JP2009153442A - Method for pretreatment of herbal biomass and method for producing ethanol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for the pretreatment of a herbal biomass, capable of producing a sugar liquid usable for ethanol fermentation in high efficiency at a low cost by using a herbal biomass raw material such as malt feed, and a method for producing ethanol by using the sugar liquid produced by the pretreatment method. <P>SOLUTION: The pretreating method for a herbal biomass includes the treatment of a herbal biomass raw material in a dilute sulfuric acid having a concentration of 0.5-10% under normal pressure at 50-100°C for 1-24 hr, the neutralization of the treated product with an alkali and the hydrolysis of the neutralized product with an enzyme to obtain a sugar liquid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a herbaceous biomass pretreatment method for obtaining a sugar solution that can be used for ethanol fermentation using a herbaceous biomass material such as beer lees and a method for producing ethanol using the sugar solution. .

Background art

  Attempts to produce ethanol from renewable biomass such as rice straw and bagasse and use it as energy and chemical raw materials are underway in Japan and overseas.

  The types of sugars constituting such plant-based or wood-based biomass raw materials include pentoses such as xylose and arabinose and hexoses such as glucose, galactose and mannose. Usually, these ratios vary depending on the type of biomass.

  Such biomass raw materials include hemicellulose, cellulose, and lignin, and hemicellulose and cellulose are decomposed into pentose and hexose. However, there are differences in their decomposition conditions. Hemicellulose is decomposed relatively easily under acid, alkali, or hot water conditions, and a sugar can be obtained with a high recovery rate of 90% or more. On the other hand, cellulose is required to be decomposed under more severe conditions, but under such conditions, sugar overdegradation occurs at almost the same rate, so the recovery rate of sugar decreases. End up.

  For example, after obtaining a hemicellulose-derived sugar using an acid or an alkali, the residue can be treated with an acid or an enzyme to obtain a cellulose-derived sugar (glucose). When dilute sulfuric acid is used, the glucose yield is as low as about 40%, and sugar hyperdegradable substances such as formic acid, levulinic acid, and hydroxymethylfurfural (HMF) tend to be generated. Such substances can act as fermentation inhibitors and affect fermentation.

Therefore, a pretreatment method using hydrolysis by an enzyme has been studied as an alternative to acid and alkali. In general, such a pretreatment method is a method in which a biomass raw material is primarily hydrolyzed by acid, alkali, hot water treatment or the like, and then hydrolyzed by an enzyme.
However, primary hydrolysis with acid, alkali, etc. also serves as a pretreatment for enzyme hydrolysis, and appropriate methods and conditions depending on the raw materials and the like so that the enzyme can work effectively in the enzyme hydrolysis treatment. Must be selected.

  As the pretreatment for enzymatic hydrolysis, acid, alkali, hot water treatment, or treatment using explosion is known. Among these, the treatment using an alkali is likely to decompose the sugar under alkaline conditions, and therefore, the treatment using an acid, hot water treatment, or explosion is considered to be more advantageous as the pretreatment.

  In the treatment using an acid, the fermentation inhibitor described above may be generated depending on the conditions, and as a result, the sugar recovery rate may decrease. In the treatment using hot water treatment or blasting, it is possible to prevent excessive decomposition of sugar, but the recovery rate of sugar is not very high. In fact, all of the treatments using acid, hydrothermal treatment, and blasting that have been reported so far have to be performed under high temperature and pressure conditions during the treatment (for example, Applied Biochemistry and Biotechnology, Vol. 129-132, 496-508, 2006 (Non-patent Document 1), Sugar Industry, Vol. 131, No. 11, 766-769, 2006 (Non-Patent Document 2), and Master Brewers Association of the Americas, Vol. 43, No. 3, 189-193, 2006 (Non-patent Document 3)), it seems that there is still a possibility of excessive decomposition of sugar.

  Further, for example, JP 2007-151433 A (Patent Document 1) discloses a pretreatment method for lignocellulose using waste building materials as a raw material, and a method for producing ethanol using a sugar solution obtained thereby. Yes. However, here, the raw material needs to be processed at a high temperature (for example, 140 to 220 ° C.) and in multiple steps, and it is considered that the possibility of a decrease in the recovery rate of sugar due to excessive decomposition of sugar cannot be eliminated. .

