JP2011087478A - Method for producing ethanol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing ethanol, including fermenting a saccharification solution containing hexoses and pentoses originated from biomass with yeasts, by which the yield of the ethanol can sufficiently be improved. <P>SOLUTION: The method for producing ethanol includes treating biomass having a protein content of 1.5 to 12 mass% based on the dry mass of the biomass and containing cellulose with ammonia, hydrolyzing the obtained modified biomass to obtain the saccharification solution containing hexoses and pentoses, subjecting the hexoses in the saccharification solution to an ethanol fermentation using a yeast capable of converting the hexoses into ethanol, removing the ethanol in the obtained primary fermentation solution in a state holding temperature of <60°C to give an ethanol concentration of ≤4% (W/V), and then subjecting the primary fermentation solution after the removal of the ethanol to ethanol fermentation using a yeast capable of converting the pentoses into ethanol to obtain the secondary fermentation solution. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing ethanol using biomass as a raw material (hereinafter sometimes referred to as “bioethanol”).

In recent years, use of bioethanol as a transportation fuel has been studied as a CO ₂ reduction measure for preventing global warming. On the other hand, conventional bioethanol is produced from molasses and starch resources, so competition with food is regarded as a problem. In order to avoid such a problem, a method for producing ethanol from cellulosic biomass such as woody plants and herbs has been studied.

  As a method for producing ethanol from cellulosic biomass, there is a method in which polysaccharides such as cellulose and hemicellulose contained in biomass are hydrolyzed to obtain a saccharified solution, and the saccharified solution is fermented with yeast. In this method, cellulose contained in cellulosic biomass is converted into glucose, which is hexose, and hemicellulose is converted to glucose, mannose, galactose, or pentose, which is xylose, arabinose, etc., by hydrolysis. The And ethanol can be obtained by carrying out ethanol fermentation of the saccharified liquid containing these hexose and pentose using yeast.

  Saccharomyces cerevisiae (Saccharomyces cerevisiae) is known as a yeast capable of converting hexose to ethanol (Patent Document 1). However, Saccharomyces cerevisiae does not have the ability to convert pentose to ethanol.

  On the other hand, in recent years, microorganisms capable of producing ethanol from pentose have been created by genetic recombination technology. Examples of such microorganisms include bacteria capable of producing ethanol from pentose and hexose by incorporating an ethanol conversion gene into Escherichia coli and other bacteria (Patent Documents 2 and 3). In addition, it is known that there are yeasts that can utilize xylose and produce ethanol, such as Pichia stipitis and Candida shihatae (Non-patent Document 1).

JP 59-135896 A Japanese translation of PCT publication No. 5-502366 JP-T 6-504436

Delgenes et al. Enzyme Microb. Technol. , Vol. 19 220-225 (1996)

  However, microorganisms produced by applying genetic recombination technology to E. coli and the like are subject to strict regulations from the viewpoint of safety. Moreover, in the conventional method for producing bioethanol, ethanol can be produced in a sufficient yield even if yeast capable of converting hexose to ethanol and yeast capable of converting pentose to ethanol are used in combination. Can not.

  Therefore, the present invention provides a method capable of sufficiently improving the yield of ethanol when fermenting saccharified liquid containing hexose and pentose derived from biomass with yeast to produce ethanol. For the purpose.

  As a cause of insufficient ethanol yield in a conventional bioethanol production method using a yeast capable of converting 6-carbon sugar to ethanol and a yeast capable of converting 5-carbon sugar to ethanol, 5-carbon sugar is ethanol. It is considered that the yeast that can be converted into ethanol has low ethanol resistance. Furthermore, according to the study of the present inventors, the function of yeast capable of transforming proteins contained in biomass between the pretreatment step and the pentose fermentation step and converting the pentose to ethanol by the altered product. Was also found to be inhibited.

  Then, when this inventor further examined based on said knowledge, after performing the modification | reformation by the ammonia process about the biomass containing a specific concentration of protein, the saccharification of reformed biomass and the primary which converts hexose into ethanol It has been found that the above-mentioned problems can be solved and the yield of ethanol can be drastically improved by sequentially performing fermentation, removal of ethanol from the primary fermentation liquor, and secondary fermentation for converting pentose to ethanol. The present invention has been completed.

