JPH038759B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for producing citric acid by contacting and reacting microorganisms capable of producing citric acid immobilized on a gel-like carrier with a sugar solution. This is a method in which the cells or spores of a bacterial strain capable of producing citric acid, such as Aspadillas or Candida, are immobilized in a gel carrier such as polyacrylamide, carrageenan, or alginic acid, and the immobilized microorganism is brought into contact with sugars. . In this method, unlike the so-called fermentation method, there are very few complicated by-products, and the process can be managed in a state where purification is easy.

In recent years, methods for producing useful substances using immobilized microorganisms have been attempted, and a typical example is a method for producing alcohol from glucose using yeast. However, there has been no attempt to obtain citric acid, which is produced through a complex biological reaction, as efficiently as in the case of alcohol, but also through a biological reaction that easily produces by-products such as α-ketoglutaric acid and gluconic acid. be the first.

The present inventor discovered that Aspergillus niger, which is generally used for citric acid fermentation production,
niger) and Candida lipolyteica (Candida
Cultured microbial cells or spores such as M. lipolytica are entrapping immobilized in various gels of polyacrylamide, Katsupa carrageenan, or calcium alginate, molded into spheres or blocks, and the molded material is used as a solid catalyst to form a fluidized bed column. It was discovered that citric acid can be obtained by using glucose or sucrose as a substrate and performing the reaction while aerating air or oxygen. The present invention is constituted by such novel biotechnological findings. That is, the present invention provides citric acid production using an immobilized microorganism, which is characterized in that a microorganism having the ability to produce citric acid selected from the genus Aspergillus or Candida immobilized on a gel-like carrier is brought into contact with a sugar solution. This is a method for producing acid.

In the present invention, the microorganism having the ability to produce citric acid may be a microorganism belonging to any genus known for citric acid fermentation, but especially microorganisms of the genus Aspagillus and Candida, which have a strong ability to produce citric acid. For example, Aspadillas niger G.
−011ATCC1015, Candida lipolyteica
OUT6340 or the like can be suitably used. Furthermore, among bacteria, Brevibacterium flavum ATCC14067, Bacillus licheniformis
licheniformis) ATCC21667 etc. are suitable.

These culture conditions may be those suitable for each bacterial species in the case of generally known citric acid fermentation. For example, for aspadillas, 10%
In a medium based on sugar beet or cane sugar concentrate,
Cultivate deep under aeration conditions at 30°C at pH 5.8. A nutrient source involved in citric acid production is added to the medium as necessary. Also for yeast, 10% glucose, 0.2
% ammonium chloride, 0.05% monobasic potassium phosphate,
0.05% Magnesium Sulfate, 0.2% Yeast Extract, 0.01
A typical example is a method using a medium to which % thiamine and 2% calcium carbonate are added. In this case, aerated culture conditions of PH7.0 and 30°C are used. These microorganisms are collected, washed, and then subjected to entrapping immobilization; however, the culture method is merely an example and does not limit the present invention in any way. In the case of filamentous fungi (molds), it is of course possible to collect spores formed by solid-state culture and use them in the present invention in the same manner as the deep-cultured fungi.

The entrapping immobilization of the microorganisms capable of producing citric acid can be carried out by a commonly known method for immobilizing microbial cells, but it is particularly effective to employ the gelling method described below.

(1) When polyacrylamide is used as the immobilizing agent A suspension of bacterial cells or spores in physiological saline is added with acrylamide monomer, N,N'-methylenebisacrylamide, beta (β) dimethylaminopropionitrile, and peroxide. Add potassium sulfuric acid and leave at room temperature to gel.

(2) Method for gelling carrageenan The same bacterial cells or spores as above are heated together with 4% Katupa carrageenan, and the slurry is dropped into a 2% potassium chloride solution using a syringe, and formed into a spherical gel.

(3) Alginic acid gelation method The bacterial cells obtained in the same manner as described in the polyacrylamidation method were added to a spore suspension with sodium alginate to a concentration of 2%, and heated to 30°C to form a slurry. The material is dropped into a 0.1M calcium chloride solution using a syringe and coagulated to form a spherical gel.

The gelation methods mentioned above are also just examples,
In addition to these gelling bases, collagen, cellulose succinate, casein succinate, methyl acrylate, methacrylic acid copolymers, etc. can also be used to sufficiently obtain the effects of the present invention.

