JP2015181387A - D-lactic acid-producing microorganisms and production methods thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism that produces D-lactic acid in a high concentration and at a high sugar-based yield, and to provide a method for efficiently producing D-lactic acid using the microorganism of the genus Erwinia.SOLUTION: Provided is a D-lactic acid-producing microorganism named Erwinia M3 and deposited as NITE P-01579 or a method for fermentation-producing D-lactic acid using the microorganism and a culture medium not containing molasses being a colored component or a culture medium having yeast extract at a concentration of less than 0.1%, where pH and/or redox potential is maintained within a certain range.

Description

  The present invention relates to a D-lactic acid-producing microorganism and a method for producing D-lactic acid using the same.

Biodegradable resins produced from biomass are carbon neutral and can be produced without using petroleum as a raw material, and are therefore expected to play a major role in solving environmental and energy / resource problems.
Among the biodegradable resins, polylactic acid, which uses lactic acid as a raw material, consumes less energy in the manufacturing process than other biodegradable resins, and has high performance as a plastic. Be expected.

However, the use of polylactic acid resin composed only of poly-L-lactic acid is limited because its mechanical strength and heat resistance are insufficient. On the other hand, stereocomplex polylactic acid obtained by melt-kneading poly-L-lactic acid and poly-D-lactic acid is excellent in heat resistance and other physical properties, and therefore is expected to be used in a wide range of applications.
In order to obtain stereocomplex polylactic acid, L-lactic acid and D-lactic acid are required as raw materials. Although many production techniques have been established and L-lactic acid has been produced at low cost on a commercial basis, it cannot be said that a method for producing highly optically active D-lactic acid at low cost has been sufficiently established. The low-cost fermentation production technology is extremely important in the practical application of polylactic acid resin.

  As industrial D-lactic acid fermentation methods, methods using recombinant microorganisms and methods using non-recombinant microorganisms have been studied. As an example of research using recombinant microorganisms, Ishida, N. et al., Saccharomyces cerevisiae subsp. It was disclosed that when D-lactic acid dehydrogenase gene derived from mesenteroides NBRC3426 strain was introduced to produce D-lactic acid from glucose, D-lactic acid having an optical purity of 99.9% was produced (Non-patent Document 1). Patent Document 1). However, it was a problem to require expensive components in the culture medium.

On the other hand, as a method using a non-recombinant microorganism, a microorganism belonging to the genus Erwinia can ferment D-lactic acid only with glucose and an inorganic salt without using a coloring medium component such as yeast extract or polypeptone with glucose as a carbon source. It is disclosed (Patent Document 2). Since no coloring medium component is used, the wastewater treatment cost is expected to be reduced.
However, the genus Erwinia ferments D-lactic acid at a low cost with a fermentation time of a constant quality using a fermentation medium with a constant quality without adding colored components such as molasses and yeast extract to the medium. However, it was a problem that the yield to sugar was low and the concentration of D-lactic acid was low.

JP 2005-102625 A JP 2009-291132 A

Ishida, N .; et al, J. et al. Biosci. Bioeng. , 101 (2), 172-177 (2006)

  An object of the present invention is to solve the above-mentioned conventional problems and to provide a microorganism having a high yield to sugar and a high concentration of D-lactic acid. Furthermore, it is providing the D-lactic acid manufacturing method using the said microorganisms.

The inventors of the present invention have conducted extensive studies on microorganisms with high sugar yield and high D-lactic acid production concentration. By mutating microorganisms belonging to the genus Erwinia, colored components such as waste molasses and yeast extract can be obtained. It has been found that it is possible to fermentatively produce D-lactic acid with high efficiency using a fermentation medium having a constant quality without adding to the medium and with a fermentation time equivalent to the conventional one.
Further, as a result of further studies, formic acid, which is a by-product of lactic acid fermentation, is produced by controlling the oxidation-reduction potential (ORP) in the culture solution when producing D-lactic acid by fermentation using the microorganism. It was found that the production amount of was reduced. Moreover, it discovered that the yield to sugar and the production | generation of D-lactic acid increased by controlling pH in a culture solution.

