JP5724876B2 - Method for producing succinic acid - Google Patents

Description

  The present invention relates to a method for producing an aliphatic dicarboxylic acid such as succinic acid using a microorganism.

  In addition, the present invention particularly relates to aliphatic dicarboxylic acids from aqueous solutions containing aliphatic dicarboxylic acids (salts) and saccharides, amino acids, proteins, inorganic salts, and the like obtained from microorganisms such as glucose, sucrose, and cellulose, which are biological materials. Relates to a method for producing an aliphatic dicarboxylic acid.

  Aliphatic dicarboxylic acids are widely used as raw materials for polymers such as polyesters and polyamides, particularly biodegradable polyesters, and as raw materials for foods, pharmaceuticals and cosmetics. In particular, when an aliphatic dicarboxylic acid is used as a polymer raw material, a high-purity aliphatic dicarboxylic acid is required for maintaining the degree of polymerization of the polymer and preventing coloring. Of these, succinic acid is expected in recent years as a raw material for biodegradable polymers together with lactic acid.

  Aliphatic dicarboxylic acids have heretofore been produced industrially from petroleum-derived raw materials. For example, succinic acid has been produced by a hydrogenation reaction of maleic acid, a raw material derived from petroleum. On the other hand, in recent years, various aliphatic dicarboxylic acids can be produced from plant-derived materials with a high carbon yield from a wide range of biological materials. For example, succinic acid, adipic acid and the like can be produced by fermentation.

  In the fermentation production of aliphatic dicarboxylic acid, for example, the fermentation broth is treated by centrifugation and filtration to remove solids such as bacterial cells, and then desalted with an ion exchange resin or the like, and crystallized from the solution. A method of purifying an aliphatic dicarboxylic acid by column chromatography is used.

As a method using an ion exchange resin, Patent Document 1 discloses a method of purifying an aliphatic dicarboxylic acid with an H ⁺ type strongly acidic cation exchange resin after centrifuging cells. In Patent Document 2, as a method for removing microbial cells, the microbial cell is aggregated by adjusting the suspension of the microorganism to pH 4 or lower and dissolving sodium polyacrylate in the microbial suspension. A method for separating bacterial cell agglomerates is disclosed.

  In addition, in the production of aliphatic dicarboxylic acid by fermentation, a raw material is generally a saccharide, such as glucose, sucrose, and cellulose. However, when the polysaccharide is contained as an impurity, the raw material saccharide may not be completely assimilated by the microorganism, and the saccharide may be mixed into the aliphatic dicarboxylic acid.

  Further, when ammonia is used as a neutralizing agent in fermentation using microorganisms, amino acids are by-produced and amino acids are mixed into aliphatic dicarboxylic acids. Furthermore, proteins derived from microorganisms and inorganic salts used in fermentation may be mixed in aliphatic dicarboxylic acids.

  However, it has been difficult to remove these impurities by an efficient and inexpensive method.

  As a purification method of aliphatic dicarboxylic acid produced by fermentation, a method of decomposing a calcium salt of aliphatic dicarboxylic acid with sulfuric acid (for example, see Patent Document 3), a method using an ion exchange resin (for example, see Patent Document 4). )It has been known. However, these methods not only provide a sufficient degree of purification, but also generate a large amount of by-product salt, and the treatment becomes a problem.

  Furthermore, a method has been proposed in which ammonium hydrogen sulfate and / or sulfuric acid is added to an ammonium salt of an aliphatic dicarboxylic acid to produce a dicarboxylic acid and an ammonium sulfate salt, and the generated ammonium sulfate salt is thermally decomposed into ammonium hydrogen sulfate and ammonia for recycling. (For example, see Patent Document 5). However, in this method, there is no problem in treating the salt because the by-product salt is decomposed and recycled, but decomposition of the by-product salt requires high temperature and is difficult. Further, the degree of purification was insufficient.

  A method using electrodialysis (see, for example, Patent Document 6) is also generally known. However, since electrodialysis increases in proportion to the production scale, the scale merit is small and the cost is high even in industrial scale production.

  As a method for solving these problems, a method of adding a solvent to an aqueous solution containing an organic acid to extract the organic acid (for example, see Patent Document 7) is known.

International Publication No. 2005/030973 Japanese Patent Laid-Open No. 5-3780 Japanese Patent Laid-Open No. 3-030885 Japan Special Table 2002-505310 Japanese Special Table 2001-514900 Japanese Unexamined Patent Publication No. 2005-333886 Japanese National Table No. 9-500649

  However, since the methods described in Patent Documents 1 and 2 use chemicals and resins, it is time-consuming and a simpler and more efficient method for purifying aliphatic dicarboxylic acids has been desired. Accordingly, an object of the present invention is to improve the purification efficiency of an aliphatic dicarboxylic acid from a suspension containing an aliphatic dicarboxylic acid obtained by reacting a biological material and a microorganism with microbial cells, thereby producing an aliphatic dicarboxylic acid. The object is to provide a method capable of efficiently producing an acid.

  In addition, when the aliphatic dicarboxylic acid is purified, the extraction method is performed by adding a phase-separable solvent to the aqueous solution containing the aliphatic dicarboxylic acid. In the process, a solid content that does not dissolve in either the aqueous phase or the solvent phase is generated. However, there are cases in which not only the extraction is inhibited, but also the subsequent processes are adversely affected and the purity of the aliphatic dicarboxylic acid is deteriorated.

  In particular, an aliphatic solution obtained by microbial conversion from glucose, sucrose, cellulose, or the like, which is a biological material, is brought into contact with an aqueous solution composed of an aliphatic dicarboxylic acid (salt), saccharide, amino acid, protein, inorganic salt, etc., and a solvent. When extracting dicarboxylic acid, extraction operation and phase separation operation may be particularly difficult.

  Furthermore, the extraction method described in Patent Document 7 does not provide a method for recovering the target organic acid as crystals.

  Therefore, the present invention eliminates the inhibition of phase separation due to the generation of the above-mentioned solids and the obstacles to the production operation associated therewith, and is effective and stable from an aqueous solution containing an aliphatic dicarboxylic acid obtained by microbial conversion from a biological material. It is another object of the present invention to provide a method for producing an aliphatic dicarboxylic acid.

  As a result of intensive studies to solve the above problems, the present inventors have found that the problems of the present invention can be solved by the method for producing an aliphatic dicarboxylic acid of the present invention described below.

That is, according to the present invention, the following inventions are provided.
1. From an aqueous solution including succinate obtained by reacting a biological raw material and a microorganism, a method for producing a succinic acid, a manufacturing method of succinic acid comprising the steps of (1) to (4).
(1) Aliphatic dicarboxylic acid generating step for accumulating succinic acid in the reaction solution by reacting biological material and microorganisms (2) Liquid containing succinic acid and microorganisms obtained in the aliphatic dicarboxylic acid generating step PH adjustment step (3) for separating microorganisms from the liquid obtained in the pH adjustment step (4) microorganism separation step 1. A contact step in which an aqueous solution obtained by separating microorganisms in step 1 is contacted with a solvent capable of phase separation with the aqueous solution . 2. The method according to item 1 above, wherein the microorganism is a coryneform bacterium.
3. Solvent inorganic value / ratio of organic value (I / O value) is 0.2 to 2.3, a boiling point at normal pressure (1 atm) is 40 ° C. or more organic solvents, item 1 or 2. The method according to 2 .
4). A method for producing succinic acid from an aqueous solution containing succinic acid obtained by reacting a biological material and a microorganism, and comprising the following steps (1) to (6).
(1) An aliphatic dicarboxylic acid production step for accumulating succinic acid in a reaction solution by reacting a biological material with a microorganism.
(2) pH adjusting step of obtaining a liquid having a pH of 1.0 or more and 5.0 or less by mixing a liquid containing succinic acid and microbial cells obtained in the aliphatic dicarboxylic acid generating step with an acid.
(3) Bacterial cell separation step for separating microbial cells from the liquid obtained in the pH adjustment step ( 4 ) Aqueous solution obtained by separating the bacterial cells in the bacterial cell separation step, and a solvent capable of phase separation from the aqueous solution ( 5 ) Solid content removal step for removing solids generated by contact of the aqueous solution and the solvent ( 6 ) Phase separation step for separating the solvent by phase separation
5. The method for producing succinic acid according to 4 above, wherein in the solid content removal step, the solid content is removed using a settler.
6). 6. The method for producing succinic acid according to item 4 or 5 , wherein in the contacting step, the aqueous solution and the solvent are contacted using a static mixer.
7). The method for producing succinic acid according to any one of the preceding items 4 to 6 , wherein in the solid content removing step, the solid content is removed together with at least one liquid selected from the aqueous solution and the solvent. .
8). The method for producing succinic acid according to item 7 above, wherein the solid content extracted is subjected to solid-liquid separation, and the obtained liquid is returned to at least one of the step before the contact step between the aqueous solution and the solvent and the step after the phase separation step. .
9. 9. The method for producing succinic acid according to 8 above, wherein the solid-liquid separation is performed by a centrifugal sedimentation method.
10. 9. The method for producing succinic acid according to item 8 , wherein the solid-liquid separation is performed using a filter.
11. The remaining liquid after separation by phase separating the solvent, and a solvent for liquid phase separation, is contacted in a countercurrent multistage extraction column, comprising the step of separating by phase separation the solvent later, preceding the preceding paragraph 4 The method for producing succinic acid according to any one of 10
12 The manufacturing method of the succinic acid of any one of the preceding clause 4 to the preceding clause 11 which performs the said contact process at 30-60 degreeC.
13. A method for producing succinic acid from an aqueous solution containing succinic acid obtained by reacting a biological raw material and a microorganism, the method comprising producing the following steps (1) to (8).
(1) An aliphatic dicarboxylic acid production step for accumulating succinic acid in a reaction solution by reacting a biological material with a microorganism.
(2) pH adjusting step of obtaining a liquid having a pH of 1.0 or more and 5.0 or less by mixing a liquid containing succinic acid and microbial cells obtained in the aliphatic dicarboxylic acid generating step with an acid.
(3) Cell separation step for separating microorganism cells from the liquid obtained in the pH adjustment step
(4) A contact step in which the aqueous solution obtained by separating the cells in the cell separation step and a solvent capable of phase separation are brought into contact with the aqueous solution. ( 5 ) In the step of concentrating the succinic acid recovered in the contact step. there are, the extract phase concentration step (6) crystallization step (7) to precipitate the succinate from the liquid after the extraction phase concentration step crystallization of succinic acid precipitated in about crystallization step in which the water concentration in the extract phase by the concentration increases Solid-liquid separation step to recover ( 8 ) Crystallization mother liquor recycling step for returning at least part of the crystallization mother liquor after recovering the succinic acid obtained in the solid-liquid separation step to any step before the crystallization step
14 14. The method for producing succinic acid according to 13 above, further comprising a protonation step of adding an acid to an aqueous solution containing succinic acid obtained from the biological raw material before the contacting step.
15. 15. The method for producing succinic acid according to 13 or 14 above, wherein the concentration of the solvent contained in the solution containing succinic acid after the extraction phase concentration step supplied to the crystallization step is 10% by weight or less.
16. 16. The method for producing succinic acid according to 15 above, wherein the concentration of the solvent contained in the solution containing succinic acid supplied to the crystallization step is 1% by weight or less.
17. After removing components other than succinic acid from the mother liquor after recovering succinic acid in the solid-liquid separation process, at least a part of the liquid is recycled to at least one of the processes from the contact process to the crystallization process. The method for producing succinic acid according to any one of items 13 to 16 above.
18. 18. The method for producing succinic acid according to any one of items 13 to 17 , wherein water is added in any step before the extraction phase concentration step.
19. An acid having a pKa of less than 4 is used as an acid to be added in the protonation step, and the pH of the aqueous solution containing succinic acid obtained from the biological material in the protonation step is maintained in the range of more than 1 and less than 4. 19. The method for producing succinic acid according to any one of items 14 to 18 above, wherein:
20. The solvent used in the contact step has a ratio of an inorganic value to an organic value of 0.2 or more and 2.3 or less, and a boiling point of 40 ° C. or more, any one of the items 13 to 19 above 2. A method for producing succinic acid according to item 1.
21. 21. The method for producing succinic acid according to any one of items 13 to 20 , further comprising a step of removing microorganisms before the contacting step.
22. [ 22] The method for producing succinic acid according to any one of [ 13] to [ 21] above, comprising a step of removing the protein before the contacting step.
23. 23. The method according to any one of items 13 to 22 above, wherein a mixer settler is used in the contacting step, and an extraction phase, a residual extraction phase, and a phase containing a solid content are recovered from a settler portion of the mixer settler. A method for producing succinic acid .
24. 24. The method for producing succinic acid according to 23 , wherein the phase containing the solid content recovered from the settler part is subjected to solid-liquid separation, and the liquid phase is recovered.
25. 25. The method for producing succinic acid according to item 23 or 24 , wherein succinic acid is further recovered from the extracted residual phase obtained in the settler section using a countercurrent multistage extraction tower in the contacting step.
26. 26. The method for producing succinic acid according to any one of items 13 to 25 , wherein cooling is performed in the crystallization step.
27. 27. The method for producing succinic acid according to any one of items 13 to 26 , wherein the crystallization step includes a decompression operation.

  According to the method of the present invention, an aliphatic dicarboxylic acid can be efficiently and stably produced from an aqueous solution containing an aliphatic dicarboxylic acid obtained from a biological material.

  In other words, microbial cells can be efficiently separated even by low-speed centrifugation, so that the equipment load can be reduced, and the separation time in solvent extraction and the intermediate insoluble matter can be reduced. Aliphatic dicarboxylic acid can be purified efficiently.

It is drawing which shows one form of the manufacturing method of aliphatic dicarboxylic acid which has the contact process of the aqueous solution and solvent containing aliphatic dicarboxylic acid, a phase-separation process, and a solid content removal process. The figure which shows the construction procedure of plasmid pMJPC17.2. The underlined number indicates a primer consisting of the sequence of SEQ ID NO. It is drawing which shows the composition change in dilution / distillation operation of the extract in Example 2-1 and Example 3-1. It is drawing which shows the contact process of the aqueous solution containing a succinic acid and solvent in Example 2-4, a phase-separation process, and a solid content removal process. It is drawing which shows the contact process of the aqueous solution containing a succinic acid and solvent in Example 2-8, a phase-separation process, and a solid content removal process.

Hereinafter, embodiments of the present invention will be described in detail.
I. 1st this invention 1st this invention is a method of manufacturing aliphatic dicarboxylic acid from the aqueous solution containing aliphatic dicarboxylic acid obtained by making a biological raw material and microorganisms react, Comprising: The following processes (1 ) To (4).
(1) Aliphatic dicarboxylic acid production step for accumulating aliphatic dicarboxylic acid in the reaction solution by reacting biological material and microorganisms (2) Aliphatic dicarboxylic acid and microorganism obtained in the aliphatic dicarboxylic acid production step PH adjustment step (3) for obtaining a solution having a pH of 1.0 or more and 5.0 or less by mixing a solution containing a solution and an acid, a microorganism separation step (4) for separating microorganisms from the solution obtained in the pH adjustment step ) A contact process in which an aqueous solution obtained by separating microorganisms in the microorganism separation process is contacted with a solvent.

  The steps other than the above (1) to (3) may include any conventionally known steps. Hereafter, it explains in detail according to each process.

I- (1) Aliphatic dicarboxylic acid production step (biological raw material) in which an aliphatic dicarboxylic acid is accumulated in a reaction solution by reacting a biological raw material with a microorganism
Examples of biological materials include wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil cass, persimmon, buckwheat, soybean, fat, waste paper, paper Residue, marine product residue, livestock excrement, sewage sludge, food waste and the like.

