JP2008054676A - Method for producing ethanol from biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing ethanol from a biomass. <P>SOLUTION: The method for producing the ethanol by using a lignocellulose-based biomass as a raw material, and carrying out a part or the whole of culturing, saccharifying and fermenting processes of saccharogenic amylase-producing microorganisms in the same vessel includes the following steps: preparing fine powder by subjecting the lignocellulose-based biomass to finely grinding treatment; culturing the microorganisms producing the saccharogenic amylase in a liquid medium containing cellulose or the like as a carbon source to induce/secrete the saccharogenic amylase and to prepare a culture liquid containing the saccharogenic amylase, regulating the temperature of the obtained culture liquid in the same vessel so as to match the set reaction condition of the saccharogenic amylase, carrying out the saccharification treatment under the set reaction condition by using the culture liquid, and converting the obtained saccharified liquid into the ethanol by adding a yeast and carrying out alcohol fermentation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a technology for converting ethanol from lignocellulosic biomass into ethanol by enzymatic saccharification and fermentation processes. More specifically, the present invention relates to a saccharification and alcohol fermentation process, that is, a saccharification enzyme is produced. The present invention relates to a new ethanol conversion process for lignocellulosic biomass that enables practical use of culturing microorganisms, saccharification using the saccharifying enzyme source, and part or all of the alcohol fermentation process in the same tank. The present invention is a new saccharification process of lignocellulosic biomass that can be substituted for the acid saccharification method that consumes a large amount of sulfuric acid and the like used in the saccharification process of lignocellulosic biomass containing a large amount of hemicellulose and lignin in addition to cellulose, And a new ethanol conversion technology for lignocellulosic biomass using the saccharification process.

  The cell structure that forms lignocellulosic biomass consists mainly of cellulose consisting of glucose (hexose), xylose (pentose), mannose (hexose), galactose (hexose), arabinose (pentose) ) Is composed of hemicellulose and lignin, and if they are compared to reinforced concrete, cellulose is the main reinforcing rod, hemicellulose is the wire that bundles them, and lignin is the concrete (binder) that fills the voids It is said that he plays a role like As a method for recovering sugars such as glucose from these lignocellulosic biomass raw materials, conventionally, lignocellulosic biomass raw materials are treated with a strong acid such as sulfuric acid, and hemicellulose and lignin that are first dissolved are separated and filtered. The residue was further treated with sulfuric acid or the like to recover sugars such as glucose from the remaining cellulose. However, since these methods consume a large amount of sulfuric acid, their disposal is an environmental problem.

  Thus, conventionally, in an ethanol production process from lignocellulosic biomass, saccharification is usually performed by hydrolysis with an acid. For example, a two-stage hydrolysis method using dilute sulfuric acid (dilute sulfuric acid method), etc. Has been proposed. In this method, after recovering the sugar solution obtained by the first stage hydrolysis, the second stage hydrolysis is performed on the undegraded residue, and the hydrolysis is performed in two stages, thereby allowing the monosaccharide to be decomposed excessively. It is said that it is possible to efficiently obtain monosaccharides from both hemicellulose and cellulose, but in the first stage hydrolysis, about 140-190 ° C., in the second stage hydrolysis, , Very severe conditions of about 180-240 ° C. are required.

  In general, many ethanol production processes using fermentation methods have been proposed for biomass raw materials. In this case, conventionally used biomass raw materials are limited to carbohydrate raw materials such as starch or sugarcane molasses. Yes. On the other hand, in the case of lignocellulosic biomass, as described above, it is mainly composed of cellulose and hemicellulose, and is the same sugar as starch and molasses, but the sugar binding state and the constituent monosaccharides. The types are different, and further, there is a feature that a lot of lignin is contained. Until now, pentoses such as xylose and arabinose obtained by decomposition of hemicellulose have not been able to produce practical ethanol by microorganisms in the natural world.

  In the process of producing ethanol from lignocellulosic biomass, in the fermentation process, hemicellulose-derived sugars are fermented, for example, in a genetically modified Escherichia coli with an alcohol-fermenting bacterial gene inserted into an E. coli host. Fermentation is known to be carried out using general yeast, but this type of method involves a multi-stage fermentation process, which means that the fermentation method, fermentation means, management of fermentation conditions, etc. are expensive and complicated. There was a problem, and further improvement was required to improve the productivity of the lignocellulosic biomass ethanol conversion process and to establish a practical technology.

  In addition, the development of practical technology for producing ethanol for fuel from construction waste wood, etc., a method of performing a fermentation process of lignocellulosic biomass with one type of fungus, and an enzymatic method replacing the dilute sulfuric acid method were used. Development of a saccharification process, development of a method for performing saccharification and alcohol fermentation in the same tank, and development of a technology for simultaneously performing saccharification and fermentation with one type of fungus are strongly expected. The fact is that it has not been developed yet.

