JP2003235592A - Organic acid production method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing an organic acid at high production rate and in high yield relative to a raw material saccharide using aerobic bacteria. <P>SOLUTION: The method for producing the organic acid by reaction of aerobic bacterial cells comprises culturing the aerobic bacteria at ≥10.5 wt.% but <25 wt.% in the maximum concentration of the raw material saccharide in the culture liquid. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the production of organic acids using aerobic bacteria.

[0002]

2. Description of the Related Art In the case of producing succinic acid and the like by fermentation, it is usual to use Anaerobiospirillu.
Anaerobic bacteria such as genus m) and genus Actinobacillus are used. When anaerobic bacteria are used, the yield of the product is high, but on the other hand, it is necessary to add a large amount of an organic nitrogen source such as CSL to the medium because it requires a large amount of nutrients to grow. . Addition of a large amount of these organic nitrogen sources not only leads to an increase in culture medium cost, but also leads to an increase in purification cost when the product is taken out, which is not economical.

Further, aerobic bacteria are once cultivated under aerobic conditions to grow the cells, which are then collected and washed and used as stationary cells to produce organic acids without aeration of oxygen. It is also known how to do it. In this case, when growing cells, it is economical to add a small amount of organic nitrogen and sufficient growth can be achieved with a simple medium, but it is economical, but the production amount of the target organic acid and the production rate per cell are high. Is still insufficient, and establishment of a better method was desired.

[0004]

An object of the present invention is to provide a method for producing an organic acid having higher fermentation efficiency.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that the production rate and the yield can be controlled by controlling the concentration of sugar as a raw material within a specific range. The inventors have found that the product can be accumulated at a high concentration without lowering the temperature, and have completed the present invention.

That is, the gist of the present invention is that when an organic acid is produced by a microbial cell reaction of an aerobic bacterium, the culture is performed at a maximum concentration of raw sugar of 10.5% or more and less than 25%. And a method for producing an organic acid.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. The bacterium used in the present invention is not particularly limited as long as it has the ability to produce organic acids. Among them, Bacillus
Genus, Rizobium, Corynebacterium, Breviba
Aerobic bacteria such as microorganisms belonging to the genus cterium and the genus Arthrobacter are preferable.

Of the above aerobic bacteria, coryneform bacteria are preferred, and the coryneform bacteria are preferably microorganisms belonging to the genus Corynebacterium, microorganisms belonging to the genus Brevibacterium or microorganisms belonging to the genus Arthrobacter. Among them, preferably, those belonging to the genus Corynebacterium or the genus Brevibacterium,
More preferably, Corynebacterium glutamicum (Corynebacterium glutamic)
um), Brevibacterium flavum (Brevib)
Activum flavum), Brevibacterium ammoniagenes Brevibacterium a
mmoniagenes) or Brevibacterium lactofermentum (Brevibacterium)
Lactofermentum).

As a particularly preferred specific example of the above-mentioned microorganism, Brevibacterium flavum (Brevibac)
terium flavum) MJ-233 (FERM
BP-1497), the same MJ-233AB-41 (FE
RM BP-1498), Brevibacterium ammoniagenes (Brevibacterium ammo)
niagenes) ATCC 6872, Corynebacterium glutamicum (Corynebacterium)
m glutamicum) ATCC31831, Brevibacterium lactofermentum (Brevi
Bacterium lactofermentum)
ATCC 13869 may be mentioned.

The above-mentioned microorganisms used in the production method of the present invention are not only wild strains but also mutant strains obtained by ordinary mutation treatment such as UV irradiation and NTG treatment, and genetics such as cell fusion or gene recombination method. Any strain such as a recombinant strain derived by the method may be used. The host of the genetically modified strain may be the same genus species as the parent strain or different genus species as long as it is a transformable microorganism. It is preferable to use bacteria as a host.

Among these, in this reaction, it is more effective to use a mutant strain lacking lactate dehydrogenase. As a specific method for producing the mutant strain of coryneform bacterium lacking lactate dehydrogenase, the method described in JP-A No. 11-205385 can be mentioned, which can be easily prepared in accordance with this method. When using the bacterium in the present reaction, it is possible to use the one that is slant-cultured in a solid medium such as agar medium for the direct reaction, but it is preferable to use the one that is previously cultivated (seed culture) in the liquid medium. .

[0012] The main carbon source of the medium used for culturing these bacteria is not particularly limited as long as it is a carbon source that can be assimilated by the present microorganism, but usually galactose, lactose, glucose, fructose, glycerol,
Carbohydrates such as sucrose, sucrose, starch, and cellulose; fermentable sugars such as polyalcohols such as glycerin, mannitol, xylitol, and ribitol are used. Of these, glucose, fructose, and glycerol are preferable, and glucose is particularly preferable.

Further, starch saccharified solution, molasses and the like containing the above fermentable sugar are also used. These fermentable sugars can be used alone or in combination. In this reaction,
The maximum concentration of the saccharide is less than 25% (W / V), preferably 20% (W / V) or less. On the other hand, if the concentration of the raw material sugar is too low, it is not preferable in terms of the pot efficiency during industrial production. Therefore, it is usually 10.5% (W / V) or more, preferably 11% (W / V) or more. To react.

