Method for producing ethanol using thermotolerant yeast

Abstract

To provide a method for efficiently producing ethanol by using yeast.SOLUTION: According to the method for producing ethanol including simultaneously performing a step of performing multiple parallel fermentation of a substrate using a saccharifying enzyme and a thermostable yeast under a condition of 37 °C to 47 °C and a step of distilling ethanol produced by the multiple parallel fermentation, ethanol can be efficiently produced.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing ethanol using a thermotolerant yeast. [Background technology]

[0002] In recent years, growing environmental awareness has led to an increased focus on biofuels obtained from biomass. Bioethanol is one such type of biofuel, produced by fermenting biomass feedstocks using yeast.

[0003] However, the bioethanol production process is a harsh environment for yeast. For example, the heat generated during fermentation causes the temperature in the fermenter to rise, causing high-temperature stress for the yeast. Therefore, the use of thermotolerant yeast that can withstand high temperatures in ethanol production is being investigated. High-temperature fermentation using thermotolerant yeast offers advantages such as reduced cooling costs during fermentation and savings in cooling water, and is expected to be one of the next-generation fermentation technologies that will lead to reduced running costs.

[0004] However, yeast is exposed to various stresses other than high temperature during fermentation, and in particular, the fermentation product ethanol and by-product carbon dioxide are thought to inhibit fermentation production. Additionally, high temperatures increase membrane fluidity, which causes electrons to leak from the respiratory chain electron transport system in mitochondria, which then transfer to oxygen, generating reactive oxygen species (ROS). ROS are thought to damage macromolecules such as DNA, lipids, and proteins, thereby inhibiting fermentation production.

[0005] As an example of a fermentation technique using thermotolerant yeast, Non-Patent Document 1 describes a method in which rice is hydrolyzed with an enzyme, followed by ethanol fermentation using thermotolerant yeast at 40°C, and then the ethanol is distilled under reduced pressure after fermentation is completed (see, for example, Fig. 4 in Non-Patent Document 1). On the other hand, in order to enjoy the benefits of high-temperature fermentation as described above, it is preferable to carry out fermentation under higher temperature conditions. However, the present inventors recognized the problem that poor fermentation occurs when the method described in Non-Patent Document 1 is carried out under higher temperature conditions, around 45°C. This is thought to be due in part to the various stresses that the yeast is subjected to as described above.

[0006] Thus, even in fermentation techniques using thermotolerant yeast, there is a continuing need for methods for efficiently producing ethanol by, for example, reducing stress on the yeast. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Journal of the Japan Institute of Energy ,94, 1154-1162(2015) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for efficiently producing ethanol using yeast. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have found that ethanol can be efficiently produced by simultaneously carrying out a step of parallel multiple fermentation of a substrate using a saccharifying enzyme and a thermotolerant yeast under high-temperature conditions and a step of distilling the ethanol produced thereby. The present invention was completed based on this finding.

[0010] That is, the present invention is as specified by the following items. [1] A method for producing ethanol, comprising simultaneously carrying out the following steps A and B under conditions of 37 to 47°C: (Step A) A step of performing parallel multiple fermentation of the substrate using saccharifying enzymes and thermotolerant yeast (Step B) A step of distilling the ethanol produced in Step A [2] The method according to [1] above, characterized in that (i) step A is carried out, and then (ii) step A and step B are carried out simultaneously. [3] The method according to [1] or [2] above, characterized in that (i) step A is carried out, and after the ethanol concentration in the fermentation liquid in which parallel fermentation is carried out reaches 0.06% (w / v) or more, (ii) step A and step B are carried out simultaneously. [4] The method according to any one of [1] to [3] above, characterized in that (i) step A is carried out under conditions of 37 to 42°C, and then (ii) step A and step B are carried out simultaneously under conditions of 43 to 47°C. [5] The method according to [1] above, characterized in that step A and step B are initiated simultaneously. [6] The method according to any one of [1] to [5] above, wherein the thermotolerant yeast is Kluyveromyces marxianus. [7] The method according to any one of the above [1] to [6], wherein the distillation is reduced pressure distillation. [Effects of the Invention]

[0011] According to the present invention, ethanol can be efficiently produced using yeast. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a diagram showing a schematic diagram of an ethanol production apparatus for carrying out the present invention. [Figure 2] FIG. 1A is a diagram showing an outline of the time series in Example 1, Example 2, and Comparative Example 3. FIG. 1B is a diagram showing an outline of the time series in Example 4, Example 5, and Example 6. [Figure 3] FIG. 1 is a graph showing the change over time in the number of viable bacteria in the fermentation broth in Examples 1 and 2 and Comparative Example 3. [Figure 4] FIG. 1 is a graph showing the change in glucose concentration in the fermentation broth over time in Examples 1 and 2 and Comparative Example 3. [Figure 5] FIG. 1 is a graph showing the change over time in the acetic acid concentration in the fermentation broth in Examples 1 and 2 and Comparative Example 3. [Figure 6] FIG. 1 is a graph showing the change over time in the ethanol concentration in the fermentation broth in Examples 1 and 2 and Comparative Example 3. [Figure 7] FIG. 1 shows the change over time in the number of viable bacteria in the fermentation broth in Examples 4, 5, and 6. [Figure 8] FIG. 1 shows the change in glucose concentration in the fermentation broth over time in Examples 4, 5, and 6. [Figure 9] FIG. 1 shows the change in acetic acid concentration in the fermentation broth over time in Examples 4, 5, and 6. [Figure 10] FIG. 1 shows the change in ethanol concentration in the fermentation broth over time in Examples 4, 5, and 6. DETAILED DESCRIPTION OF THE INVENTION

