JP2524287B2 - Method for regenerating microorganism-immobilized carrier - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a biotechnology in which microorganisms are grown inside a carrier for immobilizing porous granular chitosan to produce useful substances such as ethanol fermentation, various organic acid fermentations, and physiologically active substances such as enzymes and peptides. The present invention relates to a method for regenerating a reactor system.

[0002]

2. Description of the Related Art As a method for immobilizing growing microorganisms, a method of encapsulating with natural polysaccharides such as sodium alginate and κ-carrageenan, and a method of encapsulating with a photocurable resin are generally known. However, such a comprehensive immobilization method
The sterilization operation is complicated, and since the gel structure of the carrier contains microorganisms, the substance permeation is poor, and the carrier cannot be regenerated, which is not suitable for industrial use on a large scale. Met.

In order to solve the above-mentioned drawbacks, in recent years, methods for retaining microorganisms inside fiber aggregates or porous carriers have been actively studied. This is an immobilization method that is extremely effective for sterilization and has excellent substance permeation and is very effective for immobilizing aerobic microorganisms.

In such an immobilization method, the microorganisms invade from the pores existing on the surface of the carrier and actively proliferate inside the carrier, while efficiently producing various target substances. However, if the inside of the carrier is contaminated due to mutation or deterioration of the microorganism or invasion of undesired microorganisms, it is necessary to remove and wash the microorganisms inside the carrier to regenerate the carrier. No method has been developed to deal with it, and the carrier is currently discarded.

[0005]

[0008] The present invention, physical strength of the immobilization responsible <br/> body, porous structure, without impairing the physical properties of the carrier for immobilizing such fixed Kano microorganisms, carrier It is intended to provide a method for efficiently removing and washing the internal microorganisms and regenerating the carrier in a reusable state.

[0006]

The present invention relates to a regeneration method in which a porous granular chitosan microorganism-immobilized carrier is treated with hot alkali and then with an enzyme. It is known that the cell walls of microorganisms are mainly composed of the substances shown in Table 1. ("New Edition Microbiology I" p85-116, Asakura Shoten, 1981)

[0007]

[Table 1]

The present inventors treated the microorganisms grown inside the carrier for immobilizing porous microorganisms in a hot alkali, washed them with water, and then treated them with an enzyme that dissolves the constituents of the cell wall of the microorganisms. It was found that the microorganism-immobilized carrier can be regenerated under mild conditions in which the microorganisms inside the carrier are dissolved and removed, and the physical properties of the carrier are not impaired.

The carrier for immobilizing porous granular microorganisms of the present invention is an organic substance-based immobilization carrier such as porous granular chitosan produced by the method disclosed in Japanese Patent Laid-Open No. 2-225539 previously disclosed by the applicant. Is preferred.

The method for regenerating the microorganism-immobilized carrier of the present invention comprises:
For the carrier to be regenerated, the carrier volume and double the amount of 0.1 to
An aqueous solution of 2N sodium hydroxide or potassium hydroxide is added, and immersion treatment is performed at 50 ° C to 80 ° C for 1 to 24 hours. After the microorganisms that have grown inside the carrier are killed and the enzyme is made to work easily, it is washed thoroughly with water to remove the alkali.

Next, the carrier treated with alkali is placed in an aqueous solution of cell wall lysing enzyme for treatment. Although it is preferable that the amount of enzyme to be added is large, it is usually possible to treat by adding an aqueous solution having an enzyme concentration of 100 u / ml or more to the carrier volume. The pH and temperature of the aqueous solution are adjusted to the optimum conditions for the enzyme, and the mixture is immersed and shaken for 1 to 24 hours.

As the enzyme to be used, mannosidase, glucanase, protease, chitinase and lichenase are used for yeast, chitinase, chitosanase, cellulase, glucanase, mannosidase and lichenase are used for filamentous fungi, and bacteria and actinomycetes are used. , Murein degrading enzyme that decomposes mucopeptide, lysozyme, chitinase,
Chitosanase, grayed Rukanaze, proteases are effective, used in combination with one or several.

After lysing the cell wall of the microorganism, the carrier is regenerated by completely removing the cell wall lysate, undissolved residue and enzyme by washing with water.

