Method for producing ethanol from lignocellulosic biomass

Abstract

The present invention provides a method for improving a defoaming effect of a fermentation liquid when lignocellulosic biomass is used. More specifically, the present invention provides a method for producing ethanol from lignocellulosic biomass, the method involving a multiple parallel fermentation step, a multiple semi-parallel fermentation step, or a sequential fermentation step, wherein when the multiple parallel fermentation step is involved, the multiple parallel fermentation step involves a step for adding a defoaming agent to a fermentation liquid containing lignocellulosic biomass, a diastatic enzyme, and yeast; when the multiple semi-parallel fermentation step or sequential fermentation step is involved, the multiple semi-parallel fermentation step or sequential fermentation step involves a step for adding a defoaming agent to a saccharified solution containing lignocellulosic biomass and a diastatic enzyme and / or to a fermentation liquid containing the saccharified solution and yeast; and the defoaming agent includes a nonionic surfactant.

Description

Method for producing ethanol from lignocellulosic biomass REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority based on Japanese Patent Application No. 2024-116145 filed on July 19, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method for producing ethanol from lignocellulosic biomass.

[0003] The technology to produce sugar from non-edible lignocellulosic biomass is extremely beneficial for creating a recycling-oriented society, as it can be used as a fermentation substrate for microorganisms to produce fuels such as alcohol that can serve as an alternative to gasoline, and it can prevent competition with food sources.

[0004] Enzymatic saccharification, which involves hydrolysis using saccharifying enzymes such as cellulolytic enzymes or microorganisms that produce these saccharifying enzymes, is known as a method for producing sugars such as monosaccharides and oligosaccharides that can be used as fermentation substrates from polysaccharides in lignocellulosic biomass.

[0005] Also, methods for producing ethanol from lignocellulose by applying enzymatic saccharification include multiple parallel fermentation, semi-parallel multiple fermentation, and sequential saccharification and fermentation. In multiple parallel fermentation (also called simultaneous saccharification and fermentation), for example, the saccharifying enzyme and an ethanol-fermenting microorganism are coexisted, and enzymatic saccharification and ethanol fermentation are carried out simultaneously. In semi-parallel multiple fermentation, for example, lignocellulosic biomass and a saccharifying enzyme are added to one reactor and an enzymatic saccharification reaction is carried out, and then an ethanol-fermenting microorganism is added to the same reactor and an enzymatic saccharification reaction and ethanol fermentation are carried out. In sequential saccharification and fermentation, for example, lignocellulosic biomass and a saccharifying enzyme are first added to a reactor and saccharification is carried out, and the resulting saccharified solution is transferred to another reactor, where an ethanol-fermenting microorganism is added and ethanol fermentation is carried out.

[0006] The fermentation process involves the problem of foaming. As described above, the culture solution in the reaction tank (fermenter) contains sugars such as oligosaccharides, which makes the viscosity of the culture solution high. In addition, the ethanol-fermenting microorganisms such as yeast synthesize carbon dioxide gas along with ethanol from the sugars in the fermenter, creating an environment in which foaming is likely to occur.

[0007] Here, foaming undesirably causes problems such as outflow of the fermentation liquid from the fermenter, poor transport due to foam entrapment in the pump, a decrease in the effective volume of the fermenter due to an increase in foam, a decrease in ethanol productivity due to difficulty in pH control because the upper layer of the culture liquid is covered with foam, and inactivation of saccharifying enzymes when the foam bursts.

[0008] As a means for solving the problem of foaming, Patent Document 1 describes a method for producing ethanol, which includes a first fermentation step in which yeast capable of xylose assimilation are cultured in a fermenter while a saccharified solution derived from lignocellulosic biomass is added to the fermenter, and a second fermentation step in which the addition of the saccharified solution is stopped when a certain amount of the added saccharified solution is reached, and fermentation is carried out by batch culture, in which the rate of feeding the saccharified solution to the yeast in the first fermentation step is adjusted based on the sugar consumption rate of the yeast, and the sugar consumption rate of the yeast is equal to or greater than the maximum sugar consumption rate at which glucose and xylose do not remain in the culture solution, but is equal to or less than the maximum sugar consumption rate of the yeast. Patent Document 1 describes waste paper as lignocellulosic biomass, along with softwood and hardwood, but does not provide any specific examples.

[0009] Patent Document 2 describes a method for enhancing yeast growth for the production of biological products, which comprises culturing at least one yeast in a growth medium containing a nutritional composition comprising corn steep liquor and at least one of at least one antifoaming agent (a) or at least one surfactant (b), or any combination thereof, to obtain a grown yeast culture.

[0010] Patent Document 3 describes a foam control agent suitable for bioethanol fermentation treatment, which contains a polyglycerol ether having a specific structure.

[0011] Patent No. 6097869 Publication Special Publication No. 2018-537075 Publication Special Publication No. 2023-522839

[0012] When ethanol is produced using lignocellulosic biomass such as waste paper, foaming occurs more intensely than with ordinary bleached pulp. However, Patent Documents 1 to 3 do not specifically mention the use of waste paper as lignocellulosic biomass.

[0013] As a result of extensive investigations aimed at solving the above problems, the present inventors have found that the defoaming effect of the fermentation liquor can be improved by using a specific defoaming agent. The present invention is based on this finding. Examples of lignocellulosic biomass used in the fermentation liquor include waste paper.

[0014] Therefore, the present invention provides a method for improving the defoaming effect of a fermentation liquor when using lignocellulosic biomass (e.g., recycled paper pulp or unbleached pulp).

[0015] The present invention includes the following [1] to

[10] .

