JP2010130908A - Method for producing glycerate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To newly provide a method for efficiently, simply and inexpensively purifying and recovering a high-purity glycerate from a fermented solution of glyceric acid. <P>SOLUTION: A fermented solution of glyceric acid is previously subjected to electrodialysis, and an obtained aqueous solution containing glyceric acid is mixed with an alkaline earth metal salt to be crystallized as an alkali metal glycerate, so that the glycerate is recovered. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method characterized by purification and recovery of glyceric acid as an alkaline earth metal salt from an obtained glyceric acid fermentation broth in a method for producing glycerate by a bioprocess using microorganisms or the like.

  In recent years, biorefinery has attracted attention as a raw material conversion policy that does not depend only on soaring petroleum resources, or as a technical concept that addresses global warming issues such as carbon dioxide reduction. Biomass is carbon neutral among renewable energy, and the introduction of biofuels such as bioethanol and biodiesel is progressing on a global scale. In biodiesel, triglycerides, the main component of fats and oils, are converted into fatty acid methyl esters by transesterification and used as fuel, but the development of effective use of glycerin produced as a by-product of this reaction is the key to process development. . Considering the rapid increase in biodiesel usage in recent years, the solution of the glycerin problem is urgent. Also in the oleochemical industry, the effective use of glycerin is also a big problem because processes using vegetable oils and fats, which are alternative and reproducible resources for petroleum, have been introduced.

So far, many attempts have been made to produce useful substances from glycerin using biological and chemical catalyst systems. Regarding the method for producing glyceric acid by chemical catalyst using glycerin as a raw material, D, L-glycerin has been proposed. Methods for producing acids or salts thereof (for example, Patent Documents 1, 2, and 3) are known.
Among D, L-glyceric acids, optically active D-glyceric acid is used as an amino acid raw material such as L-serine (Patent Document 4), or as an intermediate for producing pharmaceuticals and agricultural chemicals (Patent Document 5), and It is an industrially useful chemical substance used as an additive to a resin or the like or a raw material thereof (Patent Document 6). D-glycerate is also known to accelerate the detoxification of alcohol and acetaldehyde derived from it in vivo (Peter Eriksson et al., (2007), Metabolism, 56: 895- 898), a very promising chemical in the future.
However, since the racemic form of D, L-glyceric acid is produced in the methods using chemical catalysts (Patent Documents 1, 2, and 3), for example, in order to obtain D-glyceric acid, the racemic form is converted into chemical means or microbiology. Therefore, it is necessary to obtain D-glyceric acid by racemic resolution by a conventional means (Patent Document 7). Further, in the method using a chemical catalyst (Patent Documents 1, 2, and 3), tartronic acid and glycolic acid are also generated as byproducts of the oxidation reaction in addition to glyceric acid. In addition, when manufacturing glyceric acid in large quantities, development of a manufacturing method that is more environmentally friendly and cheaper than chemical catalyst methods that use Pt, Au, platinum-based catalysts, etc. (Patent Documents 1, 2, and 3) is required. Accordingly, development of a method for directly, selectively and inexpensively producing D-glyceric acid from a raw material glycerol-containing solution by a bioprocess using microorganisms and the like, and efficient D-glyceric acid ( And the development of a method for purifying the salt) is very important.

So far, as a bioprocess for directly and selectively producing optically active glyceric acid using glycerol as a raw material, the only production of D-glyceric acid using a bacterium belonging to the genus Gluconobacter, which is a kind of acetic acid bacteria. A method (Patent Document 8) is known. In this method for producing D-glyceric acid, as a method for separating and purifying D-glyceric acid from a medium containing D-glyceric acid, a method using an ion exchange resin packed column is used. The salt adsorption method requires regeneration of the ion exchange resin, pH adjustment, and the like, and the operation becomes complicated during scale-up for mass production, which is not satisfactory.

  In addition to the adsorption method using an ion exchange resin, precipitation with an alkaline earth metal salt such as calcium can be considered as a simple method for recovering glycerate from a medium containing glyceric acid (Lee et al., (1998 ), Journal of Membrane Science, 145: 53-66), unlike calcium citrate, which has relatively low water solubility (25 ° C, solubility of about 0.1 g / 100 g water), the solubility of calcium glycerate in water is comparable. (25 ° C., solubility of about 6 g / 100 g water), it cannot be applied at the accumulation concentration of glyceric acid in the medium (10-50 g / L) as currently known.

