JP2016077262A - Production method of 2,3-butanediol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an economical production method of 2,3-butanediol by a microbial fermentation using biologically safe microorganisms.SOLUTION: The invention provides a production method of 2,3-butanediol comprising a step A of culturing Zymobacter microorganisms, such as Zymobacter palmae having an ability to utilize pentose in a fermentation raw material containing pentose such as xylose; and a step (B) of purifying 2,3-butanediol from the culture medium obtained in the step.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing 2,3-butanediol by microbial fermentation.

  2,3-butanediol (hereinafter sometimes referred to as “2,3-BDO”) is an intermediate material for pharmaceuticals and cosmetics, ink, perfume, liquid crystal, insecticide, softening reagent, explosive, plasticizer, etc. As a specific example, it can be a raw material for methyl ethyl ketone (Non-Patent Document 1) and 1,3-butadiene (Non-Patent Document 2). 2,3-BDO is industrially produced by a method in which 2-butene oxide is hydrolyzed in an aqueous perchloric acid solution. In recent years, there has been an urgent need to shift to a sustainable recycling society in response to the depletion of petroleum resources and global warming, and the chemical industry is actively researching the shift from petroleum materials to biomass-derived materials. In particular, a method for producing 2,3-butanediol by a microbial fermentation method is drawing attention.

  As fermentation raw materials for microbial fermentation, in addition to sugars derived from conventional edible biomass, sugars derived from non-edible biomass have attracted attention. When using sugars derived from non-edible biomass as fermentation raw materials, cellulose, hemicellulose, etc. contained in non-edible biomass are decomposed into sugars by saccharifying enzymes. At this time, not only hexose sugars such as glucose are used. In addition, pentose sugars such as xylose can be obtained simultaneously. Therefore, development of an efficient fermentation technology for raw materials containing pentose is required.

  Enterobacteria such as Klebsiella oxytoca (Non-patent Document 3) are known as microorganisms that efficiently fermentatively produce 2,3-butanediol from pentose sugars. Since these microorganisms do not require specific vitamins or amino acids for fermentation, there is an advantage that the number of steps required when purifying 2,3-butanediol from the fermentation broth is reduced. However, these microorganisms are pathogenic and are known to cause respiratory infections and the like. Therefore, when it is going to culture these fermenting bacteria in large quantities for the purpose of industrial 2, 3- butanediol production, the installation for strict management is required and it leads to the increase in cost.

  On the other hand, as microorganisms that are biologically safe and ferment pentoses to 2,3-butanediol, for example, Bacillus bacteria (Non-patent Document 4) and Paenibacillus polymyxa (Non-patent Document 5). ) Is disclosed. However, in 2,3-butanediol fermentation using these microorganisms, expensive auxiliary materials such as yeast extract and animal protein hydrolyzate are used, which increases costs.

  In addition, a transformed microorganism of the genus Zymobacter having the ability to assimilate pentose by introducing a foreign gene encoding xylose isomerase, xylulokinase, transaldolase and transketolase into a microorganism of the genus Zymobacter is known. (Patent Document 1, Patent Document 2). However, although it is described in Patent Documents 1 and 2 that this transformed microorganism has ethanol productivity, there is no description or suggestion about the ability to ferment pentose to 2,3-butanediol. .

JP 2005-261421 A US Patent Application Publication No. 2007/0298476

A. N. Bourns, The Catalytic Action Of Aluminum Silicates, Canadian J. Org. Res. (1947) Nathan Shlchter, Pyrolysis of 2,3-butylene Glycol Dicarbonate to Butadiene, Indu. Eng. Chem. 905 (1945) Jansen NB, Production of 2,3-butanediol from D-xylos by Klebsiella oxytoca ATCC 8724. Biotechnol Bioeng. 1984 Apr; 26 (4): 362-9. Wang Q, Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2, 3-butanediol production. Biotechnol Bioeng. 2012 Jul; 109 (7): 1610-21. Marwoto B, Metabolic analysis of acetic acid accumulation xyrose consumption by Paenibacillus polymyxa. Appl Microbiol Biotechnol. 2004 Mar; 64 (1): 112-9.

  As described above, in the conventional method for producing 2,3-butanediol from pentose by microbial fermentation, biological safety and the cost of auxiliary materials have been problems. Accordingly, an object of the present invention is to establish an economical method for producing 2,3-butanediol by microbial fermentation using biologically safe microorganisms.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a genus Zymobacter, which has not been known as a 2,3-butanediol-fermenting microorganism, is capable of fermenting 2,3-butanediol. Furthermore, the present inventors have found that the above-mentioned problems can be solved by imparting xylose assimilation property to the microorganism, and have completed the present invention.

That is, the present invention is as follows (1) to (13).
(1) A step of cultivating a microorganism belonging to the genus Zymobacter having the ability to assimilate pentose in a fermentation raw material containing pentose (step A), and from the culture solution obtained in the step 2, The manufacturing method of 2, 3- butanediol including the process (process B) which refine | purifies 3-butanediol.
(2) The method according to (1), wherein the step A is culturing under aerated conditions.
(3) The method according to (1) or (2), wherein the step A is culturing under a condition of an oxygen transfer capacity coefficient (kLa) 9h ⁻¹ or more.
(4) The microorganism is a transformed microorganism of the genus Zymobacter into which a foreign gene encoding at least one enzyme selected from xylose isomerase, xylulokinase, transaldolase, and transketolase is introduced (1) to ( The method according to any one of 3).
(5) The method according to (4), wherein the microorganism is a transformed microorganism of the genus Zymobacter into which a foreign gene encoding xylose isomerase, xylulokinase, transaldolase, and transketolase has been introduced.
(6) The method according to any one of (1) to (5), wherein the microorganism is Zymobacter palmae.
(7) The method according to any one of (1) to (6), wherein the pentose contained in the fermentation raw material of Step A is xylose.
(8) The method according to any one of (1) to (7), wherein the abundance ratio of xylose to total sugar in the fermentation raw material is 5 to 100%.
(9) The method according to any one of (1) to (8), wherein the step A is culturing in a medium having a total sugar concentration of 20 g / L or more.
(10) The method according to any one of (1) to (9), wherein the fermentation raw material includes a biomass-derived sugar solution.
(11) The method according to any one of (1) to (10), wherein the step B includes a distillation step.
(12) The method according to (11), including a desalting treatment step before the distillation step.
(13) The method according to (12), wherein the desalting treatment step includes at least an ion exchange treatment step.

