JP4930650B1 - Method for producing ethanol from lignocellulose-containing biomass - Google Patents

Abstract

In a method for producing ethanol from lignocellulose raw material, an ethanol production method having high production efficiency is provided.
[Solution]
The culture solution after saccharification and fermentation using lignocellulose as a raw material is solid-liquid separated with a screw press having a screen size of 1.0 to 2.0 mm, and fine fibers that cannot be removed in the solid-liquid separation step are sieved with 80 to 600 mesh. It is possible to improve the ethanol production amount by selectively recovering and re-saccharifying or simultaneous saccharification and fermentation using the recovered fine fiber as a raw material. Furthermore, the ethanol production can be improved by adding an electrolyte in the process.
[Selection] Figure 1

Description

  In the method for producing ethanol from biomass containing lignocellulose, the present invention recovers the fine fibers remaining in the suspension after the concurrent saccharification and fermentation treatment, and the collected fine fibers are again saccharified or the concurrent saccharification and fermentation treatment. The present invention relates to a method for producing ethanol from biomass containing lignocellulose, which can improve ethanol production.

The technology to produce sugar from lignocellulose raw material that has been treated suitable for saccharification is the use of this sugar as a fermentation substrate for microorganisms to convert alcohol as an alternative fuel for gasoline, succinic acid and lactic acid as plastic raw materials. Since it can produce raw materials for products, it is a useful technology for the formation of a recycling society.
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. The lignocellulose material from which lignin has not been removed is less susceptible to degradation by enzymes than the lignocellulose material from which lignin has been removed, and remains as a residue in the saccharified solution together with impurities such as resin and metal without being saccharified. In general, the residue is separated and discarded by screen, centrifugation, or the like. The problem is to collect and effectively use the residue to reduce the cost of the enzymatic saccharification method. As a technique for reusing the residue recovered in the enzymatic saccharification method, a method of obtaining thermal energy by burning the residue (Patent Document 1), hydrolyzing the residue into hydrothermal gas, and synthesizing ethanol from the generated synthesis gas with an ethanol synthesis catalyst A method (Patent Document 2), a method using a residue as fuel or fertilizer (Patent Document 3), and a method using a residue as thermal energy (Patent Document 4) have been reported. However, these methods are not sufficient as a method for solving the problem of cost reduction when devising a practical facility because the cost increase accompanying the addition of the processing step is great.
Moreover, the technique which returns the undegraded residue which the enzyme after the saccharification fermentation process adsorb | sucked to a saccharification fermentation process again, and reuses an enzyme is reported (patent document 5). However, in this method, since the undecomposed residue itself is in a state that is not easily decomposed even if mixed with the enzyme solution again, it is a problem to make the undegraded residue easily saccharified. The present inventors have found that the amount of ethanol produced is increased by mechanically treating the undecomposed residue recovered by solid-liquid separation and again performing saccharification and fermentation (Patent Document 6). However, there is a problem that the residue used as a raw material in this method is a residue collected by a 420 mesh (38 μm) screen and contains a wide range of fibers. Large fibers with a large amount of adsorbed lignin are difficult to be decomposed by enzymes, so they cannot be sufficiently saccharified unless pretreated (such as mechanical treatment). If only small-sized fibers with a small amount of lignin adsorbed are selectively collected and pretreated as a raw material and can be enzymatically saccharified again, an efficient increase in ethanol production can be expected.

Patent No. 4447148 JP 2005-168335 A JP 2008-54676 A JP 2009-106932 A JP 2010-98951 A JP 2011-41493 A

  The subject of this invention is providing the ethanol manufacturing method with a high ethanol yield in the ethanol manufacturing process which uses lignocellulose as a raw material.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors selectively recovered only fine fibers that are easily saccharified from the residue contained in the culture solution of the concurrent saccharification and fermentation process, and the recovered fine fibers It was found that by performing saccharification or parallel saccharification and fermentation again as a raw material, the amount of ethanol produced was improved and the amount of residue discharged in the process was reduced, and the following invention was completed.