  As prior art relating to saccharification and fermentation of lignocellulosic biomass, for example, a method of enzymatic saccharification of wood or the like (Patent Documents 1 to 4), and an alcohol via hexose such as glucose using wood or the like as a starting material Methods for producing ethanol by fermentation (Patent Documents 5 to 7), methods for producing ethanol via pentoses such as xylose (Patent Documents 8 and 9), and the like have been proposed.

  As another prior art, for example, hydrogen peroxide containing a small amount of tungstic acid or molybdate catalyst is used to selectively decompose lignin, and the resulting lignocellulose-based cellulose is efficiently treated with cellulase enzymes. A method for producing saccharified well and producing glucose, which is a raw material for ethanol fermentation, with high efficiency has been proposed (Patent Document 10).

  In addition, as a method and apparatus for efficiently decomposing components of cellulose, hemicellulose, and lignin using biomass resources as starting materials, for example, a cell treatment process with basidiomycetes or decaying fungi, The manufacturing method of the monosaccharide and / or oligosaccharide which has a high-pressure hot-water treatment process processed with hot water is proposed (patent document 11).

  In addition, wood is used to produce basic industrial chemicals and carbon materials such as methanol, methyl formate, ethanol, lactic acid, and propanediol, and at the same time, the heat, effective utilization of heat recovery, and power, steam, and hot water minus the in-house power As a method of effective utilization of wood zero emission type, which has high heat utilization in terms of equipment cost and operation cost, for example, after using wood as a raw material, hydrothermal treatment as pretreatment, and enzymatic saccharification A method for obtaining ethanol via a pentose such as xylose has been proposed (Patent Document 12).

  Thus, it has been strongly demanded to obtain biomass ethanol by saccharification (glucosylation) of lignocellulosic biomass, and various technologies for producing biomass ethanol using woody biomass as a raw material have been developed. In fact, a method for obtaining glucose as a raw material for ethanol fermentation with high purity from woody biomass has not been established. In particular, in the process of converting woody biomass to ethanol, a practical technology for simultaneous saccharification and fermentation of saccharification and alcohol enzymes in the same tank has not yet been established. It was a major cause that hindered full-scale use.

JP 2004-171799 A Japanese Patent No. 3041539 JP 2002-186938 A JP 2002-238590 A JP 2003-289893 A JP 2003-310246 A JP 2002-537848 A Special table 2001-51658 gazette US Pat. No. 6,582,944 JP 2006-149343 A JP 2005-27541 A JP 2006-111593 A

  Under such circumstances, in view of the prior art, the present inventors perform enzymatic saccharification and alcoholic fermentation in the same tank in place of acid hydrolysis technology such as dilute sulfuric acid method currently performed. As a result of intensive research with the goal of developing new saccharification and fermentation technologies that can be used, lignocellulosic biomass can be used as saccharification enzyme reaction tanks or ethanol fermentation tanks as they are. The present inventors have succeeded in developing a new ethanol conversion method and completed the present invention. An object of the present invention is to provide a novel ethanol production technology from lignocellulosic biomass in which a part or all of the culture, saccharification and alcohol fermentation processes can be performed in the same tank.

The present invention for solving the above-described problems comprises the following technical means.
(1) A method for producing ethanol by using a lignocellulosic biomass as a raw material and carrying out part or all of the cultivation, saccharification and fermentation processes of microorganisms producing saccharifying enzymes in the same tank, and 1) lignocellulosic biomass 2) Microorganisms that produce saccharifying enzymes are cultured in a liquid medium containing cellulose, hemicellulose, or sugars as a carbon source. 3) Inducing and secreting saccharifying enzymes 4) Prepare a culture solution containing saccharifying enzyme, 4) Adjust the temperature of the obtained culture solution in the same tank according to the set reaction conditions of saccharifying enzyme, 5) Set reaction using the culture solution 6) Ethanol, characterized in that saccharification is carried out under conditions, 6) ethanol is added to the obtained saccharified solution and alcohol fermentation is carried out to convert it into ethanol The method of production.
(2) A microorganism that produces saccharifying enzyme is cultured in a liquid medium using cellulose, hemicellulose, or saccharide as a carbon source, and a liquid medium using an inducer capable of inducing / secreting saccharifying enzyme as a carbon source; The method for producing ethanol according to (1) above, wherein the enzyme-induced culture is performed at a temperature of 40 ° C. to 40 ° C. in conformity with a set pH condition of the saccharifying enzyme.
(3) The method for producing ethanol according to (1), wherein the reaction temperature is adjusted to a range of 40 to 60 ° C. according to the set reaction conditions of the saccharifying enzyme.
(4) The method for producing ethanol according to (1), wherein the residue of the saccharification treatment is collected and reused as fuel and / or fertilizer.
(5) The method for producing ethanol according to (1), wherein the lignocellulosic biomass material is finely pulverized to 200 microns or less.
(6) The method for producing ethanol according to (5), wherein the apparent crystallinity of cellulose in the raw fine powder obtained by the fine pulverization is 10% or less.
(7) The method for producing ethanol according to (1) above, wherein a filamentous fungus producing cellulose-degrading enzyme is cultured to prepare a culture solution containing a saccharifying enzyme.
(8) The method for producing ethanol according to (1) above, wherein Trichoderma or Aspergillus-related bacteria are used as a microorganism that produces a saccharifying enzyme.