That is, the method for producing ethanol of the present invention comprises:
A first step of obtaining a modified biomass by treating a biomass containing cellulose with a protein content of 1.5 to 12% by mass based on the dry mass of the biomass and containing cellulose;
A second step of hydrolyzing the modified biomass to obtain a saccharified solution containing hexose and pentose;
About this saccharified liquid, a 1st yeast fermentation liquid is obtained by performing ethanol fermentation using the 1st yeast which can convert 6 carbon sugar into ethanol, The state which kept this primary fermentation liquid at the temperature lower than 60 degreeC And the third step of removing ethanol in the primary fermentation broth so that the ethanol concentration is 4% (W / V) or less,
About the primary fermentation liquid after a 3rd process, the 4th process of obtaining a secondary fermentation liquid by performing ethanol fermentation using the 2nd yeast which can convert pentose into ethanol,
It is characterized by providing. Here, “W / V” means “weight / volume”.

  In the conventional production method, pretreatment such as ammonia treatment, dilute sulfuric acid treatment, hydrothermal treatment and the like may be performed from the viewpoint of increasing the hydrolysis treatment efficiency when hydrolyzing the polysaccharide to obtain a saccharified solution. is there. However, in the production method of the present invention, even if pretreatment such as hydrothermal treatment or dilute sulfuric acid treatment is performed instead of the ammonia treatment, the effect of improving the ethanol yield is not recognized. When these pretreatments are performed, excessive decomposition of sugar occurs during the treatment, which becomes an inhibitor during fermentation. From this, the ammonia treatment in the production method of the present invention is not a simple pretreatment for hydrolysis, but an essential element for suppressing the production of an inhibitor of the function of the second yeast in the fourth step. Conceivable. And the contribution by this ammonia treatment and the contribution by the ethanol removal in the third step are combined, and the function of the second yeast in the fourth step can be exerted to the maximum. It is considered that the yield can be dramatically improved.

  Furthermore, in the conventional manufacturing method, the dilute sulfuric acid treatment has a problem that environmental problems such as waste liquid treatment are large. In addition, in the case of hydrothermal treatment, since a large amount of water is used, there is a problem that the sugar concentration of the obtained saccharified solution is lowered. On the other hand, the ammonia treatment in the present invention does not have the above problems, and the production method of the present invention is useful also in this respect.

  In the production method of the present invention, the biomass may be herbaceous biomass.

  In the production method of the present invention, Saccharomyces cerevisiae can be suitably used as the first yeast, and Pichia stipitis or Candida shihatae can be suitably used as the second yeast.

  Moreover, in the said 3rd process, it is preferable to remove ethanol from a primary fermentation liquid by distillation under reduced pressure. Thereby, ethanol can be efficiently and reliably removed in a state where the primary fermentation broth is kept at a temperature lower than 60 ° C.

  In the second step, the modified biomass is preferably hydrolyzed with an enzyme. Thereby, biomass can be decomposed into monosaccharides under mild conditions.

  As described above, according to the method for producing ethanol of the present invention, when ethanol is produced by fermenting a saccharified solution containing hexose and pentose derived from biomass with yeast, the yield of ethanol is sufficiently increased. It becomes possible to improve.

It is a graph which shows the time-dependent change of the ethanol concentration in the fermentation liquid in the primary fermentation using each saccharified liquid obtained in Examples 1 and 2. It is a graph which shows the time-dependent change of the ethanol concentration in the fermentation liquid in the secondary fermentation using each saccharified liquid obtained in Example 1 and 2. It is a graph which shows a time-dependent change of the ethanol concentration in a fermented liquid in fermentation equivalent to secondary fermentation using a model sugar liquid.

  Hereinafter, a preferred embodiment of the present invention (hereinafter referred to as “the present embodiment”) will be described in detail.

The method for producing ethanol according to this embodiment is as follows:
A first step of obtaining a modified biomass by treating a biomass containing cellulose with a protein content of 1.5 to 12% by mass based on the dry mass of the biomass and containing cellulose;
A second step of hydrolyzing the modified biomass to obtain a saccharified solution containing hexose and pentose;
About this saccharified liquid, a 1st yeast fermentation liquid is obtained by performing ethanol fermentation using the 1st yeast which can convert 6 carbon sugar into ethanol, The state which kept this primary fermentation liquid at the temperature lower than 60 degreeC And the third step of removing ethanol in the primary fermentation broth so that the ethanol concentration is 4% (W / V) or less,
About the primary fermentation liquid after a 3rd process, the 4th process of obtaining a secondary fermentation liquid by performing ethanol fermentation using the 2nd yeast which can convert pentose into ethanol,
Is provided.