(1) Pretreatment step of treating lignocellulose raw material as a raw material suitable for enzymatic saccharification reaction, enzymatic saccharification of pretreated lignocellulose raw material with enzyme, and saccharide produced by enzymatic saccharification treatment as a substrate A primary parallel saccharification and fermentation process performed in parallel with the fermentation process, and a solid suspension that separates the treated suspension from the primary parallel saccharification and fermentation process into a residue and a liquid fraction with a screw press having a screen size of 1.0 to 2.0 mm. A liquid separation step, a sieving treatment step for separating the liquid fraction separated in the solid-liquid separation step into a fine fiber and a liquid fraction by a sieving treatment of 80 to 600 mesh, a liquid fraction excluding the fine fiber after the sieving treatment In a method for producing ethanol from lignocellulosic raw materials having a distillation step of separating and recovering fermentation products by distillation under reduced pressure, the separated fine fibers are saccharified or simultaneously saccharified Ethanol production process from lignocellulosic feedstock, characterized by.

(2) The method for producing ethanol from a lignocellulosic raw material according to (1), wherein the separated fine fibers are transferred to a primary saccharification and fermentation step and subjected to saccharification and fermentation.

(3) The method for producing ethanol from a lignocellulosic raw material according to (1), wherein the separated fine fibers are transferred to a secondary saccharification and fermentation step and subjected to saccharification and fermentation.

(4) The pretreatment of the lignocellulosic raw material is a method wherein the lignocellulosic raw material is one or more alkaline chemicals selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogencarbonate, or sodium sulfite Any one of the items (1) to (3), which is a pretreatment including a chemical treatment immersed in a solution containing one or more alkaline chemicals selected from the alkaline chemicals. A method for producing ethanol from the lignocellulosic raw material described.

(5) In the primary parallel saccharification and fermentation step, an electrolyte composed of a water-soluble salt is added to the raw material suspension, and saccharification or parallel saccharification and fermentation is performed with the electric conductivity of the raw material suspension being 5 to 25 mS / cm. The method for producing ethanol from a lignocellulosic material according to any one of items (1) to (4).

  According to the present invention, the amount of ethanol produced by selectively recovering the fine fibers contained in the culture solution after the simultaneous saccharification and fermentation using lignocellulose as a raw material, and performing saccharification or parallel saccharification and fermentation again using the recovered fine fibers as a raw material Can be improved.

The figure which shows the manufacturing process flow of Example 1. The figure which shows the manufacturing process flow of Example 2. The figure which shows the manufacturing process flow of the comparative example 1 The figure which shows the manufacturing process flow of Example 5. The figure which shows the manufacturing process flow of Example 6.

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

<Lignocellulose raw material>
As the lignocellulosic raw material used as a raw material in the method of the present invention, as woody material, chips or bark of papermaking trees, forest land residual materials, thinned wood, sawdust or sawdust generated from lumber mills, etc., pruning roadside trees Branches and leaves, building waste, etc. are listed. Herbaceous products include agricultural waste such as kenaf, rice straw, straw, bagasse, herbaceous energy crops Elianthus, Miscanthus and Napiergrass. In addition, as the lignocellulosic material in the present invention, paper derived from wood, waste paper, pulp, pulp sludge and the like can be used.

Among the woody lignocellulosic raw materials, the bark of wood is hardly used at present and is available in large quantities at a lumber factory or chip factory, and from the wood part of the wood Since it is flexible and has many soluble components, it is particularly preferred as a raw material for saccharification treatment and concurrent saccharification and fermentation treatment.
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.

<Pretreatment suitable for parallel saccharification and fermentation>
The lignocellulose that has been subjected to pretreatment suitable for the enzymatic saccharification treatment of the present invention is lignocellulose that has been subjected to the following pretreatment on the lignocellulose-based raw material so that the lignocellulose can be subjected to concurrent saccharification and fermentation.
Mechanical treatment, chemical treatment, hydrothermal treatment, pressurized hot water treatment, carbon dioxide added hydrothermal treatment, steaming treatment, wet grinding treatment, dilute sulfuric acid treatment, steam explosion treatment, ammonia explosion treatment, carbon dioxide explosion treatment, ultrasonic irradiation Treatment, microwave irradiation treatment, electron beam irradiation treatment, gamma ray irradiation treatment, supercritical treatment, subcritical treatment, organic solvent treatment, phase separation treatment, wood decay fungus treatment, green solvent activation treatment, various catalyst treatments, radical reaction Treatment, ozone oxidation treatment.
These processes may be either single processes or a combination of a plurality of processes. Among these, it is preferable to perform one or more pretreatments selected from alkali treatment, pressurized hot water treatment, and mechanical treatment on the lignocellulose-containing biomass.

