JPH0638754A - Production of immobilized biocatalyst using polyvinyl alcohol - Google Patents

Abstract

PURPOSE: To readily produce a immobilized biocatalyst having high water resistance and mechanical strength and excellent in biological activity in a short time at a low cost.

CONSTITUTION: In production of immobilized biocatalyst of an microorganism or an enzyme by polyvinyl alcohol by subjecting a mixture comprising polyvinyl alcohol and a microorganism or an enzyme to gelation treatment in 3 wt.% to saturated aqueous solution of boric acid to produce gel globes, the resultant gel globes are immersed into 3-20 wt.% phosphoric acid or phosphoric acid salt aqueous solution to cure these gel globes.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing an immobilized biocatalyst using polyvinyl alcohol, which is useful in the field of wastewater treatment and the biochemical industry.

[0002]

2. Description of the Related Art Techniques for encapsulating microbial cells or enzymes using various natural or synthetic polymer materials have been attracting attention since the 1980s, and are actually applied to industrial production. There are quite a few examples that have been successful. For example, high fructose syrup,
Examples include production of biochemical products such as 6-APA and L-amino acid. Typical polymer materials that are commonly used for immobilization carriers include polyacrylamide, κ-carrageenan, sodium alginate, and agar. Polyacrylamide is cheaper than other polymers and is often used, but it is difficult to make spherical granules suitable for general continuous reactors, and both the single molecule and the polymerization accelerator are toxic, Not suitable for immobilizing microbial cells. κ-Carrageenan is easy to mold and has low toxicity, but its main drawback is that it is expensive. Although sodium alginate is low in cost and easy to be spherical, the reaction solution containing cations such as phosphate, sodium and potassium may cause the gel strength to be considerably unstable and may be severely disintegrated. Also, the mechanical strength of agar gel is too weak,
Not suitable for long-term operation. Therefore, in order to effectively apply the immobilization technology of microbial cells to the industrialization process of biochemical products, especially in the field of wastewater treatment, which has recently attracted attention, it is necessary to deactivate the biochemical activity of microorganisms and enzymes. A low cost, high mechanical strength carrier material and its manufacturing method must be developed.

Polyvinyl alcohol (PVA) is a water-soluble resin produced by polymerizing and alcoholizing vinyl acetate alone. Polyvinyl alcohol is recognized as non-toxic and harmless to the human body, is an industrialized polymer raw material that is easy to mold, has high mechanical strength, and can be mass-produced at low cost, and is therefore very suitable as a carrier for immobilized biocatalysts.

In recent years, a number of microorganism immobilization techniques using PVA have been disclosed in European and American patent publications. For example, a method in which a PVA aqueous solution and a microorganism are mixed and then gel immobilization is carried out by freeze-vacuum drying or freeze-thawing (JP-A-57-14129 and JP-A-61-13938).
5), a method of forming a gel having a photocrosslinking structure by irradiation with ultraviolet rays (JP-A-1-454372), a method of mixing a PVA aqueous solution and a microorganism and bringing the mixture into contact with a saturated boric acid aqueous solution to form a gel (JP-A-61-161). 100193) and the like. According to the above method, a gel having high strength can be obtained and can be used as a carrier for immobilizing microorganisms and enzymes, but there are still many drawbacks to be improved. In the freeze-drying method, the material is kept in a frozen state at a low temperature of -30 to -80 ° C, and then dehydrated and dried to a certain water content. Not only it takes time, but the operation is complicated and consumes energy. The photocrosslinking method is often used when forming a film, and is not suitable in the general immobilization process of cells and enzymes. Also, PV
In the A-boric acid method, a gel of considerable strength can be obtained only by contact-immersing the PVA-microorganism mixture in an aqueous boric acid solution for 12 to 24 hours, and only a weak gel can be obtained in a short time. .

The disadvantages common to the prior arts are that (1) the immobilization process requires a long time, the operation is complicated, and the cost increases and the productivity decreases unless mass production is performed in a large production plant. And (2) low temperature and vacuum conditions and boric acid are both poorly suited to the microbial survival environment. In particular, boric acid is toxic and its microbial activity decreases during the long manufacturing process.