  Among them, plant resources such as wood, rice straw, rice husk, rice bran, old rice, corn, sugar cane, cassava, sago palm, okara, corn cob, tapioca cass, bagasse, vegetable oil residue, rice cake, buckwheat, soybean, oil, waste paper and paper residue More preferred are wood, rice straw, rice husk, old rice, corn, sugar cane, cassava, sago palm, straw, oil, waste paper and papermaking residue, and most preferred are corn, sugar cane, cassava and sago palm.

  The biological material generally contains many alkali metals such as nitrogen element, Na, K, Mg, and Ca, and alkaline earth metals.

  The biological raw material is not particularly limited. For example, the biological raw material is converted into a carbon source through a known pretreatment such as a chemical treatment such as acid and alkali, a biological treatment using a microorganism, a physical treatment, and a saccharification step. Be guided.

  The process is not particularly limited, but includes, for example, a miniaturization process by pretreatment such as chipping, scraping, and crushing a biological material. If necessary, a grinding step by a grinder or a mill is further included.

  The biological raw material thus refined is further guided to a carbon source through pretreatment and saccharification steps. Specific methods thereof include, for example, chemical methods such as acid treatment with strong acids such as sulfuric acid, nitric acid, hydrochloric acid and phosphoric acid, alkali treatment, ammonia freeze steaming explosion method, solvent extraction, supercritical fluid treatment and oxidant treatment, Examples include physical methods such as fine pulverization, steam explosion, microwave treatment, and electron beam irradiation, and biological treatment such as hydrolysis by microorganisms or enzyme treatment.

(Microorganism)
The type of microorganism is not particularly limited, but examples include enteric bacteria such as Escherichia coli, Bacillus bacteria, coryneform bacteria, etc., and it is preferable to use aerobic microorganisms, facultative anaerobic microorganisms, or microaerobic microorganisms. .

  Specific examples of aerobic microorganisms include, for example, Coryneform Bacteria, Bacillus bacteria, Rhizobium bacteria, Escherichia bacteria, Lactobacillus bacteria, and Lactobacillus bacteria. Bacteria of the genus Succinobacillus, bacteria of the genus Anaerobiospirillum, bacteria of the genus Actinobacillus, bacteria of the genus filamentous fungi, bacteria of the genus Rhodococcus, bacteria of the genus Nocardiaceto ) Genus bacteria. Among these, coryneform bacteria are preferable.

  Examples of Escherichia bacteria include, for example, Escherichia coli. Examples of Lactobacillus bacteria include, for example, Lactobacillus helveticus (J Appl Microbiol, 2001, 91, p846-852).

  Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus stearothermophilus, and the like. Examples of the genus Rhizobium include Rhizobium etli.

  The coryneform bacterium is not particularly limited as long as it is classified as such. For example, bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Arthrobacter, myco Examples include bacteria belonging to the genus Mycobacterium, the genus Microbacterium, the bacteria belonging to the genus Micrococcus, and the like.

  Among these, bacteria belonging to the genus Corynebacterium and bacteria belonging to the genus Brevibacterium are preferable, and the bacteria classified as Corynebacterium glutamicum are classified into Brevibacterium flavum. More preferred are bacteria, bacteria classified as Brevibacterium ammoniagenes, and bacteria classified as Brevibacterium lactofermentum.

  Microorganisms that convert carbon sources into aliphatic dicarboxylic acids include wild strains of microorganisms as described above, mutant strains obtained by normal mutation treatment such as UV irradiation and NTG treatment, and genetics such as cell fusion and genetic recombination methods. And recombinants derived by a conventional method.

  As the gene recombinant, those obtained by known methods such as enhancing the expression of biosynthetic enzyme genes and decreasing the expression of degrading enzyme genes are used for each aliphatic dicarboxylic acid.

  Specifically, for example, as a recombinant microorganism that converts a carbon source into succinic acid, a microorganism that has been genetically modified so that pyruvate carboxylase activity is enhanced compared to an unmodified form, and a lactodehydrogenase activity that is unmodified form Examples include microorganisms that have been genetically modified so as to be reduced.

  Here, as a particularly preferable specific example of the parent strain of coryneform bacterium used for recombinant production, Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872, Corynebacterium glutamicum ATCC31831, Brevibacterium lactofermentum ATCC13869, etc. are mentioned.

  Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl, W., Ehrmann, M., Ludwig, W. and Schleiffer, K. H., International Journal of Systematic). Bacteriology, 1991, vol. 41, p255-260).

  Therefore, in the present invention, Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 strain are the same as Corynebacterium glutamicum MJ-233 strain and MJ-233 AB-41 strain, respectively. Stocks.

  Brevibacterium flavum MJ-233 was established on April 28, 1975, by the Ministry of International Trade and Industry, Institute of Industrial Science, Microbial Industrial Technology Research Institute (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (〒305-8565 Japan) Deposited as deposit number FERM P-3068 in Tsukuba City, Ibaraki Prefecture, 1-chome, 1-chome, 1st 6th), transferred to an international deposit under the Budapest Treaty on May 1, 1981, deposited under the deposit number of FERM BP-1497 Has been.

  Bacteria genetically modified so that pyruvate carboxylase (hereinafter also referred to as “PC” or “pc”) activity is enhanced as compared with the unmodified type are described in, for example, Japanese Patent Application Laid-Open No. 11-196888. Similarly to the method, it can be constructed by highly expressing the pc gene in a host bacterium with a plasmid in a microorganism such as a coryneform bacterium.

  Moreover, it may be integrated on the chromosome by homologous recombination, or the expression of the pc gene can be enhanced by promoter replacement. Transformation can be performed, for example, by an electric pulse method (Res. Microbiol., Vol. 144, p. 181-185, 1993).

  Enhancement of PC activity can be achieved by known methods such as those described in J. Org. Bacteriol. , 158, 55-62, (1984), and can be confirmed by measuring PC activity.

  In addition, a microorganism that has been genetically modified so that lactodehydrogenase (hereinafter sometimes referred to as “LDH”) activity is reduced as compared with the unmodified type is described in, for example, Japanese Patent Application Laid-Open No. 11-206385. It can be constructed by disrupting the LDH gene on the chromosome by the method using homologous recombination or the method using the sacB gene (Schaffer, A. et al. Gene 145 (1994) 69-73).

  LDH activity may be completely lost. The decrease or disappearance of the LDH activity can be confirmed by measuring the LDH activity by a known method (L. Kanarek, et al., J. Biol. Chem. 239, 4202 (1964), etc.).

  The microorganism used in the method for producing succinic acid is preferably a microorganism in which both of the enhancement of PC activity and the reduction of LDH activity are modified, as described in Examples below. Examples of such microorganisms include the Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain described in Japanese Patent Application Laid-Open No. 2008-259451.

  In addition, the microorganisms which convert a carbon source into aliphatic dicarboxylic acid are not limited to the said microorganisms, Any well-known microorganisms can be used.

  By reacting a microorganism that converts the carbon source to succinic acid with a carbon source in a reaction solution such as a medium containing the carbon source, succinic acid can be accumulated in the reaction solution.

  As the microorganism used for the reaction, a slant cultured in a solid medium such as an agar medium may be directly used for the reaction. However, it is preferable to use a microorganism that has been grown in advance (seed culture) in a liquid medium.

  As a medium used for seed culture, a normal medium used for culturing the above-mentioned microorganism can be used. For example, a general medium in which natural nutrient sources such as meat extract, yeast extract and peptone are added to a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate and magnesium sulfate can be used.

  In the case of a coryneform bacterium, seed culture is usually performed while aeration and agitation are performed and oxygen is supplied in the range of 25 ° C. to 35 ° C. which is the optimum temperature for growth. The culture time may be a time for obtaining a certain amount of cells, but is usually 6 to 96 hours.

(Aliphatic dicarboxylic acid)
Examples of the aliphatic dicarboxylic acid include lactic acid, succinic acid, malic acid, fumaric acid, oxaloacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid, cis-aconitic acid, pyruvic acid, and acetic acid. Among these, dicarboxylic acids such as succinic acid, malic acid and fumaric acid are preferable, and succinic acid is particularly preferable. A plurality of types of aliphatic dicarboxylic acids may be included.

(Reaction solution)
In a reaction solution containing a carbon source derived from the biological material, a reaction between the carbon source and a microorganism that converts the carbon source to an aliphatic dicarboxylic acid is carried out to react the aliphatic dicarboxylic acid in the reaction solution. Can be accumulated.

  As the carbon source derived from the above-mentioned biological materials, hexoses such as glucose, mannose, galactose, fructose, sorbose and tagatose, pentoses such as arabinose, xylose, ribose, xylulose and ribulose, maltose, sucrose, lactose, trehalose , Disaccharides or polysaccharides such as starch and cellulose, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, monoctinic acid, Fatty acids such as arachidic acid, eicosenoic acid, arachidonic acid, behenic acid, erucic acid, docosapentaenoic acid, docosahexaenoic acid, lignoceric acid and ceracolonic acid, and glycerin, mannitol, xylose It is preferable to use a fermentable saccharides polyalcohols such as tall and ribitol. Among these, glucose, maltose, fructose, sucrose, lactose, trehalose and cellulose are more preferable.

  When succinic acid is produced as the aliphatic dicarboxylic acid, the concentration of the carbon source used in the reaction solution is not particularly limited, but it is advantageous to make it as high as possible within the range not inhibiting the production of succinic acid, It is preferable to set it as 30% (W / V), and it is more preferable to set it as 10 to 20% (W / V).

  It does not specifically limit as a reaction liquid containing the said carbon source, For example, the culture medium for culture | cultivating microorganisms may be sufficient, and buffer solutions, such as a phosphate buffer solution, may be sufficient. The reaction solution is preferably an aqueous solution further containing a nitrogen source and an inorganic salt.

  Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by microorganisms to generate succinic acid. Specifically, for example, ammonium salt, nitrate, urea, soybean hydrolysate, casein decomposition Various organic or inorganic nitrogen compounds such as food, peptone, yeast extract, meat extract and corn steep liquor.

  As the inorganic salt, for example, metal salts such as various phosphates, sulfates, magnesium, potassium, manganese, iron and zinc are used. Moreover, it is preferable to add factors that promote growth such as vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids as necessary. In order to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the culture solution.

  The reaction solution preferably contains carbonate ion, bicarbonate ion, or carbon dioxide gas (carbon dioxide gas) in addition to the above-described carbon source, nitrogen source, inorganic salt, and the like.

  Carbonate ion or bicarbonate ion is supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate, which can also be used as a neutralizing agent. Or it can also supply from these salts or carbon dioxide gas.

  Specific examples of the carbonate or bicarbonate salt include magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate and potassium bicarbonate.

  The amount added to the carbonate ion or bicarbonate ion reaction solution is preferably 1 to 500 mM, more preferably 2 to 300 mM, and even more preferably 3 to 200 mM. When carbon dioxide gas is contained in the reaction solution, the content of carbon dioxide gas per liter of the solution is preferably 50 mg to 25 g, more preferably 100 mg to 15 g, and even more preferably 150 mg to 10 g.

  In microbial conversion to obtain aliphatic dicarboxylic acid by reacting biological materials and microorganisms, the metabolic activity of microorganisms decreases or the activity of microorganisms stops when pH is lowered, and the production yield deteriorates. Ordinarily, a neutralizing agent is used because microorganisms die.

  Usually, the pH in the reaction system is measured by a pH sensor, and the pH is adjusted by adding a neutralizing agent so as to be in a predetermined pH range. The pH value is adjusted to a range in which the activity is most effectively exhibited according to the type of microorganisms such as bacteria and mold used. There is no restriction | limiting in particular about the addition method of a neutralizing agent, Continuous addition or intermittent addition may be sufficient.

  Examples of the neutralizing agent include ammonia, ammonium carbonate, urea, alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, and alkaline earth metal carbonate. Among these, ammonia, ammonium carbonate, and urea are preferable.

Examples of the alkali (earth) metal hydroxide include NaOH, KOH, Ca (OH) ₂ and Mg (OH) ₂ , and mixtures thereof. As the alkali (earth) metal carbonates, _{e.g., Na 2 CO 3, K 2} CO 3, CaCO 3, etc. MgCO ₃ and NaKCO _3, etc. and mixtures thereof.

  When succinic acid is produced as an aliphatic dicarboxylic acid, the pH in the production reaction is usually preferably from 5 to 10, more preferably from 6 to 9.5. During the reaction, it is preferable to adjust the pH of the reaction solution within the above range with an alkaline substance, carbonate, urea, or the like, if necessary.

  When succinic acid is produced as an aliphatic dicarboxylic acid, the temperature of the production reaction is preferably 37 to 45 ° C, more preferably 39 ° C to 45 ° C, further preferably 39 ° C to 43 ° C, and particularly preferably 39 to 41 ° C. .

  When succinic acid is produced as the aliphatic dicarboxylic acid, the reaction time is preferably 1 hour to 168 hours, and more preferably 3 hours to 72 hours.

  When producing succinic acid as an aliphatic dicarboxylic acid, the amount of microorganisms used in the reaction is not particularly limited, but is preferably 1 to 700 g / L, more preferably 10 to 500 g / L, and even more preferably 20 to 400 g / L. .

  When succinic acid is produced as the aliphatic dicarboxylic acid, the production reaction may be carried out with aeration and stirring, but it may be carried out in an anaerobic atmosphere without aeration and without supplying oxygen. Under the anaerobic atmosphere here, for example, the container is sealed and reacted without aeration, supplied with an inert gas such as nitrogen gas and reacted, or the inert gas containing carbon dioxide gas is vented. Can be obtained.

  By the reaction as described above, the aliphatic dicarboxylic acid is generated and accumulated in the reaction solution, and a “liquid containing the aliphatic dicarboxylic acid and the microorganism” is obtained.

  As described above, the microorganism that converts the carbon source into succinic acid and the method for obtaining the liquid containing the succinic acid and the microorganism using the microorganism have been described as examples. However, the microorganism that converts the carbon source into an aliphatic dicarboxylic acid other than succinic acid. There are also many known methods for producing aliphatic dicarboxylic acids using them.

  Therefore, reaction conditions such as reaction temperature and pressure in producing aliphatic dicarboxylic acid by microorganisms depend on the activity of microorganisms such as selected cells and molds, but suitable conditions for obtaining aliphatic dicarboxylic acid May be selected according to each case.

  In the present invention, the fermentation broth after microbial conversion may be appropriately concentrated in consideration of operability and efficiency in the subsequent purification step. Although it does not specifically limit as a concentration method, For example, the method of distribute | circulating an inert gas, the method of distilling off water by heating, the method of distilling off water by pressure reduction, the method of combining these, etc. are mentioned. Further, the concentration operation may be performed by a batch operation or a continuous operation.

  By applying the following purification steps (2) to (4) to the “liquid containing aliphatic dicarboxylic acid and microorganism” obtained as described above, the aliphatic dicarboxylic acid can be produced.

I- (2) pH adjusting step for obtaining a liquid having a pH of 1.0 or more and 5.0 or less by mixing an acid with a liquid containing the aliphatic dicarboxylic acid obtained in the aliphatic dicarboxylic acid generating step and a microorganism. The pH of the fermentation suspension when separating cells from a liquid (fermentation suspension) containing an aliphatic dicarboxylic acid and a microorganism is usually preferably 1.0 or more and 5.0 or less, and preferably 1.5 or more and 4. 0 or less is more preferable, and 2.0 or more and 3.5 or less is more preferable.

  When adjustment of the pH of the fermentation suspension is necessary, it is not particularly limited as long as it is an acid, but the pH can be adjusted using an acid such as hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid.