  Examples of the mechanical treatment include arbitrary mechanical means such as crushing, cutting, and grinding, and making lignocellulose easy to be saccharified in the subsequent saccharification and fermentation treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader etc. can be used.

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 lignocellulosic from the standpoint of reducing chemical costs and preventing yield loss due to elution and overdegradation of cellulose. It is desirable that it is 50 mass parts or less with respect to 100 mass parts of absolutely dry 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.

For lignocellulosic raw materials that have been subjected to pretreatment suitable for enzymatic saccharification reaction, it is preferable to perform sterilization treatment before use in the preparation of lignocellulosic raw material suspension. 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.

<Primary parallel saccharification and fermentation process>
A lignocellulosic raw material that has been subjected to pretreatment suitable for saccharification and fermentation is mixed with appropriate amounts of water and enzymes, and microorganisms such as yeast necessary for fermentation, and supplied to the primary saccharification and fermentation step. A typical process of the concurrent saccharification and fermentation treatment method is shown in FIG.
In FIG. 1, the lignocellulosic raw material treated in a state suitable for saccharification and fermentation treatment in the pretreatment step is saccharified by an enzyme (cellulose → glucose) and then fermented by yeast (glucose → ethanol).

  The suspension concentration of the lignocellulosic raw material is preferably 1 to 30% by mass. If it is less than 1% by mass, there is a problem in that the concentration of the product is ultimately too low and the cost for concentrating the product becomes high. Moreover, as the concentration exceeds 30% by mass, it becomes difficult to stir the raw materials, resulting in a problem that productivity is lowered.

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 in the saccharification process or the concurrent saccharification and fermentation process is preferably maintained in the range of 3.5 to 10.0, more preferably in the range of 4.0 to 7.5.

  The temperature of the saccharification step or the concurrent saccharification and fermentation step is not particularly limited as long as it is within the optimum temperature range of the enzyme, preferably 25 to 50 ° C, and more preferably 30 to 40 ° C. The reaction is preferably continuous, but may be semi-batch or batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, it is 10 to 240 hours, more preferably 15 to 160 hours. Also in the case of a continuous type, the average residence time is 10 to 150 hours, more preferably 15 to 100 hours.

As the microorganism used for fermentation, yeast or the like is used, and a medium or the like may be added simultaneously. As yeast, Saccharomyces cerevisiae (Saccharomyces cerevisiae) etc. can be used.
Moreover, the microorganisms may be immobilized. By immobilizing microorganisms, it is possible to omit the process of sending the microorganisms together with the liquid and recovering them again in the next process, or at least reduce the burden on the recovery process and reduce the risk of losing microorganisms. You can also In addition, although there is no merit as to immobilize the microorganism, it is possible to facilitate the recovery of the microorganism by selecting an aggregating microorganism.

<Solid-liquid separation process>
The culture solution that has exited the primary saccharification and fermentation step is transferred to a solid-liquid separation step and separated into a liquid component (filtrate) and a residue (primary residue). A screw press having a screen size of 1.0 to 2.0 mm is used as an apparatus for performing solid-liquid separation. The screw press is a device that is structurally resistant to fiber clogging and can be efficiently solid-liquid separated with relatively little energy. A back pressure may be applied to improve the solid-liquid separation efficiency.
The residue separated in the solid-liquid separation step contains lignin, hemicellulose, and cellulose, and lignin and the like are adsorbed on the cellulose, which makes it difficult for the enzyme to be saccharified. The residue after the solid-liquid separation step contains a large amount of fiber that has not been decomposed in the primary parallel saccharification and fermentation step, and saccharification is facilitated by performing mechanical treatment or chemical treatment (Patent Document 6).
The filtrate (liquid component) separated in the solid-liquid separation process is transferred to the next sieving process.

