio of the solution volume in the primary culture tank and the secondary culture tank is preferably in the range of 7: 3 to 3: 7, and more preferably in the range of 6: 4 to 4: 6.

As shown in FIG. 1, the pretreated lignocellulosic material is added continuously or intermittently from the supply port 1 of the primary culture tank A. The raw material to be added may be in a solid state or suspended in an aqueous solution. In the primary culture tank A, the raw saccharification and fermentation treatment of the raw material is performed, the lignocellulosic raw material is saccharified by an enzyme, and the produced saccharide is simultaneously converted into ethanol. The raw material suspension after the concurrent saccharification and fermentation is continuously or intermittently discharged from the discharge port 2 of the primary culture tank A.

The raw material suspension discharged from the discharge port 2 of the primary culture tank A is supplied into the secondary culture tank B from the supply port of the secondary culture tank B. Undegraded fibers and polysaccharides contained in the raw material suspension supplied into the secondary culture tank B are further subjected to parallel saccharification and fermentation treatment in the secondary culture tank B. The raw material suspension after the concurrent saccharification and fermentation is discharged continuously or intermittently from the discharge port 3 of the secondary culture tank B.

In the present invention, it is preferable to perform parallel saccharification and fermentation at a temperature of the raw material suspension in the secondary culture tank higher by 1 ° C. or more than the temperature of the raw material suspension in the primary culture tank. It is more preferable to perform parallel saccharification and fermentation at a temperature of the suspension of 36 to 38 ° C and a temperature of the raw material suspension in the secondary culture tank of 39 to 43 ° C.
By maintaining the temperature of the raw material suspension in the primary culture tank A and the secondary culture tank B in the above range, glycerol produced as a by-product is converted to ethanol, so that ethanol productivity is increased.

Cellulolytic enzymes used in parallel saccharification and fermentation are enzymes collectively called cellulases having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity.
Each cellulolytic enzyme may be added with an appropriate amount of an enzyme having the respective activity. However, commercially available cellulase preparations have the above-mentioned various cellulase activities and also have hemicellulase activity. Since many products are available, a commercially available cellulase preparation may be used.

Commercial cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, the genus Humicola, and the like. There are cellulase formulations derived from Commercially available products of such cellulase preparations are all trade names, for example, cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor) ), GC220 (manufactured by Genencor).
0.5-100 mass parts is preferable and, as for the usage-amount of the cellulase formulation with respect to 100 mass parts of raw material solid content, 1-50 mass parts is especially preferable.

The pH of the reaction solution in the concurrent saccharification and fermentation step is preferably maintained in the range of 3.5 to 10.0, more preferably in the range of pH 4.0 to 7.5.

  The reaction in the concurrent saccharification and fermentation process is preferably a continuous type, but may be a semibatch type or a batch type.

The residence time of the reaction solution in each culture tank (primary culture tank A or secondary culture tank B) in the concurrent saccharification and fermentation step is preferably 3 to 100 hours, and more preferably 5 to 50 hours.

Isatchenkia orientalis used as yeast may be a genetically modified microorganism prepared using a gene recombination technique. As the genetically modified microorganism, a microorganism capable of simultaneously fermenting hexose and pentose can be used without particular limitation.
Microorganisms may be immobilized. By immobilizing microorganisms, the process of separating and re-recovering microorganisms in the next process can be omitted, so that at least the burden required for the recovery process can be reduced and the loss of microorganisms can be reduced. is there. Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.

  In the present invention, a water-soluble salt can be added as an electrolyte in the concurrent saccharification and fermentation process. It is preferable to add an electrolyte to the raw material suspension used in the concurrent saccharification and fermentation step to maintain the electric conductivity of the raw material suspension in the range of 5 to 25 mS / cm. By maintaining the electric conductivity in the range of 5 to 25 mS / cm, the adsorption of the enzyme to the unreacted components and reaction residues of the lignocellulose raw material is suppressed, so the enzyme circulation rate in the process is high for a long period of time. Can be maintained. The electrolyte can be added without limitation in any process as long as the electrolyte can be added in the operation in the sugar production process or ethanol production process, but it is added in the concurrent saccharification and fermentation process. This is desirable because it is easy to operate.

  As the water-soluble salt, salts selected from alkali metal salts and alkaline earth metal salts are preferable. Alkali metal salts and alkaline earth metal salts include alkali metal and alkaline earth metal halides, sulfates, sulfites, thiosulfates, carbonates, bicarbonates, phosphates, dihydrogen phosphates, Examples thereof include water-soluble salts selected from the group consisting of hydrogen phosphate di-salt, acetate and citrate.