[0013] (Method of the present invention) The method of the present invention is an ethanol production method characterized by comprising simultaneously carrying out the following steps A and B under conditions of 37 to 47°C. (Step A) A step of performing parallel multiple fermentation of the substrate using saccharifying enzymes and thermotolerant yeast (Step B) A step of distilling the ethanol produced in Step A

[0014] (Process A: Parallel multiple fermentation) In step A, the substrate undergoes simultaneous parallel saccharification and fermentation (SSF). Simultaneous saccharification and fermentation (SSF) is a type of fermentation in which two processes simultaneously occur in the same vessel: saccharification (hydrolysis), in which the substrate is converted into sugars by saccharifying enzymes, and fermentation, in which the sugars are converted into ethanol by the action of yeast (i.e., ethanol fermentation), in which multiple processes simultaneously occur in the same vessel to ultimately obtain ethanol from a substrate such as starch.

[0015] (Saccharification enzymes) The saccharifying enzyme used in step A is not particularly limited, as long as it can saccharify the substrate into sugars that can be used for ethanol fermentation by thermotolerant yeast under conditions of 37 to 47°C. For example, when the substrate is starch-based biomass, the saccharifying enzyme may be one or more selected from the group consisting of α-amylase, β-amylase, glucoamylase, pullulanase, etc., with α-amylase and glucoamylase being preferred. Commercially available saccharifying enzymes may be used, such as Uniase S (manufactured by Yakult Pharmaceutical Co., Ltd.) and Gluc SBG (manufactured by Amano Enzyme Inc.), with Uniase S being preferred. Furthermore, koji such as rice koji may also be used as the saccharifying enzyme. Furthermore, when the substrate is cellulosic biomass, cellobiohydrolase, endoglucanase, β-glucosidase, etc. may be used. For example, when starch such as that from rice is used as a substrate as described below, dextrin is produced from the starch by the action of α-amylase (a liquefying enzyme) (liquefaction), and glucose is further produced from the dextrin by the action of glucoamylase (a saccharifying enzyme) (saccharification). Therefore, the saccharifying enzyme used in step A may be a liquefying / saccharifying enzyme. The amount of saccharifying enzymes used is not particularly limited as long as it is a necessary and sufficient amount, but for example, 0.05 to 0.1% (w / v) of the enzymes can be added to a fermentation broth containing 15% (w / v) rice. For example, 150 to 300 U of α-amylase and glucoamylase can be added per gram of rice.

[0016] (heat-resistant yeast) The thermotolerant yeast used in step A is not particularly limited as long as it has the ability to perform ethanol fermentation at 37 to 47° C. Examples of thermotolerant yeast include Kluyveromyces marxianus, Pichia kudriavzevii, Candida tropicalis, and Ogataea polymorpha, with Kluyveromyces marxianus being preferred. In particular, preferred examples of the Kluyveromyces marxianus strain DMKU3-1042 (accession number: NITE BP-283), Kluyveromyces marxianus strain DMKU3-118 (accession number: NITE BP-289), Kluyveromyces marxianus strain DMKU3-p106 (accession number: NITE BP-290), Kluyveromyces marxianus strain DMKU3-p1042 (accession number: NITE BP-291), which are deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (NITE) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), and derivative strains thereof are also preferred. Yeasts such as Saccharomyces cerevisiae are commonly used for ethanol fermentation from sugars, but the optimum temperature for growth and fermentation is around 30°C, making them unsuitable for use under high-temperature conditions. However, the present invention uses a thermotolerant yeast, enabling ethanol production even under high-temperature conditions that are difficult for Saccharomyces cerevisiae to use. The amount of the thermotolerant yeast used is not particularly limited. For example, a pre-culture solution of the thermotolerant yeast is added to the fermentation liquid at 0.1 to 1% (v / v), and the amount of the thermotolerant yeast in the fermentation liquid after the addition of the thermotolerant yeast is 1 × 10 6 ~1×10 7 Examples include expressing the number of CFU / mL.

[0017] (substrate) The substrate used in step A is not particularly limited, and may be any material that can be used as a raw material for parallel multiple fermentation. Examples include starch and cellulose, with starch being preferred due to its high saccharification and fermentation efficiency. For example, when starch is used as a substrate, saccharification takes place, in which glucose is produced from starch by the action of liquefaction and saccharification enzymes such as α-amylase or β-amylase and glucoamylase, and ethanol fermentation takes place, in which two molecules of ethanol and two molecules of carbon dioxide are produced from one molecule of glucose by the action of yeast. Furthermore, as the substrate, in addition to starch, cellulose, etc. themselves, it is also possible to use, for example, biomass containing starch, cellulose, etc. The biomass is not particularly limited, and examples thereof include food resources (blackstrap molasses, food processing residues, animal and plant residues, food waste, etc.), industrial resources (paper, pulp waste, etc.), forestry resources (forestry residues, etc.), agricultural resources (rice straw, rice husks, wheat straw, etc.), and resource crops (rice, sugarcane, sugar beet, potatoes, corn, etc.). These substrates can be used by adding them to an appropriate medium. The amount of the substrate to be added is not particularly limited, but examples include 5 to 25% (w / v) or 10 to 20% (w / v) in the medium.