<Sieving process>
The filtrate after solid-liquid separation is subjected to a sieving process to separate it into fine fibers and filtrate (liquid component). As a method of the sieving process, any sieving apparatus that can separate fine fibers can be used without particular limitation. As the sieving apparatus, a screen, a filter press, a belt press, a rotary press, or the like can be used. The sieve mesh (mesh) is preferably 80 mesh to 600 mesh (28 to 182 μm), more preferably 150 mesh to 400 mesh (39 to 97 μm). In order to improve processing efficiency, vibration may be applied by attaching a vibration device to the sieve. The fine fibers separated by the above treatment have a low lignin content as compared with primary residues and secondary residues, and are easily saccharified by enzymes. Further, by removing fine fibers by sieving, the amount of solid content adhering to the vacuum distillation apparatus used in the subsequent distillation step can be reduced, and there is an advantage that the apparatus can be operated for a long time. The recovered fine fibers may be transferred to the primary saccharification and fermentation process and used as a raw material for saccharification and fermentation (see FIG. 1). Moreover, the recovered fine fibers can be transferred to a secondary saccharification and fermentation process (a saccharification and fermentation process different from the primary saccharification and fermentation process) described later and used as a raw material for saccharification and fermentation (see FIG. 2). Furthermore, only saccharification may be performed in another step. Thus, by saccharifying or saccharifying and fermenting the fine fiber, the enzyme adsorbed on the fine fiber can be used effectively.
On the other hand, the filtrate separated by the sieving process is transferred to the distillation step.

<Secondary concurrent saccharification and fermentation process>
The secondary parallel saccharification and fermentation process is a saccharification and fermentation process independent of the primary parallel saccharification and fermentation process. It can be saccharified and fermented using new lignocellulose as a raw material, or saccharified and fermented using the residue discharged in the process as a raw material. You can also In addition, sugar that has not been fermented to ethanol in the primary parallel saccharification and fermentation process can be fermented in the secondary parallel saccharification and fermentation process. In the primary parallel saccharification and fermentation process, hexose derived from cellulose, ie, glucose, mannose, galactose, etc. is ethanol-fermented, but xylose, a pentose derived from hemicellulose, may remain unreacted. . In such a case, the pentose can be fermented by adding yeast that more reliably ferments the pentose in the secondary parallel saccharification and fermentation treatment step.
In this invention, the fine fiber collect | recovered by the said sieving process can be transferred to a secondary parallel saccharification fermentation process process, and can be saccharified and fermented.

<Distillation process>
The filtrate after the sieving treatment or the treatment solution (culture solution) after the secondary concurrent saccharification and fermentation treatment step is transferred to the distillation step (see FIGS. 1 and 2).
In the distillation step, the fermentation product is separated by distillation using 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 to be inactivated, and the amount of newly added enzyme is increased, so that economical efficiency is deteriorated.
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.

<Centrifuge separation process>
The distillation residue is transferred to the centrifugal separation step, and the remaining residue (secondary residue) is removed by centrifugation, and then the liquid fraction is circulated to the primary parallel saccharification and fermentation step (see FIG. 1). This liquid fraction contains an enzyme and is reused in the primary concurrent saccharification and fermentation process. On the other hand, the residue contains lignin and can be recovered as a combustion raw material and used as energy, or lignin can be recovered and used effectively.

    In the present invention, a water-soluble salt can be added as an electrolyte in the enzymatic saccharification treatment step. In the enzymatic saccharification treatment step, it is preferable to add an electrolyte to the raw material suspension to maintain the electric conductivity of the raw material suspension in the range of 5 to 25 mS / cm. By maintaining the electrical 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 that the enzyme circulation rate in the enzymatic saccharification treatment process is long. Over high levels. In the enzymatic saccharification treatment step, the electrolyte can be added without limitation in any step as long as it is an operation that can add an electrolyte. It is desirable to add it in the primary saccharification and fermentation process because the operation is easy.

  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.

Next, the present invention will be described in more detail with reference to examples.