Further, if the maximum concentration does not exceed 25% (W / V), it is also preferable to add the above-mentioned saccharides as the reaction progresses, in order to improve the accumulated amount of the product. The nitrogen source is not particularly limited as long as it is a carbon source that the microorganism can assimilate, but specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein hydrolyzate, peptone, yeast extract, meat extract, Examples include various organic and inorganic nitrogen compounds such as corn steep liquor.

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

The pH of the culture broth is usually adjusted to pH 5 to 9, preferably pH 6.5 to 8.5, and the pH of the culture broth may be adjusted with alkaline substances, carbonates, urea, etc., if necessary during the reaction. Adjust within the range. Since aerobic bacteria are used in this reaction, oxygen is required for the growth of bacterial cells. On the other hand, in this reaction, after contacting the culture solution with the bacterial cells, the bacterial cells first undergo logarithmic growth and then reach a stationary phase.
Therefore, the amount of oxygen required also changes during the logarithmic growth phase or stationary phase, so it is necessary to adjust the aeration rate and stirring rate after considering the difference in stirring efficiency due to the difference in reaction scale and blade shape. .

In the method of the present invention, the reaction temperature is not particularly limited as long as it is within a range where the microbial cell reaction is carried out, but the reaction temperature after the microbial cell growth reaches the stationary phase is set to that of the bacterium. It is preferable that the temperature is higher than the optimum growth temperature, and the temperature in the logarithmic growth phase is the optimum growth temperature of the bacterial cells ± 2 ° C.
It is more preferable to carry out the reaction at a temperature of, and after the stationary phase is reached, the temperature is raised above the temperature. Here, the temperature after the stationary phase is particularly preferably a temperature increased by 2 to 10 ° C. from the temperature in the logarithmic growth phase.

The optimum temperature for growth of the microorganism used in this reaction is
Usually, it is 25 ° C to 35 ° C. This reaction is usually terminated when the raw sugar in the culture medium is consumed. At this time, organic acids such as malic acid, fumaric acid, and succinic acid are generated in the reaction solution. Of these, succinic acid has the highest degree of accumulation and is preferred as a product. The organic acid thus accumulated in the culture broth is separated and purified from the culture broth according to a conventional method. Specifically, after removing solid matter such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin etc., the organic acid can be separated and purified from the solution by crystallization or column chromatography. it can.

[0019]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Example 1 Brevibacterium flavum MJ233AB-41
(FERM BP-1498) to JP-A-11-206
According to 385, strains lacking lactate dehydrogenase (LDH) were prepared. That is, a PCR reaction was carried out on a partial fragment of the lactate dehydrogenase gene using the total DNA extracted from the MJ233AB-41 strain by a conventional method as a template and two primers described as SEQ ID NOS: 5 and 6 in the patent. 3 μl of the obtained reaction solution and PC
Cloning vector pGEM-T (PRO for R products
MEGA) 1 μl, and mixed with 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 m
Each component of M ATP, 10 mM MgCl ₂ and 1 unit of T4 DNA ligase was added and allowed to react at 4 ° C. for 15 hours for binding. Using the obtained plasmid mixture,
Escherichia coli JM109 by calcium chloride method
(Takara Shuzo) was transformed and a medium containing 50 mg of ampicillin (10 g of tryptone, 5 yeast extract).
g, NaCl 5 g and agar 16 g dissolved in 1 L of distilled water)
Was applied to. The strain grown on this medium was liquid-cultured by a conventional method, and plasmid DNA was prepared from the culture medium. 20 μl of this plasmid DNA was added to 50 mM Tris buffer (p
H7.5), 1 mM dithiothreitol, 10 mM M
Add each component of gCl ₂ , 100 mM NaCl, restriction enzymes SphI and SalI 1 unit, and add 1 at 37 ° C.
Reacted for hours. Gene C from the obtained DNA solution
A 300 bp fragment was recovered using leanII (manufactured by Funakoshi), mixed with 10 μl of the DNA solution and 1 μl of a chloramphenicol-resistant cloning vector pHSG396 (Takara Shuzo Co., Ltd.) decomposed with SphI and SalI to obtain a 50 mM Tris buffer. (PH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgC
Each component of ₁₂ and T4 DNA ligase 1 units was added and reacted at 4 ° C. for 15 hours to bind. Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method, and a medium containing 50 mg of ampicillin (tryptone 10 g, yeast extract 5 g, NaCl 5 g and agar 16).
g was dissolved in 1 L of distilled water). The strain grown on this medium is liquid-cultured by a conventional method, and about 300 bp, which is a partial fragment of the lactate dehydrogenase gene, is obtained from the culture.
The plasmid DNA into which the fragment was introduced was prepared. The plasmid was introduced into Brevibacterium flavum MJ233AB-41 by the electric pulse method, and the medium containing 5 mg of chloramphenicol (urea: 2 g, ammonium sulfate: 7 g, potassium phosphate 1: 0.5 g, potassium phosphate 0: 0.5 g, magnesium sulfate heptahydrate: 0.5
g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate
Hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid:
1 g and 16 g of agar were dissolved in 1 L of distilled water). Among the strains grown on this medium, LDH activity was 1 as a strain in which the gene was disrupted by homologous recombination.
Strains that became less than 1/0 were selected.

