JP6167758B2 - Ethanol production method - Google Patents

Description

  The present invention relates to a method for efficiently producing ethanol by fermenting sugars in a medium containing acetic acid using yeast.

2. Description of the Related Art In recent years, technology development for producing energy and useful substances that have been conventionally produced from petroleum by using biomass, which is a renewable resource, has been promoted. By using biomass as an alternative to petroleum products, it is expected that the accumulation of CO ₂ that is one cause of global warming can be suppressed. Non-food edible biomass includes plants such as algae, herbs, and woods, building waste, and agricultural waste, but the most abundant on the planet is plant-derived cellulosic biomass . Cellulosic biomass is mainly composed of cellulose, hemicellulose and lignin, and the ratio varies depending on the raw materials. In woody biomass, cellulose is about 50%, hemicellulose is 20 to 25%, and lignin is 25 to 30%.

  In order to produce alcohols such as ethanol from cellulosic biomass, biomass pretreatment step, saccharification step of hydrolyzing cellulose and hemicellulose as pretreatment raw material to break down into monosaccharides, monosaccharides using microorganisms such as yeast It goes through a fermentation process that uses alcohol fermentation. Enzymatic saccharification that hydrolyzes cellulose and hemicellulose using enzymes and microorganisms that produce them as a fermentation substrate from polysaccharides in biomass resources is a method that has a low environmental impact. As being considered. On the other hand, concurrent saccharification and fermentation has attracted attention as a method for shortening the time required for saccharification and fermentation and increasing the efficiency of saccharification and fermentation.

  In parallel saccharification and fermentation, it is desirable that the optimum temperature and pH of the saccharifying enzyme coincide with the optimum temperature and pH of yeast growth, but the optimum temperature of commercially available fungal saccharifying enzymes is generally 50-60 ° C. In contrast, yeast growth is only around 30 ° C. Therefore, the function of saccharifying enzyme cannot be fully exhibited. Therefore, it is important to develop microorganisms that can grow and ferment at higher temperatures than sake yeast. As such a microorganism, Issatchenkia orientalis has been isolated (Patent Document 1). Patent Document 1 describes Isachenchia orientalis MF-121 strain having a combination of acid resistance, salt resistance, sugar resistance and alcohol resistance. Patent Document 2 describes a method of continuously producing ethanol by treating a cellulosic biomass-containing slurry in a parallel saccharification and fermentation treatment tank that simultaneously performs a saccharification reaction by cellulase and a fermentation reaction by Isachenchia microorganisms. ing.

  In Patent Document 3, a lignocellulosic raw material is added to water containing cellulose saccharifying enzyme together with water-soluble salts, and an enzymatic saccharification treatment is performed in an enzymatic saccharification treatment step as a raw material suspension adjusted to have an electric conductivity of 5 to 25 mS / cm. A method is described.

  In addition, Patent Document 4 includes the step of fermenting a raw material liquid containing monosaccharides using a yeast named Candida intermedia 4-6-4T2, and producing ethanol characterized in that A method is described.

  On the other hand, liquor yeast, which is a general yeast, and the above Isachenchia orientalis cannot assimilate xylose, and hemicellulose mainly composed of xylose contained in cellulosic biomass has not been used. In order to efficiently produce alcohols from cellulosic biomass, it is necessary to modify yeast so that xylose can be used, and various studies have been conducted. Among them, it has become clear that organic acids such as acetic acid and formic acid generated in the pretreatment process and saccharification process of cellulosic biomass have an adverse effect on the growth and fermentability of microorganisms. In addition, it has been reported that the fermentability of xylose decreases particularly in the fermentation process. In contrast, methods for imparting resistance to organic acids by modifying genes in the pentose phosphate cycle by genetic manipulation, methods for destroying genes that have the function of incorporating soluble small molecules into cells, and the like have been studied. It was. However, acetic acid resistance can be imparted according to these methods, but the degree of acetic acid resistance is not sufficient.

