JP3959403B2 - Purification method of organic acid - Google Patents

Description

  The present invention relates to a technique for purifying organic acids by electrodialysis. More specifically, it is a purification method that enables a culture solution having a specific composition to be converted into an organic acid with high current efficiency by electrodialysis under a specific condition.

  Currently, many organic acids and their derivatives are produced by petrochemical synthesis and are used in a wide range of fields such as foods, pharmaceuticals, cosmetics, chemicals and polymer raw materials. Since organic acids can be produced from an inexpensive carbon source by a room temperature and atmospheric pressure method, various organic acids such as lactic acid and amino acids have been studied by a cultivation method instead of a petrochemical synthesis method.

  Culture Method An organic acid production method is a method in which an organic acid-producing microorganism is cultured, and the microorganism is allowed to act on sugars such as glucose in a reaction medium to produce an organic acid salt. Conventionally, aerobic coryneform bacteria have been widely used to produce useful substances such as amino acids under aerobic conditions, but are anaerobic in a reaction solution containing carbonate ion, bicarbonate ion, or carbon dioxide gas. It is also used to produce an oxygen-containing compound by acting on an organic raw material under conditions (see Patent Document 1).

  The organic acid salt produced by the culture method is separated and recovered from the culture solution by a salting-out method, a recrystallization method, an organic solvent extraction method, an esterification distillation separation method, a chromatographic separation method, an electrodialysis method, or the like. Specifically, organic acid is produced by separating the organic acid calcium from the culture solution and then recovering the organic acid by sulfuric acid decomposition, or by esterifying and distilling the organic acid salt in the culture solution and then hydrolyzing the organic acid. There are methods such as recovery, but these methods have high-cost problems due to generation of by-products and complicated processes and operations.

  In recent years, organic acid separation / recovery technology using electrodialysis has been actively studied in view of its relatively simple apparatus and process and no generation of by-products, and has been introduced into some industrial processes. The electrodialysis method includes desalting electrodialysis in which an electrolyte such as an organic acid salt and a non-electrolyte are separated to concentrate or remove the electrolyte, and a salt such as an organic acid salt by protons and hydroxide ions generated by the decomposition of water. There is a hydrolytic electrodialysis that produces acid and alkali from the water. When electrodialysis technology is used for culture method organic acid purification, the organic acid salt produced by the culture method is separated and recovered from the culture solution by desalting electrodialysis method, and then the organic acid salt is converted to organic acid by water splitting electrodialysis method There are a method and a culture method in which an organic acid salt is directly introduced into a water-splitting electrodialyzer and converted into an organic acid.

In the water-splitting electrodialysis apparatus, bipolar membranes (composite ion exchange membranes in which a cation exchange membrane and an anion exchange membrane are bonded) and cation exchange membranes or anion exchange membranes are alternately arranged between the anode and cathode facing each other. In this structure, a base chamber is formed on the anode side of the bipolar film and an acid chamber is formed on the cathode side. In the case of an electrodialyzer consisting of a bipolar membrane / cation exchange membrane, an organic acid salt is introduced into the acid chamber, while in the case of an electrodialyzer consisting of a bipolar membrane / anion exchange membrane, the organic acid salt is converted into a base. By introducing into the chamber, the organic acid and the base are separated and recovered.
Regarding water-splitting electrodialysis technology, recovery of acid and alkali solutions from salts of weak acids such as acetic acid by bipolar membrane / cation exchange membrane method, and weak bases such as ammonium by bipolar membrane / anion exchange membrane method A proposal has been made on the recovery of an acid and an alkali solution from a salt formed (see Patent Document 2).

  When purifying an organic acid from an organic acid salt using a water-splitting electrodialyzer, a bipolar membrane / cation exchange membrane method has been used exclusively so far (see Patent Documents 2 to 6). The reason for this is that in the case of a salt made of a weak acid such as an organic acid salt, good current efficiency can be obtained by blocking the movement of hydroxide ions to the acid chamber by the cation exchange membrane, so the bipolar membrane / cation exchange membrane method is In the bipolar membrane / anion exchange membrane method, since the ionization degree of the organic acid generated in the acid chamber is very low, the electric resistance of the acid chamber is increased and the organic acid is increased. This is because it becomes difficult to proceed with purification.

