JP2010000024A - New microorganism and method for producing ethanol using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a yeast that efficiently produces ethanol from glucose and xylose in the presence of the glucose and xylose, and a method for producing ethanol using the same. <P>SOLUTION: There is provided a microorganism belonging to Pachysolen tannophilus producing ethanol from glucose and xylose in the presence of the glucose and xylose. Among these, Pachysolen tannophilus MKY0802OK (Deposit No.FERM ABP-10965) which does not accept catabolite repression from glucose is preferably used. Ethanol can be efficiently produced using the microorganism from glucose and xylose which can be contained in a lignocellulose-based biomass hydrolyzate. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel microorganism and a method for producing ethanol using the same. Specifically, the present invention relates to a novel microorganism capable of efficiently producing ethanol from glucose and xylose in the presence of glucose and xylose, and a method for producing ethanol using the same.

  Since the industrial revolution, fossil fuels such as oil, coal, and natural gas have been used in large quantities as energy resources. As a result, global warming due to greenhouse gases such as carbon dioxide is becoming increasingly serious today. Furthermore, it has been predicted that oil, which has been a major energy resource so far, has been available for several decades, and there is an urgent need to secure a new earth-friendly energy resource to replace these fossil fuels. It has become. One solution to these problems is to produce ethanol from biomass (renewable, bio-derived organic resources excluding fossil resources) and use this ethanol as a new energy resource. (Bioethanol) is being researched and developed around the world. Since carbon contained in bioethanol is derived from carbon dioxide in the atmosphere fixed by photosynthesis, the total amount of carbon dioxide in the atmosphere does not increase even when burned. Therefore, the future potential as renewable natural energy is expected.

  Under such circumstances, ethanol production using starch or sugar contained in edible parts such as corn and sugarcane as biomass is performed on a large scale in the United States, Brazil and the like. However, using these food resources as raw materials for bioethanol has started to raise concerns about rising food prices and serious food shortages in developing countries. Therefore, in the United States, in order to make effective use of lignocellulosic biomass in non-edible parts of crops, the country is working on research and development of cellulase, an enzyme that breaks down cellulose into glucose. In view of such a situation, the present inventor has developed an ethanol production technology using lignocellulosic biomass contained in leaves (eg, soft biomass) and wood (hard biomass), which are non-edible parts of crops. I have been working on development.

  In order to produce ethanol from lignocellulosic biomass, first, the lignocellulose is saccharified by hydrolysis. Glucose and xylose are mixed in the lignocellulose hydrolyzate obtained here. However, only a few types of microorganisms that can assimilate xylose and produce ethanol are known at present.

Non-Patent Document 1 reports Pichia stipilis, Candida shehatae, and Pachysolen tanophilus as microorganisms that can convert both glucose and xylose into ethanol. In addition, as described in Non-Patent Document 1, attempts have been made to introduce a series of enzyme genes involved in the reaction from xylose to ethanol into Saccharomyces cerevisiae having high conversion efficiency from glucose to ethanol.
Jeffries, W.M. J. et al. , And Jin Y. S. , Advanced in Applied Microbiology, 47, 221-268 (2000).

  However, when fermentation is performed in the coexistence of glucose and xylose using the microorganism described in Non-Patent Document 1, xylose is substantially reduced until almost all glucose is consumed due to catabolite repression (catabolic metabolite suppression) by glucose. Is not metabolized. Even if fermentation is performed using a microorganism into which an enzyme gene has been introduced, catabolite repression cannot be avoided in the presence of glucose and xylose. Therefore, even when the above-described conventional technique is used, when ethanol is produced from both sugars using a medium in which glucose and xylose coexist, there is a problem that productivity is low because fermentation takes a long time.

  Accordingly, an object of the present invention is to provide a microorganism that efficiently produces ethanol from glucose and xylose in the presence of glucose and xylose, and a method for producing ethanol using the microorganism.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that ethanol can be efficiently produced from glucose and xylose by using a microorganism belonging to Pachisolen tanophilus, which is excellent in ethanol-producing ability in the presence of glucose and xylose.

  That is, the present invention is a microorganism belonging to Pachisolen tanophilus that can produce ethanol from glucose and xylose in the presence of glucose and xylose.

  According to the present invention, ethanol can be efficiently produced from glucose and xylose in the presence of glucose and xylose.

  Hereinafter, preferred embodiments of the present invention will be described. The technical scope of the present invention should be determined based on the description of the scope of claims, and is not limited to the following embodiments.

  This embodiment relates to a microorganism belonging to Pachisolen tanophilus that can produce ethanol from glucose and xylose in the presence of glucose and xylose.