[0014]

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to this range. Various physical properties of the (A) immobilization carrier were measured by the following methods. Measurement of compression modulus rheometer NRM-2010J-CW (immobility Industry Co., Ltd.
The sample was packed in an adapter having a diameter of 1 cm and a depth of 2 cm, and a compressive stress when a rod having a diameter of 0.8 cm was pushed at a speed of 2 cm / min was measured and expressed in the form of elastic modulus. Measurement of Pore Diameter After freeze-drying the sample, measurement was performed with a scanning electron microscope, and the average value of 100 pores was calculated. Measurement of Pore Volume After freeze-drying the sample, the pore volume per 1 g of the dried sample was measured with a mercury press-in type Poiserizer Model 9310 (manufactured by Shimadzu Corporation).

(B) The number of yeasts in the yeast-immobilized carrier was measured by the following method. 1) takes support 5ml yeast immobilized graduated cylinder 50 ml, added 0.9% of chloride sodium c anhydrous solution 30 ml. 2) After crushing the carrier well with a glass rod, add 0.9% aqueous sodium chloride solution to bring the total volume to 50 ml. 3) After stirring well, wait for the carrier debris to settle, take the supernatant and count with a Tohma hemocytometer. The number of immobilized yeasts in the carrier and the number of yeasts remaining in the regenerated carrier were calculated by the following formula.

[0016]

[Equation 1] Number of yeasts in carrier [piece / ml-carrier] = (count value x 50 x
10 ⁶ ) / 5

(C) Yeast residual rate was calculated from the following formula.

[0018]

[Equation 2]

Example 1 To 350 g of chitosan having a deacetylation degree of 80% and an average molecular weight of 35,000, polyethylene glycol (molecular weight 2
50,000, 500 g of Sanyo Chemical Industry Co., Ltd. was added,
It was dissolved in a 3.5% acetic acid aqueous solution so that the total amount became 5.000 ml. This chitosan solution is 1% ammonia water, 20%
After a certain amount was dropped into a mixed solution of ethanol and 79% water to coagulate and regenerate, it was thoroughly washed with water until it became neutral, and porous granular chitosan 5,000 having an average particle diameter of 1.2 mm was used.
0 ml (wet) was obtained. After removing the water adhering porous granular chitosan, 3 at 25 ° C. in 0.5% acetic acid aqueous solution 5,000ml
After the immersion treatment for 0 seconds, it was immediately washed with water until it became neutral. The volume of porous granular chitosan after washing with water is 3,000.
ml.

To 1,000 ml of porous granular chitosan,
After adding 2,000 ml of water containing 0.06 epoxy equivalent / l of ethylene glycol diglycidyl ether, the mixture was reacted at 80 ° C. for 3 hours and then thoroughly washed with water.

Next, after thoroughly replacing the water contained in the porous granular chitosan with ethanol, the mixture was stirred in 2,000 ml of ethanol containing 1 mol of acetic anhydride at 25 ° C. for 12 hours,
The amino group was acetylated. In order to remove the acylating agent that has reacted with the hydroxyl groups of chitosan, after saponifying with 1N sodium hydroxide in the same volume as the carrier for 2 hours at 40 ° C, wash with water and remove the desorbed acylating agent sufficiently. Removed to 90
0 ml of porous granular chitosan microbial immobilization carrier (Sample 1) was obtained.

Then, using the sample 1, a yeast immobilization carrier was prepared by the following method. 1) Glucose 50 g / l, potassium dihydrogen phosphate 1 g
/ L, polypeptone 1 g / l, yeast extract 5 g / l, ammonium sulfate 1 g / l, magnesium sulfate heptahydrate 0.5 g / l in a yeast growth medium 100 ml adjusted to pH 7.0 Saccharomyces cerevisiae IFO-
0224 was inoculated. 2) Reciprocal shaking culture was carried out at 30 ° C. and 160 rpm for 20 hours. When the number of yeasts was measured with the hemacytometer of Thoma,
It was 1.5 × 10 9 cells / ml. 3) To an Erlenmeyer flask containing 500 ml of yeast growth medium and 100 ml of porous granular chitosan microorganism immobilization carrier, 25 ml of the yeast suspension obtained above is added. 4) After culturing with rotary shaking at 30 ° C. and 150 rpm for 24 hours, the medium was removed by suction. 5) Glucose 150 g / l, potassium dihydrogen phosphate 1
g / l, yeast extract 0.2 g / l, ammonium sulfate 1
g / l, magnesium sulfate heptahydrate 0.1 g / l, calcium chloride 0.05 g / l, copper sulfate pentahydrate 0.005
g / l, ferrous sulfate heptahydrate 0.005 g / l, potassium chloride 0.1 g / l, zinc sulfate heptahydrate 0.1 g / l
Add ml. 6) Shake culture at 30 ° C. and 50 rpm for 48 hours. 7) After 48 hours, the medium is removed by suction, 500 ml of a new medium for ethanol fermentation is added, and the same culture is performed for 48 hours. This operation is repeated once more. The yeast immobilization carrier (Sample 2) was obtained by the above operation.