  Regarding the problem of increasing the electrical resistance of the acid chamber in the bipolar membrane / anion exchange membrane electrodialysis method, it has been proposed to lower the electrical resistance by supplying the acid chamber with a solution containing a strong acid or a salt that dissociates in water. It is described that the produced ammonium lactate was supplied to the base chamber and a 0.1N hydrochloric acid aqueous solution was supplied to the acid chamber to efficiently convert it into lactic acid (see Patent Document 7).

Non-Patent Document 1 describes that commercially available chemical ammonium lactate was converted to lactic acid at a current efficiency of 90% by bipolar membrane / anion exchange membrane electrodialysis. Here, only lactic acid is supplied to the acid chamber, and no mention is made of the conductivity of the acid chamber.
Bipolar membrane / anion exchange membrane water-splitting electrodialysis has been proposed for application to electrodialysis culture (see Patent Documents 8 to 9). The bipolar membrane / anion exchange membrane method is adopted to introduce the culture solution into the electrodialysis tank and sequentially extract the organic acid from the culture solution. That is, in the case of the bipolar membrane / anion exchange membrane method, the organic acid salt introduced into the base chamber is ionized, and the organic acid ions permeate the anion exchange membrane to produce an organic acid in the acid chamber. Organic acids can be separated and recovered from organic substances and polymer compounds, and electrodialysis culture utilizes the characteristics of the bipolar membrane / anion exchange membrane method. However, the electrodialysis culture technology using these bipolar membranes / anion exchange membranes has problems such as low conversion rate from organic acid salt to organic acid and addition of strong acid to acid chamber to improve current efficiency. There are many.

  The organic acid salt-containing culture solution contains various substances mixed from microorganisms and reaction media. When purifying organic acid from such a culture solution using a hydrolyzed electrodialyzer, solid substances such as microorganisms may adhere to the ion exchange membrane and cause deterioration of the membrane function. Is performed by separating and removing solid substances from the culture solution by a method such as membrane separation or centrifugation before electrodialysis (see Patent Document 9).

In addition, when a polyvalent cation such as Mg or Ca contained in the culture solution degrades the ion exchange membrane, the culture solution is brought into contact with a chelating resin before electrodialysis to adsorb and remove the polyvalent cation. (See Patent Documents 6 and 9).
JP-A-11-113588 Japanese Patent Publication No.33-2023 JP-A-2-286090 Japanese Patent No. 2872723 JP-A-8-24587 JP-A-9-135698 Japanese Patent No. 3337587 JP-A-11-137286 US Pat. No. 5,814,498 Monthly Food Chemical, 6, p30-35 (1996)

  In the production of organic acid using coryneform bacteria, when the organic acid is purified from a culture solution containing an organic acid salt using bipolar membrane / cation exchange membrane electrodialysis, the current efficiency may decrease and the conversion reaction may stop. is there. As a result of examining this cause, in addition to the solid substances reported so far and ion-exchange membrane contamination by polyvalent cations such as Mg and Ca, impurities in the culture solution prevent organic acid purification. There was found.

Usually, a salt made of a strong acid such as sulfate, nitrate, hydrochloride or phosphate is added to the reaction medium, and therefore a culture solution containing an organic acid salt contains a salt made of a strong acid. When such a culture solution is treated by a bipolar membrane / cation exchange membrane electrodialysis method, when the culture solution is introduced into the acid chamber, the organic acid salt in the culture solution generates an organic acid in the acid chamber. This salt also produces strong acid in the acid chamber, and the strong acid is ionized again into acid ions and protons, and the protons pass through the cation exchange membrane and combine with the hydroxide ions in the base chamber to generate water. It was thought to decline. When the content of the salt made of a strong acid increases, the current efficiency is remarkably deteriorated, and the conversion reaction of the organic acid salt to the organic acid is eventually stopped. Here, the strong acid means that the ionization constant as an acid is 1 ×.
This refers to those having 10 ⁻³ or more, or those having a pK of 3 or less, specifically sulfuric acid, nitric acid, hydrochloric acid or phosphoric acid.