Next, the present invention will be described in more detail.
The present invention is a method for producing ethanol by using a lignocellulosic biomass as a raw material, and performing culture or saccharification and fermentation of microorganisms that produce saccharifying enzymes in the same tank, and producing ethanol. Finely pulverizing to prepare fine powder, culturing microorganisms producing saccharifying enzyme in liquid medium using cellulose, hemicellulose, or saccharide as carbon source, thereby inducing and secreting saccharifying enzyme, saccharifying enzyme Preparing a culture solution containing sucrose, adjusting the temperature of the obtained culture solution in the same tank according to the set reaction conditions of the saccharifying enzyme, and performing saccharification treatment using the culture solution under the set reaction conditions In addition, yeast is added to the resulting saccharified solution and alcohol fermentation is performed to convert it into ethanol. Examples of lignocellulosic biomass raw materials include, but are not limited to, corn residues, sugarcane bagasse, inawara, etc., as well as residual materials such as sawmills, forest land residuals, and building wood. If it is not lignocellulosic biomass, it can be used similarly.

  In the present invention, a microorganism producing a saccharifying enzyme is cultured in a liquid medium using cellulose or the like as a carbon source at 20 to 40 ° C. in conformity with the set pH condition of the saccharifying enzyme, or in accordance with the set reaction condition of the saccharifying enzyme. Thus, adjusting the reaction temperature in the range of 40 to 60 ° C. is a preferred embodiment. Cultivation of microorganisms that produce saccharifying enzymes is preferably carried out at, for example, 20 to 40 ° C. and pH 4 to 5. In this case, the pH conditions of the culturing are optimum pH conditions for saccharifying enzymes (set pH conditions). For this purpose, it is necessary to select a saccharogenic enzyme-producing microorganism that satisfies the conditions.

  In addition, since the temperature condition of the saccharification treatment is preferably several tens of degrees Celsius higher than the culture temperature of 20 to 40 ° C., the temperature of the obtained culture solution is the optimum reaction temperature of the saccharifying enzyme. According to (set reaction conditions), it adjusts in the range of 40-60 degreeC suitably, for example. In this case, the temperature of the culture solution is adjusted in the same tank without moving the culture solution. Therefore, at this stage, it is not always necessary to separate the bacterial cells. In this process, the residue is not collected, but if necessary, the operation of separating the bacterial cells cannot be prevented. Absent.

  As described above, in the present invention, a combination of a saccharifying enzyme-producing microorganism and a saccharifying enzyme is selected, and a suitable reaction condition (temperature and / or pH condition) of a process for preparing a saccharified solution by saccharification treatment is adjusted. The saccharifying enzyme-producing microorganism is cultured under the above-mentioned conditions. Subsequently, for example, the temperature condition for saccharification treatment is shifted to the high temperature side, and the temperature condition for fermentation is shifted to the low temperature side. In addition, since it can be used as it is as a fermentation broth, it is characterized in that part or all of the culture, saccharification, and fermentation processes are performed in the same tank. In order to satisfy such conditions, it is necessary to select and combine them so that the culture conditions of the microorganism producing saccharifying enzyme and the reaction conditions of the saccharification treatment with the saccharifying enzyme are well matched. The present invention realizes simultaneous culture, saccharification, and fermentation by combining microorganisms, saccharification enzymes, and saccharification treatment conditions that satisfy these requirements.

  The present invention is characterized in that a saccharified solution is prepared by a saccharification method of lignocellulosic biomass by an enzyme method instead of the conventional acid saccharification, and the saccharified solution is subjected to alcohol fermentation to obtain ethanol. In the present invention, four fungi (filamentous fungi) strains having the ability to saccharify biomass such as cellulose were isolated from the soil and used. These microorganisms are the biomass research center in the National Institute of Advanced Industrial Science and Technology, China Center. It is saved so that it can be sold at.