[0016] [1] A method for producing ethanol from lignocellulosic biomass, comprising: a multiple parallel fermentation process, a semi-parallel multiple fermentation process, or a sequential fermentation process; wherein, in the case of the multiple parallel fermentation process, the multiple parallel fermentation process comprises adding an antifoaming agent to a fermentation liquor containing lignocellulosic biomass, a saccharifying enzyme, and yeast; and, in the case of the semi-parallel multiple fermentation process or the sequential fermentation process, the semi-parallel multiple fermentation process or the sequential fermentation process comprises adding an antifoaming agent to a saccharification solution containing lignocellulosic biomass and a saccharification enzyme and / or a fermentation liquor containing the saccharification solution and yeast, wherein the antifoaming agent comprises a nonionic surfactant. [2] The method for producing ethanol according to [1], wherein the lignocellulosic biomass is at least one selected from the group consisting of recycled paper pulp and unbleached pulp. [3] The method for producing ethanol according to [1] or [2], comprising adding the antifoaming agent continuously or intermittently. [4] The method for producing ethanol according to any one of [1] to [3], wherein the waste paper is selected from the group consisting of paper cups, paper containers, and miscellaneous paper. [5] The method for producing ethanol according to any one of [1] to [4], wherein the antifoaming agent comprises a polyoxyalkylene ether-type nonionic surfactant. [6] The method for producing ethanol according to any one of [1] to [5], wherein the specific gravity of the antifoaming agent at 25°C is 0.95 to 1.00. [7] The method for producing ethanol according to any one of [1] to [6], wherein the cloud point of a 10% by mass aqueous solution of the antifoaming agent is 10°C or lower. [8] The method for producing ethanol according to any one of [1] to [7], wherein the mass ratio of the total amount of antifoaming agent to the total amount of lignocellulosic biomass (total amount of antifoaming agent:total amount of lignocellulosic biomass) throughout the entire culture is 1:100 to 1:10,000. [9] The method for producing ethanol according to any one of [1] to [8], wherein the multiple parallel fermentation step, the multiple semi-parallel fermentation step, or the sequential saccharification and fermentation step is carried out continuously.

[10] A defoaming agent for use in a method for producing ethanol from at least one lignocellulosic biomass selected from recycled paper pulp and unbleached pulp, the defoaming agent comprising a nonionic surfactant.

[0017] According to the present invention, there is provided a method for producing ethanol from lignocellulosic biomass (e.g., waste paper pulp or unbleached pulp), the method comprising: a multiple parallel fermentation process, a semi-parallel multiple fermentation process, or a sequential fermentation process; in the case of the multiple parallel fermentation process, the multiple parallel fermentation process comprises a step of adding an antifoaming agent to a fermentation liquor containing lignocellulosic biomass, a saccharifying enzyme, and yeast; and in the case of the semi-parallel multiple fermentation process or the sequential fermentation process, the semi-parallel multiple fermentation process or the sequential fermentation process comprises a step of adding an antifoaming agent to a saccharification liquor containing lignocellulosic biomass and a saccharifying enzyme and / or a fermentation liquor containing the saccharification liquor and yeast; and when the antifoaming agent comprises a nonionic surfactant, the antifoaming effect of the fermentation liquor can be improved.

[0018] 1 shows the change in ethanol concentration over time in Test Plot 2-1 and Test Plot 2-2. Specific Description of the Invention

[0019] The method for producing ethanol from lignocellulosic biomass of the present invention includes a multiple parallel fermentation process, a semi-parallel multiple fermentation process, or a sequential fermentation process. In the case of the multiple parallel fermentation process, the multiple parallel fermentation process includes a step of adding an antifoaming agent to a fermentation liquor (preferably a saccharification and fermentation liquor) containing lignocellulosic biomass, a saccharification enzyme, and yeast. In the case of the semi-parallel multiple fermentation process or the sequential fermentation process, the semi-parallel multiple fermentation process or the sequential fermentation process includes a step of adding an antifoaming agent to a saccharification liquor containing lignocellulosic biomass and a saccharification enzyme and / or a fermentation liquor containing the saccharification liquor and yeast, wherein the antifoaming agent includes a nonionic surfactant. Preferably, the lignocellulosic biomass is at least one selected from the group consisting of recycled paper pulp and unbleached pulp. In this specification, "a saccharification liquid containing lignocellulosic biomass and a saccharification enzyme, and / or a fermentation liquid containing the saccharification liquid and yeast" refers to the saccharification liquid, the fermentation liquid, or a combination of the saccharification liquid and the fermentation liquid.

[0020] The method for producing ethanol from lignocellulosic biomass of the present invention includes multiple parallel fermentation, semi-parallel multiple fermentation, or sequential fermentation. In the case of the multiple parallel fermentation, the multiple parallel fermentation comprises adding an antifoaming agent to a fermentation liquor containing lignocellulosic biomass, a saccharifying enzyme, and yeast. In the case of the multiple semi-parallel fermentation or sequential fermentation, the multiple semi-parallel fermentation or sequential fermentation comprises adding an antifoaming agent containing a nonionic surfactant to a saccharification liquor containing lignocellulosic biomass and a saccharifying enzyme and / or a fermentation liquor containing the saccharification liquor and yeast. The lignocellulosic biomass is preferably at least one selected from the group consisting of recycled paper pulp and unbleached pulp. Here, the "fermentation liquor containing lignocellulosic biomass, saccharifying enzymes, and yeast" of the present invention includes not only the fermentation liquor produced by fermentation using lignocellulosic biomass, saccharifying enzymes, and yeast, but also the culture liquor before fermentation (e.g., a culture liquor containing lignocellulosic biomass, saccharifying enzymes, and yeast in a multiple parallel fermentation process, and a culture liquor containing yeast and a saccharified liquid obtained by mixing lignocellulosic biomass with saccharifying enzymes and performing a saccharification reaction). Furthermore, the "saccharified liquid containing lignocellulosic biomass and saccharifying enzymes" of the present invention includes not only the saccharified liquid obtained by mixing lignocellulosic biomass with saccharifying enzymes and performing a saccharification reaction, but also the mixed liquid before the saccharification reaction (e.g., a mixed liquid containing lignocellulosic biomass and saccharifying enzymes before the saccharification reaction in the saccharification process of a semi-parallel multiple fermentation process or a sequential fermentation process).

[0021] According to one embodiment of the present invention, the method for producing ethanol from lignocellulosic biomass of the present invention may include at least one of the following steps: if the method includes a multiple parallel fermentation step, the method includes adding an antifoaming agent containing a nonionic surfactant to a fermentation liquor containing lignocellulosic biomass, a saccharifying enzyme, and yeast in the multiple parallel fermentation step; and if the method includes a semi-parallel multiple fermentation step or a sequential fermentation step, the method includes adding an antifoaming agent containing a nonionic surfactant to a saccharification liquor containing lignocellulosic biomass and a saccharifying enzyme and / or a fermentation liquor containing the saccharification liquor and yeast in the semi-parallel multiple fermentation step or the sequential fermentation step. (1) Multiple parallel fermentation step, Semi-parallel multiple fermentation step, or Sequential saccharification and fermentation step, (2) Solid-liquid separation step, (3) Ethanol concentration step, and (4) Solids removal step

