JP2012139211A - Method for producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ethanol from lignocellulosic biomass at low cost, using yeast with a cellulose hydrolysis enzyme group presented on a cell surface.SOLUTION: The production method of the ethanol from the lignocellulosic biomass includes: a process for obtaining cellulose fractions from the lignocellulosic biomass; a process for producing a saccharification biomass by mixing 100 g-dry mass/L or more of the solid content of the cellulose fractions with a cellulose hydrolysis enzyme preparation, and treating the mixture with enzyme for 0.5-10 hours; and a process for mixing the yeast with the saccharification biomass and culturing the mixture.

Description

  The present invention relates to a method for producing ethanol. More specifically, the present invention relates to a method for producing ethanol from biomass.

  In recent years, alternative fuels are being developed in the face of fear of exhaustion of fossil fuels. In particular, bioethanol derived from biomass has attracted attention. This is because biomass is a renewable resource, exists in large quantities on the earth, and does not increase carbon dioxide in the atmosphere even if it is used (carbon neutral), thereby contributing to the prevention of global warming.

  However, currently produced bioethanol is mainly made from corn and sugarcane, and competition with food is a problem. Therefore, in the future, production of bioethanol using lignocellulosic biomass such as rice straw, wheat straw and waste wood that does not compete with food will be required.

  Lignocellulosic biomass is mainly composed of three components, cellulose, hemicellulose, and lignin. Among these, cellulose can be used for ethanol fermentation by yeast Saccharomyces cerevisiae that can assimilate glucose when it is degraded (saccharified) to glucose by hydrolysis.

  Conventionally, hydrothermal decomposition methods, acid treatment methods, and alkali treatment methods are known as pretreatment methods. As an acid treatment method, a method using a dilute acid at a high temperature (200 ° C. or higher) and a method using concentrated sulfuric acid are known. However, the hydrothermal decomposition method and acid treatment method partially decompose cellulose under extreme conditions, resulting in excessive decomposition products (by-products), low glucose yield (saccharification rate), and ethanol fermentation. The problem is that it can also produce inhibiting substances.

  The pretreated product of lignocellulosic biomass is subjected to enzyme treatment. In the enzyme treatment, the cellulose component and hemicellulose component constituting the pretreated product are hydrolyzed to generate oligosaccharides and monosaccharides. However, since the commercially available enzyme used for saccharification has a low titer, a large amount of enzyme is required for sufficient saccharification.

  On the other hand, attempts have been made to produce ethanol directly from pretreated biomass by modifying yeast Saccharomyces cerevisiae, which cannot inherently assimilate cellulose, hemicellulose, etc., using biotechnological techniques. A cell surface presentation technique is used as such a biotechnological technique. For example, an enzyme group that hydrolyzes cellulose, that is, yeast that surface-displays endoglucanase, cellobiohydrolase, β-glucosidase, and the like has been produced by a cell surface display technique (Patent Document 1).

International Publication No. 2010/032762

  In view of industrial practicality, it is necessary to study to improve ethanol productivity by combining the above methods. In particular, a method in which ethanol fermentation is carried out using yeast that presents a cellulosic hydrolase group on the cell surface while proceeding to a certain extent with saccharification of lignocellulosic biomass pretreatment products by an enzyme treatment method has been studied as a promising technique. However, reducing the cost of enzyme treatment has become an issue.

  An object of this invention is to provide the method of producing ethanol from lignocellulosic biomass at low cost using yeast.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention have been able to reduce the cost of lignocellulosic biomass at low cost by subjecting the pretreated product of lignocellulosic biomass to a yeast fermentation substrate for a short time. The inventors have found that ethanol can be produced and completed the present invention.

  The present invention provides a method for producing ethanol from lignocellulosic biomass, the method comprising a step of obtaining a cellulose fraction from lignocellulosic biomass, the cellulose fraction having a solid content of 100 g-dry mass / L or more. Is mixed with a cellulose hydrolase preparation, and subjected to an enzyme treatment for 0.5 to 10 hours to produce saccharified biomass, and a step of mixing yeast with the saccharified biomass and culturing.

  In one embodiment, the enzyme treatment time is 0.5 to 8 hours.