  In the present invention, the lignocellulosic biomass raw material is used after being pulverized. The raw material is pulverized by, for example, pulverizing wood chips to about 2 mm with an appropriate pulverizer and further pulverizing to about 20 microns with a planetary ball mill or the like. For example, using a planetary ball mill (planet ball mill model PULSEROSETE5, etc., manufactured by FRITSCH), 25 zirconium balls having a diameter of 16 mm and 25 g of wood chips are sealed in a container with a reaction capacity of 450 mL, and pulverized for 4 hours at 250 rpm. A fine powder of 20 microns and an apparent crystallinity of 10% or less of cellulose is prepared.

  In the present invention, useful microorganisms producing saccharifying enzymes were isolated and used by the following method. That is, a diluted suspension of soil collected from nature is applied to an agar plate medium containing cellulose as the only carbon source and a nitrogen source and mineral source appropriately added, and cultured at 30 ° C. for several days to several weeks. did. Microorganism colonies appearing on the agar plate are fished on a fresh sterile agar plate. Furthermore, in a liquid medium containing cellulose as the sole carbon source and appropriately adding a nitrogen source and a mineral source, a few at 30 ° C etc. Culture was performed for about one week from the day to obtain a culture solution.

  The above culture broth was reacted with wood flour such as pulverized eucalyptus as a substrate, and microorganisms capable of decomposing them to produce sugars such as glucose were screened. By this method, KIF-62 filamentous fungi (Trichoderma genus), KIF-68 filamentous fungus (Aspergillus genus), KIF-77 filamentous fungus (Trichoderma genus), KIF-78 filamentous fungus (Aspergillus genus) 4 strains were isolated.

  The above-mentioned microorganisms are cultured to prepare an enzyme culture solution. However, the present invention is not limited to these microorganisms, and is a saccharogenic enzyme-producing bacterium that is equivalent or similar to these microorganisms and is compatible with the method of the present invention. It can be used similarly if it is obtained. When the separated and selected microorganisms are further cultured in a liquid medium containing cellulose as a carbon source, saccharifying enzymes such as cellulase accumulate inductively in a high-concentration culture solution, and this is used as an enzyme solution (culture solution). . In this case, if necessary, the enzyme culture filtrate can be obtained by centrifuging and separating it from the cells.

Examples of the liquid medium for saccharifying enzyme induction production using cellulose as a carbon source include 15 g / L of polypeptone, 1 g / L of yeast extract, 5 g / L of sodium nitrate, 10 g / L of potassium dihydrogen phosphate, 1 g of magnesium sulfate · 7H ₂ O / L, including ferrous sulfate · 7H ₂ O 0.01 g / L, zinc sulfate · 7H ₂ O 0.01 g / L, manganese sulfate · 5H ₂ O 0.01 g / L, copper sulfate · 5H ₂ O 0.01 g / L A culture medium is illustrated as a suitable thing. The culture temperature is preferably 20 to 40 ° C. and the pH is preferably 4 to 5.

  Next, the saccharification reaction is carried out by adjusting the culture solution to the set reaction conditions. For example, when pulverized wood flour is mixed at about 10% to 30% with respect to the enzyme culture solution and reacted at 40 ° C. to 60 ° C. for several days at pH 3 to 6, a saccharified solution is obtained. In this case, if necessary, the saccharification reaction solution can be separated into a sugar-containing solution and an undecomposed residue such as lignin by centrifugation.

  Next, ethanol fermentation is performed by adding alcohol-fermenting bacteria (yeast) to the saccharified solution. Examples of ethanol-fermenting bacteria using hexose such as glucose as a carbon source include yeasts typified by Saccharomyces. Examples of ethanol-fermenting bacteria using pentoses such as xylose as a carbon source include yeasts typified by Pichia. However, it is not limited to these, and an appropriate alcohol-fermenting bacterium can be used.

  In the present invention, for example, yeast for ethanol fermentation is liquid-cultured, and 5 to 10% thereof is mixed with a saccharified solution containing sugar. In the case of Saccharomyces yeast, 30 ° C. with slow stirring under anaerobic anaerobic conditions. Incubate for 48 hours. In addition, when a pentose is used as a main carbon source, Pichia spp. Is used, and in this case, cultivation is performed at 30 ° C. with aeration and agitation for 24 to 100 hours.

  Purified ethanol can be obtained by directly introducing the fermentation broth into a predetermined ethanol distillation apparatus and distilling it at a high concentration. By omitting the microbial cell separation operation from the fermentation broth, it is possible to improve work efficiency and reduce costs. The fermentation broth can be centrifuged to separate into an ethanol-containing liquid and a yeast residue, and can be distilled with a distillation apparatus. Distiller residue such as dead cells discharged from the distillation apparatus or culture residues such as yeast discharged by centrifugation are collected and recycled as fertilizer or fuel raw material to be used as fertilizer and fuel. Can be

  The ethanol fermentation process of the present invention is shown in FIG. The optimal growth temperature of cellulase-related enzyme-producing bacteria such as KIF78 is around 30 ° C., whereby the optimum temperature for the saccharification reaction of enzymes such as cellulase produced is around 50 ° C. Therefore, when the enzyme reaction is carried out at 50 ° C., the enzyme is activated and the saccharification reaction is promoted to obtain a high-concentration saccharified solution, but the saccharifying enzyme-producing bacteria cannot survive at 50 ° C. and die. To do.

