JP2014187905A - Ethanol production method at low temperature using south pole-originated yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique to produce biomass ethanol that enables ethanol fermentation and simultaneous saccharification fermentation from biomass in a low-temperature environment.SOLUTION: This invention provides an ethanol fermentation method and a simultaneous saccharification fermentation method at a low temperature by using Mrakia fungi comprising Mrakia blollopis, South Pole-originated basidiomycete yeast, upon ethanol fermentation and simultaneous saccharification fermentation from biomass.

Description

  The present invention relates to a method for producing ethanol from sugar or biomass enzyme saccharified solution using Antarctic basidiomycetous yeast at a low temperature. Furthermore, this invention relates to the manufacturing method of the ethanol which carries out simultaneous saccharification fermentation from biomass by low temperature using the Antarctic basidiomycetous yeast.

  Japan, which has a lush green land with approximately 70% of it surrounded by forests, is rich in unused lignocellulosic biomass such as cedar, rice straw and wheat straw. Such unused lignocellulosic biomass not only contributes to the production of biomass ethanol from non-food resources, but also the earth for producing sugar as food raw materials, chemical industrial raw materials, or fermentation raw materials after depletion of petroleum resources. It is the most important carbon source above (Non-Patent Document 1). However, the lignocellulosic biomass that exists in nature has a complex structure in which cellulose, hemicellulose, and lignin are intertwined. Therefore, the structure of lignin and cellulose / hemicellulose is destroyed by physical pulverization using a cutter mill or alkali treatment. After the pretreatment (Non-patent Document 2) or a combination of the aforementioned pretreatments, enzyme treatment is performed. Despite these pretreatments, the enzymatic saccharified lignocellulosic biomass has not reached a level that can be actually used in terms of price and fermentation efficiency in conjunction with subsequent ethanol fermentation, etc. References 3, 4). This is because the activity of cellulase and β-glucosidase is inhibited by accumulation of glucose derived from cellulose, which is a product of saccharification by cellulase, and mannose, galactose and xylose derived from cellobiose and hemicellulose (Non-Patent Document 5). ). There is a technique called simultaneous saccharification and fermentation as a countermeasure for inhibiting the activity of the enzyme due to the accumulation of these saccharides. This technology is expected as a method to improve the saccharification reaction and fermentation efficiency because the saccharification reaction by cellulase and the fermentation by yeast etc. can be performed at the same time, so that the sugar concentration produced by the enzyme can be suppressed to a low concentration. (Non-Patent Document 6).

  In general, the optimum activity temperature of cellulase is around 50 ° C to 60 ° C, and the optimum fermentation temperature of yeast is around 30 ° C. (Non-Patent Document 7). So far, chilling basidiomycetous yeasts have been reported from various polar regions such as Arctic, Siberia, Alaska, Alps, Patagonia, Antarctica (Non-patent Document 8). Although Murachia is also known as a basidiomycete that undergoes ethanol fermentation at low temperatures, there has never been a report that high concentrations of 2% (v / v) or more ethanol were produced from sugars (Non-patent Document 9). ).

  By the way, more than 20% of Japan's land is occupied by areas where temperatures drop to around 0 ° C in winter. In order to perform fermentation and simultaneous saccharification and fermentation in such areas in winter, the saccharification / fermentor must be kept warm, which entails significant energy costs and increases the price of biomass ethanol.

Patent Publication 2011-115063 Yuko Igarashi et al: Saccharification method of bark and simultaneous saccharification and fermentation method Patent Publication 2011-55715 Shunsuke Hayashi et al.: Ethanol Production Method by Simultaneous Saccharification and Fermentation Patent Publication 2012-179021 Shunsuke Hayashi et al: Method for producing ethanol from waste