  According to the present invention, in a method for producing 2,3-butanediol from a fermentation raw material containing pentose by microbial fermentation, 2,3-butanediol can be obtained more safely and economically than in the prior art. it can.

Step A: 2,3-butanediol fermentation step In the method of the present invention, a microorganism belonging to the genus Zymobacter having the ability to assimilate pentose is cultured in a medium containing a fermentation raw material containing pentose.

  The zymobacter genus bacterium used may be isolated from the natural environment, or may be partially modified in nature by mutation or genetic recombination. As a specific example of the bacterium belonging to the genus Zymobacter having the fermentation ability of 2,3-butanediol, Zymobacter palmae is preferably used from the viewpoint of 2,3-butanediol fermentation ability. Zymobacter palme normally does not assimilate pentose, but can be granted by introducing a gene involved in assimilation of pentose.

  As a gene to be introduced in the present invention, an enzyme gene that isomerizes aldopentose into ketopentose can be used. Examples of the gene to be used include pentose isomerase, pentose reductase and / or pentol dehydrogenase.

  Pentose isomerase is defined as an enzyme that catalyzes the isomerization of aldopentose and ketopentose, which are structural isomers of pentose. The pentose isomerase used in the present invention is not particularly limited as long as it has an activity of catalyzing the direct isomerization of pentose. For example, xylose isomerase (EC 5.3.1.5) and arabinose isomerase (EC 5. 3.1.3). Of these, for example, xylose isomerase is an enzyme that catalyzes the direct isomerization of xylose (aldopentose) to xylulose (ketopentose) and / or vice versa, also known as xylose ketoisomerase. Direct isomerization refers to isomerization in one step catalyzed by pentose isomerase, as opposed to two-step conversion via sugar alcohol intermediates catalyzed by pentose reductase and pentol dehydrogenase.

  The pentose reductase is one of the reductases and means an enzyme having an activity of converting aldopentose into a sugar alcohol using NADH or NADPH as a coenzyme. For example, xylose reductase corresponds to, for example, EC 1.1.1.307 and EC 1.1.1.21, and is an enzyme that converts xylose into xylitol. The arabinose reductase corresponds to EC1.1.1.21, for example, and is an enzyme that converts arabinose into arabitol. Moreover, even if it is not the enzyme classified by said EC number, if it is an enzyme which has the said activity, it will be contained in the pentose reductase in this invention.

  Pentol dehydrogenase is one of the dehydrogenases and is an enzyme that converts pentol into ketopentose using NAD + as a coenzyme. For example, xylitol dehydrogenase corresponds to, for example, EC 1.1.1.9 or EC 1.1.1.10, and is an enzyme that converts xylitol into xylulose. The arabitol dehydrogenase corresponds to, for example, EC 1.1.1.11 or EC 1.1.1.12. It is an enzyme that converts arabitol into ribose. Even an enzyme that is different from pentol dehydrogenase in terms of classification is included in the pentol dehydrogenase in the present invention as long as it has the above activity.

  The reaction catalyzed by pentose reductase and pentol dehydrogenase isomerizes aldopentose into ketopentose, similar to pentose isomerase.

  Of the enzymes catalyzing the isomerization of aldopentose and ketopentose, pentose pentose isomerase can be preferably used, and xylose isomerase can be more preferably used.

  The xylose isomerase gene to be used is not particularly limited, and may be cDNA or the like in addition to genomic DNA. Further, it may be derived from any organism such as animals, plants, fungi (yeasts, molds, etc.) and bacteria. Information on such genes can be easily obtained by searching a database published on the web such as NCBI. For example, the base sequence of the Escherichia coli xylose isomerase gene that can be preferably used in the present invention is described in, for example, GenBank Accession No. NC — 007779 REGION: 3909650..3910972.

  The activity of xylose isomerase can be confirmed by carrying out an enzymatic reaction in the presence of xylose and measuring isomerized xylulose using HPLC or the like. Xylose isomerase activity can be measured, for example, by the method disclosed in JP-A-2008-79564.

  Examples of organisms to be donors of xylose isomerase include Enterobacteriaceae such as Enterobacter genus, Escherichia genus, Klebsiella genus, Erwinia genus, Salmonella genus, etc. , Actinoplanes genus, Arthrobacter genus, Streptomyces genus actinomycetes, or Bacillus genus, Paenibacillus genus, Lactobacillus genus, Lactobacillus The genus Staphylococcus, Thermoanaerobacter (Thermoan) genus Erobacter, Thermus, Xanthomonas, Rhizobium, Pseudomonas, Clostridium, Bacteroides, Bacteroides can be used, A microorganism belonging to the genus Escherichia, more preferably Escherichia coli.

  In addition, as a gene to be introduced in the present invention, an enzyme gene having an activity of catalyzing phosphorylation of ketopentose can also be used. Examples of the enzyme having an activity of catalyzing the phosphorylation of ketopentose include xylulokinase (EC 2.7.1.17) and librokinase (EC 2.7.1.16). Can be preferably used.

  The xylulokinase gene to be used is not particularly limited, and may be cDNA or the like in addition to genomic DNA. Further, it may be derived from any organism such as animals, plants, fungi (yeasts, molds, etc.) and bacteria. Information on such genes can be easily obtained by searching a database published on the web such as NCBI. For example, the base sequence of the Escherichia coli xylulokinase gene that can be preferably used in the present invention is described in, for example, GenBank Accession No. NC — 007779 REGION: 3911044..3912498.

  The activity of xylulokinase is measured by measuring the phosphorylated xylulose-5-phosphate using HPLC or by using an auxiliary enzyme reaction in the presence of xylulose. For example, Feldmann SD, Sahm H, Springer GA. Cloning and expression of the genes for xylose isomerase and xylokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12. Mol Gen Genet. 1992 Aug; 234 (2): 201-10. Can be measured by the method disclosed in.

  Examples of organisms to be donors of xylulokinase include Enterobacteriaceae such as Enterobacter genus, Escherichia genus, Klebsiella genus, Erwinia genus, Salmonella genus, etc. Bacteria, lactic acid bacteria such as Bacteroides genus, Lactobacillus genus, Actinoplanes genus, Arthrobacter genus, Streptomyces genus Streptomyces genus Bacillus fungi, Bacillus genus, Paenibacillus genus, Staphylococcus Occus, Thermoanaerobacter, Xanthomonas, Rhizobium, Pseudomonas, Clostridium, and microorganisms belonging to the genus Clostridium can be used. And more preferably Escherichia coli.

