JP5742102B2 - Method for producing alcohol from oil pods - Google Patents

Description

  The present invention relates to a method for producing alcohol from oil pods, and more particularly to a method for producing ethanol from corn germ cake.

In ethanol production using sugar, starch, etc. as raw materials, in order to compete with food problems in the future, development of a new bioethanol production method independent of sugar and starch is required. Production of corn, which is a raw material for bioethanol, has been increasing year by year, and production of bioethanol from cellulosic waste other than starch has become increasingly important. For example, a method of hydrolyzing waste such as corn stover, such as skin, core, and stem by dilute sulfuric acid method to decompose hemicellulose and then converting it into sugar by cellulase is known (Non-patent Document 1).
Moreover, it can be converted to ethanol by hydrolyzing cellulose with cellulase and fermenting with yeast via glucose. As a cellulase high-producing bacterium, the filamentous fungus Trichoderma reesei is known, and it is known to have a plurality of cellulases capable of completely degrading cellulose into glucose (Non-patent Document 2).

  Moreover, the manufacturing method of the bioethanol which produces | generates ethanol by carrying out the enzymatic decomposition of the cellulose and hemicellulose which are contained in biomass, and then fermenting the saccharide produced | generated by the enzymatic decomposition of cellulose and hemicellulose is disclosed. The bioethanol production method includes a pretreatment step for destroying lignin contained in biomass and a structure decomposition treatment of cellulose and hemicellulose contained in the biomass, and cellulose subjected to structure decomposition treatment in this pretreatment step. And an enzymatic decomposition step for enzymatically decomposing hemicellulose, a fermentation step for fermenting the saccharide produced in the enzymatic decomposition step, a solution containing the saccharide produced in the enzymatic decomposition step, and ethanol produced in the fermentation step It has the heat exchange process which heat-exchanges with the solution which contains, and the fermentation bacteria isolation | separation process which isolate | separates fermentation bacteria from the solution containing the ethanol produced | generated at the said fermentation process (patent document 1).

  Moreover, as arming yeast capable of metabolizing xylose, XYL1 (INSD accession number X59465) and XYL2 (INSD accession number X55392), both xylose reductase (XR) gene and xylitol dehydrogenase (XDH) gene derived from Pichia stipitis, In addition, the construction of plasmid pIUX1X2XK for intracellular expression of XKS1 (INSD accession number X82408) gene, which is a xylulokinase (XK) gene derived from Saccharomyces cerevisiae, is also known (Patent Document 2).

On the other hand, corn germ cake after corn germ oiling is used as a feed material, but further processing such as adsorption of protein solution is performed to increase the content of crude protein as feed material. Yes.
For this reason, oil pods such as corn germ meal are not high-value-added materials, and their effective use has been desired.

JP2009-100713 JP2008-193935

http://sugar.lin.go.jp/japan/view/jv_0610b.html http://pelican.nagaokaut.ac.jp/GER/study/e15.html

  An object of the present invention is to develop a bioethanol production technique with low environmental load and low energy by saccharifying and alcoholic fermentation, particularly ethanol fermentation, by an enzymatic hydrolysis reaction of an oilseed meal such as corn germ meal.

The method for producing alcohol from oil mash according to the present invention includes a saccharification / decomposition step of saccharifying / degrading oil basin with bacterial cells, and a step of subjecting the sugar obtained in the saccharification / decomposition step to alcohol fermentation, The cocoon is a corn germ cocoon generated in the edible oil production process, and the cell is a filamentous fungus. In particular, the alcohol is ethanol.
The corn germ cake is an acidic germ cake after oil extraction.
The filamentous fungus is Trichoderma reesei and is characterized by performing saccharification / decomposition in an acidic region.
The alcohol fermentation is alcohol fermentation using an arming yeast, and the arming yeast is an arming yeast that can assimilate xylose and cellooligosaccharide.

  In the alcohol production method of the present invention, corn germ cake as an oil pod is used as it is for saccharification and fermentation, so that saccharification and fermentation can be carried out in a short time without requiring extreme acid treatment or heat energy. Therefore, oil pods with low added value can be effectively used as biomass resources.