However, with respect to an aqueous solution of 5 to 20% by weight of an alkaline earth metal glycerate (eg, a salt of magnesium, calcium, strontium, barium, etc.), a water-miscible solvent such as methanol, ethanol and propanol is added to the aqueous solution. It is known that when 1 to 10 times the amount is added, the alkaline earth metal glycerate can be crystallized and recovered (Patent Document 9).
Although this method uses a solvent, it is considered to be a very useful method because of the relatively simple purification method of glycerate. However, although this method is suitable as a method for recovering glycerate with relatively few impurities produced by chemical synthesis, it is actually adapted to a glycerate-containing medium obtained by fermentation using microorganisms. In addition, the alkaline earth metal glycerate has a problem that the purity is low and the recovered amount is small.

On the other hand, organic acid separation and recovery technology using electrodialysis has been studied extensively in recent years because it has relatively simple equipment and processes and does not involve the production of by-products, and has been introduced into some industrial processes. Yes. The electrodialysis method includes desalting electrodialysis in which an electrolyte such as an organic acid salt and a non-electrolyte can be separated to concentrate or remove the electrolyte, and an organic acid salt such as an organic acid salt is generated by protons and hydroxide ions generated by the decomposition of water. There is hydrolytic electrodialysis that produces acid and alkali from salts. However, there has been no report on the technology for separating and recovering glyceric acid and glycerate using electrodialysis so far, so that an efficient pretreatment method for a medium containing glyceric acid can be established using electrodialysis. I couldn't have predicted at all.
JP-A-5-331100 Special Table 2004-529894 JP 60-226842 A Japanese Unexamined Patent Publication No. 3-91489 Special Table 2006-507268 JP 2004-67725 A Japanese Unexamined Patent Publication No. 1-225486 Japanese Patent Publication No. 7-51069 JP-A-5-339200

  An object of the present invention is to eliminate the above-mentioned problems of the prior art, and specifically, to construct a new technology for efficiently producing glycerate by a bioprocess using microorganisms. In particular, the present invention intends to provide a new means for purifying and recovering high-purity glycerate from glyceric acid fermentation broth efficiently, simply and inexpensively.

  In order to achieve the above-mentioned object, the present inventors have conducted intensive research, and as a result of combining electrodialysis and crystallization methods in the step of recovering glyceric acid from the glyceric acid fermentation broth as an alkaline earth metal salt. The inventors have found that not only the purity is improved but also the amount of recovered alkaline earth metal glycerate is actually increased, and the present invention has been completed.

That is, the present invention has been completed by obtaining the above knowledge, and specifically, is as follows.
(1) The glyceric acid fermentation broth is subjected to electrodialysis, and after mixing an alkaline earth metal salt with the obtained concentrated glyceric acid solution, a water-miscible solvent is added and mixed to obtain an alkaline earth metal salt of glyceric acid. A method for producing an alkaline earth metal glycerate, characterized by crystallization and recovery.
(2) The method according to (1) above, wherein the glyceric acid fermentation broth is obtained by culturing a microorganism having glyceric acid-producing ability in a glycerol-containing culture broth.
(3) The method according to (2) above, wherein the cultivation of the microorganism having the ability to produce glyceric acid is performed while controlling the pH of the culture solution to be equal to or higher than the pKa value (3.55) of glyceric acid.
(4) a microorganism having a glycerin acid-producing ability, characterized in that the Gluconobacter (Gluconobacter) genus Acetobacter (Acetobacter) genus, or a microorganism selected from Gluconacetobacter (Gluconacetobacter) genus bacteria, The method according to any of (2) or (3) above.

  According to the present invention, a glycerol salt having a high industrial use value as an intermediate raw material for manufacturing pharmaceuticals, cosmetics, chemicals, etc., from a microbial culture solution containing a large amount of impurities, a highly pure, efficient, simple and inexpensive means. Therefore, it is possible to reduce the downstream cost.