[0018] (Culture medium) The medium used in step A is not particularly limited, as long as it allows the thermotolerant yeast used to grow and perform ethanol fermentation. Examples of media include those containing, in addition to the substrate, water, a nitrogen source, trace amounts of inorganic substances and minerals, etc. Specific examples include YP medium, YM medium, YNB medium, etc., with YP medium being preferred. Examples of YP medium include those containing about 1% (w / v) yeast extract and about 2% (w / v) peptone. The medium may be commercially available or not. Such a medium can be used as a fermentation liquid by adding (inoculating) the above-mentioned substrate, saccharifying enzyme (which may be a liquefying / saccharifying enzyme), and thermotolerant yeast.

[0019] (Time to perform process A) In the method of the present invention, the time for performing step A is not particularly limited, but examples of the lower limit include 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, and 20 hours or more, and examples of the upper limit include 40 hours or less, 36 hours or less, 32 hours or less, 30 hours or less, 28 hours or less, 26 hours or less, 24 hours or less, 22 hours or less, and 20 hours or less, and these lower and upper limits can be arbitrarily combined, and examples thereof include preferably 12 to 28 hours, more preferably 16 to 24 hours, and even more preferably 18 to 22 hours. These times are the total of the time spent performing step A alone and the time spent performing step A together with step B and / or other steps simultaneously. The initiation of step A can be, for example, by adding (inoculating) a saccharifying enzyme (which may be a liquefaction / saccharifying enzyme) and a thermotolerant yeast to an appropriate medium containing a substrate, followed by incubation under appropriate conditions. The end of step A can be, for example, when, after fermentation has progressed, the substrate in the fermentation broth is depleted, or fermentation is inhibited by various stresses, causing ethanol to (substantially) cease to be produced, resulting in the (substantially) end of step A. Step A can also be (substantially) ended by intentionally inducing such events.

[0020] (Step B: Distillation) In step B, the ethanol produced in step A is distilled. The distillation method is not particularly limited as long as it allows ethanol to be distilled, and examples include vacuum distillation, atmospheric distillation, and steam distillation. However, from the viewpoint of avoiding yeast death or activity reduction, vacuum distillation is preferred at least while the parallel multiple fermentation in step A and the distillation in step B are carried out simultaneously. During vacuum distillation, the pressure in the experimental apparatus can be reduced to, for example, ½ atmosphere or less, ⅓ atmosphere or less, or ¼ atmosphere or less, and preferably to ⅓ to ⅕ atmosphere. The method of reducing the pressure is not particularly limited, and examples include operating a vacuum pump directly or indirectly connected to the saccharification, fermentation, and distillation tank.

[0021] (Time to perform process B) In the method of the present invention, the time for performing step B is not particularly limited, and examples of the lower limit include 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, 20 hours or more, 22 hours or more, 24 hours or more, 26 hours or more, 28 hours or more, 30 hours or more, and 32 hours or more; examples of the upper limit include 40 hours or less, 36 hours or less, 32 hours or less, 30 hours or less, 28 hours or less, 26 hours or less, 24 hours or less, 22 hours or less, and 20 hours or less; and these lower and upper limits can be arbitrarily combined; for example, 12 to 28 hours is preferred, 14 to 26 hours is more preferred, and 16 to 24 hours is more preferred. These times are the total of the time spent performing step B alone and the time spent performing step B together with step A and / or other steps simultaneously.

[0022] (Perform steps A and B simultaneously) The method of the present invention involves simultaneously carrying out step A and step B. The time for simultaneously carrying out step A and step B is not particularly limited, but examples of the lower limit include 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, and 16 hours or more, and examples of the upper limit include 30 hours or less, 28 hours or less, 26 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, and 12 hours or less, and these lower and upper limits can be arbitrarily combined, and examples thereof include 8 to 24 hours, preferably 10 to 22 hours, more preferably 12 to 20 hours, and more preferably 12 to 20 hours.

[0023] According to the method of the present invention, even if ethanol, carbon dioxide, and / or oxygen (substances that cause ROS), which are stressful for yeast, are generated or present in the fermentation liquor during the multiple parallel fermentation in step A, these substances are removed from the fermentation liquor by simultaneously carrying out the distillation in step B. This suppresses fermentation inhibition by these substances, and it is believed that multiple parallel fermentation can be carried out efficiently. Furthermore, even under high temperature conditions of 37 to 47°C, no glucose remains in the fermentation liquor, and a high ethanol recovery rate can be achieved. Furthermore, by simultaneously carrying out the multiple parallel fermentation in step A and the distillation in step B, the time required from the start of the multiple parallel fermentation to the recovery of ethanol can be shortened compared to when these processes are carried out separately.