It is a figure which shows the classification process of a corn grain. It is TLC which shows glucose, cellooligosaccharide etc. which are contained in the solution after saccharification decomposition. It is a measurement result of the reducing sugar contained in the solution after saccharification / decomposition. It is a figure which shows the relationship between the density | concentration of hemicellulase in saccharification and reaction time. It is a figure which shows the production | generation ratio of a reducing sugar in the ratio in corn germ rice cake (solid content conversion). It is a plasmid map of the arming yeast which can metabolize xylose. It is a plasmid map of the arming yeast which can assimilate cellooligosaccharide. It is a liquid chromatography figure which shows the composition change before and behind alcohol fermentation.

  Corn germ rice is one of the by-product resources obtained from the production of vegetable oils. It is an oil pod produced after squeezing and extracting oil contained in corn germ when producing corn edible oil. Unlike soybeans and rapeseed, which are also known as oil cereals, corn has oil localized in the germ, so the corn is not used as a raw material, but only the germ is separated and used for corn oil production. The corn grain fractionation process is shown in FIG.

  Corn grains are used as starch raw materials by starch makers. Corn grains are roughly divided into pericarp (about 5% by mass (hereinafter abbreviated as%)), endosperm (75-80%), germ (10-15%), and oil (about 5%). Since starch is contained in the endosperm portion, starch makers currently separate endosperm from germ by a process called wet milling. In this step, corn kernels are immersed in a dilute sulfite solution for about 48 hours to swell and separate the corn kernels. By soaking, the separated endosperm contains water and settles in the lower part, and the germ containing much oil gathers in the upper part. After collecting and drying this due to a difference in specific gravity, the endosperm portion is used as a raw material for producing corn starch, and the germ portion is used as a vegetable oil raw material as a by-product. Germ originally contains about 35% oil, but by wet milling, soluble proteins contained in the germ are extracted and the oil is concentrated, resulting in an oil raw material containing about 50% oil. Compared to soybeans and rapeseed, the number of germs generated is small, but since it is a raw material containing a large amount of oil per unit, it is a raw material with good production efficiency of vegetable oils. About half of the oil contained in the germ is extracted by a process called pressing, and the rest is extracted with an organic solvent n-hexane to obtain crude corn oil. Become oil.

The corn germ from which the oil has been removed becomes a corn germ bud, similar to soybeans and rapeseed. Table 1 shows the results of analyzing the components of corn germ meal. Table 1 shows the analytical values of corn germ cakes A, B and C of different production lots and their average values.
As shown in Table 1, the nitrogen (N) content contained in corn germ cake or the crude protein value (N% × 6.25) based thereon is as low as about 20%. Moreover, since an acid is used in wet milling in the classification of endosperm and germ, the germ shows acidity, and the germ bud is confirmed to have an acidity of pH 3.5 to 4.0 even after oil extraction. Further, corn germ contains almost no starch. For this reason, corn germ rice is less useful as a feed material or fertilizer material than soybean meal or rapeseed meal.
In addition, the average content of cellulose contained in the corn germ cake is 10.9% cellulose, 23.3% hemicellulose, and 0.6% lignin, and the crude fibers and soluble nitrogen-free materials are combined. About 50 to 55% of the content of about 63.5% corresponds to the cellulose.

Next, the corn germ cake may be influenced by other microorganisms contained in the germ cake in the saccharification / decomposition process by microorganisms described later. Therefore, the following microorganism test method was used to confirm what kind of microorganisms were contained in corn germ buds. The results are shown in Table 2.
(1) The general viable cell count is as follows. 1 g of corn germ cake is suspended in 10 ml and 100 ml of sterilized water, and after light centrifugation, the supernatant is added to a standard agar medium and cultured at 37 ° C. for 48 hours. The number of colonies formed on the agar was counted.
(2) For the number of coliforms, 1 g of corn germ cake was suspended in 10 ml and 100 ml of sterilized water, and after light centrifugation, the supernatant was added to X-gal agar medium and cultured at 37 ° C. for 24 hours. Later, it was confirmed whether a blue colony (positive) was formed on the agar.
(3) Mold / yeast is suspended in 10 ml and 100 ml of sterilized water with 1 g of corn germ cake, lightly centrifuged, and then the supernatant is added to potato dextrose agar medium at 30 ° C. for 5 days. The number of colonies on the agar was measured.
(4) Salmonella was tested by partially improving the test method published in the Food Sanitation Inspection Guidelines (Microbiology). That is, 10 g of corn germ cake is taken as a sample amount, pre-cultured with BPW medium, then selectively enriched with HTT medium, then separated and cultured with DHL agar medium and BG agar medium, and formed on agar. Colonies were observed.

