JP5311398B2 - Method for producing glycerin derivative - Google Patents

Description

  The present invention relates to a method for producing a glycerin derivative by a microbial reaction process, which is characterized by a step of culturing while controlling the pH of a culture solution constant with a glycerin-containing alkaline solution.

  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. Biodiesel fuel (BDF) is obtained by converting triglycerides, which are the main components of fats and oils, into fatty acid methyl esters by a transesterification reaction. The development of effective utilization of glycerin produced as a by-product of this reaction is the key to process development. It has become. Considering the rapid increase in BDF 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.

  Since the transesterification reaction of fats and oils is carried out with an alkaline catalyst such as NaOH or KOH and methanol at the industrial level, the by-product glycerin (crude glycerin) discharged during this reaction is methanol, soap, and plant-derived lignin. In many cases, it contains impurities such as polyphenols and is alkaline. Therefore, in order to use crude glycerin as a raw material, it has been necessary to perform pretreatment at high cost.

Until now, many attempts have been made to produce useful substances from glycerin using a chemical catalytic process (for example, Non-Patent Documents 1 and 2), and glycerin such as epichlorohydrin and propylene glycol (1,2-propanediol). Derivatives are being produced at the industrial level. These chemicals are originally manufactured using propylene derived from fossil fuels, and technological development using glycerin as a raw material has been promoted due to the price reduction due to surplus glycerin. These are mainly used as general-purpose chemicals such as resins and polymer raw materials.
However, in the process of utilizing glycerin by chemical catalyst, it is necessary to use glycerin with a relatively high purity (purity) as a raw material, and it is still expensive to use crude glycerin actually discharged from BDF production etc. It was necessary to perform pretreatment.
In addition, for example, when the price of glycerin is soaring with the increase in oil and fat prices as seen from the end of 2007 to 2008, the production processes of these low-priced general-purpose chemicals are severely costly. Therefore, it is expected to produce a highly functional and high value-added chemical substance by a microbial reaction process (bioprocess).

Many attempts have been made to produce useful substances from glycerin using a bioprocess (for example, Non-Patent Documents 3 and 4), dihydroxyacetone has already been industrialized, and 1,3-propanediol has also been produced at an industrial level. Among these, dihydroxyacetone is used for cosmetics and pharmaceuticals and is known as a functional glycerin derivative. By using the bioprocess in this way, it is also possible to produce a chemical product having high added value from glycerin and having a certain price.
However, as in the case of chemical catalysts, when crude glycerin actually discharged from BDF production, etc. is used as a raw material, impurities in it may have an inhibitory effect on the growth of many microorganisms, and the reaction may not proceed well. Since there are many, it was still necessary to pre-process crude glycerol (for example, nonpatent literature 5). On the other hand, a method for producing D-glyceric acid from glycerin using a microorganism belonging to the genus Gluconobacter from glycerin is also known (see Patent Document 1). The production amount of D-glyceric acid by this method is as follows: It was about 12-57 g / L, which was not satisfactory.

Japanese Patent Publication No. 7-51069

M. Pagliaro, R. Ciriminna, H. Kimura, M. Rossi, C. Della Pina: Angew. Chem. Int. Ed., 46, 2-20 (2007). C.-H. Zhou, J. N. Beltramini, Y.-X. Fan, G. Q. Lu: Chem. Soc. Rev., 37, 527-549 (2008). G. P. da Silva, M. Mack, J. Contiero: Biotechnol. Adv., 27, 30-39 (2009) T. Willke, K. Vorlop: Eur. J. Lipid Sci. Technol., 110, 831-840 (2008). A. ur-Rehman, R. G. S. Wijesekara, N. Nomura, S. Sato, M. Matsumura: J. Chem. Technol. Biotechnol., 83, 1072-1080 (2008).

  The object of the present invention is to solve the above-mentioned problems of the prior art. Specifically, a new technology for inexpensively and efficiently producing useful glycerin derivatives such as glyceric acid and dihydroxyacetone by a bioprocess is constructed. In particular, the present invention intends to provide a new means for producing a glycerin derivative using crude glycerin as a raw material.