[0024] (Under conditions of 37-47℃) In the method of the present invention, step A and step B are simultaneously carried out under conditions of 37 to 47° C. There are no particular limitations as long as these temperature conditions are met, but the lower limit includes, in addition to 37° C. or higher, for example, 38° C. or higher, 39° C. or higher, 40° C. or higher, 41° C. or higher, 42° C. or higher, 43° C. or higher, 44° C. or higher, 45° C. or higher, and 46° C. or higher, and the upper limit includes, in addition to 47° C. or lower, for example, 46° C. or lower, 45° C. or lower, and the like. These lower and upper limits can be arbitrarily combined, and for example, 40 to 47° C. is preferred, 43 to 47° C. is more preferred, and 44 to 46° C. is even more preferred. In the present invention, unless otherwise specified, the temperature refers to the liquid temperature of the fermentation liquid in the saccharification, fermentation, and distillation tank, and is expressed as a value rounded to the nearest whole number.

[0025] In the method of the present invention, the parallel multiple fermentation in step A and the distillation in step B are simultaneously carried out under high-temperature conditions of 37 to 47°C, which offers many advantages, including reduced cooling costs and energy, reduced amounts of saccharification enzymes, and suppression of contamination and proliferation of unwanted bacteria. Here, the "reduction of the amount of saccharifying enzymes" is based on the fact that the optimum temperature for saccharifying enzymes is relatively high (for example, about 70°C for α-amylase and about 55°C for glucoamylase). Therefore, by performing multiple parallel fermentation under high-temperature conditions closer to their optimum temperatures, the amount of saccharifying enzymes required can be reduced compared to performing multiple parallel fermentation at lower temperatures. For example, it is estimated that the amount of saccharifying enzymes required can be reduced by about one-quarter when performing multiple parallel fermentation at 45°C compared to when performing it at 40°C. Therefore, performing multiple parallel fermentation under higher temperature conditions is more advantageous from the perspective of reducing the amount of saccharifying enzymes required. Furthermore, with regard to "suppressing the introduction and proliferation of undesirable bacteria," fermentation is generally carried out after removing undesirable bacteria contained in the raw material substrate or adhering to the inside of the fermenter through heat sterilization. However, if fermentation is carried out under higher temperature conditions, the proliferation of undesirable bacteria (especially mesophilic bacteria) can be suppressed, and therefore fermentation can be started quickly without heat sterilization, particularly when using raw material substrates that are originally low in undesirable bacteria (for example, packaged discarded food or food processing residues), thereby saving the energy and costs required for heat sterilization.

[0026] (Combination of processes) As long as the effects of the present invention can be enjoyed, the method of the present invention may or may not include, for example, performing step A or step B separately in addition to simultaneously performing step A and step B, or may include performing a step other than step A or step B in addition to simultaneously performing step A and step B. Furthermore, the order and number of times that these steps are performed are not particularly limited. Furthermore, there are also no particular limitations on whether step A and / or step B and other steps are performed separately or simultaneously.

[0027] The method of the present invention preferably includes, for example, (i) carrying out step A, and then (ii) simultaneously carrying out step A and step B. For example, (i) step A is first started, and then step B is started while step A is continued, thereby (ii) carrying out step A and step B simultaneously. In this way, by not initially performing the distillation in step B, it is possible to maintain a state in which sufficient oxygen is present in the tank, and therefore, when using yeast with a high oxygen requirement, the yeast can grow more rapidly in the initial stage. After the yeast has grown rapidly, step A and step B can be performed simultaneously, allowing for more efficient multiple parallel fermentation.

[0028] The time for performing (i) step A before simultaneously performing (ii) step A and step B is not particularly limited, but examples of the lower limit include 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, and 8 hours or more, and examples of the upper limit include 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, and 8 hours or less, and these lower and upper limits can be arbitrarily combined, and examples thereof include preferably 2 to 10 hours, more preferably 3 to 9 hours, and even more preferably 4 to 8 hours.

[0029] Furthermore, the time for performing (i) step A before (ii) simultaneously performing step A and step B, or the time for performing (i) step A and then (ii) simultaneously performing step A and step B (starting to perform) step A and step B thereafter, may be determined, for example, using the viable cell count of yeast in the fermentation broth undergoing multiple parallel fermentation as an indicator. For example, (i) step A may be performed, and the viable cell count of yeast in the fermentation broth undergoing multiple parallel fermentation has increased to at least a certain multiple compared to the start of step A, and then (ii) step A and step B are performed simultaneously. In this case, examples of at least a certain multiple include at least 2 times, at least 4 times, at least 8 times, at least 16 times, or at least 32 times. In addition, examples of at least a certain number of viable cells may include at least a certain number of viable cells in the fermentation broth undergoing multiple parallel fermentation, and then (ii) step A and step B are performed simultaneously. In this case, examples of at least a certain number of viable cells include at least 2 x 10 7 CFU / mL or more, 3×10 7 CFU / mL or more, 4×10 7 CFU / mL or more, 8×10 7 CFU / mL or more, 16×107 Examples include CFU / mL or more.

[0030] In the method of the present invention, the time for performing (i) step A before (ii) simultaneously performing step A and step B, or the time for performing (i) step A and then (ii) simultaneously performing step A and step B (to start performing) step A and step B, can also be preferably determined using the ethanol concentration in the fermentation broth in which multiple parallel fermentation is performed as an indicator. For example, it is preferable to include performing (i) step A, and then simultaneously performing (ii) step A and step B after the ethanol concentration in the fermentation broth in which multiple parallel fermentation is performed reaches 0.06% (w / v) or more. In this case, the lower limit of the ethanol concentration in the fermentation broth in which multiple parallel fermentation is performed is 0.06% (w / v) or more, and examples thereof include 0.08% (w / v) or more, 0.1% (w / v) or more, 0.5% (w / v) or more, and 1.0% (w / v) or more. The upper limit is not particularly limited, but examples include 2.5% (w / v) or less, 2.0% (w / v) or less, 1.5% (w / v) or less, etc. These lower and upper limits can be combined arbitrarily, and preferred examples include 0.1 to 1.5% (w / v).