  Since the optimum culture temperature of yeast for converting glucose, xylose, etc. obtained by saccharification reaction to ethanol is around 30 ° C., ethanol is added to the saccharification culture solution containing dead KIF bacteria obtained by saccharification reaction. Even if yeast having fermentability is added and fermented at 30 ° C., sugar is converted to ethanol without any inhibition in ethanol fermentation.

  Table 1-2 describes the optimum pH, pH stability, optimum temperature, and thermal stability of cellulase and hemicellulase produced by the KIF strain.

  In the present invention, the saccharification and fermentation processes are partly or entirely performed in the same tank from the microorganism culture solution itself by utilizing the characteristics such as the optimum pH and temperature difference of the microorganism culture, enzyme, saccharification reaction, etc. Thus, lignocellulosic biomass can be efficiently converted to ethanol, which is the target substance. Further, in the present invention, unreacted residues and residues such as dead cells generated by saccharification reaction or ethanol fermentation can be recovered as a residue after the distillation operation and recycled as much as possible, such as fertilizer and fuel.

  Conventionally, in order to convert lignocellulosic biomass into ethanol, saccharification with acid and alcohol fermentation of the saccharified liquid must be performed in multiple stages. Even if enzymatic saccharification and alcoholic fermentation are combined, saccharification Since treatment and fermentation conditions differed greatly, it was necessary to perform these in separate steps. On the other hand, the present invention consolidates the conditions of microbial culture, saccharification treatment, and fermentation process so that at least other conditions excluding temperature conditions can be matched as much as possible. It implement | achieves implementing culture | cultivation of biomass, saccharification, and part or all of a fermentation process in the same tank.

  The present invention relates to culture, saccharification, and liquefaction using culturing, saccharification, and fermentation processes for lignocellulosic biomass raw materials, with at least temperature conditions as variable factors and other conditions (for example, pH conditions) as fixed factors. By constructing a fermentation system, some or all of these processes can be carried out in the same tank. In the present invention, it is important to include at least the temperature condition as the variable factor. For example, in the final fermentation process, there is no problem even if the nutrient composition is additionally added to change the medium composition. Moreover, in this invention, if each process of the said culture | cultivation, saccharification, and fermentation can be implemented by the same tank, without inhibiting other processes, in addition to the said temperature conditions, a variable factor can be increased suitably. Is possible.

  The present invention consolidates the above culture, saccharification and fermentation processes, and uses one tank provided with a temperature control device provided with a heater and / or cooling means, so that part or all of these processes can be used. Can be carried out in the same tank, thereby reducing the energy and cost of these processes and building a method for ethanol conversion of lignocellulosic biomass that can be put into practical use with a low environmental load. , To provide.

  The present invention includes at least a culturing process of a saccharifying enzyme-producing microorganism targeting lignocellulosic biomass raw material, a saccharification process using the saccharifying enzyme, and a part or all of the saccharification and fermentation processes of culturing saccharification and fermentation. Using common culture, saccharification, and fermenter that can be controlled and set to any temperature condition as the main variable factor, the temperature conditions of the above culture, saccharification, and fermentation are sequentially adjusted to suitable levels for each process. By adjusting and adjusting the composition of culture, saccharification and fermentation broth as needed, a part or all of these processes are carried out in the same tank. In the ethanol conversion process of lignocellulosic biomass feedstock, there has been no example that has made it possible to carry out each process of culturing, saccharification, and fermentation in a common culture tank or the like whose temperature is the main variable.

The following effects are exhibited by the present invention.
(1) It is possible to construct and provide a new ethanol conversion process for lignocellulosic biomass in which a part or all of the saccharification and fermentation processes can be carried out with high efficiency in the same tank from the cultivation of microorganisms producing saccharifying enzymes.
(2) It is possible to provide a practical and practical ethanol production technology capable of realizing ethanol production from lignocellulosic biomass raw material at high efficiency and low cost using the ethanol conversion process.
(3) It is possible to provide a co-cultivation and saccharification process by an enzymatic method that can replace an acid saccharification process that is a conventional saccharification method of lignocellulosic biomass.
(4) The lignocellulose system can be pulverized without chemical treatment, and wood can be saccharified under mild conditions using a microorganism-derived saccharifying enzyme without using chemicals.
(5) Glucose (hexose) from the cellulose component and hexose such as mannose and pentose such as xylose can be simultaneously saccharified and recovered from the hemicellulose component.
(6) Ethanol can be fermented from both glucose and xylose using an appropriate ethanol-fermenting microorganism as appropriate, and ethanol can be produced without waste.
(7) Since strong acids such as sulfuric acid are not used, waste liquid treatment is simple and environmental impact is low.

  EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.

In this example, KIF62 strain is used as a microorganism that produces saccharifying enzyme, and polypeptone 15 g / L, yeast extract 1 g / L, sodium nitrate 5 g / L, potassium dihydrogen phosphate 10 g are used as nutrient sources for enzyme-induced production. / L, magnesium sulfate _· 7H 2 O1g / L, ferrous _· 7H 2 _O0.01g / L sulfuric acid, zinc sulfate _· 7H 2 _O0.01g / L, manganese sulfate _· 5H 2 _O0.01g / L, copper sulfate, Disperse 100 mL of medium containing 0.01 g / L of 5H ₂ O and adjusting the pH condition to pH 5.0 according to the set pH condition of the saccharification reaction, and 4 g of pulverized eucalyptus powder for enzyme induction into a 500 mL Erlenmeyer flask. These were shake-cultured at 30 ° C. for 5 days to prepare a culture solution in which enzymes such as cellulase and hemicellulase were induced.

To 100 mL of this culture solution, 20 g of finely pulverized eucalyptus powder is added, the temperature of the culture solution is adjusted according to the set reaction temperature, and saccharification treatment by enzymatic saccharification reaction is carried out at pH 5.0 and 50 ° C. for 4 days. As a result, 100 mL of a saccharified solution containing 8 g of glucose and 2 g of xylose was prepared. The temperature of the saccharified solution is returned to 30 ° C., and as a nutrient source for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, 10 mL of a 10-fold concentration solution of pH 5.0 was added, and 10 mL of a yeast Saccharomyces cerevisiae seed solution having glucose utilization performance was added, followed by culturing under anaerobic conditions for 24 hours. Obtained 5 g of ethanol.

In this example, KIF68 strain is used as a microorganism producing saccharifying enzyme, and polypeptone 15 g / L, yeast extract 1 g / L, sodium nitrate 5 g / L, potassium dihydrogen phosphate 10 g are used as nutrient sources for enzyme-induced production. / L, magnesium sulfate _· 7H 2 O1g / L, ferrous _· 7H 2 _O0.01g / L sulfuric acid, zinc sulfate _· 7H 2 _O0.01g / L, manganese sulfate _· 5H 2 _O0.01g / L, copper sulfate, Disperse 1000 mL of medium containing 0.01 g / L of 5H ₂ O and adjusting the pH condition to pH 5.0 according to the set pH condition of the saccharification reaction, and 40 g of finely ground bay pine powder for enzyme induction into a 5000 mL Erlenmeyer flask. These were shake-cultured at 30 ° C. for 5 days to prepare a culture solution in which enzymes such as cellulase and hemicellulase were induced.

200 g of finely ground eucalyptus powder was added to 1000 mL of this culture solution, the temperature of the culture solution was adjusted according to the set reaction temperature, and saccharification treatment was performed by enzymatic saccharification while stirring at 50 ° C. for 6 days. 1000 mL of a saccharified solution containing 71 g of glucose and 8.2 g of xylose was prepared. The temperature of the saccharified solution is returned to 30 ° C., and as a nutrient source for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, Add 100 mL of 10 times concentration solution of pH 5.0, add 10% of the amount of saccharified solution (100 mL in this example), and further add 100 mL of Saccharomyces cerevisiae seed solution having glucose utilization performance, Culturing was carried out for 24 hours under anaerobic conditions to obtain 35 g of ethanol in the culture solution.

In this example, KIF77 strain is used as a microorganism producing saccharifying enzyme, and corn steep liquor 5 g / L, potassium dihydrogen phosphate 10 g / L, potassium nitrate 5 g / L, phosphorus as nutrient sources for enzyme-induced production. Acid 2ammonium 5g / L, Magnesium sulfate 7H ₂ O 1g / L, Ferrous sulfate 7H ₂ O 0.01g / L, Zinc sulfate 7H ₂ O 0.01g / L, Manganese sulfate 5H ₂ O 0.01g / L , 1000 mL of medium containing 0.01 g / L of copper sulfate · 5H ₂ O and 0.1 g / L of antifoaming agent SAG471, adjusted to pH 4.5 according to the pH conditions set for the saccharification reaction, and for enzyme induction Then, 40 g of finely ground bay pine powder was dispensed into a 2000 mL jar fermenter, and these were cultured with aeration and stirring at 30 ° C. for 5 days to produce cellulase and hemicellular The culture enzyme has been induced, such as zero were prepared.