  As shown in Table 2, the number of general viable bacteria was 40, and the number of molds and yeasts was 10 by measurement at 10-fold dilution. Coliform bacteria and Salmonella were not detected. Because corn germ cake is acidic, the number of bacteria measured is considered to be less than that of soybean meal and rapeseed meal. Moreover, after autoclaving the corn germ buds at 121 ° C. for 20 minutes, the same microbial test was performed. As a result, the above bacterial count was not detected at all.

As described above, corn germ cake is rich in cellulosic matter and obtained in an acidic state, has low utility value as a feed raw material or fertilizer raw material, and is not a high value-added material, but its effective use is desired.
In addition, although FIG. 1 demonstrated the fractionation of the corn germ rice cake as a preferable example of this invention as a raw material of an oil candy, it is similarly an oil potato with a low protein content and a low utility value as a feed raw material or a fertilizer raw material. The effective use is also desired.

The method for producing alcohol, particularly ethanol, of the present invention undergoes a saccharification / decomposition step of degrading the cellulose contained in oil pods, particularly corn germ buds, into glucose, cellooligosaccharides and the like through culturing microorganisms.
The microorganism used for saccharification / decomposition is preferably a filamentous fungus, a bacterium, or a radioactive bacterium, and among these, a filamentous fungus is preferable. Of the filamentous fungi, Trichoderma reesei is preferable.
The reason for this is that Trichoderma reesei is (1) a cellulase enzyme-producing bacterium, (2) expresses and produces a plurality of cellulase enzymes, (3) grows fast and has strong proliferative power, (4) growth inhibition Strong growth, wide growth area, (5) active research on gene recombination, etc. For example, the expression and production of the enzyme having the highest cellulose decomposition efficiency can be controlled by genetically manipulating the production ratio of the cellulase family to be expressed.

The filamentous fungus Trichoderma reesei was obtained from the Biological Genetic Resource Department (NBRC) of the National Institute for Product Evaluation and Technology, and the saccharification and decomposition of cellulosic substances in corn germ buds were examined.
As described above, corn germ buds have a pH in the range of 3.5 to 4.0. Therefore, when cultivated with Trichoderma reesei, it was examined whether this filamentous fungus can grow in this pH region.
Potato extract 4 g, Glucose 20 g, and Agar 15 g were dissolved in 1 L of sterilized water, and then autoclaved and sterilized in PDA medium using sulfuric acid, pH 3.9, pH 4.2, pH 4.5, pH 4.7, pH 4.9. The pH was adjusted so that Trichoderma reesei was cultured with shaking at 30 ° C. for 5 days. As a result, a slight delay in growth was observed at pH 3.9, but conidia formation was observed in all medium liquids. On the fifth day, sufficient growth was confirmed. From this result, it was found that Trichoderma reesei can grow in the same pH range as that of corn germ cake (pH 3.85).

The concentration of the corn germ pod as a substrate in the culture was 5%, and shaking culture was performed at 30 ° C. for 5 days under the following 6 conditions, and the culture state of Trichoderma reesei and the degradation degree of corn germ bud were investigated.
(1) Medium + corn germ sterilized, pH 6.0
(2) No medium + corn germ sterilization treatment, pH 6.0
(3) Water + corn germ sterilized, pH 6.5
(4) No water + corn germ sterilization treatment, pH 6.5
(5) Water + corn germ sterilized, pH 3.5
(6) No water + corn germ sterilization treatment, pH 3.5
The medium used under the conditions (1) and (2) was prepared by adding water to 5.26 g of sodium glutamate, 5.26 g of dipotassium hydrogen phosphate, 1.05 g of magnesium sulfate heptahydrate, and 2.11 g of sodium chloride. In addition, the composition was made up to 1 L and autoclaved at 121 ° C. for 20 minutes. (3) to (6) were cultured only with water without using a medium.
After shaking culture at 30 ° C. for 5 days, the culture state is visually observed, and the supernatant solution is collected by centrifugation, and glucose, cellooligosaccharides, etc. contained in this solution are qualitatively analyzed by thin layer chromatography (TLC). Analysis was performed. The results are shown in FIG.