[0031] In the method of the present invention, the temperature conditions may be constant throughout all steps, but they do not have to be constant. For example, the temperature may be increased one or more times at any step and / or decreased one or more times at any step. Such changes in temperature conditions may be made for each step or during the steps.

[0032] For example, the method of the present invention preferably includes (i) performing step A, and then (ii) simultaneously performing step A and step B under a temperature condition higher than the temperature at which step A was performed. Furthermore, for example, more preferably includes (i) performing step A under a condition of 37 to 42°C, and then (ii) simultaneously performing step A and step B under a condition of 43 to 47°C, and even more preferably includes (i) performing step A under a condition of 38 to 41°C, and then (ii) simultaneously performing step A and step B under a condition of 44 to 46°C. In this way, by carrying out (i) at a lower temperature and then carrying out (ii) at a higher temperature, (i) when carrying out step A, high temperature stress and oxidative stress due to ROS can be alleviated, allowing the yeast to grow more rapidly, and then (ii) when carrying out steps A and B simultaneously, parallel multiple fermentation and distillation can be carried out more efficiently under more energetically advantageous temperature conditions while alleviating fermentation inhibition due to ethanol, ROS, and / or carbon dioxide, etc.

[0033] In the case where the method of the present invention comprises carrying out (i) step A and then (ii) simultaneously carrying out steps A and B as described above, other steps may or may not be included between (i) and (ii). However, it is preferable that no other steps are included, i.e., (ii) is carried out consecutively after (i).

[0034] In the method of the present invention, step A and step B do not have to be started simultaneously as described above, but step A and step B may be started simultaneously. In this case, the workload involved in starting the steps can be reduced.

[0035] Furthermore, the method of the present invention may further include simultaneously performing step A and step B, and then performing step B. By continuing step B even after step A is completed, a larger amount of ethanol can be distilled from the fermentation liquor. The time for simultaneously performing step A and step B followed by step B is not particularly limited, and examples of the lower limit include 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, 16 hours or more, 18 hours or more, and examples of the upper limit include 20 hours or less, 18 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less ... lower limit and upper limit can be arbitrarily combined, and examples thereof include 1 to 11 hours, preferably 2 to 10 hours, more preferably 4 to 8 hours. When step B is continued after step A is completed, the distillation may be vacuum distillation, atmospheric distillation, steam distillation, etc. However, when the yeast is used as a seed culture for the next fermentation after ethanol recovery as described below, it is preferable to use vacuum distillation until the completion of step B in order to avoid the death or decrease in activity of the yeast.

[0036] (Ethanol recovery) The method of the present invention may further include a step of recovering ethanol. The method for recovering ethanol is not particularly limited, but examples include recovering ethanol in an ethanol recovery tank connected to the saccharification, fermentation, and distillation tank of the ethanol production apparatus via a cooling pipe, and / or a trap tank provided on the exhaust side of a vacuum pump. Ethanol recovery may be carried out once, or may be carried out two or more times. Furthermore, ethanol may be recovered in one ethanol recovery tank or trap tank, or in two or more ethanol recovery tanks and / or trap tanks. In the method of the present invention, the timing of recovering ethanol is not particularly limited, and may be after a certain time from the start of step A. As the certain time, the lower limit may be, for example, 12 hours or more, 14 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 22 hours or more, 24 hours or more, 26 hours or more, 28 hours or more, 30 hours or more, 32 hours or more, 34 hours or more, 36 hours or more, 38 hours or more, etc.; as the upper limit, for example, 40 hours or less, 39 hours or less, 38 hours or less, 37 hours or less, 36 hours or less, 35 hours or less, 34 hours or less, 33 hours or less, 32 hours or less, 30 hours or less, 28 hours or less, 26 hours or less, 24 hours or less, etc.; these lower limit and upper limit values ​​can be arbitrarily combined, and for example, 14 to 38 hours is preferred, 22 to 30 hours is preferred, and 24 to 28 hours is more preferred.

[0037] (Stirring of fermented liquid) The method of the present invention may further include a step of stirring the fermentation liquor. While there are no particular limitations on the timing or duration of stirring the fermentation liquor, it is preferable to stir the fermentation liquor throughout step A and / or step B. The stirring conditions are not particularly limited, but examples include stirring at 50 to 250 rpm (rounds per minute). The stirring conditions may be constant throughout all steps included in the present invention, but do not have to be constant. For example, (i) when performing step A, stirring may be performed at a slightly faster speed of approximately 160 to 200 rpm, thereby allowing oxygen to be introduced into the highly viscous fermentation liquor and promoting yeast growth. Furthermore, for example, (ii) when performing steps A and B simultaneously, stirring may be performed at a slightly slower speed of approximately 80 to 120 rpm, thereby suppressing the generation of bubbles. Stirring can be performed, for example, using a stirrer installed in the saccharification, fermentation, and distillation tank of the ethanol production apparatus.