Ethanol was produced according to the production flow shown in FIG.
[Preprocessing]
Chip-shaped eucalyptus globula bark was crushed with a uniaxial crusher (Seiho Kiko Co., Ltd., SC-15) equipped with a 20 mm round hole screen and used as a raw material.
After adding a calcium hydroxide solution suspended in water to 12.5% by mass of calcium hydroxide with respect to 100 kg (absolute dry weight) of the above raw material (liquid ratio to raw material 8), 1 at 120 ° C. Heated for an hour (alkali treatment). The raw material after the alkali treatment was ground with a refiner (manufactured by Kumagai Riki Kogyo, KRK high concentration disk refiner: clearance 0.5 mm). After adding the same amount of pure water to the raw material after the grinding treatment, the pH was adjusted to 5 with sulfuric acid under stirring. 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. The solid (separated product) after solid-liquid separation was used as a raw material for the saccharification and fermentation process.
[Primary parallel saccharification and fermentation]
100 kg of raw material (absolute dry weight), polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, and water are added to the primary saccharification and fermentation tank so that the raw material concentration becomes 10% by mass. Was added to adjust the final volume to 1 m ³ . Liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, pH
5.6) A culture solution containing yeast cells pre-cultured at 50 ° C. for 30 hours at 30 ° C. and 50 L of commercially available cellulase (Accelerase DUET, manufactured by Genencor) are added to the fermentor, and the primary culture is performed at 30 ° C. for 24 hours. Saccharification and fermentation were performed. The pH of the culture solution during saccharification and fermentation was adjusted to 5.0.
[Solid-liquid separation]
The culture solution obtained by the first concurrent saccharification and fermentation is separated into solid and liquid by a screw press (SHX-200 x 1500 L, manufactured by Togoku Industry Co., Ltd., screen size 1.2 mm) to separate the residue (primary residue) and the filtrate. did. The recovered primary residue was 19.4 kg (absolute dry weight).
[Sieving process]
The filtrate after the solid-liquid separation was passed through a 400 mesh (39 μm) screen to collect fine fibers in the culture solution. The total recovered amount of fine fibers was 15.6 kg (absolute dry weight). The total amount (15.6 kg) of the collected fine fibers was returned to the primary saccharification and fermentation tank.
[Ethanol production]
The filtrate obtained by the sieving treatment is concentrated with an aqueous solution containing ethanol under the conditions of distillation temperature: 40 ° C., heating temperature: 80 ° C., supply liquid amount: 150 L / h, using a vacuum distillation apparatus (Evapor PEP-1, Okawara Seisakusho). Separated into culture. The volume and ethanol concentration of the obtained aqueous solution containing ethanol were measured, and the amount of ethanol recovered was calculated. The ethanol concentration in the solution was measured with a glucose sensor (BF-400 manufactured by Oji Scientific Instruments).
[Centrifuge]
The concentrated culture solution separated from the vacuum distillation apparatus was separated into a residue (secondary residue) and a filtrate by operating a decanter centrifuge (made by IHI, HS-204L type) at a rotational speed of 4500 rpm and a differential speed of 5.0 rpm. . The filtrate was transferred to a primary parallel saccharification and fermentation tank. The recovered secondary residue was 18.6 kg (absolute dry weight).

Ethanol was produced according to the production flow shown in FIG.
[Preprocessing]
The same method as in Example 1 was performed.
[Primary parallel saccharification and fermentation]
The same method as in Example 1 was performed.
[Solid-liquid separation]
The same method as in Example 1 was performed. The recovered primary residue was 19.2 kg (absolute dry weight).
[Sieving process]
The same method as in Example 1 was performed. A total of 15.5 kg (absolute dry weight) of recovered fine fibers obtained by treating 100 kg of raw materials by primary parallel saccharification and fermentation.
[Secondary parallel saccharification and fermentation]
15.5 kg (absolute dry weight) of fine fibers obtained by sieving was added as a raw material to the secondary saccharification and fermentation tank. Each was added to a secondary parallel saccharification and fermentation tank so as to be 5 g / L of polypeptone, 3 g / L of yeast extract, and 3 g / L of malt extract, and the final volume was adjusted to 150 L with water. Commercially available yeast (trade name: Maurivin: Mauri Yeast Australia Pty Limited) at 30 ° C. at 50 ° C. 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) Incubate for hours. 50 L of a culture solution containing yeast after culturing and 10 L of commercially available cellulase (Accelerase DUET, manufactured by Genencor Corp.) were added to the fermentor, and secondary saccharification and fermentation was performed at 30 ° C. for 24 hours. The pH of the culture solution during saccharification and fermentation was adjusted to 5.0.
[Ethanol production]
The same method as in Example 1 was performed.
[Centrifuge]
The same method as in Example 1 was performed. The recovered secondary residue was 18.6 kg (absolute dry weight).

<Comparative Example 1>
Ethanol was produced according to the production flow shown in FIG. A test in which [Sieving treatment] in Example 1 was omitted was defined as Comparative Example 1 (below).
[Preprocessing]
The same method as in Example 1 was performed.
[Primary parallel saccharification and fermentation]
The same method as in Example 1 was performed.
[Solid-liquid separation]
The same method as in Example 1 was performed. The recovered primary residue was 19.3 kg (absolute dry weight).
[Ethanol production]
The filtrate obtained by the solid separation was separated into an aqueous solution containing ethanol and a concentrated culture solution in the same manner as described in Example 1. The volume of the aqueous solution containing ethanol and the ethanol concentration were measured, and the amount of ethanol recovered was calculated.
[Centrifuge]
The same method as in Example 1 was performed. The recovered secondary residue was 34.2 kg (absolute dry weight).