  On the other hand, when an organic acid is purified from a culture solution containing an organic acid salt using a bipolar membrane / anion exchange membrane electrodialysis method, the electric resistance value of the acid chamber increases and the conversion reaction may be stopped. There is a problem that alkali metal derived from a medium or the like generates a strong base in the base chamber, and current efficiency decreases due to ionized hydroxide ions, thereby hindering efficient organic acid purification.

  As a result of studying a solution to such a problem, the present inventor obtained an organic acid from a culture solution containing an organic acid salt obtained by reacting coryneform bacteria with saccharides in a reaction medium using a water-splitting electrodialyzer. In the purification method, a water-splitting electrodialysis apparatus composed of a bipolar membrane and an anion exchange membrane is used, and the acid resistance value of the acid chamber is adjusted by adjusting a salt made of a strong acid and an alkali metal in the culture solution to a specific concentration. The present inventors have found that the current efficiency can be improved by lowering the current and the present invention has been completed.

That is, the present invention
(1) After producing a culture solution containing an organic acid salt by allowing coryneform bacteria to act on sugars in a reaction medium in a culture tank, the organic acid is purified from the culture solution using a water-splitting electrodialyzer. In the method
(A) The water splitting electrodialysis apparatus is composed of a bipolar membrane and an anion exchange membrane,
(B) the culture solution contains a salt composed of a strong acid, the concentration is 0.03 mol / liter to 0.5 mol / liter, and
(C) the alkali metal concentration in the culture solution is 0.03 mol / liter or less,
A method for purifying an organic acid characterized by the following.
(2) The method for purifying an organic acid according to (1), wherein the salt made of a strong acid contains at least one of sulfate, nitrate, hydrochloride and phosphate.
(3) The method for purifying an organic acid according to (1), wherein the alkali metal contains at least one of lithium, sodium and potassium.
(4) The method for purifying an organic acid according to (1), wherein the cells are separated and removed from the culture solution and then introduced into a water-splitting electrodialysis apparatus.
(5) The organic acid according to (1), wherein the coryneform bacterium is Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, or Micrococcus Purification method.
(6) The method for purifying an organic acid according to (1), wherein the organic acid salt to be produced is selected from succinate and lactate.

  According to the present invention, when purifying an organic acid from an organic acid salt-containing culture solution using a hydrolyzed electrodialyzer, the hydrolyzed electrodialyzer comprises a bipolar membrane and an anion exchange membrane. Strong acid produced from a salt made of a strong acid stays in the acid chamber, so the current efficiency does not decrease. Conversely, the increase in the electric resistance of the acid chamber due to the low ionization degree of the organic acid is mitigated, and the current efficiency is increased. Is obtained. Furthermore, in the present invention, the strong acid is contained in the culture solution, and the concentration thereof is a specific concentration. Therefore, if the amount of the salt composed of strong acid in the reaction medium is increased, the amount of strong acid in the acid chamber increases, and the low There is no need for special operations such as supplying a strong acid or a salt that dissociates in water into an acid chamber that can be operated at a high current density at a voltage.

  On the other hand, when using a bipolar membrane / anion exchange membrane water-splitting electrodialysis apparatus, alkali metal may generate a strong base and reduce current efficiency. However, in the present invention, the alkali metal concentration in the culture solution is low. Since it has a specific concentration, it is possible to purify organic acids while maintaining high current efficiency.

  As described above, in the present invention, an organic acid is stably and highly efficient without requiring an operation for removing impurities such as a salt made of a strong acid or an alkali metal before water-splitting electrodialysis and without increasing the number of purification steps. Can be purified and an inexpensive product can be provided.

  Organic acid salt purified by the action of coryneform bacteria on saccharides in the reaction medium has a specific concentration of strong acid salt and alkali metal present in the culture solution, and the culture solution is a bipolar membrane / anion exchange membrane type. It is purified by converting it into an organic acid using a water splitting electrodialyzer.

The coryneform bacterium used in the present invention is a group of microorganisms defined in the Bargeys Manual of Determinative Bacteriology (8, 599, 1974) under normal aerobic conditions. There is no particular limitation as long as it grows and produces the desired organic acid salt under reduced conditions.
Specific examples of coryneform bacteria include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus.