J. Nowak et al .: Composite structure of wood cells in petrified wood. Materials Science and Engineering: C, 25, 119-130, 2005 Y. Yamashita et al: Effective enzyme saccharification and ethanol production from Japanese cedar using various pretreatment methods. Journal of Bioscience and Bioengineering, 110, 79-86, 2010 Kosuke Kimoto: Bioethanol production process from wood-based raw materials. Mitsui Engineering & Shipbuilding Technical Report, 201, 24-29, 2010 Kenji Asano et al.: Bioethanol production cost and CO2 reduction cost analysis in Japan. [Online] AIST / BTRC DISCUSSION PAPER, July, 2007, Internet https://unit.aist.go.jp/btrc/research_result/documents/DISCUSSIONPAPER .pdf Z. Xiao et al .: Effect of Sugar Inhibition on Cellulases and β- Glucosidase During Enzymatic Hydrolysis of Softwood Substrates. Applied Biochemistry and Biotechnology. 113-116, 1115-1126, 2004 I. Ballesteros et al .: Selection of ThermotolerantYeasts for Simultaneous Saccharification and Fermentation (SSF) of Cellulase to Ethanol. Applied Biochemistry and Biotechnologu. 28-29, 307-315, 1991 L. Dwiarti et al: Simultaneous saccharification and fermentation of paper sludge without pretreatment using cellulase from Acremonium cellulolyticus and thermotolerant Saccharomyces cerevisiae. Biomass and Bioenergy. 42, 114-122, 2012 E. Branda et al: Yeast and yeast-like diversity in the southernmost glacier of Europe (Calderone Glacier, Appenines, Italy). FEMS Microbiology Ecology. 72, 354-369, 2010 SR Thomas-Hall et al: Cold-adaped yeasts from Antarcticaand the Italian Alps-description of three novel species: Mrakia robertii sp. Nov., Mrakia blollopis sp. Nov. And Mrakiella niccombii sp. Nov. Extremophiles (2010) Vol. 14, pp.47-59

The present invention includes the following inventions.
(1) A method for producing high-concentration ethanol at low temperatures using fungi belonging to the genus Murachia.
(2) A method of producing bioethanol from a biomass enzyme saccharified solution at low temperatures using fungi belonging to the genus Murachia.
(3) A method for producing bioethanol from biomass by simultaneous saccharification and fermentation at low temperatures using fungi belonging to the genus Murachia.
(4) A method for producing bioethanol from biomass by simultaneous fermentation with saccharification at low temperatures by adding a nonionic surfactant.
(5) The bioethanol production method according to any one of (2) to (4) above, wherein the biomass is derived from eucalyptus.

  An object of the present invention is to prevent a saccharification / fermenter from being heated in the winter when the outside air temperature is low, or to keep the energy cost used for heating low. It is to provide a microbial conversion method for efficiently performing ethanol fermentation from a biomass saccharified liquid such as lignocellulosic biomass. A second object of the present invention is to provide a microbial conversion method for efficiently performing simultaneous saccharification and fermentation of biomass such as lignocellulosic biomass.

  By the way, in recent years, a fast-growing eucalyptus-derived biomass has attracted attention as a lignocellulosic biomass raw material. However, eucalyptus wood saccharified liquid has also been reported to be insufficient in ethanol production by the fermentation microorganism method. Accordingly, it is a third object of the present invention to provide a method capable of efficiently performing ethanol fermentation at a low temperature even using biomass derived from eucalyptus.

  As a result of intensive research to achieve the above-mentioned problems, the present inventors have found that basidiomycetous yeasts such as Antarctic Murachia brolopis produce high concentrations of ethanol from high concentrations of sugars at low temperatures. . And, the present inventors can also saccharified liquid prepared from biomass, including eucalyptus-derived biomass, and ethanol fermentation at low temperature. Furthermore, when nonionic surfactant is added and lignocellulosic biomass is co-fermented It discovered that fermentation efficiency improved further and came to complete this invention.