Urea: 4 g, ammonium sulfate: 14 g,
1 potassium phosphate: 0.5 g, 2 potassium phosphate 0.5
g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate hydrate: 20
mg, D-biotin: 200 μg, thiamine hydrochloride: 20
100 mL of a medium containing 0 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 ml was put in a 500 mL Erlenmeyer flask and sterilized by heating at 120 ° C. for 20 minutes.
This was cooled to room temperature, 4 ml of 50% glucose aqueous solution that had been sterilized in advance and 5 ml of 0.1% chloramphenicol aqueous solution that had been sterile filtered were added, and the lactate dehydrogenase gene-disrupted strain was inoculated for 24 hours and 30 hours.
Seed culture was performed at ° C.

Urea: 8 g, ammonium sulfate: 28 g,
Potassium phosphate 1 g, dipotassium phosphate 1 g, magnesium sulfate heptahydrate: 1 g, ferrous sulfate heptahydrate: 40 mg, manganese sulfate hydrate: 40 mg, D-
Biotin: 400 μg, thiamine hydrochloride: 400 μg, yeast extract 2 g, casamino acid 2 g, antifoaming agent (Adecanol LG294: manufactured by Asahi Denka): 1 ml and distilled water: 1000
5 ml of fermentation broth was added with ml of medium, and sterilized by heating at 120 ° C for 20 minutes. After cooling this to room temperature, 1000 ml of a previously sterilized 22% glucose aqueous solution was added to make the glucose concentration in the system 11%, and the above seed culture solution was added in total to keep it at 30 ° C. pH is 2M
Maintained at 8.0 with sodium carbonate, aeration is 400m / min
The reaction was performed at L and stirring at 300 rpm. After 25 hours from the start of the reaction, glucose was almost consumed and succinic acid was accumulated at 37 g / L. At this time, the yield of succinic acid with respect to sugar was 53%, and the production rate was 1.40 g / L / hr.

Example 2 The procedure of Example 1 was repeated except that the concentration of glucose to be added was changed to 30%, that is, the glucose concentration in the system was changed to 15%, and glucose was almost consumed after 35 hours. Succinic acid was accumulated at 48 g / L.
At this time, the yield of succinic acid with respect to sugar was 55%, and the production rate was 1.37 g / L / hr.

Example 3 The procedure of Example 1 was repeated except that the concentration of glucose to be added was changed to 40%, that is, the glucose concentration in the system was changed to 20%, and glucose was almost consumed after 48 hours. And 65 g / L of succinic acid was accumulated.
At this time, the yield of succinic acid with respect to sugar was 60%, and the production rate was 1.35 g / L / hr.

COMPARATIVE EXAMPLE 1 The procedure of Example 1 was repeated except that the concentration of glucose to be added was changed to 50%, that is, the glucose concentration in the system was changed to 25%, and glucose was almost consumed after 85 hours. And 72 g / L of succinic acid was accumulated.
At this time, the yield of succinic acid with respect to sugar was 54%, and the production rate was 1.04 g / L / hr.

Comparative Example 2 Example 1 except that the concentration of glucose added was changed to 10%, that is, the glucose concentration in the system was changed to 5%.
I went the same way. After 18 hours, glucose was almost consumed and succinic acid was accumulated at 18 g / L. At this time, the yield of succinic acid based on sugar was 40%, and the production rate was 1.03 g.
/ L / hr

[0026]

EFFECTS OF THE INVENTION According to the method of the present invention, the target substance can be obtained at a high reaction rate and a high yield in the production of organic acid using aerobic bacteria.

Claims (8)

[Claims] 1. When producing an organic acid by a microbial cell reaction of an aerobic bacterium, the maximum concentration of raw sugar in a culture solution is 1
A method for producing an organic acid, which comprises culturing at 0.5% or more and less than 25%. 2. The maximum concentration of raw sugar is 11% or more and 25%.
It is less than, The manufacturing method of Claim 1 characterized by the above-mentioned. 3. The production method according to claim 1, wherein the raw material sugar is glucose. 4. The method for producing an organic acid according to claim 1, wherein the organic acid is malic acid, fumaric acid or succinic acid. 5. The method for producing an organic acid according to claim 4, wherein the organic acid is succinic acid. 6. The method for producing an organic acid according to claim 1, wherein the aerobic bacterium is a coryneform bacterium. 7. The method for producing an organic acid according to claim 1, wherein the aerobic bacterium is a bacterium belonging to the genus Corynebacterium. 8. The method for producing an organic acid according to claim 1, wherein the aerobic bacterium is a mutant strain in which the lactic acid-producing ability of the bacterium is 90% or more lower than that of the wild type.