[0022] <Parallel fermentation process> A parallel fermentation process generally refers to a process in which saccharification of a feedstock and ethanol production by fermentation proceed simultaneously. According to one embodiment of the present invention, the parallel fermentation process is a process in which saccharification (also referred to as enzymatic saccharification) and ethanol fermentation are simultaneously carried out in the presence of an ethanol-fermenting microorganism together with a saccharifying enzyme. For example, the parallel fermentation process is a process in which saccharification and ethanol fermentation are simultaneously carried out using a fermentation broth containing lignocellulosic biomass, a saccharifying enzyme, and yeast. A specific method for the parallel fermentation process involves, for example, first supplying lignocellulosic biomass, a saccharifying enzyme, and yeast to a reactor. In the present invention, the lignocellulosic biomass, the saccharifying enzyme, and the yeast may be premixed and supplied to the reactor, or they may be supplied separately. However, it is preferable to add the saccharifying enzyme and yeast to the lignocellulosic biomass already supplied to the reactor. The lignocellulosic biomass is saccharified by the saccharifying enzyme, and the resulting sugars are converted to ethanol by fermentation with yeast.

[0023] Semi-parallel multiple fermentation process: A semi-parallel multiple fermentation process generally refers to a process in which ethanol production by fermentation is simultaneously performed after the saccharification of a feedstock has partially progressed. According to one embodiment of the present invention, the semi-parallel multiple fermentation process involves mixing lignocellulosic biomass with a saccharifying enzyme to perform a saccharification reaction to obtain a saccharified solution (also referred to as a saccharification process), and then mixing the resulting saccharified solution with an ethanol-fermenting microorganism (preferably yeast) to perform a saccharification reaction and ethanol fermentation using the resulting fermentation solution (also referred to as a saccharification and fermentation process). In this semi-parallel multiple fermentation process, the reaction vessel for the saccharification step and the reaction vessel for the saccharification and fermentation step may be the same or different. Preferably, when the semi-parallel multiple fermentation process is performed in a batch system and the ethanol-fermenting microorganism is yeast, the reaction vessel for the saccharification step and the reaction vessel for the saccharification and fermentation step are the same. When the semi-parallel multiple fermentation process is performed in a continuous system, the reaction vessel for the saccharification step and the reaction vessel for the saccharification and fermentation step are different. In a specific example of a semi-parallel simultaneous fermentation process, lignocellulosic biomass and saccharification enzymes are first supplied to a reactor. The lignocellulosic biomass is first saccharified by the saccharification enzymes (saccharification process). Yeast is then added to the same reactor, where enzymatic saccharification and ethanol fermentation are carried out. The sugars produced are then converted to ethanol by fermentation with the yeast (saccharification and fermentation process).

[0024] <Sequential Saccharification and Fermentation Process> The sequential saccharification and fermentation process generally refers to a process in which saccharification of a raw material and ethanol production by fermentation are carried out in a stepwise manner. According to one embodiment of the present invention, the sequential saccharification and fermentation process involves mixing lignocellulosic biomass with a saccharifying enzyme to saccharify the resulting saccharified solution (also referred to as the saccharification process), and then mixing the resulting saccharified solution with an ethanol-fermenting microorganism (preferably yeast) to perform ethanol fermentation using the resulting fermentation solution (also referred to as the fermentation process). Preferably, the reaction vessel for the saccharification process and the reaction vessel for the fermentation process are separate, and the saccharified solution obtained in the saccharification process is transferred to a reaction vessel separate from the reaction vessel for the saccharification process. A specific method for the sequential saccharification and fermentation process includes, for example, first supplying lignocellulosic biomass and a saccharifying enzyme to a reaction vessel. The lignocellulosic biomass is saccharified by the saccharifying enzyme (saccharification process). The resulting saccharified solution is then transferred to another reaction vessel, where yeast is added, and the resulting sugars are converted to ethanol by fermentation with the yeast (fermentation process).

[0025] (Lignocellulose Biomass) According to one embodiment of the present invention, the lignocellulose biomass is not particularly limited, but examples thereof include pulp or a raw material containing lignocellulose other than pulp, and is preferably biomass containing lignocellulose that produces more foam than in normal fermentation. Here, normal fermentation includes, for example, fermentation using hardwood bleached kraft pulp (LBKP). Lignocellulose biomass may be used singly or in combination of two or more types. Here, "produces more foam than in normal fermentation" means that when cultured using approximately the same amount of fermentation liquid, the foam height is higher than in normal fermentation. For example, when 600 mL of culture liquid is placed in a 1 L culture tank (a cylindrical culture tank with a bottom diameter of 8 cm) and the number of yeast cells is 10 ８ After the cell density reached 100 cells / mL or more, when the foam height was compared 30 minutes after adding 5% by mass of pulp, the foam height was less than 1 cm in the normal fermentation using LBKP, while it was 3 to 4 cm or more in the fermentation using recycled paper pulp.

[0026] Pulp as the lignocellulosic biomass that foams more than that obtained by normal fermentation includes, for example, waste paper pulp and unbleached pulp, preferably waste paper pulp. Specific examples of waste paper in the waste paper pulp include waterproof waste paper such as paper cups, pressed postcards, cartocans, and wraps, ordinary printed waste paper such as catalogs (e.g., colored wood-free paper), folding cartons, and shredded paper (e.g., shredded office paper), special printed waste paper such as carbonless paper and foil-stamped folding cartons, and lignin-rich waste paper such as magazines, preferably waterproof waste paper or ordinary printed waste paper, more preferably paper cups and folding cartons. The unbleached pulp (also referred to as unbleached pulp or unbleached pulp) includes chemical pulp and mechanical pulp, and more specifically includes chemical pulp such as hardwood kraft pulp (e.g., unbleached kraft pulp, oxygen-bleached unbleached kraft pulp), softwood kraft pulp (e.g., unbleached kraft pulp, oxygen-bleached unbleached kraft pulp), sulfite pulp, soda pulp, semi-chemical pulp, chemi-ground wood pulp, and waste wood-derived pulp. Hardwood unbleached kraft pulp, hardwood oxygen-bleached unbleached kraft pulp, softwood unbleached kraft pulp, softwood oxygen-bleached unbleached kraft pulp, sulfite pulp, and soda pulp are preferred. The unbleached pulp is pulp that has not been bleached. The bleaching treatment is not particularly limited, and various methods such as chlorine bleaching and hydrogen peroxide bleaching can be used.

[0027] Lignocellulosic biomass other than pulp includes waste paper, pulp sludge, sludge, sewage sludge, food waste, etc., with waste paper being preferred. The types of waste paper are as described above. These biomasses can be used alone or in combination. The biomass may be a dry solid, a wet solid, or a slurry.