That is, according to the present invention,
1. A microorganism named Erwinia M3 and deposited as NITE P-01579 is provided.
The present invention also includes the following inventions 2 to 4.
2. Using the microorganism described in 1 above and a medium that does not contain molasses, which is a colored component, or a medium that has a yeast extract concentration of less than 0.1%, the pH during culture is within the range of 7.0 to 8.0. A method for producing D-lactic acid while controlling.
3. Using the microorganism described in 1 above and a medium that does not contain molasses, which is a colored component, or a medium that has a yeast extract concentration of less than 0.1%, an oxidation-reduction potential (ORP) during culture is -250 mV to -100 mV. A method for producing D-lactic acid while controlling within the range.
4). Using the microorganism described in 1 above and a medium that does not contain molasses, which is a colored component, or a medium that has a yeast extract concentration of less than 0.1%, the pH during culture is within the range of 7.0 to 8.0. A method for producing D-lactic acid while controlling an oxidation-reduction potential (ORP) within a range of −250 mV to −100 mV.

  According to the microorganism of the present invention, fermentation production with a high sugar yield and high D-lactic acid production concentration can be achieved using a medium that does not contain molasses, which is a colored component, or a medium that has a yeast extract concentration of less than 0.1%. It can be carried out. Further, by controlling the pH and / or redox potential in the culture solution during the production of D-lactic acid, it is possible to provide a fermentation production method having a high yield to sugar and a high concentration of D-lactic acid.

It is the bar graph showing the D-lactic acid production density | concentration of each mutant about 10 types of mutant strains (TDLNo.1-10) of the genus Erwinia used in the Example of this application.

Hereinafter, the present invention will be described in detail.
<Microorganism named Erwinia M3 and deposited as NITE P-01579>
The microorganism of the present invention is a mutant strain of a microorganism belonging to the genus Erwinia, and has been deposited as a new strain on March 25, 2013 with the Patent Microorganism Depositary, National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation ( The accession number is NITE P-01579), which is readily available.

Erwinia M3 that specifically produces D-lactic acid of the present invention is obtained by mutating a microorganism belonging to the genus Erwinia that is also present in the soil and other places, and Erwinia isolated from the natural environment. It can be obtained by performing mutation using a genus by a generally known method.
Examples of mutation methods include mutagenesis using chemical mutagens such as alkylating agents and physical mutagens such as X-rays, gamma rays, and neutrons, and mutation introduction by genetic manipulation.

The Erwinia M3 of the present invention preferably uses mutagenesis with ethyl methanesulfonic acid from the viewpoint of improving the yield to sugar and the concentration of D-lactic acid production.
Here, the yield to sugar is a value obtained from the following formula (i), and the D-lactic acid production concentration is a value obtained from the area obtained by analyzing the fermentation broth using HPLC.
[Equation 1]
Yield to sugar (%) = [{(D-lactic acid concentration after fermentation) − (D-lactic acid concentration before fermentation)} / {(glucose concentration after fermentation−glucose concentration before fermentation)}] × 100 ( i)

<Method for producing D-lactic acid>
The method for producing D-lactic acid of the present invention can be generally performed by a known method. Examples of the production method include batch culture, fed-batch culture, continuous culture method, and the like. Here, fed-batch culture refers to a culture method in which a medium is fed continuously or intermittently to a container during the culture, and the medium is not removed from the container until the end of the culture. Continuous culture refers to a method in which a medium is fed continuously or intermittently to a container during culture and the medium (usually equivalent to the medium to be fed) is extracted from the container.

  Generally well-known things can be used as a carbon source in the culture medium used for fermentation production. Examples thereof include sugars such as glucose, fructose, galactose, sucrose, inulin, maltose, arabinose, cellobiose, lactose, melibiose, raffinose, trehalose, salicin, mannitol, sorbitol, mannose, starch, starch hydrolyzate, and molasses.