  In one embodiment, the cellulose fraction has a solid content of 200 g-dry weight / L or more.

  In one embodiment, the cellulose hydrolase preparation is an enzyme concentration of 1-4 FPU / g-dried biomass pretreatment product.

  In one embodiment, the yeast is a yeast transformed to present at least two cellulose hydrolases on the cell surface.

  According to the method of the present invention, ethanol can be produced at low cost from lignocellulosic biomass using yeast.

Changes in glucose concentration and ethanol concentration in the fermentation broth from the start of enzyme treatment when enzyme treatment is performed at the enzyme concentration of 1 FPU / g-dried biomass pretreatment product and fermentation is started 2 hours after the start of enzyme treatment It is a graph which shows. It is a graph which shows a time-dependent change of the ethanol density | concentration in the fermentation liquid from the enzyme treatment / fermentation start at the time of starting fermentation simultaneously with an enzyme treatment and making a substrate concentration increase by the fed-batch method. It is a graph which shows the ethanol density | concentration produced in the fermented liquid 72 hours after the fermentation start. The graph which shows the time-dependent change of the ethanol density | concentration in the fermentation liquid from the start of enzyme treatment at the time of performing enzyme treatment with the enzyme concentration of 4FPU / g-dry biomass pretreatment substance, and starting fermentation after progress of enzyme treatment 2 hours It is. When the enzyme treatment is performed with the enzyme concentration of 1 FPU / g-dried biomass pre-treated product or 10 FPU / g-dried biomass pre-treated product, and fermentation is started after 2 hours or 24 hours from the start of enzyme treatment, It is a graph which shows a time-dependent change of the ethanol concentration in a fermented liquor. Fermentation liquid from the start of fermentation when enzyme treatment is performed at an enzyme concentration of 1 to 10 FPU / g-dried biomass pretreatment using wild strain yeast and fermentation is started after 0 to 24 hours have elapsed from the start of the enzyme treatment. It is a graph which shows a time-dependent change of the ethanol concentration in it.

  Biomass refers to a carbohydrate material derived from biological resources. Examples thereof include starch obtained from corn and the like, and molasses (waste molasses) obtained from sugarcane and the like. Lignocellulosic biomass refers to biomass composed mainly of three types of components, cellulose, hemicellulose, and lignin. Use of lignocellulosic biomass is preferable because it does not compete with food. Examples of lignocellulosic biomass include wastes generated when processing biological materials such as rice, wheat, corn, sugarcane, and wood (pulp). For example, rice straw, wheat straw, and waste material are listed.

  Saccharification of biomass refers to, for example, decomposing cellulose (polysaccharide) having glucose (monosaccharide) as a structural unit to glucose. Examples of the saccharification method include a hydrothermal decomposition method, an acid treatment method, and an enzyme treatment method. The dilute sulfuric acid method and hydrothermal decomposition method are preferable in terms of cost. Yeast Saccharomyces cerevisiae and the like produce ethanol by fermentation using glucose as a substrate.

  The saccharified biomass refers to a composition obtained by saccharifying a biomass pretreatment product, and includes, for example, glucose (monosaccharide) produced by the decomposition of cellulose (polysaccharide) as a main component.

  Cellulose refers to a fibrous polymer in which glucopyranose (glucose) is linked by β1,4-glucoside bonds.

  Cellulose hydrolase hydrolyzes the β1,4-glucoside bond of cellulose. Examples of cellulose hydrolase include, but are not limited to, endo β1,4-glucanase (hereinafter simply referred to as “endoglucanase”), cellobiohydrolase, and β-glucosidase.

  Endoglucanase is an enzyme usually called cellulase, which cleaves cellulose from the inside of the molecule, and glucose, cellobiose, and cellooligosaccharide (degree of polymerization is 3 or more, and usually 10 or less, Non-limiting). Endoglucanase is highly reactive to low crystallinity or amorphous cellulose, such as non-crystallized cellulose, soluble cellooligosaccharides, and cellulose derivatives such as carboxymethylcellulose (CMC), but has a crystalline structure Reactivity to cellulose microfibrils is low. Endoglucanase is a representative example of an enzyme that hydrolyzes amorphous cellulose. Examples of endoglucanase include, but are not limited to, Trichoderma reesei-derived endoglucanase (particularly EGII).