[0038] (Measurement of viable bacteria count) The method of the present invention may further include a step of measuring the number of viable bacteria in the fermentation broth. More specifically, for example, the method may include sampling the fermentation broth at appropriate times and measuring the colony forming units (CFU) to determine the number of viable bacteria in the fermentation broth. By measuring and monitoring the number of viable bacteria in the fermentation broth, it is possible to confirm the progress of each step and also to use the number of viable bacteria as an indicator for determining the timing and / or duration of each step.

[0039] (Measurement of glucose concentration, acetic acid concentration, and ethanol concentration) The method of the present invention may further include a step of measuring the glucose concentration, acetic acid concentration, and / or ethanol concentration in the fermentation broth. More specifically, the method may include sampling the fermentation broth at appropriate times and measuring the glucose concentration, acetic acid concentration, and ethanol concentration in the fermentation broth by HPLC (high performance liquid chromatography) or the like. By measuring and monitoring the glucose concentration, acetic acid concentration, and / or ethanol concentration in the fermentation broth, it is possible to confirm the progress of each step and also to use the results as an indicator for determining the timing and / or duration of each step.

[0040] (Ethanol production equipment) The method of the present invention can be carried out, for example, using an ethanol production apparatus whose schematic diagram is shown in Figure 1. In Figure 1, the arrows indicate the flow of ethanol, and when the vacuum pump is activated, the ethanol produced in step A travels from the saccharification and fermentation / distillation tank through a cooling pipe and is captured in a primary ethanol recovery tank and a trap tank (not shown).

[0041] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples. [Example]

[0042] 1. Example 1, Example 2, and Comparative Example 3 First, the effects of simultaneously performing multiple parallel fermentation and distillation were compared in Examples 1 and 2 (simultaneous performance of multiple parallel fermentation and distillation) and Comparative Example 3 (not simultaneously performing multiple parallel fermentation and distillation) as described below.

[0043] [Example 1] Simultaneous multiple parallel fermentation and distillation (distillation begins 4 hours after the start of multiple parallel fermentation): YP medium (1% (w / v) of yeast extract, 2% (w / v) of peptone) containing 15% (w / v) steamed rice was supplemented with 0.1% (w / v) of a liquefaction and saccharification enzyme (Uniase S, manufactured by Yakult Pharmaceutical Co., Ltd.) (approximately 200 U each of α-amylase and glucoamylase per gram of rice) and 1% (v / v) (1 × 10) of pre-cultured thermotolerant yeast Kluyveromyces marxianus DMKU3-1042 strain (accession number: NITE BP-283). 7 A fermentation broth was prepared (pH 6.5) by adding ethanol to a concentration of approximately 100 CFU / mL. Five liters of this mixture was added to the saccharification, fermentation, and distillation tank (Mitsuwa Frontech's small microbial culture device, Lab Scale Jar Fermenter, NBC-10000) of the apparatus shown in Figure 1. Initially, the mixture was incubated for 4 hours at 40°C with an agitation speed of 180 rpm. After 4 hours, the pressure in the saccharification, fermentation, and distillation tank was reduced to 1 / 4 atmosphere while the mixture was still in the fermentation tank. The mixture was then incubated for 20 hours at 45°C with an agitation speed of 100 rpm. This allowed for simultaneous fermentation and distillation. After 24 hours, ethanol was recovered from the primary ethanol recovery tank and a trap tank (not shown) on the exhaust side of the vacuum pump. For the purpose of obtaining experimental data, distillation was continued at 45°C for 40 hours after the start of the fermentation. The total amount of ethanol recovered from the primary ethanol recovery tank and trap tank was defined as the total ethanol volume. During operation, the fermentation broth was sampled periodically from the saccharification, fermentation, and distillation tank. The colony-forming units (CFUs) were measured to determine the viable cell count. The glucose, acetic acid, and ethanol concentrations were measured by HPLC (high-performance liquid chromatography). Specifically, measurements were performed at 60 °C using an HPLC system consisting of an L-2130 pump, an L-2490 refractive index detector, an L-2200 autosampler, an L-2350 column oven, and a Hitachi Model D-2000 Elite HPLC System Manager. This system was equipped with a GLC610-S Gelpack column (Hitachi Chemical, Tokyo, Japan). Water distilled by an RFD240NA water distillation unit (Aquarius, Advantec, Japan) was used as the mobile phase, and measurements were performed at a flow rate of 0.3 mL / min. Note that all temperatures listed above refer to the liquid temperature of the fermentation broth in the saccharification, fermentation, and distillation tank.

[0044] [Example 2] Parallel multiple fermentation and distillation are carried out simultaneously (distillation begins 8 hours after the start of parallel multiple fermentation): The conditions were the same as in Example 1 above, except that the incubation was initially carried out under conditions of 180 rpm stirring at 40°C for 8 hours and that ethanol was collected 28 hours after the start of parallel fermentation.

[0045] Comparative Example 3 Distillation is carried out simultaneously after the end of multiple parallel fermentation (distillation begins 20 hours after the start of multiple parallel fermentation): First, the mixture was incubated for 8 hours at an agitation speed of 180 rpm and 40°C, followed by another 12 hours at an agitation speed of 180 rpm and 45°C, thereby carrying out multiple parallel fermentation. After 20 hours from the start of multiple parallel fermentation, when the mixture was essentially complete (as judged by the absence of any decrease in glucose concentration in the fermentation broth, as shown in Table 2 and Figure 4), the pressure in the experimental apparatus was reduced to ¼ atmosphere, and the mixture was incubated for 20 hours at an agitation speed of 100 rpm and 45°C, followed by distillation. Then, 40 hours after the start of multiple parallel fermentation, ethanol was recovered and the distillation was completed. Other than these, the mixture was subjected to the same conditions as in Example 1 above.