  The microorganism of the present invention may be any microorganism as long as it is a microorganism belonging to Pachisolen tanophilus that can produce ethanol from glucose and xylose in the presence of glucose and xylose. Of the microorganisms belonging to Pachisolene / Tanofilus, it is preferable to be a microorganism that does not receive catabolite repression by glucose. In the present specification, “not subject to catabolite repression by glucose” (hereinafter also simply referred to as “not subject to catabolite repression”) means that a microorganism assimilate glucose and xylose substantially simultaneously. To do. In other words, it means that the microorganism assimilate xylose in the presence of glucose. Specifically, the concentration of glucose present in the medium is 0.1 g / L or more, preferably 0.5 to 20 g / L, more preferably 1 to 20 g / L, still more preferably 2 to 18 g / L, particularly It means that assimilation of xylose is started at a time point of preferably 3 to 15 gL, most preferably 5 to 13 g / L.

  The microorganism not subjected to such catabolite repression is a newly discovered microorganism by the present inventor, Pachisolen Tanophilus MKY0802OK (Pachysolen tanophilus MKY0802OK) (hereinafter also simply referred to as “the strain of the present invention”). Is more preferable. Hereinafter, Pachisolene and Tanofilus MKY0802OK will be described.

[Primary screening]
100 samples of 1 mL of wastewater were collected from 15 drains in Fujisawa City, Kanagawa Prefecture. A sample stock solution was prepared by suspending 1 mL of the waste water of the sample in 5 mL of sterile distilled water. The final diluted solution obtained by appropriately diluting this was applied to an agar medium containing rice straw hydrolyzate having the composition shown in Table 1 below using a corn large rod in 0.1 mL, and cultured in an incubator at 32 ° C. did. On the third day from the start of culture, colonies formed on the agar medium were picked up using platinum ears and transplanted to a slant medium (slant) containing rice straw hydrolyzate having the composition shown in Table 1 below. And the strain estimated to be 280 strains of yeast was isolated from all 100 samples.

[Secondary screening]
A 100 mL Erlenmeyer flask was charged with 10 mL of a culture medium containing rice straw hydrolyzate having the composition shown in Table 2 below, and 1 platinum loop of the strain isolated in the primary screening was inoculated into this culture medium. This was cultured with shaking at 32 ° C. for 5 days using a constant temperature shaking culture apparatus (rotating radius: 2 cm, rotating speed: 220 rpm). After completion of the culture, the culture solution was centrifuged to separate the cells and the supernatant, and total sugar, glucose, and ethanol contained in the supernatant were quantified. The quantification of total sugar is described in Michinori Nakamura, Junji Kakinuma, “Biochemical Experimental Method (25) Starch / Related Glycoenzyme Experimental Method”, published by the Japan Society for Publishing Center, October 1989, page 206. It carried out according to the sulfuric acid phenol method. Glucose was quantified using Wako Pure Chemical Industries, Ltd., glucose CII test Wako. The ethanol was quantified using an F-kit manufactured by JK International. The above operation was performed for all 280 strains obtained in the primary screening, and one strain having the best ethanol productivity was selected.

[Taxonomic properties]
The taxonomic properties of the strains screened above are described below.

  (A) Morphological properties

(B) Spore formation <Day 7 from the start of culture at 25 ° C. on a YM plate medium>
A tube that grew from the vegetative cell and became thicker, and an ascending sac-like organ was observed at the tip of the tube, but no ascospore formation was observed.

(C) Physiological properties In the table below, “+” indicates positive, “−” indicates negative, “W (weak)” indicates weak positive, and “S (slow)” indicates 2 to 3 weeks after the start of the test. “L (latant)” indicates that a positive reaction was rapidly recognized after 2 weeks before the start of the test.

(D) Identification As a result of BLAST (Altschul et al., 1997) homology search for Apollon DB-FU, the 26S rDNA-D1 / D2 base sequence of this strain is a reference strain of Pachsolen tanophilus, a kind of ascomycetous yeast. It showed 100% homology to NRRL Y-2460 ^T (Accession Number: U76346). The 26S rDNA-D1 / D2 nucleotide sequence of this strain is also found in P. cerevisiae in the results of homology searches against international nucleotide sequence databases such as GenBank / DDBJ / EMBL. It showed 100% homology to the tanophilus reference strain NRRL Y-2460 ^T (Accession Numbers: EU011641, U76346). In a phylogenetic tree created based on the top 10 base sequences obtained by homology search for apolone DB-FU, The same phylogenetic branch was formed with the tanophilus reference strain NRRL Y-2460 ^T (accession number: U76346), and the bootstrap value indicating the reliability of this phylogenetic branch was supported at 100%. From the above, in the result of the 26S rDNA-D1 / D2 nucleotide sequence analysis, It was presumed to be a bacterial species belonging to tanophilus.