  Cellobiohydrolase degrades from either the reducing end or non-reducing end of cellulose to release cellobiose. Cellobiohydrolase degrades crystalline cellulose such as cellulose microfibrils with a crystalline structure, but is not reactive to low crystallinity or amorphous cellulose such as cellulose derivatives such as carboxymethylcellulose (CMC). Low. Cellobiohydrolase is a representative example of an enzyme that hydrolyzes crystalline cellulose. Hydrolysis of crystalline cellulose by cellobiohydrolase is slower than hydrolysis of amorphous cellulose by endoglucanase due to the strong structure due to the intermolecular and intramolecular dense hydrogen bonds of crystalline cellulose. . There are two types of cellobiohydrolase, which are called cellobiohydrolase 1 and cellobiohydrolase 2, respectively. Examples of cellobiohydrolase include, but are not limited to, Trichoderma reesei cellobiohydrolase (particularly CBH2).

  β-glucosidase is an exo-type hydrolase that separates glucose units from non-reducing ends in cellulose. β-glucosidase cleaves a β1,4-glucoside bond between an aglycon or sugar chain and β-D-glucose, and hydrolyzes cellobiose or cellooligosaccharide to produce glucose. β-glucosidase is a representative example of an enzyme that hydrolyzes cellobiose or cellooligosaccharide. One type of β-glucosidase is currently known and is referred to as β-glucosidase 1. Examples of β-glucosidase include, but are not limited to, Aspergillus acreatas-derived β-glucosidase (particularly BGL1).

  In the method of the present invention, a cellulose fraction is first obtained from lignocellulosic biomass.

  The method for obtaining the cellulose fraction from lignocellulosic biomass is not particularly limited. An example is a hydrothermal decomposition method. In the hydrothermal decomposition method, for example, lignocellulosic biomass is pulverized as necessary, and mixed with water so as to have a content of, for example, about 20% by mass (dry mass), and this mixture is heat-treated. The heat treatment is performed at 120 to 300 ° C., preferably 150 to 280 ° C., more preferably 180 to 250 ° C., for 15 seconds to 1 hour. The treatment temperature and time can vary depending on the biomass used, and increasing the treatment temperature can shorten the treatment time. In addition, you may pressurize during heat processing.

  When lignocellulosic biomass is hydrothermally decomposed, a water-soluble hemicellulose fraction and a water-insoluble cellulose fraction are separated. The water-insoluble cellulose fraction can be easily separated as a solid content by a centrifugal separation method or the like.

  In the method of the present invention, the above cellulose fraction having a solid content of 100 g-dry mass / L or more is then mixed with a cellulose hydrolase preparation and subjected to enzyme treatment for 0.5 to 10 hours to produce saccharified biomass.

  Generally, in the preparation of saccharified biomass, the hydrolysis reaction does not proceed even if a high-content lignocellulosic biomass pretreatment product is mixed with a cellulose hydrolase preparation. The content is usually 10 to 50 g-dry mass / L. However, in the method of the present invention, even if a high content lignocellulosic biomass pretreatment product is used, there is no hindrance to saccharification and ethanol fermentation in the subsequent process, but rather a high content lignocellulosic biomass pretreatment from the viewpoint of cost reduction. It is preferable to use a product. The solid content of the lignocellulosic biomass pretreatment product is 100 g-dry mass / L or more, preferably 200 g-dry mass / L or more.

  The cellulose hydrolase preparation used in the present invention is not particularly limited as long as it is a preparation containing the cellulose hydrolase. Commercially available enzyme preparations are used, but those prepared by culturing enzyme-producing microorganisms or the like may be used. The activity of cellulose hydrolase is expressed as the amount of enzyme (1 FPU) that liberates 1 μmol of glucose from filter paper (Filter Paper: No. 1 filter manufactured by Whatman) per minute. In general, in preparation of saccharified biomass, an enzyme preparation used for hydrolysis of cellulose needs to be used at a higher enzyme concentration than 30 FPU / g-dried biomass pretreatment product in order to efficiently hydrolyze cellulose. However, in the method of the present invention, the required cellulose hydrolase concentration is 1 to 4 FPU / g-dried biomass pretreatment product.