After culturing, separation was observed in the supernatant and the precipitate by centrifugation (3000 rpm, 5 minutes). Under the culture conditions (1), (2), (3) and (6), the tissue disruption of the corn germ bud was clearly confirmed in the precipitate. That is, it is considered that the cellulosic material of corn germ cake was decomposed by the filamentous fungus Trichoderma reesei. Furthermore, the appearance of the supernatant was divided into a clear one and a cloudy one, and a clear supernatant was obtained under the culture condition (6).
Moreover, from the TLC analysis result of the supernatant solution shown in FIG. 2, qualitative spots of glucose and cellooligosaccharide, which are cellulosic degradation products, were confirmed in the supernatant solution.
Similar to the visual result of tissue disintegration, the concentration of spots confirmed under the culture conditions (1), (2), (3) and (6) was high, and saccharification / degradation into glucose / cellooligosaccharide was an appropriate condition. Conceivable.

Moreover, it is thought that the turbidity of the supernatant liquid visually confirmed indicates the degree of contamination of other fungi in the culture. That is, since the filamentous fungus Trichoderma reesei is larger than other bacteria and settles easily by centrifugation, it is hardly observed in the supernatant. However, if other bacteria are present, they are in the supernatant. Causes turbidity. In the above experiment, the clearest supernatant was obtained under the culture condition (6).
As a result, in the saccharification / decomposition step of the present invention, the culture condition (6) in the acidic region where the supernatant is clear and the decomposition into saccharides by TLC analysis is excellent is a preferable culture condition.
Furthermore, the culture condition (6) in the acidic region does not require sterilization of corn germ buds, and glucose and cellooligosaccharides, which are cellulosic degradation products, can be obtained simply by adding water and culturing. It is an excellent saccharification / decomposition process that can achieve a sufficient cost reduction.

In the saccharification and decomposition, cellulose contained in corn germ cake was examined. However, corn germ cake contains not only 10.9% cellulose but also 23.3% hemicellulose. For this reason, when the microbe which saccharifies and decomposes this hemicellulose was examined, it turned out that the hemicellulase derived from Aspergillus niger is suitable.
In order to confirm the production amount of reducing sugar and glucose when hemicellulase derived from Trichoderma reesei and Aspergillus niger and both coexist in the saccharification / decomposition process of corn germ buds, (1) 2) When only Trichoderma reesei is added to the corn germ pod, (3) When only the above hemicellulase is added, (4) The following four conditions are used when both Trichoderma reesei and hemicellulase are added. The saccharification degradation experiment shown was conducted.

Add 10 g of corn germ cake to 100 ml of distilled water so that the corn germ cake becomes 10% (w / v) with respect to distilled water in a 500 ml baffled Erlenmeyer flask.
In the case of (1) corn germ cocoon alone, (2) 0.05 g corn germ cocoon cultured with Trichoderma reesei, (3) 0.01% hemicellulase derived from Aspergillus niger, (4) Trichoderma reesei The reaction was carried out for 2 days while rotating the stirrer at 37 ° C. and 160 rpm under 4 conditions of 0.05 g of cultured corn germ cake and 0.01% of the above hemicellulase.
After completion of the reaction, the supernatant was collected by centrifugation. In order to confirm what kind of oligosaccharide is contained in the supernatant, it was confirmed by thin layer chromatography (TLC). As a result, glucose was remarkably confirmed under the above conditions (3) and (4). . In addition, the reducing sugar concentration contained in the supernatant after the reaction was measured by the DNS method, and the glucose concentration was measured by the gluco-oxidase method. The results obtained by converting the measurement results into the proportion of corn germ cake are shown in FIGS. 3 (a) and 3 (b). FIG. 3 (a) is a diagram showing the proportion of reducing sugar, and FIG. 3 (b) is a diagram showing the proportion of glucose contained in the reducing sugar.

  From the results of FIGS. 3 (a) and (b), compared to the case where only Trichoderma reesei or hemicellulase was added alone, more of Trichoderma reesei and hemicellulase coexisted at the stage of the second day of the reaction. Reducing sugar and glucose could be obtained. 23.8% reducing sugar and 18.4% glucose were obtained from corn germ straw. Furthermore, the ratio of glucose to the produced reducing sugar was as high as 77.3%, and it was found that most of the obtained reducing sugar was glucose. The fact that 23.8% of reducing sugar was obtained from corn germ cake means that 69.6% of the total cellulose contained in corn germ cake could be decomposed into reducing sugar. From these results, it is more effective to add both Trichoderma reesei and hemicellulase, rather than adding Trichoderma reesei and hemicellulase alone, in order to obtain more sugar from corn germ buds.