  Moreover, as a result of simple morphological observation, the vegetative cells of this strain were observed to have a wide oval shape to an oval shape, and vegetative growth was observed to be due to multipolar budding. Consistent with the morphological characteristics of tanophilus (Kurtzman and Fell, 1998). Ascospore formation was not observed, but a thickened tube extending from the vegetative cells that characterize the genus Pachsolen, and formation of an ascending organ at the tip of the tube were observed.

  Furthermore, the results of the physiological property test were estimated from the results of 26S rDNA-D1 / D2 nucleotide sequence analysis to which the present strain was attributed. When compared with tanophilus (Kurtzman and Fell, 1998), this strain exhibits galactose fermentability and does not assimilate succinic acid. Although it showed different properties from Tanophilus, P. It showed properties similar to tanophilus and supported the results of 26S rDNA-D1 / D2 nucleotide sequence analysis.

  From the results of the above 26S rDNA-D1 / D2 nucleotide sequence analysis, simple morphology observation, and physiological property test, although ascospore formation was not observed in the simple morphology observation, The thickened tube extending from vegetative cells, which is the characteristic of tannophilus, and the ascending sac-like organ at the distal end of the tube were observed. Considering comprehensively from the fact that it was almost supported, this strain was judged to be a novel bacterium belonging to Pachysolen tanophilus, and named Pachysolen tanophilus MKY0802OK (Pachysolen tanophilus MKY0802OK).

  In addition, this strain was deposited on May 13, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center under the name MKY0802OK under the accession number FERM ABP-10965.

[Ethanol production method]
Ethanol can be produced by culturing the above-described microorganism of the present invention in a medium. Hereinafter, a method for producing ethanol using the microorganism of the present invention will be described.

  As a medium for culturing the microorganism of the present invention, a conventionally known medium can be appropriately employed as long as it is a medium capable of growing the microorganism. The nutrient sources contained in the medium include commonly used carbon sources, nitrogen sources, inorganic substances, and the like.

  Examples of the carbon source include saccharides such as monosaccharides such as glucose, galactose, fructose, and xylose, disaccharides such as maltose, lactose, and sucrose, and sugar alcohols such as glycerin and xylitol. Can be used in combination with more than one species. In the present invention, among these carbon sources, glucose and xylose are preferably included. According to the microorganism of the present invention, ethanol is efficiently produced from glucose and xylose because it does not undergo catabolite repression even in the presence of glucose and xylose. Therefore, the effect of the present invention becomes more remarkable by including glucose and xylose as carbon sources in the medium.

  Microorganisms that undergo conventional catabolite repression do not start assimilating xylose until the concentration of residual glucose in the medium is approximately 0 g / L. In addition, microorganisms that are subject to conventional catabolite repression, like general microorganisms, have a fermentation rate that decreases as the concentration of assimilable nutrients decreases, so almost all glucose is consumed after the glucose concentration decreases. It takes a long time to do. For example, in a microorganism that receives general catabolite repression, it takes 3 to 4 days for the glucose concentration to reach approximately 0 g / L after reaching 2 g / L. However, since the microorganism of the present invention is not subject to catabolite repression and can assimilate xylose even in the presence of glucose, the glucose concentration decreases from the above to a glucose concentration of almost 0 g / L. I don't need time. Therefore, even in a medium containing glucose and xylose, ethanol can be efficiently produced from glucose and xylose in a short time.

  As the nitrogen source, for example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium nitrate, amino acids such as urea and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, corn steep liquor and the like may be used as the nitrogen source. Among these, amino acids such as ammonium chloride, ammonium sulfate, urea, and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid, nitrogen-containing natural products such as peptone, meat extract, and yeast extract are preferable. These nitrogen sources can be used alone or in combination of two or more.

  Examples of inorganic substances include sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate, magnesium sulfate, ammonium sulfate, and other phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. Hydrochloride, sulfate, acetate and the like are used. In addition, vitamins such as thiamine and biotin, and if necessary, nucleic acid-related substances such as adenine and uracil may be used. These inorganic substances can be used alone or in combination of two or more.