In the present specification, the glyceric acid fermentation broth means a culture broth obtained by culturing microorganisms capable of producing glyceric acid and performing glyceric acid fermentation. In addition to glyceric acid, various metabolites such as organic acids and medium components Etc.
Any microorganism can be used as the microorganism used for the glyceric acid fermentation as long as it is a microorganism capable of producing glyceric acid.
Typically, an acetic acid bacterium belonging to the genus Acetobacter, Glucon acetobacter, and Gluconobacter and having the ability to convert glycerol into glyceric acid can be used. Specific examples of acetic acid bacteria include Acetobacter aceti , Acetobacter cibinongensis , Acetobacter estunensis , Acetobacter indonesiensis , Acetobacter lovaniensis , Acetobacter orientalis , Acetobacter orleanensis , Acetobacter pasteurianus , Acetobacter pasteurianus , Acetobacter peroxyd ( Acetobacter syzygii ), Acetobacter tropicalis , other Acetobacter sp. NBRC3283 strains and the like. Specific examples of the bacterium belonging to the genus Gluconacetobacter include Gluconacetobacter europaeus , Gluconacetobacter hansenii , Gluconacetobacter liquefaciens , and Gluconacetobacter liquefaciens. -Obodies ( Gluconacetobacter oboediens ), gluconacetobacter xylinus ( Gluconacetobacter xylinus), other gluconeacetobacter genus bacteria ( Gluconacetobacter sp.) NBRC14815 strain etc. are mentioned. On the other hand, specific examples of Gluconobacter bacteria include Gluconobacter albidus , Gluconobacter cerinus , Gluconobacter frateurii , Gluconobacter frateurii , and Gluconobacter frateurii. ( Gluconobacter kondonii ), Gluconobacter oxydans , Gluconobacter thailandicus , other Gluconobacter sp. NBRC3259 strains, and the like.
Although most of the microorganisms produce D-glyceric acid, Gluconobacter sp. NBRC3259 strain produces both D-glyceric acid and L-glyceric acid. The glyceric acid in the fermentation liquid can be applied regardless of the mixture with D-glyceric acid or L-glyceric acid.
Furthermore, the microorganisms used in the glyceric acid fermentation of the present invention include mutant strains that belong to the same genus of the above microorganisms as long as glyceric acid can be produced. This may be due to spontaneous mutation, or artificial addition or deletion, substitution, etc. of a gene by applying some physical or chemical treatment such as ultraviolet irradiation or chemical mutagen treatment. It may be triggered.

  Cultivation for producing glyceric acid can be performed using a normal liquid nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, and the like, but glycerol is preferably added to the medium. As the carbon source, for example, glucose, etc., in addition to the above-mentioned glycerol, and as the nitrogen source, for example, sodium nitrate, ammonium sulfate, ammonium chloride, urea, etc. can be used alone or in combination. Further, as the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, or the like can be used. In addition to these, nutrients such as yeast extract, peptone, polypeptone, meat extract, corn steep liquor and the like can be appropriately added to the medium as necessary, and these nitrogen-containing organic substances can be substituted for the nitrogen source.

As the culture conditions for the microorganisms used in the present invention, the temperature conditions may be those under which the microorganisms can act. On the other hand, the pH of the culture solution is about 3 to 10 at the start of the reaction, preferably pH 4 The range near ˜8 is good, and it can be carried out by adding sodium hydroxide, potassium hydroxide or the like in order to adjust the pH in the fermenter to a certain range during the reaction.
In particular, the pH value is preferably a pKa value of glyceric acid, that is, pH 3.55 or more, and specifically, it is preferably adjusted to pH 4 to 8, more preferably pH 4.5 to 7.
By using such pH conditions of the culture solution, glyceric acid is ionized (anion) in the fermentation solution, which is advantageous in that electrodialysis in the next step can be performed as it is.
However, after completion of the glycerin fermentation, the pH of the fermentation broth may be adjusted so as to be equal to or higher than the pKa value of glyceric acid.