In addition, glucose recovered using cellulase from cellulose components contained in algae, wood, waste, and the like can be used as a carbon source in a fermentation production medium.
As a medium composition, generally known ones can be used. For example, a medium that has been sterilized by adding 20 to 100 g / l of a carbon source component to a W medium composed of ammonium sulfate, phosphate, and inorganic salt can be used.
Moreover, you may use the culture medium which added phosphate and urea to tap water, and added 20-100 g / l of carbon sources.
As the sterilization treatment, autoclave sterilization or filtration sterilization can be used.

  The production of D-lactic acid is performed, for example, by culturing a mutant strain of a microorganism belonging to the genus Erwinia using a W medium containing glucose 20 to 100 g / l for 24 hours at 30 ° C. It can be carried out by adding about 5% of the volume and fermenting culture. Further, the cells are cultured for 24 hours at 30 ° C., and the bacterial cells are mixed so as to have a glycerol concentration of 10 to 20%, and stored frozen at −80 ° C. or in liquid nitrogen, and can be used as inoculum.

The temperature during fermentation is preferably controlled in the range of 20 ° C. to 50 ° C., and more preferably 25 ° C. to 40 ° C. from the viewpoint of D-lactic acid production rate, yield to sugar, and D-lactic acid production concentration. More preferably, the temperature is from 35C to 35C. When it is lower than 20 ° C., D-lactic acid production by microorganisms may hardly occur. On the other hand, if it is higher than 50 ° C., it may cause a problem in the growth of microorganisms.
Although it may be stirred or unstirred during the fermentation, stirring is preferred from the viewpoints of D-lactic acid production rate, yield to sugar, and D-lactic acid production concentration.

  The mutant strain of the microorganism belonging to the genus Erwinia of the present invention, in particular, the microorganism deposited under the deposit number NITE P-01579, controls the pH and / or redox potential in the culture solution during D-lactic acid fermentation. Fermentative production with a high sugar yield and a high concentration of D-lactic acid can be performed.

  It is preferable to control pH in the culture solution at the time of fermentation of this invention in the range of 7.0-8.0. From the viewpoint of sugar yield and D-lactic acid production concentration, it is more preferable to control in the range of 7.4 to 7.7. The pH in the culture solution can be controlled by neutralizing the produced D-lactic acid, and a generally known method can be used for neutralization. For example, the method of adding alkaline components, such as ammonia, calcium carbonate, sodium hydroxide, to a culture solution is mentioned. The pH in the culture solution is generally a value that is desired to be measured by a known method, and is a value that is confirmed with a pH meter or pH test paper.

  The oxidation-reduction potential in the culture solution during fermentation of the present invention is preferably controlled in the range of −250 mV to −100 mV. From the viewpoint of sugar yield and D-lactic acid production concentration, it is more preferable to control in the range of -200 mV to -100 mV. If it is lower than −250 mV, the production of by-products during fermentation, particularly formic acid, increases, and the yield to sugar may decrease. On the other hand, if it is higher than −100 mV, the sugar component is consumed for growth due to the characteristics of the cells, and the D-lactic acid production concentration may be lowered. The oxidation-reduction potential in the culture solution can be adjusted by aeration of air or oxygen, carbon dioxide and nitrogen. The oxidation-reduction potential in the culture can be measured by a generally known method using a noble metal such as platinum or carbon as an indicator electrode and a silver / silver chloride electrode as a counter electrode.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. Each physical property was measured by the following methods.

(1) D-lactic acid, glucose, formic acid concentration, D-lactic acid production concentration:
The concentration of lactic acid, glucose and formic acid, and the D-lactic acid production concentration were measured under the following conditions using HPLC, and each concentration was determined from the area.
HPLC: GL-7410 manufactured by GL Sciences Inc.
Analysis column: Fermentation monitoring column (manufactured by Bio Rad)
Flow rate: 0.8 ml / min
Temperature: 70 ° C
Developing solvent: 1 mM sulfuric acid Detection: A210 and differential refraction Sample amount: 10 μl