The culture solution discharged from the concurrent saccharification and fermentation step can be transferred to a solid-liquid separation step and separated into a liquid component (filtrate) and a solid component (residue). As an apparatus for performing solid-liquid separation, a screw press, a screen, a filter press, a belt press, a rotary press, or the like can be used. As the screen, a vibrating screen to which a vibrating device is added can be used.
The recovered solid content (residue) can be transferred to a parallel saccharification and fermentation process and used as a raw material for the parallel saccharification and fermentation.

The liquid component separated in the solid-liquid separation step can be transferred to a distillation step and a fermentation product (ethanol or the like) can be distilled and separated by a vacuum distillation apparatus. Since the fermentation product can be separated at a low temperature under reduced pressure, inactivation of the enzyme can be prevented. As the vacuum distillation apparatus, a rotary evaporator, a flash evaporator, or the like can be used.
The distillation temperature is preferably 25 to 60 ° C. If it is lower than 25 ° C., it takes time to distill the product, and the productivity is lowered. On the other hand, when the temperature is higher than 60 ° C., the enzyme is heat-denatured and deactivated, and the amount of newly added enzyme increases, resulting in poor economic efficiency.
The concentration of the fermentation product remaining in the distillation residue fraction after distillation is preferably 0.1% by mass or less. By setting it as such a density | concentration, the amount of fermentation products discharged | emitted with a solid substance in a subsequent solid-liquid separation process can be reduced, and a yield can be improved.

  The distillation residue after separating the fermentation product (such as ethanol) after distillation can be transferred to a residue separation step and separated into a liquid fraction and a residue. The liquid fraction separated in the residue separation step contains an enzyme and can be circulated to the parallel saccharification and fermentation tank BR1. Moreover, it can also transfer to a secondary parallel saccharification and fermentation process (The above-mentioned parallel saccharification and fermentation process is different from the primary parallel saccharification and fermentation process). In the secondary concurrent saccharification and fermentation step, a new lignocellulose raw material can be added to cause saccharification and fermentation, or fermentation for the purpose of fermentation of pentoses such as xylose can be performed.

Residues separated in the residue separation step include enzymes, lignin, and fermenting microorganisms. Lignin can be recovered as a combustion raw material and used as energy, or lignin can be recovered and used effectively. Moreover, fermenting microorganisms (yeast etc.) can be isolate | separated from a residue, and can also be reused in a parallel saccharification fermentation process. In addition, since the enzyme is adsorbed on the residue, water-soluble salts are added to the residue (residue suspension), the enzyme is released from the residue, the enzyme is recovered, and the recovered enzyme is reused in the process. You can also.

  When performing secondary concurrent saccharification and fermentation, a distillation step may be installed after the secondary concurrent saccharification and fermentation step in order to recover the fermentation product produced by the secondary concurrent saccharification and fermentation. Moreover, in order to remove the residue contained in the culture solution discharged | emitted from the secondary parallel saccharification and fermentation tank, solid-liquid separation can also be performed after a secondary parallel saccharification and fermentation process, and a residue can also be removed.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these Examples.

[Experimental Example 1]
The test was carried out in the manufacturing process shown in FIG.
[Preprocessing]
Chip-like eucalyptus and globula woodland residues (70% bark, 30% branches and leaves) were crushed with a uniaxial crusher (SC-15, manufactured by Saiho Kiko Co., Ltd.) equipped with a 20 mm round hole screen and used as a raw material.
After adding 20 kg of 97% sodium sulfite and 1 kg of sodium hydroxide to 100 kg (absolute dry weight) of the raw material, water was added to adjust the volume of the aqueous solution to 800 L. The raw material suspension was mixed and then heated at 170 ° C. for 1 hour. The raw material suspension after the heat treatment was crushed with a refiner (manufactured by Kumagai Riki Kogyo Co., Ltd., KRK high concentration disc refiner) with the disc (plate) clearance set to 1.0 mm. Next, solid-liquid separation (dehydration) was performed using a 20-mesh (847 μm) screen, and the solution was washed with water until the electric conductivity of the solution reached 30 μS / cm. A saccharification test was carried out using the solid (pretreated product) after solid-liquid separation as a raw material.