  The above saccharides as a carbon source can be obtained by hydrolyzing carbohydrates such as starch and cellulose (hereinafter also referred to as “saccharification”). It can also be obtained by hydrolyzing biomass containing sugar, starch, and cellulose mentioned in the above carbon source. Examples of biomass include saccharide-based biomass mainly composed of sugar, starch-based biomass mainly composed of starch, and lignocellulose-based biomass mainly composed of lignocellulose. Specifically, sugar-based biomass includes sugarcane, sugar beet, sweet sorghum, and starch-based biomass includes rice, wheat, barley, corn, potato, sweet potato, cassava, and ligno. Examples of cellulosic biomass include rice straw, rice husk, wheat straw, bagasse, coconut husk, corn cob, weed, wood, pulp, and paper. Among these, it is preferable to use a hydrolyzate of lignocellulosic biomass as a carbon source. Since lignocellulosic biomass produces glucose and xylose by hydrolysis, the effect of the present invention can be further enhanced by adding a hydrolyzate of lignocellulosic biomass to the medium for culturing the microorganism of the present invention. Shall. In addition to the carbon sources such as glucose and xylose, the hydrolyzate of lignocellulosic biomass also contains a nitrogen source and an inorganic salt necessary for the growth of the microorganism of the present invention. It is not necessary to add salt or the amount added can be greatly reduced. Furthermore, since lignocellulosic biomass is not a food resource, there is no risk of increasing the food crisis.

  The said biomass hydrolyzes by a conventionally well-known method, and produces saccharide | sugar which is a carbon source. The hydrolysis method mainly includes acid decomposition and enzymatic decomposition, but it is preferable to use enzymatic decomposition from the viewpoint of environmental conservation. Examples of the acid used for the acid decomposition include sulfuric acid, hydrochloric acid, and nitric acid, and these can be used alone or in combination of two or more. Examples of the enzyme used for enzymatic degradation include amylase, cellulase, glucoamylase, xylanase, hemicellulase and the like. These hydrolases can be used alone or in combination of two or more depending on the type of polysaccharide contained in the biomass. For example, since the above lignocellulosic biomass mainly includes cellulose, hemicellulose, lignin and the like, cellulase or hemicellulase can be used alone or in combination with cellulase and hemicellulase. In addition, hydrolases such as Maycellase (manufactured by Meiji Seika Co., Ltd.) and Sumiteam C (manufactured by Shin Nippon Chemical Industry Co., Ltd.) having the functions of cellulase and hemicellulase can also be used. The specific hydrolysis method is not particularly limited, but in the hydrolysis of lignocellulosic biomass, pretreatment such as pulverization is preferably performed before the hydrolysis reaction. As a specific method for the pretreatment, a sample such as rice straw or corn cob is dried for 2 to 3 days and cut into about 2 to 3 cm using a cutter or a mixer. There is a method in which this is crushed with a ball mill or the like and sieved with a shifter or the like to form a powder of about 2 mm. Further, the method can be carried out on an industrial scale by using a continuous vibration mill or the like. By crushing the sample using such a method, the reaction efficiency of the subsequent hydrolysis reaction can be improved.

  The culture conditions can be appropriately adjusted by those skilled in the art without particular limitation as long as the growth of the microorganism of the present invention is not substantially inhibited and ethanol can be produced. The culture temperature is usually 20 to 40 ° C. Preferably it is 30-35 degreeC. Moreover, although the pH of a culture medium is 4-7 normally, in order to suppress the proliferation of various germs in the culture medium after sterilization, it is preferable that it is 4-5. When the pH is less than 4 before or during the culture, it is preferable to control the pH using ammonia or the like.

  As the culture method, any one of anaerobic conditions and aerobic conditions can be used, and can be appropriately selected by those skilled in the art depending on the composition of the nutrient source contained in the medium. The microorganism of the present invention can produce ethanol from glucose and xylose under either anaerobic condition or aerobic condition, but the rate of ethanol production from glucose is better under anaerobic conditions. Slightly faster. On the other hand, the rate of ethanol production from xylose is much faster under aerobic conditions. Therefore, by using multi-stage culture or multi-stage culture in which anaerobic conditions and aerobic conditions are combined in the presence of glucose and xylose, ethanol production efficiency and ethanol yield can be significantly improved over conventional culture methods. In addition, in this specification, multistage culture means culturing using a plurality of culture vessels having different culture conditions. Moreover, multistage culture means culturing using a plurality of culture conditions in one culture tank. That is, multistage culture or multistage culture includes a plurality of culture conditions (stages), and the number of stages is preferably 2 to 3 stages, more preferably 2 stages. The plurality of culture conditions are not limited to anaerobic conditions and aerobic conditions, but include, for example, cases where aging conditions are provided, and also include various culture conditions relating to medium composition, temperature, pH, and the like.