エタノールの生産量の結果を表１に示す。実施例１（微細繊維を回収し一次併行糖化発酵槽に返送した場合）及び実施例２（微細繊維を回収し二次併行糖化発酵槽に移送した場合）では、比較例１（微細繊維を回収しない場合）と比較しエタノール生産量が向上した。

The results of ethanol production are shown in Table 1. In Example 1 (when fine fibers are collected and returned to the primary parallel saccharification and fermentation tank) and Example 2 (when fine fibers are collected and transferred to the secondary parallel saccharification and fermentation tank), Comparative Example 1 (fine fibers are collected) The amount of ethanol produced was improved compared to the case of

[Saccharification and fermentation test]
A saccharification and fermentation test was conducted in a test tube using the fine fiber obtained in Example 1 as a raw material, and the ethanol production was measured by the following method.
Liquid medium A (polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, glucose 30 g / L, dissolved in distilled water, pH 5.6) 100 ml and liquid medium B (polypeptone 15 g / L, yeast extract 10 g / L, malt extract 10 g / L: dissolved in distilled water) Commercial yeast (trade name: Maurivin: Mauri Yeast Australia Pty Limited) was cultured at 30 ° C. for 24 hours in a medium mixed with 20 ml. Centrifugation (5000 rpm, 20 minutes) of the culture solution after culturing was performed, and the supernatant was removed, and the volume of the culture solution was adjusted to 10 ml (yeast was collected) (concentrated yeast cells).
It added so that the final concentration of a raw material (fine fiber) might be 5 mass% to a 300 ml Erlenmeyer flask (sterilized). Next, 10 ml of concentrated yeast cells and 2.5 ml of commercially available cellulase (Accelerase DUET, Genencor) were added, and the final volume was made up to 100 ml with distilled water. This mixed solution was cultured at 30 ° C. for 24 hours (saccharification and fermentation). The culture solution after the culture was centrifuged (5000 rpm, 20 minutes), and the ethanol concentration of the supernatant was measured. Further, the kappa number (index of lignin content) of the fine fiber was measured by a measuring method based on JISP8211.

<Comparative example 2>
Using the primary residue obtained in Example 1 as a raw material, a saccharification and fermentation test was conducted in the same manner as in Example 3 to measure the ethanol production and the primary residue kappa number.

<Comparative Example 3>
Using the secondary residue obtained in Example 1 as a raw material, a saccharification and fermentation test was performed in the same manner as in Example 3, and the ethanol production and the secondary residue kappa number were measured.

表２にエタノール濃度及びカッパー価を示す。微細繊維（実施例３）を原料とした場合、一次残渣（比較例２）及び二次残渣（比較例３）を原料とした場合と比較し培養液中のエタノール濃度が高かった。また、微細繊維（実施例３）のカッパー価は、一次残渣（比較例２）及び二次残渣（比較例３）のカッパー価と比較し値が低かった。以上の結果から、微細繊維は一次残渣及び二次残渣と比較しリグニン含量が低く、その結果として微細繊維を糖化発酵の原料として用いた場合、一次残渣及び二次残渣と比較しエタノール生産量が高まることがわかった。微細繊維は前処理（機械的処理等）を施さなくても糖化発酵が容易に進行し、糖化発酵の原料として適していることが判明した。

Table 2 shows the ethanol concentration and the kappa number. When the fine fiber (Example 3) was used as the raw material, the ethanol concentration in the culture broth was higher than when the primary residue (Comparative Example 2) and the secondary residue (Comparative Example 3) were used as the raw material. Moreover, the kappa number of the fine fiber (Example 3) was lower than that of the primary residue (Comparative Example 2) and the secondary residue (Comparative Example 3). From the above results, the fine fiber has a low lignin content compared to the primary residue and the secondary residue, and as a result, when the fine fiber is used as a raw material for saccharification and fermentation, the ethanol production is lower than the primary residue and the secondary residue. I found it to increase. It turned out that saccharification and fermentation proceed easily even if the fine fiber is not subjected to pretreatment (such as mechanical treatment), and is suitable as a raw material for saccharification and fermentation.