  The present invention has an excellent effect that ethanol can be efficiently provided from biomass at a low temperature. More specifically, for example, according to the present invention, 45 g / l or more ethanol can be provided from 120 g / l saccharide at low temperature. Furthermore, according to the present invention, ethanol can be efficiently provided at low temperature from biomass derived from eucalyptus, which has been difficult in the past. Further, according to the present invention, for example, by performing simultaneous saccharification fermentation from 100 g / l lignocellulosic biomass, it becomes possible to provide at least 11 g / l ethanol.

Residual glucose concentration (▲) and ethanol concentration (●) when ethanol fermentation with Mrakia blollopis was performed under the conditions of Example 1. Residual glucose concentration (▲) and ethanol concentration (●) when ethanol fermentation with Mrakia blollopis was performed under the conditions of Example 2. Glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation with Mrakia blollopis was performed under the conditions of Comparative Example 1. The glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation with Mrakia blollopis was performed under the conditions of Example 3. The glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation with Mrakia blollopis was performed under the conditions of Example 4. The glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation with Mrakia blollopis was performed under the conditions of Example 5. The glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation with Mrakia blollopis was performed under the conditions of Example 6.

1. TECHNICAL FIELD The present invention provides a method for producing a low-temperature high-concentration alcohol capable of producing a high-concentration alcohol at a low temperature. More specifically, the present invention provides a method for producing a high-concentration alcohol from a sugar solution at a low temperature by using Murachia fungi.

  The genus Murchia used in the present invention may be any strain as long as it is capable of ethanol fermentation at low temperatures, but is preferably a strain capable of growth and ethanol fermentation at a low temperature of 10 ° C. or lower. Specific examples of such strains include Murachia brolopis, a fungus belonging to the genus Murchia belonging to the family Basidiomycota agaricus Agaricus phyllum fungus, Cystophilobasidium, Cystophilobasidium, and most preferably Murachia. -Brolopis SK-4 strain (Mrakia blollopis) FERM P-22126 strain is mentioned.

  In the present invention, the low temperature means a low temperature of room temperature of 20 ° C. or less, preferably a range of −1 ° C. to 20 ° C., more preferably a range of 4 ° C. to 15 ° C. Can do.

  In the method for producing high-concentration alcohol at low temperature, the raw material is sugar, specifically, one or more selected from the group consisting of glucose, sucrose, fructose, galactose, maltose, lactose, raffinose, trehalose, and melibiose. Sugar can be used. In any case of glucose, fructose, and sucrose, a sugar concentration of, for example, 1 to 200 g / l can be used. As ingredients other than sugar, starch, wheat bran, and / or whey may be included in the raw material.

In the method for producing low-temperature and high-concentration alcohol of the present invention, for example, about 48 g / l ethanol can be obtained at 10 ° C. by adding Murachia brolopis to about 120 g / l glucose solution.
During low temperature ethanol fermentation, the sugar solution can be stirred at about 10 to 200 rpm.

2. Low temperature ethanol production method using biomass Moreover, this invention provides the method of producing alcohol at low temperature from biomass.
More specifically, the present invention provides a method for producing a low-temperature alcohol from lignocellulosic biomass by using Murachia fungi.

  The genus Murchia used in the present invention may be any strain as long as it is a strain capable of ethanol fermentation at a low temperature, as in the above-mentioned 1. Low temperature high concentration alcohol fermentation method, for example, Muracia brolopis, More preferably, for example, Murakia brolopis SK-4 strain (Mrakia blollopis) (FERM P-22126 strain).

2-1. Biomass The biomass that is the subject of the present invention may be any biomass as long as a sugar solution can be prepared from the biomass. Examples thereof include cellulose-containing biomass such as lignocellulosic biomass.

  In the present specification, cellulose refers to a polymer in which glucose is polymerized by β-1,4 glucoside bonds and derivatives thereof. The degree of polymerization of glucose in cellulose is not particularly limited, but is preferably 200 or more. Examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. Cellulose may also contain cellooligosaccharide and / or cellobiose, which are partially decomposed products. Further, the cellulose may be crystalline cellulose or non-crystalline cellulose, but preferably contains crystalline cellulose. Furthermore, the cellulose is not particularly limited as long as it is derived from biomass. It may be derived from plants, fungi, and / or bacteria.