  Furthermore, as a gene to be introduced in the present invention, an enzyme gene having transketolase and / or transaldolase activity can also be used. Transketolase is a reaction that converts xylulose-5-phosphate and erythrose-4-phosphate to fructose-6-phosphate and glyceraldehyde-3-phosphate, and vice versa, and / or ribose 5- Defined as an enzyme that catalyzes the reaction of converting phosphate and xylulose 5-phosphate into sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate and vice versa. The transketolase used in the present invention is not particularly limited as long as it has the above-mentioned catalytic activity. For example, EC 2.2.1.1 is applicable.

  The transketolase and / or transaldolase gene to be used is not particularly limited, and may be cDNA or the like in addition to genomic DNA. Further, it may be derived from any organism such as animals, plants, fungi (yeasts, molds, etc.) and bacteria. Information on such genes can be easily obtained by searching a database published on the web such as NCBI. For example, the base sequence of the Escherichia coli transketolase gene that can be preferably used in the present invention is described in, for example, GenBank Accession No. NC — 007779 REGION: complement (3078300..3080291). For example, the base sequence of the Escherichia coli transaldolase gene that can be preferably used in the present invention is described in, for example, GenBank Accession No. NC — 007779 REGION: 8238..9191.

  The transketolase activity is determined by performing an enzymatic reaction in the presence of xylulose-5-phosphate and erythrose-4-phosphate, and measuring the produced glyceraldehyde-3-phosphate using HPLC or the like. It can be confirmed by measuring the decrease of NADH using a typical enzyme reaction. For example, Sprenger GA, Schorken U, Sprenger G, Sahn H. et al. Transketolase A of Escherichia coli K12. Purification and properties of the enzyme from recombinant strains. Eur J Biochem. 1995 Jun 1; 230 (2): 525-32. Can be measured by the method disclosed in.

  Transaldolase is defined as an enzyme that catalyzes the reaction of converting sedheptulose 7-phosphate and glyceraldehyde 3-phosphate into erythrose-4-phosphate and fructose-6-phosphate and vice versa. The transketolase used in the present invention is not particularly limited as long as it has the above-mentioned catalytic activity. For example, EC 2.2.1.2 is applicable.

  Transaldolase activity can be determined by conducting an enzymatic reaction in the presence of fructose-6-phosphate and erythrose-4-phosphate and measuring the resulting glyceraldehyde-3-phosphate using HPLC or the like. Can be confirmed by measuring a decrease in NADH using a simple enzyme reaction, for example, GA. Springer, U., et al. Schorken, G.M. Springer & H.C. Sahm TransolaseB of Escherichia coli K-12: cloning of its gene, talB, and charactarization of the enzyme from recombinant strains). J. et al. Bacteriol. 1995, 177: 20: 5930-6).

  Examples of organisms serving as transaldolase and / or donor of transaldolase include, for example, Enterobacter genus, Escherichia genus, Klebsiella genus, Erwinia genus, Salmonella genus, etc. Enterobacteriaceae, Actinoplanes, Arthrobacter, Streptomyces, and other actinomycetes, or Bacillus, Paenibacillus, Lactobacillus, Lactobacillus, Lactobacillus, ) Genus, Staphylococcus genus, Thermore The genus can be used in the genus Thermoanaerobacter, thermus (Thermus), the genus Xanthomonas, the genus Rhizobium, the genus Pseudomonas, the genus Clostridium, and the genus Bacteroides , Preferably a microorganism belonging to the genus Escherichia, and more preferably Escherichia coli.

  As long as the microorganism belonging to the genus Zymobacter has the ability to assimilate the pentose, it may be introduced with at least one of the above genes, but (1) xylose isomerase, (2) xylulokinase and (3 (1) a gene into which a foreign gene encoding at least one of transaldolase and transketolase has been introduced, and (1) a xylose isomerase gene, (2) xylulokinase, and (3) a transaldolase and transketase. Those into which a foreign gene encoding both trase is introduced are preferred.

  Microorganisms other than the above, those having xylose resolving power, and further having no xylose resolving power due to abnormality of promoter site or ribosome binding site, etc., but xylose isomerase, xylulokinase, transaldolase or transketolase on the DNA Microorganisms encoding genes can also be used as donors.

  The genes to be introduced may be a combination of genes from a plurality of different donors. In addition, these genes may be incorporated into genomic DNA in the host genus Zymobacter or may be present on a vector.

  The method for introducing a gene is not particularly limited, but as a method for introducing a gene present on a vector, for example, methods disclosed in Patent Document 1 and Patent Document 2 can be used. That is, each gene is inserted into a multicloning site of a commercially available cloning plasmid vector for Escherichia coli (for example, pUC118), and a transformant that transforms Escherichia coli to produce a product of each gene is grown. The plasmid was recovered from the grown transformant, the region containing each introduced gene was excised, and a broad host range vector plasmid (for example, pMFY31 (Agric. Biol. Chem., Vol. 49 (9), 2719- 2724, 1985, etc.), a Zymobacter genus transformant having the ability to assimilate pentose can be produced by transforming Zymobacter bacterium with a recombinant plasmid vector inserted into the multicloning site of No. 2724, 1985, etc. As the broad host range vector plasmid, a commercially available one can also be used, and the transformation method itself is well known, for example, as described in Patent Document 1 and Patent Document 2, a T medium containing magnesium ions ( 2.0 wt% glucose, 1.0 wt% Bacto-yeast extract, 1.0 wt% potassium dihydrogen phosphate, 0.2 wt% ammonium sulfate, 0.05 wt% magnesium sulfate After culturing in a medium such as heptahydrate (pH 6.0), the cells can be transformed into competent cells and then transformed by conventional methods such as electroporation. The body can be obtained by culturing and selecting the transformed bacteria in a medium containing xylose as the sole carbon source.

  In the case where the transgene is incorporated into genomic DNA, for example, Kirill A. et al. Datsenko and Barry L. Wanner. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Proc Natl Acad Sci USA. Jun 6, 2000; 97 (12): 6640-6645. Can be used.

  Although the fermentation raw material used by this invention contains pentose, it may contain hexose in addition to pentose.

  The pentose sugar has five carbons constituting the sugar, and is also called pentose. There are aldopentoses with an aldehyde group at the 1-position and ketopentoses with a ketone group at the 2-position. Examples of aldopentose include xylose, arabinose, ribose, and lyxose. Examples of ketopentose include ribulose and xylulose. The pentose used in the present invention may be any pentose as long as it can assimilate microorganisms, but is preferably xylose or arabinose, more preferably from the viewpoint of the natural ratio or availability. Is xylose.