  More specifically, Corynebacterium glutamicum FERM P-18976, ATCC13032, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13745, ATCC13746, ATCC13746, ATCC13746, ATCC13746, , ATCC 31831 and the like.

Examples of the Brevibacterium include Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum MJ-233 (FERM BP-1497) or MJ-233AB-41 (FERM BP-1498), Brevibacterium ammoniagenes ATCC6872 etc. are mentioned.
Examples of the genus Arthrobacter include Arthrobacter globiformis ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738, ATCC35698 and the like.

  As the genus Micrococcus, Micrococcus freudenreichii no. 239 (FERM P-13221), Micrococcus luteus No. 240 (FERM P-13222), Micrococcus ureae IAM1010, Micrococcus roseus IFO3764 and the like.

  Of the various coryneform bacteria described above, Corynebacterium glutamicum R (FERM P-18976), Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 13869 and the like are particularly preferable.

  The coryneform bacterium used in the present invention may be a wild-type mutant strain (for example, FERM P-18777, FERM P-18978 strain, etc.) existing in nature, and uses biotechnology such as gene recombination. Artificial strains (for example, FERM P-17878, FERM P-17888, FERM P-18879, etc.) may be used.

  Coryneform bacteria can be cultured using a normal nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, and the like. As the carbon source, for example, glucose, molasses and the like can be used, and as the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination. In addition, as the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, manganese sulfate and the like can be used. In addition to these, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, various vitamins (for example, biotin, thiamine, etc.) can be appropriately added to the medium.

  Culturing can usually be performed at a temperature of about 20 ° C. to about 40 ° C., preferably about 25 ° C. to about 35 ° C. under aerobic conditions such as aeration stirring or shaking. The pH during the culture is in the range of about 5 to 10, preferably about 7 to 8. The pH during the culture can be adjusted by adding an acid or an alkali. The carbon concentration at the start of the culture is about 1 to 20% (W / V), preferably about 2 to 5% (W / V). The culture period is usually about 1 to 7 days.

  Subsequently, the cultured cells of coryneform bacteria are separated and recovered from the culture obtained as described above. The method for separating and recovering the cultured cells is not particularly limited, and for example, known methods such as centrifugation and membrane separation can be used.

  The collected cultured microbial cells are usually used as they are in the next step, but the cultured microbial cells may be treated and the resulting treated microbial cells may be used in the next step. The microbial cell treated product may be any cultivated microbial cell that has been subjected to some kind of treatment. Examples thereof include an immobilized microbial cell in which the microbial cell is fixed with acrylamide, carrageenan, or the like while being alive.

  Next, the cultured cells of coryneform bacteria recovered or separated from the culture obtained as described above or the treated cells thereof are subjected to a reaction for producing a target organic acid salt in a reaction medium in a reduced state. . The organic acid salt production method can be either a batch production method or a continuous production method.

  The reaction medium contains saccharides that are raw materials for organic acid salt production. Any sugar can be used as long as it is metabolizable by coryneform bacteria. Examples thereof include polysaccharides. Of these, glucose is preferable.

  More preferably, the composition of the reaction medium used for the organic acid salt formation reaction is necessary for the coryneform bacterium or its processed product to maintain its metabolic function, that is, the carbon source of various sugars and protein synthesis. Including nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride or ammonium nitrate, and inorganic salts such as potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate or sodium chloride, and also Li, Ca, Fe, Contains trace metal salts such as Mn, Cu or Zn. These addition amounts can be appropriately determined depending on the required reaction time, the type of target organic acid salt product, the type of coryneform bacterium used, and the like. Depending on the coryneform bacterium used, the addition of specific vitamins may be preferred. Further, depending on the target organic acid salt, it may be effective to add carbon dioxide or an inorganic carbonate such as various carbonates or bicarbonates to the reaction medium in addition to an organic carbon source such as a saccharide.

  The reaction between coryneform bacteria (including cultured bacteria and treated products thereof) and saccharides is preferably carried out under temperature conditions where coryneform bacteria can act, and the pH during the reaction is around 5 to 10, preferably The range near 7-8 is good, and pH adjustment during the reaction can be carried out by adding ammonia or amine.