  In the present embodiment, biomass having a protein content of 1.5 to 12% by mass based on the dry mass of the biomass and containing cellulose is used as the raw material biomass. The protein content is preferably 2 to 8% by mass. When using biomass with a protein content less than the lower limit as a raw material, the modified biomass obtained by ammonia treatment of the biomass raw material is hydrolyzed, and in particular, the saccharified solution is obtained by hydrolysis using an enzyme. In the obtaining step, saccharification tends not to proceed sufficiently. On the other hand, the protein content of the cellulosic biomass is approximately 12% by mass or less. As this raw material biomass, what satisfies the said conditions among herbaceous biomass and woody biomass can be used suitably. Here, a combustion method is used as a method for measuring the protein content of biomass. That is, each dried biomass sample is pyrolyzed in oxygen gas (purity 99.9%), the produced nitrogen oxide (NOx) is reduced to nitrogen gas, and this nitrogen gas is quantified by gas chromatography. The total nitrogen content in the dry biomass is calculated from this value and the mass of the biomass sample. Next, the nitrate nitrogen content and nitrite nitrogen content in the same biomass sample are measured by liquid chromatography, and the total amount of these is subtracted from the total nitrogen content described above. The nitrogen content is obtained. Further, the organic nitrogen content is multiplied by a coefficient of 6.25 to obtain the protein content in the dry biomass.

  Herbaceous biomass is preferable in that it can achieve a high ethanol yield or has a shorter growth period. Examples of herbaceous biomass include switchgrass, Eliansus, and napiergrass.

  On the other hand, woody biomass generally has a low protein content and is often less than the above lower limit. When such woody biomass is used, the enzymatic saccharification of the modified biomass after the ammonia treatment tends to hardly proceed. Of woody biomass, conifers such as pine and cedar tend to be less prone to enzymatic saccharification of the modified biomass after ammonia treatment. On the other hand, compared with conifers, broad-leaved trees tend to promote enzymatic saccharification of the modified biomass after ammonia treatment. In particular, willow-derived biomass such as Ezo nokinu willow has a low protein content compared to herbaceous biomass, but woody biomass has a relatively high protein content. When such willow-derived biomass is used, an effect equal to or higher than that obtained when using herbaceous biomass can be obtained, which is particularly preferable. In addition, the reason why an excellent effect is obtained when using willow-derived biomass is because the protein content is lower than that of herbaceous biomass, so that the production of an inhibitor of secondary fermentation due to protein alteration is more reliably performed. This is thought to be because it can be suppressed.

  In the present embodiment, first, the raw material biomass is treated with ammonia to obtain modified biomass (first step). Such ammonia treatment can be performed using ammonia or ammonia and an organic amine. The amount of ammonia used is preferably 10 mg to 300 g with respect to 1 g of raw material biomass. As a result, the reforming of the raw material biomass proceeds, and it becomes possible to increase the recovery rate of sugar after enzymatic hydrolysis.

  Next, the modified biomass is hydrolyzed to obtain a saccharified solution containing 6-carbon sugars and 5-carbon sugars (second step). The method for hydrolysis of the modified biomass is not particularly limited, and a chemical method using an acid catalyst such as concentrated sulfuric acid, a biochemical method using an enzyme, or the like can be used. Among them, mild conditions can be employed, and enzymatic hydrolysis is preferably employed from the standpoint of suppressing the production of inhibitors against fermentation by yeast. As an enzyme used in this case, a general cellulolytic enzyme (cellulase) can be used.

  In the present embodiment, since the use of water is not essential in the first step, which is a pretreatment step, unlike the hydrothermal treatment in the conventional manufacturing method, It is possible to adjust the slurry to an arbitrary concentration. Therefore, the sugar concentration of the saccharified solution after the hydrolysis treatment with an enzyme can be kept high, and the ethanol concentration after the subsequent fermentation step can be increased, so that an energy-saving ethanol distillation process can be achieved.