  The cultured cells are separated and collected from the culture obtained as described above. The method for separating / recovering the cultured cells is not particularly limited, and known methods such as centrifugation and membrane separation can be used. The collected cultured cells are usually used as they are as a microorganism source for the production of the next glycerate.

In the present invention, the glyceric acid fermentation broth obtained by appropriately separating and removing cells and the like from the culture in this manner is subjected to an electrodialysis treatment.
The electrodialysis apparatus used in the present invention is a normal desalting electrodialysis apparatus including an anion exchange membrane 1 and a cation exchange membrane 2 exemplified in FIG. This electrodialyzer has a structure in which a cation exchange membrane 2, an anion exchange membrane 1, and a cation exchange membrane 2 are arranged in this order between an anode and a cathode to form a salt recovery chamber 3 and a desalting chamber 4. Yes. In such an electrodialysis apparatus, the organic acid salt that has escaped from the desalting chamber 4 cannot reach the anode due to the voltage applied to both electrodes, and remains in the salt recovery chamber 3. During this time, an aqueous solution such as sodium nitrate as an electrode solution is supplied to the electrode chamber 5 by a pump and circulated.

In addition, as the anion recovery type cartridge including an anion exchange membrane / cation exchange membrane used in the present invention, a commercially available membrane cartridge, for example, a cartridge AC-122-10 manufactured by Sanactis Co., Ltd. or the like is used. Can do. In the electrodialysis according to the present invention, generally, the measurement voltage is 3.6 to 4.7 V (electrode voltage is about 3.5 V), the current density is 1 to 100 mA / cm ² , and the solution temperature is in the range of 5 to 40 ° C. It is preferable to carry out with.

Under such electrodialysis conditions, when the glyceric acid fermentation broth is supplied to the electrodialysis apparatus equipped with the anion exchange membrane, non-electrolytes such as glycerin and glucose added as a carbon source remain in the desalting chamber 4, The glycerate is concentrated in the salt recovery chamber 3 (FIG. 1). According to such electrodialysis, an aqueous solution containing about 1 to 20% by weight of glyceric acid in which considerable impurities are removed and concentrated is obtained.
However, salts such as sulfate ions, nitrate ions, phosphate ions, etc. contained in the culture solution have also moved to the salt recovery chamber 3, and in the present invention, the glyceric acid recovered from the salt recovery chamber is further contained. In an aqueous solution, an alkaline earth metal salt is used to crystallize glyceric acid as an alkaline earth metal salt.

  That is, an alkaline earth metal salt is added in an equimolar amount or more with glycerate to the glyceric acid-containing aqueous solution from which the impurities have been removed and concentrated. Examples of the alkaline earth metal in this case include salts of magnesium, calcium, strontium, barium and the like.

  Subsequently, a water-miscible solvent is added to and mixed with the glycerate-containing aqueous solution, and an alkaline earth metal salt of glyceric acid is crystallized by using a filtrate obtained by filtering off insoluble matter that immediately precipitates. Thereafter, the alkaline earth metal glycerate is recovered by filtration or drying. In this case, the amount of the solvent that can be mixed with water is preferably 1 to 8 times the volume, more preferably 3 to 5 times the volume of water. Examples of the water miscible solvent include alcohols such as methanol, ethanol and propanol.

  It is also possible to produce glyceric acid by using an aqueous solution in which the obtained alkaline earth metal glycerate is dissolved in a bipolar membrane electrodialyzer or adding a strong acid.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
In the following Examples, glyceric acid was quantified by high performance liquid chromatography (HPLC) using a column (SH1011 manufactured by Shodex) that simultaneously analyzes sugar and organic acid.

Example 1
First, a model solution of glyceric acid fermentation broth was prepared, and glycerate was purified according to the present invention. The model solution was prepared by dissolving distilled water in which 40 g of glyceric acid, 100 g of glycerin, 0.9 g of KH ₂ PO ₄ , 0.1 g of K ₂ HPO _{4, 1} g of MgSO ₄ · 7H ₂ O and 25 g of yeast extract (manufactured by DIFCO) with a pH of 5 After being adjusted to 1 L, the volume up to 1 L was used.