  Organic salts that can be produced by the reaction of coryneform bacteria with sugars include succinate, lactate, acetate, malate, fumarate, citrate, maleate, tartrate, oxaloate. Examples thereof include acetate, cisaconite, isocitrate and 2-oxoglutarate, but the present invention can be particularly preferably applied to succinate or lactate.

  The culture solution containing the organic acid salt produced by the reaction with the saccharide can be directly introduced into the water-splitting electrodialyzer to separate and recover the organic acid, but the microbial cells in the culture solution (including the immobilized cells) When solid substances such as) adhere to and contaminate the dialysis membrane or the like, it is preferable to separate and remove the solid substance using membrane separation or centrifugation before introducing the culture solution into the electrodialyzer. In addition, only an electrolyte substance such as an organic acid salt can be separated and recovered from the culture solution in advance using a desalting electrodialysis method. The separated solid matter such as microorganisms can be returned to the culture tank for use.

  Thus, the organic acid salt-containing culture solution obtained by appropriately separating and removing impurities from the culture solution can be subjected to hydrolysis electrodialysis, but the inorganic electrolyte substance derived from the reaction medium and the like is separated and removed from the organic acid salt. Usually, the organic acid salt-containing culture solution contains a salt composed of a strong acid of sulfate, nitrate, hydrochloride or phosphate. In the culture medium used in the present invention, the concentration of the salt made of a strong acid is 0.03 M (mol / liter) to 0.5 M (mol / liter), and the alkali metal concentration is 0.03 M (mol / liter). Must be:

  The electrodialysis apparatus used in the present invention is a two-chamber water splitting electrodialysis apparatus composed of a bipolar membrane and an anion exchange membrane shown in FIG. This electrodialyzer has a structure in which a bipolar membrane 1 and an anion exchange membrane 2 are arranged in order between an anode and a cathode to form an acid chamber 3 and a base chamber 4. In such an electrodialysis apparatus, the organic acid salt supplied to the base chamber 4 is ionized into an organic acid ion and a base ion at a voltage applied to both electrodes, and the organic acid ion permeates through the anion exchange membrane to form the acid chamber 3. And combines with protons supplied from the bipolar membrane 1 to generate an organic acid. The base ions remain in the base chamber 4 and combine with the hydroxide ions supplied from the bipolar membrane to generate a base. The organic acid aqueous solution generated in the acid chamber 3 is circulated and concentrated by the pump 11 between the acid solution tank 8 connected to the acid chamber 3 and the organic acid solution is connected to the salt solution tank 7 connected to the base chamber 4. Generally, a method of performing electrodialysis while circulating a mixed solution of a salt and a base with a pump 10 is employed. During this time, an aqueous solution such as sodium hydroxide, sodium sulfate, sulfuric acid, or sodium chloride is supplied to the electrode chambers 5 and 6 from the electrode liquid tank 9 connected to the electrode chamber by the pump 12 and circulated.

  The bipolar membrane used in the present invention is an ion exchange membrane having a structure in which a cation exchange membrane and an anion exchange membrane are bonded together, and is a commercially available ion exchange membrane such as Neocepta BP- manufactured by Tokuyama Corporation. 1 can be used.

In addition, as the anion exchange membrane used in the present invention, a commercially available ion exchange membrane, for example, Neoceptor AHA manufactured by Tokuyama Corporation can be used.
In the electrodialysis according to the present invention, it is generally preferable that the cell voltage is 1.5 to 3.5 V, the current density is 20 to 200 mA / cm ² , and the solution temperature is 10 to 50 ° C.

  When an organic acid salt-containing culture solution is supplied to the bipolar membrane / anion exchange membrane electrodialyzer under such electrodialysis conditions, an organic acid is generated in the acid chamber. A strong acid is also generated in the acid chamber from a salt composed of a strong acid such as sulfate, nitrate, hydrochloride, phosphate, etc. derived from the reaction medium contained in the culture solution, and the strong acid is ionized again into acid ions and protons. Since it stays in the chamber, the current efficiency is not lowered. Rather, the electric resistance value of the acid chamber is lowered, and the organic acid can be purified by a low voltage and high current reaction. In order to reduce the electric resistance value of the acid chamber with a strong acid and proceed with the purification of the organic acid with high efficiency, the concentration of the salt made of the strong acid in the organic acid salt-containing culture solution must be 0.03 M or more. In order to further increase the current efficiency, the concentration of the salt made of a strong acid is preferably 0.05M or more. In the case of less than 0.03M, in the bipolar membrane / anion exchange membrane method, the electric resistance value of the acid chamber becomes high and the conversion reaction cannot proceed efficiently, and a strong acid or the like is added to the acid chamber to lower the electric resistance. Operation is required.