Furthermore, since the treatment using the acid, hydrothermal treatment, or blasting described above requires a high temperature of 100 ° C. or higher, usually a pressure device is essential, which not only increases the cost of the equipment, It becomes difficult to scale up. Moreover, since the load by pressurization is large, there exists a concern that the lifetime of an apparatus may become short. Furthermore, when an acid is used at a high temperature, there is a high possibility that a substance that inhibits fermentation of microorganisms is generated.
For this reason, it can be said that the pre-processing method which does not require severe conditions, such as high temperature and pressurization, is still desired.

  On the other hand, by-products generated during the brewing process such as beer lees and food waste materials generated during the food manufacturing process have been used for livestock feeds, but as raw materials for ethanol production. As far as the present inventors are aware of the use, it cannot be said that sufficient studies have been conducted yet.

JP 2007-151433 A Ignacio Ballesteros, Ma Jose Negro, Jose Miguel Oliva, Araceli Cabanas, Paloma Manzanares, Mercedes Ballesteros, “Ethanol Production From Steam-Explosion Pretreated Wheat Straw”, Applied Biochemistry and Biotechnology, Vol. 129-132, 496-508, 2006 Sven Fleishcer, Thomas Senn, “Cellulosic starch-based raw materials in ethanol production”, Sugar Industry, Vol. 131, No. 11, 766-769, 2006 Sho Shindo, Tadanori Tachibana, “Production of Bioethanol from Spent Grain-A By product of Beer Production”, Master Brewers Association of the Americas, Vol. 43, No. 3, 189-193, 2006

Summary of the Invention

  The present inventors have succeeded in efficiently obtaining a sugar solution that can be used for ethanol fermentation under a relatively mild condition, using beer lees as an example of a herbaceous biomass raw material. Further, by using the obtained sugar solution, it was possible to achieve an improvement in ethanol yield at a low cost. The present invention is based on such knowledge.

  Therefore, the present invention provides a herbaceous biomass pretreatment method capable of obtaining a sugar solution that can be efficiently and efficiently used for ethanol fermentation using a herbaceous biomass material such as beer lees, and a sugar solution obtained thereby. An object of the present invention is to provide an ethanol production method for producing ethanol using the same.

  In the pretreatment method of herbaceous biomass according to the present invention, a herbaceous biomass raw material is treated in a dilute sulfuric acid having a concentration of 0.5 to 10% under normal pressure at 50 to 100 ° C. for 1 to 24 hours. The treated product is neutralized with an alkali and hydrolyzed with an enzyme to obtain a sugar solution.

  According to one preferable aspect of the present invention, in the pretreatment method, β-glucosidase and cellulase are used as the enzymes used in the enzymatic hydrolysis.

  According to another preferred embodiment of the present invention, in the pretreatment method, the herbaceous biomass raw material is beer lees.

  Moreover, according to another aspect of the present invention, there is provided a method for producing ethanol, characterized in that ethanol is produced by microorganism ethanol fermentation using the sugar solution obtained by the pretreatment method according to the present invention. Is done. According to another preferred embodiment of the present invention, in the production method, the microorganism used for ethanol fermentation is a yeast strain belonging to the genus Saccharomyces, the genus Pichia, or the genus Candida.

  According to the pretreatment method for herbaceous biomass of the present invention, a sugar solution as a raw material that can be used for ethanol fermentation at low cost and efficiently can be obtained from the herbaceous biomass material. In addition, according to the ethanol production method of the present invention, ethanol fermentation is performed using the sugar solution obtained by the herbaceous biomass pretreatment method of the present invention, and ethanol is produced at low cost. The rate can be improved. Furthermore, since the present invention uses herbaceous biomass raw materials such as beer lees as raw materials, it can be expected to suppress the discharge of waste, while the obtained ethanol can be expected to use so-called biofuels, etc. A contribution can be expected.

Detailed description of the invention

Pretreatment method of herbaceous biomass As described above, the pretreatment method of herbaceous biomass according to the present invention is a method of treating herbaceous biomass raw material in diluted sulfuric acid having a concentration of 0.5 to 10% at normal pressure and 50 to 100 ° C. After treatment for 1 to 24 hours under the conditions, the treated product is neutralized with an alkali and hydrolyzed with an enzyme to obtain a sugar solution. Here, the pretreatment means a treatment performed for obtaining a sugar solution as a raw material prior to ethanol fermentation.