  Subsequently, about the saccharified liquid obtained at the 2nd process, primary fermentation liquid is obtained by performing ethanol fermentation using the 1st yeast which can convert hexose into ethanol, and this primary fermentation liquid is obtained. While maintaining the temperature lower than 60 ° C., ethanol in the primary fermentation broth is removed so that the ethanol concentration is 4% (W / V) or less (third step).

  The first yeast is not particularly limited as long as hexose can be converted into ethanol, and known ones can be used. Among them, Saccharomyces cerevisiae is preferable. The primary fermentation is preferably performed at a pH of 5.0 to 7.0 and a temperature of 20 to 37 ° C.

  In addition, the removal of ethanol from the primary fermentation liquid in the third step may be performed in parallel with the primary fermentation (simultaneously with the primary fermentation) or after the primary fermentation is completed. Good. The method for removing ethanol is not particularly limited as long as it is carried out in a state where the primary fermentation liquid is kept at a temperature lower than 60 ° C., but vacuum distillation is preferred. Further, stripping may be performed by blowing an inert gas such as carbon dioxide or nitrogen. When removing ethanol from the primary fermentation broth, the temperature of the primary fermentation broth is lower than 60 ° C, and preferably 50 ° C or lower. If it is the said temperature, at the time of removal of ethanol, it will be thought that the production | generation of the substance which inhibits the fermentation function of the yeast used for secondary fermentation is suppressed, and secondary fermentation advances efficiently. Moreover, it is preferable that the temperature of a primary fermentation liquid is 0 degreeC or more from a viewpoint of the efficiency of ethanol removal. Further, the ethanol concentration in the primary fermentation liquid after ethanol removal varies depending on the ethanol tolerance of the yeast used in the subsequent secondary fermentation, but it is 4% or less, preferably 3% or less, more preferably 2 % Or less, particularly preferably 1% or less. By setting the ethanol concentration to the above value, the efficiency of ethanol production from pentose by yeast in secondary fermentation is improved.

  Subsequently, about the primary fermentation liquid from which ethanol was removed, a secondary fermentation liquid is obtained by performing ethanol fermentation using the 2nd yeast which can convert pentose into ethanol (4th process).

  The second yeast is not particularly limited as long as it can convert pentose to ethanol, and a known pentose-utilizing yeast can be used. Among them, Pichia stippitis or Candida shihatae is preferable. The secondary fermentation is preferably performed at a pH of 5.0 to 7.0 and a temperature of 20 to 37 ° C.

  Separation and recovery of ethanol from the secondary fermentation broth can be performed in the same manner as ethanol removal in the third step, but can also be performed by heating and distillation under normal pressure.

  In this way, by using specific raw material biomass and producing ethanol through the first to fourth steps, a saccharified solution containing hexose and pentose derived from biomass is fermented with yeast to produce ethanol. It is possible to achieve a high ethanol yield, which has been difficult to achieve with conventional manufacturing methods.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example at all.

[Reference Example: Measurement of protein content in biomass material]
Each dried biomass sample is pyrolyzed in oxygen gas (purity 99.9%), and the produced NOx is converted to nitrogen gas for conversion. The nitrogen gas is quantified by gas chromatography, The total nitrogen content in the dry biomass was calculated from the mass of the biomass sample. Next, the nitrate nitrogen content and the nitrite nitrogen content in the same biomass sample are measured by liquid chromatography, and the total value of these is subtracted from the total nitrogen content described above, so that the organic content in the dry biomass is reduced. The nitrogen content was obtained. This value was multiplied by a coefficient of 6.25 to obtain the protein content in each dry biomass. The results are shown in Table 1.