[0028] The above-mentioned lignocellulosic raw materials other than pulp are preferably used after being subjected to pretreatment (delignification treatment, etc.).

[0029] According to one embodiment of the present invention, the concentration of lignocellulosic biomass in the fermentation liquor in the reaction tank is preferably 5 to 30% by mass, and more preferably 10 to 20% by mass. A lignocellulosic biomass concentration of 5% by mass or more is advantageous in avoiding the problem of the final product having an excessively low concentration, resulting in high costs for ethanol concentration. Furthermore, a lignocellulosic biomass concentration of 30% by mass or less is advantageous in avoiding the problem of increased productivity due to difficulty in stirring the raw material as the concentration increases.

[0030] (Saccharification enzyme) The saccharification enzyme is not particularly limited as long as it is a cellulose- or hemicellulose-degrading enzyme. Examples of the cellulose-degrading enzyme include enzymes collectively known as cellulases, which have cellobiohydrolase activity, endoglucanase activity, beta-glucosidase activity, etc. Each cellulose-degrading enzyme may be added in an appropriate amount, but many commercially available cellulose-degrading enzyme preparations have the above-mentioned various cellulase activities as well as hemicellulase activity, so commercially available cellulose-degrading enzyme preparations may also be used.

[0031] Commercially available cellulolytic enzyme agents include those derived from the genera Trichoderma, Acremonium, Aspergillus, Phanerochaete, Trametes, Humicola, Bacillus, and Irpex. Commercially available cellulolytic enzyme agents include, for example, Cellulosin AL8 (manufactured by HI Corporation), Cellic CTec2 (manufactured by Novozymes), Cellulase Y-NC (manufactured by Yakult Pharmaceutical Co., Ltd.), and Meicelase (manufactured by Meiji Seika Pharma), all of which are trade names.

[0032] The saccharifying enzymes used can be used alone or in combination, taking into consideration the properties of the cellulolytic enzyme preparation.

[0033] The activity of a saccharifying enzyme as used herein is defined as follows.

[0034] 375 μL of enzyme solution and 0.5 g of bone dry weight of hardwood bleached kraft pulp (LBKP) were added to 8 mL of 1.5 mL of 1 M acetate buffer (pH 4.8) to make a total of 10 mL of the system. The reaction was carried out at 33 ° C for 4 hours, and then heated to 95 ° C for 10 minutes to stop the reaction. This was analyzed using a refractive index (RI) detector by high performance liquid chromatography (High performance liquid chromatography: HPLC) (Prominence, manufactured by Shimadzu Corporation), and the glucose concentration was measured. Based on the measurement results, the amount of enzyme protein that produces 1 μmol of glucose per minute was defined as 1 unit (U).

[0035] The HPLC measurement conditions are as follows: Column: Shodex SUGAR SP0810 (Showa Denko K.K.) Mobile phase: ultrapure water Flow rate: 0.8 mL / min Temperature: 80°C According to one embodiment of the present invention, the amount of saccharifying enzyme added to the fermentation liquid in the reaction tank is not particularly limited, but is preferably 50 to 500 U / L, and more preferably 100 to 300 U / L.

[0036] According to one embodiment of the present invention, the ratio of the amount of saccharification enzyme input to the lignocellulosic biomass in the fermentation liquid in the reaction tank in the saccharification process (initial saccharification enzyme content (U) / initial lignocellulosic biomass content (kg)) is preferably 500 to 5,000 U / kg, and more preferably 1,000 to 3,000 U / kg. According to another embodiment of the present invention, the ratio of the amount of saccharification enzyme input to the lignocellulosic biomass in the fermentation liquid in the reaction tank in the saccharification process of the semi-parallel saccharification process or the saccharification process of the sequential saccharification and fermentation process is the same as above.

[0037] (Yeast) The yeast is not particularly limited, but is preferably capable of fermenting sugars (hexose, pentose). Specific examples of yeast include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Scheffersomyces such as Scheffersomyces stipitis (modified from Pichia stipitis), yeasts of the genus Candida such as Candida shihatae, and yeasts of the genus Pachysolen such as Pachysolen tannophilus. Yeasts belonging to the genus Saccharomyces are preferred, and Saccharomyces cerevisiae is more preferred. Yeast produced using genetic engineering techniques can also be used. As the yeast produced using genetic engineering techniques, any yeast capable of simultaneously fermenting hexose and pentose can be used without particular limitation. Preferred examples of yeast produced using genetic engineering techniques include yeast with improved ability to produce ethanol from xylose, more preferably yeast into which a gene encoding xylose reductase, a gene encoding xylulose phosphorylase, a gene encoding xylitol dehydrogenase, a gene encoding transaldolase, a gene encoding transketolase, and a gene encoding alcohol dehydrogenase have been introduced in an expressible manner, and even more preferably yeast described in WO 2016 / 060171. Here, the genes may be endogenous genes of the yeast.

[0038] (Antifoaming Agent) According to one embodiment of the present invention, the antifoaming agent of the present invention is not particularly limited as long as it has a defoaming effect and / or foam suppressing effect on, for example, a fermentation broth (including a culture broth before fermentation) used for cell growth and / or fermentation. However, from the viewpoint of the defoaming effect, a preferred example is an antifoaming agent containing a nonionic surfactant (preferably, an antifoaming agent containing a nonionic surfactant as a main component, hereinafter also referred to as a nonionic surfactant-based antifoaming agent). Therefore, the antifoaming agent of the present invention may be referred to as an antifoaming agent composition. Here, the content of the nonionic surfactant in the antifoaming agent is, for example, 80 to 100% by mass, preferably 90 to 100% by mass, and more preferably 95 to 100% by mass.

[0039] The nonionic surfactant is preferably a polyether type, and more preferably a polyoxyalkylene ether type. Examples of commercially available polyether type nonionic surfactants include those manufactured by Adeka Corporation under the trade names "ADEKA NOL LG-109," "ADEKA NOL LG-126," "ADEKA NOL LG-294," "ADEKA NOL LG-295S," and "ADEKA NOL LG-299" (each a registered trademark), those manufactured by San Nopco under the trade name "NOPTAMU 300EZ," those manufactured by Kao Corporation under the trade name "Antifoam No. 1," and those manufactured by Fujifilm Wako Pure Chemical Industries under the trade name "Antifoam PE-L," "Antifoam PE-M," and "Antifoam PE-H." Examples of specially designed nonionic surfactants include those manufactured by Kao Corporation under the trade name "FERMOL 1000." Commercially available polyoxyalkylene ether type nonionic surfactants include those manufactured by Adeka Corporation under the trade names "ADEKA NOL LG-109," "ADEKA NOL LG-126," "ADEKA NOL LG-294," and "ADEKA NOL LG-299" (each a registered trademark), and those manufactured by San Nopco under the trade name "NOPTAMU 300EZ."