(2) Sugar yield:
The sugar yield was determined from the following formula (ii).
[Equation 2]
Yield to sugar (%) = [{(D-lactic acid concentration after fermentation) − (D-lactic acid concentration before fermentation)} / {(glucose concentration after fermentation−glucose concentration before fermentation)}] × 100 ( ii)

(3) Optical purity of lactic acid:
The optical purity of lactic acid was measured under the following conditions using HPLC, and determined from the following formula (iii) using the area.
HPLC: GL-7410 manufactured by GL Sciences Inc.
Analytical column: Sumichiral OA-5000 (Sumitomo Chemical Co., Ltd.)
Flow rate: 1 ml / min
Temperature: 40 ° C
Developing solvent: 1 mM CuSO4 (0.22 μ filtration)
Detection: A254
Sample volume: 10 μl
[Equation 3]
Optical purity (%) = {(D−L) / (D + L)} × 100 (iii)

(4) The amount of cells in the culture solution:
The amount of bacterial cells in the culture solution was determined by measuring the turbidity (OD) at an absorbance of 600 nm using a spectrophotometer (U2000, manufactured by Hitachi High-Technologies Corporation).
The pH and redox potential were controlled by the following method.

(5) pH control:
Control of pH in the culture solution was performed using 28% aqueous ammonia (Nacalai Tesque) as a neutralizing agent and “DJ-1023P” (manufactured by Biot) as a pH controller.

(6) Control of redox potential (ORP):
Control of the oxidation-reduction potential in the culture solution was performed by using an ORP control device “DJ-1083” (manufactured by Biot) and adding compressed air (5 L / min) so as to obtain a desired ORP.

Hereinafter, mutant strains of microorganisms belonging to the genus Erwinia used in this example will be described.
A mutant strain of a microorganism belonging to the genus Erwinia was obtained by mutation treatment according to the following method.

[Preparation of mutant strain]
Wild type Erwinia sp. 2% glycerol-containing W medium (ammonium sulfate 2 g / l, magnesium sulfate 0.25 g / l, calcium chloride dihydrate 0.015 g / l, sodium chloride 0.5 g / l, sodium hydrogenphosphate dodecahydrate 14 0.3 g / l, potassium hydrogen phosphate 5.4 g / l, zinc sulfate heptahydrate 2 mg / l, ammonium molybdate tetrahydrate 0.15 mg / l, copper sulfate pentahydrate 0.2 mg / l, (Including cobalt chloride 0.4 mg / l, manganese sulfate pentahydrate 1.5 mg / l), and shaking culture was performed at 30 ° C. and 150 rpm for 24 hours.
After culturing, 1 ml of a culture solution containing microbial cells was added to a 1.5 ml tube, collected using a centrifuge, and washed twice with distilled water. After washing, ethyl methanesulfonic acid (Nacalai Tesque) was added to a final concentration of 0.3% and 3%, and mutation treatment was performed at room temperature for 30 minutes and 60 minutes.

After the mutation treatment, the cells were washed twice with sterilized water and suspended in 1 ml of sterilized water. 100 μL of the suspended bacterial cell solution was applied to 2% glucose-containing W medium and cultured at 30 ° C. for 24 hours. A D-lactic acid fermentation test was performed for 72 hours on a 3 ml test tube scale using colonies grown on the plate, and a fermentation test using a 20 ml flask scale was further performed for 72 hours for strains with high D-lactic acid production.
In the fermentation test, the medium was 2% glucose-containing W medium, the neutralizing agent was calcium carbonate having a concentration of 2% with respect to the medium, and the initial cell amount was 30 ° C., 24% in 2% glucose-containing W medium. Using an inoculum that has been cultured for a long time (in the subsequent fermentation, an inoculum prepared by the same method was used), adjusted to OD = 5, and the fermentation broth was shaken at 30 ° C. and 50 rpm. And fermented for 72 hours.
The D-lactic acid production concentration of the 20 mL mutant strain is shown in FIG. As a result, TDL no. Three strains were obtained. In the following examples, this TDL No. 3 strains were used (TDL No. 3 strain was named Erwinia M3 and deposited with the deposit number of NITE P-01579).

