JPH0710228B2 - Electrodialysis method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an electrodialysis method for separating a product obtained by fermentation from a fermentation broth using an ion exchange membrane.

[Problems to be Solved by Prior Art and Invention]

In the microbial industry, a cation exchange membrane and an anion exchange membrane are arranged between the anode and the cathode to separate the target substance produced by fermentation from the fermentation broth, and the fermentation broth is placed in the cathode chamber. Attempts have been made to separate the target substance produced by fermentation by supplying it with electrodialysis. [Applied and Environmental microliology Vol. 52 No. 2
Pages 314-319, 1986)]. This method has the advantage that the target substance can be separated from the fermentation liquor very quickly and easily. However, when the actual method described above is performed, excellent effects are obtained immediately after startup, but with the passage of time, the increase in the production amount of the target substance reaches a peak, and The problem was that the amount of material produced did not increase.

[Means for solving problems]

Therefore, the inventors of the present invention have conducted intensive studies to solve the problems in the method for separating the product by fermentation using the above-mentioned ion exchange membrane and to measure the increase in the production amount of the target substance. . As a result, they have found that the above problems are due to the decrease in the growth of microorganisms existing in the fermentation broth. Then, as a result of further research on a method for preventing a decrease in the growth of microorganisms, an excellent effect was obtained by using polyphosphoric acid or a salt thereof as a phosphate ion source added to the fermentation liquid for culturing the microorganisms. It was found that the above was obtained, and the present invention was completed.

That is, the present invention provides an electrodialysis method for separating a product obtained by fermentation from a fermentation broth in an electrodialysis tank in which a cation exchange membrane and an anion exchange membrane are arranged between an anode and a cathode. In the electrodialysis method, polyphosphoric acid or a salt thereof is allowed to exist in the fermentation liquid.

As the polyphosphoric acid used in the present invention, known polyphosphoric acid can be used without any limitation. Specific examples of the polyphosphoric acid used in the present invention include pyrophosphoric acid, triphosphoric acid, 4-phosphoric acid, 5-phosphoric acid, chain polyphosphoric acid such as 6-phosphoric acid; 3 metaphosphoric acid, 4
Examples thereof include cyclic polyphosphoric acids such as metaphosphoric acid, 5-metaphosphoric acid, 6-metaphosphoric acid. As the salt of polyphosphoric acid, the above-mentioned metal salts of polyphosphoric acid can be used without any limitation. Particularly, alkali metal salts such as sodium salts and potassium salts; or alkali salts such as magnesium salts, calcium salts and barium salts. Earth metal salts are preferred. As the polyphosphoric acid or salt thereof used in the present invention, chain-like or cyclic polyphosphoric acid of 3 phosphoric acid or 3 metaphosphoric acid or more or a salt thereof is preferable, and particularly, polyphosphoric acid of 4 phosphoric acid or 4 metaphosphoric acid or more or a salt thereof is preferable. Cyclic polyphosphoric acid or a salt thereof is preferable because the growth of microorganisms and the production of the target substance are good.

The amount of the above-mentioned polyphosphoric acid or a salt thereof is not particularly limited, but from the viewpoint of increasing production by fermentation, 0.007 g / dl to 10 g / dl and 0.02 g / dl to the fermentation liquor.
It is preferably present in the range of 0.2 g / dl.

The electrodialysis method of the present invention can be applied to any known fermentation as long as the product is an electrolyte. For example, glutamic acid fermentation, lysine fermentation, valine fermentation, ornithine fermentation, homoserine fermentation, threonine fermentation, isoleucine fermentation and other amino acid fermentation; inosinic acid fermentation, guanylic acid fermentation and other nucleic acid fermentation; citric acid fermentation, fumaric acid fermentation, lactic acid fermentation, Organic acid fermentation such as succinic acid fermentation, acetic acid fermentation, gluconic acid fermentation, pyruvic acid fermentation, α-ketoglutaric acid fermentation, itaconic acid fermentation, malic acid fermentation; fatty acid fermentation; antibiotic fermentation and the like.