  Examples of the lignocellulosic biomass to be subjected to the method of the present invention include those obtained by converting a polysaccharide composed of cellulose and hemicellulose into a tree by lignin. The lignocellulosic biomass in the present invention includes hardwoods, conifers, bagasse, rice straw, corn stover, oil palm empty fruit bunches, other agricultural, forestry and fishery resources and wastes thereof, and wood fibers and wood derived therefrom. Also included are cellulosic biomass, cellulosic biomass, and wastes thereof with low or no lignin content such as chips, wood chips, pulp and waste paper. These biomass resources may be used in combination of two or more.

  Preferred lignocellulosic biomass is, for example, rice straw, bagasse, corn stover, oil palm empty fruit bunch, eucalyptus wood flour, and cedar wood flour.

2-2. Biomass saccharified liquor fermentation method In the biomass saccharified liquor fermentation method of the present invention, (1) first, biomass is saccharified to produce a biomass saccharified liquor, and then this biomass saccharified liquor is ethanol-fermented at a low temperature using the above Murcia fungus. And (2) a simultaneous saccharification and fermentation method in which low-temperature ethanol fermentation is carried out using the above-mentioned Murcia fungus that simultaneously saccharifies biomass.

2-2-1. Biotreatment of biomass Biomass for ethanol production is obtained in the form of waste materials, for example, if it is lignocellulosic biomass, but in order to efficiently saccharify it, it is usually crushed and dropped by an enzymatic method. In this case, a pretreatment is performed in which lignocellulose is decomposed by an enzyme.

  As the pretreatment in the present invention, any pretreatment method usually used in biomass saccharification may be employed. Examples of the pretreatment include (1) pulverization treatment, (2) steaming / explosion treatment or hydrothermal treatment, (3) dilute sulfuric acid treatment, (4) alkali treatment, (5) organosolv treatment, (6) biology Target pretreatment methods are known, and these can be combined. Preferably, mechanochemical treatment can be performed as pretreatment.

2-2-2. Saccharification of biomass Cellulose and hemicellulose in biomass are first decomposed into monosaccharides such as oligosaccharides or glucose by saccharification treatment. Examples of the saccharification treatment step include hydrolysis treatment with sulfuric acid and the like, and enzyme treatment.

  Examples of the enzyme used for the enzyme treatment include cellulase and hemicellulase, and the saccharification conditions of biomass cellulase and / or hemicellulase can be based on general conditions for saccharification of conventional biomass.

  Cellulase is a series of enzymes that decompose cellulose into glucose, and the cellulase is selected from these enzymes. Preferably, endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91) , And β-glucosinase (EC 23.2.4.1, EC 3.2.1.21).

  Although it does not specifically limit as a cellulase, It is preferable that it is a cellulase with high activity itself. Examples of such cellulases include Acremonium, Trichoderma, Fusarium, Phanerochaete, Tremetes, and Penicillium. In addition to Humicola, Aspergillus, and Murakia, Clostridium, Pseudomonas, Cellulomonas, and Luminococcus Examples include cellulases derived from actinomycetes such as (Ruminococcus) bacteria, Bacillus bacteria, Sulfolobus bacteria, Streptomyces bacteria, Thermoactinomyces bacteria, etc. . One or a combination of two or more of these cellulases can be used.

  Any hemicellulase may be used as long as it is a hemicellulase conventionally used for saccharification of biomass. Hemicellulase is a general term for an enzyme group that catalyzes an enzyme reaction system that hydrolyzes hemicellulose into xylose, arabinose, mannose, galactose, and the like. There are enzymes called by various names such as xylosidase and mannosidase. Although it does not specifically limit as hemicellulase, It is preferable that it is hemicellulase with high activity itself. One or a combination of two or more of these various hemicellulases can be used.