  As a preferred embodiment of the above culture method, when the microorganism of the present invention is cultured in the presence of glucose and xylose, the culture is performed under anaerobic conditions where the ethanol production rate from glucose is high, and the ethanol production rate from xylose. And a step of culturing under an aerobic condition that is fast, and a method using multi-stage culture that switches the culture condition from an anaerobic condition to an aerobic condition. Switching the culture conditions from anaerobic conditions to aerobic conditions is performed by program control that calculates the respiratory rate by online monitoring the concentration of carbon dioxide (carbon dioxide) discharged and determines the value based on the value of the respiratory rate. It can be broken. In this culture method, the respiration rate usually increases rapidly immediately after the start of the culture under anaerobic conditions, and reaches a peak about 12 to 24 hours after the start of the culture. In the culture under the anaerobic condition, glucose is mainly assimilated, but some xylose can be assimilated at the same time. And as glucose is consumed and the glucose concentration in the medium decreases, the respiration rate also decreases. When the respiration rate reaches a predetermined lower limit, the dissolved oxygen concentration is preferably maintained at about 10 to 20% with respect to the dissolved oxygen concentration before the start of culture by increasing the number of agitation in the culture tank or the aeration rate. Switch to temper conditions. In the culture under the aerobic condition, xylose is assimilated. In such multi-stage culture, switching from anaerobic conditions to aerobic conditions can be performed by monitoring the respiratory rate. The respiration rate is calculated according to the following formula 1 from the carbon dioxide concentration in the exhaust gas in the medium measured online.

(In the above Equation 1, RQ represents the respiration rate [gmolCO ₂ / Lh], Q represents the current ventilation rate [L / min], C represents the carbon dioxide concentration [% (v / v)], and V _L Represents the amount of culture medium (L) [L].
The carbon dioxide concentration can be measured using an exhaust gas analyzer generally used in this technical field. In general, the respiration rate increases with the start of culture, rapidly decreases after reaching a peak, and decreases to near zero. Therefore, after the respiration rate reaches the peak, when the respiration rate value becomes 0 to 40%, preferably 0 to 20%, the number of agitation or aeration in the culture tank is increased. To switch from anaerobic conditions to aerobic conditions. Further, switching from the anaerobic condition to the aerobic condition can also be performed by monitoring the glucose concentration in the medium using a glucose sensor or the like. In this case, switching to the aerobic condition is performed when the glucose concentration becomes 0 to 3%, preferably 0 to 1%. By performing switching when the respiration rate or glucose concentration falls within the above range, ethanol can be produced in a high yield in a short time.

  A conventionally well-known thing can be employ | adopted suitably for the culture tank used for culture | cultivation. When using a hydrolyzate of biomass as the carbon source, particularly a hydrolyzate of lignocellulosic biomass, it is preferable to use a culture tank equipped with a stirrer. In the medium containing the suspension obtained by hydrolyzing lignocellulosic biomass, insoluble components are likely to settle to the bottom of the tank, so that stirring is effective for improving the ethanol production rate. Further, in the culture in the presence of xylose, an aerobic condition is preferable as described above. Therefore, an aeration-stirring type culture tank is preferable. It may be a tank.

  As the culture method, a conventionally known method can be appropriately selected according to the ethanol production environment and production scale. Examples of the culture method include batch culture, continuous culture, fed batch culture, and repeated batch culture. Among these, it is preferable to use continuous culture in view of the culture characteristics of the microorganism of the present invention. As a preferred embodiment, there is a two-stage continuous culture system comprising a culture tank set with conditions suitable for glucose utilization and a culture tank set with conditions suitable for xylose utilization. For example, the first tank is set to anaerobic conditions suitable for glucose utilization, the second tank is set to conditions suitable for xylose utilization, and glucose is completely consumed in the first tank. By setting the conditions, it enables high-speed and highly efficient continuous ethanol production. In such continuous culture, the rate of supply and recovery of the medium can be expressed by the dilution rate as shown in Equation 2 below.

(In Equation 2 above, D represents the dilution rate [1 / day], F represents the flow rate [L / day], and V represents the volume [L] of the medium in each culture tank.)
For example, when the medium volume is 1.5 L and the flow rate is 0.75 L / day, the dilution rate is 0.5 [1 / day]. The dilution rate can be appropriately selected by those skilled in the art within the range of 0.1 to 1.0 [1 / day] as long as no washout occurs. Such a continuous culture method can be applied to ethanol production on an industrial scale.

  The effect of this invention is demonstrated using a following example and a comparative example. However, the technical scope of the present invention is not limited only to the following examples.

[Comparison of culture characteristics]
The culture characteristics of Pachisoren tanophilus MKY0802OK, which is a strain of the present invention, and standard ethanol-fermenting yeast (Saccharomyces cerevisiae NBRC2018) were compared.