Glycerate was concentrated by electrodialysis using 10 mL of the model solution having the above composition. The electrodialysis apparatus used is a tabletop electrodialysis apparatus Micro Acylizer S1 (manufactured by Astom Co., Ltd.) equipped with an anion recovery type cartridge AC-122-10 (manufactured by San Actis Co., Ltd.) (FIG. 1). Same configuration). 0.5 M NaNO ₃ was used as the electrode solution, and 2 mL of pure water was used as the salt recovery solution, and the end of electrodialysis was set when the conductivity of the sample solution reached 1000 μS. Data on voltage, current, conductivity, quantity of electricity, and milliequivalents were measured and recorded every minute. The result is as shown in FIG. 2, and it was shown that glycerate was recovered and concentrated even in the model fermentation broth containing impurities. The recovery rate of glycerate from the model solution was 99.4% by electrodialysis for 78 minutes, and the energy required to recover 1 kg of glyceric acid at this time was 0.313 kWh / kg.

(Example 2)
Next, glycerate was concentrated by electrodialysis using an actual glyceric acid fermentation broth by acetic acid bacteria Acetobacter tropicalis NBRC16470. NBRC16470 strain was precultured with 5% glucose, 0.5% yeast extract, 0.5% polypeptone, 0.1% MgSO ₄ · 7H ₂ O (5 mL / tube) And reciprocally shake culture at 30 ° C. for 48 hours. Then, glycerol 15 wt%, yeast extract 2% by weight, KH ₂ PO ₄ 0.09 wt%, K ₂ HPO ₄ 0.01 _{wt%, MgSO 4 · 7H 2 O} 0.1 wt% of the medium (pH 7) 500 mL 25 mL of the above culture solution was inoculated into a 1 L mini jar containing. The culture was performed at a temperature of 30 ° C., an aeration rate of 1 vvm, stirring at 500 rpm, and 6 days. During the culture period, sodium hydroxide was added by automatic control so that the pH did not drop below pH 5.

  The reaction culture solution was centrifuged to obtain a glycerate-containing culture solution from which solid substances such as bacterial cells were separated and removed. The amount of glycerate was determined to be 14.6 g / L. 10 mL of this glycerate-containing culture solution was subjected to electrodialysis under the same conditions as in Example 1. The result is as shown in FIG. 3, and it was shown that glycerate was concentrated also in the actual glyceric acid fermentation broth. The recovery rate was 99.4% after 80 minutes of electrodialysis, and the energy required to recover 1 kg of glyceric acid at this time was 0.24 kWh / kg.

(Example 3)
Next, using a glyceric acid fermentation broth of acetic acid bacteria Gluconobacter NBRC3259 strain, the glyceric acid calcium salt was purified by a combination of electrodialysis and crystallization. The NBRC3259 strain was precultured in the same manner as in Example 2. Subsequently, glycerol 15% by weight, yeast extract 0.1% by weight, polypeptone 2.5% by weight, KH ₂ PO ₄ 0.09% by weight, K ₂ HPO ₄ 0.01% by weight, MgSO ₄ .7H ₂ O 25 mL of the above culture solution was inoculated into a 1 L miniger containing 500 mL of 0.1% by weight medium (pH 7). The culture was performed at a temperature of 30 ° C., an aeration rate of 1 vvm, stirring at 500 rpm, and 6 days. During the culture period, sodium hydroxide was added by automatic control so that the pH did not drop below pH 5.

  The reaction culture solution was centrifuged to obtain a glycerate-containing culture solution from which solid substances such as bacterial cells were separated and removed. The amount of glycerate was determined to be 25 g / L. When 500 mL of this glycerate-containing culture solution was subjected to electrodialysis under the same conditions as in Example 1, 48 mL of a 97.9 g / L glycerate-containing aqueous solution was obtained.

To the glycerate-containing aqueous solution obtained above, 3.3 g of CaCl ₂ .2H ₂ O was mixed, and about 200 mL of ethanol was further added and mixed. Immediately precipitated insoluble matter was removed by filtration, and the filtrate was allowed to stand at 4 ° C. to precipitate calcium glycerate. The upper liquid was removed and dried to obtain 3.1 g of calcium glycerate. When the obtained calcium glycerate was dissolved in pure water and the purity was examined by HPLC, it was almost 100%.