  When the aliphatic dicarboxylic acid is recovered as a salt aqueous solution in the aqueous solution containing the aliphatic dicarboxylic acid when the aliphatic dicarboxylic acid is recovered in the solvent in the contact step described later, the aliphatic dicarboxylic acid and / or the aliphatic dicarboxylic acid In some cases, the amount of the salt of the aliphatic dicarboxylic acid extracted in the solvent that is phase-separated from the aqueous solution containing the aliphatic dicarboxylic acid is small. Therefore, it is preferable to protonate by adjusting the pH by adding an acid to the aqueous solution.

  For example, when an aliphatic dicarboxylic acid is obtained from a bio-derived raw material, if it is obtained using fermentation by microorganisms, the hydrogen ion concentration (pH) of the fermentation liquid may be adjusted in order to advance the fermentation efficiently. .

  When alkali neutralization is performed, the aliphatic dicarboxylic acid is present as an aqueous salt solution, and thus it is particularly preferable to perform protonation. For example, when ammonia is used as a neutralizing agent in the fermentation operation, the aliphatic dicarboxylic acid is present as an ammonium salt, so that protonation with an acid is preferably performed.

  The protonation may be performed at any stage as long as it is before the contact step described later.

  Since the acid used for protonation needs to be salt-exchanged with an aliphatic dicarboxylate, it is usually stronger than an aliphatic dicarboxylic acid, that is, an acid having an acid dissociation constant pKa smaller than that of an aliphatic dicarboxylic acid, usually a pKa of 4 It is preferable to use less acid.

  The acid used for protonation may be an organic acid, an inorganic acid, a monovalent acid or a polyvalent acid, but an inorganic acid is preferred. .

  When sulfuric acid is used for protonation, ammonium sulfate is generated as a by-product salt. In the production method of the present invention, when the inorganic salt is a by-product salt, it is preferable because an improvement in liquid-liquid separation property due to a salting-out effect by the by-product salt can be expected in the contact step.

  The amount of acid to be added depends on the strength of the acid to be used, but it is usually preferable to add the acid in an amount of 0.1 to 5 times the amount of the cation constituting the aliphatic dicarboxylate. Usually, acid addition is adjusted by pH.

  The pH of the acid used for protonation is preferably at least pKa or less, although it depends on the acid strength pKa of the aliphatic dicarboxylic acid. Further, the pH is preferably in the range of more than 1 and less than 4.

  On the other hand, even if an acid is added excessively, the way of lowering the pH gradually decreases, and the excess acid does not undergo salt exchange with the aliphatic dicarboxylate and exists in the system as an acid. The surplus acid is finally recovered as a residual extraction phase in the subsequent contact step, and the treatment again requires neutralization and the like, which is inefficient. Therefore, it is preferable to control the pH at 1 or more.

  In addition, you may use together a pH adjustment process and the protonation process mentioned later by one process. That is, the protonation step may be included in the pH adjustment step, or the protonation step may be performed in the pH adjustment step.

I- (3) Microbe separation step for separating microorganisms from the liquid obtained in the pH adjustment step The methods for separating and removing microorganisms from the fermentation suspension include sedimentation separation, centrifugal separation and filtration separation, and a combination thereof. Is used. Industrially, it is carried out by methods such as centrifugation and membrane filtration separation. In centrifugation, centrifugal sedimentation, centrifugal filtration, or the like can be used.

  The centrifugation method is not limited to the type of separator and the operating conditions as long as it can remove microorganisms of usually 70% or more, preferably 80% or more, more preferably 90% or more. It is preferable to separate with a centrifugal force of 100,000 G.

  The separation operation can be carried out either batchwise or continuously.

  The membrane used for membrane filtration is not particularly limited as long as it is a pore size membrane through which aliphatic dicarboxylic acid can pass and microorganisms and aggregates thereof cannot pass through, but microfiltration membranes and ultrafiltration membranes are preferred, and microfiltration membranes are preferred. More preferred.

  The material of the film is not particularly limited, and for example, an organic film such as polyolefin, polysulfine, polyacrylonitrile, and polyvinylidene fluoride, or a film made of an inorganic material such as ceramic can be used.

  As an operation method, any of a dead end type and a cross flow type can be used. In membrane filtration separation, since microorganisms often clog the membrane, a method of performing membrane filtration after roughly removing microorganisms by centrifugation or the like is also used.

I- (4) Contacting step in which an aqueous solution obtained by separating microorganisms in the microorganism separation step is brought into contact with a solvent. An aqueous solution containing an aliphatic dicarboxylic acid after separation of microorganisms in the microorganism separation step is brought into contact with the solvent. To extract the aliphatic dicarboxylic acid. You may make it contact with a solvent, after concentrating the said aqueous solution. By the contacting step, the aliphatic dicarboxylic acid can be selectively extracted into the solvent, and saccharides, amino acids and inorganic salts having high water solubility are mainly distributed in the aqueous phase.

  By-product salt generated by protonation of the aliphatic dicarboxylate is distributed to the aqueous solution phase and can be easily separated from the aliphatic dicarboxylic acid. For example, when the by-product salt is ammonium sulfate, almost all is recovered in the aqueous solution phase. At the same time, ammonium sulfate can be recovered as ammonium sulfate containing organic components of amino acid and saccharide together with the amino acid and saccharide recovered in the aqueous phase by a treatment such as concentration, crystallization, and drying. This ammonium sulfate is useful as a fertilizer because it contains an organic substance moderately.

(solvent)
The solvent used in the contacting step is not particularly limited as long as it is an organic solvent that is phase-separated from an aqueous solution containing an aliphatic dicarboxylic acid, but the ratio of inorganic value / organic value (hereinafter abbreviated as I / O value). Is preferably 0.2 or more and 2.3 or less, and more preferably 0.3 or more and 2.0 or less.

  By using the solvent, the aliphatic dicarboxylic acid can be selectively extracted and efficiently separated from impurities.

  In addition, the solvent used in the contacting step preferably has a normal pressure (1 atm) and a boiling point of 40 ° C. or higher, more preferably 60 ° C. or higher. The boiling point at normal pressure is preferably 120 ° C. or lower, more preferably 100 ° C. or lower, and preferably 90 ° C. or lower.

  By using the solvent, it is possible to avoid the risk that the solvent evaporates and ignites, the problem that the solvent evaporates and the extraction efficiency of the aliphatic dicarboxylic acid decreases, and the problem that the solvent is difficult to recycle. . In addition, there is an advantage that the amount of heat required when the used solvent is separated by a method such as distillation or purified and reused can be reduced.

  Inorganic values and organic values have been proposed by organic conceptual diagrams ("systematic organic qualitative analysis", Satoshi Fujita, Kazama Shobo (1974)) and have been set in advance for functional groups constituting organic compounds. The organic value and the inorganic value are calculated based on the numerical values, and the ratio is obtained.

  Examples of the organic solvent having an I / O value of 0.2 or more and 2.3 or less and a boiling point of 40 ° C. or more at normal pressure include ketone solvents such as methyl ethyl ketone, methyl isobutyl ketone and acetone, tetrahydrofuran, dioxane and the like. Examples include ether solvents, ester solvents such as ethyl acetate, nitrile solvents such as acetonitrile, and alcohols having 3 or more carbon chains such as propanol, butanol and octanol.

  Table 1 shows the I / O value and boiling point of each organic solvent.

  The solvent as described above is brought into contact with an aqueous solution obtained by separating microorganisms in the microorganism separation step to extract the aliphatic dicarboxylic acid. Here, the solvent is preferably added in a volume of 0.5 to 5 with respect to the volume 1 of the “aqueous solution obtained by separating microorganisms in the microorganism separation step”, more preferably in a volume of 1 to 3. preferable.

(Contact operation)
Although the temperature of a contact process will not be specifically limited if it is the temperature from which aliphatic dicarboxylic acid is extracted, 10-50 degreeC is preferable and 20-40 degreeC is more preferable.

  The aliphatic dicarboxylic acid is recovered in the solvent by the contacting step. When the aqueous solution contains an amino acid or a saccharide, both the amino acid and the saccharide are distributed in the aqueous solution phase, so that the aliphatic dicarboxylic acid can be separated from the saccharide and the amino acid.

  The operation of contacting the aqueous solution containing the aliphatic dicarboxylic acid and the aqueous solution with the solvent in the contacting step may be performed in one step or in multiple steps, but is preferably performed in multiple steps. Further, the solvent may be flowed in parallel or countercurrent with respect to the aqueous solution containing the aliphatic dicarboxylic acid.

  Further, the aliphatic dicarboxylic acid obtained in the solvent may be subjected to further purification operations such as column treatment and crystallization treatment. Further, when a plurality of aliphatic dicarboxylic acids are contained, an operation of separating only the target aliphatic dicarboxylic acid may be further performed.

  The time for contacting is not particularly limited as long as the aliphatic dicarboxylic acid is sufficiently extracted, and is usually 1 second to 5 hours although it depends on the contact device and the contact conditions.

  By setting the contact time to 1 second or longer, extraction of the aliphatic dicarboxylic acid into the solvent phase becomes sufficient. On the other hand, by making the contact time 5 hours or less, it is efficient to prevent the contact device from becoming unnecessarily large, while suppressing the denaturation of the proteins coexisting with the aliphatic dicarboxylic acid by the solvent, Can be reduced.

  The pressure at the time of contact is not particularly limited as long as the aliphatic dicarboxylic acid is sufficiently extracted, but when it is continuously carried out, it is usually operated at atmospheric pressure.

  When the aliphatic dicarboxylic acid is succinic acid, a succinic acid derivative can be produced using the obtained succinic acid after producing succinic acid by the method of the present invention.

  Here, as the succinic acid derivative, for example, succinic acid salt, succinic anhydride, maleic anhydride, succinic acid ester, succinimide, 1,4-diaminobutane, succinic acid nitrile, 2-pyrrolidone, N-methylpyrrolidone (NMP), N-ethylpyrrolidone (NEP), 1,4-butanediol (1,4-BG), tetrahydrofuran (THF), γ-butyrolactone (GBL) and polytetramethine ether glycol (PTMG). .

  Moreover, a succinic acid containing polymer can be manufactured by performing a polymerization reaction using the succinic acid manufactured by the manufacturing method of this invention as a raw material.

  In recent years, with the increase in the number of environmentally friendly industrial products, attention has been focused on polymers using plant-derived raw materials. In particular, succinic acid produced in the present invention is processed into polymers such as polyester and polyamide. Can be used.

  Specific examples of the succinic acid-containing polymer include a succinic acid polyester obtained by polymerizing a diol such as butanediol and ethylene glycol and succinic acid, and a diamine such as hexamethylenediamine and succinic acid. And succinic acid polyamide obtained.

II. 2nd invention 2nd invention is a method of manufacturing aliphatic dicarboxylic acid from the aqueous solution containing aliphatic dicarboxylic acid obtained from biological origin, Comprising: Fat containing the following processes (1)-(3) It is a manufacturing method of a group dicarboxylic acid.

(1) A contact step in which an aqueous solution containing an aliphatic dicarboxylic acid obtained from a biological raw material is brought into contact with a solvent that can be phase-separated with the aqueous solution. (2) Solid content generated by contact of the aqueous solution and the solvent in the contact step. (3) Phase separation step of separating the solvent by phase separation

  The steps other than the above (1) to (3) may include any conventionally known steps. Hereafter, it explains in detail according to each process.

II- (1) Contacting process in which an aqueous solution containing an aliphatic dicarboxylic acid obtained from a biological raw material is brought into contact with a solvent that can be phase-separated with the aqueous solution The aliphatic dicarboxylic acid is selectively extracted into the solvent by the contacting process Highly water-soluble saccharides, amino acids and inorganic salts are mainly distributed in the aqueous phase.

  The solvent is not particularly limited as long as it is phase-separated from an aqueous solution containing an aliphatic dicarboxylic acid, and the solvents described above in I- (4) can be used.

  The contact operation between the aqueous solution containing the aliphatic dicarboxylic acid and the solvent is performed as described above in I- (4). In addition, the temperature at the time of contact will not be specifically limited if it is the temperature from which aliphatic dicarboxylic acid is extracted, but 30-60 degreeC is preferable.

  By setting the contact temperature to 30 ° C. or higher, it is possible to suppress the increase in the viscosity of the solvent, to shorten the time required for sedimentation of solids described later, and to prevent the solids from floating in the solvent phase. It is possible to prevent solid content from being mixed into the phase. Moreover, by making a contact temperature 60 degrees C or less, the extraction rate of aliphatic dicarboxylic acid can be improved and efficiency is good.

  In the phase separation step described later, the aqueous solution and the solvent are phase-separated, but in some cases, a phase containing a solid content at the phase interface (hereinafter sometimes referred to as an intermediate phase) may be formed. The intermediate phase makes it difficult to separate the solvent phase (hereinafter sometimes referred to as the extraction phase) and the aqueous phase (hereinafter sometimes referred to as the extraction residual phase), or impurities to the extraction phase There is a risk of increasing the amount of contamination. Therefore, it is preferable to remove the intermediate phase.

  As shown in FIG. 1, a particularly preferable form of the contacting step is to separate an extraction phase liquid, an intermediate phase, and an extracted residual phase by mixing an aqueous solution containing an aliphatic dicarboxylic acid and a solvent after mixing with a mixer settler. The intermediate phase is recovered and separated into solid and liquid, and the separated and recovered liquid is phase-separated as necessary, and then returned to at least one of the step before the contact step and the step after the phase separation step.

(Contact device)
The contact device may be any device as long as it can contact the aqueous solution containing the aliphatic dicarboxylic acid and the solvent and the solvent phase, recover the aqueous solution phase, and remove the solid content in the solid content removal step described later. The above-mentioned mixer-settler type extraction device is preferable because the device is simple and easy to operate.

  The mixer may be of any type as long as an aqueous solution containing an aliphatic dicarboxylic acid and a solvent that can be phase-separated with the aqueous solution are sufficiently mixed. Examples thereof include a container having a stirring device and a static mixer.

  However, in the case of using a container having a stirring device, it adheres to the solid content in which bubbles such as air entrained in the container are generated by stirring, and remarkably inhibits the phase separation of the solid content in the subsequent settler part. It is preferable to stir under conditions that do not involve air or the like. The mixer is preferably a static mixer from the viewpoint of wide operating tolerance and equipment cost.

(Contact process and phase separation process)
The method for producing an aliphatic dicarboxylic acid according to the second aspect of the present invention includes a contact step in which an aqueous solution containing an aliphatic dicarboxylic acid is contacted with a solvent capable of phase separation with the aqueous solution, and the liquid is phase-separated after the contact step. A phase separation step described later is included.

  When the contacting step is carried out by batch operation, a solvent capable of phase separation from the aqueous solution is added to an aqueous solution containing an aliphatic dicarboxylic acid, and after sufficiently mixing, in the phase separation step, an extraction phase, an intermediate phase, and a residual extraction phase are added. These can be separated and recovered by a method of taking out from the vicinity of each phase through a discharge port, a method of taking out sequentially from the bottom of the contacted container, and the like. The intermediate phase containing a large amount of solids can be taken out together with the extraction phase, or can be taken out together with the extracted residual phase.

  Further, for example, when the contact step is carried out by continuous operation, it is obtained by contacting and mixing with an aqueous solution containing an aliphatic dicarboxylic acid and a mixer part having a mixer for contacting and mixing the aqueous solution and a solvent capable of phase separation. A contact device (hereinafter referred to as a mixer-settler type extractor) comprising a settler part having a settler applied to a step (hereinafter sometimes referred to as a phase separation step) of phase separation by allowing the mixed liquid to stand. In the phase separation step using (which may be described), the extractor phase, the intermediate phase and the extracted residual phase can also be recovered in the settler part.