In addition, various culture conditions such as the type of microorganism used in the fermentation in the present invention, the composition of the medium, the culture temperature, the pH of the fermentation broth, and the degree of aeration and stirring are known conditions in the above-mentioned various fermentations. Are adopted without any restrictions.

At this time, as the medium to be used, either a synthetic medium or a natural medium can be used as long as it is a medium containing a main carbon source, a nitrogen source, an inorganic substance and other growth promoting substances, but when performing electrodialysis. In order to prevent the ion exchange membrane from being clogged or deteriorated to cause an abnormal increase in voltage and a decrease in current effect, it is desirable to use a synthetic medium as much as possible.

For example, carbon sources often used include sugars such as sucrose, galactose, fructose, glucose, starch, maltose, rhamnose, xylose and lactose; methanol, ethanol, n-propyl alcohol,
There are alcohols such as glycerin; chain hydrocarbons such as n-paraffin and the like, and these may be mixed and used. As the nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate,
Inorganic or organic nitrogen compounds such as phosphorous ammonium, urea, ammonium acetate and ammonium citrate are used. Usually, these are phosphoric acid source, potassium source, magnesium source,
In addition to these metal salts as a sulfur source, iron source, and manganese source, growth promoting substances such as biotin and thiamine, and calcium carbonate as a pH lowering agent may be added.

In the present invention, as the microorganism to be used for fermentation, the culture solution of the bacterial cell can be directly used as it is, but it is more preferable to immobilize it by a known method. For example, as a method of immobilization, the bacteria collected from the culture solution by centrifugation or filtration are suspended in a saline solution, and polyvinyl alcohol, polyacrylamide, kupper carrageenan, calcium alginate, A method for obtaining immobilized microbial cells by adding a carrier such as a photo-crosslinkable resin prepolymer, collagen, cellulose succinate, casein succinate, methyl acrylate, methacrylic acid copolymer, a crosslinking agent, or an encapsulating material Can be mentioned.

In the present invention, it is a preferable embodiment that a substance that reversibly undergoes redox is present in the fermentation liquid. Examples of such substances include metal ions such as iron, manganese, copper, zinc, cobalt, and lead; iron-containing complex compounds such as red blood salt and yellow blood salt; methyl viologen, benzyl viologen, neutral red, neutral bio. Examples thereof include redox dyes such as let. The amount of these substances used is not particularly limited, but in consideration of the amount of fermentation production, it is generally preferable to use it in the range of 0.1 mg / dl to 10 mg / dl in the fermentation broth.

As the electrodialysis tank used in the electrodialysis method of the present invention, the electrodialysis tank used in ordinary electrodialysis can be used without any limitation. An example of the electrodialysis tank used in the present invention is shown in FIG. In the figure, 1 is an electrodialysis tank in which a cation exchange membrane 4 and an anion exchange membrane 5 are alternately arranged between an anode 2 and a cathode 3. This electrodialysis tank 1
It is divided into five chambers by two pairs of cation exchange membrane 4 and anion exchange membrane 5. These five chambers are an anode chamber 6, a concentrating chamber 7, a diluting chamber 8, a concentrating chamber 7 and a cathode chamber 9 in order from the chamber in which the anode 2 exists. Although FIG. 1 shows an example in which two pairs of cation exchange membranes and anion exchange membranes are used, these ion exchange membranes may be one pair or three or more pairs.

As the cation exchange membrane and the anion exchange membrane, known ones can be used without any limitation. In this case, it is preferable to use a medium excellent in selective permeability of the fermentation product with respect to various medium components necessary for the growth of bacteria existing in the fermentation broth, for example, various salts such as magnesium, manganese, iron, potassium and sodium. Further, it is desirable that the material can be sterilized by ultraviolet rays, .gamma.-rays, alcohol, a surfactant, a chlorine-based bactericide, heating, etc. and can be washed. The cation exchange membrane preferred in the present invention has a sulfonic acid group, and the anion exchange membrane has a quaternary ammonium salt group. Further, as the anion exchange membrane, one having an average pore diameter of 100 Å or less is preferably used in the present invention.