  In general, in the preparation of saccharified biomass, the enzyme treatment requires 2 days to several days in order to completely hydrolyze cellulose to glucose. However, in the method of the present invention, it is 0.5 to 10 hours, preferably 0.5 to 8 hours, more preferably 0.5 to 6 hours. The temperature of the enzyme treatment is not particularly limited as long as the enzyme works. Preferably, it is 40-80 degreeC. In the present invention, the cellulose fraction becomes a slurry by enzyme treatment.

  In the present invention, the yeast is then mixed with the saccharified biomass and cultured. Yeast is not particularly limited as long as ethanol can be fermented with glucose as a substrate. It may be a wild-type yeast, preferably a yeast transformed so as to present cellulose hydrolase on the cell surface, more preferably three kinds of celluloses of the endoglucanase, cellobiohydrolase and β-glucosidase. Among the hydrolases, the yeast is transformed so as to present at least two types of cellulose hydrolase on the cell surface, and more preferably transformed so as to present the three types of cellulose hydrolase on the cell surface. Yeast. As long as the enzyme treatment is performed using a preparation containing the cellulose hydrolase, the enzyme presented on the cell surface layer among the three types of cellulose hydrolase is not particularly limited.

  A method for producing such a transformed yeast is not particularly limited. For example, the method shown by patent document 1 is mentioned. More simply, the gene is produced by introducing the endoglucanase, cellobiohydrolase, and β-glucosidase genes into a wild strain such as yeast Saccharomyces cerevisiae that can be ethanol-fermented with glucose as a substrate.

  The concentration of yeast cells when mixed with the saccharified biomass is not particularly limited. Preferably, it is about 25-100 g-wet weight / L. Yeast culture conditions are not particularly limited. Usually, the conditions for ethanol fermentation using glucose as a substrate may be used. The culture temperature is, for example, 30 to 35 ° C.

  In the method of the present invention, since the fermentation can be completed in a short time, the culture time is usually 2 to 3 days.

  EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1
(Process 1: Hydrothermal treatment of rice straw)
Rice straw is mixed with water to a content of about 20% by mass (dry mass), and this is put into a hydrothermal treatment apparatus (manufactured by Mitsubishi Heavy Industries, Ltd.) and treated at about 180 ° C. and about 3 MPa for 5 to 20 minutes. did. The solid was then separated and used as a fermentation substrate.

(Step 2: Enzyme treatment)
The enzyme treatment liquid containing the rice bran hydrothermal solid content obtained in step 1 was prepared. The composition is shown in Table 1.

  This enzyme treatment solution is put into a 50 mL capacity plastic test tube (Corning), and the enzyme treatment is performed at a rotation speed of 35 rpm at 50 ° C. using a thermoblock rotator (SN-06BN manufactured by Nisshin Rika Corporation). went.

(Process 3: Ethanol fermentation 1)
For fermentation, yeast Saccharomyces cerevisiae NBRC1440 strain (wild strain), and transformed yeast displaying cell surface layers of cellulose hydrolases, ie, endoglucanase (EG), cellobiohydrase (CBH2) and β-glucosidase (BGL) ( NBRC1440 / EG-CBH2-3c / BGL: Patent Document 1) was used. The NBRC1440 / EG-CBH2-3c / BGL chromosome incorporates 3 copies of endoglucanase II, 3 copies of cellobiohydrolase 2, and 1 copy of β-glucosidase 1 gene.

  For the above yeast, seed culture is performed overnight in a tester containing 5 mL of YPD liquid medium (yeast extract 10 g / L, polypeptone 20 g / L, glucose 20 g / L), and then the culture solution is a flask containing 500 mL of YPD liquid medium. The main culture was performed for 2 days. This culture solution is centrifuged (3000 rpm, 4 ° C., 10 minutes), the yeast cells are washed twice with sterilized distilled water, and then suspended in sterilized distilled water to a cell concentration of 500 g-wet weight / L. did.