[0040] According to another embodiment of the present invention, the defoaming agent of the present invention is not particularly limited as long as it has a defoaming effect and / or a foam-suppressing effect on a fermentation broth used for cell growth and / or fermentation, but is preferably a polyether-type defoaming agent, and more preferably a polyoxyalkylene ether-type defoaming agent.

[0041] According to one embodiment of the present invention, the specific gravity of the defoaming agent of the present invention at 25°C (the ratio of the density of the defoaming agent to the density of water) is 0.90 to 1.05, and if it is smaller than the specific gravity of the fermentation liquor (approximately 1.00 to 1.016), the defoaming agent is likely to be present at the liquid surface, and therefore, from the viewpoint of being highly effective against bubbles on the liquid surface, the specific gravity is preferably 0.93 to 1.01, more preferably 0.95 to 1.00, and even more preferably 0.95 to 0.99. The specific gravity of the defoaming agent at 25°C can be measured by methods known in the art, such as the hydrometer method, pycnometer method, oscillating density meter method, balance method, etc., as described in JIS K0061:2001 "Methods for measuring density and specific gravity of chemical products," and the specific gravity of the fermentation liquor can be measured by the hydrometer method.

[0042] According to one embodiment of the present invention, the cloud point of the defoaming agent of the present invention may be, for example, 25°C or lower when a 10% by mass aqueous solution of the defoaming agent is prepared. From the viewpoint that the defoaming function is significantly reduced when the temperature is below the cloud point, the cloud point is preferably 15°C or lower, more preferably 5°C or lower. The lower limit of the cloud point is not particularly limited, but a value below the measurement limit or above 0°C is preferred. The cloud point of the defoaming agent can be measured by a method known in the art. A known method for measuring the cloud point involves, for example, preparing an aqueous solution containing 1 or 10% by mass of the defoaming agent in ion-exchanged water, or an aqueous solution containing 10% by mass of the defoaming agent in a 25% by mass aqueous solution of diethylene glycol monobutyl ether, heating the aqueous solution, and visually measuring the temperature at which the aqueous solution begins to become cloudy. For example, if the temperature at which clouding begins to occur is below 20°C, the measurement result using ion-exchanged water (10% by mass aqueous solution) is used as the cloud point; if the temperature is between 20 and 90°C, the measurement result using a 25% by mass aqueous solution of diethylene glycol monobutyl ether or ion-exchanged water (1% by mass aqueous solution) is used as the cloud point. Regarding the measurement results for ion-exchanged water, since the water freezes at temperatures below 0°C and the cloud point cannot be measured (measurement limit), the cloud point when the water is cloudy at 0°C is considered to be below 0°C (below the measurement limit).

[0043] According to one embodiment of the present invention, the viscosity (mPa s) of the defoaming agent of the present invention at 25°C can be 100 to 1,000, and from the viewpoint of workability, it is preferably 150 to 700, and more preferably 200 to 300. The viscosity of the defoaming agent is measured according to a method known in the technical field, for example, JIS K5101-6-2:2004.

[0044] According to one embodiment of the present invention, the solubility (g / 100 mL) in water of the defoaming agent of the present invention at 25°C is either insoluble or almost insoluble in water, and includes substances that fall under the categories of "slightly soluble," "very slightly soluble," and "almost insoluble" described in the 18th Edition of the Japanese Pharmacopoeia. The defoaming agent of the present invention has a solubility in water of, for example, about 5 g / 100 mL or less at 25°C. From the viewpoint that the defoaming effect is better when the defoaming agent is dispersed, the solubility is preferably 1 g / 100 mL or less, more preferably 0.1 g / 100 mL or less, and even more preferably 0.01 g / 100 mL or less. The lower limit of the solubility in water is not particularly limited, but 0.001 g / 100 mL or more is preferred.

[0045] According to one embodiment of the present invention, the pH of the fermentation broth in the reaction tank in the parallel fermentation step is not particularly limited, but is preferably maintained in the range of 3 to 10, and more preferably in the range of 4 to 8. According to another embodiment of the present invention, the pH of the fermentation broth in the reaction tank in the saccharification and fermentation step of the semi-parallel simultaneous fermentation step or the fermentation step of the sequential saccharification and fermentation step is the same as described above.

[0046] According to one embodiment of the present invention, the temperature of the fermentation liquor in the reaction tank in the multiple parallel fermentation process is not particularly limited as long as it is within the optimal temperature range for the saccharifying enzyme and / or yeast, but is preferably 20 to 40°C, and more preferably 30 to 40°C. According to another embodiment of the present invention, the temperature of the fermentation liquor in the reaction tank in the saccharification and fermentation process in the semi-parallel multiple fermentation process or the fermentation process in the sequential saccharification and fermentation process is the same as described above. According to another embodiment of the present invention, the temperature of the saccharified liquor in the reaction tank in the saccharification process in the semi-parallel multiple fermentation process or the saccharification process in the sequential saccharification and fermentation process is not particularly limited as long as it is within the optimal temperature range for the saccharifying enzyme, but is preferably 30 to 70°C, and more preferably 40 to 60°C.

[0047] According to one embodiment of the present invention, the number of bacteria in the reaction tank in the parallel multiple fermentation process is not particularly limited, but is preferably 10 ７ cells / mL or more is preferred, ８ The preferred range of the number of cells is 10 ７ ~10 ９ cells / mL, and a more preferred range of bacterial cell count is 10 ８ ~10 ９ According to another embodiment of the present invention, the number of bacterial cells in the reaction tank in the saccharification and fermentation step of the semi-parallel saccharification and fermentation process or the fermentation step of the sequential saccharification and fermentation process is the same as above.

[0048] According to another embodiment of the present invention, the number of bacteria to be inoculated into the reaction tank in the parallel multiple fermentation process is not particularly limited, but the final concentration is preferably 10 ６ cells / mL or more is preferred, ７ The preferred range of inoculation number is 10 ６ ~10 ８ cells / mL, and a more preferred range of inoculation number is 10 ７ ~10 ８ According to another embodiment of the present invention, the number of bacteria inoculated into the reaction tank in the saccharification and fermentation step of the semi-parallel saccharification and fermentation process or the fermentation step of the sequential saccharification and fermentation process is the same as above.