Next, platinum, graphite, copper or the like is preferably used as the anode 2, and platinum, iron, stainless steel or the like is preferably used as the cathode 3. The anolyte and the catholyte may be known ones,
Generally, an aqueous solution of sulfuric acid is used as the anolyte and an aqueous solution of sodium hydroxide is used as the catholyte. Further, as described later, the fermented liquid can be passed through the cathode chamber without using the catholyte. A DC power supply 10 is connected between the anode 2 and the cathode 3.

The fermented liquid cultivated in the fermenter 11 is used as a diluting chamber 8 and a cathode chamber 9.
The product produced by fermentation moves through the anion exchange membrane 5 to the concentration chamber 7. On the other hand, the fermented liquid is collected in the fermenter 11. The product of the fermentation that has moved to the concentration chamber 7 is recovered in the recovery tank 12 together with the concentrated liquid that is circulated to the concentration chamber 7. In FIG. 1, the fermentation tank 11 and the electrodialysis tank 1 are provided independently, and fermentation and electrodialysis are performed in separate tanks. It is also possible to carry out the fermentation in the cathode compartment 9.

Although FIG. 1 shows an electrodialysis tank in which the fermented liquid is introduced into both the diluting chamber and the cathode chamber, either one of the fermented liquids may be introduced.

Next, FIG. 2 shows an example of an electrodialysis tank that is preferably used for continuous electrodialysis for a long period of time. This electrodialysis tank incorporates a pH controller 13 that measures the pH of the fermented liquid in the fermentation tank 11 and controls the DC power supply 10 between the anode and the cathode on / off or controls the current according to the measured value. As a result, the concentration of the target substance due to fermentation is higher than the concentration that causes the growth of bacteria and the inhibition of radiation.
When the pH is out of the range, electrodialysis is started, or the current density is increased to separate the target substance from the fermentation broth, which enables control.

The operation of the electrodialysis cell as described above is generally performed by selecting a voltage in the range of 0.1 to 35 V and a current density in the range of 1.6 to 10 amps / dm ² . By adopting such a condition, stable operation is usually possible for 3 to 30 days. Further, when the above-mentioned immobilized microbial cells and the electrodialysis tank capable of pH control shown in FIG. 2 are used, continuous operation for 3 to 8 months is also possible.

[Effect]

By adopting the electrodialysis method of the present invention, it becomes possible to carry out stable fermentation over a long period of time, and it is possible to increase the production amount of the desired fermentation product. That is, as is clear from Example 1 and Comparative Example 1 described later, in electrodialysis using an ion exchange membrane, polyphosphoric acid (Wako Paper Co., Ltd.) was added to the medium as a phosphate ion necessary for the growth of bacterial cells. In Example 1, the amount of cells after 72 hours of culture was 0.4 at the absorbance of 660 nm, and the total amount of L-lactic acid produced by fermentation reached 8.0 g per 1 dl of the medium used. On the other hand, in Comparative Example 1 in which potassium dihydrogen phosphate, which has been conventionally used in a medium, was used as a phosphate ion source, and electrodialysis was performed under the same conditions as in Example 1, except that the amount of cells after culturing was high. Is suppressed to 0.2, and the amount of L-lactic acid produced is only 2.0 g / dl.

The value of Example 1 is 10 for potassium dihydrogen phosphate and pH adjuster.
% Of calcium carbonate, which is a conventional fermentation method rather than electrodialysis using an ion exchange membrane. It is almost the same as the value obtained in Comparative Example 2.

As described above, according to the electrodialysis method of the present invention, potassium dihydrogen phosphate or sodium, dipotassium monohydrogen phosphate or sodium, which has been added to a normal medium as a phosphate ion source, or a combination thereof is conventionally used. Compared with the method described in 1), the amount of cells after culturing for a certain period of time and the amount of the target substance produced by fermentation are large, and an excellent effect is obtained. Moreover, the present invention, while performing the fermentation, because it is possible to quickly and easily separate the target substance from the fermentation liquor, release the feed back inhibition of the fermentation by the target substance, and prevent the pH change and viscosity increase of the fermentation liquor. ,
It is possible to prevent the fermentation production capacity of the cells from decreasing.