  In order to achieve the above-mentioned object, the present inventors have conducted intensive research. As a result, in the process of producing a glycerin derivative by a bioprocess, the production ratio of glyceric acid and dihydroxyacetone is greatly increased depending on the composition of the glycerin solution fed to the medium. It was found that glyceric acid and dihydroxyacetone can be selected and produced simply by selecting a feed solution. In particular, when a glycerin-containing alkaline solution is used as the glycerin solution to be fed, the amount of glycerin acid produced increases dramatically, and the glycerin-containing alkaline solution is used as a crude material to be replicated during the transesterification of fats and oils. It has been found that glycerin can be used without complicated pretreatment, 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) A method for selectively producing glyceric acid or dihydroxyacetone by culturing a microorganism having the ability to produce glyceric acid from glycerin in a glycerin-containing medium, and a glycerin-containing alkaline solution containing no methanol, and A method for selectively producing glyceric acid or dihydroxyacetone, wherein the microorganism is cultured while selecting any one of methanol-containing glycerin solutions and feeding the solution to a medium.

(2) A microorganism having the ability to produce glyceric acid from glycerin is cultured while feeding a glycerin-containing alkaline solution not containing methanol to a glycerin-containing medium, and glyceric acid is collected from the medium, The method according to (1) above.

(3) The method according to (2) above, wherein the culture is performed while maintaining the medium pH at 5 to 7 by feeding a methanol-free alkaline solution containing glyceric acid.

(4) The method according to (3) above, wherein the glycerin-containing alkaline solution not containing methanol is derived from the crude glycerin that is replicated during the transesterification reaction of fats and oils.

(5) The microorganism having the ability to produce glyceric acid from glycerin is cultured while feeding the methanol-containing glycerol solution to the glycerol-containing medium, and dihydroxyacetone is collected from the medium. Production method.

(6) The method according to claim 5, wherein the methanol-containing glycerin solution is alkaline.

(7) The method according to (6) above, wherein the methanol-containing glycerin solution is derived from the crude glycerin that is replicated during the transesterification of the oil.

(8) The microorganism according to any one of (1) to (7) above, wherein the microorganism having the ability to produce glyceric acid from glycerin is a microorganism selected from bacteria belonging to the genus Gluconobacter. The method described.

  According to the present invention, glyceric acid and dihydroxyacetone, which are highly industrially useful as intermediate materials for pharmaceuticals, cosmetics, chemical production, etc., can be produced by simply selecting one of them by changing the feed components to the medium. In addition, the production of these products, especially glyceric acid, is dramatically increased. In addition, according to the present invention, for example, crude glycerin, which is an inexpensive raw material that is by-produced and treated as waste during the transesterification of fats and oils, can be used without complicated pretreatment, and manufactured. Cost reduction can be expected.

It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 1 with time. It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 2 with time. It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 3 with time. It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 4 with time. It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 5 with time. It is drawing which represented each quantity of glycerol, glyceric acid, and dihydroxyacetone in Example 6 with time. 4 is a drawing showing amounts of glycerol, glyceric acid, and dihydroxyacetone in Comparative Example 1 over time.

As the microorganism used in the present invention, any microorganism can be used as long as it can produce glycerin derivatives such as glyceric acid and dihydroxyacetone.
Typically, an acetic acid bacterium belonging to the genus Acetobacter, Glucon acetobacter, or Gluconobacter and having the ability to convert glycerin to glyceric acid can be used. In particular, a gluconobacter bacterium having a high production amount of dihydroxyacetone in addition to glyceric acid is desirable. Specific examples include Gluconobacter albidus , Gluconobacter cerinus , Gluconobacter cerinus , Arthrobacter Furateuri (Gluconobacter frateurii), Gluconobacter-Kondoni (Gluconobacter kondonii), Gluconobacter oxydans (Gluconobacter oxydans), Gluconobacter, Thailand Randy dregs (Gluconobacter thailandicus), other Gluconobacter bacteria belonging to the genus (Gluconobacter sp .) NBRC3259 strain and the like.
Furthermore, as long as it has the capability to convert glycerol into glyceric acid, it is included as long as it is a mutant strain of the grade which belongs to the same genus of the said microorganism as microorganisms used for this invention. 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 of microorganisms for producing glycerin derivatives can be performed using a normal liquid nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, and the like. As the carbon source, for example, glycerin and glucose can be used, and as the nitrogen source, for example, sodium nitrate, ammonium sulfate, ammonium chloride, urea and the like 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.
The glyceric acid produced by the microorganism is a mixture of D-glyceric acid and L-glyceric acid, but is not a racemic mixture of equal amounts. The proportion of D-form and L-form varies depending on the type of microorganism, but the proportion of D-form is high. It is.