  The hexose has 6 carbons constituting the sugar and is also called hexose. There are aldoses with an aldehyde group at the 1-position and ketoses with a ketone group at the 2-position. Examples of aldose include glucose, mannose, galactose, allose, growth, and talose. Examples of ketose include fructose, psicose, and sorbose. The hexose used in the present invention may be any as long as it can assimilate microorganisms, but from the viewpoint of the natural ratio, availability, etc., preferably glucose, mannose, galactose, More preferred is glucose.

  Although it does not specifically limit regarding the fermentation raw material containing pentose sugar and hexose sugar, The cellulose containing biomass origin sugar liquid known to contain both hexose sugar and pentose sugar is used preferably. Examples of the cellulose-containing biomass include plant biomass such as bagasse, switchgrass, corn stover, rice straw, and straw, and woody biomass such as trees and waste building materials. Cellulose-containing biomass contains cellulose or hemicellulose, which is a polysaccharide obtained by dehydrating and condensing sugar, and a sugar solution that can be used as a fermentation raw material is produced by hydrolyzing such a polysaccharide. The method for preparing a cellulose-containing biomass-derived sugar solution is well known, and any method may be used. Such a sugar is produced by acid hydrolysis of biomass using concentrated sulfuric acid to produce a sugar solution. A method for producing a sugar solution by hydrolyzing biomass with dilute sulfuric acid and further treating with an enzyme such as cellulase, etc. (Japanese Patent Application Laid-Open No. 11-506934, JP-A-2005-229821) (A. Aden et al., “Lignocellulosic Biomass to Ethanol Process Design and Economics Optimized Co-Current Dirty Acid Physiology Hydrology and Enzymological Hydrology.) tober "NREL Technical Report (2002)). As a method not using acid, a method of hydrolyzing biomass using subcritical water at about 250 to 500 ° C. to produce a sugar solution (Japanese Patent Laid-Open No. 2003-212888), or treating biomass with subcritical water Later, a method for producing a sugar solution by further enzyme treatment (Japanese Patent Laid-Open No. 2001-95597), a biomass solution by hydrolyzing biomass with pressurized hot water at 240 to 280 ° C. and then further enzyme treatment Is disclosed (Japanese Patent No. 3041380). After the treatment as described above, the obtained sugar solution may be purified. The method is disclosed in WO2010 / 067875, for example.

  The weight ratio of the pentose and hexose contained in the mixed sugar is not particularly limited, but the weight ratio of the pentose and hexose in the mixed sugar is expressed as (pentose): (hexose). And 1: 9 to 9: 1 are preferred. This is a sugar ratio when a cellulose-containing biomass-derived sugar solution is assumed as a mixed sugar.

  The total sugar concentration contained in the fermentation raw material used in the present invention is not particularly limited. From the viewpoint of production efficiency, it may be as high as possible as long as it does not inhibit the production of 2,3-BDO by microorganisms. preferable. Specifically, the total sugar concentration in the fermentation raw material that is a medium is preferably 15 to 500 g / l, and more preferably 20 to 300 g / l. When the total sugar concentration is 15 g / l or less, the effect of improving the yield from pentose may be reduced. Moreover, if the total sugar concentration is low, the production efficiency of 2,3-BDO will also decrease.

  The concentration of hexose contained in the fermentation raw material used in the present invention is not particularly limited within the above-mentioned total sugar concentration and the range of the ratio of pentose and hexose, but the method for producing 2,3-BDO of the present invention Can be used to obtain a good yield even in a mixed sugar solution containing hexose at a concentration of 5 g / L or more.

  As a fermentation raw material, in addition to the sugar, a carbon source, a nitrogen source, inorganic salts, and organic micronutrients such as amino acids and vitamins as needed that favorably act on fermentation of 2,3-butanediol-fermenting microorganisms are appropriately used. You may make it contain. Moreover, you may contain an antifoamer suitably for the purpose of performing fermentation efficiently.

  Specific examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, amino acids, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysate, casein digest, corn Steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, zinc salts and the like can be appropriately added and used. In order to produce 2,3-butanediol by pentose fermentation, among these, corn steep liquor, ammonium salts such as ammonium sulfate and ammonium hydrogen phosphate, potassium dihydrogen phosphate, hydrogen phosphate monobasic Phosphate such as potassium, ammonium monohydrogen phosphate, calcium salt such as calcium chloride, iron salt such as ferrous sulfate, manganese salt such as manganese sulfate, zinc salt such as zinc sulfate, magnesium sulfate It is preferable to contain EDTA such as magnesium salt, EDTA disodium or a salt thereof.

  In addition, nicotinic acid may be appropriately added to the medium depending on the growth of microorganisms, and an antifoaming agent may be appropriately added and used.

  The culture temperature is not particularly limited as long as microorganisms grow, but the temperature is usually 20 to 50 ° C, preferably 25 to 40 ° C.

  The culture medium pH at the time of culture is usually maintained within the range of pH 4 to 8 with inorganic or organic acids, alkaline substances, urea, calcium carbonate, ammonia gas, etc., but is adjusted to pH 5 to 7 and cultured. It is preferable that pH is 5 or more and less than 6.

  There is no restriction | limiting in particular in the culture | cultivation form of microorganisms, It can carry out by batch culture, fed-batch fermentation, continuous culture, etc.

  In the present invention, it is preferable to culture microorganisms under aerated conditions. The aeration in the present invention means that a gas containing oxygen flows into the culture tank. Although there is no restriction | limiting in particular as an aeration method, For example, the upper surface ventilation | gas_flowing which replaces the air layer on a culture solution with the gas containing oxygen other than the method of allowing the gas containing oxygen to pass through in a culture solution is also included. In addition to the method of blowing oxygen-containing gas into the culture tank for the top ventilation, the culture fluid is stirred in an open atmosphere to generate an air flow over the culture fluid, so that oxygen is introduced into the culture fluid. A method of supplying is also included. In the present invention, aeration is preferably performed by a method of allowing a gas containing oxygen to pass through the culture solution.