  In the present invention, the herbaceous biomass raw material refers to a herb itself, a part of the herb, a product obtained by treating the herb or a herb-derived product, such as rice straw, rice husk, various weeds, bagasse, soybean hulls, and Examples include fermented potatoes. Here, fermented rice cakes include not only fermented rice cakes produced by various alcohol fermentation such as beer rice cakes and shochu rice cakes, but also rice cakes produced in the process of producing various fermented foods. In addition, beer lees here mean not only wort but also wort-filtered residues of wort derived from various wheats produced in the process of preparing various alcoholic beverages such as happoshu and new genres. In the present invention, beer lees can be used in a wet state or a dry state. Therefore, in the present invention, when the herbaceous biomass raw material is beer lees, the residue obtained by filtering the wort derived from various oats produced in the preparation process during the production of alcoholic beverages, that is, the lees discharged from the saccharification tank is left as it is. It may be used.

  According to a preferred embodiment of the present invention, the herbaceous biomass raw material is fermented rice cake, more preferably beer rice cake.

  In the pretreatment method of the present invention, herbaceous biomass such as beer lees is first heat-treated in acid. Dilute sulfuric acid is used as the acid. Here, acids other than sulfuric acid, such as hydrochloric acid, can be used. For example, hydrochloric acid is highly volatile and may corrode the reaction apparatus, possibly shortening the life of the reaction apparatus itself.

  The conditions for the hydrolysis treatment with dilute sulfuric acid are typically a sulfuric acid concentration of 0.5 to 10% by volume, preferably about 3 to 5% by volume, and most preferably about 3% by volume. If the sulfuric acid concentration is less than 0.5%, the amount of recovered sugar may not be sufficient, and if the sulfuric acid concentration is too high, there may be a problem of excessive sugar decomposition sugar.

The temperature of the solution during the hydrolysis is typically 50 to 100 ° C., preferably 70 to 90 ° C., and most preferably about 90 ° C. If the temperature is less than 50 ° C., the amount of recovered sugar may not be sufficient. For temperatures exceeding 100 ° C., the necessity of pressure treatment may occur when the method is actually carried out.
In the present invention, the pressure during the hydrolysis is normal pressure.

  The reaction time of the hydrolysis treatment using dilute sulfuric acid is preferably 1 to 9 hours, more preferably 3 to 9 hours, and most preferably about 3 hours. If the reaction time is shorter than 1 hour, the sugar recovery may not be sufficient, and if the reaction time is too long, the sugar recovery rate may decrease due to generation of by-products that inhibit the enzyme reaction.

  This hydrolysis treatment with sulfuric acid facilitates the subsequent enzymatic hydrolysis reaction.

  After the hydrolysis treatment with sulfuric acid, an alkali is added to the treated product (treatment solution) to adjust its pH to or near a pH value that is preferable for the subsequent enzymatic reaction. The pH value is preferably about pH 4-6, more preferably about pH 5. As the alkali used at this time, any alkaline substance can be used as long as the treatment solution acidified with sulfuric acid can be in a predetermined pH range and does not affect the subsequent enzyme treatment or fermentation process. May be. As such an alkali, for example, calcium hydroxide, potassium hydroxide and the like can be used. If necessary, a pH buffer solution (for example, sodium acetate buffer solution) may be added to the treated product to stabilize the pH of the treated product.

Preferably, the enzyme is added to the pH-adjusted product without stirring, or while stirring, preferably at pH 4-6, more preferably at pH 5, preferably at temperature 40-60 ° C, more preferably at temperature 50 ° C. The enzyme hydrolysis is performed by reacting, preferably for 1 to 100 hours, more preferably for 24 to 48 hours.
Any enzyme can be used as long as it can decompose cellulose, hemicellulose, and the like contained in the herbaceous biomass raw material and convert them into sugars, and the decomposition reaction and the saccharification reaction are separately performed. These enzymes may be used, or a plurality of enzymes may be used in combination as necessary.

  According to a preferred embodiment of the present invention, the enzyme is used by mixing β-glucosidase and cellulase. Of these, commercially available β-glucosidase can be used, and specific examples include NS50010 (available from Novozymes), OPTIMASH BG (available from Genencor Kyowa), and the like. Commercially available cellulases can also be used, and specific examples include NS50013 (Novozymes), Cellulase A “Amano” 3 (Amano Enzymes), and the like. It is desirable to use both enzymes from Novozymes.