When Trichoderma reesei coexists with hemicellulase, the reaction time for obtaining the most reducing sugar and glucose is changed. In the presence of Trichoderma reesei, the hemicellulase concentration is changed. I examined it.
A 500 ml baffled Erlenmeyer flask was taken so that the corn germ cake was 10% (w / v) with respect to distilled water, and 0.05 g of corn germ cake cultured with Trichoderma reesei was added thereto. Further, add hemicellulase so that the hemicellulase concentration becomes 0.1%, 0.01%, 0.005%, 0.001%, and react at 37 ° C. and 160 rpm. About 1 ml was recovered. Moreover, it carried out similarly in the case of adding only Trichoderma reesei without adding hemicellulase without adding hemicellulase. In order to confirm what kind of oligosaccharide is contained in the supernatant, it was confirmed by thin layer chromatography (TLC) (developing solvent: ethyl acetate: methanol: water = 12: 7: 3, color development method: sulfuric acid spray) ). Next, the reducing sugar concentration of the supernatant was measured by the DNS method. The glucose concentration was measured by the gluco-oxidase method. The measurement results are shown in FIGS. 4 (a) and (b). FIG. 4 (a) is a diagram showing the production rate of reducing sugar, and FIG. 4 (b) is a diagram showing the production rate of glucose.

As shown in FIGS. 4 (a) and (b), the reducing sugar and glucose production ratios both increased with increasing hemicellulase concentration. It was also found that reducing sugars and glucose were most produced in 48 hours after the reaction. Since then, the sugars produced by Trichoderma reesei are thought to be metabolized and reduced.
In the saccharification / decomposition step of the present invention, the concentration of hemicellulase is preferably 0.1% or more with respect to corn germ meal.
The saccharification / decomposition time is preferably a time at which the production rate of reducing sugar or glucose is maximized, and this time can be determined by preliminary saccharification / decomposition experiments.

In order to investigate the optimal water content of corn germ cake in the saccharification and decomposition process, the difference in the amount of reducing sugar produced by changing the water content contained in corn germ cake was measured.
Considering that corn germ cake as a raw material contains 10% water, the water content of the raw material will be 50%, 60%, 70%, 80%, 90%, 91.8% Corn germ cake was prepared. The control was a water content of 10%. Hemicellulase was added so that it might become 0.1% with respect to a corn germ bud, and it was left still at 37 degreeC and made it react. Sampling was performed every 24 hours after the start of the reaction, and 1 ml of the supernatant was collected by centrifugation. The reducing sugar concentration contained in the obtained sample was measured by the DNS method. The results are shown in FIG. FIG. 5 is a diagram showing the production rate of reducing sugar as a proportion in corn germ meal (in terms of solid content).

As can be seen from FIG. 5, as the water content was increased, most of the corn germ cake could be decomposed into reducing sugar. This is thought to be because the lower the moisture content, the higher the concentration of reducing sugar, and the easier the inhibition of the enzyme reaction by the product. In order to decompose corn germ cake more and efficiently obtain reducing sugar and glucose, it is important not only to increase the amount of corn germ cake added, but also to increase the water content, thereby decomposing more corn germ cake . On the other hand, considering the capacity of the reactor, it can be said that it is necessary to reduce the water content in order to add more corn germ cake per volume. Furthermore, a certain water content is required for efficient decomposition.
As shown in FIG. 5, the moisture content is preferably 60% to 90%. If it is less than 60%, the saccharification / decomposition reaction is not sufficient, and if it exceeds 90%, the capacity of the reaction apparatus becomes too large.

The alcohol fermentation, particularly the ethanol fermentation process will be described.
Saccharomyces cerevisiae is known as a yeast excellent in ethanol production used in the ethanol fermentation process. However, this yeast can efficiently ferment glucose to ethanol, but C5 sugars such as xylose and cellooligosaccharides cannot be assimilated. By constructing a yeast that can perform ethanol fermentation from these sugars, ethanol fermentation can be efficiently performed from the saccharification and decomposition product of corn germ meal. Therefore, the following arming yeast was constructed.