  The upper limit of the concentration of the salt made of a strong acid is not particularly limited from the viewpoint of organic acid purification efficiency, but considering the removal of the strong acid after the organic acid purification, about 0.5M is sufficient. Therefore, the concentration of the salt consisting of the strong acid in the organic acid salt-containing culture solution in the present invention is 0.03M to 0.5M, more preferably 0.05M to 0.5M. Since the composition of the reaction medium varies depending on the type of microorganism used for culture, the culture conditions, and the like, the type and concentration of a salt made of a strong acid contained in the culture solution also changes. In addition, strong acids such as sulfuric acid, nitric acid, hydrochloric acid, and phosphoric acid produced from salts of strong acids in hydrolytic electrodialysis do not give the same conductivity in the acid chamber, but the effect of increasing the conductivity by each acid is added. In order to purify the organic acid with high current efficiency, the total sum of the salts composed of the strong acids is 0.03M to 0.5M, preferably 0.05M to 0.5M. It is possible to adjust the concentration of the salt composed of a strong acid by devising the kind and amount of the inorganic salt when preparing the reaction medium.

  Furthermore, when bipolar membrane / anion exchange membrane electrodialysis is used, the alkali metal concentration in the culture medium to be supplied must be 0.03 M or less. That is, alkaline metals such as Li, Na or K generate strong bases in the base chamber, but strong bases are easily ionized, and hydroxide ions permeate the anion exchange membrane and react with protons in the adjacent acid chamber. Reduces current efficiency to produce water. If the alkali metal concentration in the culture solution is high, control the pH of the base chamber so that it does not increase by supplying the culture solution to the base chamber in the course of electrodialysis or removing the base solution from the system. As a result, it is possible to prevent a decrease in current efficiency to some extent, but stable operation of electrodialysis is difficult. Therefore, it is necessary to keep the alkali metal in the culture medium introduced into the bipolar membrane / anion exchange membrane electrodialysis at a low concentration, and the preferable alkali metal concentration is 0.03M or less, more preferably 0.02M or less. As long as stable purification is performed with high current efficiency, the lower limit is not particularly limited. Adjustment to the alkali metal concentration is possible by devising the kind and blending amount of the inorganic salt when preparing the reaction medium.

In addition, since ammonium salt may gasify ammonia and cause swelling of the bipolar membrane, it is necessary to adjust so that the ammonia concentration in the base solution is not increased too much.
The organic acid can be separated and recovered by electrodialysis by either a batch or continuous method, and can be used as an electrodialysis culture.

  Thus, the organic acid and the base can be separated from the organic acid salt by the bipolar membrane / anion exchange membrane method electrodialysis, and the organic acid can be recovered. The recovered organic acid can be removed by using a chelate resin treatment, an ion exchange method, a crystallization method, or the like so as to satisfy the required quality depending on the application. The separated base can be reused as a pH adjuster in culturing organic acid production.

Example 1
EXAMPLES Hereinafter, although this invention is demonstrated with an Example, this invention is not limited to such an Example.
(1) Corynebacterium glutamicum R (FERM P-18976) cultured under aerobic conditions (preparation of culture 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 · 7H ₂ O 5 mg, biotin 200 μg, thiamine hydrochloride 200 μg, yeast extract 2 g, casamino acid 7 g, 500 ml of a medium consisting of 1000 ml of distilled water was dispensed into a 1 liter flask, heated and sterilized at 120 ° C. for 10 minutes, and then cooled to room temperature as a seed culture medium. Similarly, 1000 ml of the medium having the same composition was placed in a 2 liter glass jar fermenter and sterilized by heating at 120 ° C. for 10 minutes to obtain a main culture medium.