Next, the yeast-immobilized carrier was regenerated by the following method. 1) Add 40 ml of 1N-NaOH to 20 ml of immobilized yeast. 2) Heat at 60 ° C. for 2 hours to heat the immobilized cells. 3) After removing the treatment liquid and thoroughly washing with water, 20 ml of pure water was added to the yeast cell wall lysing enzyme YL-15 (manufactured by Amano Pharmaceutical Co., Ltd.) 2
Add 00 mg and adjust pH to 7. 4) Allow to react at 30 ° C. for 12 hours. 5) After removing the reaction solution, thoroughly wash with water to regenerate the carrier (Sample 3)
I got Using 50 ml of the regenerated carrier thus obtained, a yeast-immobilized carrier was again prepared in the same manner as described above (Sample 4).

The physical properties of sample 1 are as follows. Compressive modulus 5.5 × 10 5 dyn / cm 2 Pore diameter 75 μm Pore volume 3.3 ml / g The number of immobilized yeasts in Sample 2 was as follows. Number of immobilized yeasts 1.1 × 10 10 cells / ml-Carrier The physical properties of the sample 3 and the yeast residual rate were as follows. Compressive elastic modulus 5.3 × 10 5 dyn / cm 2 Pore diameter 75 μm Pore volume 3.3 ml / g Yeast residual rate 10.1% The number of immobilized yeasts in Sample 4 was as follows. Number of immobilized yeast 1.0 × 10 10 cells / ml-carrier

As is clear from the above results, the carrier for immobilizing microorganisms according to the present regeneration method shows that the physical properties of the carrier can be determined from the results of the immobilizing carrier sample 1 and the regenerating carrier sample 3 before use. This is an excellent method because there is no deterioration and no change in the yeast immobilization capacity was observed between the yeast immobilization carrier sample 2 and the regenerated yeast immobilization carrier sample 4.

Comparative Example 1 After preparing an immobilized yeast using the porous granular chitosan obtained by the same method as in Example 1, the enzyme was simply used without the hot alkali treatment. The reproduction processing was performed by the method. 1) To 40 ml of immobilized yeast, 40 ml of pure water was added, and 400 mg of yeast cell wall lysing enzyme YL-15 was added to adjust the pH to 7. 2) React at 30 ° C. for 12 hours. 3) After removing the reaction solution, thoroughly wash with water. The compressed elastic modulus of the carrier after regeneration is 5.5 × 10 5 dyn /
cm 2, pore diameter is 75 μm, pore volume is 3.3 ml / g
The residual rate of yeast was 59%, and regeneration was not performed sufficiently. When the immobilized yeast was prepared again using 20 ml of the regeneration carrier, the number of immobilized yeast after regeneration was 6.5 × 10.
It was as low as 9 cells / ml-carrier.

Comparative Example 2 Immobilized yeast was prepared using the granular porous chitotosan obtained by the same method as in Example 1, and then only hot alkali treatment was performed without using an enzyme, and the following method was used. A regeneration process was performed. 1) Add 80 ml of 1N NaOH to 40 ml of immobilized yeast. 2) Heat at 60 ° C. for 2 hours to heat the immobilized yeast. 3) Remove the treatment liquid and wash thoroughly with water. Compressive elastic modulus after reproduction is 5.4 × 10 5 dyn / cm 2
The pore diameter was 75 μm, the pore volume was 3.3 ml / g, and the yeast residual rate was 65%, indicating that regeneration was not carried out sufficiently. When the immobilized yeast was prepared again using 20 ml of the regenerated carrier, the immobilized yeast after regeneration was 7.1 × 10 9 cells / ml.
-Low on carrier.

[0028]

EFFECTS OF THE INVENTION According to the present invention, since microorganisms grown inside the microorganism-immobilized carrier are treated with hot alkali and then treated with an enzyme that dissolves the cell wall of the microorganisms, most of the unnecessary microorganisms are removed, This is an excellent regeneration method in which the immobilization ability of microorganisms does not change before and after the regeneration and the physical properties of the carrier do not change at all.

Claims (1)

(57) [Claims] 1. A method for regenerating a microorganism-immobilized carrier, which comprises treating a porous granular chitosan microorganism-immobilized carrier with a hot alkali and then treating it with an enzyme.