  In the present invention, when culturing a microorganism having the ability to produce glyceric acid from glycerin, culturing is performed while intermittently or continuously feeding methanol-containing glycerin-containing alkaline solution or methanol-containing glycerin solution to the medium. If a glycerin-containing alkaline solution containing no methanol is used, glyceric acid is produced more, and if a methanol-containing glycerin solution is used, more dihydroxyacetone is produced. The methanol-containing glycerin solution used in the production of dihydroxyacetone may be a solution in which methanol is added to a glycerin-containing alkaline solution in addition to a solution in which methanol is added to glycerin. The production rate of glyceric acid and dihydroxyacetone depends on the proportion of methanol contained. Therefore, glycerin, dihydroxyacetone, or both can be selectively and efficiently produced by adjusting the presence or absence of methanol or adjusting its concentration.

  The glycerin-containing alkaline solution may be a solution obtained by adding an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, or an alkaline earth metal hydroxide such as calcium hydroxide or magnesium hydroxide to glycerin. However, in the present invention, as the glycerin-containing solution, a crude glycerin solution that is produced as a by-product when the fats and oils are transesterified using an alkali catalyst for the production of a more useful fatty acid ester can be used. This crude glycerin solution is alkaline derived from the catalyst.

Examples of such a crude glycerin solution include a crude glycerin solution by-produced in the production of biodiesel fuel (BDF). For example, when producing fatty acid methyl as biodiesel fuel (BDF), a raw material is used. As the by-product crude glycerin solution is mixed with methanol, but when dihydroxyacetone is selectively produced, the crude glycerin solution containing such methanol Can be used. Moreover, when manufacturing glyceric acid, it removes and uses methanol from the crude glycerol solution containing such methanol, but methanol can be easily distilled off by heating.
Microorganism culture conditions may be performed under temperature conditions where microorganisms can act. On the other hand, the pH of the culture solution is about 3 to 10, preferably about 4 to 8, at the start of the reaction.

As the microorganism grows, an organic acid such as glyceric acid accumulates in the medium as the metabolic reaction proceeds. Therefore, although the pH of the medium gradually decreases, since the optimum growth pH exists for microorganisms, it is desirable to control the pH in the culture tank to be constant around the optimum growth pH. The methanol-containing or non-containing glycerin-containing alkaline solution includes the crude glycerin solution produced as a by-product during the transesterification, and is used intermittently or continuously as a pH regulator to keep the pH of the medium constant. Feeded. The pH of the medium depends on the optimum growth pH of the microorganism, but is preferably in the range of pH 4 to 8, more preferably pH 5 to 7.
In the pH control of such a medium, when a simple alkaline aqueous solution is used and glycerin is not used, the production amounts of glyceric acid and dihydroxyacetone are both reduced (FIGS. 2 and 7).

  The cultured cells of the microorganism 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 recovered cultured cells are usually used as they are as a microorganism source for the production of the next glyceric acid or dihydroxyacetone.

  Isolation and purification of glyceric acid or dihydroxyacetone produced and accumulated in the culture solution can be performed by any method. For example, the reaction solution after removing the cells by centrifugation or the like can be used for various chromatographies. It can be isolated and purified by a separation method by graphy, a separation method by crystallization, a separation method using a membrane, or the like.

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, glycerin and dihydroxyacetone are quantified using a column that analyzes sugar alcohol (SC1011 manufactured by Shodex), and glyceric acid is quantified using a column that simultaneously analyzes sugar and organic acid (SH1011 manufactured by Shodex). Chromatography (HPLC) was performed.

反応液中のpHは6に制御し、pH調整剤として、グリセリン50容量％を含む5M NaOHを使用した。前述した方法による本培養開始から、６日目までに生成したグリセリン酸およびジヒドロキシアセトンを経時的に定量したところ、下記の図１の通りであり、６日間の反応でグリセリン酸量は144 g/lであった。この反応の間、pH調整剤として反応液中に供給されたグリセリンは286 gであった。反応期間中に消費されたグリセリン１モルに対する、生成グリセリン酸は0.82モルであった。 Example 1
Inoculate Gluconobacter frateurii NBRC103465 obtained from a microorganism preservation organization into 5 test tubes containing 5 mL of preculture medium having the composition of 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, and 0.5% MgSO ₄ 7H ₂ O. Then, reciprocal shaking culture was performed at 30 ° C. for 48 hours. Subsequently, the whole amount (25 mL) of the above preculture was inoculated into a 1-liter miniger containing 500 mL of the main culture medium having the composition shown in Table 1. The culture was performed at a temperature of 30 ° C., an aeration rate of 0.5 vvm, and stirring at 500 rpm.