  A test using Eucalyptus globulus forest land remnant (70% bark, 30% branch leaves) as a raw material instead of the bark of eucalyptus globulus used as a raw material in Example 1 was taken as Example 4. Except for using the remaining forest land, all tests were performed in the same manner as in Example 1 (the manufacturing flow is the same as in FIG. 1).

<Comparative example 4>
A test in which [Sieving treatment] in Example 4 was omitted was referred to as Comparative Example 4. All tests were performed in the same manner as in Example 4 except that the sieving treatment was omitted (the manufacturing flow is the same as in FIG. 3).

エタノールの生産量の結果を表３に示す。実施例４（微細繊維を回収し一次併行糖化発酵槽に返送した場合）では、比較例４（微細繊維を回収しない場合）と比較しエタノール生産量が向上した。

The results of ethanol production are shown in Table 3. In Example 4 (when fine fibers were collected and returned to the primary parallel saccharification and fermentation tank), ethanol production was improved as compared with Comparative Example 4 (when fine fibers were not collected).

Ethanol was produced according to the production flow shown in FIG.
[Preprocessing]
The same method as in Example 1 was performed.
[Primary parallel saccharification and fermentation]
It implemented by the method similar to Example 1 except adding sodium chloride as electrolyte in a culture solution. To the culture solution prepared in the same manner as in Example 1, sodium chloride (electrolyte) was added so that the final concentration was 100 mM (electrical conductivity of the raw material suspension: 12.2 mS / cm). Next, yeast cells and commercially available cellulase were added to the fermentor in the same manner as in Example 1 to perform primary saccharification and fermentation.
[Solid-liquid separation]
The same method as in Example 1 was performed. The recovered primary residue was 15.3 kg (absolute dry weight).
[Sieving process]
The same method as in Example 1 was performed. The collected fine fibers totaled 13.4 kg (absolute dry weight). The total amount (13.4 kg) of the collected fine fibers was returned to the primary saccharification and fermentation tank.
[Ethanol production]
The same method as in Example 1 was performed.
[Centrifuge]
The same method as in Example 1 was performed. The recovered secondary residue was 14.7 kg (absolute dry weight).

エタノール生産量の結果を表４に示す。培養液中に塩化ナトリウムを添加した場合（実施例５）では、塩化ナトリウムを添加しない場合(実施例１)と比較しエタノール生産量が向上した。

The results of ethanol production are shown in Table 4. In the case where sodium chloride was added to the culture solution (Example 5), the ethanol production was improved as compared to the case where sodium chloride was not added (Example 1).

Ethanol was produced according to the production flow shown in FIG.
[Preprocessing]
The same method as in Example 1 was performed.
[Primary parallel saccharification and fermentation]
It implemented by the method similar to Example 1 except adding sodium chloride as electrolyte in a culture solution. To the culture solution prepared in the same manner as in Example 1, sodium chloride (electrolyte) was added so that the final concentration was 100 mM (electrical conductivity of the raw material suspension: 12.2 mS / cm). Next, yeast cells and commercially available cellulase were added to the fermentor in the same manner as in Example 1 to perform primary saccharification and fermentation.
[Solid-liquid separation]
The same method as in Example 1 was performed. The recovered primary residue was 14.8 kg (absolute dry weight).
[Sieving process]
The same method as in Example 1 was performed. The collected fine fibers totaled 13.0 kg (absolute dry weight). The total amount (13.0 kg) of the collected fine fibers was returned to the primary saccharification and fermentation tank.
[Secondary parallel saccharification and fermentation]
The same method as in Example 2 was used.
[Ethanol production]
The same method as in Example 1 was performed.
[Centrifuge]
The same method as in Example 1 was performed. The recovered secondary residue was 14.2 kg (absolute dry weight).

エタノール生産量の結果を表５に示す。培養液中に塩化ナトリウムを添加した場合（実施例６）では、塩化ナトリウムを添加しない場合(実施例２)と比較しエタノール生産量が向上した。

The results of ethanol production are shown in Table 5. When sodium chloride was added to the culture solution (Example 6), the amount of ethanol produced was improved compared to the case where sodium chloride was not added (Example 2).