II- (2) Solid content removal process for removing solid content generated by contact of the aqueous solution and the solvent in the contact process In the solid content removal process, an aqueous solution containing an aliphatic dicarboxylic acid obtained from a biological material, The solid content generated by contact with the aqueous solution and the phase-separable solvent is removed.

  Usually, when a fermentation method using microorganisms is used when obtaining from a biological material, an aqueous solution containing an aliphatic dicarboxylic acid often contains a polymer having a higher order structure such as a biological protein. Proteins and the like are usually highly water-soluble, and most of them are distributed to the aqueous solution phase in the extraction operation. However, when they come into contact with the solvent in the contact process, the higher-order structure is destroyed and denatured, so that both water and solvent Some of them do not dissolve and become solids.

  Therefore, in the solid content removal process according to the present invention, the solid content that is not dissolved in the water or the solvent generated in the contact process is removed.

  The solid content generated in the contact process tends to gather mainly in the vicinity of the liquid-liquid interface, and there is a problem that makes phase separation difficult. Even if solid content is generated near the liquid-liquid interface, if it is contacted by a batch method, it will not cause a major problem in operation if the generated solid content is removed each time and the extracted phase and the extracted residual phase are recovered. There is a case.

  On the other hand, in continuous extraction, particularly in a countercurrent multistage extraction tower, solids are continuously generated, so liquid dispersion and liquid separation are hindered, which not only prevents stable operation but also makes extraction impossible.

  Moreover, when the liquid containing solid content moves to the subsequent process, it may adversely affect the subsequent process. For example, the extract containing aliphatic dicarboxylic acid recovered in the contact process may be concentrated due to the low concentration of aliphatic dicarboxylic acid, but if there is a solid content, the solid content adheres to the heating surface such as a reboiler and is burnt. , Deteriorate the heat transfer efficiency.

  Furthermore, depending on the subsequent process, there may be a problem in quality. For example, in the case of using aliphatic dicarboxylic acid as a polyester raw material, it has been found that nitrogen content is greatly involved in the color tone of the polymer. Since the solid content contains a large amount of protein-denatured products and a high content of nitrogen, if the solid content is mixed in the final product, the color tone of the polyester may be affected.

  For the reasons described above, these problems are solved by removing the solid content generated in the contact process in the solid content removal process.

  The solid content removal method is not particularly limited, and for example, it may be performed by a method of filtering the solid content or a method of separating with a device having a function of phase-separating each phase after bringing incompatible liquids into contact with each other. it can.

  For example, in batch extraction, a solvent is added to a fermentation broth containing an aliphatic dicarboxylic acid, and after sufficient mixing, an extraction phase, an intermediate phase rich in solids, and a residual extraction phase can be separated and recovered.

  Further, in continuous extraction, in a mixer-settler type extractor comprising a mixer part for mixing the fermentation liquor and the solvent and a settler part for separating the liquid mixture into liquids, the extractor, the intermediate phase containing a large amount of solids, and the residue Each phase can be recovered.

  The mixer may be of any type as long as the fermented liquid and the solvent are sufficiently mixed, and examples thereof include a stirring tank and a static mixer. However, in the case of using a stirrer, the bubbles that have been entrained in the stirrer by stirring to the generated solid matter adhere to the solid matter, and the settling of the solid matter in the subsequent settler is significantly inhibited. Be careful.

  The mixer is preferably a static mixer from the viewpoint of the wide allowable operating range and the equipment cost.

  On the other hand, the type of settler is not particularly limited. For example, a type in which the extraction phase, the intermediate phase, and the extracted residual phase are respectively collected in a single tank type, and a type in which the extraction phase, the intermediate phase, and the extracted residual phase are respectively recovered in a multi-tank type, and the like.

  Since the intermediate phase rich in solids also includes an extraction phase and a residual phase, solid-liquid separation is performed to separate the solid and the extracted phase and / or the residual phase, and the extracted phase and / or the residual phase is recovered. be able to.

  The solid-liquid separation method is not particularly limited, and any method of sedimentation separation and filtration separation can be used. In sedimentation separation, the solid content may be separated by sedimentation in a gravitational field, or the solid content may be separated by sedimentation in a centrifugal field.

  Among these, centrifugal sedimentation is preferable because of the sedimentation speed. The method may be batch operation or continuous operation. Examples of the continuous centrifugal sedimentator include a screw decanter and a separation plate centrifugal sedimentator.

  In filtration separation, the method is classified according to the filter medium, filtration pressure, continuous operation / batch operation, etc., and any method is not particularly limited as long as the solid content can be separated from the extraction phase and / or the extraction residual phase.

  However, the aperture of the filter medium is preferably 0.1 μm or more and 10 μm or less. By setting the thickness to 0.1 μm or more, the permeation flux becomes sufficient, and the filtration time can be shortened. Further, when the thickness is 10 μm or less, the solid content is sufficiently separated.

  Moreover, since the material of the filter medium needs to be insoluble in a solvent, it is preferable to use Teflon (registered trademark) or the like. For the filtration, any of a vacuum type, a pressure type, and a centrifugal type can be used. Furthermore, the system may be a continuous system or a batch system.

  The solid content may be removed by a method of selectively separating the solid content, or may be removed together with the liquid used in the contact step. However, in order to increase the recovery efficiency of the aliphatic dicarboxylic acid, only the solid content is removed. It is preferable to remove it selectively. When it is removed together with the liquid used in the contacting step, it is preferable to selectively remove only the solid content by solid-liquid separation in the subsequent step.

  More specifically, the solid content is taken out and removed together with the liquid used in the contact step, and the solid content is further separated from the mixture of the taken out liquid and the solid matter, and then only the liquid is again the step before the contact step and the phase separation. It is preferable to return to at least one of the steps after the step.

II- (3) Phase separation step for separating the solvent by phase separation (phase separation operation)
The phase separation operation in the phase separation step can be performed by standing in a tank for a certain period of time or can be performed by a centrifugal separator. The mixer-settler type extractor as described above has a settler part that causes phase separation by allowing the mixed liquid obtained by contact mixing to stand, and by allowing the liquid to stand for a certain time in the settler part. Can be phase separated.

  The phase separation step may be performed continuously or batchwise.

  Although the temperature at the time of phase separation will not be specifically limited if each phase is a temperature which can be isolate | separated, it is preferable to process at 30-60 degreeC and the temperature comparable as contact operation.

  By setting the phase separation temperature to 30 ° C. or higher, the liquid viscosity is prevented from becoming high, the solid content is easily separated, and the solid content in the solvent phase and the increase in the amount of the solvent mixed in the solid content are suppressed. Can do. In addition, by setting the phase separation temperature to 60 ° C. or lower, it is possible to prevent succinic acid from being back-extracted into the aqueous solution during the phase separation process.

  The time for phase separation is not particularly limited as long as each phase is phase-separated, and it is usually 1 minute to 5 hours, although it depends on the contact device, contact conditions, and phase separation method.

  By setting the phase separation time to 1 minute or longer, phase separation becomes sufficient, and mixing of the aqueous solution and solid content into the solvent phase and the mixing of the solvent and solid content into the aqueous solution phase can be prevented. Further, by setting the phase separation time to 5 hours or less, it is possible to prevent the phase separation apparatus from becoming unnecessarily large and efficient.

  Further, the pressure at the time of phase separation is not particularly limited as long as the aliphatic dicarboxylic acid is sufficiently extracted, but in the case of continuous operation, it is usually preferable to operate at atmospheric pressure.

(Phase separator)
The settler used in the phase separation step may be any system as long as it can phase-separate the liquid after bringing the aqueous solution containing the aliphatic dicarboxylic acid into contact with the solvent capable of phase separation. May be.

  Specifically, for example, one that collects the extraction phase, the intermediate phase, and the residual phase in a single tank, one that collects the extraction phase, the intermediate phase, and the residual phase in a multi-tank system, and centrifugal separation using a rotating device And the like for recovering each phase.

  Since the intermediate phase containing a large amount of the solid usually contains at least one liquid selected from the liquid of the extraction phase and the liquid of the extraction residual phase, the intermediate phase is obtained by the same method as described above in II- (2). Can be separated into solid and liquid, and at least one liquid selected from the liquid of the extraction phase and the liquid of the extraction residual phase can be separated and recovered.

<Aliphatic dicarboxylic acid>
The aliphatic dicarboxylic acid of the second aspect of the present invention is not particularly limited as long as it is produced from an aqueous solution containing an aliphatic dicarboxylic acid obtained from a biological raw material, and is an aliphatic hydrocarbon having two carboxy groups. Any one can be adopted.

  The number of carbon atoms of the aliphatic dicarboxylic acid is preferably 2 or more and 40 or less, more preferably 3 or more and 20 or less, and particularly preferably 3 or more and 10 or less. By making the carbon number of the hydrocarbon group 40 or less, it becomes easy to exist as a stable aqueous solution. Moreover, it becomes easy to exist stably in the solvent which can be phase-separated with aqueous solution by making C2 or more into carbon number. The aliphatic hydrocarbon group portion of the aliphatic dicarboxylic acid may be linear or cyclic.

  More specifically, for example, oxalic acid, succinic acid, glutaric acid, adipic acid, sebacic acid, malic acid, fumaric acid, oxaloacetic acid, 2-oxoglutaric acid, cis-aconitic acid, dodecanedioic acid, dimer acid, etc. Can be mentioned. Among these, adipic acid, succinic acid and dimer acid are preferable, and succinic acid and adipic acid are particularly preferable.

  In the second aspect of the present invention, an aliphatic dicarboxylic acid is derived from a carbon source derived from a biological material. Specifically, for example, using these carbon sources, fermentation methods by microbial conversion, chemical conversion methods including reaction steps such as hydrolysis, dehydration reaction, hydration reaction, oxidation reaction, reduction reaction, and combinations thereof The dicarboxylic acid is preferably synthesized by the above.

  Among these, a fermentation method by microbial conversion using a microorganism having an ability to produce an aliphatic dicarboxylic acid is preferable. Fermentation by microbial conversion using microorganisms capable of producing aliphatic dicarboxylic acids is performed as described above in I- (1).

  In addition, when using a fermented liquid in the method of this invention, it is preferable to use the fermented liquid after removing microorganisms. The microorganism removal method is performed as described above in I- (3).

  As described above, the aliphatic dicarboxylic acid obtained by microbial conversion from the carbon source derived from the biological material is usually dissolved in the aqueous solution, but before the subsequent contact step, the dicarboxylic acid is present. A part of may be present as a solid, for example, by precipitation.

  In addition, the aqueous solution containing an aliphatic dicarboxylic acid obtained by microbial conversion usually contains saccharides, amino acids, etc. used in the microbial conversion, and this aqueous solution and solvent are used in a subsequent contact step. In particular, when it is brought into contact, solids are easily generated, the problems of the present invention become remarkable, and the effect of the solution according to the present invention is also increased.

<Subsequent process>
In order to finally collect the aliphatic dicarboxylic acid selectively recovered in the extraction phase as an aliphatic dicarboxylic acid crystal, a concentration step and a crystallization step are usually required. Here, a general method for recovering the aliphatic dicarboxylic acid crystals from the extraction phase will be described, but the subsequent steps that can be employed in the present invention are not limited thereto.

(Extraction phase concentration process)
The extraction phase concentration step is performed as described later in III- (2).

(Crystallization process)
The crystallization step is performed as described later in III- (3).

(Solid-liquid separation process)
As described later in III- (4), the aliphatic dicarboxylic acid slurry obtained in the crystallization step separates the aliphatic dicarboxylic acid crystal and the mother liquor by solid-liquid separation operation.

III. 3rd this invention 3rd this invention is a method of manufacturing aliphatic dicarboxylic acid from the aqueous solution containing the aliphatic dicarboxylic acid obtained from the biological origin raw material, Comprising: The following processes (1)-(5) It is a manufacturing method of the aliphatic dicarboxylic acid containing this.
(1) A contact step in which an aqueous solution containing an aliphatic dicarboxylic acid is brought into contact with a solvent that is phase-separated from the aqueous solution. (2) A step of concentrating the aliphatic dicarboxylic acid recovered in the contact step, and extracting by the concentration. Extraction phase concentration step in which water concentration in phase increases (3) Crystallization step for depositing aliphatic dicarboxylic acid from liquid after extraction phase concentration step (4) Solid recovery for recovering aliphatic dicarboxylic acid precipitated in crystallization step Liquid separation step (5) Crystallization mother liquor recycling step for returning at least part of the crystallization mother liquor after recovering the aliphatic dicarboxylic acid obtained in the solid-liquid separation step to any step prior to the crystallization step

  The steps other than the above (1) to (5) may include any conventionally known steps. Hereafter, it explains in detail according to each process.

III- (1) Contacting step of contacting an aqueous solution containing an aliphatic dicarboxylic acid with a solvent that is phase-separated from the aqueous solution The contacting step is performed as described above in II- (1). Moreover, it is preferable to include the process of removing microorganisms or protein by an above-described method before a contact process.

  In the contacting step, as described above, it is preferable to recover the phase including the extraction phase, the extracted residual phase, and the solid content from the settler portion of the mixer settler using the mixer settler, and the solid content recovered from the settler portion. More preferably, the phase containing is separated into solid and liquid and the liquid phase is recovered.

  Further, it is more preferable to recover the aliphatic dicarboxylic acid from the extracted residual phase obtained in the settler part using a countercurrent multistage extraction tower.

III- (2) A step of concentrating the aliphatic dicarboxylic acid recovered in the contacting step, wherein the concentration of water in the extraction phase increases due to the concentration. Generally, the concentration of the aliphatic dicarboxylic acid in the extraction phase is Since it is dilute, a concentration operation is required. The concentration is not particularly limited, but it is preferable that the solubility of the aliphatic dicarboxylic acid in the final concentrated solution is not more than the saturation solubility and is as close to the saturation solubility as possible.

  The solvent used for extraction of the aliphatic dicarboxylic acid often forms the lowest azeotropic composition with water, and the azeotropic composition is often a solvent-rich composition. Therefore, with the concentration operation, a large amount of the solvent is distilled off, and the solvent concentration in the concentrated solution is lower than that before the concentration.

  Aliphatic dicarboxylic acids obtained from biological raw materials generally contain many water-soluble impurities, and therefore, in the subsequent crystallization process, a system in which water is present has a higher purification effect than a system in which a solvent coexists. I can expect.

  In addition, since the recovery of the solvent until the subsequent step becomes more difficult, the concentration of the solvent contained in the solution containing the aliphatic dicarboxylic acid after the extraction phase concentration step is preferably 10% by weight or less. It is more preferable to set the weight% or less.

  In order to set the solvent concentration of the final concentrated solution to 10% by mass or less and to make the aliphatic dicarboxylic acid concentration close to the saturation solubility, water is added in at least one of the steps before concentration of the extraction phase and the concentration operation. It is preferable.

III- (3) Crystallization step for precipitating aliphatic dicarboxylic acid from the liquid after the extraction phase concentration step The crystallization step is usually performed by converting the aliphatic dicarboxylic acid in the solution containing the extraction phase to the solubility difference of the dicarboxylic acid, etc. Is a step of precipitating solid aliphatic dicarboxylic acid using

  The crystallization method may be any method as long as the aliphatic dicarboxylic acid is precipitated as a solid from the solution containing the extraction phase.

  More specifically, for example, a cooling crystallization method in which crystallization is performed by utilizing the temperature dependence of solubility by changing the solution temperature, and a solvent in the solution is volatilized by evaporating the solvent from the solution by operations such as heating and decompression. A method of crystallization by increasing the group dicarboxylic acid concentration, a method of a combination thereof, and the like.

  In cooling crystallization, cooling methods include, for example, a method in which a solution containing an extraction phase is circulated through an external heat exchanger and the like, and a pipe through which a refrigerant flows is placed in the solution containing the extraction phase. There are ways to do it.