Conditions for supplying oxygen to the culture medium in the culture of microorganisms can be appropriately set by combining aeration and agitation, and an oxygen transfer capacity coefficient kLa (h ⁻¹ ) (hereinafter, referred to as 30 ° C. water at normal pressure). Simply abbreviated as kLa). kLa indicates the ability to move dissolved oxygen from the gas phase to the liquid phase per unit time during aeration stirring, and is defined by the following formula (1). (Biotechnological Experiments, Japan Biotechnology Society, Bafukan, p. 310 (1992)).
dC / dt = kLa × (C ^* −C) (1)
Here, C: dissolved oxygen concentration DO (ppm) in the culture solution, C ^* : dissolved oxygen concentration DO (ppm) in equilibrium with the gas phase when oxygen is not consumed by microorganisms, kLa: oxygen transfer capacity coefficient (hr ^-1 ). Since the following equation (2) is derived from the above equation (1), kLa can be obtained by plotting the logarithm of C ^* -C against the aerated time.
In (C ^* −C) = − kLa × t (2).

  KLa in the present invention is a numerical value measured by a gas displacement method (dynamic method). In the gas displacement method (dynamic method), water is put into an aeration and agitation culture apparatus into which a dissolved oxygen concentration electrode is inserted, and oxygen in these liquids is replaced with nitrogen gas to reduce the oxygen concentration of the liquid, This is a method of calculating kLa by switching the nitrogen gas to compressed air and measuring the rising process of dissolved oxygen under a predetermined aeration rate, stirring rate and temperature.

The kLa set in the culture in the present invention is not particularly limited, but is preferably 9h ⁻¹ or more. The upper limit of kLa is not particularly limited, but is preferably 200 h ⁻¹ or less.

  The culture solution obtained in the 2,3-butanediol fermentation step contains 2,3-butanediol as well as impurities derived from fermentation raw materials and ethanol as a fermentation byproduct. It is necessary to purify the 2,3-butanediol contained.

Purification Step The 2,3-butanediol purification step from the 2,3-butanediol culture solution is not particularly limited and may be carried out by a technique known to those skilled in the art. In the present invention, the 2,3-butanediol culture is performed. It is preferable to include a step of distilling the liquid.

  In distillation of 2,3-butanediol culture, 2,3-butanediol is recovered from the vapor side. The distillation method is not particularly limited, but any of simple distillation, precision distillation, atmospheric distillation, and vacuum distillation, which are generally applicable, may be selected from a thin film distillation apparatus, a plate distillation apparatus, a packed distillation apparatus, or the like. Can do. Moreover, it is applicable to this invention even if it is a batch type or a continuous type. Among them, vacuum distillation is preferable, and the boiling point can be lowered, so that generation of impurities can be suppressed. Specifically, the heating temperature is preferably 60 ° C. or higher and 150 ° C. or lower. When the temperature is lower than 60 ° C., the degree of decompression needs to be remarkably lowered, so that it is very difficult to maintain the apparatus at an industrial level. On the other hand, when the temperature is higher than 150 ° C., a trace amount of sugar remaining in the 2,3-butanediol solution is decomposed, and coloring substances are produced as by-products, which is not preferable. Therefore, it is preferable to adjust the degree of vacuum so that 2,3-butanediol distills within the heating temperature range.

  In addition, although the 2,3-butanediol culture solution may be directly subjected to the distillation step, it is preferably subjected to a desalting step or a concentration step prior to the distillation step.

  A specific example of the desalting step is an ion exchange treatment. The ion exchange treatment is a method for removing ion components in the 2,3-butanediol culture solution using an ion exchanger. As the ion exchanger, ion exchange resin, ion exchange membrane, ion exchange fiber, ion exchange paper, gel ion exchanger, liquid ion exchanger, zeolite, carbonaceous ion exchanger, montmorillonite can be used. A treatment using an ion exchange resin is preferably employed.

  Ion exchange resins include strong anion exchange resins, weak anion exchange resins, strong cation exchange resins, weak cation exchange resins, and chelate exchange resins, depending on the functional groups. For example, strong anion exchange resins include “Amberlite” IRA410J, IRA411, IRA910CT manufactured by Organo Corporation and “Diaion” SA10A, SA12A, SA11A, NSA100, SA20A, SA21A, UBK510L, UBK530, UBK550 manufactured by Mitsubishi Chemical Corporation. , UBK535, UBK555, etc. Examples of weak anion exchange resins include “Amberlite” IRA478RF, IRA67, IRA96SB, IRA98, and XE583 manufactured by Organo Corporation and “Diaion” WA10, WA20, WA21J, and WA30 manufactured by Mitsubishi Chemical Corporation. On the other hand, strong cation exchange resins include “Amberlite” IR120B, IR124, 200CT, 252 manufactured by Organo Corporation and “Diaion” SK104, SK1B, SK110, SK112, PK208, PK212, PK216 manufactured by Mitsubishi Chemical Corporation. , PK218, PK220, PK228, etc., and weak cation exchange resins selected from “Amberlite” FPC3500, IRC76 manufactured by Organo Corporation, “Diaion” WK10, WK11, WK100, WK40L manufactured by Mitsubishi Chemical Corporation, etc. can do.

  In the present invention, it is preferable to use an anion exchange resin and a cation exchange resin in combination as the ion exchange resin, and among them, it is better to use a strong anion resin and a strong cation resin capable of removing various ions. The anion exchange resin is preferably regenerated with a dilute alkaline aqueous solution such as sodium hydroxide and used as an OH type. In the case of a cation exchange resin, it is preferably regenerated with a dilute acidic aqueous solution such as hydrochloric acid and used as an H type. . The desalting method using an ion exchange resin may be a batch method or a column method, and is not particularly limited as long as it is a method capable of efficiently desalting. However, in the manufacturing process, a column method that can be easily used repeatedly is preferably employed. The liquid passing speed is usually controlled by SV (space velocity), SV = 2 to 50 is preferable, and it is preferable to pass the liquid at SV = 2 to 10 which can be purified to a higher degree. Various types of resins such as gel type, porous type, high porous type, and MR type are commercially available, and these ion exchange resins may have any shape, and an optimal shape may be selected in accordance with the quality of the solution.

  In addition, nanofiltration membrane treatment is a specific example of the desalting step. The nanofiltration membrane treatment of the 2,3-butanediol culture solution is performed by filtering the 2,3-butanediol culture solution through the nanofiltration membrane as disclosed in, for example, Japanese Patent Application Laid-Open No. 2010-150248. Thus, 2,3-butanediol can be efficiently separated on the permeate side, and inorganic salts, saccharides, and coloring components can be efficiently separated on the non-permeate side.