200 g of finely ground bay pine powder is added to 1000 mL of this culture solution, the temperature of the culture solution is adjusted according to the set reaction temperature, and saccharification treatment is performed by enzymatic saccharification while stirring at 50 ° C. for 6 days. 1000 mL of a saccharified solution containing 84 g of glucose, 16 g of mannose, and 4 g of xylose was prepared. The temperature of the saccharified solution is returned to 30 ° C., and as a nutrient source for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, 100 mL of a 10-fold concentrated solution of pH 5.0 was added, and 100 mL of a yeast Saccharomyces cerevisiae seed solution having glucose utilization performance was added, followed by culturing under anaerobic conditions for 24 hours, and 48 g in the culture solution. Of ethanol was obtained.

In this example, KIF78 strain is used as a microorganism producing saccharifying enzyme, and corn steep liquor 5 g / L, potassium dihydrogen phosphate 10 g / L, potassium nitrate 5 g / L, phosphorus as nutrient sources for enzyme-induced production. Acid 2ammonium 5g / L, Magnesium sulfate 7HO 1g / L, Ferrous sulfate 7H ₂ O 0.01g / L, Zinc sulfate 7H ₂ O 0.01g / L, Manganese sulfate 5H ₂ O 0.01g / L, Sulfuric acid 5000 mL of medium containing 0.01 g / L of copper 5H ₂ O and 0.1 g / L of antifoaming agent SAG471, adjusted to pH 4.5 according to the pH conditions set for saccharification reaction, Disperse 250 g of pulverized eucalyptus powder into a 10 L jar fermenter, and culture by aeration and stirring at 30 ° C. for 8 days to obtain cellulase, hemicellulase, etc. An enzyme-induced culture broth was obtained.

To 5 L of this culture solution, 1000 g of finely pulverized eucalyptus powder was added, the temperature of the culture solution was adjusted according to the set reaction temperature, and saccharification treatment was performed by enzymatic saccharification reaction while stirring at 50 ° C. for 6 days. 5 L of a saccharified solution containing 400 g of glucose and 80 g of xylose was prepared. The temperature of the saccharified solution is returned to 30 ° C., and as a nutrient source for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, 500 mL of a 10-fold concentration solution of pH 5.0 is added, and further 500 mL of a yeast Saccharomyces cerevisiae seed solution having glucose utilization performance is added, cultured for 24 hours under anaerobic conditions, and fermented with 190 g of ethanol. A liquid 6L was obtained.

  Thereafter, 500 mL of the same concentrated nutrient source as above is added to the culture solution, and 500 mL of Pichia stititis seedling solution having xylose fermentation ability is added to the culture solution, and the mixture is aerobically stirred at 30 ° C. As a result of cultivation for 72 hours, 10 g of ethanol was further accumulated, and a total of 200 g of ethanol was accumulated in about 7 L of the culture solution.

In this example, KIF77 strain is used as a microorganism that produces saccharifying enzyme, and polypeptone 15 g / L, yeast extract 1 g / L, sodium nitrate 5 g / L, potassium dihydrogen phosphate 10 g as nutrient sources for enzyme-induced production. / L, magnesium sulfate _· 7H 2 O1g / L, ferrous _· 7H 2 _O0.01g / L sulfuric acid, zinc sulfate _· 7H 2 _O0.01g / L, manganese sulfate _· 5H 2 _O0.01g / L, copper sulfate, Disperse 1000 mL of medium containing 0.01 g / L of 5H ₂ O and adjusting the pH condition to pH 5.0 according to the pH conditions set for the saccharification reaction, and 50 g of pulverized eucalyptus powder for enzyme induction into a 5000 mL Erlenmeyer flask. These were shake-cultured at 30 ° C. for 5 days to prepare a culture solution in which enzymes such as cellulase and hemicellulase were induced.

100 g of birch-derived xylan is added to 1 L of this culture solution, the temperature of the culture solution is adjusted according to the set reaction temperature, and saccharification treatment is performed by enzymatic saccharification while stirring at pH 4.0 and 50 ° C. for 5 days. Thus, 1000 ml of a saccharified solution containing xylose 63 was prepared. The temperature of the saccharified solution is returned to 30 ° C., and further, as nutrients for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, A 10% concentration solution of pH 5.0 was added in a volume of 10% of the amount of saccharified solution (100 ml in this example), and 100 mL of a yeast Pichia stipitis seedling solution having xylose-assimilating performance was added at 30 ° C. The cells were cultured for 72 hours under aerobic conditions with aeration and agitation, and 12 g of ethanol was obtained in the culture solution.