[0049] According to one embodiment of the invention, the ratio of the initial content of antifoaming agent in the fermentation broth in the reaction tank to the initial volume of the fermentation broth (volume on day 1) (initial content of antifoaming agent:initial content of fermentation broth) is not particularly limited, but may be, for example, 1:500 to 1:40,000, preferably 1:850 to 1:20,000, and more preferably 1:1,000 to 1:15,000. According to another embodiment of the invention, the ratio of the initial content of antifoaming agent in the fermentation broth in the reaction tank to the initial volume of the fermentation broth in the saccharification and fermentation step of the semi-parallel multiple fermentation step or the fermentation step of the sequential saccharification and fermentation step is the same as above. According to yet another embodiment of the present invention, the ratio of the initial content of antifoaming agent in the saccharified solution to the initial amount of saccharified solution (amount on day 1) in the reaction tank in the saccharification step of a semi-parallel simultaneous fermentation process or a sequential saccharification and fermentation process (initial content of antifoaming agent:initial content of saccharified solution) is not particularly limited, but may be, for example, 1:200 to 1:500,000, preferably 1:500 to 1:400,000, and more preferably 1:1,000 to 1:200,000. Here, a preferred saccharified solution in this embodiment is a mixed solution containing lignocellulosic biomass and a saccharifying enzyme before the saccharification reaction.

[0050] According to one embodiment of the present invention, the ratio of the initial content of antifoaming agent to the initial content of lignocellulosic biomass in the fermentation liquor in the reaction tank in the parallel multiple fermentation process (initial content of antifoaming agent:initial content of lignocellulosic biomass) is not particularly limited, and may be, for example, 1:50 to 1:2,000, preferably 1:75 to 1:1,000, and more preferably 1:100 to 1:800. According to another embodiment of the present invention, the ratio of the initial content of antifoaming agent to the initial content of lignocellulosic biomass in the fermentation liquor in the reaction tank in the saccharification and fermentation step of the semi-parallel multiple fermentation process or the fermentation step of the sequential saccharification and fermentation process is the same as above. According to another embodiment of the present invention, the ratio of the initial content of antifoaming agent to the initial content of lignocellulosic biomass (initial content of antifoaming agent:initial content of lignocellulosic biomass) in the saccharification solution in the reaction tank in the saccharification step of the semi-parallel simultaneous fermentation process or the saccharification step of the sequential saccharification and fermentation process is not particularly limited, and may be, for example, 1:50 to 1:40,000, preferably 1:75 to 1:20,000, and more preferably 1:100 to 1:10,000.

[0051] According to one embodiment of the invention, the amount of antifoaming agent added per time can be appropriately determined by a person skilled in the art, but for example, in a multiple parallel fermentation process, the mass ratio of the amount of antifoaming agent added per time to the amount of fermentation broth added (antifoaming agent:fermentation broth) is not particularly limited, and may be, for example, 1:200 to 1:50,000, preferably 1:500 to 1:40,000, and more preferably 1:1,000 to 1:20,000. According to another embodiment of the present invention, the mass ratio of the amount of antifoaming agent added per time to the amount of fermentation broth added in the saccharification and fermentation process of the semi-parallel multiple fermentation process or the fermentation process of the sequential saccharification and fermentation process is the same as above. According to another embodiment of the present invention, the mass ratio of the amount of antifoaming agent added each time to the amount of saccharified solution added (antifoaming agent:saccharified solution) in the saccharification step of the semi-parallel multiple fermentation process or the saccharification step of the sequential saccharification and fermentation process is not particularly limited, and may be, for example, 1:200 to 1:500,000, preferably 1:500 to 1:400,000, and more preferably 1:1,000 to 1:200,000.

[0052] According to one embodiment of the present invention, the timing of adding the antifoaming agent is not particularly limited, but may include, for example, when preparing the fermentation broth, when foaming occurs, or when adding lignocellulosic biomass. Here, "when preparing the fermentation broth" includes adding the antifoaming agent to a fermentation broth containing lignocellulosic biomass, saccharification enzymes, and yeast, or adding the antifoaming agent to a fermentation broth containing a saccharification broth and yeast. Note that "adding the antifoaming agent to a fermentation broth containing a saccharification broth and yeast" includes not only adding the antifoaming agent to a fermentation broth containing a saccharification broth and yeast, but also adding the antifoaming agent to the saccharification broth and then mixing it with yeast to form a fermentation broth. Furthermore, "when foaming occurs" includes adding the antifoaming agent when foam reaches a certain height. The foam height can be appropriately determined by those skilled in the art. According to another embodiment of the present invention, the antifoaming agent may be added continuously or intermittently during the culture period, with intermittent addition being preferred. According to another embodiment of the present invention, in a semi-parallel multiple fermentation process or a sequential saccharification and fermentation process, the defoaming agent may be added to a saccharified solution in the saccharification step of the semi-parallel multiple fermentation process or the saccharification step of the sequential saccharification and fermentation process. Here, use of the defoaming agent of the present invention is advantageous in improving the defoaming effect of the saccharified solution.

[0053] According to one embodiment of the present invention, the multiple parallel fermentation process is preferably a continuous process, but may also be a semi-batch or batch process. Here, the continuous process refers to, for example, a process in which raw material supply and ethanol production are continuous. According to another embodiment of the present invention, the semi-parallel multiple fermentation process or the sequential saccharification and fermentation process may be the same as described above.

[0054] In the parallel combined fermentation process, it is preferable to adjust the concentration of lignocellulosic biomass in the fermentation liquor so that it is maintained within a predetermined range. Examples of the predetermined range include 5 to 30% by mass, preferably 10 to 20% by mass. This adjustment is performed, for example, by supplying lignocellulosic biomass to the reaction vessel. Here, the supply rate of lignocellulosic biomass to the reaction vessel is not particularly limited. However, when the parallel combined fermentation process is continuous and the fermentation liquor is discharged from the reaction vessel, it is preferable that the supply rate of lignocellulosic biomass to the reaction vessel and the discharge rate of the fermentation liquor from the reaction vessel are approximately the same. Furthermore, as described below, when the solid-concentrated fermentation liquor after separation of the aqueous ethanol solution is recovered and transferred to the reaction vessel in the solid-liquid separation process, it is preferable that the combined supply rate of the solid-concentrated fermentation liquor and lignocellulosic biomass to the reaction vessel and the discharge rate of the fermentation liquor from the reaction vessel are approximately the same. According to another embodiment of the present invention, the concentration of lignocellulosic biomass in the saccharification and fermentation step of the semi-parallel co-fermentation process or the fermentation step of the sequential saccharification and fermentation process is the same as described above.