JP 2004-344084 A JP2011-139686A JP 2012-213375 A JP2013-94123A

  Cellulose-based biomass pretreatment products contain organic acids such as acetic acid and cause inhibition of ethanol fermentation. Therefore, means such as devising the pretreatment process and imparting acetic acid resistance are required. Further, compared to fermentation of hexoses such as glucose, fermentation of pentoses such as xylose is known to be more strongly inhibited by fermentation of acetic acid, and countermeasures are required. Problem to be solved by the present invention is to provide a method for producing ethanol that uses yeast that does not undergo fermentation inhibition of acetic acid in ethanol production and can improve ethanol production by the presence of acetic acid in the fermentation broth. It was.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventor has a sufficient ability of acetic acid resistance in yeast Isachenchia orientalis having ethanol fermentation ability, and further increases the ethanol production by adding acetic acid to the fermentation broth. I found out. Furthermore, the present inventor has found that even in a modified strain obtained by imparting pentose fermentation ability to Isachenchia orientalis by gene recombination, ethanol production increases due to the presence of acetic acid in the fermentation broth. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A method for producing ethanol, comprising a step of fermenting sugar in a medium containing acetic acid using Issatchenkia orientalis yeast.
(2) The ethanol production method according to (1), wherein the acetic acid concentration in the medium is 0.1 to 1.0% (w / v).
(3) The method for producing ethanol according to (1) or (2), wherein the Issatchenkia orientalis yeast is a yeast modified so that a xylose metabolic gene can be expressed.
(4) The method for producing ethanol according to (3), wherein the xylose metabolism gene is at least one gene of a xylose reductase gene, a xylitol dehydrogenase gene or a xylulokinase gene.

(5) The method for producing ethanol according to any one of (1) to (4), wherein the saccharide is glucose and / or xylose.
(6) The method for producing ethanol according to any one of (1) to (5), wherein the saccharide is a saccharide obtained by saccharifying cellulosic biomass using cellulase.
(7) The method for producing ethanol according to (6), wherein saccharification and fermentation of cellulosic biomass are simultaneously performed.

  According to the ethanol production method of the present invention, ethanol fermentation can be efficiently performed from hexose and pentose without being inhibited by fermentation even when the raw material for ethanol fermentation contains acetic acid. Moreover, in the ethanol production method of the present invention, ethanol production from hexose and pentose can be increased by adding an appropriate amount of acetic acid. That is, according to the present invention, it is not necessary to remove the organic acid from the cellulosic biomass pretreatment product, and it is not necessary to go through complicated steps such as imparting acetic acid resistance by genetic manipulation. Good ethanol production can be performed from hexose and pentose.

  Hereinafter, the present invention will be described in detail. Note that the descriptions of materials, methods, and numerical ranges described in this specification are not intended to be limited to the materials, methods, and numerical ranges, and other materials, methods, and numerical ranges are not intended. It does not exclude the use of such as.

  The ethanol production method of the present invention is a method comprising a step of fermenting a saccharide in a medium containing acetic acid using Issatchenkia orientalis yeast. The fermentation raw material obtained by pretreating cellulosic biomass contains an organic acid such as acetic acid, and the ethanol yield decreases due to fermentation inhibition. In the method of the present invention, by using Isachenchia orientalis yeast, it is possible to avoid yield reduction due to fermentation inhibition and to increase the ethanol yield by keeping the acetic acid concentration at an appropriate concentration.

  The concentration of acetic acid in the medium is not particularly limited, but is preferably 0.1 to 1.0% (w / v), more preferably 0.1 to 0.7% (w / v), Preferably it is 0.2 to 0.5% (w / v), and particularly preferably 0.3 to 0.5% (w / v).