[Reference example: Production of saccharified liquid using various biomass (preliminary test)]
Various biomasses were hydrolyzed with enzymes after ammonia treatment, and the progress of saccharification was investigated.
Elianthus, switchgrass (above herbs), Ezo nokinu willow, cedar (more woods) were pulverized with a cutter mill and a hammer mill to an average particle size of about 200 μm.
Next, about 10 g of each pulverized biomass is put into a high-pressure container having an internal volume of 250 ml, and after cooling the container to −40 ° C., about 25 g of liquid ammonia is introduced, and 80 ° C., 4.3 MPa or 100 ° C., 6 After reaching 3 MPa, treatment was performed for 2 hours.
Each biomass after the ammonia treatment was suspended in 0.05 M-acetate buffer (pH 4.5) to make the slurry concentration 2.5%. Celluclast 1.5 L and Novozyme 188 (both registered trademarks, Novozymes) were added to the biomass suspension as saccharification enzymes to a protein content of 0.1%, and shaken at 37 ° C. for 72 hours (200 rpm). )did.
The enzyme-saccharified solution was heat-treated at 95 ° C. for 15 minutes to inactivate the enzyme, and then the glucose concentration of the solution obtained by filtration of the suspension was measured by HPLC (column Shodex SUGAR 0810, detector RI). And the enzyme saccharification rate (glucose) was calculated. In addition, the amount of glucose which raw material biomass has was measured by the sulfuric acid hydrolysis method. The enzyme saccharification rate was calculated with the amount of glucose as 100%. The results are shown in Table 2.

  In Table 2, in switchgrass, Elianthus (above, herbaceous) and Ezo nokinu willow (woody) having a protein content of 1.5% by mass or more, 70% or more is obtained by performing ammonia treatment. In contrast, woody cedars having a protein content of less than 1.5% by mass had extremely low saccharification rates even after ammonia treatment. From this, it is clear that when using biomass with a protein content of less than 1.5% by mass, the modification effect by the ammonia treatment is extremely small, and saccharification by the hydrolysis treatment using an enzyme does not proceed easily. It became. From this result, subsequent experiments were conducted on biomass excluding cedar.

[Comparative Example 1: Pichia stipitis, ethanol production from various biomass saccharified liquids by one-stage fermentation]
(Manufacture of enzymatic saccharified liquid from various biomass)
Elianthus, switchgrass, napiergrass (above herbs) and Ezo nokinu willow (wooden) were ground using a cutter mill and a hammer mill to an average particle size of about 200 μm.
Next, about 200 g of each pulverized biomass is put into a high-pressure vessel having an internal volume of 5 L, and about 500 g of liquid ammonia preheated to about 80 ° C. in another vessel is introduced, and the conditions of 100 ° C. and 6.3 MPa are introduced. Below, it was treated with ammonia in liquid phase for 2 hours.
The modified biomass after the ammonia treatment was suspended in 0.01 M-acetate buffer, adjusted to pH 4.5 with 1% -hydrochloric acid, and the slurry concentration was adjusted to 5%. Celluclast 1.5 L and Novozyme 188 (both registered trademarks, Novozymes) as saccharification enzymes were added to the biomass suspension so that the protein concentration was 0.2%, respectively, and shaken at 37 ° C. for 72 hours ( 200 rpm), and the modified biomass was saccharified.
The suspension after enzymatic saccharification was subjected to solid-liquid separation by centrifugation and filtration, and the resulting solution was concentrated by a rotary evaporator so that the xylose concentration became 3% or more. Table 3 shows the contents of glucose and xylose in each obtained saccharified solution. Moreover, it analyzed by the substance considered to produce | generate by the hyperdegradation of saccharide | sugar and lignin in the pre-processing stage of biomass, and inhibits the fermentation function with respect to the whole yeast, The result was combined with Table 3, and was described. In any of the saccharified solutions, these inhibitors were hardly detected.
In addition, as a comparison target (control), there is also a synthetic medium composed of an aqueous solution obtained by adding the reagent yeast extract (1%) and peptone (2%) to the reagent glucose (5.6%) and xylose (3.0%). In addition, the evaluation was performed.
(Ethanol fermentation)
Fermentation of each saccharified solution was performed by a one-stage fermentation method using Pichia stipitis. Pichia stipitis was inoculated into a 100 ml Erlenmeyer flask containing 25 ml of saccharified solution, and fermentation was performed while shaking at 28 ° C. and 80 rpm. Table 4 shows the ethanol yield when the glucose and xylose in the saccharified solution are all converted to ethanol.