  The saccharification reaction may be performed at a low temperature within the working temperature range of the enzyme, for example, within the working temperature range of the enzyme. For example, a range of 40 ° C. or lower can be employed.

2-2-3. Ethanol fermentation of biomass saccharified liquid In the present invention, the biomass saccharified liquid is alcohol-fermented at a low temperature. The low temperature means a low temperature of room temperature of 20 ° C. or lower, similar to the conditions described in 1. Low-temperature high-concentration ethanol fermentation method, preferably in the range of −1 ° C. to 20 ° C., more preferably The range of 4 ° C to 15 ° C can be adopted. The genus Murchia used in the present invention is the same as the strain described in 1. Low temperature high concentration ethanol fermentation method. Specifically, the fungi belonging to the genus Murachia brolopis SK-4 (Merakia blollopis) (FERM P-22126) are included in the family Basidiomycota agaricus Agaricus phyllum fungus net cystophilobasidium cystophylobasidium family Murachia. The most preferred strain.

  For example, in a fermentation method using lignocellulosic biomass enzyme saccharified solution, about 17 g / l ethanol can be obtained at 10 ° C by adding Murachia broropis to a saccharified solution having a glucose concentration of about 41 g / l. it can.

2-2-4. Simultaneous saccharification of biomass As described above, it is known that the activity of cellulase is inhibited by the accumulation of glucose and cellobiose derived from cellulose, which is a product of the saccharification reaction by cellulase, in the reaction solution. In order to prevent this inhibition, saccharification and ethanol fermentation are performed simultaneously. Can be prevented. A method of simultaneously performing saccharification treatment and ethanol fermentation in this manner is called a simultaneous saccharification and fermentation method.

  Also in this invention, saccharification from biomass and ethanol fermentation can also be performed simultaneously. In the present invention, there is no particular limitation on the method and apparatus for simultaneous saccharification and fermentation of biomass such as lignocellulosic biomass, and known methods and apparatuses can be applied. The simultaneous saccharification reaction apparatus may be a batch type, a fixed bed reaction type, a continuous type, or the like.

  Moreover, in the simultaneous saccharification of biomass, the production amount of ethanol can be increased by further adding a nonionic surfactant. As the addition amount of the nonionic surfactant, for example, 0.1% (v / v) to 10% (v / v) can be added per 100 g / l lignocellulosic biomass. Preferably, by adding nonionic surfactant and Murakia broropis to 1% (v / v), it is about 1.1-1.6 times compared to simultaneous saccharification and fermentation of biomass without surfactant. The amount of ethanol produced can be obtained.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

[Example 1]
Ethanol fermentation from high-concentration glucose is 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l KH to 120 g / l glucose in 20 mM citrate buffer. ₂ Fermentation medium containing PO ₄ , 0.3 g / l MgSO ₄ 7H ₂ O in YPD liquid medium (10 g / l yeast extract, 20 g / l peptone, 20 g / l glucose) for 96 hours at 10 ° C, 8 ml of Murachia brolopis (FERM P-22126 strain) that had been pre-cultured under the condition of 120 rpm was centrifuged at 3000 × g 4 ° C. for 10 minutes to recover the cells. Bacteria collected after washing twice with sterilized distilled water and centrifuging again and suspending in 1 ml of sterilized distilled water were inoculated as an inoculation source and subjected to a fermentation test for 19 days at 10 ° C and 120 rpm. . A portion of the fermentation test solution was collected immediately after the start of the fermentation test, centrifuged at 12100 × g 4 ° C. for 5 minutes, and the supernatant was collected. The collected supernatant was then measured for glucose concentration and ethanol concentration by high-speed chromatography. The quantitative conditions for glucose were as follows: detector: JASCO RI-2031, column: Aminex HPX87P, mobile phase: ion-exchanged water, flow rate: 0.6 mL / min, column temperature: 80 ° C. The ethanol quantification conditions were as follows: detector: JASCO RI-2031, column: Aminex HPX87H, mobile phase: 5 mM H ₂ SO ₄ , flow rate: 0.6 mL / min, column temperature: 65 ° C. The results are shown in Figure 1. It was confirmed that the ethanol concentration was gradually produced from the start of fermentation, and the glucose concentration gradually decreased. Glucose was completely consumed 19 days after the start of fermentation. Under this condition, ethanol having an ethanol concentration of about 48 g / l was obtained after the fermentation.