[Concurrent saccharification and fermentation]
In advance, in a liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, pH 5.6) as yeast, Isachenchia orientalis (Issatchencia orientalis, this strain is May 2003) The deposit was made at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on the 22nd, and the accession number FERM P-19368) was cultured at 37 ° C. for 24 hours.
As shown in FIG. 1, two culture tanks (culture tank A and culture tank B) were connected to perform parallel saccharification and fermentation. The volume of the solution in the culture tank A was 1.0 m ³ , and the volume of the solution in the culture tank B was 1.0 m ³ (the ratio of the solution volume was culture tank A: culture tank B = 1: 1).
Polypeptone 5 g / L in the culture vessel A, and adjusted yeast extract 3 g / L, after the addition of each such that the malt extract 3 g / L, water was added to a final volume of 0.8 m ^3. A culture solution containing yeast cells was added to the culture tank A and cultured for 24 hours. When the yeast density grew to 1 × 10 ⁸ / ml, 50 L of a commercially available cellulase solution (Accelerase DUET, Genencor) was added to the culture tank A. Next, water was added to the culture tank A to adjust the final volume of the culture solution to 1 m ³ .
Next, a raw material suspension having a raw material concentration of 10% by mass was continuously added from the supply port 1 of the culture tank A. The saccharification and fermentation treatment was carried out by setting the retention time of the culture solution in the culture tank A (time for the raw material suspension to pass through the culture tank A: capacity / flow rate of the culture tank A) to 15 hours. That is, the raw material suspension was continuously added from the raw material supply port 1 of the culture tank A at a flow rate of 66.6 L / h from the time when the concurrent saccharification and fermentation treatment was started. On the other hand, the raw material suspension was discharged at 66.6 L / h from the discharge port 2 of the culture tank A simultaneously with the start of the supply of the raw material, and transferred to the culture tank B. Also in the culture tank B, the saccharification and fermentation treatment was performed by setting the residence time of the raw material suspension (time for the raw material suspension to pass through the culture tank B: capacity / flow rate of the culture tank B) to 15 hours. The cellulase solution was continuously added to the culture tank A at 3.3 L / h. The culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 38 ° C. Further, the pH of the raw material suspension was maintained at 5.0 in both the culture tank A and the culture tank B. In addition, when the volume of the raw material suspension decreased during the continuous operation, the final volume of the culture solution was maintained at 1 m ³ by automatically adding the medium. The pH of the raw material suspension during the culture was maintained at 5.0.
The concentration of ethanol contained in the raw material suspension discharged from the discharge port 3 of the culture tank B 30 hours after the raw material suspension is discharged from the culture tank B (when it reaches a steady state) is measured with a glucose sensor (Oji Measured with a measuring instrument BF-400 type). The results are shown in Table 1.

[Experiment 2]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 3]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 4]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 5]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 6]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 7]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 8]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 9]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 10]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 11]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 12]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 13]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 14]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 15]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 16]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 17]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 18]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 19]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 20]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 21]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 22]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 23]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 24]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 25]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 38 ° C and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 26]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 27]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 28]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 29]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 30]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 31]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experiment 32]
In Experimental Example 1, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 33]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental example 34]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

[Experimental Example 35]
In Experimental Example 1, culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 1. The results are shown in Table 1.

As shown in Table 1, when the temperature of the culture tank A is 36 to 38 ° C. and the temperature of the culture tank B is 39 to 43 ° C. (Experimental Examples 9 to 13, Experimental Examples 16 to 20, Experimental Examples) 23-27), when tested in a temperature range other than the above (Experimental Examples 1-8, Experimental Examples 14 and 15, Experimental Examples 21 and 22, Experimental Examples 28-35), the ethanol concentration was higher. Moreover, when the temperature of the culture tank B was tested by 1 degree C or more higher than the temperature of the culture tank A, the ethanol concentration was high.

[Experimental Example 36]
In Experimental Example 1, the volume of the solution in the culture tank A was 1.4 m ³ , and the volume of the solution in the culture tank B was 0.6 m ³ (the ratio of the solution volume is the culture tank A: the culture tank B = Except for the change to 7: 3), all tests were performed in the same manner as in Experimental Example 1. The flow rate of the raw material suspension passing through the culture tank A and the culture tank B was set at 66.6 L / h as in Experimental Example 1. The results are shown in Table 2.

[Experimental Example 37]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experiment 38]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 39]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 40]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental example 41]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 42]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 43]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 44]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 45]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental example 46]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental example 47]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 48]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 49]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 50]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 51]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 52]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 53]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 54]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 55]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 56]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 57]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 58]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experiment 59]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 60]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 61]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 62]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 63]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 64]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 65]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 66]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 67]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Experimental Example 68]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 69]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

[Example 70]
In Experimental Example 36, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 36. The results are shown in Table 2.