  As the amount of the enzyme used in the enzyme treatment, the β-glucosidase is preferably 0.01 to 0.2% by volume, more preferably 0.05 to 0.1% by volume as a volume% with respect to the pH-adjusted processed product. Moreover, about cellulase, Preferably it is 0.05-0.5 volume%, More preferably, it is 0.1-0.25 volume%.

  In the present invention, the obtained enzyme hydrolyzate may be subjected to solid-liquid separation before fermentation, if necessary. Filtration, centrifugation, etc. can be used for the method of solid-liquid separation, and it is preferable to use filtration from a viewpoint of small energy consumption.

  The obtained enzyme hydrolyzate or solid-liquid separated filtrate usually contains glucose, xylose, arabinose, galactose, and mannose. In the present invention, the obtained enzyme hydrolyzate and the solid-liquid separated filtrate are sometimes referred to as “sugar solution”.

  As described above, the pretreatment method according to the present invention has a simpler process than the conventional method and does not require high-pressure treatment, and therefore does not require equipment or equipment for high pressure. For this reason, cheap and efficient processing is possible.

Production method of ethanol According to another aspect of the present invention, as described above, ethanol is produced by microorganism ethanol fermentation using the sugar solution obtained by the pretreatment method of the present invention. A method for producing ethanol is provided.

  Microorganisms for ethanol fermentation, such as yeast, are added to the sugar solution obtained by the pretreatment method, and the microorganisms are cultured under conditions of appropriate temperature, pH, etc., and subjected to alcoholic fermentation to convert sugar to ethanol. To produce ethanol. At this time, if necessary, a microbial fermentation substrate such as nitrogen or phosphorus may be further added to the sugar solution.

  Here, the microorganisms used for ethanol fermentation include various known ethanol-fermenting microorganisms belonging to the genus Zymomonas, yeasts of the genus Saccharomyces, yeasts of the genus Pichia, yeasts of the genus Candida, etc. The microorganisms imparted can be used. These may be combined as necessary. Specific examples of the microorganism include Saccharomyces cerevisiae DV10 strain (available from LALLEMAND [Montereal, Canada]) as Saccharomyces genus yeast, and Pichia stipitis NBRC10063 strain (NBRC stock) as Pichia genus yeast.

According to a preferred embodiment of the present invention, the microorganism used for ethanol fermentation is a yeast strain belonging to the genus Saccharomyces, Pichia, or Candida, more preferably Pichia stipitis .

  FIG. 6 shows a flow chart showing one embodiment of the herbaceous biomass pretreatment method and ethanol production method according to the present invention.

  In this specification, expression of a value using “about” or “degree” includes a variation in a value that can be allowed by those skilled in the art to achieve the purpose by setting the value. Meaning.

  The present invention will be described in detail by the following examples, but the present invention is not limited thereto.

The herbaceous biomass raw material was pretreated under the following experimental conditions.
Common conditions
Herbaceous biomass raw materials used:
Dry beer lees: According to a conventional method, the beer lees residue discharged from the wort filter in the wort production process was press-dehydrated and then subjected to rotary drying to obtain dry beer lees.
Conditions for enzymatic hydrolysis:
Enzymes: NS50010 (β-glucosidase) manufactured by Novozymes and NS50013 (cellulase) manufactured by Novozymes.
pH: A sodium acetate buffer solution was added to adjust the pH to 5.0.
Temperature: 50 ° C
Stirring: 0 or 100 rpm (shaking device: horizontal shaking was performed with BIOSHAKER BR-15 manufactured by TAITEC)
Time: 24 hours

Example 1 Comparison of Sulfuric Acid Concentration The following experiment was conducted to examine the optimum sulfuric acid concentration used in the pretreatment.
experimental method:
5 g of dried beer cake is put into a 200 ml eggplant flask, and 0, 0.5, 1, 2, 3, 4, 5, 7.5, and 20% sulfuric acid is added to each flask. 50 ml was added and immersed in an oil bath. The oil bath was set to 150 ° C. (because of the positive pressure state, the sample temperature was about 100 ° C.) and heated for 2 hours. Calcium hydroxide was added to the heated sample and adjusted to pH 5.0, and then enzymatic hydrolysis was performed according to the common conditions described above (with stirring).
The hydrolyzed sample solution was filtered and a supernatant was obtained. The total amount of sugar collected in each supernatant obtained was measured by the phenol-sulfuric acid method.