Furthermore, the calcium salt that is often used as a pH adjuster is one that promotes adsorption to the cells of the phage that lyses the grown cells, but according to the present invention, the target substance produced by fermentation is used. Since it can be immediately separated from the fermentation broth, it is not necessary to use such a pH adjuster.
Therefore, according to the present invention, it is possible to prevent contamination of the bacterial cells by the charge. Furthermore, since polyphosphoric acid has an action of suppressing the infiltration of the phage into the fungus body, it is possible to prevent the contamination of the phage from this point as well.

As a result of these, the present invention has an excellent effect that enables fermentation to be stably carried out for a long time continuously.

〔Example〕

As an inoculum of Example 1 and Comparative Examples 1 and 2, L-lactic acid producing bacterium Lactobacillus delbrutskii-IFO3534 was used. Glucose 1.0 g / dl,
10 ml of liquid medium (pH 7.0) consisting of 5.0 g / dl of polypeptone and 0.5 g / dl of yeast extract was dispensed into a medium-sized test tube, and autoclaved at 121 ° C for 15 minutes. One platinum loop of the inoculum was inoculated into this and static culture was carried out at 45 ° C. for 24 hours. 10 ml of this culture
100 g of similarly sterilized glucose 1.0 g / dl, polypeptone 1.0 g / dl, yeast extract 0.5 g / dl, sodium acetate 1.0 g / d
A seed culture was prepared by inoculating a liquid medium (pH 6.8) consisting of 1 and statically culturing at 45 ° C for 15 hours.

As the medium for the main culture, glucose 10.0 g / dl, yeast extract 2.0 g / dl, polypeptone 0.8 g / dl, tetraphosphoric acid 0.02 g / dl, magnesium sulfate heptahydrate 0.05 g / dl, salt 0.01 g / dl Use, pH
Was set to 7.2.

When performing electrodialysis using the electrodialysis tank shown in FIG. 2, 500 ml of the above medium was dispensed into a 750 ml glass fermentor, and after sterilization and cooling to room temperature, 50 ml of the seed mother
Was inoculated and cultured at 45 ° C. with gentle stirring.

In order to perform electrodialysis, a pH controller (Oriental Electric Co., Ltd.) in which a pH electrode for fermentation (Advantech FX-180T), which was previously attached to the fermenter, was connected to a DC power source (MPO35-2 manufactured by Takasago Seisakusho KK) ) Manufactured by pHD II). In addition, the DC power supply is connected to each electrode of the electrodialysis tank so that when the pH of the fermented liquid falls below the specified value, the electric current will automatically flow to the electrodialysis tank to enable electrodialysis. I chose Anion exchange membrane (Tokuyama Soda Co., Ltd.)
The electrodialysis cell that combines Neoceptor ACH-45T (manufactured by Tokuyama Soda Co., Ltd., Neocepta CH-45T) and a cation exchange membrane uses copper as the anode and platinum as the cathode. 0.1N NaOH was used in the ₂ SO ₄ cathode chamber, and deionized water was circulated in the concentrating chamber and the recovery tank with a microtube pump (MP-3 manufactured by Tokyo Rika Co., Ltd.).

About 7 hours after the start of main culture, electrodialysis showed that the pH of the fermentation broth was 4.7.
The fermentation liquor was circulated to the diluting chamber with a microtube pump when the temperature dropped to 0. Fig. 3 shows the values obtained by converting the total amount of L-lactic acid produced in the fermentation liquor injection and recovery tank per 1 dl of the medium used and the amount of cells in the fermentation liquor.

The amount of L-lactic acid in the fermentation liquor and the recovery tank was quantified by the Barker-Somerson method. The culture volume is 1N
After 11-fold dilution with hydrochloric acid, the absorbance at 660 nm was measured using a spectrophotometer (UV-240, manufactured by Shimadzu Corporation).

As Comparative Example 1, instead of tetraphosphate in the medium of the main culture,
Electrodialysis was performed in the same manner as in Example 1 except that 0.2 g / dl of potassium dihydrogen phosphate was used.