Ethanol was produced according to the production flow shown in FIG.
[Preprocessing]
Chip-shaped eucalyptus globula bark was crushed with a uniaxial crusher (Seiho Kiko Co., Ltd., SC-15) equipped with a 20 mm round hole screen and used as a raw material.
After adding 700 ml of water containing 20 g of 97.0% sodium sulfite and 1 g of sodium hydroxide to 100 kg (absolute dry weight) of the raw material, the mixture was heated at 1700C for 1 hour. The raw material after the heat treatment was ground with a refiner (manufactured by Kumagai Riki Kogyo, KRK high concentration disk refiner: clearance 0.5 mm). After adding the same amount of pure water to the raw material after the grinding treatment, the pH was adjusted to 5 with sulfuric acid under stirring. 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. The solid (separated product) after solid-liquid separation was used as a raw material for the saccharification and fermentation process.
[Primary parallel saccharification and fermentation]
The same method as in Example 1 was performed.
[Solid-liquid separation]
The same method as in Example 1 was performed. The recovered primary residue was 14.7 kg (absolute dry weight).
[Sieving process]
The same method as in Example 1 was performed. The collected fine fibers totaled 12.9 kg (absolute dry weight). The total amount (12.9 kg) of the collected fine fibers was returned to the primary saccharification and fermentation tank.
[Ethanol production]
The same method as in Example 1 was performed.
[Centrifuge]
The same method as in Example 1 was performed. The recovered secondary residue was 14.0 kg (absolute dry weight).

エタノール生産量の結果を表６に示す。実施例７（微細繊維を回収し一次併行糖化発酵槽に返送した場合）では、比較例１（微細繊維を回収しない場合）と比較しエタノール生産量が向上した。以上の結果から、原料を亜硫酸ナトリウム及び水酸化ナトリウムを含む溶液で処理した場合においても篩い処理により微細繊維を回収し一次併行糖化発酵槽に返送することによりエタノール生産量が向上することが判明した。

The results of ethanol production are shown in Table 6. In Example 7 (when fine fibers were collected and returned to the primary parallel saccharification and fermentation tank), ethanol production was improved as compared with Comparative Example 1 (when fine fibers were not collected). From the above results, it was found that even when the raw material was treated with a solution containing sodium sulfite and sodium hydroxide, ethanol production was improved by collecting fine fibers by sieving and returning them to the primary saccharification and fermentation tank. .

According to the present invention, it is possible to improve ethanol production by reusing fine fibers contained in a culture solution after saccharification and fermentation as raw materials for saccharification and fermentation. Moreover, ethanol production can be improved by adding an electrolyte to the culture solution.

Claims (5)

Pretreatment process for treating lignocellulose raw material as a raw material suitable for enzymatic saccharification reaction, enzymatic saccharification of pretreated lignocellulose raw material with enzyme and fermentation treatment using saccharide produced by enzymatic saccharification treatment as substrate Primary saccharification and fermentation step performed in parallel, solid-liquid separation step of separating the treated suspension from the primary saccharification and fermentation step into a residue and a liquid fraction with a screw press having a screen size of 1.0 to 2.0 mm , A sieving process for separating the liquid fraction separated in the solid-liquid separation process into a fine fiber and a liquid fraction by a sieving process of 80 to 600 mesh, a liquid fraction excluding the fine fiber after the sieving process is distilled under reduced pressure In the method for producing ethanol from a lignocellulosic raw material having a distillation step for separating and recovering the fermentation product, the separated fine fiber is saccharified or concurrently saccharified and fermented Ethanol production process from lignocellulosic feedstock characterized and. The method for producing ethanol from a lignocellulosic material according to claim 1, wherein the separated fine fibers are transferred to a primary saccharification and fermentation step and subjected to saccharification and fermentation. The method for producing ethanol from a lignocellulosic raw material according to claim 1, wherein the separated fine fibers are transferred to a secondary saccharification and fermentation process and subjected to saccharification and fermentation. The pretreatment of the lignocellulose raw material is one or more alkaline chemicals selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogencarbonate, or sodium sulfite and the alkaline chemical. From the lignocellulosic raw material according to any one of claims 1 to 3, wherein the raw material is a pretreatment including a chemical treatment immersed in a solution containing at least one alkaline chemical selected from Ethanol production method.   In the primary saccharification and fermentation step, an electrolyte composed of a water-soluble salt is added to the raw material suspension, and saccharification or parallel saccharification and fermentation is performed with the electric conductivity of the raw material suspension being 5 to 25 mS / cm. The ethanol manufacturing method from the lignocellulose raw material of any one of Claims 1-4 to do.

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