  The yeast used in the present invention is not particularly limited as long as it is an Isachenchia orientalis yeast, but preferably a strain that can grow in the range of 37 ° C to 45 ° C and does not substantially produce a protease is preferable. Specific examples of Isachenchia orientalis yeast strains include the following yeast strains available from NBRC (Biological Resource Center, NITE). For example, yeast strains represented by NBRC numbers 0011, 0012, 0013, 0155, 0201, 0584, 0841, 1162, 1279, 1395, 1664, 10737. Particularly preferred is alcohol fermentative yeast MF-121 of Isachenchia orientalis yeast. This strain was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on May 22, 2003, and was assigned the deposit number FERM P-19368.

  Further, Isachenchia orientalis yeast may be immobilized. If the yeast is immobilized, the step of recovering the yeast in the next step can be omitted, or at least the burden on the recovery step can be reduced, and the risk of losing the yeast can also be reduced. In addition, although there is no merit as to immobilize the yeast, the yeast can be easily recovered by selecting the yeast having aggregability.

  As Isachenchia orientalis yeast, yeast modified so as to be able to express a xylose metabolism gene can also be used. In order for yeast that does not have pentose fermentability to use xylose, a xylose metabolic pathway is required. When it is necessary to use xylose, it is preferable to use yeast modified so that xylose metabolism gene can be expressed. Xylose metabolism genes require xylose reductase that converts xylose to xylitol, xylitol dehydrogenase that converts xylitol to xylulose, and xylulokinase that converts xylulose to xylulose 5-phosphate.

  The xylose metabolic gene may be used after being isolated from the alcohol-fermenting yeast strain MF-121 of Isachenchia orientalis yeast. Alternatively, the xylose metabolism gene may be used after being isolated from a pentose-fermenting species such as Pichia stipitis or Candida shehatae. For example, an xylose metabolic gene can be isolated from the yeast described above, an expression vector linked to a promoter can be constructed, and the expression vector can be introduced into Isachenchia orientalis yeast as a host. Thereby, yeast modified so that a xylose metabolism gene can be expressed can be produced.

  The promoter for controlling expression is not particularly limited, and a promoter capable of highly expressing a target gene can be used. For example, a glyceraldehyde 3-phosphate dehydrogenase gene (TDH3) promoter, a 3-phosphoglycerate kinase gene (PGK1) promoter, a hyperosmotic response 7 gene (HOR7) promoter, and the like are preferable. These promoters may be used after being isolated from the alcohol-fermenting yeast strain MF-121 of Isachenchia orientalis yeast. However, the organism species is not particularly limited as long as it is transformed into Isachenchia orientalis yeast to express the target gene.

  A method for introducing and expressing a xylose metabolic gene in a host yeast can be performed using a general molecular biological technique known to those skilled in the art. That is, it can be carried out by incorporating the target gene into an appropriate vector and transforming the host yeast using the vector. Further, a gene fragment containing an arbitrary region can be excised with a restriction enzyme or amplified by PCR and used for transformation.

  As a method for introducing a vector or gene fragment into a host cell, a conventionally known method can be used. Examples thereof include a calcium phosphate method or a calcium chloride / rubidium chloride method, an electroporation method, an electroinjection method, and a lithium acetate method. A yeast introduced with a vector can obtain a transformant by selection using a marker gene and selection by active expression.

  In order to grow the yeast of the present invention, any can be used as long as it contains a monosaccharide as an organic substrate. Glucose is preferred as the monosaccharide. The nitrogen source is not particularly limited, and ammonium salts such as ammonium sulfate, urea, corn steep liquor and the like can be used. It can also be grown on media containing yeast extract, malt extract, and the like. The pH of the medium is preferably weakly acidic and grows even at pH 3 to 2.

  The saccharide used as a fermentation raw material in the present invention means a monosaccharide such as glucose and / or xylose, and may be a saccharide obtained by saccharifying cellulosic biomass using cellulase.