[Example 1] D-lactic acid fermentation using Erwinia M3:
Using a 500 mL scale flask, 100 mL of W medium containing 2% glucose (0.01 g / L each of glutamic acid, glutamine, aspartic acid, asparagine, arginine, and lysine) and 10% concentration of calcium carbonate in the medium Was added thereto so that Erwina M3 strain inoculum was OD = 5 in the medium, and the fermentation solution was fermented for 72 hours while shaking at 30 ° C. and 50 rotations. After 72 hours, the D-lactic acid production concentration was 8.7 g / L, and the yield to sugar was 94.6%.

[Comparative Example 1] Erwinia sp. D-lactic acid fermentation using Erwinia M3 strain as a wild strain Erwinia sp. Fermentation was carried out for 72 hours in the same manner as in Example 1 except that the change was made. The D-lactic acid production concentration after 72 hours was 0.25 g / l, and the corresponding yield was 61.4%.

[Example 2] Effect of redox potential-1:
Using a 5 L scale jar fermenter, inoculate Erwinia M3 seed medium in 2% glucose-containing W medium (adding 0.01 g / L each of glutamic acid, glutamine, aspartic acid, asparagine, arginine, and lysine) in the medium. = 12.5 and the total amount of the fermented liquid made up to 3 l was fermented while stirring at 30 ° C. and 50 rpm. During fermentation, sugar was added so that the glucose concentration would be 20 g / L when the glucose concentration reached 5 g / L, so that the total glucose charge was finally 4%.
Furthermore, the pH was controlled to 6.8 using 28% aqueous ammonia as a neutralizing agent. The reached ORP was -380 mV. The concentration of D-lactic acid produced after 72 hours was 17.7 g / L, and the yield relative to sugar at that time was 41.4%. The production concentration of formic acid was 8.2 g / L.

[Example 3] Effect of redox potential-2:
Fermentation was carried out in the same manner as in Example 2 except that ORP was controlled to -150 mV with compressed air. During fermentation, sugar was added so that the glucose concentration would be 20 g / L when the glucose concentration reached 5 g / L, so that the total glucose charge was finally 4%. Furthermore, the pH was controlled to 6.8 using 28% aqueous ammonia as a neutralizing agent. The D-lactic acid production concentration after 72 hours was 19.6 g / L, and the yield to sugar was 62.4%. Moreover, the production concentration of formic acid was not confirmed.

Example 4 Effect of pH:
Fermentation was carried out in the same manner as in Example 3 except that the pH to be controlled was 7.6. During fermentation, sugar was added so that the glucose concentration would be 20 g / L when the glucose concentration reached 5 g / L, so that the total glucose charge was finally 4%. Furthermore, the pH was controlled to 7.6 using 28% ammonia water as a neutralizing agent. The D-lactic acid production concentration after 72 hours was 36.8 g / L, and the yield to sugar was 92.0%. Moreover, the production concentration of formic acid was not confirmed.

  According to the present invention, since D-lactic acid as a polylactic acid raw material can be produced efficiently at low cost, D-lactic acid can be industrially produced at low cost.

Claims (4)

  A D-lactic acid producing microorganism named Erwinia M3 and deposited as NITE P-01579.   Using the microorganism according to claim 1 and a medium not containing waste molasses which is a colored component or a medium having a yeast extract concentration of less than 0.1%, the pH during cultivation is within the range of 7.0 to 8.0. A method for producing D-lactic acid while being controlled.   Using the microorganism according to claim 1 and a medium not containing waste molasses which is a colored component or a medium having a yeast extract concentration of less than 0.1%, an oxidation-reduction potential (ORP) during culture is -250 mV to- A method for producing D-lactic acid while controlling it within a range of 100 mV.   Using the microorganism according to claim 1 and a medium not containing waste molasses which is a colored component or a medium having a yeast extract concentration of less than 0.1%, the pH during cultivation is within the range of 7.0 to 8.0. And a method for producing D-lactic acid while controlling the oxidation-reduction potential (ORP) within a range of −250 mV to −100 mV.