[0055] According to one embodiment of the present invention, in the parallel fermentation process, it is preferable to adjust the concentration of the saccharifying enzyme in the fermentation liquor so that it is maintained within a predetermined range. The predetermined range is 50 to 500 U / L, preferably 100 to 300 U / L. The adjustment is carried out, for example, by supplying the saccharifying enzyme to the reaction vessel. Furthermore, in the solid-liquid separation process, the solids-enriched fermentation liquor may be recovered and transferred to the reaction vessel, thereby supplying the recovered saccharifying enzyme. According to another embodiment of the present invention, the concentration of the saccharifying enzyme in the saccharification and fermentation process of the semi-parallel fermentation process or the saccharification process of the sequential saccharification and fermentation process is the same as described above.

[0056] <Solid-liquid separation step> The fermentation liquor discharged from the reaction tank can be separated into a solids-enriched fermentation liquor and an aqueous solution, for example, a solids-enriched fermentation liquor and an aqueous ethanol solution, using a separation device such as a vacuum distillation device or a membrane separator in the solid-liquid separation step. Examples of vacuum distillation devices that can be used include a rotary evaporator and a flash evaporator. Under reduced pressure, ethanol can be separated at a low temperature, preventing enzyme deactivation. The resulting aqueous ethanol solution can be concentrated in the ethanol concentration step described below.

[0057] The solid-concentrated fermentation liquid obtained after separation of the aqueous ethanol solution may be recovered and transferred directly to a reaction tank, or may be separated into a residue and a supernatant using a solid removal device as described below.

[0058] <Ethanol Concentration Step> According to one embodiment of the present invention, the aqueous ethanol solution obtained in the solid-liquid separation step can be further concentrated by distillation or using various separation membranes, for example, zeolite membranes, to produce high-purity ethanol. In addition, the aqueous ethanol solution may be transferred to a reaction tank after removing the ethanol.

[0059] <Solid Removal Step> The solids-enriched fermentation liquid obtained after separation of the aqueous ethanol solution in the solid-liquid separation step may be transferred to a solid removal device to separate it into a residue and a supernatant. The resulting supernatant may be transferred to a reaction tank. Examples of solid removal devices that can be used include a disk centrifuge, a screw press, a screen, a filter press, a belt press, and a rotary press. The separated residue contains enzymes, lignin, and yeast. The enzymes adsorbed to the residue can be liberated, recovered, and reused. Lignin can be recovered as a combustion material and used as energy, or the lignin can be recovered and effectively utilized. The yeast can also be separated from the residue and reused in a parallel multiple fermentation process, a semi-parallel multiple fermentation process, or a sequential saccharification and fermentation process.

[0060] According to the ethanol production method of the present invention, ethanol can be efficiently produced from lignocellulosic raw materials. The ethanol obtained by the ethanol production method can be suitably used, for example, as ethanol for fuel, industrial use, or food additive.

[0061] According to another aspect of the present invention, there is provided a defoaming agent for use in a method for producing ethanol from lignocellulosic biomass selected from the group consisting of recycled paper pulp and unbleached pulp, the defoaming agent comprising a nonionic surfactant.

[0062] Any of the above-mentioned embodiments of the antifoaming agent can be produced in accordance with the description of the production method of the present invention.

[0063] The present invention will be specifically explained below using test examples, but the present invention is not limited to these test examples. Furthermore, unless otherwise specified, the units and measurement methods described in this specification are in accordance with Japanese Industrial Standards (JIS).

[0064] Method for measuring cell count The cell count of the yeast Saccharomyces cerevisiae used in the test examples was observed and measured under an optical microscope (magnification 400x) using a Thoma hemocytometer.

[0065] Method for measuring ethanol concentration The ethanol concentration in the fermentation broth was measured by heating the fermentation broth at 95°C for 10 minutes to inactivate the yeast and enzymes, and then analyzing and measuring the ethanol concentration in the fermentation broth using a refractive index (RI) detector on an HPLC (Prominence, manufactured by Shimadzu Corporation). The HPLC measurement conditions were as follows: Column: Shodex SUGAR SP0810 (manufactured by Showa Denko K.K.) Mobile phase: ultrapure water Flow rate: 0.8 mL / min Temperature: 80°C

[0066] Test Example 1: Preparation of Paper Cup-Derived (Waste Paper-Derived) Pulp Paper cup-derived pulp was prepared by a general preparation method. Specifically, the method was as follows. First, commercially available waterproofed paper cups were cut into pieces 8 mm wide or less using a cutter. The cut paper cups were placed in a Waring blender so that the weight was 4% by mass (equivalent to 40 g on a dry weight basis), and water was added to make the total weight 1000 g. After adjusting to 1000 g, the paper cups were disintegrated by intermittent rotation at a rotation speed of 19,000 rpm for 30 minutes or more. The paper cup disintegration liquid was placed in a laundry net, and the net was washed with at least four times the amount of water and dehydrated twice to obtain paper cup-derived pulp with a moisture content of approximately 70% by mass.

[0067] Study of the type of antifoaming agent The foam-inhibiting and antifoaming effects on foaming during ethanol production using pulp derived from paper cups (recycled paper) were confirmed. First, 5% by mass (equivalent to 30 g on a dry weight basis) of the paper cup-derived pulp obtained above, 79 U of saccharifying enzyme, 80 ppm of antibacterial agent, and 60 ppm of antibiotic were added to a 1 L culture tank, and the liquid volume was adjusted with sterilized water to a reaction weight of approximately 500 g. After adjustment, the temperature was controlled to be constant at 33 °C, a stirring speed of 370 rpm, and pH 4.8, and the reaction was carried out for 24 hours. After 24 hours, an additional 5% by mass (equivalent to 30 g on a dry weight basis) of paper cup-derived pulp was added, and the reaction was carried out for 2 hours at 33 °C, a stirring speed of 370 rpm, and pH 4.8. 10 mL of the reaction solution (saccharified solution) obtained by the above reaction was dispensed into 50 mL conical tubes. In each test group, the antifoaming agent shown in Table 1 was added to the conical tube containing the reaction solution at concentrations of 5, 10, 20, 50, or 100 ppm. The conical tube containing the reaction solution and antifoaming agent was vigorously shaken. Immediately after foaming, the conical tube was left to stand, and the state of the foam was observed after 10 seconds.