  Examples of the cellulosic biomass used in the present invention include cellulosic biomass such as conifers, hardwoods, forest residue, building waste, pruning waste, agricultural waste such as sawdust, kenaf, rice straw, and wheat straw. Also, use cellulose or hemicellulose-based fibers that have been treated so that the enzyme is likely to act by chemical pulp production methods such as alkali extraction and alkali digestion, organosolv, etc. from the above cellulose biomass. It is preferable. Furthermore, it is also preferable to use a processed product obtained by subjecting cellulosic biomass to mechanical grinding and thermal and chemical pretreatment. For example, chemical pulp, waste paper, pulp factory sludge, treated forest residue, mechanochemical treated eucalyptus bark, and the like can be mentioned.

  As saccharification and fermentation of cellulosic biomass, the saccharification / fermentation separation method (SHF) that performs enzymatic saccharification of biomass and fermentation of saccharified solution in two stages may be used, or concurrent saccharification and saccharification and fermentation performed simultaneously Fermentation (SSF) may be used.

  In the case of the SHF method, the saccharification and fermentation pH is preferably 2 to 7, and the reaction temperature is preferably 30 to 60 ° C, more preferably 35 to 50 ° C. In the SSF method, the pH of saccharification and fermentation is preferably 2 to 7. The reaction temperature is preferably 30 to 60 ° C, more preferably 35 to 50 ° C.

  The type of cellulolytic enzyme used for saccharification of cellulosic biomass is not particularly limited as long as it can decompose cellulose. Cellulose degrading enzyme is an enzyme generically called so-called cellulase having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity. Each cellulose-degrading enzyme may be mixed with an enzyme having each activity in an appropriate amount. However, since many commercially available cellulase preparations contain hemicellulase, it is preferable to use a commercially available product. Commercial cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, the genus Humicola, and the like. There are cellulase preparations derived from, but not limited to. Commercially available products of such cellulase preparations are all trade names, for example, cellulosine T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor) ) And the like. Although the usage-amount of a cellulase formulation with respect to 100 mass parts of raw material solid content is not specifically limited, 0.5-100 mass parts is preferable and 1-70 mass parts is especially preferable.

  The culture solution discharged from the saccharification and fermentation steps is not particularly limited, but may be transferred to a solid-liquid separation step and separated into a liquid component (culture solution) and a solid component (residue). However, the solid-liquid separation step is not essential in the present invention. As an apparatus for performing solid-liquid separation, a screw press, a screen, a filter press, a belt press, a rotary press, a drum filter, a disk filter, or the like can be used. As the screen, a vibrating screen to which a vibrating device is added can be used. The liquid component (culture solution) separated in the solid-liquid separation step can be subsequently transferred to the distillation step.

  In the distillation step, ethanol, which is a fermentation product, can be separated by distillation using a vacuum distillation apparatus. However, the distillation step is not essential in the present invention. Since ethanol can be separated at a low temperature under reduced pressure, inactivation of the enzyme can be prevented. As the vacuum distillation apparatus, a rotary evaporator, a flash evaporator, or the like can be used.

  The distillation temperature is preferably 25 to 60 ° C. If it is lower than 25 ° C., it takes time to distill ethanol and productivity is lowered. On the other hand, when the temperature is higher than 60 ° C., the enzyme is heat-denatured and deactivated, and the amount of newly added enzyme increases, resulting in poor economic efficiency.

  The concentration of ethanol remaining in the liquid fraction (culture solution) after distillation is preferably 0.1% by mass or less. By setting it as such a density | concentration, the ethanol amount discharged | emitted with a solid substance in a subsequent solid-liquid separation process can be reduced, and a yield can be improved.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

<Acetic acid addition test 1>
A fermentation test using glucose as a substrate was performed using Isachenchia orientalis yeast strain MF-121.
The MF121 strain was cultured in 1200 ml of YMG medium with shaking at 37 ° C. for 24 hours under aerobic conditions. The composition of YMG medium is 0.5% (w / v) peptone, 0.3% (w / v) yeast extract, 0.3% (w / v) malt extract, 3% (w / v) glucose, pH 5.6 is there. The cells were collected by centrifugation, washed with sterile water, and suspended in 240 ml of sterile water. 0.1-0.7% (w / v) to fermentation medium (1% (w / v) corn steep liquor, 0.2% (w / v) urea, 3% (w / v) glucose) containing 10 ml of this cell suspension ) Acetic acid was added to adjust the pH to 5.0. Shaking culture was performed at 37 ° C. for 8 hours under microaerobic conditions. As a control, a test without acetic acid was performed in the same manner. The test was performed with n = 3.