<Example 1>
1 L of a medium (YPXD medium) was placed in a 3 L jar fermenter (3MD, manufactured by Maruhishi Bioengineer Co., Ltd.), adjusted to pH 4 with L-lactic acid, and sterilized at 121 ° C. for 20 minutes in an autoclave. This was inoculated with a preculture solution of Pachisol / Tanofilus MKY0802OK that had been shaken and cultured in a 500 mL Erlenmeyer flask (YPXD medium 100 mL) for 2 days in advance. The aeration amount was 0.25 vvm, and the cells were cultured at 30 ° C. with gentle agitation at 50 rpm so that the microorganisms did not settle. The xylose concentration, glucose concentration, and ethanol concentration in the medium before the start of culture (day 0) and after 2, 4, 6, 8, and 10 days were measured. The YPXD medium contains glucose and xylose as a carbon source, like the hydrolyzate of lignocellulosic biomass. The composition of the YPXD medium is shown in Table 10 below.

<Example 2>
A 500 mL Erlenmeyer flask was charged with 100 mL of medium (YPD medium), adjusted to pH 4 with L-lactic acid, and sterilized with an autoclave at 121 ° C. for 20 minutes. This was inoculated with a pre-culture solution of Pachisoren / Tanofilus MKY0802 which was previously shake-cultured for 2 days in a 500 mL Erlenmeyer flask (YPD medium 100 mL). Two of the above culture solutions were prepared, and anaerobic fermentation by stationary culture or aerobic fermentation by reciprocal shaking culture was performed at 30 ° C., respectively. The composition of the YPD medium is shown in Table 11 below.

<Example 3>
Culturing was performed in the same manner as in Example 2 except that YPX medium was used as the medium. The composition of the YPX medium is shown in Table 12 below.

<Comparative Example 1>
The culture was performed in the same manner as in Example 1 except that standard ethanol-fermenting yeast (Saccharomyces cerevisiae NBRC2018) was used as the microorganism.

<Comparative example 2>
The culture was performed in the same manner as in Example 2 except that standard ethanol-fermenting yeast (Saccharomyces cerevisiae NBRC2018) was used as the microorganism.

<Comparative Example 3>
Culturing was performed in the same manner as in Example 3 except that standard ethanol-fermenting yeast (Saccharomyces cerevisiae NBRC2018) was used as the microorganism.

  The results of Example 1 and Comparative Example 1 are shown in FIG. The Pachisolene tanofilus MKY0802OK of Example 1 assimilated glucose at a high rate immediately after the start of the culture, and consumed almost all glucose after 6 days. In addition, after 2 days, it was shown that xylose was assimilated simultaneously with glucose. Then, the utilization rate of xylose increased from about 4 days after the utilization rate of glucose slowed down. After 8 days of culture, almost all xylose was consumed. The ethanol yield was 38% by mass (73% of the theoretical yield) with respect to the total amount of glucose and xylose. On the other hand, in the standard ethanol-fermenting yeast of Comparative Example 1, as in Example 1, assimilation of glucose started immediately after the start of culture, and almost all glucose was assimilated after 4 days. However, xylose was not assimilated at all even after 10 days of culture. The yield of ethanol was 40% by mass with respect to the amount of glucose and 25% by mass with respect to the total amount of glucose and xylose.

  The results of Examples 2 and 3 and Comparative Examples 2 and 3 are shown in Table 13 below.

[Comparison of catabolite repression effects]
The culture characteristics of the strain of the present invention, Pachisoren tanophilus MKY0802OK, and Pachisoren tanophilus NBRC1007 (Pachisolen tanophilus Bodyin & Adzet NBRC1007), which is a typical microorganism subjected to catabolite repression, were compared. In addition, about the Pachisoren-Tanofilus MKY0802OK which is a strain of this invention, it compared using the same data as the said Example 1.

<Comparative example 4>
Culturing was carried out in the same manner as in Example 1 except that Pachisolen tanofilus NBRC1007 (Pachysolen tanophilus NBRC1007), which is a typical microorganism that receives catabolite repression, was used as the microorganism.

  The results are shown in FIG. As described above, in Example 1, about 70% of xylose was assimilated by 6 days after the culture when almost all glucose was consumed. That is, it was shown that glucose and xylose are assimilated simultaneously. Almost all the sugar consisting of glucose and xylose was consumed after 8 days, and the fermentation was complete. On the other hand, in Comparative Example 4, xylose was not assimilated until 6 days after at least 90% or more of glucose was consumed. And even after 10 days of culture, 10 g / L of xylose still remained.