[Example 1; ethanol production by two-stage fermentation from willow-derived saccharified solution using Saccharomyces cerevisiae and Pichia stipitis (distillation under reduced pressure)]
Ethanol was produced by a two-stage fermentation method using Saccharomyces cerevisiae and Pichia stippitis, using a saccharified solution derived from Ezo nokinu willow as a raw material. As the saccharified solution, a saccharified solution derived from Ezo nokinu willow obtained in the same manner as that used in Comparative Example 1 was used. In addition, the control synthetic medium used in Comparative Example 1 was also evaluated.
In the fermentation of each saccharified solution, first, as a primary fermentation, Saccharomyces cerevisiae was inoculated into the saccharified solution in a 100 ml Erlenmeyer flask, and fermentation was performed while shaking at 28 ° C. and 80 rpm. After glucose in the fermentation broth was consumed, the fermentation broth was subjected to vacuum distillation (heated water bath temperature 40 ° C.), and ethanol in the fermentation broth was removed until the concentration became 0.5% or less. Next, the distillation residue is filtered aseptically, 25 ml of the resulting liquid is collected in a 100 ml Erlenmeyer flask, and as a secondary fermentation, Pichia stepitis is inoculated here, and fermentation is performed while shaking at 28 ° C. and 80 rpm. went.
In FIG. 1, the time-dependent change of the ethanol concentration in the fermented liquor in primary fermentation is shown. Moreover, the time-dependent change of the ethanol concentration in the fermented liquor in secondary fermentation is shown in FIG. Furthermore, Table 4 shows the ethanol yield with 100% when all the glucose and xylose in each saccharified solution are converted to ethanol.

[Example 2; ethanol production by two-stage fermentation from various herbaceous biomass-derived saccharified liquids using Saccharomyces cerevisiae and Pichia stipitis (distillation under reduced pressure)]
Using the saccharified liquid derived from various herbaceous biomass as a raw material, ethanol was produced and produced by a two-stage fermentation method using Saccharomyces cerevisiae and Pichia stipitis by the same operation as in Example 1. Three types of saccharified solutions derived from Elianthus, Switchgrass and Napiergrass obtained in the same manner as those used in Comparative Example 1 were used as saccharified solutions.
In FIG. 1, the time-dependent change of the ethanol concentration in a fermented liquid in the primary fermentation using each saccharified liquid is shown. Moreover, the time-dependent change of the ethanol concentration in the fermented liquor in secondary fermentation is shown in FIG. Furthermore, Table 4 shows the ethanol yield with 100% when all the glucose and xylose in each saccharified solution are converted to ethanol.

From the results shown in Table 4, in the one-stage fermentation using Pichia stipitis, the ethanol yield is low, whereas in the two-stage fermentation using Saccharomyces cerevisiae and Pichia stipitis, the primary fermentation and 2 It can be seen that the ethanol yield is greatly improved when ethanol is removed without heating by vacuum distillation during the subsequent fermentation.
In addition, from the results shown in FIG. 2, Ezo nokinu willow gave an ethanol production rate equivalent to that of the control in the secondary fermentation, but the saccharified liquid derived from other biomass tended to have a slightly lower ethanol production rate than the control. is there. This suggests that in the process of saccharification liquid production, a substance that gives weak inhibition to the ethanol production from xylose by Pichia stipitsis occurs.

[Evaluation of effects of fermentation and heating by Pichia stipitis using a saccharified solution modeled after primary fermentation]
As a model of the fermentation broth after the end of primary fermentation, a 5% (W / V) ethanol aqueous solution containing 100 ml of YPX (0.5% by mass of yeast extract, 1.0% by mass of polypeptone, 5.0% by mass of xylose) is used. This was prepared and subjected to vacuum distillation in the same manner as in Example 2. Distillation under reduced pressure was heated in a hot water bath at 40 ° C., and was carried out under reduced pressure of 60 to 100 HPa for 50 minutes to obtain a residual liquid having a volume of 72 ml and an ethanol concentration of 0.55% (W / V).
On the other hand, 100 ml of 5% (W / V) ethanol aqueous solution containing the same YPX as described above was subjected to atmospheric distillation. Heating was performed with a mantle heater, and distillation was performed at a liquid temperature of about 100 ° C. for 24 minutes to obtain a residual liquid having a volume of 83 ml and an ethanol concentration of 0.5% (W / V).
Distilled residue of each of vacuum distillation and atmospheric distillation was made up to 100 ml with distilled water and filtered through a polyethersulfone filter having a pore size of 0.45 μm. Each of the obtained liquids was inoculated with pre-cultured Pichia stippitis yeast, and cultured with shaking at 28 ° C. and 80 rpm for 30 to 43 hours, and the ethanol concentration in the fermentation broth was measured over time. The results are shown in FIG.
The residual liquid from which ethanol has been removed by atmospheric distillation has a lower ethanol concentration increase rate than the residual liquid from which ethanol has been removed by vacuum distillation. This is presumed to be caused by fermentation inhibition by melanoidin or the like caused by the reaction of the yeast extract and / or peptone-derived protein, peptide, or amino acid contained in the fermentation broth with sugar by heating during ethanol removal. The On the other hand, it can be seen that the residual liquid from which ethanol is removed without heating by reduced-pressure distillation has a relatively low inhibitory effect.