  The result was as shown in FIG. 1 (N = 1).

  As shown in the results, the amount of recovered total sugar was greatly improved when sulfuric acid having a concentration of 3% or more was used. A sulfuric acid concentration of 3-5% was judged to be preferred. In the following examples, a sulfuric acid concentration of 3% was adopted in consideration of cost and safety.

Example 2: Comparison of treatment temperatures The following experiments were conducted to investigate the optimum temperature for pretreatment.
experimental method:
50 g of dried beer koji was put into a 1000 ml glass reaction vessel (manufactured by Buch Glass Worcester, Switzerland), 500 ml of 3% sulfuric acid was further added, and the experiment was started by heating. Heating was performed separately for heating temperatures (treatment temperatures) of 50, 60, 70, 80, and 90 ° C.
After starting the experiment, 1 ml was taken from each of the samples at 0, 1, 3, 9, and 24 hours (treatment time) and adjusted to pH 5.0 by adding potassium hydroxide. Enzymatic hydrolysis was performed according to 100 rpm stirring).
The hydrolyzed sample solution was filtered and a supernatant was obtained. The total amount of sugar collected in each supernatant obtained was measured by the phenol-sulfuric acid method.

  The result was as shown in FIG. 2 (N = 1).

  As shown in the results, when 3% concentration of sulfuric acid is used, the recovery amount of total sugar is greatly improved under the conditions of a treatment temperature of 90 ° C. and a treatment time of 3 to 9 hours. The amount of sugar recovered was most improved. On the other hand, when treated at 90 ° C. for 24 hours, a decrease in the total amount of recovered sugar was observed. In the following examples, a processing temperature of 90 ° C. and a processing time of 3 hours were adopted in view of energy cost.

Example 3: Examination of the amount of β-glucosidase used for enzyme hydrolysis The following experiment was conducted to examine the optimum amount of β-glucosidase for enzyme hydrolysis.
experimental method:
After putting 50 g of dried beer cake into a 1000 ml glass reaction vessel (manufactured by Buch Glass Worcester, Switzerland), adding 500 ml of 3% sulfuric acid and heating at 90 ° C. for 3 hours, the sample solution was adjusted to pH 5.0. Adjusted. Next, prepare 1 ml of the sample solution obtained by adding β-glucosidase to 0, 0.01, 0.05, 0.1, and 0.2% by volume, respectively. The enzyme hydrolysis treatment was performed without stirring at 50 ° C. for 1, 3, 24, and 48 hours.
The hydrolyzed sample solution was filtered and a supernatant was obtained. About each obtained supernatant, the amount of glucose was measured using the biochemistry analyzer by YSI.

  The result was as shown in FIG. The figure shows the amount of glucose collected (g / l) at each time. The numerical value was expressed as an average value ± standard error (N = 3).

  As shown in the results, glucose could be efficiently recovered according to the amount of β-glucosidase added. In the following examples, a β-glucosidase concentration of 0.05% was adopted in consideration of the balance between β-glucosidase concentration and treatment time (saccharification time) and cost.

Example 4: Examination of the amount of cellulase used for enzyme hydrolysis The following experiment was conducted to examine the optimum amount of cellulase for carrying out enzyme hydrolysis.
experimental method:
After putting 50 g of dried beer cake into a 1000 ml glass reaction vessel (manufactured by Buch Glass Worcester, Switzerland), adding 500 ml of 3% sulfuric acid and heating at 90 ° C. for 3 hours, the sample solution was adjusted to pH 5.0. Adjusted. Next, β-glucosidase is added to 1 ml of the obtained pretreatment solution so that the concentration becomes 0.05%, and the concentration becomes 0, 0.05, 0.1, 0.25, 0.5%. In this way, cellulase was added, and these were maintained at 50 ° C. for 1, 3, 24, and 48 hours, and subjected to enzymatic hydrolysis without stirring.
The hydrolyzed sample solution was filtered and a supernatant was obtained. About each obtained supernatant, the amount of glucose was measured using the biochemistry analyzer by YSI.

  The result was as shown in FIG. The figure shows the amount of glucose collected (g / l) at each time. The numerical value was expressed as an average value ± standard error (N = 3).