<Construction of arming yeast capable of metabolizing xylose>
By incorporating a xylose metabolic system into the yeast Saccharomyces cerevisiae, a yeast capable of simultaneously fermenting C5 and C6 sugars can be constructed. Thus, yeast yeast was used to construct Pichia stipitis-derived xylose reductase and xylitol dehydrogenase in the cells, and to overexpress S. cerevisiae-derived xylulokinase. A yeast was constructed in which the vector pIUX1X2XK into which the above three types of enzymes had been incorporated was incorporated into the yeast MT8-1. FIG. 6 shows a plasmid map. After confirming that each enzyme activity was present, a yeast strain in which three kinds of enzymes were incorporated was obtained.
The arming yeast can be constructed by the method described in Example 1 of Japanese Patent Application Laid-Open No. 2008-193935.

<Construction of arming yeast that can assimilate cellooligosaccharides>
By presenting cellooligosaccharide-degrading enzyme on the surface layer of yeast, a yeast capable of directly fermenting cellooligosaccharide can be constructed. Therefore, a yeast was constructed in which β-glucosidase was further displayed on the surface of the yeast having the ability to metabolize xylose. A plasmid pIBG13 that displays β-glucosidase-1 (BGL1) derived from Aspergillus acculeatus with α-agglutinin was incorporated into yeast, and yeast that assimilated both xylose and cellooligosaccharide was constructed. FIG. 7 shows a plasmid map. It was also confirmed that there was BGL activity.
The arming yeast can be constructed by the method described in Example 1 of Japanese Patent Application Laid-Open No. 2008-193935.

A saccharide solution obtained from corn germ cake is diluted twice with sterilized water, arming yeast capable of assimilating the above-mentioned cellooligosaccharide is added so that the yeast cell mass becomes 40 g / L, and fermentation is performed at 30 ° C. for 4 hours. It was. The results are shown in FIG. FIG. 8 is a liquid chromatography diagram showing composition changes before and after alcoholic fermentation.
The degradation product after the saccharification / decomposition process of corn germ cake contains approximately 5 g / L of glucose. It can be seen that in 4 hours of fermentation, glucose disappears and ethanol is produced. The ethanol yield was approximately 80% of the theoretical yield. Moreover, there was no contamination by other microorganisms during the culture, and no inhibition on ethanol fermentation was confirmed.

3000 g of corn germ cake after squeezing (pH 3.8, moisture 9.2%), 1 g of corn germ cake cultivated with Trichoderma reesei, 3 g of hemicellulase (hemocellulase “Amano” 90 manufactured by Amano Enzyme), distilled water 6000 ml was added, and the saccharification / decomposition reaction was performed while stirring the contents at 37 ° C. for 48 hours. In addition, the corn germ bud which culture | cultivated Trichoderma reesei used what was cultured on 37 degreeC and the conditions for 2 days.
As a result of the saccharification / decomposition reaction, 13.8% reducing sugar was obtained from the corn germ cake (solid content) in the presence of Trichoderma reesei and hemicellulase, and 4.69% Glucose was obtained.

The saccharide solution obtained by the saccharification / decomposition reaction is diluted twice with distilled water, and the above-described arming yeast capable of assimilating the cellooligosaccharide is added so that the amount of yeast cells becomes 40 g / L, followed by fermentation at 30 ° C. for 4 hours. It was.
As a result, 2.15% ethanol was recovered. This represents that 90% of the sugars were converted to ethanol in theoretical yield.

  The alcohol production method of the present invention can be used as an ethanol production method with a low environmental load because an oil pod having low added value can be effectively used.

Claims (5)

An alcohol production method comprising: a saccharification / decomposition step of saccharifying / degrading oil pods with bacterial cells; and a step of alcohol-fermenting the sugar obtained in the saccharification / decomposition step,
The oil pod is a corn germ pod generated in the edible oil production process,
The method for producing alcohol from oil pods, wherein the cells are filamentous fungi.   The method for producing alcohol according to claim 1, wherein the alcohol is ethanol.   The alcohol production method according to claim 1, wherein the corn germ cake is an acidic germ cake after oil extraction. The method for producing an alcohol according to any one of claims 1 to 3 , wherein the filamentous fungus is Trichoderma reesei, and saccharification is performed in an acidic region. The alcohol production according to any one of claims 1 to 4 , wherein the alcohol fermentation is alcohol fermentation using an arming yeast, and the arming yeast is an arming yeast capable of assimilating xylose and cellooligosaccharide. Method.