[0046] The time series in Example 1, Example 2, and Comparative Example 3 are outlined in FIG. 2(A).

[0047] [result] (a) Number of viable bacteria in the fermentation liquid The results are shown in Table 1 and Figure 3. Example 1 showed a fairly high viable cell count even 20 hours after the start of multiple parallel fermentation. In comparison, Example 2 and Comparative Example 3 showed low viable cell counts, but Example 2 showed a higher viable cell count. A high viable cell count is advantageous when used as seed culture for the next fermentation. In Comparative Example 3, the viable cell count increased significantly 40 hours after the start of multiple parallel fermentation, which is thought to be due to the proliferation of yeast in the fermentation liquid that had become more resistant to stress (a similar phenomenon occurred in two separate experiments (data not shown)).

[0048] [Table 1]

[0049] (b) Glucose concentration in the fermentation liquid The results are shown in Table 2 and Figure 4. In Examples 1 and 2, 20 hours after the start of multiple parallel fermentation, most of the glucose in the fermentation broth had been consumed. This indicates that multiple parallel fermentation had essentially finished 20 hours after the start of multiple parallel fermentation. However, in Comparative Example 3, approximately 4% (w / v) of glucose was still present in the fermentation broth 20 hours after the start of multiple parallel fermentation, and this glucose remained unconsumed thereafter, indicating that fermentation had been inhibited. In addition, in all of Example 1, Example 2, and Comparative Example 3, it was shown that glucose was contained in the fermentation liquid 0 hours after the start of parallel fermentation (immediately after the start), but this is thought to be because saccharification had progressed to some extent between the time the fermentation liquid was sampled and the time the glucose concentration was measured.

[0050] [Table 2]

[0051] (c) Acetic acid concentration in the fermentation liquid The results are shown in Table 3 and Figure 5. Acetic acid in the fermentation broth is produced from ethanol, and it is known that the accumulation of ROS in the fermentation broth leads to the production of large amounts of acetic acid. However, since the amount of acetic acid accumulated in the fermentation broth was small in all of Examples 1, 2, and Comparative Example 3, it is inferred that the fermentation proceeded smoothly.

[0052] [Table 3]

[0053] (d) Ethanol concentration in the fermentation liquid The results are shown in Table 4 and Figure 6. In Examples 1 and 2, vacuum distillation was started when the ethanol concentration in the fermentation broth was low, but even after the start of vacuum distillation, a decrease in the glucose concentration and an increase in the ethanol concentration in the fermentation broth were observed, indicating that fermentation was continuing. In Comparative Example 3, vacuum distillation was started 20 hours after the start of parallel fermentation, at which point the ethanol concentration had reached its maximum.

[0054] [Table 4]

[0055] (e) Ethanol recovery rate The theoretical amount of ethanol produced was calculated from the amount of glucose produced by the saccharifying enzyme from steamed rice. The total amount of ethanol recovered was divided by this theoretical amount to calculate the ethanol recovery rate, assuming that the theoretical value was 100%. The results are shown in Table 5. Higher ethanol recovery rates were observed in Examples 1 and 2 compared to Comparative Example 3. The low ethanol recovery rate in Comparative Example 3 is thought to be due to the presence of unreacted glucose.

[0056] [Table 5]

[0057] (f) Summary In Examples 1 and 2, unlike Comparative Example 3, glucose was consumed with almost no residue remaining in the fermentation broth, and a high ethanol recovery rate was achieved. This is thought to be the result of fermentation inhibition being suppressed because ethanol, carbon dioxide, oxygen (causing substances of ROS), etc. were removed from the fermentation broth by distillation. The time from the start of parallel fermentation to ethanol recovery was 24 hours in Example 1, 28 hours in Example 2, and 40 hours in Comparative Example 3. That is, in Examples 1 and 2, the time until ethanol recovery was significantly reduced (specifically, by 60% or 70%) compared to Comparative Example 3. Furthermore, in Examples 1 and 2, the viable cell count was particularly high from the latter half of the parallel multiple fermentation onwards compared to Comparative Example 3, and it was found that this is advantageous when used as seed bacteria for the next fermentation. Thus, in Examples 1 and 2, by simultaneously carrying out multiple parallel fermentation and distillation, ethanol could be produced more efficiently overall, compared to Comparative Example 3, in which these processes were carried out separately.

[0058] 2. Examples 4, 5, and 6 Next, the effects of different conditions were compared and examined in Examples 4, 5, and 6 as follows.