  Above all, in the method of volatilizing the solvent in the solution by reducing the pressure inside the apparatus and cooling with the heat of vaporization of the solvent, it is possible to prevent inhibition of heat transfer due to precipitation of aliphatic dicarboxylic acid at the heat exchange interface. In addition to the concentration of the aliphatic dicarboxylic acid in the solution, it is preferable from the viewpoint of the crystallization yield.

  The crystallization operation may be a batch operation or a continuous operation. The continuous operation is preferable because the variation in the particle size of the solid aliphatic dicarboxylic acid obtained can be reduced, and the energy required for crystallization can be reduced efficiently during mass production.

  The crystallization apparatus does not need to be a special crystallization tank, and a known stirring tank can be used.

III- (4) Solid-liquid separation step for recovering aliphatic dicarboxylic acid precipitated in the crystallization step The aliphatic dicarboxylic acid slurry obtained by crystallization separates the aliphatic dicarboxylic acid crystal and the mother liquor by solid-liquid separation operation. The separation method is not particularly limited, and examples thereof include filtration separation and sedimentation separation.

  The solid-liquid separation operation may be batch or continuous. Examples of the efficient solid-liquid separator include a continuous centrifugal filter and a centrifugal sedimentator such as a decanter.

  Depending on the desired purity of the aliphatic dicarboxylic acid, the wet cake recovered by the solid-liquid separation operation can be rinsed with cold water or the like.

III- (5) Crystallization mother liquor recycling step in which at least a part of the crystallization mother liquor after recovering the aliphatic dicarboxylic acid obtained in the solid-liquid separation step is returned to any step prior to the crystallization step. Solid-liquid separation At least a part of the mother liquor and / or cleaning liquid obtained in the process can be recycled to a process prior to the crystallization process.

  The process for recycling the crystallization mother liquor is not particularly limited, but can be recycled to the contact process and the concentration process. When recycled to the contact process, the extraction tower becomes larger, but impurities that are easily distributed to the aqueous phase (having a small distribution coefficient) can be selectively removed from within the recycling system.

  On the other hand, when it is recycled to the concentration process, the extraction tower can be made small, but all the nonvolatile impurities are accumulated in the recycling system.

  Although it is possible to recycle all of the mother liquor and the rinsing liquid, it is preferable that at least a part of the mother liquor and the rinsing liquid be purged outside the system because impurities accumulate in the recycle system by continuing operation for a long period of time.

  Purge water is usually discharged after treating organic substances such as activated sludge, but purge water contains aliphatic dicarboxylic acid and has a low pH. It is effective as

  In the crystallization mother liquor recycling, the amount of recycling and the recycling place can be determined according to the desired purity, yield and yield of the aliphatic dicarboxylic acid.

  The aliphatic dicarboxylic acid produced by the production method of the third invention is the same as the aliphatic dicarboxylic acid of the second invention. In the third aspect of the present invention, as described above in the second aspect of the present invention, an aliphatic dicarboxylic acid is derived from a carbon source derived from a biological material.

<Protonation process>
Further, when the aliphatic dicarboxylic acid is recovered in the solvent in the contact step, and the aliphatic dicarboxylic acid is present as an aqueous salt solution in the aqueous solution containing the aliphatic dicarboxylic acid, it is described above in I- (2). It is preferable to protonate the salt by a method similar to protonation.

  The protonation step of adding an acid to an aqueous solution containing an aliphatic dicarboxylic acid may be a step performed at any stage as long as it is a step prior to the contact step.

<Other processes>
Furthermore, other steps, such as drying and purification, can be applied to the aliphatic dicarboxylic acid obtained by the method of the present invention, depending on its use. For example, a decolorization process using an adsorbent such as activated carbon, an ion exchange process for removing coexisting ions with an ion exchange resin, a process for hydrogenating coexisting unsaturated dicarboxylic acid, and a crystallization process for further purification. Can be considered.

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

Reference Example 1: Preparation of a succinic acid fermenting strain (A) Extraction of genomic DNA of Brevibacterium flavum MJ233 Brevibacterium flavum MJ233 was established on April 28, 1975 at the Institute of Microbial Industrial Technology, Ministry of International Trade and Industry. (Currently, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (deposited as FERM P-3068, 1-6 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 305-8565, Japan), May 1981 It was transferred to an international deposit based on the Budapest Treaty on the 1st, and deposited with a deposit number of FERM BP-1497.

A medium [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.5 g, FeSO ₄ · 7H ₂ O 6 mg, MnSO _4. 4-5H ₂ O 6 mg, biotin 200 μg, thiamine 200 μg, yeast extract 1 g, casamino acid 1 g, glucose 20 g, dissolved in 1 L distilled water] In 10 mL, the Brevibacterium flavum MJ233 strain is cultured until the late logarithmic growth phase The cells were collected by centrifugation (10000 g, 5 minutes).

  The obtained cells were suspended in 0.15 mL of a 10 mM NaCl / 20 mM Tris buffer solution (pH 8.0) / 1 mM EDTA · 2Na solution containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis.

  After adding an equal amount of phenol / chloroform solution to the lysate and shaking gently at room temperature for 10 minutes, the whole amount is centrifuged (5,000 × g, 20 minutes, 10-12 ° C.) The fraction was collected and sodium acetate was added to a concentration of 0.3 M, and then twice as much ethanol was added and mixed. The precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol and then air-dried. To the obtained DNA, 5 mL of a 10 mM Tris buffer (pH 7.5) -1 mM EDTA · 2Na solution was added and allowed to stand at 4 ° C. overnight, and used as template DNA for subsequent PCR.

(B) Construction of a plasmid for PC promoter substitution The DNA fragment of the N-terminal region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) above as a template and the entire genome sequence was reported. This was performed by PCR using synthetic DNA (SEQ ID NO: 1 and SEQ ID NO: 2) designed based on the sequence of the gene of Corynebacterium glutamicum ATCC13032 strain (Cgl0689 of GenBank Database Accession No. BA00000036). The DNA of SEQ ID NO: 1 used was phosphorylated at the 5 ′ end.

Composition of reaction solution: Template DNA 1 μL, Pfx DNA polymerase (manufactured by Invitrogen) 0.2 μL, 1 × concentration attached buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 minutes.

  Confirmation of the amplified product was carried out by separating by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and then visualized by ethidium bromide staining to detect a fragment of about 0.9 kb.

  Recovery of the target DNA fragment from the gel was carried out using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and this was used as the PC gene N-terminal fragment.

  On the other hand, the TZ4 promoter fragment derived from Brevibacterium flavum MJ233 strain is constitutively highly expressed using plasmid pMJPC1 (Japanese Patent Application Laid-Open No. 2005-95169) as a template, and the synthetic DNAs described in SEQ ID NO: 3 and SEQ ID NO: 4 It was prepared by PCR using The DNA of SEQ ID NO: 4 used was phosphorylated at the 5 'end.

Reaction solution composition: Template DNA 1 μL, PfxDNA polymerase (manufactured by Invitrogen) 0.2 μL, 1 × concentration attached buffer, 0.3 μM primer, 1 mM MgSO ₄ , 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

Reaction temperature condition: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 30 seconds was repeated 25 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 3 minutes.

  The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected.

  Recovery of the target DNA fragment from the gel was performed using QIAQuick Gel Extraction Kit (manufactured by QIAGEN), and this was used as the TZ4 promoter fragment.

  The PC gene N-terminal fragment prepared above and the TZ4 promoter fragment were mixed, and ligation kit ver. 2 (manufactured by Takara Shuzo Co., Ltd.), cleaved with restriction enzyme PstI, separated by 1.0% agarose (SeaKem GTG agarose: FMC BioProducts) gel electrophoresis, and a DNA fragment of about 1.0 kb was isolated from QIAQuick Gel Extraction Kit (Manufactured by QIAGEN) was used as a TZ4 promoter: PC gene N-terminal fragment.

  Further, the DNA fragment was mixed with DNA prepared by cutting E. coli plasmid pHSG299 (Takara Shuzo) with PstI, and ligation kit ver. 2 (Takara Shuzo) were used for connection.

  Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal.

  Clones that formed white colonies on the medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. The obtained plasmid DNA was cleaved with the restriction enzyme PstI, whereby an inserted fragment of about 1.0 kb was recognized, and this was named pMJPC17.1.

  The DNA fragment of the 5 ′ upstream region of the pyruvate carboxylase gene derived from Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in Reference Example 1 (A) as a template, and Corynebacterium It was carried out by PCR using synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) designed based on the sequence of the gene of Glutamicum ATCC13032 strain (GenBank Database Accession No. BA00000036).

Composition of reaction solution: 1 μL of template DNA, 0.2 μL of Pfx DNA polymerase (manufactured by Invitrogen), 1 × concentrated buffer, 0.3 μM each primer, 1 mM MgSO ₄ and 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  The amplification product was confirmed by separation by 1.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.7 kb was detected.

  The DNA fragment of interest was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN). The recovered DNA fragment was phosphorylated at the 5 ′ end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then ligated kit ver. 2 (Takara Shuzo) was used to bind to the SmaI site of E. coli vector pUC119 (Takara Shuzo), and Escherichia coli (DH5α strain) was transformed with the resulting plasmid DNA.

  The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. The obtained plasmid DNA was subjected to a PCR reaction using the synthetic DNAs shown in SEQ ID NO: 7 and SEQ ID NO: 6 as primers.

Composition of reaction solution: 1 ng of the above plasmid, Ex-Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) 0.2 μL, 1 × concentration attached buffer, 0.2 μM each primer, 0.25 μM dNTPs were mixed to make a total volume of 20 μL.

Reaction temperature conditions: DNA thermal cycler PTC-200 (manufactured by MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds and 72 ° C. for 50 seconds was repeated 20 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  As a result of confirming the presence or absence of the inserted DNA fragment in this way, a plasmid that recognized an amplification product of about 0.7 kb was selected and named pMJPC5.1.

  Next, the above-mentioned pMJPC17.1 and pMJPC5.1 were cleaved with restriction enzyme XbaI and mixed, and ligation kit ver. 2 (Takara Shuzo) were used for connection. The DNA fragment cleaved with restriction enzyme SacI and restriction enzyme SphI was separated by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis, and a DNA fragment of about 1.75 kb was prepared by QIAQuick Gel Extraction Kit (manufactured by QIAGEN Gel Extraction Kit). ).

  A DNA fragment in which the TZ4 promoter is inserted between the 5 ′ upstream region and the N-terminal region of the PC gene, and a DNA prepared by cleaving the plasmid pKMB1 (JP-A-2005-95169) containing the sacB gene with SacI and SphI Mixed and ligation kit ver. 2 (Takara Shuzo) were used for connection.

  Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kana machine and 50 μg / mL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified.

  By cutting the obtained plasmid DNA with restriction enzymes SacI and SphI, an insert fragment of about 1.75 kb was observed, which was named pMJPC17.2 (FIG. 2).

(C) Production of PC enhanced strain
Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / ΔLDH (strain with reduced LDH activity: Japanese Patent Application Laid-Open No. 2005-95169) was obtained using the plasmid DNA of pMJPC17.2 using the calcium chloride method (Journal of Molecular Biology, 53, 159, 1970) and re-prepared from E. coli strain JM110.

  The Brevibacterium flavum MJ233 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., Vol. 144, p. 181-185, 1993), and the obtained transformant contained 25 μg / mL of kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water] was smeared.

  Since the strain grown on the medium is a plasmid in which pMJPC17.2 cannot replicate in the Brevibacterium flavum MJ233 strain, the PC gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination with the kanamycin, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was eliminated by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

  Among the strains thus obtained, those in which the TZ4 promoter derived from pMJPC17.2 is inserted upstream of the PC gene and those that have returned to the wild type are included. Confirmation of whether the PC gene is promoter-substituted type or wild type can be easily confirmed by subjecting the cells obtained by liquid culture in LBG medium to direct PCR reaction and detecting the PC gene. .

  When analyzed using primers (SEQ ID NO: 8 and SEQ ID NO: 9) for PCR amplification of the TZ4 promoter and PC gene, a 678 bp DNA fragment should be observed in the promoter-substituted form. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / PC-5 / ΔLDH.

Reference Example 2: Preparation of succinic acid fermentation broth by jar fermenter (A) Seed culture Urea: 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g, 2 potassium phosphate 0.5 g, magnesium sulfate 7 hydrate Product: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g Into a 500 mL Erlenmeyer flask was added 100 mL of the medium dissolved in 1000 mL and sterilized by heating at 121 ° C for 20 minutes. This was cooled to room temperature, 4 mL of a 50% glucose aqueous solution sterilized in advance was added, the Brevibacterium flavum MJ233 / PC-5 / ΔLDH strain constructed above was inoculated, and shaken at 30 ° C. for 16 hours (160 rpm) Cultured.

(B) Main culture Ammonium sulfate: 3.0 g, 85% phosphoric acid: 6.7 g, potassium chloride: 4.9 g, magnesium sulfate heptahydrate: 1.5 g, ferrous sulfate heptahydrate: 120 mg , Manganese sulfate hydrate: 120 mg, corn steep liquor (manufactured by Oji Cornstarch) 30.0 g, 10N aqueous potassium hydroxide solution: 11.0 g, antifoaming agent (CE457: manufactured by NOF Corporation): 2.5 g of distilled water 2.0 L of the medium prepared by dissolving in the solution was placed in a 5 L fermenter and sterilized by heating at 121 ° C. for 20 minutes. After cooling this to room temperature and adjusting the pH to 7.0 by adding 28% aqueous ammonia, D-biotin sterilized in advance, 0.2 g / L aqueous solution of thiamine hydrochloride: 15 mL, and 720 g / sterilized in advance. L Sucrose aqueous solution: 110 mL was added, and 100 mL of the above seed culture solution was added thereto, and the mixture was kept at 30 ° C.

  The main culture was started using a 28% aqueous ammonia so that the pH was kept at 7.2 or less, aeration was 3.0 L / min, back pressure was 0.05 MPa, and stirring was 750 rpm. After the dissolved oxygen concentration dropped to almost 0, when it started to rise again and reached 1 ppm, about 5.3 g of a 72% sucrose aqueous solution sterilized in advance was added. Each time the dissolved oxygen concentration increased again, the addition of the aqueous sucrose solution was repeated by the above method, and continued until 19 hours after the start of the culture.

(C) Succinic acid production reaction 85% Phosphoric acid: 1.6 g, Magnesium sulfate heptahydrate: 1.1 g, Ferrous sulfate heptahydrate: 43 mg, Manganese sulfate / hydrate: 43 mg, 10N Aqueous potassium hydroxide solution: 2.86 g was dissolved in distilled water, adjusted to 42 mL, and then heat sterilized at 121 ° C. for 20 minutes to prepare a reaction concentrated medium.

  The reaction concentrated medium cooled to room temperature: 42 mL, and sterilized 720 g / L sucrose aqueous solution: 530 mL, sterilized water: 1.2 L, D-biotin and thiamine hydrochloride each 0.2 g / L aqueous solution: 20 mL sterilized in advance. Then, 675 mL of the culture solution obtained by the above main culture was added to a 5 L jar fermenter to start the reaction.

  Reaction temperature is 40 ° C., stirring speed is 150 per minute, pH is adjusted to 7.35 by sequential addition of neutralizing agent (ammonium bicarbonate: 171 g, 28% ammonia water: 354 g, distilled water: 529 g). The process was terminated when the residual sucrose in the reaction solution became 0.1 g / L or less.

  The succinic acid fermentation liquor thus prepared (containing the bacterial cells) was used in the following Examples and Comparative Examples.