The ethanol concentration after the culture was measured using a biosensor (Oji Scientific Instruments).
The results are shown in Table 1. Assuming that the ethanol production in the test group cultured without addition of acetic acid was 100%, the ethanol production increased in the test group added with 0.1 to 0.7% (w / v) acetic acid. In the test group cultured in the fermentation medium added with 0.4% (w / v) acetic acid, the ethanol production increased by 15% compared to the test group cultured without adding acetic acid.

<Acetic acid addition test 2 (pentose sugar fermentation strain)>
A xylose-fermenting Isachenchia orientalis yeast was prepared using Isachenchia orientalis yeast strain MF-121 according to the method described in the following document. The following documents describe methods practiced in yeasts such as the budding yeast Saccharomyces cerevisiae.

Ho, N. et al., Advances in Biochemical Engineering Biotechnology, Vol. 65, pages 163-192, 1999;
Eliasson, A. et al., Applied and Environmental Microbiology, Vol. 66, No. 8, pages 3381-3386, 2000; and
Matsushika, A. et al., Bioresource Technology, Vol. 100, pages 2392-2398, 2009).

  A xylose reductase gene (XR), a xylitol dehydrogenase gene (XDH) and a xylulokinase gene (XK), which are genes of the xylose metabolic pathway, were isolated from Isachenchia orientalis yeast strain MF-121 by PCR. In order to force the three genes to be expressed, the promoter regions of 3-phosphoglycerate kinase gene (PGK) and glyceraldehyde 3-phosphate dehydrogenase gene (TDH) as constant expression promoters were isolated from the MF121 strain by PCR. Using molecular biological techniques, DNA fragments were prepared by replacing the promoter regions of the XR gene, XDH gene, and XK gene with the promoter region of the PGK gene or TDH gene, and introduced into the multicloning site of the cloning vector. Orotidine-5′-phosphate decarbonase gene (URA3) isolated from MF121 strain was introduced into the cloning vector as a selection marker for selecting transformants. By the above operation, a cloning vector containing the URA3 gene as a marker gene and expressing the XR, XDH, and XK genes by the PGK or TDH promoter was prepared. As the host strain, a uracil auxotrophic strain, which was prepared by the UV method and complemented by the URA3 gene, was used. The MF121 strain was transformed by the lithium acetate method, and the target gene was introduced into the endogenous URA3 gene region by homologous recombination. A strain that grows in the minimum medium was isolated as a transformant, and after confirming that the target gene was introduced by PCR, a fermentation test using xylose as a substrate was performed, and a strain having a high xylose potency was selected.

  Using the produced xylose fermentable strain, a fermentation test using glucose and xylose as substrates was performed. Pre-culture was performed in the same manner as in <Acetic acid addition test 1>. The composition of the fermentation medium is 1% (w / v) corn steep liquor, 0.2% (w / v) urea, 0.5% (w / v) glucose, 3% (w / v) xylose), 0.1-0.7% ( w / v) acetic acid was added to adjust the pH to 5.0. As a control, a test without acetic acid was performed in the same manner. The test was performed with n = 3.

The ethanol concentration after the culture was measured using a biosensor (Oji Scientific Instruments).
The results are shown in Table 2. Assuming that the ethanol production in the test group cultured without adding acetic acid was 100%, the ethanol production increased in the test group added with 0.1 to 0.7% (w / v) acetic acid. In the test group cultured in the fermentation medium added with 0.4% (w / v) acetic acid, the ethanol production increased by 23% compared to the test group cultured without adding acetic acid.