[0059] [Example 4] The conditions were the same as in Example 1, except that: 1) all steps were performed with a defoaming net in the saccharification, fermentation, and distillation tank; 2) ethanol recovery was performed 24 hours after the start of multiple parallel fermentation and also 20, 28, 32, and 40 hours after the start of multiple parallel fermentation; and 3) a sample of the fermentation broth to which only the saccharifying enzymes had not been added was taken and used as the fermentation broth sample (for measuring the viable cell count, glucose concentration, acetic acid concentration, and ethanol concentration) 0 hours after the start of multiple parallel fermentation. Immediately after this sampling, the saccharifying enzymes were added to the fermentation broth to start multiple parallel fermentation. Note that 1) was added because, in Example 1, bubbles generated in the saccharification, fermentation, and distillation tank may have entered the vacuum distillation line, potentially reducing the efficiency of vacuum distillation. 2) was added to obtain more detailed experimental data. 3) As mentioned above, in Examples 1 and 2 and Comparative Example 3, when the fermentation broth to which the saccharifying enzyme had also been added was sampled and used for measurement 0 hours after the start of the multiple parallel fermentation (immediately after the start), it was found that the fermentation broth contained a certain amount of glucose. This was thought to be because some saccharification had progressed between the time the fermentation broth was sampled and the time the glucose concentration was measured.

[0060] [Example 5] The conditions were the same as in Example 4, except that all steps from 4 hours after the start of parallel fermentation were carried out at 46°C instead of 45°C.

[0061] [Example 6] Five liters of the prepared fermentation broth was placed in a saccharification, fermentation, and distillation tank, and incubation was started for 4 hours at a stirring speed of 180 rpm and 40°C. At the same time, the vacuum pump was turned on to reduce the pressure in the saccharification, fermentation, and distillation tank to 1 / 4 atmosphere, and distillation was also started, thereby performing parallel multiple fermentation and distillation simultaneously. After 4 hours from the start of parallel multiple fermentation and distillation, all steps were performed at 45°C. Other than this, the conditions were the same as in Example 4.

[0062] The time series in Examples 4, 5, and 6 is outlined in FIG. 2(B).

[0063] [result] (a) Number of viable bacteria in the fermentation liquid The results are shown in Table 6 and Figure 7. In Example 4, a fairly large viable cell count was observed even after 24 hours from the start of multiple parallel fermentation. In comparison, in Examples 5 and 6, a decrease in viable cell count was observed after 24 hours from the start of multiple parallel fermentation, but the drastic decrease seen in Comparative Example 3 was not observed, and a generally sufficient viable cell count was observed.

[0064] [Table 6]

[0065] (b) Glucose concentration in the fermentation liquid The results are shown in Table 7 and Figure 8. 20 hours after the start of multiple parallel fermentation, almost all of the glucose in the fermentation broth had been consumed in all of Examples 4, 5, and 6. In other words, it can be seen that 20 hours after the start of multiple parallel fermentation, the multiple parallel fermentation had essentially finished.

[0066] [Table 7]

[0067] (c) Acetic acid concentration in the fermentation liquid The results are shown in Table 8 and Figure 9. During the process, the acetic acid concentration increased slightly 28 hours after the start of multiple parallel fermentation in Example 4, and increased slightly 20 hours after the start of multiple parallel fermentation in Example 5. However, no significant decrease in the viable cell count was observed after these times, suggesting that fermentation proceeded smoothly in all of Examples 4, 5, and 6.

[0068] [Table 8]

[0069] (d) Ethanol concentration in the fermentation liquid The results are shown in Table 9 and Figure 10. In all of Examples 4, 5, and 6, the ethanol concentration in the fermentation broth reached a maximum value 12 hours after the start of parallel fermentation.

[0070] [Table 9]

[0071] (e) Ethanol recovery rate The theoretical amount of ethanol produced was calculated from the amount of glucose produced by the saccharifying enzyme from steamed rice. The total amount of ethanol recovered was divided by this theoretical amount to calculate the ethanol recovery rate, assuming that the theoretical value was 100%. The results are shown in Table 10. Examples 4, 5, and 6 all showed high ethanol recovery rates.

[0072] [Table 10]

[0073] (f) Summary In all of Examples 4, 5, and 6, glucose was consumed with almost no residue remaining in the fermentation broth, and a sufficiently high ethanol recovery rate was achieved 24 hours after the start of multiple parallel fermentation. Therefore, it was demonstrated that ethanol can be produced efficiently overall by simultaneously performing multiple parallel fermentation and distillation, regardless of whether multiple parallel fermentation and distillation are started simultaneously (Example 6) or not (Examples 4 and 5), or whether multiple parallel fermentation is carried out under higher temperature conditions (Example 5).

Claims

1. A method for producing ethanol, comprising simultaneously carrying out the following steps A and B under conditions of 37 to 47°C: (Step A) A step of performing parallel multiple fermentation of a substrate using a saccharifying enzyme and a thermotolerant yeast (Step B) A step of distilling the ethanol produced in step A

2. 2. The method according to claim 1, wherein (i) step A is carried out, and then (ii) step A and step B are carried out simultaneously.

3. The method according to claim 2, characterized in that (i) step A is carried out, and after the ethanol concentration in the fermentation liquid in which parallel fermentation is carried out reaches 0.06% (w / v) or more, (ii) step A and step B are carried out simultaneously.

4. The method according to claim 2, characterized in that (i) step A is carried out under conditions of 37 to 42°C, and then (ii) step A and step B are carried out simultaneously under conditions of 43 to 47°C.

5. 2. The method of claim 1, wherein steps A and B are initiated simultaneously.

6. The method according to any one of claims 1 to 5, wherein the thermotolerant yeast is Kluyveromyces marxianus.

7. The method according to any one of claims 1 to 5, wherein the distillation is vacuum distillation.

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