The production of citric acid in the present invention can be carried out either batchwise or by continuous contact reaction using a column. That is, immobilized bacterial cells or immobilized spores are suspended in a buffer solution adjusted to a pH suitable for the reaction, and this is packed into a cylindrical fluidized bed column, and fermentable sugars such as glucose and molasses are added to the column. 1
% to 20% concentration and ventilate from the bottom of the column through a glass filter, etc. If the aeration is too strong, the reaction efficiency tends to deteriorate, so try not to disturb the immobilized microorganism layer too much. The reaction temperature is
It varies slightly depending on the strain being tested, but a temperature around 30°C is best. Replace the reaction solution with fresh solution every 24 hours.
Continue the reaction.

In the case of a continuous method, a substrate solution containing sugars is continuously fed into a column filled with immobilized microorganisms using a peristaltic pump or the like, and the reaction solution is flowed out from the other side of the reaction column at the same rate as the injection rate. Good results can be obtained by adjusting the temperature of the column as necessary to maintain the reaction at optimal conditions.

The actual state of the present invention will be shown below with examples.
The citric acid production ability of the immobilized microorganism was determined from the amount of citric acid produced by the reaction.

Example 1 Spores of Aspagillus niger G-011 preserved in wort agar were mixed with 100 ml of 10% sugar beet waste molasses with pH 5.8.
The cells were cultured with shaking in a 500 ml Sakaguchi flask containing 48 hours of high citric acid production activity, and the cells were separated from the culture solution and washed with water. 0.9 g of the washed bacterial cells
Dissolve and mix 6 g of acrylamide monomer and 0.32 g of N,N'-methylenebisacrylamide suspended in 32 ml of % saline solution, add 4 ml of 5% β-dimethylaminopropionitrile, and then
Mix 4 ml of 2.5% potassium persulfate (K ₂ S ₂ O ₈ ) thoroughly and leave to stand at 25° C. for 15 minutes to obtain immobilized Aspagillus cells. The prepared gel was cut into blocks of 4 mm ³ to 5 mm ³ and packed in a sterile gauze bag in a cylindrical reactor with aeration while adding 100 ml of 20 mM tartrate buffer containing 10% sucrose (PH 3.0). )
The reaction solution was exchanged three times every 24 hours at 30°C. After 24 hours of reaction, each
Citric acid solutions of 430 mg/dl, 435 mg/dl and 428 mg/dl were obtained. No other organic acid by-products that would interfere with purification were observed in the reaction solution.

Example 2 Candida lipolytica OUT6340 strain was treated with glucose 10%, NH ₄ Cl 0.2%, KH ₂ PO ₄ 0.05%,
_MgSO4・_7H2O0.05 %, yeast extract 0.2%, 0.01%
It is inoculated into a culture solution containing thiamine and 2% CaCO ₃ and cultured in a conventional manner at 30°C under aerated conditions for 48 hours. Bacterial cells are collected from this culture solution by centrifugation, washed with water, and then subjected to an immobilization step.

Suspend the cultured washed bacterial cells in a 2% sodium alginate solution so that the bacterial cell concentration is 20%, fill this solution into a 50 ml syringe, and drip. This suspended bacterial body fluid was mixed with 0.1M CaCl ₂ that had been stirred in advance.
Immobilize in solution. The obtained immobilized Candida cells were shaken for 2 hours in a 0.1M CaCl ₂ solution and then subjected to reaction. The obtained immobilized microorganism was added to 100 ml of glucose at a concentration of 10%.
Example 1 using 20mM phosphate buffer (PH7.0)
The reaction was carried out according to the following. The reaction solution was replaced every 24 hours. The average citric acid concentration in each exchange solution is
It was 100mg/dl.

Example 3 The immobilized Aspagillus cells obtained according to Example 1 were packed into a continuous fluidized bed column, and 100 ml of 20 mM tartrate buffer (PH 3.0) containing 6% sucrose was added.
The reaction was operated at a rate of 4 ml/hr under aerated conditions. As a result of collecting the effluent reaction product liquid from 4 hours after the start of the reaction operation, a solution with a citric acid concentration of 3 mg/ml was obtained.

Claims (1)

[Scope of Claims] 1. An immobilized microorganism characterized in that a microorganism having a citric acid-producing ability selected from the genus Aspergillus or Candida immobilized on a gel-like carrier is brought into contact with a sugar solution. Method for producing citric acid by. 2. A method for producing citric acid using an immobilized microorganism according to claim 1, which uses a carrier selected from polyacrylamide, carrageenan, and alginic acid. 3. A method for producing citric acid using an immobilized microorganism according to claim 1, which uses glucose, sucrose, maltose, and extracts of natural products containing these sugars as the sugar solution. 4. A method for producing citric acid using an immobilized microorganism according to claim 1 or 3, in which the immobilized microorganism is subjected to a contact reaction while continuously or intermittently replenishing a sugar solution.