[0068] Table 2 shows the minimum concentration of the antifoaming agent at which the foam disappeared within 10 seconds after standing for various antifoaming agents.

[0069] Test plots 1-7 (ADEKA NOL LG-294) and 1-8 (ADEKA NOL LG-299) showed high defoaming effects.

[0070] Test Example 2: Confirmation of antifoaming agent effect in continuous saccharification and fermentation Based on the results obtained in Test Example 1, the antifoaming effects of silicone antifoam agent KM-70 (Test Area 2-1) and ADEKA NOL LG-294 (Test Area 2-2) in continuous saccharification and fermentation were compared. Note that in Test Example 1, ADEKA NOL LG-294 had a shorter defoaming time than ADEKA NOL LG-299, and therefore LG-294 was used in Test Example 2.

[0071] A 1-L culture vessel was charged with 5% by mass of paper cup-derived pulp (equivalent to 30 g on a dry weight basis), 79 U of saccharifying enzyme, 0.22% by mass of urea, 1% by mass of CSL (corn steep liquor), and 10 ppm of antibacterial agent, and the yeast Saccharomyces cerevisiae was cultured at a final concentration of 1.0 × 10 ７ The cells were inoculated at a concentration of 1000 cells / mL. The liquid volume was adjusted with sterilized water to a reaction weight of approximately 500 g. 100 ppm of antifoaming agent was added to each of the prepared fermentation broths, and the temperature was controlled to a constant 33°C, stirring speed of 370 rpm, and pH 4.8, for 24 hours. After 24 hours of reaction, 5% by mass (equivalent to 30 g on a dry weight basis) of pulp derived from paper cups was added to bring the total volume to 600 g (fermentation broth surface height: 11 cm), and the reaction was continued for another 24 hours. After the reaction, approximately half of the fermentation broth was removed from the culture tank, and the fermentation broth was centrifuged. 300 g of the supernatant of the centrifuged fermentation broth was completely evaporated to remove ethanol, and the evaporator fraction and centrifugal residue were returned to the fermentation broth, which was then returned to the culture tank. An additional 5% by mass (equivalent to 30 g on a dry weight basis) of pulp derived from paper cups was added, and the reaction was resumed. This procedure was repeated every 24 hours, and the culture was continued for 10 days. During the culture period, when foaming reached a height of 5 cm above the fermentation liquid surface, an antifoaming agent was added, and the amounts added were compared.

[0072] The conditions for preparing the fermentation liquid are shown below.

[0073] The composition of the fermentation liquid is shown below.

[0074] Table 5 shows the total amount of antifoaming agent added over 10 days of culture in Test Example 2. In Test Plot 2-1, 100 ppm of antifoaming agent was added 168, 192, and 216 hours after the start of culture. In Test Plot 2-2, 100 ppm of antifoaming agent was added 168 hours after the start of culture.

[0075] It was confirmed that the amount of antifoaming agent used in Test Plot 2-2 (ADEKA NOL LG-294) was less than half that used in Test Plot 2-1 (KM-70). Here, even when the antifoaming agent was added to Test Plot 2-1, cultivation was possible, but the foam did not completely disappear.

[0076] The ethanol concentrations in the fermentation broth in experimental plots 2-1 and 2-2 are shown in Figure 1. The ethanol concentrations in the fermentation broth in experimental plot 2-1 were 47.6 g / L at 48 hours, 46.0 g / L at 96 hours, 44.3 g / L at 144 hours, and 42.0 g / L at 192 hours. The ethanol concentrations in the fermentation broth in experimental plot 2-2 were 47.5 g / L at 48 hours, 45.5 g / L at 96 hours, 44.1 g / L at 144 hours, and 42.0 g / L at 192 hours. The ethanol concentrations in the fermentation broth were comparable between experimental plots 2-1 and 2-2.

[0077] In experimental plot 2-3, cultivation was carried out under the same conditions as experimental plot 2-1, except that bleached hardwood kraft pulp (LBKP) was used as the lignocellulosic biomass. As a result, the total amount of antifoaming agent (KM-70) added over the 10-day cultivation period was only 100 ppm, which was the amount at the start of cultivation.

Claims

1. A method for producing ethanol from lignocellulosic biomass, comprising: a multiple parallel fermentation process, a semi-parallel multiple fermentation process, or a sequential fermentation process; when the multiple parallel fermentation process is included, the multiple parallel fermentation process comprises a step of adding an antifoaming agent to a fermentation liquor containing lignocellulosic biomass, a saccharifying enzyme, and yeast; when the semi-parallel multiple fermentation process or the sequential fermentation process is included, the semi-parallel multiple fermentation process or the sequential fermentation process comprises a step of adding an antifoaming agent to a saccharification liquor containing lignocellulosic biomass and a saccharifying enzyme, and / or a fermentation liquor containing the saccharification liquor and yeast, wherein the antifoaming agent comprises a nonionic surfactant.

2. The method for producing ethanol according to claim 1, wherein the lignocellulosic biomass is at least one selected from recycled paper pulp and unbleached pulp.

3. The method for producing ethanol according to claim 1 or 2, wherein the waste paper is selected from the group consisting of paper cups, paper containers, and miscellaneous paper.

4. The method for producing ethanol according to claim 1 or 2, wherein the antifoaming agent comprises a polyoxyalkylene ether type nonionic surfactant.

5. The method for producing ethanol according to claim 1 or 2, wherein the specific gravity of the antifoaming agent at 25°C is 0.95 to 1.

00.

6. The method for producing ethanol according to claim 1 or 2, wherein the cloud point of the antifoaming agent is 10°C or lower in a 10% by mass aqueous solution of the antifoaming agent.

7. The method for producing ethanol according to claim 1 or 2, wherein the mass ratio of the total amount of antifoaming agent to the total amount of lignocellulosic biomass (total amount of antifoaming agent:total amount of lignocellulosic biomass) throughout the entire culture is 1:100 to 1:10,000.

8. The method for producing ethanol according to claim 1, wherein the multiple parallel fermentation process, the multiple semi-parallel fermentation process, or the sequential saccharification and fermentation process is carried out continuously.

9. A defoaming agent for use in a method for producing ethanol from at least one lignocellulosic biomass selected from recycled paper pulp and unbleached pulp, the defoaming agent comprising a nonionic surfactant.

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