[Production of ethanol using hydrolyzate of lignocellulosic biomass]
<Example 4>
1L of tap water and air-dried rice straw were pulverized in a 3L jar fermenter (3MD, manufactured by Maruhishi Bioengineering Co., Ltd.) using a pot mill (manufactured by Nippon Ceramic Science Co., Ltd., ANZ-50S). The suspension was suspended, and the pH was adjusted to 4.5 using L-lactic acid. Mecelase (manufactured by Meiji Seika Co., Ltd.) was added thereto and hydrolyzed at 40 ° C. for 24 hours with stirring to obtain a saccharified solution having a total sugar concentration of 95 g / L (glucose concentration of 40 g / L). 10 g of dry yeast (Asahi Breweries, ID1409012) was added thereto, and sterilized at 121 ° C. for 10 minutes in an autoclave. This was inoculated with a preculture solution of Pachisol / Tanofilus MKY0802OK that had been shaken and cultured in a 500 mL Erlenmeyer flask (YPXD medium 100 mL) for 2 days in advance. The aeration amount was 0.2 vvm, and the cells were cultured at 32 ° C. with gentle stirring at 50 rpm so that insoluble matters in the medium did not settle. The total sugar concentration, glucose concentration, ethanol concentration, dissolved oxygen (DO) concentration, and respiration rate were measured before the start of culture (day 0) and 1 to 4 days after the start of culture. The respiration rate reached its peak after 1 day of culture and then began to decrease. Simultaneously with the start of the decrease, the number of stirring was controlled, and the dissolved oxygen concentration in the culture solution was adjusted to be maintained at around 20% with respect to the dissolved oxygen concentration before the start of the culture.

  In addition, the addition amount of the said mecerase was added so that the initial enzyme concentration in a reaction liquid might be 20 u / mL (avicerase activity). The avicelase activity at that time was measured by the following method.

  First, a sample solution was prepared by diluting the culture supernatant to an appropriate amount with 0.1 M acetate buffer (pH 4.5). 2 mL of the above-mentioned acetate buffer and 2.5 mL of 2% Avicel solution were added and placed in a constant temperature bath (50 ° C.) with a shaker to stabilize, then 0.5 mL of the sample solution was added and reacted for 30 minutes. After completion of the reaction, 0.5 mL of 0.5N aqueous sodium hydroxide solution was added to stop the reaction, and unreacted Avicel was removed by centrifugation. Then, the reducing sugar in the reaction solution was measured by an enzymatic method. Activity was sought.

  The results are shown in FIG. As glucose was rapidly consumed immediately after the start of culture, the respiration rate increased and reached a peak after one day. Almost all glucose in the medium was consumed after 2 days. Eventually, 4 days after almost all of the associable sugar was consumed, 33.0 g / L ethanol was produced from 200 g / L rice straw. The yield of ethanol was 17.0% by mass for rice straw (air-dried product) and 37.2% by mass for total sugar.

<Example 5>
Hydrolysis and ethanol production were carried out in the same manner as in Example 4, except that corn cob (a crushed corn cob) was used instead of rice straw. In addition, the composition of the saccharide in the saccharified solution after hydrolysis of corn cob was 39 g / L of glucose, 26 g / L of xylose, and 45 g / L of other saccharides out of 110 g / L of total saccharide.

  The results are shown in FIG. As in Example 2, as the glucose was rapidly consumed immediately after the start of culture, the respiration rate increased and reached a peak one day later. Almost all glucose in the medium was consumed after 2 days. Finally, 34.0 g / L ethanol was produced from 200 g / L corn cob 4 days after almost all of the associable sugar was consumed. The ethanol yield was 17.0% by mass for corn cob (air-dried) and 30.9% by mass for total sugar.

<Example 6>
Air-dried weeds (variety unknown) collected in a residential area in Kiyosato, Fujieda City, Shizuoka Prefecture were cut into 2 to 3 cm with a cutting machine, and this was made into a powder of 2 mm or less using a ball mill. Hydrolysis and ethanol production were performed in the same manner as in Example 4 except that this weed powder was used. In addition, the composition of the saccharide in the saccharified solution after hydrolyzing the weeds was 38 g / L of glucose, 30 g / L of xylose, and 56 g / L of other sugars out of 120 g / L of total sugar.

  The results are shown in FIG. As in Examples 2 and 3, as glucose was rapidly consumed immediately after the start of culture, the respiration rate increased and reached a peak after 1 day. Almost all glucose in the medium was consumed after 2 days. Nearly all the assimilable sugar was consumed by 3 days, and finally, after 4 days, 35.1 g / L ethanol was produced from 200 g / L weeds. The ethanol yield was 17.5% by weight for weeds (air-dried) and 29.1% by weight for total sugars.