Example 4
Next, using the glyceric acid fermentation broth of acetic acid bacteria Gluconobacter NBRC3259 strain, the crystallization method was applied to the sample that had been subjected to the electrodialysis method and the sample that had not been electrodialyzed as a comparison. The recovery efficiency of glyceric acid calcium salt was compared. The NBRC3259 strain was precultured in the same manner as in Example 2, and then cultured in a 1 L miniger with the same medium and conditions as in Example 3.

  The reaction culture solution was centrifuged to obtain a glyceric acid-containing culture solution from which solid substances such as bacterial cells were separated and removed. When the amount of glyceric acid was quantified, it was 22 g / L.

50 mL of each culture solution was taken in two sample bottles, and 50 mL of one culture solution was subjected to electrodialysis under the same conditions as in Example 1. The sample solution after electrodialysis was diluted with pure water to obtain about 50 mL of a 22 g / L glycerate-containing solution (hereinafter referred to as dialysate). 0.77 g of CaCl ₂ · 2H ₂ O was mixed with the culture solution and dialysate having the same concentration and the same volume, and further 150 mL of ethanol was added and mixed.

  Immediately precipitated insoluble matter was removed by filtration, and the dry weight of the insoluble matter separated by filtration was 1.14 g in the culture solution and 0.624 g in the dialysis solution. Since these are insoluble in water, they are insolubles other than calcium glycerate. This result clearly shows that the sample (dialysis solution) subjected to electrodialysis has less impurities than the fermentation solution.

  On the other hand, the glyceric acid calcium salt was crystallized by allowing the filtrate to stand at 4 ° C. The upper solution was removed and dried to obtain 0.381 g and 0.511 g of glyceric acid calcium salt from the culture solution and dialysate, respectively. This result clearly shows that the sample (dialysis solution) subjected to electrodialysis has a better recovery rate of calcium glycerate than the fermentation solution.

  Further, when looking at the crystals of the obtained calcium glycerate, the crystals obtained from the fermentation broth have a flesh color and are still less pure than the crystals obtained from the dialysate as shown in FIG. It has been shown. When the purity was actually examined by HPLC, the purity of the dialysate crystals was almost 100%, whereas the fermentation crystal was detected with an unidentified substance peak. This result shows that by applying electrodialysis, impurities in the culture solution are clearly removed and the recovery rate of glycerate by crystallization is improved.

BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which represented roughly the desalting electrodialysis apparatus comprised by an anion exchange membrane / cation exchange membrane. It is drawing which showed the collection | recovery efficiency of the glycerol salt by the electrodialysis using the model solution of glycerol fermented liquor. It is drawing which showed the collection | recovery efficiency of the glycerol salt by the electrodialysis using an actual glycerol acid fermented liquor. It is a photograph of glyceric acid calcium salt obtained from glyceric acid fermentation broth (left) and glyceric acid fermentation broth (right) subjected to electrodialysis.

Explanation of symbols

1. Anion exchange membrane 2. Cation exchange membrane Salt recovery room 4. 4. Desalination chamber Electrode chamber 6. 6. Sample liquid pump Electrode solution pump 8. Salt recovery syringe

Claims (4)

  The glyceric acid fermentation broth is subjected to electrodialysis, and after mixing an alkaline earth metal salt with the resulting concentrated glyceric acid solution, a water-miscible solvent is added and mixed to crystallize the alkaline earth metal salt of glyceric acid. A method for producing an alkaline earth metal glycerate, characterized in that it is collected.   2. The method according to claim 1, wherein the glyceric acid fermentation broth is obtained by culturing a microorganism having glyceric acid-producing ability in a glycerol-containing culture broth.   The method according to claim 2, wherein the culture of the microorganism having the ability to produce glyceric acid is performed while controlling the pH of the culture solution to be equal to or higher than the pKa value (3.55) of glyceric acid. Microorganism having glycerate producing ability, characterized in that Gluconobacter (Gluconobacter) genus Acetobacter (Acetobacter) genus, or a microorganism selected from Gluconacetobacter (Gluconacetobacter) genus bacteria, according to claim 2 Or the method in any one of 3.