As shown in Table 2, when the temperature of the culture tank A was 36 to 38 ° C. and the temperature of the culture tank B was 39 to 43 ° C. (Experimental Examples 44 to 48, Experimental Examples 51 to 55, Experimental Examples) 58-62), when tested in a temperature range other than the above (Experimental Examples 36-43, Experimental Examples 49 and 50, Experimental Examples 56 and 57, Experimental Examples 63-70), the ethanol concentration was higher. Moreover, when the temperature of the culture tank B was tested by 1 degree C or more higher than the temperature of the culture tank A, the ethanol concentration was high.

[Experimental Example 71]
In Experimental Example 1, the volume of the solution in the culture tank A was 0.6 m ³ , and the volume of the solution in the culture tank B was 1.4 m ³ (the ratio of the solution volume is the culture tank A: the culture tank B = All tests were performed in the same manner as in Experimental Example 1 except that the ratio was changed to 3: 7). The flow rate of the raw material suspension passing through the culture tank A and the culture tank B was set at 66.6 L / h as in Experimental Example 1. The results are shown in Table 3.

[Experimental Example 72]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 73]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 74]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 75]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 76]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 77]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 35 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 78]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 79]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 80]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 81]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experiment 82]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 83]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 84]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 36 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 85]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 86]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experiment 87]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 88]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 89]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 90]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Example 91]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 37 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Example 92]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 93]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 94]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 95]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 96]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 97]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 98]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 38 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 99]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 38 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 100]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 39 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 101]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 40 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental example 102]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 41 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 103]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 42 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 104]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 43 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

[Experimental Example 105]
In Experimental Example 71, the culture was performed while maintaining the temperature of the culture tank A at 39 ° C. and the temperature of the culture tank B at 44 ° C. All other operations were tested in the same manner as in Experimental Example 71. The results are shown in Table 3.

As shown in Table 3, when the temperature of the culture tank A was 36 to 38 ° C and the temperature of the culture tank B was 39 to 43 ° C (Experimental examples 79 to 83, Experimental examples 86 to 90, Experimental examples) 93-97), when tested in a temperature range other than the above (Experimental Examples 71-78, Experimental Examples 84 and 85, Experimental Examples 91 and 92, Experimental Examples 96-105), the ethanol concentration was higher. Moreover, when the temperature of the culture tank B was tested by 1 degree C or more higher than the temperature of the culture tank A, the ethanol concentration was high.

The present invention provides a method for producing ethanol with high production efficiency from lignocellulose.

1: Raw material supply port 2: Cultivation tank A discharge port 3: Cultivation tank B discharge port A: Primary culture tank B: Secondary culture tank

Claims (4)

The lignocellulosic raw material suspension, which has been pretreated as a raw material suitable for enzymatic saccharification reaction, is supplied to two culture tanks (primary culture tank and secondary culture tank) connected in series. In a method for producing ethanol from lignocellulosic raw materials in which simultaneous saccharification and fermentation is carried out using cellulose saccharifying enzyme and Isatchenkia orientalis as yeast in the culture tank of the above, the solution volume in the primary culture tank and the secondary culture tank The saccharification and fermentation is performed in a ratio of the solution volume of 7: 3 to 3: 7, and the temperature of the raw material suspension in the secondary culture tank is 1 ° C. or higher than the temperature of the raw material suspension in the primary culture tank. A method for producing ethanol from a lignocellulosic raw material, characterized by carrying out concurrent saccharification and fermentation at a high temperature. The parallel saccharification and fermentation is carried out at a temperature of the raw material suspension in the primary culture tank of 36 to 38 ° C and a temperature of the raw material suspension in the secondary culture tank of 39 to 43 ° C. Item 2. A method for producing ethanol from a lignocellulosic material according to Item 1. One or more lignocellulosic raw materials that have been pretreated as raw materials suitable for the enzymatic saccharification reaction are selected from chemical treatment, pressurized hot water treatment, and mechanical treatment for the lignocellulosic raw materials. The method for producing ethanol from a lignocellulosic raw material according to claim 1 or 2, comprising a lignocellulose-containing biomass that has been subjected to a pretreatment including the above treatment. The method for producing ethanol from a lignocellulosic raw material according to claim 1, wherein the lignocellulosic raw material is forest land residue.

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