[Production of ethanol by multi-stage continuous culture using rice straw hydrolyzate]
<Example 7>
Ethanol was produced by multistage continuous culture using rice straw hydrolyzate. The multistage continuous culture will be described with reference to the schematic diagram of the multistage continuous culture apparatus shown in FIG. First, the medium in the medium supply tank 100 is sent to the anaerobic fermenter 300 that is the first fermenter via the liquid feed pump 200. The medium subjected to anaerobic fermentation in the anaerobic fermentation tank 300 is subsequently sent to the aerobic fermentation tank 400 which is the second fermentation tank via the liquid feed pump 210. The aerobic fermenter 400 is supplied with air from the compressor 500 that has passed through the air filter 600. Then, the medium subjected to aerobic fermentation in the aerobic fermentation tank 400 is finally collected via the liquid feeding pump 220.

  Using the same medium containing rice straw hydrolysis as in Example 1, multistage continuous culture using the continuous culture apparatus was performed. First, 1.5 L each of the medium was fed from the medium supply tank 100 to the anaerobic fermenter 300 and the aerobic fermenter 400. Both the anaerobic fermenter 300 and the aerobic fermenter 400 are stirred so that insoluble components do not settle, and the dissolved oxygen concentration in the medium is in a state of almost 0% with respect to the dissolved oxygen concentration before the start of culture. The culture was carried out at 30 ° C. for 2 days with aeration. Then, a new culture medium is supplied by supplying the culture medium from the culture medium supply tank 100 to the anaerobic fermentation tank 300 and from the anaerobic fermentation tank 300 to the aerobic fermentation tank 400 so that the dilution rate is 0.5 (1 / day). The culture medium was supplied from the culture medium supply tank 100, and the cultured medium was collected from the aerobic fermentation tank 400 at the same rate. Thus, multistage continuous culture was performed for 1 month, and the total sugar concentration, ethanol concentration, and dissolved oxygen concentration in each fermenter were measured.

  The results are shown in FIGS. After switching from batch culture to multistage continuous culture two days after the start of culture, the total sugar concentration, ethanol concentration, and dissolved oxygen concentration are almost steady in 8 to 10 days after the start of culture in the anaerobic fermenter 300 as the first fermenter. After 10 days, the average total sugar concentration was maintained at 40 g / L and the average ethanol concentration was 20 g / L, and the average ethanol yield relative to the total sugar was 41% by mass. The dissolved oxygen concentration was almost 0% with respect to the dissolved oxygen concentration before the start of culture. On the other hand, in the aerobic fermenter 400 which is the second fermenter, after switching to the multistage continuous culture, it enters a steady state 8 to 10 days after the start of the culture, and after 10 days, the average total sugar concentration is 20 g / L. The average ethanol concentration was 28 g / L, and the average ethanol yield based on the total sugar was 39% by mass. In addition, the dissolved oxygen concentration transitioned around 20% with respect to the dissolved oxygen concentration before the start of culture. This result showed that ethanol fermentation was performed mainly using glucose in the anaerobic fermenter 300 and mainly using xylose in the aerobic fermenter.

It is the graph which compared the culture | cultivation characteristic of the strain of this invention and standard ethanol fermentation yeast. It is the graph which compared the culture | cultivation characteristic of the typical yeast which receives the strain of this invention, and a catabolite repression. It is a graph showing ethanol production using a rice straw hydrolyzate. It is a graph showing ethanol production using a corn cob hydrolyzate. It is a graph showing ethanol production using a weed hydrolyzate. It is a schematic diagram of a multistage continuous culture apparatus. It is a graph showing ethanol production in an anaerobic fermenter in multistage continuous culture. It is a graph showing ethanol production in an aerobic fermenter in multistage continuous culture.

Explanation of symbols

100 medium supply tank,
200, 210, 220 Liquid feed pump,
300 anaerobic fermenters,
400 aerobic fermenter,
500 compressors,
600 Air filter.

Claims (9)

  A microorganism belonging to Pachisolen tanophilus, capable of producing ethanol from glucose and xylose in the presence of glucose and xylose.   The microorganism according to claim 1, which is not subjected to catabolite repression by glucose.   The microorganism according to claim 1 or 2, which is MKY0802 OK (accession number: FERM ABP-10965).   A method for producing ethanol by culturing the microorganism according to any one of claims 1 to 3 in a medium.   The method for producing ethanol according to claim 4, wherein the medium contains glucose and xylose.   The method for producing ethanol according to claim 4 or 5, wherein the medium contains a lignocellulosic biomass hydrolyzate.   The lignocellulosic biomass hydrolyzate comprises at least one hydrolyzate selected from the group consisting of rice straw, rice husk, wheat straw, bagasse, coconut husk, corn cob, weed, wood, pulp, and paper. Item 7. The method for producing ethanol according to Item 6.   The method for producing ethanol according to any one of claims 4 to 7, wherein the culture is a multi-stage culture or a multi-stage culture including a stage using an aerobic condition and a stage using an anaerobic condition.   The method for producing ethanol according to any one of claims 4 to 8, wherein the culture is continuous culture.

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