The pH in the reaction solution was controlled at 6, and 5M NaOH containing 50% by volume of glycerin was used as a pH adjuster. The glyceric acid and dihydroxyacetone produced from the start of the main culture by the above-described method until the sixth day were quantitatively determined as shown in FIG. 1 below. The amount of glyceric acid after the 6-day reaction was 144 g / l. During this reaction, glycerin supplied to the reaction solution as a pH adjuster was 286 g. The produced glyceric acid was 0.82 mol per 1 mol of glycerin consumed during the reaction period.

(Example 2)
Gluconobacter frateurii NBRC103465 was inoculated into a test tube containing 5 mL of preculture medium having a composition of 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, and 0.5% MgSO ₄ · 7H ₂ O. Time reciprocal shaking culture was performed. Next, 1.5 mL each of the culture solution was inoculated into three Erlenmeyer flasks containing 30 mL of the same preculture medium, followed by reciprocal shaking culture at 30 ° C. for 24 hours. Subsequently, a total amount (90 mL) of the preculture was inoculated into a 5-liter jar containing 2.5 L of a main culture medium having the composition shown in Table 1. The culture was performed at a temperature of 30 ° C., an aeration rate of 0.5 vvm, and stirring at 500 rpm.
The pH in the reaction solution was controlled at 6, and 200 mL of 5M NaOH containing 50% by volume of glycerol was first used as a pH adjuster, and 7.5M NaOH 200 containing 25% by volume of glycerin was used after all of this alkaline solution was used. mL was used in the order of 100 mL of 10M NaOH after all this alkaline solution had been used. The produced glyceric acid and dihydroxyacetone were quantified as shown in FIG. 2 below. The amount of glyceric acid was 137 g / L after 7 days of reaction. During this reaction, 200 g of glycerin was supplied to the reaction solution as a pH adjuster. The produced glyceric acid was 0.66 mol per 1 mol of glycerin consumed during the reaction period.

(Example 3)
Furthermore, with respect to Gluconobacter frateurii NBRC103465, jar culture was performed in the same manner as in Example 2. However, the culture was started in a 5-liter jar containing 2 L of the main culture medium having the composition shown in Table 1. In addition, the pH of the reaction solution is controlled to 6, and 500 ml of crude glycerin (glycerin 40 wt%) that does not contain methanol discharged from the BDF production process is used as a pH adjuster without pretreatment. After all was used, 150 mL of 10M NaOH was used. The produced glyceric acid and dihydroxyacetone were quantified as shown in FIG. 3 below, and the amount of glyceric acid was 81 g / L after 6 days of reaction. During this reaction, 200 g of glycerin was supplied to the reaction solution as a pH adjuster. The produced glyceric acid was 0.46 mol per 1 mol of glycerin consumed during the reaction period.

Example 4
For Gluconobacter frateurii NBRC103465, jar culture was performed in the same manner as in Example 1. The pH in the reaction solution was controlled at 6, and crude glycerin containing methanol discharged from the BDF production process (glycerin 66 wt%, methanol 30 wt%) was used as a pH adjuster without pretreatment. When the produced | generated glyceric acid and dihydroxyacetone were quantified, compared with FIG. 1 of Example 1, glyceric acid was hardly produced, but the production amount of dihydroxyacetone increased instead (FIG. 4). In the reaction for 2 days, the amount of dihydroxyacetone was 54 g / L. During this reaction, glycerin supplied into the reaction solution as a pH adjuster was 74 g. The produced glyceric acid was 0.68 mol per 1 mol of glycerin consumed during the reaction period.

(Example 5)
For Gluconobacter frateurii NBRC103465, jar culture was performed in the same manner as in Example 4. However, the aeration volume during culture was 1 vvm. When the produced glyceric acid and dihydroxyacetone were quantified, production of dihydroxyacetone was observed as in FIG. 4 of Example 4 (FIG. 5). After 4 days of reaction, the amount of dihydroxyacetone was 55 g / L. During this reaction, glycerin supplied to the reaction solution as a pH adjuster was 0.94 mol of glycerin acid per 1 mol of glycerin consumed during the reaction period of 120 g.