  In addition to polymer materials such as piperazine polyamide, polyamide, cellulose acetate, polyvinyl alcohol, polyimide, and polyester, inorganic materials such as ceramics are used as materials for the nanofiltration membrane. The nanofiltration membrane is generally used as a flat membrane or a hollow fiber membrane in addition to the spiral membrane element, but the nanofiltration membrane used in the present invention is preferably a spiral membrane element.

  Preferable specific examples of the nanofiltration membrane element used in the present invention include, for example, “GEsepa” of a nanofiltration membrane manufactured by GE Osmonics, which is a cellulose acetate-based nanofiltration membrane, and a nanolayer manufactured by Alfa Laval, which uses polyamide as a functional layer. NF99 or NF99HF of filtration membrane, NF-45, NF-90, NF-200 or NF-400 of Nanotec membrane manufactured by Filmtec Co., Ltd. with functional layer of cross-linked piperazine polyamide, or UTC60 manufactured by Toray Industries, Inc. Nanofiltration membrane element SU-210, SU-220, SU-600 or SU-610 can be mentioned. Among them, NF99 or NF99HF of a nanofiltration membrane made of Alfa Laval Corporation having a functional layer of polyamide, and a functional layer of crosslinked piperazine polyamide are preferable. Nanofiltration made by Filmtech NF-45, NF-90, NF-200 or NF-400 of the company, nano-filtration membrane module SU-210, SU-220, SU-600 or SU-610 manufactured by Toray Industries, Inc. Preferably, the nanofiltration membrane element SU-210, SU-220, SU-600 or SU-610 made by the same company including UTC60 made by Toray Industries, Inc., which is mainly composed of a crosslinked piperazine polyamide.

  The nanofiltration membrane may be filtered, and the filtration pressure is preferably used in the range of 0.1 MPa to 8 MPa. If the filtration pressure is lower than 0.1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane may be damaged. Moreover, if the filtration pressure is 0.5 MPa or more and 7 MPa or less, the membrane permeation flux is high, so that the 2,3-butanediol fermentation solution can be efficiently permeated and the membrane damage can be affected. It is more preferable because of its low property, and it is particularly preferable to use at 1 MPa or more and 6 MPa or less.

  The concentration of 2,3-butanediol in the 2,3-butanediol solution filtered through the nanofiltration membrane is not particularly limited, but if the concentration is high, the concentration of 2,3-butanediol contained in the permeate is also high. Therefore, energy can be reduced when concentrating, which is suitable for cost reduction.

  In the desalting step, either ion exchange treatment or nanofiltration membrane treatment may be used, and these may be used in combination, but at least ion exchange treatment is preferred. In addition, there is no particular limitation on the order in the case where ion exchange treatment and nanofiltration membrane treatment are used in combination, but the inorganic salt obtained from the permeate side is reduced by nanofiltration membrane treatment of 2,3-butanediol culture solution 2 It is preferable to ion-exchange the 3-butanediol culture solution. Thereby, the removal rate of inorganic salts can be raised by removing the inorganic salt and organic acid which pass a part of nanofiltration membrane with an ion exchange resin.

  As a method for concentrating the 2,3-butanediol culture solution, a general method known to those skilled in the art may be used. For example, a method using a reverse osmosis membrane or a heat concentration using an evaporator may be used. The method is preferably applied.

  In the method using a reverse osmosis membrane, the 2,3-butanediol solution is filtered through the reverse osmosis membrane to allow water to permeate the permeate side and keep 2,3-butanediol on the non-permeate side of the membrane. It is a method of concentrating by doing. As a reverse osmosis membrane preferably used in the present invention, a composite membrane using a cellulose acetate-based polymer as a functional layer (hereinafter also referred to as a cellulose acetate-based reverse osmosis membrane) or a composite membrane using a polyamide as a functional layer (hereinafter, Polyamide-based reverse osmosis membrane). Here, as the cellulose acetate-based polymer, organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and the like, or a mixture thereof and those using mixed esters can be mentioned. It is done. The polyamide includes a linear polymer or a crosslinked polymer having an aliphatic and / or aromatic diamine as a monomer. As the membrane form, an appropriate form such as a flat membrane type, a spiral type, and a hollow fiber type can be used.

  Specific examples of the reverse osmosis membrane used in the present invention include, for example, low pressure type SU-710, SU-720, SU-720F, SU-710L, SU-, which are polyamide-based reverse osmosis membrane modules manufactured by Toray Industries, Inc. 720L, SU-720LF, SU-720R, SU-710P, SU-720P, high pressure type SU-810, SU-820, SU-820L, SU-820FA, the company's cellulose acetate reverse osmosis membrane SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100, SC-8200, NTR-759HR, NTR-729HF, NTR manufactured by Nitto Denko Corporation -70 SWC, ES10-D, ES20-D, ES20-U, ES15-D, ES1 -U, LF10-D, Alfa Laval RO98pHt, RO99, HR98PP, CE4040C-30D, GE "GESepa", Filmtec BW30-4040, TW30-4040, XLE-4040, LP-4040, LE-4040, SW30- 4040, SW30HRLE-4040, and the like.

  Concentration with a reverse osmosis membrane is carried out under pressure, but if the filtration pressure is lower than 1 MPa, the membrane permeation rate decreases, and if it is higher than 8 MPa, the membrane damage is affected, so in the range of 1 MPa to 8 MPa. Preferably there is. Moreover, if the filtration pressure is in the range of 1 MPa or more and 7 MPa or less, the membrane permeation flux is high, so that the 2,3-butanediol solution can be concentrated efficiently. Most preferably, it is in the range of 2 MPa or more and 6 MPa or less because there is little possibility of affecting the film damage. In a low concentration 2,3-butanediol solution, a method using a reverse osmosis membrane is preferable because of its low cost.

  In addition, it is preferable to include the process of isolate | separating the microbial cell contained in a 2, 3- butanediol culture solution prior to the said process. As the cell separation step, for example, a method such as centrifugation or filtration separation can be used. Only one of these methods may be used or a combination thereof may be used.

  The 2,3-butanediol produced according to the present invention is characterized by high purity. As a method for measuring impurities contained in 2,3-butanediol, evaluation is performed by using purity measurement by gas chromatography (GC) or purity measurement by UV detection using high performance liquid chromatography (HPLC). Since these are evaluated by the ratio of the area of 2,3-butanediol in the total detection peak area, the higher the ratio, the higher the purity of 2,3-butanediol.