Furthermore, as Comparative Example 2, calcium phosphate 10.0 was used, in which 0.2 g / dl of potassium dihydrogen phosphate was used in place of tetraphosphoric acid in the medium of the main culture, and further dry heat sterilization was performed as a pH adjusting agent at 160 ° C. for 90 minutes. Example 1 and Comparative Example 1 except that g / dl was added
Culturing was carried out in the same manner as in Example 1, but Example 1 and Comparative Example 1
Unlike, does not perform electrodialysis using an ion exchange membrane,
Performed conventional fermentation.

The amount of L-lactic acid produced and the amount of cells in Comparative Examples 1 and 2 are also shown in FIG.

In Example 1 in which 4-phosphate was used as the phosphate ion, the amount of cells after 72 hours of culture was 0.4 (OD660nm), and the total amount of L-lactic acid produced was 8.0 in terms of 1 dl of the medium used. g
It was. On the other hand, in Comparative Example 1 in which potassium dihydrogen phosphate was used as the medium, the growth of the bacterial cells was suppressed to 0.2, and the L-lactic acid amount was also low at 2.0 g / dl.

Similarly, in Comparative Example 2 in which potassium dihydrogen phosphate was used as the phosphate ion, L-lactic acid was produced to the same extent as in Example 1, and the growth of bacterial cells was also observed. However, in this case
Calcium carbonate and L added to neutralize L-lactic acid
-Since calcium lactate is formed with lactic acid, the viscosity of the fermentation liquor increases with the increase of the concentration, and it was necessary to stop the fermentation in 72 hours. In this respect, Example 1
Then, the concentration of L-lactic acid in the fermentation broth was 0.5 g by electrodialysis.
Since it was controlled to / dl or less (pH 5.5 or more) and no neutralizing agent was required, the fermentation could be stably performed even after 72 hours.

Example 2 Cells were centrifuged from the yeast culture solution used in Example 1 and washed with water. The washed bacterial cells were suspended in physiological saline, sodium alginate was added to the bacterial cell suspension to make the concentration 2%, and the mixture was heated to 30 ° C and slurried in a 0.1 molar calcium chloride solution using a syringe. Then, the mixture was dropped and coagulated to obtain spherical immobilized cells. Using this immobilized microbial cell, electrodialysis was carried out in the same manner as in Example 1 except that a part of the medium was replaced, and it was possible to continuously operate for about 8 months. L-lactic acid was 8.0 g / dl in the conventional electro-fermentation, whereas the present invention produced 60 times that amount.

Example 3 After koji soup was sterilized under high pressure and cooled, ethanol 4.
Add 0 g / dl and add acetic acid bacteria (Acetobacter aceti IFO
3281) was inoculated with 1 platinum loop, and static culture was performed at 30 ° C. for 3 to 5 days. Next, glucose 0.1 g / dl, glycerin 0.1 g / dl,
Ammonium sulfate 0.1 g / dl, 4-metaphosphoric acid 0.06 g / dl, magnesium sulfate heptahydrate 0.01 g / dl, salt 0.01 g / dl, yeast extract 0.
Acetic acid (1.5 g / dl) and ethanol (8.0 g / dl) were added to a sterilized medium (pH 7.0) consisting of 05 g / dl, 10% of the above culture solution was inoculated into this, and static culture was carried out at 30 ° C for 4 days. I got used to it. 10% by volume of an aqueous solution of polyvinyl alcohol was added to the thus obtained culture solution to suspend it, and the suspension was freeze-dried to prepare immobilized cells. For the main culture, the same medium was used, 10% (V / V) of the immobilized cells were added, and electrodialysis pH was performed in the same manner as in Example 1 while controlling the pH to 4.5. In addition, 0.2 g / dl of potassium dihydrogen phosphate was used instead of 4-metaphosphoric acid. The time-dependent changes in the concentration of total acetic acid (g / dl) produced are shown in Table 1.

Also, electrodialysis could be operated continuously for about 3 months.