  The succinic acid fermentation broth was subjected to centrifugal separation (15,000 G, 5 minutes), and the resulting supernatant was subjected to composition analysis. The composition shown in Table 2 was obtained.

[Example 1]
[Example 1-1]
<Cell separation>
(Example 1-1-1)
After adjusting the pH of the succinic acid fermentation liquor obtained in Reference Example 2 (C) above to 2.5 with 95% sulfuric acid, 10 mL was placed in a centrifuge tube, centrifuged at 500 G for 8 minutes, and then centrifuged. The turbidity (OD660) of the supernatant was measured. The turbidity (OD660) before cell separation was 24.

(Example 1-1-2)
The cells were separated in the same manner as in Example 1-1-1 except that the pH was adjusted to 3.0, and the turbidity (OD660) of the supernatant after centrifugation was measured.

(Example 1-1-3)
The cells were separated in the same manner as in Example 1-1-1 except that the pH was adjusted to 4.0, and the turbidity (OD660) of the supernatant after centrifugation was measured.

(Example 1-1-4)
The cells were separated in the same manner as in Example 1-1-1 except that the pH was adjusted to 5.0, and the turbidity (OD660) of the supernatant after centrifugation was measured.

(Comparative Example 1-1-1)
Bacterial cells were separated from the succinic acid fermentation broth (pH 7.6) in the same manner as in Example 1-1-1, and the turbidity (OD660) of the supernatant after centrifugation was measured.

  Moreover, centrifugation was performed at 4200G for 8 minutes, and the same experiment as Example 1-1-1 to 1-1-4 and Comparative example 1-1-1 was conducted.

  As a result, as shown in Table 3, when the pH was adjusted to 2.5 to 5.0 under any of the centrifugation conditions, the cell separation efficiency was improved as compared with the centrifugation at pH 7.6. A decrease in the turbidity of the separated supernatant was observed.

  In particular, when centrifugation is carried out after adjusting to pH 2.5 and 3.0, cell separation efficiency superior to that of high-speed centrifugation (4200G) at pH 7.6 is achieved even under low-speed centrifugation conditions (500G). Indicated.

[Example 1-2]
<Cell separation + MEK extraction>
(Example 1-2-1)
50 mL of the succinic acid fermentation broth obtained in Reference Example 2 (C) above was placed in a centrifuge tube, adjusted to pH 2.5 with 95% sulfuric acid, and then centrifuged at 9500 G for 10 minutes. About the obtained supernatant liquid, turbidity (OD660) was measured using a part, and MEK extraction was performed by the following procedure using the rest.

  After adding 10 g of MEK to 20 g of the supernatant and suspending for 5 minutes, the mixture was allowed to stand and the phase separation time (time until the aqueous phase and the organic phase were separated) was measured. Moreover, the abundance of the intermediate insoluble component existing between the aqueous phase and the organic phase was confirmed, and the region (organic phase clearing rate) where no suspended solids existed in the organic phase was visually confirmed.

  The region where the suspended matter does not exist in the organic phase (organic phase clear rate) can be regarded as a substantial proportion of the organic phase that can be recovered when extracting an organic acid such as succinic acid. In addition, light scattering measurement was performed using a part of the supernatant.

  Light scattering measurement was performed using an ELS-800 type apparatus manufactured by Otsuka Electronics Co., Ltd., ELS-8000 ver. Performed with 4.0 software. The incident slit was set to Φ0.2 and Φ0.1. The light source used was a He-Ne laser 10 mW, and the detector used was a photomultiplier tube for photocounting. The measurement was carried out using a 1 cm square cell at room temperature and using each treatment solution stock solution. The measurement angle was 90 ° C. and the solvent was water.

  All other measurement parameters were set automatically and the scattering intensity and particle size were measured. As the scattering intensity, an average value of n = 1 to 100 scans described in the particle size monitor table was used. Since the amount of incident light is automatically adjusted by the attenuator filter in the thick sample, it is divided by the filter value and the corrected numerical value is used for evaluation.

(Example 1-2-2)
The cells were separated in the same manner as in Example 1-2-1 except that the pH of the succinic acid fermentation liquor obtained in Reference Example 2 (C) was adjusted to 3.0, and the resulting supernatant was obtained. After adjusting the pH to 2.5 with 95% sulfuric acid, measurement of OD660 and MEK extraction were performed in the same manner as in Example 1-2-1, phase separation time, amount of intermediate insoluble matter, organic phase clear rate, average The particle size and light scattering intensity were measured.

(Example 1-2-3)
Except that the pH before cell separation was adjusted to 4.0, cell separation and MEK extraction were performed in the same manner as in Example 1-2-2, and OD660, phase separation time, amount of intermediate insoluble matter, and organic phase clear The rate, average particle size, and light scattering intensity were measured.

(Example 1-2-4)
Cell separation and MEK extraction were performed in the same manner as in Example 1-2-2 except that the pH before cell separation was adjusted to 5.0, and OD660, phase separation time, amount of intermediate insoluble matter, and organic phase clear The rate, average particle size, and light scattering intensity were measured.

(Comparative Example 1-2-1)
Except that the pH before cell separation was adjusted to 7.6, cell separation and MEK extraction were carried out in the same manner as in Example 1-2-2, OD660, phase separation time, amount of intermediate insoluble matter, organic phase clear The rate, average particle size, and light scattering intensity were measured.

  As a result, as shown in Table 4, when the pH was adjusted to 2.5 and 3.0, the phase separation time was short, the amount of intermediate insolubles was small, and the organic phase clearing rate was greatly improved. It was found that the efficiency of was good.

  Moreover, when adjusting to pH 4.0, although the phase separation time became long, the amount of intermediate | middle insoluble matters decreased somewhat, and the effect that the organic phase clear rate improved was recognized. When the pH was adjusted to 5.0, the phase separation time was long, but the amount of intermediate insoluble matter was slightly increased, but the effect of improving the organic phase clear rate was recognized. On the other hand, when the pH was adjusted to 7.6, the phase separation time was long, the amount of intermediate insolubles was large, and the organic phase clearing rate was poor, so the efficiency of MEK extraction was poor.

  In addition, it was considered that an insoluble component having a small average particle size deteriorates the liquid separation speed, and generation of an intermediate insoluble component increases under the condition that the light scattering intensity is large. Amino acids such as alanine, valine and glutamic acid were not recovered in MEK.

[Example 1-3]
Bacterial cell separation and MEK extraction were performed in the same manner as in Example 1-2-1 except that centrifugation was performed at 1000 G for 8 minutes, and OD660, phase separation time, and amount of intermediate insoluble matter were measured. As a result, OD660 was 0.191, phase separation time was 26 seconds, and the amount of intermediate insoluble matter was small. From this, it was found that MEK extraction can be performed efficiently even at low speed centrifugation.

[Example 1-4]
(A) Preparation of succinic acid fermentation broth by jar fermenter A succinic acid fermentation broth was prepared according to Reference Example 2 using the strain prepared in Reference Example 1. However, the temperature during the succinic acid production reaction was 39 ° C. and the pH was 7.6. The obtained succinic acid fermentation broth was subjected to centrifugal separation (15,000 G, 5 minutes), and the resulting supernatant was subjected to composition analysis. The results are shown in Table 5.

(B) pH adjustment and centrifugal separation of the succinic acid fermentation broth prepared with 95% sulfuric acid were adjusted to pH 7.6, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, After adjusting to 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0 and 0.5, 35 mL each was dispensed into a centrifuge tube and 10 to 40 ° C. Incubated for 30 minutes.

  However, the samples adjusted to pH 1.0 and 0.5 were kept at 50 ° C. because crystals were precipitated. In addition, about pH1.5, in order to confirm the influence by the difference in heat retention temperature, it carried out in two ways, 40 degreeC heat retention or 50 degreeC heat retention.

  After the incubation, the samples of each pH were centrifuged at 500G for 8 minutes. Of the obtained centrifugal supernatant, the pH 2.0-0.5 sample was readjusted to pH 2.5 using 28% ammonia water, and the pH 7.6-3.0 sample was 95. Each was readjusted to pH 2.5 using% sulfuric acid.

  Turbidity (OD660) was measured using a portion of the supernatant adjusted to pH 2.5. The measurement results are shown in Table 6 (centrifugation conditions: 500 G, 8 minutes). In addition, the turbidity (OD660) of the succinic acid fermentation liquid before cell separation was 25.

(C) MEK extraction MEK extraction was performed using a part of the fermentation broth supernatant adjusted to pH 2.5. 10 g of MEK was added to 20 g of the supernatant of the fermentation broth, suspended at 40 ° C. for 5 minutes, allowed to stand, and the phase separation time (time until the aqueous phase and the organic phase were separated) was measured. The amount of intermediate insolubles present between them was confirmed, and the region in the organic phase where there was no suspended matter (organic phase clear rate) was visually confirmed.

  Furthermore, light scattering measurement was performed using a part of the supernatant, and the average particle size and light scattering intensity were measured. The results of MEK extraction and light scattering measurement are shown in Table 6 (centrifugation conditions: 500 G, 8 minutes). The light scattering measurement was performed according to the measurement method described in Example 1-2-1.

  As a result, as shown in Table 6, the turbidity of the supernatant was higher in the case of centrifugation at pH 7.0 to 0.5 than in the case of centrifugation at pH 7.6 in any of the centrifugation conditions. Decline was confirmed. In particular, when centrifugation was performed at pH 5.5 or lower, a decrease in intermediate insoluble content was observed in MEK extraction compared with the case of centrifugation at pH 7.6, confirming that the extractability was improved.

  In addition, when centrifuged at pH 3.5 or lower, it was confirmed that the phase separation time during MEK extraction was shortened and the extractability was further improved as compared with the case of centrifugation at pH 7.6.

  On the other hand, the temperature before centrifugation was increased from 40 ° C. to 50 ° C. for the crystals having a pH of 1.0 or less because crystals were precipitated. About this, the result (Table 6, Examples 1-4-3 and 1-4-4) which carried out by two methods, 40 degreeC and 50 degreeC, using the sample of pH1.5, supernatant liquid turbidity The MEK extractability (phase separation time, intermediate insoluble content, organic phase clear rate) was almost unchanged.

[Example 1-5]
(A) Preparation of succinic acid fermentation broth by jar fermenter (A-1) Seed culture and main culture Using the strain prepared in Reference Example 1, according to the method described in Reference Example 2 (A) and (B), seed Culture and main culture were performed.

(A-2) Succinic acid production reaction 85% phosphoric acid: 0.3 g, magnesium sulfate heptahydrate: 0.2 g, ferrous sulfate heptahydrate: 9 mg, manganese sulfate hydrate: 9 mg 10N aqueous potassium hydroxide solution: 0.57 g was dissolved in distilled water, adjusted to 8.4 mL, and then heat-sterilized at 121 ° C. for 20 minutes to prepare a reaction concentrated medium.

  Reaction concentrated medium cooled to room temperature: 8.4 mL, pre-sterilized 720 g / L sucrose aqueous solution: 106 mL, sterile water: 178 mL, pre-filter sterilized D-biotin, thiamine hydrochloride 0.2 g / L aqueous solution: 0 4 mL, 0.2 L of the culture solution obtained by the above main culture was added to a 1 L jar fermenter to start the reaction.

  While the reaction temperature is 40 ° C., the stirring rotation speed is 200 rotations per minute, and the pH is adjusted to 7.35 by sequential addition of a neutralizing agent (ammonium bicarbonate: 171 g, 28% aqueous ammonia: 354 g, distilled water: 529 g). The reaction was continued and terminated when the residual sucrose in the reaction solution became 0.1 g / L or less.

  The succinic acid fermentation broth was subjected to centrifugal separation (15,000 G, 5 minutes), and the resulting supernatant was subjected to composition analysis. The results shown in Table 7 were obtained. Moreover, the result of measuring the turbidity (OD660) of the succinic acid fermentation broth was 30.

(B) Membrane filtration and pH adjustment The obtained succinic acid fermentation broth was adjusted to pH 2.5 with 95% sulfuric acid, and then 40 mL of cells were separated by membrane filtration (membrane pore diameter 0.22 μm, 5 μm). On the other hand, 40 mL each of succinic acid fermentation broth obtained as a comparative example was subjected to membrane filtration (membrane pore diameter 0.22 μm, 5 μm), and then the filtrate was adjusted to pH 2.5 using 95% sulfuric acid. Turbidity (OD660) measurement was performed using a part of the obtained membrane filtrate adjusted to pH 2.5. Table 8 shows the measurement results.

(C) MEK extraction MEK extraction was performed using a part of the membrane filtrate. Add 1 g of MEK to 2 g of membrane filtrate, suspend at room temperature for 5 minutes, let stand, measure the phase separation time (time until the water phase and the organic phase separate), and between the water phase and the organic phase The amount of the intermediate insoluble component present was confirmed, and the region in the organic phase where there was no suspended matter (organic phase clear rate) was visually confirmed. The MEK extraction results are shown in Table 8.

  As a result, as shown in Table 8, when membrane separation was performed using a membrane of any pore size, when membrane separation was performed at pH 2.5, it was compared with membrane separation at pH 7.4. Thus, a decrease in the turbidity of the filtrate was observed. As for the MEK extractability, when membrane separation is performed at pH 2.5, the intermediate insoluble matter is remarkably reduced and the phase separation time is shortened in any condition as compared with pH 7.4. There has been a significant improvement.

[Example 1-6]
(A) pH adjustment, centrifugation, and membrane separation The succinic acid fermentation broth obtained in Example 1-5 (A-2) was adjusted to pH 2.5 with 95% sulfuric acid. 35 mL of this was dispensed into a centrifuge tube, centrifuged at 4200 G for 8 minutes, and the resulting supernatant was subjected to membrane filtration (membrane pore size 0.22, 5 μm or no membrane filtration).

  On the other hand, as a comparative example, 35 mL of the succinic acid fermentation broth obtained in Example 1-5 (A-2) was dispensed into a centrifuge tube, centrifuged at 4200 G for 8 minutes, and the resulting supernatants were respectively membranes. Filtration (membrane pore size 0.22, 5 μm or no membrane filtration). Thereafter, the filtrate was adjusted to pH 2.5 with 95% sulfuric acid. Moreover, turbidity (OD660) measurement was performed on a portion of the membrane filtrate whose pH was adjusted to 2.5. Table 9 shows the measurement results.

(B) MEK extraction MEK extraction was performed using the above membrane filtrate. 10 g of MEK was added to 20 g of the above filtrate and suspended at 40 ° C. for 5 minutes, then allowed to stand, and the phase separation time (time until the aqueous phase and the organic phase were separated) was measured, and between the aqueous phase and the organic phase. The amount of the intermediate insoluble component present was confirmed, and the region in the organic phase where there was no suspended matter (organic phase clear rate) was visually confirmed. Moreover, light scattering measurement was performed using a part of the filtrate, and the average particle diameter and light scattering intensity were measured. The results of MEK extraction and light scattering measurement are shown in Table 9. The light scattering measurement was performed according to the measurement method described in Example 1-2-1.

  As a result, as shown in Table 9, even when centrifugation and membrane separation were combined, when centrifugation and membrane separation were performed at a pH of 2.5, compared to when centrifugation and membrane separation were performed at pH 7.4 Thus, a decrease in the turbidity of the filtrate was observed. In addition, regarding the MEK extractability, the intermediate insoluble content was remarkably reduced, the phase separation time was shortened, and the extractability was greatly improved.

[Example 2]
In this example, high-performance liquid chromatography (LC) was used for quantitative analysis of acids and saccharides, and amino acid quantitative analysis was performed using an amino acid analyzer under the following conditions. For protein quantification, the sample was hydrolyzed with hydrochloric acid, and the increment of the total amino acid content before and after hydrolysis was regarded as the protein amount.