In this example, KIF77 strain is used as a microorganism that produces saccharifying enzyme, and polypeptone 15 g / L, yeast extract 1 g / L, sodium nitrate 5 g / L, potassium dihydrogen phosphate 10 g as nutrient sources for enzyme-induced production. / L, magnesium sulfate _· 7H 2 O1g / L, ferrous _· 7H 2 _O0.01g / L sulfuric acid, zinc sulfate _· 7H 2 _O0.01g / L, manganese sulfate _· 5H 2 _O0.01g / L, copper sulfate, 500 mL of medium containing 0.01 g / L of 5H ₂ O and adjusted to pH 5.0 according to the pH conditions set for the saccharification reaction, and 25 g of pulverized sugarcane bagasse powder for enzyme induction in a 5000 mL Erlenmeyer flask These were dispensed and cultured with shaking at 30 ° C. for 5 days to prepare a culture solution in which enzymes such as cellulase and hemicellulase were induced.

To 500 mL of this culture solution, 100 g of finely pulverized sugarcane bagasse powder is added, the temperature of the culture solution is adjusted according to the set reaction temperature, and the saccharification treatment is performed by enzymatic saccharification while stirring at pH 5.0 and 50 ° C. for 4 days. The saccharified solution 500 mL containing 32 g of glucose and 14 g of xylose was prepared. The temperature of the saccharified solution is returned to 30 ° C., and further, as nutrients for ethanol fermentation, yeast extract 2.5 g / L, diammonium phosphate 0.25 g / L, magnesium sulfate · 7H ₂ O 0.025 g / L, Add 50 mL of a 10-fold concentration solution of pH 5.0, and further add 50 mL of a yeast Saccharomyces cerevisiae seed solution having glucose assimilation performance, culture for 24 hours under anaerobic conditions, and fermentation containing 30 g of ethanol 600 mL of liquid was obtained.

  As described above in detail, the present invention relates to a method for producing ethanol from biomass, and according to the present invention, simultaneous culturing, saccharification and fermentation from biomass, that is, part of the culturing, saccharification and alcohol fermentation processes. Alternatively, a new ethanol conversion technology for lignocellulosic biomass capable of producing ethanol with a high efficiency, low cost, and low environmental load process by providing all in the same tank. it can. Further, according to the present invention, in the ethanol conversion process of lignocellulosic biomass, part or all of the saccharifying enzyme-producing microorganism culture process, saccharification process and saccharified liquid alcohol fermentation process are performed in the same tank, A new saccharification process using filamentous fungi that makes fermentation feasible can be provided.

2 shows the ethanol fermentation process of the present invention.

Claims (8)

  A method for producing ethanol by using a lignocellulosic biomass as a raw material and carrying out part or all of the cultivation, saccharification and fermentation processes of microorganisms producing saccharifying enzymes in the same tank. Fine powder is prepared by pulverization. (2) Microorganisms producing saccharifying enzymes are cultured in a liquid medium containing cellulose, hemicellulose, or saccharide as a carbon source. (3) Inducing and secreting saccharifying enzymes Producing a culture solution containing saccharifying enzyme, (4) adjusting the temperature of the obtained culture solution in the same tank according to the set reaction conditions of saccharifying enzyme, (5) using the culture solution The saccharification treatment is carried out under set reaction conditions. (6) The yeast is added to the obtained saccharified solution and subjected to alcoholic fermentation to convert to ethanol. Manufacturing method of Nord.   A microorganism producing a saccharifying enzyme is cultured in a liquid medium using cellulose, hemicellulose, or saccharide as a carbon source, and a liquid medium using an inducer capable of inducing / secreting saccharifying enzyme as a carbon source, at 20 ° C. to 40 ° C. The method for producing ethanol according to claim 1, wherein the enzyme-induced culture is performed at a temperature matching a set pH condition of the saccharifying enzyme.   The manufacturing method of ethanol of Claim 1 which adjusts reaction temperature to the range of 40-60 degreeC according to the setting reaction conditions of a saccharifying enzyme.   The method for producing ethanol according to claim 1, wherein the residue of the saccharification treatment is collected and reused as fuel and / or fertilizer.   The method for producing ethanol according to claim 1, wherein the lignocellulosic biomass raw material is finely pulverized to 200 microns or less.   The method for producing ethanol according to claim 5, wherein an apparent crystallinity of cellulose in the raw fine powder obtained by the fine pulverization is 10% or less.   The method for producing ethanol according to claim 1, wherein a culture solution containing a saccharifying enzyme is prepared by culturing a filamentous fungus that produces a cellulolytic enzyme.   The method for producing ethanol according to claim 1, wherein Trichoderma spp. Or Aspergillus spp. Is used as a microorganism that produces a saccharifying enzyme.

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