(Example 6)
Next, jar culture was performed in the same manner as in Example 2 for Gluconobacter frateurii NBRC103465. The pH in the reaction solution was controlled at 6, and crude glycerin containing methanol discharged from the BDF production process (glycerin 66 wt%, methanol 30 wt%) was used as a pH adjuster without pretreatment. When the produced | generated glyceric acid and dihydroxyacetone were quantified, compared with FIG. 2 of Example 2, almost no glyceric acid was produced, but the production amount of dihydroxyacetone increased instead (FIG. 6). The amount of dihydroxyacetone after the reaction for 3 days was 60 g / L. During this reaction, 296 g of glycerin was supplied to the reaction solution as a pH adjuster. The produced glyceric acid was 0.74 mol per 1 mol of glycerin consumed during the reaction period.

(Comparative Example 1)
As a comparative example of Example 2, with respect to Gluconobacter frateurii NBRC103465, jar culture was performed in the same manner as in Example 2 using the main culture solution having the medium composition shown in Table 2. The pH in the reaction solution was controlled at 6, and mere 10M NaOH containing no glycerin was used as a pH adjuster. When the produced | generated glyceric acid and dihydroxyacetone were quantified, compared with FIG. 2 of Example 2, the glyceric acid production amount fell (FIG. 7). The amount of glyceric acid increased only to 92 g / L in the reaction for 6 days, and it was shown that a method of adding a glycerin-containing alkaline solution as a pH adjuster is useful. The produced glyceric acid was 0.57 mol per 1 mol of glycerin consumed during the reaction period.

(Comparative Example 2)
As a further comparative example of Example 2, with respect to Gluconobacter frateurii NBRC103465, jar culture was performed in the same manner as in Example 2 using the main culture solution having the medium composition shown in Table 1. The pH in the reaction solution was controlled at 6, and mere 10M NaOH containing no glycerin was used as a pH adjuster. When the produced | generated glyceric acid and dihydroxyacetone were quantified, compared with Example 2, the glyceric acid production amount fell. The amount of glyceric acid increased only to 51 g / L after 6 days of reaction, and it was shown that a method of adding a glycerin-containing alkaline solution as a pH adjuster is useful. The produced glyceric acid was 0.35 mol relative to 1 mol of glycerin consumed during the reaction period.

● ・ ・ ・ Glycerin concentration (g / L), ○ ・ ・ ・ Glyceric acid concentration (g / L), □ ・ ・ ・ Dihydroxyacetone concentration (g / L)

Claims (6)

A method for producing glyceric acid and / or dihydroxyacetone by culturing a microorganism having an ability to produce glyceric acid from glycerin in a glycerin-containing medium, which is selected from bacteria belonging to the genus Gluconobacter, the glycerin-containing medium containing a microorganism having the ability to produce glyceric acid from glycerol, while feeding further glycerin containing organic alkaline solution containing no methanol, and culturing the microorganisms, glyceric acid from the medium or, glyceric acid A method for producing glyceric acid and / or dihydroxyacetone , which is characterized by collecting a product rich in and containing dihydroxyacetone. Methanol by keeping the medium pH at 5-7 by glycerin containing organic alkaline solution feed not containing, characterized by culturing The method of claim 1. The process according to claim 2 , characterized in that the glycerin-containing alkaline solution not containing methanol is derived from the crude glycerin that is replicated during the transesterification of the fats and oils. A method for producing glyceric acid and / or dihydroxyacetone by culturing a microorganism having an ability to produce glyceric acid from glycerin in a glycerin-containing medium, which is selected from bacteria belonging to the genus Gluconobacter, the glycerin-containing medium containing a microorganism having the ability to produce glyceric acid from glycerol, while further feeding methanol-containing glycerol solutions, and culturing the microorganisms, dihydroxyacetone from the medium, or, rich in dihydroxyacetone, glyceric acid A method for producing glyceric acid and / or dihydroxyacetone, which comprises collecting a product containing the glyceric acid. The method according to claim 4 , wherein the methanol-containing glycerin solution is alkaline. The method according to claim 5 , characterized in that the methanol-containing glycerol solution is derived from the crude glycerol that is replicated during the transesterification of the fats and oils.

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