<Analysis of acids and sugars>
Column: ULTRON PS-80H 8.0 mmI. D. × 30cm
Eluent: Water (perchloric acid) (60% aqueous solution of perchloric acid 1.8 ml / 1 L-H ₂ O)
Temperature: 60 ° C

<Amino acid analysis>
Apparatus: Hitachi Amino Acid Analyzer L-8900
Analysis conditions: biological amino acid separation conditions-ninhydrin coloring method (570 nm, 440 nm)
Standard product: PF (Wako amino acid mixed solution ANII type 0.8 ml + B type 0.8 ml → 10 ml)
Injection volume: 10 μl

<Hydrolysis for protein quantification>
A sample of 10 mg or 100 mg was precisely weighed and a 1 mL constant volume with pure water was dispensed to 200 μL, dried and heated in a hydrochloric acid atmosphere at 150 ° C. for 1 hour to hydrolyze the protein. After drying this, 200 μL of pure water was added and redissolved, and after filtration through a 0.45 μm filter, the filtrate was subjected to amino acid analysis.

[Example 2-1]
<Protonation>
98% sulfuric acid was added to 1500 g of the succinic acid fermentation broth having the composition shown in Table 2 to adjust the pH to 2.5 (hereinafter sometimes referred to as an aqueous succinic acid solution). Here, the addition amount of 98% sulfuric acid was 150 g.

<Contact process (batch operation)>
The aqueous succinic acid solution after addition of sulfuric acid is placed in a 5 L jacketed stirring tank with a bottom valve, and 2475 g of a 10% hydrous methyl ethyl ketone solution in which water has been added in advance to methyl ethyl ketone (hereinafter sometimes referred to as MEK) is added. (MEK solution / succinic acid aqueous solution = 1.5 (w / w)) The mixture was stirred for 30 minutes while controlling the internal temperature to 30 ° C. by passing warm water through the jacket. Thereafter, stirring was stopped, and the mixture was allowed to stand for about 1 hour while controlling the internal temperature at 30 ° C. An intermediate phase was present in the MEK phase near the liquid-liquid interface after standing.

  When the extraction residual phase, the intermediate phase, and the extraction phase were sequentially recovered from the stirring tank bottom valve, the respective weights were 2503 g of the extraction phase, 1490 g of the extraction residual phase, and 132 g of the intermediate phase. The composition of the extracted phase is shown in Table 10 below.

<Distillation>
The recovered extract substantially removes MEK by continuous distillation. Here, the distilled distillate is recovered as an azeotropic composition of MEK and water, that is, 11 wt% water-containing MEK, but there is a concern that succinic acid may precipitate depending on the degree of concentration of the kettle residue. Therefore, 103 g of water was added to 2503 g of the extract so that the distilled distillate was 11 wt% hydrous MEK and the kettle residue was 30 wt% succinic acid solution. Here, the amount of water added was calculated according to the following calculation.

(Dilution of extract)
FIG. 3 shows a phase diagram when the MEK and succinic acid concentrations of the extract were C _{MEK, 0} and C _{SA, 0} , respectively, and the diluted MEK and succinic acid concentrations were C _{MEK, 1} and C _{SA, 1} respectively. . FIG. 3 shows the composition change of MEK and succinic acid in the dilution of the extract with water and subsequent distillation.

In FIG. 3, the dilution operation line is a line connecting the extract composition (C _{MEK, 0} , C _{SA, 0} ) and the origin (0,0),
C _SA = (C _{SA, 0} / C _{MEK, 0} ) C _MEK
In addition, the distillation operation line is a line connecting the kettle residue (0, 0.3) and the distillate (0.89, 0),
C _SA = 0.3− (0.3 / 0.89) C _MEK
Since the diluted liquid is at the intersection of each line,
C _{SA, 1} = (C _{SA, 0} / C _{MEK, 0} ) C _{MEK, 1} ... Eq. (1)
C _{SA, 1} = 0.3− (0.3 / 0.89) C _{MEK, 1} ... Eq. (2)

By solving these, the diluent composition (C _{MEK, 1} , C _{SA, 1} ) is obtained. Further, the amount of dilution water added can be calculated as (C _{MEK, 0} −C _{MEK, 1} ) / C _{MEK, 1} for the extract.

  Here, since the composition of the extract was (0.8220, 0.0353), the composition after dilution was (0.7895, 0.0339), and the amount of diluted water added was 4.12 wt. %, That is, 2503 g × 4.12 wt% = 103 g.

  Distillation was carried out using a 500 ml round bottom flask equipped with a packed column with an inner diameter of 40 mm packed with a coil pack with a diameter of 5 mm and a height of 30 cm, and an atmospheric continuous distillation apparatus equipped with a distillation apparatus. About 300 ml of the diluted solution was added to the round bottom flask, and the flask was cooked while being heated in an oil bath, and the inside of the system was stabilized while applying total return.

  Here, the temperature of the oil bath was 120 ° C. Here, the column top temperature was 74 ° C. (azeotropic temperature), and the column bottom temperature was 101 ° C. After confirming that the inside of the tower is stable, set the return ratio to 1, continuously extract the distillate and continuously supply the feed liquid to the middle stage of the packed tower, and continuously extract the residual liquid from the kettle. Started. The feed liquid was heated to 60 ° C. in advance with a preheater and then fed to the packed tower.

  The feed liquid started to be fed at 100 ml / hour so as not to disturb the tower top temperature and tower bottom temperature, and increased to 400 ml / hour over about 1 hour. The temperature of the oil bath was gradually increased as the supply liquid increased, and finally it was 140 ° C. It took 7 hours to distill 2606 g of the diluted solution. The bottom flask after distillation was relatively clean.

  The residue in the kettle after distillation was 290 g. The results of analyzing the composition are shown in Table 11 below.

<Crystal>
The liquid from which MEK was distilled off was transferred to a jacketed 500 ml separable flask, and kept warm at 80 ° C. by passing warm water through the jacket while stirring. Thereafter, using a circulating thermostatic bath with a program, the warm water passed through the jacket was cooled to 20 ° C. over 1 hour, and succinic acid was cooled and crystallized.

  The obtained slurry was vacuum filtered and further washed with 150 g of cold water to collect a wet cake. Further, the obtained wet cake was dried at 80 ° C. under a maximum pressure using a vacuum dryer, and finally 78 g of succinic acid was recovered. The composition analysis results of the obtained succinic acid are shown in Table 12 below.

[Example 2-2]
In Example 2-1, the treatment was performed in the same manner except that the extraction temperature was 20 ° C. The extracted intermediate phase was clearly more than in Example 2-1, and the recovered extracted phase, intermediate phase, and extracted residual phase were 2322 g, 316 g, and 1485 g, respectively. The results of composition analysis of the extracted phase are shown in Table 13 below.

  It took about 7 hours to process all the extract in the distillation process. The bottom flask after distillation was relatively clean. The yield of succinic acid finally obtained was 55%, and the protein content was 29 ppm.

[Example 2-3]
In Example 2-1, the treatment was performed in the same manner except that the extraction temperature was 60 ° C.

  The extracted intermediate phase was clearly less than in Example 2-2, and the recovered extracted phase, intermediate phase, and extracted residual phase were 2498 g, 131 g, and 1495 g, respectively. The results of composition analysis of the extracted phase are shown in Table 14 below.

  It took about 7 hours to process all the extract in the distillation process. The bottom flask after distillation was relatively clean. The yield of succinic acid finally obtained was 55%, and the protein content was 35 ppm.

[Example 2-4]
<Protonation>
To 1500 g of the above succinic acid fermentation broth, 98% sulfuric acid was added to adjust the pH to 2.5. Here, the addition amount of 98% sulfuric acid was about 150 g.

<Extraction (mixer (stirring tank) / settler)>
The aqueous solution of succinic acid after the addition of sulfuric acid is mixed and separated from the MEK solution using a 500 ml jacketed stirring tank and a 600 ml, 400 ml, and 300 ml jacketed 3-settler settling tank as shown in FIG. Thus, succinic acid was continuously extracted. That is, 1650 g of succinic acid aqueous solution and 2475 g of 10% hydrous methyl ethyl ketone (MEK) solution (MEK solution / succinic acid aqueous solution = 1.5 (w / w)) to which water has been added in advance, and hot water at 30 ° C. are passed through the jacket to control the temperature In addition, the suspension was continuously extracted at a rate of 50 g / min while being vigorously stirred so that the inside of the vessel became uniform while being supplied to the 500 ml jacketed agitation vessel at a rate of 20 g / min and 30 g / min, respectively.

  The extracted suspension is supplied to the first tank of a three-tank settler whose temperature is controlled by flowing hot water of 30 ° C. through the jacket, and is separated into liquids, and the extracted residual phase is continuously extracted from the bottom of the first tank. It was. The extract overflowed the weir between the first tank and the second tank and was supplied to the second tank. A considerable amount of insoluble components were suspended in the extract supplied to the second tank.

  In the second tank, insoluble components that could not be separated in the first tank were allowed to settle to the bottom, and only the clear extract was allowed to overflow the weir between the second and third tanks and supplied to the third tank. Furthermore, in the 3rd tank, the clear extract was overflowed from the liquid interface vicinity, and the extract was collect | recovered.

  After the completion of the continuous extraction, the extract remaining in each tank, the extracted residual phase, and the insoluble components remaining in the first tank intermediate phase and the second tank bottom were recovered. The recovered extracted phase, extracted residual phase, and intermediate phase were 2370 g, extracted residual phase 1490 g, and intermediate phase 260 g, respectively. The results of composition analysis of the extracted phase are shown in Table 15 below.

<Distillation>
97 g of water was added to 2370 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1, to recover 275 g of the residue from the kettle. The distillation took 7 hours, but the bottom flask after the distillation was relatively clean. Table 16 below shows the results of composition analysis of the obtained kettle residue.

<Crystal>
The recovered pot residue was crystallized in the same manner as in Example 2-1, and finally 74 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 17 below.

[Example 2-5]

<Protonation>
To 1500 g of the above succinic acid fermentation broth, 98% sulfuric acid was added to adjust the pH to 2.5. Here, the addition amount of 98% sulfuric acid was about 150 g.

<Extraction (mixer (static mixer) / settler)>
The succinic acid aqueous solution after the addition of sulfuric acid is a jacketed static mixer (Noritake 1/4 (1) -N40-174-0 (inner diameter 5 mm, number of elements 24)) and 3 tanks with jackets of 600 ml, 400 ml and 300 ml respectively. The succinic acid was continuously extracted by mixing with the MEK solution and separating the liquid using a settling formula.

  That is, 1650 g of an aqueous succinic acid solution and 2475 g of a 10% hydrous methyl ethyl ketone (MEK) solution to which water was added in advance (MEK solution / succinic acid aqueous solution = 1.5 (w / w)) were respectively transferred at a rate of 20 g / min and 30 g / min. The temperature is controlled by flowing 30 ° C warm water through the jacket, and the extracted suspension is supplied to the first tank of a 3-tank settler whose temperature is controlled by flowing 30 ° C warm water through the jacket. The mixture was separated from the liquid and the extracted residual phase was continuously extracted from the bottom of the first tank.

  The extract overflowed the weir between the first tank and the second tank and was supplied to the second tank. In the second tank, insoluble components that could not be separated in the first tank were allowed to settle to the bottom, and only the clear extract was allowed to overflow the weir between the second and third tanks and supplied to the third tank. Furthermore, in the 3rd tank, the clear extract was overflowed from the liquid interface vicinity, and the extract was collect | recovered.

  After the completion of the continuous extraction, the extract remaining in each tank, the extracted residual phase, and the insoluble components remaining in the first tank intermediate phase and the second tank bottom were recovered. The recovered extracted phase, extracted residual phase, and intermediate phase were 2435 g, extracted residual phase 1490 g, and intermediate phase 197 g, respectively. The results of composition analysis of the extracted phase are shown in Table 18 below.

<Distillation>
100 g of water was added to 2435 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1, to recover 286 g of the residue from the kettle. The distillation took 7 hours, but the bottom flask after the distillation was relatively clean.

<Crystal>
The recovered pot residue was crystallized in the same manner as in Example 2-1, and finally 76 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 19 below.

[Example 2-6]
In accordance with the method of Example 2-1, protonation and extraction operations were performed to recover 2503 g of extracted phase, 1490 g of extracted residual phase, and 132 g of intermediate phase. The intermediate phase was centrifuged at 2000 G for 10 minutes with a centrifugal settling machine to recover 130 g of the clarified liquid. Table 20 below shows the composition analysis results of the liquid obtained by mixing the clarified liquid and the extract.

<Distillation>
108 g of water was added to 2633 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1. The distillation took 7 hours, but the bottom flask after the distillation was relatively clean.

<Crystal>
The recovered pot residue was crystallized in the same manner as in Example 2-1, and finally 82 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 21 below.

[Example 2-7]
In accordance with the method of Example 2-1, protonation and extraction operations were performed to recover 2503 g of extracted phase, 1490 g of extracted residual phase, and 132 g of intermediate phase. The intermediate phase was subjected to pressure filtration with a PTFE membrane filter having an opening of 0.5 μm to recover 130 g of the clarified liquid. Table 22 shows the composition analysis results of the liquid obtained by mixing the clarified liquid and the extract.

<Distillation>
108 g of water was added to 2503 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1, to recover 310 g of the residue from the kettle. The distillation took 7 hours, but the bottom flask after the distillation was relatively clean.

<Crystal>
The recovered pot residue was crystallized in the same manner as in Example 2-1, and finally 82 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 23 below.

[Example 2-8]
In the protonation and extraction operations of Example 5, 825 g of hydrous MEK supply ((MEK solution / succinic aqueous solution = 0.5 (w / w)) was 20 g / min and hydrous MEK to 1650 g of succinic acid aqueous solution. Except for feeding to a static mixer at a feed rate of 10 g / min, an extraction phase of 688 g, an extraction residual phase of 1613 g, and an intermediate phase of 173 g were recovered, and the intermediate phase was added with a PTFE membrane filter having an opening of 0.5 μm. 172 g of the clarified liquid was recovered by pressure filtration.

<Continuous extraction>
In the process flow as shown in FIG. 5, succinic acid was recovered in the solvent phase by a continuous extraction operation. The extracted residual phase 1613 g was continuously extracted by using a jacketed continuous extraction tower (10 theoretical plates) with an inner diameter of 20 mm and a height of 2 m to recover succinic acid. Here, the extracted residual phase was supplied from the top of the column at 200 g / hour, and continuously extracted from the bottom at 200 g / hour of MEK solution adjusted in advance to a water content of 10 wt%. The continuous phase was the extracted residual phase, and the dispersed phase was the MEK phase (light liquid dispersion). The temperature of the extraction tower was controlled at 30 ° C. by passing warm water through the jacket. Finally, 1777 g of the extract was recovered.

  The recovered extract was 2637 g in total, including the extract recovered by the mixer settler and the clarified liquid from which the solid content was removed by pressure filtration of the intermediate phase. Table 24 below shows the results of analysis of the composition of the recovered extract, the extract recovered by the mixer settler, and the mixture of the clarified liquid from which the solid content was removed by pressure filtration.

<Distillation>
190 g of water was added to 2637 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1, to recover 432 g of the residue from the kettle. The distillation took 7 hours, but the bottom flask after the distillation was relatively clean.

<Crystal>
The recovered pot residue was crystallized in the same manner as in Example 2-1, and finally 114 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 25 below.

[Comparative Example 2-1]
According to the method of Example 2-1, 2633 g of the extracted phase and 1490 g of the extracted residual phase were recovered in the same manner except that the intermediate phase was recovered as the extracted phase.