[Example 2]
Enzymatic saccharification of lignocellulosic biomass was carried out by adding mechanochemically treated cedar wood flour to a concentration of 200 g / l in 20 mM citrate buffer (pH 4.5). Cellulase (6 FPU / g) and Genencor Optimash BG (20 μl / g) were added, and a hydrolysis reaction was performed while stirring at 37 ° C. and 120 rpm for 96 hours. After completion of the reaction, the saccharified solution was collected with a 0.22 μm filter unit, and the glucose concentration was measured under the conditions of Example 1. As a result of the measurement, a saccharified solution having a glucose concentration of about 40.9 g / l was obtained. Ethanol fermentation from lignocellulosic biomass enzyme saccharified solution was obtained by adding 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l KH ₂ PO ₄ , 0.3 g / l MgSO ₄ · 7H ₂ O was added, the inoculum that was cultured under the conditions of Example 1 was inoculated, and a fermentation test was performed at 10 ° C. and 120 rpm for 120 hours. A portion of the fermentation test solution was collected immediately after the start of the fermentation test, centrifuged at 12100 × g 4 ° C. for 5 minutes, and the supernatant was collected. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The result is shown in figure 2. After 96 hours from the start of the fermentation test, glucose was completely consumed, and finally 17.0 g / l of ethanol could be obtained.

[Comparative Example 1]
Enzymatic saccharification of lignocellulosic biomass was performed by adding eucalyptus wood powder treated with mechanochemicals to 20 mM citrate buffer (pH 4.5) to a concentration of 200 g / l. Cellulase (6 FPU / g) and Genencor Optimash BG (20 μl / g) were added, and a hydrolysis reaction was performed while stirring at 37 ° C. and 120 rpm for 96 hours. After completion of the reaction, the saccharified solution was collected with a 0.22 μm filter unit, and the glucose concentration was measured under the conditions of Example 1. As a result of the measurement, a saccharified solution having a glucose concentration of about 40.9 g / l was obtained. Ethanol fermentation from lignocellulosic biomass enzyme saccharified solution is obtained by adding 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l KH ₂ PO ₄ , 0.3 g / l MgSO ₄ · 7H ₂ O was added, the inoculum that was cultured under the conditions of Example 1 was inoculated, and a fermentation test was performed at 10 ° C. and 120 rpm for 120 hours. A portion of the fermentation test solution was collected immediately after the start of the fermentation test, centrifuged at 12100 × g 4 ° C. for 5 minutes, and the supernatant was collected. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in Figure 3. Glucose was not consumed up to 120 hours from the start of the fermentation test, and ethanol production was not observed.

[Example 3]
200 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l mechanochemically treated 20 mM citrate buffer (pH 4.5) After inoculating KH ₂ PO ₄ , 0.3 g / l MgSO ₄ .7H ₂ O and inoculating the inoculum that was cultured under the conditions of Example 1, Acremozyme cellulase manufactured by Meiji Co., Ltd. was added at 50 FPU / g, Genencor 20 μl / g of Optimash BG made by the company was added, and simultaneous saccharification reaction was performed with stirring at 10 ° C. and 180 rpm for 120 hours. A portion of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was recovered. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in FIG. About 7.2 g / l of glucose was produced 24 hours after the start of the simultaneous saccharification and fermentation test, and thereafter the glucose concentration decreased with the passage of the simultaneous saccharification and fermentation time. Finally, 12.5 g / l ethanol could be obtained.