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

  The expression of four kinds of enzyme genes in the transformant cells described in Patent Document 1 and Patent Document 2 was confirmed. This transformed strain has been deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, based on the Budapest Treaty since June 30, 2004, and the deposit number is FERM BP-10048. This transformed strain is already available for sale because Patent Document 2 is US Pat. No. 7,569,379.

Reference Example 1 kLa Measurement Dynamic in which dissolved oxygen electrode (manufactured by Able Co., Ltd.) is inserted into a culture apparatus (manufactured by Able Co., Ltd., Able Co., Ltd.), the dissolved oxygen concentration is measured for each aeration stirring condition, and nitrogen gas is used KLa was determined by the method. First, 1L of water is added to the culture tank, and the water temperature is adjusted to 30 ° C while stirring at a constant speed. Nitrogen gas is sufficiently blown into the water, and when the electrode value reaches the minimum value, the dissolved oxygen electrode is zero. Calibration was performed. Thereafter, the change in dissolved oxygen concentration over time after switching the aeration gas from nitrogen gas to air or nitrogen gas at a predetermined aeration rate was measured to obtain kLa. Table 1 shows kLa for each aeration stirring condition obtained.

Examples 1 to 5: kLa
<2,3-butanediol fermentation>
Zymobacter palmae [pMFY31-xt] disclosed in Patent Documents 1 and 2 was used as a microorganism of the genus Zymobacter imparted with xylose utilization. Zymobacter palmae [pMFY31-xt] was created by transforming Zymobacter palmae with a broad host range recombinant plasmid vector introduced with foreign genes encoding xylose isomerase, xylulokinase, transaldolase and transketolase It is a publicly known transformant and, as described above, is deposited under the accession number of FERM BP-10048 based on the Budapest Treaty and can be sold. Z. palmae [pMFY31-xt] was added to TX medium (40 g / L Xylose, 10 g / L Yeast extract, 10 g / L KH ₂ PO ₄ , 2 g / L (NH ₄ ) ₂ SO ₄ , 100 μg / mL ampicillin. Static culture was performed at 30 ° C. for 24 hours in a test tube containing 5 mL of 5 g / L MgSO ₄ .7H ₂ O, pH 6.0). The whole amount of the culture solution was added to an Erlenmeyer flask containing 50 mL of TX medium, followed by stationary culture at 30 ° C. for 24 hours. The culture solution was added to 1 L of 2,3-butanediol fermentation medium having the composition shown in Table 2 and neutralized with potassium hydroxide so as to have a pH of 5.5. Culturing was performed under stirring conditions. During the cultivation, the culture solution was appropriately collected and the xylose concentration and 2,3-butanediol concentration were measured.

  Each concentration measurement condition by high performance liquid chromatography (HPLC, manufactured by Shimadzu Corporation) is shown below. The 2,3-butanediol concentration, sugar concentration, and fermentation yield after fermentation were determined by the following measurement methods.

2,3-butanediol concentration measurement column: Shodex Sugar SH1011 (manufactured by Showa Denko KK)
Column temperature: 65 ° C
Mobile phase: 0.05 M sulfuric acid aqueous solution, 0.6 mL / min
Detection: RI.

Sugar concentration measuring column: Asahipak NH2P50 4E (Showa Denko KK)
Column temperature: 30 ° C
Mobile phase: water: acetonitrile = 1: 3, 0.6 mL / min
Detection: RI.

Fermentation yield It calculated using the following formula from the 2,3-butanediol concentration and sugar concentration measured by the above-mentioned HPLC analysis.
Fermentation yield (%) = 100 × {(2,3-butanediol concentration in culture medium) − (2,3-butanediol concentration in medium)} / {(sugar concentration in medium) − (sugar concentration in culture medium) )}.

  Table 3 shows the concentrations of 2,3-butanediol and xylose and the fermentation yield in the culture solution when the xylose concentration in the culture solution is closest to zero. The obtained 2,3-butanediol culture was collected and centrifuged at 4 ° C. and 8000 rpm for 15 minutes, and then the supernatant was filtered through a microfiltration membrane (manufactured by Toray Industries, Inc.) to remove the cells. The above process was performed twice to obtain a total of 2 L of 2,3-butanediol culture solution.

<Ion-exchange purification of 2,3-butanediol culture solution>
About the culture solution obtained by the above-mentioned fermentation test, the residual ion was removed by the ion exchange process. Using strong anion exchange resin IRA120J (manufactured by Organo Co., Ltd.) and strong cation exchange resin IR410 (manufactured by Organo Co., Ltd.), they were regenerated and used in 1H sodium hydroxide and 1N hydrochloric acid, respectively. The amount of resin was calculated so that the total amount of various inorganic salts and organic acids and the exchange capacity of the ion exchange resin were equal. The above-described ion exchange resin was packed in a column, and the anion exchange and the cation exchange were carried out in this order at a liquid passing speed SV = 5.

<Distillative purification of 2,3-butanediol>
Water was removed from the ion-exchanged 2,3-butanediol solution using a thin film concentrator MF-10 (manufactured by Tokyo Rika Kikai Co., Ltd.). At this time, water was evaporated at a reduced pressure of 30 hPa and a heating temperature of 60 ° C. The concentrated 2,3-butanediol solution was distilled under reduced pressure (5 mmHg) to obtain purified 2,3-butanediol. The GC purity and distillation yield of 2,3-butanediol after distillation were determined by the following measuring method.

GC purity 2,3-butanediol after distillation was analyzed using gas chromatography (GC, manufactured by Shimadzu Corporation). From the ratio of the peak area of 2,3-butanediol in the total detected peak area, Calculated by
GC purity (%) = 100 × (2,3-butanediol peak area) / (total detection peak area).
The analysis conditions for gas chromatography are shown below.
Column: RT-BDEXM (0.25 mm × 30 m, manufactured by Restek)
Column temperature: 75 ° C
Vaporization chamber, detector temperature: 230 ° C
Carrier gas: He
Linear velocity: 35 cm / sec
Detection: Flame ionization detector (FID).

Distillation Yield Calculated from 2,3-butanediol concentration before distillation calculated from 2,3-butanediol concentration measured by HPLC analysis and the amount of charged liquid, the amount of distillate after distillation and the above-mentioned GC purity From the recovered amount of 2,3-butanediol, calculation was made according to the following formula.
Distillation yield (%) = 100 × {(distilled liquid amount after distillation) × (GC purity)} / {(2,3-butanediol concentration before distillation) × (charged liquid amount before distillation)}.