(Cultivation): One seed culture medium is inoculated with coryneform bacteria Corynebacterium glutamicum R (FERM P-18976) under aseptic conditions, and aerobic shaking culture is performed at 33 ° C. for 12 hours. A culture solution was obtained. 50 ml of this seed culture solution was inoculated into the jar fermenter, and main culture was carried out overnight at a temperature of 33 ° C. with an aeration rate of 1 vvm (Volume / Volume / Minute). In order to remove the influence caused by aerobic culture, the culture solution was allowed to stand in a nitrogen gas atmosphere for about 3 hours, and then 200 ml of the culture solution was centrifuged (5000 rpm, 15 minutes) to remove the supernatant. The wet (wet) cells thus obtained were used for the following reaction.

(2) Preparation of reduced reaction culture for reaction:
(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 .7H ₂ O 5 mg A reaction stock solution consisting of 200 μg of biotin, 200 μg of thiamine hydrochloride, and 1000 ml of distilled water was prepared, heated at 120 ° C. for 10 minutes, and immediately dissolved oxygen was removed under reduced pressure conditions (˜3 mmHg) for 20 minutes. The reduction state of the reaction stock solution was confirmed by changing the color tone of the reduction state indicator resazurin (change from blue to colorless) added to the reaction stock solution at the start of decompression. 1000 ml of this reaction stock solution was introduced into a 2 liter glass reaction vessel under a nitrogen atmosphere. This reaction vessel is equipped with a pH adjusting device, a temperature maintaining device, a reaction liquid stirring device in the vessel, and a redox potential measuring device.

(3) Implementation of reaction:
The coryneform bacterial cells (wet cells) prepared after the culture were added to 1000 ml of a reaction stock solution in a reaction vessel under a nitrogen gas atmosphere. Glucose 600 mM was added in portions, the reaction temperature was maintained at 33 ° C., and the pH was controlled at 7.5 using 5 M aqueous ammonia to carry out an organic acid production reaction. After the reaction for 10 hours, the reaction medium was analyzed using liquid chromatography. As a result, 986 mM (105 g / l) of ammonium lactate was produced.

(4) Production of lactic acid by hydrolytic electrodialysis The above-mentioned reaction culture solution was filtered using a microfilter to obtain an ammonium lactate-containing culture solution from which solid substances were separated and removed. When analyzed using ion chromatography, the total concentration of sulfate and phosphate in the ammonium lactate-containing culture solution was 0.06 M, and the potassium concentration was 0.01 M. Next, lactic acid was purified from the ammonium lactate-containing culture solution by electrodialysis. The electrodialyzer used is composed of 6 bipolar membranes (Neocepta BP-1 manufactured by Tokuyama Co., Ltd.) and 5 anion exchange membranes (Neocepta AHA manufactured by Tokuyama Co., Ltd.) having an effective membrane area of 100 cm ² / sheet shown in FIG. A water-splitting electrodialyzer having a dialysis tank composed of (Anode metal and cathode metal use nickel). 800 ml of the above ammonium lactate-containing culture solution is supplied to the base chamber of this apparatus, 800 ml of 0.5 M lactic acid aqueous solution is supplied to the acid chamber, and 1000 ml of 1N sodium hydroxide solution is supplied to the anode chamber and the cathode chamber from the tanks attached to each. As a result of adjusting the cell voltage to 2.5 V and supplying electricity for 1 hour while circulating, 104 g of lactic acid (68 g of conversion from ammonium lactate, conversion rate of 96%) was obtained. The current efficiency was 90%.

(Example 2)
The same reaction as in Example 1 was carried out except that ammonium carbonate was added during the reaction according to the cells and reaction conditions obtained in the same manner as in Example 1. After reaction for 10 hours, the reaction medium was analyzed using liquid chromatography. As a result, ammonium lactate 510 mM (55 g / l) and ammonium succinate 370 mM (56 g / l) were produced.
Next, in the same manner as in Example 1, an organic acid salt-containing culture solution in which solid substances were separated and removed from the reaction culture solution was obtained. When analyzed by ion chromatography, the total concentration of sulfate and phosphate in the culture solution containing organic acid salt was 0.06M, and the potassium concentration was 0.01M. The organic acid salt-containing culture solution was electrodialyzed under the same conditions as in Example 1 to obtain 72 g of lactic acid (conversion amount from ammonium lactate: 36 g, conversion rate: 97%) and succinic acid: 33 g (conversion rate: 98%). The current efficiency was 92%.