<Acetic acid addition test 3>
A parallel saccharification and fermentation test using pulp as a substrate was performed using Isachenchia orientalis yeast strain MF-121. Pulp is eucalyptus globula chip heat treated in a chemical solution containing 30% sodium sulfite (w / v) and 1% sodium hydroxide (w / v) at 170 ° C for 1 hour, and then the disc clearance is 0.5mm with a refiner. What was ground at setting and washed with water was used. The MF121 strain was inoculated into 50 ml of YMG medium and cultured under shaking at 37 ° C. for 8 hours under aerobic conditions to obtain a pre-cultured bacterial solution. The composition of YMG medium is 0.5% (w / v) peptone, 0.3% (w / v) yeast extract, 0.3% (w / v) malt extract, 3% (w / v) glucose, pH 5.6 is there. Pre-culture is inoculated into 2L fermentation medium (1% (w / v) corn steep liquor, 0.2% (w / v) urea, 3% (w / v) glucose) in 5L jar fermenter, Yeast was grown overnight at 37 ° C. under aerobic conditions, with stirring at 190 rpm and pH 5.0.

  After yeast grew, 60 g of cellulase and 30 g of pulp were added at an absolute dry weight, and acetic acid was added to an acetic acid concentration of 0.4% (w / v) to start parallel saccharification and fermentation. The pH was 5.0, air was sent by a pump, and the air flow was controlled to 0.01 vvm. The pulp was added at an absolute dry weight of 30 g every 12 hours and cultured for 72 hours. As a control, a test without acetic acid was performed in the same manner. The test was performed with n = 2.

The ethanol concentration after the culture was measured using a biosensor (Oji Scientific Instruments).
The results are shown in Table 3. When the ethanol production in the test section where parallel saccharification and fermentation was performed without adding acetic acid was 100%, the ethanol production increased by 21% in the test section where acetic acid was added to 0.4% (w / v). did.

<Acetic acid addition test 4 (pentose fermentation strain)>
A parallel saccharification and fermentation test using pulp as a substrate was performed using the pentose fermentation strain used in <Acetic acid addition test 2>. Yeast was cultured in the same manner as in <Acetic acid addition experiment 3>, and acetic acid was added to an acetic acid concentration of 0.4% (w / v) at the start of parallel saccharification and fermentation. As a control, a test without acetic acid was performed. The test was performed with n = 2.
The ethanol concentration after the culture was measured using a biosensor (Oji Scientific Instruments).
The results are shown in Table 4. When the ethanol production in the test section where parallel saccharification and fermentation was performed without adding acetic acid was 100%, the ethanol production increased by 17% in the test section where acetic acid was added to 0.4% (w / v). did.

  From the above results, it was found that ethanol production increased by adding acetic acid to the culture solution.

Claims (6)

A method for producing ethanol, comprising a step of fermenting a saccharide in a medium containing acetic acid using Issatchenkia orientalis yeast, wherein the concentration of acetic acid in the medium is 0.1 to 1.0% (w / v) said method . The method for producing ethanol according to claim 1, wherein the Issatchenkia orientalis yeast is a yeast modified so that a xylose metabolic gene can be expressed. The method for producing ethanol according to claim 2 , wherein the xylose metabolism gene is at least one gene selected from a xylose reductase gene, a xylitol dehydrogenase gene, and a xylulokinase gene. The method for producing ethanol according to any one of claims 1 to 3 , wherein the saccharide is glucose and / or xylose. The method for producing ethanol according to any one of claims 1 to 4 , wherein the saccharide is a saccharide obtained by saccharifying cellulosic biomass using cellulase. The method for producing ethanol according to claim 5 , wherein saccharification and fermentation of cellulosic biomass are simultaneously performed.