Example 4 As the inoculum, Kyandeida gearman IFO 0566 having citric acid producing ability was used. Sucrose 5.0 g / dl, Asparagine 0.25 g / dl, Phosphoric acid 0.02 g / dl, Magnesium sulfate 7
A platinum loop was inoculated from a slant culture medium into a liquid medium (pH 6.0) consisting of 0.05 g / dl of water salt and 20 g / dl of calcium carbonate, shake cultured at 30 ° C. for 2 days, and then collected by centrifugation. 8g of this fungus body
Acetone was prepared by suspending in 50 ml of 0.9% saline solution and dispersing 4 g of N, N-dimethylvinylbenzylamine polymer and 5 mg of 4,4'-bis (dimethylamine) benzophenone in acetone.
0 ml was added to emulsify, and this was irradiated with light from an ultra-high pressure mercury lamp at 25 ° C for 2 hours. The resulting immobilized gel was vacuum dried to form particles of 2 to ³ mm ³ to prepare immobilized cells. The main culture medium is glucose 10.0 g / dl, ammonium chloride
0.4 g / dl, 5-phosphoric acid 0.02 g / dl, magnesium sulfate heptahydrate 0.05
Using a liquid medium (pH 6.0) consisting of g / dl and yeast extract 0.1 g / dl, inoculate the immobilized cells with 10.0% (V / V),
After performing aeration and stirring at 200 rpm and performing electrodialysis for 5 days while controlling the pH to 4.8, 1.3 g / dl of citric acid was produced in terms of the medium used. Also, continue
The electrodialysis could be continuously performed for more than 3 months.

Example 5 As an inoculum, L-glutamic acid-producing bacterium, Brevibacterium flavum ATCC13826, was used. As seed medium,
Glucose 5.0g / dl, Urea 1.0g / dl, 5 Potassium metaphosphate
0.02g / dl, magnesium sulfate heptahydrate 0.04g / dl, ferrous sulfate heptahydrate 2mg / dl, biotin 0.4μg / dl, thiamine hydrochloride 20μg
/ dl, corn stay plica 0.5g / dl liquid medium (p
H7.0) was used. One platinum loop of this slant medium was inoculated into this medium, shake culture was carried out at 30 ° C. for 36 hours, and the cells were collected by centrifugation. Suspend 10 g of the cells in 50 ml of 0.9% saline solution to give 4%
What was made into a slurry by heating with Katsupar carrageenan was dropped into a 2% potassium chloride solution from a syringe and formed into a spherical gel to obtain immobilized cells.

As the medium for main culture, glucose 10.0 g / dl, urea 2.8 g / d
l, 5 Potassium metaphosphate 0.04g / dl, magnesium sulfate heptahydrate 0.04g / dl, ferrous sulfate heptahydrate 2mg / dl, manganese sulphate tetrahydrate 2mg / dl, biotin 0.4μg / dl, thiamine hydrochloride 10 μg / dl,
A liquid medium containing 0.1 g / dl of casamino acid and 0.3 mg / dl of neutral red was used. 1 immobilized microbial cell in this medium
When 0.0% (V / V) was inoculated and electrodialysis was carried out for 3 days while controlling the pH at 7.5 at 30 ° C, 4.1 g / dl of L-glutamic acid was produced in terms of the medium used.

In addition, electrodialysis could be continuously operated for 3 months or more.

[Brief description of drawings]

1 and 2 are schematic views showing a preferred example of the electrodialysis tank used in the electrodialysis method of the present invention. 1 in the figure
Is an electrodialysis tank, 2 is an anode, 3 is a cathode, 4 is a cation exchange membrane, 5 is an anion exchange membrane, 6 is an anode chamber, 7 is a concentration chamber, 8 is a dilution chamber, 9 is a cathode chamber, and 10 is a direct current. A power source, 11 is a fermenter, 12 is a recovery tank, 13 is a pH controller, and 14 is a circulation pump. FIG. 3 is a graph showing the relationship between the culture time and the amount of cells and the relationship between the culture time and the amount of total lactic acid produced in the electrodialysis method of the present invention and the conventional method.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12P 7/56 8114-4B 13/14 2121-4B

Claims (1)

[Claims] 1. An electrodialysis method for separating a product obtained by fermentation from a fermentation broth in an electrodialysis tank having a cation exchange membrane and an anion exchange membrane arranged between an anode and a cathode. And a method for electrodialysis, characterized in that polyphosphoric acid or a salt thereof is present in the fermentation broth.