  As shown in the results, the amount of glucose recovered was improved by further adding cellulase to a fixed amount (0.05%) of β-glucosidase. As the cellulase concentration and treatment (saccharification) time increased, the amount of glucose recovered also increased. In terms of cellulase concentration, 0.25% and 0.5% have no significant difference in the amount of glucose recovered, and from the viewpoint of cost, the cellulase concentration of 0.25% was employed in the following examples. Regarding the saccharification time, in consideration of the fact that the longer the saccharification time, the more the amount of recovered glucose tended to increase at a concentration of 0.25%. In the following examples, the reaction time was 5 days (120 hours). )It was adopted.

Example 5: Fermentation with enzyme hydrolyzate A fermentation test was carried out using a sample solution (pretreated sugar solution) obtained after enzyme hydrolysis as described below.
experimental method:
After putting 50 g of dried beer lees into a 1000 ml glass reaction vessel (manufactured by Buch Glass Worcester, Switzerland), adding 500 ml of 3% sulfuric acid and heating at 90 ° C. for 3 hours, the sample solution was adjusted to pH 5.0. Adjusted. Next, β-glucosidase and 0.05% concentration of cellulase were added to 10 ml of the obtained pretreatment solution at 50 ° C. for 5 days at 100 rpm. Enzymatic hydrolysis was performed with stirring.
The obtained sample solution (pretreatment solution) was filtered and fermented using yeast Pichia stipitis NBRC10063 (NBRC stock) and Saccharomyces cerevisiae DV10 (available from LALLEMAND [Montereal, Canada]).
As fermentation conditions, glass test tubes (50 ml capacity) were used, 10 ml each of the pretreatment solution was put therein, yeast was adjusted so that the initial cell mass was OD660 = 1 (/ ml), and 30 ° C. And fermented under conditions of 1 to 1.5 rpm (rotator: Rotator RT-550 manufactured by TAITEC) for 4 days. The treatment liquid after the fermentation reaction is collected, and using a gas chromatography (GC) analyzer (gas chromatography apparatus: Shimadzu GC17A, HSS4A, CR7A, capillary column: Megabore ID = 0.53mm 30m, all manufactured by Shimadzu Corporation) The ethanol concentration in the treatment liquid collected under the following conditions was measured.
・ Temperature increase program: 5 minutes, 40 ° C. → 5 minutes, 10 ° C./min. To 140 ° C. → 3 minutes, 140 ° C.
・ Vial temperature: 40 ℃
・ Syringe temperature: 140 ° C
・ Injector temperature: 200 ℃
・ Detector temperature: 200 ℃
・ Insulation time: 15 minutes

  The result was as shown in FIG. 5 (N = 1).

  As shown in the results, ethanol recovery by fermentation (about 1.2%) was confirmed in any yeast. This was considered to indicate that fermentation inhibitors were not included.

  From the above examples, as shown in FIG. 6 according to the present invention, beer koji was used as a herbaceous biomass raw material, and this was heated in dilute sulfuric acid, neutralized with alkali, and then the reaction product was enzymatically treated. It was found that ethanol can be produced by hydrolyzing to obtain a sugar solution and fermenting it with a microorganism strain.

The figure shows the results of a comparative test of sulfuric acid concentration in Example 1. The figure shows the results of a comparative test of the processing temperature of Example 2. The figure shows the examination results of the amount of β-glucosidase used for the enzymatic hydrolysis of Example 3. The figure shows the results of studying the amount of cellulase used in the enzymatic hydrolysis of Example 4. The figure shows the results of a fermentation test using the enzyme hydrolyzate of Example 5. The figure is a flow diagram illustrating one embodiment of the method according to the invention.

Claims (5)

  After treating herbaceous biomass raw material in dilute sulfuric acid with a concentration of 0.5 to 10% under normal pressure and 50 to 100 ° C. for 1 to 24 hours, the treated product is neutralized with alkali, A pretreatment method for herbaceous biomass, characterized in that a sugar solution is obtained by hydrolyzing the mixture.   The pretreatment method according to claim 1, wherein β-glucosidase and cellulase are used as the enzymes used in the enzymatic hydrolysis.   The pretreatment method according to claim 1 or 2, wherein the herbaceous biomass raw material is beer lees.   A method for producing ethanol, characterized in that ethanol is fermented by microorganisms to produce ethanol using the sugar solution obtained by the pretreatment method according to any one of claims 1 to 3.   The production method according to claim 4, wherein the microorganism used for ethanol fermentation is a yeast strain belonging to the genus Saccharomyces, the genus Pichia, or the genus Candida.

Images