<Distillation>
109 g of water was added to 2633 g of the recovered extract, and continuous distillation was performed in the same manner as in Example 2-1, to recover 300 g of the residue from the kettle. In continuous distillation, the oil bath temperature was 140 ° C, but the bottom temperature decreased several hours after the start of continuous distillation (MEK detection), so the supply amount was lowered and the bottom temperature> 100 ° C was maintained at the oil bath temperature of 140 ° C. The operation was continued under conditions that allowed it. Results Distillation took 9 hours. In addition, a large amount of adhesive was adhered to the inner surface of the bottom flask after distillation.

<Crystal>
The liquid from which MEK was distilled off was transferred to a jacketed 500 ml separable flask, and kept warm at 80 ° C. by passing warm water through the jacket while stirring. Thereafter, using a circulating thermostatic bath with a program, the warm water passing through the jacket was cooled to 20 ° C. over 1 hour, succinic acid was cooled and crystallized, and after reaching 20 ° C., the mixture was further aged at 20 ° C. for 1 hour.

  The obtained slurry was vacuum filtered and further washed with 150 g of cold water to collect a wet cake. Further, the obtained wet cake was dried with a vacuum drier at 80 ° C. and a maximum reduced pressure, and finally 83 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 26 below.

[Comparative Example 2-2]
According to the method of Example 2-1, a protonated aqueous succinic acid solution is supplied from the top of the continuous extraction tower used in Example 2-9 at 200 g / hour, and 10% hydrous MEK is supplied from the bottom at 300 g / hour. As a result, a large amount of insoluble components were generated in the tower, and liquid-liquid separation was inhibited, and continuous operation was not possible.

  Table 27 below summarizes the results of the above examples and comparative examples.

[Example 3]
In this example, the quantitative analysis of acids, saccharides, quantitative analysis of amino acids, and protein were performed in the same manner as in Example 2.

[Example 3-1]
<Protonation process>
To 1500 g of the succinic acid fermentation broth having the composition shown in Table 2, 98% sulfuric acid was added to adjust the pH to 2.5. Here, the addition amount of 98% sulfuric acid was 150 g.

<Contact process>
The succinic acid aqueous solution after the addition of sulfuric acid is composed of a jacketed static mixer (Noritake 1/4 (1) -N40-174-0 (inner diameter Φ5 mm, number of elements 24)) and three tanks with jackets of 600 ml, 400 ml and 300 ml respectively. The succinic acid was continuously extracted by mixing with the MEK solution and separating the liquid using a settling formula.

  That is, 1650 g of aqueous succinic acid solution and 825 g of a 10% hydrous methyl ethyl ketone (hereinafter sometimes abbreviated as MEK) solution to which water has been added in advance (MEK solution (weight) / succinic acid aqueous solution (weight) = 0.5 (weight / weight) ) At a rate of 20 g / min and 10 g / min, respectively, by supplying hot water of 30 ° C. to the jacket to a temperature-controlled static mixer, and the extracted suspension is heated to 30 ° C. by flowing hot water of 30 ° C. through the jacket. It supplied to the 1st tank of the controlled 3 tank type settling machine, and liquid separation was carried out, and the extraction residual phase was continuously extracted from the bottom of the 1st tank.

  The extraction phase overflowed the weir between the first tank and the second tank and was supplied to the second tank. In the second tank, insoluble components that could not be separated in the first tank were allowed to settle to the bottom, and only the clear extraction phase overflowed the weir between the second and third tanks and was supplied to the third tank. Further, in the third tank, the clear extraction phase was overflowed from the vicinity of the liquid interface to recover the extraction phase, and finally the extraction phase 688 g, the extracted residual phase 1613 g, and the intermediate phase 173 g were recovered. The intermediate phase was subjected to pressure filtration with a PTFE membrane filter having an opening of 0.5 μm to recover 172 g of the clarified liquid.

<Continuous extraction>
1613 g of the extracted residual phase was obtained by using a jacketed continuous extraction tower (10 theoretical plates) with an inner diameter of Φ20 mm and a height of 2 m (MEK solution (weight) / succinic acid aqueous solution (weight) = 1) 0.0 (weight / weight)). Here, the extracted residual phase was supplied from the top of the tower at a rate of 200 g / hour, and a MEK solution adjusted in advance to a water content of 10% by weight was passed from the bottom of the tower at a rate of 200 g / hour. The continuous phase was the extracted residual phase, and the dispersed phase was the MEK phase (light liquid dispersion).

  The temperature of the extraction tower was controlled at 30 ° C. by passing warm water through the jacket. Finally, 1777 g of the extracted phase was recovered. The recovered extracted phase was combined with the clarified liquid recovered by the mixer settler, and the total amount of the liquid containing the aliphatic dicarboxylic acid was 2637 g. When the composition was analyzed, the results were as shown in Table 28 below.

<Distillation>
The recovered extracted phase substantially removes MEK by continuous distillation. Here, the amount of water added to the distillate was calculated in the same manner as in Example 2-1. The residue in the kettle after distillation was 432 g. The results of analyzing the composition are shown in Table 29 below.

<Crystal>
The liquid from which MEK was distilled off was transferred to a jacketed 500 ml separable flask, and kept warm at 80 ° C. by passing warm water through the jacket while stirring. Thereafter, using a circulating thermostatic bath with a program, the warm water passed through the jacket was cooled to 20 ° C. over 1 hour, and succinic acid was cooled and crystallized.

  The resulting slurry was vacuum filtered to separate the crystallization mother liquor. Further, the obtained wet cake of the crystallized product was washed with 250 g of cold water to recover the washing liquid, and a wet cake containing succinic acid as a main component was obtained. Further, the obtained wet cake was dried with a vacuum dryer at a maximum pressure of 80 ° C., and finally 114 g of succinic acid was recovered. The results of composition analysis of the obtained succinic acid are shown in Table 30 below.

  The crystallization mother liquor and the cleaning liquid were mixed to obtain a recovered liquid of 562 g, and the composition thereof was as shown in Table 31 below.

[Example 3-2]
<Protonation>
98% sulfuric acid was added to 1384 g of the succinic acid fermentation broth to adjust the pH to 2.5. Here, the addition amount of 98% sulfuric acid was 138 g. To this protonated liquid, 281 g corresponding to half of the recovered liquid recovered in Example 3-1 was added to prepare 1803 g of an aqueous succinic acid solution.

<Extraction>
In the same manner as in Example 3-1, an aqueous succinic acid solution was extracted with 10% water-containing MEK. The mixer settler supplies 0.5% by weight of 10% water-containing MEK to the aqueous succinic acid solution at 20 g / min and 10 g / min, respectively. The recovered intermediate phase is subjected to pressure filtration, and the extracted residual phase is further to the extracted residual phase. Countercurrent multistage continuous extraction was performed with 1.0% by weight 10% water-containing MEK. As a result, 2882 g of the extracted phase and 1593 g of the extracted residual phase were recovered. Its composition was as follows.

<Distillation>
Distillation was performed in the same manner as in Example 3-1, and 438 g of succinic acid concentrate was recovered. The composition was as shown in Table 32 below.

<Crystal>
Crystallization was carried out in the same manner as in Example 3-1, and 568 g of a recovery liquid, which was a mixed liquid of 113 g of succinic acid, a crystallization mother liquid, and a cleaning liquid, was recovered. Table 33 below shows the composition of succinic acid obtained, and Table 34 below shows the composition of the collected liquid.

  Recovered succinic acid / newly supplied succinic acid: 113 × 0.959 / (1384 × 0.0865) = 91%

[Example 3-3]
The steps from protonation to crystallization were carried out in the same procedure as in Example 3-2 to obtain 573 g of a recovered liquid that was a mixture of 114 g of succinic acid, a crystallization mother liquor, and a washing liquid. Table 35 below shows the composition of succinic acid obtained, and Table 36 below shows the composition of the collected liquid.

[Example 3-4]
The steps from protonation to crystallization were carried out in the same procedure as in Example 2 to obtain 114 g of recovered liquid, which was a mixture of succinic acid 114 g, crystallization mother liquor and washing liquid. The composition of succinic acid obtained is shown in Table 37 below. Table 38 below shows the composition of the collected liquid.

  Even when the recovered liquid, which is a mixture of the crystallization mother liquor and the recovered liquid, is recycled, the purity of the obtained succinic acid is not greatly reduced, and there is no problem in unit operation.

  Although the invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. The present application includes a Japanese patent application filed on October 7, 2009 (Japanese Patent Application No. 2009-233820), a Japanese patent application filed on November 17, 2009 (Japanese Patent Application No. 2009-262007), and a 2009 patent application. This is based on a Japanese patent application (Japanese Patent Application No. 2009-291474) filed on May 22, which is incorporated by reference in its entirety.

  In addition, non-amino organic acids such as succinic acid, fumaric acid and malic acid are obtained by the present invention. The succinic acid or the composition containing the succinic acid can be used for food additives, pharmaceuticals, cosmetics and the like. Further, fumaric acid or a composition containing fumaric acid can be used for food additives, unsaturated polyester resins, paper sizing agents, and the like. Furthermore, malic acid or a composition containing malic acid can be used for food additives, cosmetics, deodorants, detergents and dyes.

Claims (27)

From an aqueous solution including succinate obtained by reacting a biological raw material and a microorganism, a method for producing a succinic acid, a manufacturing method of succinic acid comprising the steps of (1) to (4).
(1) Aliphatic dicarboxylic acid generating step for accumulating succinic acid in the reaction solution by reacting biological material and microorganisms (2) Liquid containing succinic acid and microorganisms obtained in the aliphatic dicarboxylic acid generating step PH adjustment step (3) for separating microorganisms from the liquid obtained in the pH adjustment step (4) microorganism separation step Contacting step of contacting an aqueous solution obtained by separating microorganisms with a solvent and a solvent capable of phase separation with the aqueous solution   The method according to claim 1, wherein the microorganism is a coryneform bacterium. The solvent is an organic solvent having an inorganic value / organic value ratio (I / O value) of 0.2 or more and 2.3 or less and a normal pressure (1 atm) and a boiling point of 40 ° C. or more. Or the method of 2 . A method for producing succinic acid from an aqueous solution containing succinic acid obtained by reacting a biological material and a microorganism, and comprising the following steps (1) to (6).
(1) An aliphatic dicarboxylic acid production step for accumulating succinic acid in a reaction solution by reacting a biological material with a microorganism.
(2) pH adjusting step of obtaining a liquid having a pH of 1.0 or more and 5.0 or less by mixing a liquid containing succinic acid and microbial cells obtained in the aliphatic dicarboxylic acid generating step with an acid.
(3) Bacterial cell separation step for separating microbial cells from the liquid obtained in the pH adjustment step ( 4 ) Aqueous solution obtained by separating the bacterial cells in the bacterial cell separation step, and a solvent capable of phase separation from the aqueous solution ( 5 ) Solid content removal step for removing solids generated by contact of the aqueous solution and the solvent ( 6 ) Phase separation step for separating the solvent by phase separation The manufacturing method of the succinic acid of Claim 4 which performs the removal of the said solid content using a settler in the said solid content removal process. The method for producing succinic acid according to claim 4 or 5 , wherein in the contacting step, the aqueous solution and the solvent are contacted using a static mixer. The succinic acid according to any one of claims 4 to 6 , wherein, in the solid content removing step, the solid content is removed together with at least one liquid selected from the aqueous solution and the solvent. Production method. The succinic acid production according to claim 7 , wherein the extracted solid content is subjected to solid-liquid separation, and the obtained liquid is returned to at least one of a step before the contact step between the aqueous solution and the solvent and a step after the phase separation step. Method. The method for producing succinic acid according to claim 8 , wherein the solid-liquid separation is performed by a centrifugal sedimentation method. The method for producing succinic acid according to claim 8 , wherein the solid-liquid separation is performed using a filter. The remaining liquid after separation by phase separating the solvent, and a solvent for liquid phase separation, is contacted in a countercurrent multistage extraction column, comprising the step of separating by phase separation the solvent later claims 4 The manufacturing method of the succinic acid of any one of Claim 10 Said contacting step at 30 to 60 ° C., the manufacturing method of succinic acid according to claims 4 to any one of claims 11. A method for producing succinic acid from an aqueous solution containing succinic acid obtained by reacting a biological raw material and a microorganism, the method comprising producing the following steps (1) to (8).
(1) An aliphatic dicarboxylic acid production step for accumulating succinic acid in a reaction solution by reacting a biological material with a microorganism.
(2) pH adjusting step of obtaining a liquid having a pH of 1.0 or more and 5.0 or less by mixing a liquid containing succinic acid and microbial cells obtained in the aliphatic dicarboxylic acid generating step with an acid.
(3) Cell separation step for separating microorganism cells from the liquid obtained in the pH adjustment step
(4) A contact step in which the aqueous solution obtained by separating the cells in the cell separation step and a solvent capable of phase separation are brought into contact with the aqueous solution. ( 5 ) In the step of concentrating the succinic acid recovered in the contact step. there are, the extract phase concentration step (6) crystallization step (7) to precipitate the succinate from the liquid after the extraction phase concentration step crystallization of succinic acid precipitated in about crystallization step in which the water concentration in the extract phase by the concentration increases Solid-liquid separation step to recover ( 8 ) Crystallization mother liquor recycling step for returning at least part of the crystallization mother liquor after recovering the succinic acid obtained in the solid-liquid separation step to any step before the crystallization step The manufacturing method of the succinic acid of Claim 13 including the protonation process which adds an acid to the aqueous solution containing the succinic acid obtained from the said biological origin raw material before a contact process. The manufacturing method of the succinic acid of Claim 13 or 14 whose density | concentration of the solvent contained in the solution containing the succinic acid after the extraction phase concentration process supplied to a crystallization process is 10 weight% or less. The manufacturing method of the succinic acid of Claim 15 whose density | concentration of the solvent contained in the solution containing the said succinic acid supplied to a crystallization process is 1 weight% or less. After removing components other than succinic acid from the mother liquor after recovering succinic acid in the solid-liquid separation process, at least a part of the liquid is recycled to at least one of the processes from the contact process to the crystallization process. The method for producing succinic acid according to any one of claims 13 to 16 . The method for producing succinic acid according to any one of claims 13 to 17 , wherein water is added in any step before the extraction phase concentration step. An acid having a pKa of less than 4 is used as an acid to be added in the protonation step, and the pH of the aqueous solution containing succinic acid obtained from the biological material in the protonation step is maintained in the range of more than 1 and less than 4. The method for producing succinic acid according to any one of claims 14 to 18 , wherein: Solvent used in the contacting step is, is the ratio of the inorganic value of 0.2 to 2.3 for the organic value, and wherein the boiling point of 40 ° C. or more, of claims 13 to claim 19 The manufacturing method of the succinic acid of any one. The method for producing succinic acid according to any one of claims 13 to 20 , further comprising a step of removing microorganisms before the contacting step. The method for producing succinic acid according to any one of claims 13 to 21 , further comprising a step of removing the protein before the contacting step. 23. The method according to any one of claims 13 to 22 , wherein a mixer settler is used in the contacting step, and an extraction phase, a residual extraction phase, and a phase including a solid content are recovered from a settling portion of the mixer settler. The manufacturing method of succinic acid of description. 24. The method for producing succinic acid according to claim 23 , wherein the phase containing the solid content recovered from the settler part is subjected to solid-liquid separation, and the liquid phase is recovered. The method for producing succinic acid according to claim 23 or 24 , wherein succinic acid is further recovered from the extracted residual phase obtained in the settler section using a countercurrent multistage extraction tower in the contacting step. The method for producing succinic acid according to any one of claims 13 to 25 , wherein cooling is performed in the crystallization step. 27. The method for producing succinic acid according to any one of claims 13 to 26 , wherein the crystallization step includes a reduced pressure operation.

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