[Example 4]
200 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l mechanochemically treated eucalyptus wood flour in 20 mM citrate buffer (pH 4.5) After inoculating KH ₂ PO ₄ , 0.3 g / l MgSO ₄ .7H ₂ O and inoculating the inoculum that was cultured under the conditions of Example 1, Acremozyme cellulase manufactured by Meiji Co., Ltd. was added at 50 FPU / g, Genencor 20 μl / g of Optimash BG manufactured by KK was added, and simultaneous saccharification reaction was performed with stirring at 10 ° C. and 180 rpm for 120 hours. A portion of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was recovered. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in FIG. About 11.9 g / l of glucose was produced 24 hours after the start of the simultaneous saccharification and fermentation test, and thereafter the glucose concentration remained almost constant. Finally, 14.2 g / l glucose remained, and a maximum of 7.1 g / l ethanol could be obtained.

[Example 5]
200 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / mechanochemically treated cedar wood flour in 20 mM citrate buffer (pH 4.5) l KH ₂ PO ₄ , 0.3 g / l MgSO ₄ · 7H ₂ O, 1% (v / v) Tween 80 as a nonionic surfactant was added, and the inoculum that was cultured under the conditions of Example 1 was planted. After sterilization, 50 FPU / g of Acremozyme cellulase manufactured by Meiji Co., Ltd. and 20 μl / g of Optimash BG manufactured by Genencor were added and subjected to simultaneous saccharification reaction while stirring at 10 ° C. and 180 rpm for 120 hours. A portion of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was recovered. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in FIG. About 1.5 g / l of glucose was produced 24 hours after the start of the simultaneous saccharification and fermentation test, and then the glucose concentration was almost digested by 120 hours of fermentation. A maximum of 13.2 g / l ethanol could be obtained.

[Example 6]
200 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l mechanochemically treated eucalyptus wood flour in 20 mM citrate buffer (pH 4.5) KH ₂ PO ₄ , 0.3 g / l MgSO ₄ · 7H ₂ O, 1% (v / v) Tween 80 as a nonionic surfactant was added, and the inoculum that was cultured under the conditions of Example 1 was inoculated Then, 50 FPU / g of Acremozyme cellulase manufactured by Meiji Co., Ltd. and 20 μl / g of Optimash BG manufactured by Genencor were added, and simultaneous saccharification reaction was performed while stirring at 10 ° C. and 180 rpm for 120 hours. A portion of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was recovered. The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in FIG. About 7.3 g / l glucose was accumulated 24 hours after the start of the simultaneous saccharification and fermentation test, and about 4.7 g / l glucose remained at the end of the reaction. Finally, a maximum of 11.6 g / l ethanol could be obtained.

  According to the present invention, high-concentration ethanol can be obtained from high-concentration glucose at a low temperature, which has been impossible in the past. In addition, for example, ethanol can be obtained from lignocellulosic biomass enzyme saccharified solution by fermentation at low temperature. Furthermore, when hydrolyzing lignocellulosic biomass, simultaneous saccharification and fermentation can be performed at low temperature by adding Murachia brolopis, an Antarctic basidiomycetous yeast.

  By adding a nonionic surfactant to the reaction vessel for simultaneous saccharification and fermentation, ethanol production can be increased 1.1 to 1.6 times. From this, not only can the cost of heating the reaction vessel be reduced at the time of bioethanol at a low temperature, but it can also be used for the production of alcoholic beverages at a low temperature.

Claims (5)

  A method for producing ethanol from biomass saccharified liquid at low temperatures using fungi belonging to the genus Murachia.   A method for producing ethanol from biomass by simultaneous saccharification and fermentation at low temperatures using fungi belonging to the genus Murachia.   A method for efficiently producing ethanol from biomass at a low temperature by simultaneous saccharification and fermentation using fungi belonging to the genus Murachia, which comprises adding a surfactant.   The method for producing ethanol according to claim 1, wherein the biomass is eucalyptus-derived biomass.   A method for producing high-concentration ethanol from sugar solution at low temperatures using fungi belonging to the genus Murachia.

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