  After 2 hours from the start of the enzyme treatment in Step 2, 2 mL of the yeast suspension was added to the enzyme treatment solution to adjust the cell concentration to 100 g-wet weight / L. After yeast addition, fermentation was started at 35 ° C. At the start of fermentation, the lid of the plastic test tube was changed to a silicon stopper (ASONE Corporation, No. 9) provided with a gas discharge hole (0.6 × 32 mm).

  The glucose concentration in the fermentation broth and the ethanol concentration produced were quantified over time by HPLC (High performance liquid chromatography system; Shimadzu Corporation 10A system). Shim-pack, SPR-Pb (manufactured by Shimadzu Corporation, 250 L × 7.8) is used as the HPLC separation column, and ultrapure water (purified water using Milli-Q from Nippon Millipore Corporation) as the mobile phase. And a refractive index detector was used as the detector. The HPLC conditions were a transfer rate of 0.6 mL / min and a column temperature of 80 ° C. The result is shown in FIG.

  As is clear from FIG. 1, from a biomass pre-treatment product having a high concentration of 200 g-dry mass / L, in a short time of 48 hours from the start of the enzyme treatment, in a wild-type yeast, an ethanol yield of 35 g / L (72% (Yield to biomass total sugar ethanol) was achieved, and in the transformed yeast in which cellulose hydrolyzing enzyme was displayed on the cell surface, an ethanol yield of 42 g / L (86% yield of biomass total sugar ethanol) was achieved. The amount of cellulose hydrolase preparation added as 1FPU / g-dried biomass pretreatment product corresponds to about 1/100 of the conventional amount, and it was found that the amount of enzyme preparation added can be greatly reduced.

(Comparative Example 1: Simultaneous start of enzyme treatment / fermentation)
An enzyme-treated solution having the same composition as in Table 1 except that 50 g-dry mass / L of rice bran hydrothermally treated solid content was used instead of 200 g-dry mass / L of rice straw hydrothermally treated solid content of Example 1. This was put into a 200 mL capacity screw mouth bottle (manufactured by Duran), 2 mL of yeast suspension prepared in the same manner as in Example 1 was added, and the bacterial cell concentration was adjusted to 100 g- Wet weight / L. After yeast addition, enzyme treatment / fermentation was started while stirring the liquid at 35 ° C. 50 g-dry mass / L of rice straw hydrothermal treatment solid content was added 6 hours, 24 hours and 48 hours after the start of enzyme treatment / fermentation (finally 200 g-dry mass / L of rice straw hydrothermal treatment). Solids) and continued fermentation. FIG. 2 shows the change over time of the ethanol concentration produced in the fermentation broth determined in the same manner as in Example 1.

  As is apparent from FIG. 2, when the enzyme treatment and fermentation are started simultaneously, 96 hours or more from the start of enzyme treatment / fermentation to achieve an ethanol yield of 35 g / L from a biomass pretreatment product of 200 g-dry mass / L. It cost.

(Example 2: Ethanol fermentation 2)
Fermentation was carried out in the same manner as in Example 1 except that 2 hours after the start of enzyme treatment in Example 1 and 4 hours or 6 hours later, 2 mL of the yeast suspension was added to the enzyme treatment solution. . The ethanol concentration produced in the fermentation broth after 72 hours from the start of fermentation determined in the same manner as in Example 1 is shown in FIG.

  As is apparent from FIG. 3, the wild-type yeast has the highest ethanol yield after 6 hours of enzyme treatment, and the transformed yeast presenting cellulolytic enzyme on the cell surface shows the highest ethanol after 2 hours of enzyme treatment. The yield was found to be high.

(Example 3: Ethanol fermentation 3)
In the same manner as in Example 1, except that the cellulose hydrolase preparation of 4 FPU / g-dried biomass pretreatment product was used instead of the cellulose hydrolase preparation of 1 FPU / g-dry biomass pretreatment product of Example 1. Fermentation was performed. FIG. 4 shows the change over time in the ethanol concentration produced in the fermentation broth determined in the same manner as in Example 1.

  As is clear from FIGS. 1 and 4, 4FPU / g-dried biomass pre-treated cellulose hydrolase preparation can be obtained by using either wild-type yeast or transformed yeast displaying cellulosic hydrolase. It was found that there was almost no difference in ethanol yield between the cellulose hydrolase preparation of 1FPU / g-dried biomass pretreatment product, and the amount of enzyme preparation added could be reduced to about 1 FPU / g-dry biomass pretreatment product.