The total yield of 2,3-butanediol was calculated from the fermentation yield calculated from the result of the fermentation step and the distillation yield calculated from the result of the purification step, according to the following formula. The results are shown in Table 3.
Total yield of 2,3-butanediol (g / g sugar) = (fermentation yield) / 100 × (distillation yield) / 100.

Examples 6-9: pH
Z. palmae [pMFY31-xt] was cultured by the method described in Examples 1-5. The culture broth cultured in a flask was added to 1 L of 2,3-butanediol fermentation medium having the composition shown in Table 2, and water was added so that the pH was 4.5, 4.8, 6.1, or 6.5, respectively. While neutralizing with potassium oxide, the cells were cultured at an aeration rate of 0.2 vvm, an agitation rate of 400 rpm, and a temperature of 30 ° C. The culture solution was appropriately collected during the culture, and the xylose concentration and 2,3-butanediol concentration were measured by the methods described in Examples 1-5. Table 4 shows the concentrations of 2,3-butanediol and xylose in the culture solution and the fermentation yield when the xylose concentration in the culture solution is closest to zero. The 2,3-butanediol culture solution after the fermentation test was collected and centrifuged at 4 ° C. and 8000 rpm for 15 minutes, and then the supernatant was filtered through a microfiltration membrane (manufactured by Toray Industries, Inc.) to remove the cells. The above process was performed twice to obtain a total of 2 L of 2,3-butanediol culture solution. The obtained culture solution was desalted and distilled by the methods described in Examples 1 to 5 to obtain purified 2,3-butanediol. Table 4 shows the results of determining the GC purity and distillation yield of 2,3-butanediol after distillation by the methods described in Examples 1-5.

Examples 10-12: Sugar concentration palmae [pMFY31-xt] was cultured by the method described in Examples 1-5. The culture broth cultured in a flask was added to 1 L of 2,3-butanediol fermentation medium having the composition shown in Table 2 adjusted so that the xylose concentration was 5.0, 15, or 100 g / L, respectively, and the pH was 5. The cells were cultured at aeration rate of 0.2 vvm, stirring speed of 400 rpm, and temperature of 30 ° C. while neutralizing with potassium hydroxide so as to be 5. The culture solution was appropriately collected during the culture, and the xylose concentration and 2,3-butanediol concentration were measured by the methods described in Examples 1-5. Table 5 shows the concentrations of 2,3-butanediol and xylose and the fermentation yield in the culture solution when the xylose concentration in the culture solution is closest to zero. The 2,3-butanediol culture solution after the fermentation test was collected and centrifuged at 4 ° C. and 8000 rpm for 15 minutes, and then the supernatant was filtered through a microfiltration membrane (manufactured by Toray Industries, Inc.) to remove the cells. The above process was performed twice to obtain a total of 2 L of 2,3-butanediol culture solution. The obtained culture solution was desalted and distilled by the methods described in Examples 1 to 5 to obtain purified 2,3-butanediol. Table 5 shows the results of determining the GC purity and distillation yield of 2,3-butanediol after distillation by the methods described in Examples 1-5.

Examples 13-15: pentose sugar ratio Z. palmae [pMFY31-xt] was cultured by the method described in Examples 1-5. The composition shown in Table 2 was prepared by adjusting the concentration of the xylose and glucose (g / L) to (40:20), (20:40), or (2.5: 40) in the culture solution cultured in the flask. Was added to 1 L of 2,3-butanediol fermentation medium and cultured at an aeration rate of 0.2 vvm, an agitation rate of 400 rpm, and a temperature of 30 ° C. while neutralizing with potassium hydroxide so that the pH was 5.5. The culture solution was appropriately collected during the culture, and the sugar concentration and 2,3-butanediol concentration were measured by the methods described in Examples 1-5. Table 6 shows the 2,3-butanediol and sugar concentrations in the culture solution and the fermentation yield when the sugar concentration in the culture solution is closest to zero. The 2,3-butanediol culture solution after the fermentation test was collected and centrifuged at 4 ° C. and 8000 rpm for 15 minutes, and then the supernatant was filtered through a microfiltration membrane (manufactured by Toray Industries, Inc.) to remove the cells. The above process was performed twice to obtain a total of 2 L of 2,3-butanediol culture solution. The obtained culture solution was desalted and distilled by the methods described in Examples 1 to 5 to obtain purified 2,3-butanediol. Table 6 shows the results of determining the GC purity and distillation yield of 2,3-butanediol after distillation by the methods described in Examples 1-5.

  2,3-butanediol (2,3-BDO) obtained by the method of the present invention is a raw material for pharmaceutical and cosmetic intermediate materials, inks, perfumes, liquid crystals, insecticides, softening reagents, explosives, plasticizers, etc. As a useful compound.

Claims (13)

A step of culturing a microorganism belonging to the genus Zymobacter having the ability to assimilate pentose in a fermentation raw material containing pentose (Step A), and 2,3-butane from the culture solution obtained in the step A method for producing 2,3-butanediol, comprising a step of purifying a diol (step B).   The method according to claim 1, wherein the step A is culturing under aerated conditions. The method according to claim 1 or 2, wherein the step A is culturing under conditions of an oxygen transfer capacity coefficient (kLa) of 9h ^-1 or more.   The microorganism according to any one of claims 1 to 3, wherein the microorganism is a transformed microorganism of the genus Zymobacter into which a foreign gene encoding at least one enzyme selected from xylose isomerase, xylulokinase, transaldolase, and transketolase is introduced. 2. The method according to item 1.   The method according to claim 4, wherein the microorganism is a transformed microorganism of the genus Zymobacter into which a foreign gene encoding xylose isomerase, xylulokinase, transaldolase and transketolase has been introduced.   The method according to any one of claims 1 to 5, wherein the microorganism is Zymobacter palmae.   The method according to any one of claims 1 to 6, wherein the pentose contained in the fermentation raw material of Step A is xylose.   The method according to any one of claims 1 to 7, wherein an abundance ratio of xylose to total sugar in the fermentation raw material is 5 to 100%.   The method according to any one of claims 1 to 8, wherein the step A is culturing in a medium having a total sugar concentration of 20 g / L or more.   The method according to any one of claims 1 to 9, wherein the fermentation raw material includes a biomass-derived sugar solution.   The method according to claim 1, wherein the step B includes a distillation step.   The method according to claim 11, comprising a desalting treatment step before the distillation step.   The method according to claim 12, comprising at least an ion exchange treatment step as the desalting treatment step.