(Comparative Example 1)
Ammonium lactate was produced under the same conditions as in Example 1 to obtain a reaction culture solution having an ammonium lactate concentration of 943 mM (101 g / l). After the solid substance was separated and removed, the ammonium lactate-containing culture was analyzed by ion chromatography. The total concentration of sulfate and phosphate was 0.06M, and the potassium concentration was 0.01M. In the electrodialysis apparatus shown in Example 1, the ion exchange membrane of the dialysis tank was changed from an anion exchange membrane to a cation exchange membrane (Neocepta CMB manufactured by Tokuyama Corporation), and 6 bipolar membranes and 5 cation exchange membranes were used. In this configuration, 800 ml of an ammonium lactate-containing culture solution was supplied to the acid chamber, and 800 ml of a 2% aqueous sodium hydroxide solution was supplied to the base chamber from the tank attached to each of them, and energization was started at a current density of 100 mA / cm ² . . The conversion reaction was stopped during the reaction, and the conversion rate after 1 hour was 72%.

(Comparative Example 2)
Ammonium lactate was produced under the same conditions as in Example 1 except that KH ₂ PO ₄ and K ₂ HPO ₄ were additionally added to the reaction medium composition of Example 1 and the respective blending amounts were 2.0 g / l. 962 mM A reaction culture solution containing (103 g / l) ammonium lactate was obtained. After the solid substance was separated and removed, the ammonium lactate-containing culture solution was analyzed by ion chromatography. The total concentration of sulfate and phosphate was 0.08M, and the potassium concentration was 0.04M. The electrodialysis treatment was carried out under the same conditions as in Example 1, but the conversion reaction was stopped during the reaction, and the conversion rate after 1 hour was 77%.

  The purification method of the present invention is useful as an industrial purification method because an organic acid can be easily purified from an organic acid salt (eg, lactate, succinate, etc.) contained in a culture solution.

It is drawing which represented roughly the two-chamber method water-splitting electrodialysis apparatus comprised by a bipolar membrane / anion exchange membrane.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Bipolar membrane, 2 ... Anion exchange membrane, 3 ... Acid chamber, 4 ... Base chamber, 5 ... Anode chamber 6 ... Cathode chamber, 7 ... Salt solution tank, 8 ... Acid solution tank, 9 ... Electrode solution tank,
10 ... Salt solution pump, 11 ... Acid solution pump, 12 ... Electrode solution pump,
13 ... Salt solution piping, 14 ... Acid solution piping, 15 ... Electrode solution piping, 16 ... Electrodialysis tank

Claims (6)

In a method of purifying an organic acid from the culture solution using a water-decomposing electrodialyzer after generating a culture solution containing an organic acid salt by allowing coryneform bacteria to act on sugars in a reaction medium in a culture tank,
(A) The water splitting electrodialysis apparatus is composed of a bipolar membrane and an anion exchange membrane,
(B) The culture solution contains a salt composed of a strong acid, the concentration thereof is 0.03 mol / liter to 0.06 mol / liter , and (C) the alkali metal concentration in the culture solution is 0.03 mol / liter. Less than a liter, and
(D) A method for purifying an organic acid, which comprises introducing a culture solution containing an organic acid salt into a base chamber of the water-splitting electrodialysis apparatus .   The method for purifying an organic acid according to claim 1, wherein the salt comprising a strong acid contains at least one selected from the group consisting of sulfate, nitrate, hydrochloride and phosphate. The method for purifying an organic acid according to claim 1, wherein the alkali metal contains at least one selected from the group consisting of lithium, sodium and potassium.   The method for purifying an organic acid according to claim 1, wherein the cells are separated and removed from the culture solution and then introduced into a water-splitting electrodialysis apparatus.   The method for purifying an organic acid according to claim 1, wherein the coryneform bacterium is Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, or Micrococcus. . The method for purifying an organic acid according to claim 1, wherein the organic acid salt to be produced is selected from succinate and lactate.

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