(Example 4: Ethanol fermentation 4)
Fermentation was carried out in the same manner as in Example 1, except that the enzyme treatment time or / and the amount of enzyme added in Example 1 were changed. The time-dependent change of the ethanol concentration produced in the fermented liquid quantified in the same manner as in Example 1 is shown in FIGS. FIG. 5 (a) shows the results of fermentation performed in the same manner as in Example 1 for comparison. FIG. 5 (b) shows that, in place of the cellulose hydrolase preparation of 10 FPU / g-dried biomass pretreatment product in Example 1 except that the cellulose hydrolase preparation of 10 FPU / g-dry biomass pretreatment product was used, The result of the fermentation performed in the same manner as in Example 1 is shown, and FIG. 5 (c) shows the yeast suspension in the enzyme treatment solution after 24 hours instead of 2 hours from the start of the enzyme treatment in Example 1. The result of the fermentation performed in the same manner as in Example 1 except that 2 mL was added is shown, and FIG. 5 (d) shows the cellulose hydrolase preparation of 1 FPU / g-dried biomass pretreatment product of Example 1. Instead, using a cellulose hydrolase preparation of 10 FPU / g-dried biomass pretreatment product, after 2 hours from the start of enzyme treatment in Example 1, after 24 hours, 2 mL of the above yeast suspension in the enzyme treatment liquid Except for adding, it shows the results of fermentation carried out in the same manner as in Example 1.

  As is clear from FIG. 5, using either a wild-type yeast or a transformed yeast on which cellulosic hydrolase is displayed on the cell surface, the ethanol yield can be increased with an enzyme treatment time of 24 hours and an enzyme treatment time of 2 hours. It was found that there was almost no difference and the enzyme treatment time could be shortened to about 2 hours. Moreover, there is almost no difference in ethanol yield between the cellulose hydrolase preparation of 10 FPU / g-dried biomass pretreatment and the cellulose hydrolase preparation of 1 FPU / g-dry biomass pretreatment, and the amount of enzyme preparation added is 1 FPU. It was found that the amount could be reduced to about / g-dried biomass pretreatment product. In particular, when transformed yeast with cellulosic hydrolase displayed on the cell surface was used, it was found that the ethanol yield was the highest in the 2-hour enzyme treatment time of the cellulose hydrolase preparation pretreated from 1 FPU / g-dried biomass. It was.

(Example 5: Ethanol fermentation 5)
Fermentation was carried out in the same manner as in Example 1 using the wild-type yeast while changing the enzyme treatment time and / or the amount of enzyme added in Example 1. 6A and 6B show the change over time of the ethanol concentration produced in the fermentation broth determined in the same manner as in Example 1. FIG.

  As is clear from FIG. 6, it was found that the wild-type yeast had the highest ethanol yield at the enzyme treatment time of 6 hours and the 2FPU / g-dried biomass pretreatment product.

  According to the method of the present invention, ethanol can be produced at low cost from lignocellulosic biomass using yeast. That is, the initial fermentation substrate concentration can be increased, the amount of enzyme preparation added for saccharification can be reduced, and the ethanol yield is significantly increased.

Claims (5)

A method for producing ethanol from lignocellulosic biomass,
Obtaining a cellulose fraction from lignocellulosic biomass,
The step of mixing the cellulose fraction having a solid content of 100 g-dry mass / L or more with a cellulose hydrolase preparation, and performing enzyme treatment for 0.5 to 10 hours to produce saccharified biomass, and yeast as the saccharified biomass A method comprising the steps of mixing and culturing.   The method according to claim 1, wherein the enzyme treatment time is 0.5 to 8 hours.   The method of Claim 1 or 2 whose solid content of the said cellulose fraction is 200 g-dry mass / L or more.   The method according to any one of claims 1 to 3, wherein the cellulose hydrolase preparation is an enzyme concentration of 1 to 4 FPU / g-dried biomass pretreatment product.   The method according to any one of claims 1 to 4, wherein the yeast is a yeast transformed to present at least two types of cellulose hydrolase on the cell surface.

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