[Reference Example: Measurement of Melanoidin]
Various biomass is treated with ammonia, and the saccharified solution and simulated saccharified solution obtained by enzymatic saccharification treatment (YPX medium, yeast extract 0.5%, polypeptone 1.0% to xylose 5.0% (W / V) (Added) were incubated so as to have a temperature history similar to that of the vacuum distillation (40 ° C., 50 minutes) in the example and the atmospheric distillation (100 ° C., 24 minutes) in the comparative example. About these saccharified liquids, the relative melanoidin production | generation density | concentration was compared by measuring the light absorbency of visible light (wavelength 430nm). Table 5 shows the absorbance of each saccharified solution untreated and incubated at each temperature.
Note that melanoidin is not a single substance defined by a specific structure, but a generic term for colored substances produced by a reaction of saccharides with proteins, polypeptides, amino acids and the like.

  When the heat treatment at 100 ° C. corresponding to the atmospheric distillation conditions for removing ethanol from the primary fermentation broth is performed, the absorbance of each saccharified liquor increases as compared to the untreated case, It is presumed that melanoidin is produced. On the other hand, in the treatment at 40 ° C. corresponding to the vacuum distillation conditions, no increase in absorbance was observed, indicating that production of melanoidin was suppressed.

  From the above results, yeast that can convert hexose into ethanol was used for the saccharified solution obtained by subjecting biomass having a protein content of 1.5 to 12% by mass to ammonia treatment and enzymatic hydrolysis. In the production of ethanol that performs secondary fermentation using yeast capable of converting primary fermentation and pentose to ethanol, the ethanol concentration is 4% (W / w) while maintaining the primary fermentation solution at a temperature lower than 60 ° C. It became clear that ethanol can be obtained with a high yield by setting it to V) or less. The reason why the efficiency of the secondary fermentation is lowered by setting the temperature of the primary fermentation broth to 60 ° C. or higher is that the protein-derived substance contained in the saccharified solution is transformed into melanoidin by heating, which is a pentose. It was presumed to inhibit the fermentation function of yeast that can be converted to ethanol.

Claims (6)

A first step of obtaining a modified biomass by treating a biomass containing cellulose with a protein content of 1.5 to 12% by mass based on the dry mass of the biomass and containing cellulose;
A second step of hydrolyzing the modified biomass to obtain a saccharified solution containing hexose and pentose;
About the said saccharified liquid, a primary fermentation liquid is obtained by performing ethanol fermentation using the 1st yeast which can convert 6 carbon sugar into ethanol, The state which hold | maintained the said primary fermentation liquid at the temperature of less than 60 degreeC Then, a third step of removing ethanol in the primary fermentation broth so that the ethanol concentration is 4% (W / V) or less,
A fourth step of obtaining a secondary fermentation broth by performing ethanol fermentation using a second yeast capable of converting pentose to ethanol with respect to the primary fermentation broth after the third step;
A method for producing ethanol, comprising:   The method for producing ethanol according to claim 1, wherein the biomass is herbaceous biomass.   The method for producing ethanol according to claim 1 or 2, wherein the first yeast is Saccharomyces cerevisiae.   The method for producing ethanol according to any one of claims 1 to 3, wherein the second yeast is Pichia stipitis or Candida shihatae.   5. The method for producing ethanol according to claim 1, wherein in the third step, ethanol is removed from the primary fermentation broth by distillation under reduced pressure.   In the said 2nd process, the said modified biomass is hydrolyzed with an enzyme, The manufacturing method of ethanol of any one of Claims 1-5 characterized by the above-mentioned.

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