On the other hand, Kiyoyuki Kitano (Japanese Patent Laid-Open Nos. 64-5460 and 64-5491) improves the above-mentioned drawbacks by a method of bringing a mixture of PVA and microbial cells into contact with a sulfuric acid solution to shorten the time required for immobilization. did. However, the concentration of the gel solution used is considerably high, and it is necessary to use 30% sodium sulfate or 70% ammonium sulfate solution. If the concentration of the gel solution is too low, it will be difficult to form into granules and the strength will be weak. It has drawbacks. That is,
The cost of the material is high and the high concentration of salts is used in the gelling process, which adversely affects the biochemical activity of the microorganism.

[0007] Therefore, by overcoming all of the above-mentioned drawbacks, microorganisms and enzymes can be easily immobilized on PVA in a short time at low cost without lowering the activity thereof, resulting in high water resistance and mechanical strength and excellent biochemical activity. There is a demand for a method of obtaining immobilized microorganisms or immobilized enzymes (hereinafter, these are collectively referred to as immobilized biocatalysts).

[0008] As a result of intensive studies conducted by the present inventors in view of such a situation, it was found that, for example, molding was carried out in a short time with a 7% boric acid solution and further strengthened with a 5% phosphate solution. In other words, the two-step gelation with boric acid and phosphoric acid or phosphate makes molding extremely easy and in a short time, and is a cheap, non-biotoxic phosphorus source of microbial energy metabolism. By using a low concentration of acid salt, it is possible to obtain an immobilized biocatalyst with a gel strength superior to the conventional sulfate method at low cost and without lowering biochemical activity of microorganisms etc. And completed the present invention.

[0009]

[Means for Solving the Problems] That is, according to the present invention, a gel sphere is produced by subjecting a mixture of polyvinyl alcohol and a microorganism or enzyme to a gelation treatment with a boric acid solution having a concentration of 3% by weight to saturated boric acid. In the method for producing a biocatalyst for immobilizing microorganisms or enzymes with polyvinyl alcohol, the gelation time is 10 minutes to 2 hours, and the obtained gel spheres are added to 3 to 20% by weight phosphoric acid or phosphate aqueous solution. The present invention relates to a method characterized by dipping for 30 minutes or more to cure.

In the present invention, a mixture of polyvinyl alcohol and a microorganism or an enzyme is subjected to gelation treatment with a boric acid solution having a concentration of 3% by weight to a saturated aqueous solution of boric acid to form a gel sphere. In the method for producing an immobilized biocatalyst, an aqueous boric acid solution containing 3 to 20% by weight of phosphoric acid or a phosphoric acid salt is used as the aqueous boric acid solution, and the gelling and curing treatment time is 30 minutes to 3 hours. The present invention relates to a method characterized by simultaneously performing curing and curing.

[0011]

EXAMPLE The present invention provides an improved method for producing an immobilized biocatalyst with polyvinyl alcohol, in which a mixture of polyvinyl alcohol and a microorganism or an enzyme is gelled with a boric acid aqueous solution having a concentration of 3 wt% to saturated. In the method for producing a biocatalyst in which a microorganism or an enzyme is immobilized by polyvinyl alcohol, which is treated to produce gel spheres,
It is characterized in that gel spheres are hardened using a 20% by weight phosphoric acid or phosphate aqueous solution. Curing of gel spheres with aqueous phosphoric acid or phosphate solution takes time for gelling treatment.
It is carried out for 10 minutes to 2 hours by a method of further immersing the obtained gel sphere in an aqueous solution of phosphoric acid or a phosphoric acid salt of 3 to 20% by weight for 30 minutes or more to cure it.

The principle and characteristics of the method of the present invention are that PVA is converted into PV
After molding by the ionic cross-linking action between the hydroxyl group in the molecule A and the boric acid molecule, the obtained gel which is not so strong is immersed in phosphoric acid or phosphate aqueous solution to obtain PVA and phosphoric acid or phosphate aqueous solution. By curing by the esterification reaction of 1), an immobilized biocatalyst having high water resistance and mechanical strength and hardly losing biochemical activity of microorganisms or enzymes can be obtained. The manufacturing flow chart of the present invention is as shown in FIG.

The PVA used in the present invention has a saponification degree of
70 ~ 98% or more, polymerization degree 1000 ~ 3000, saponification degree 95 ~ 98% or more, polymerization degree 1500 ~ 20
00 is preferable. If the degree of polymerization is too low, the gel will become unstable, and if it is too high, the viscosity will increase, making it difficult to process. PVA is mixed with microorganisms or enzymes in the form of an aqueous solution, with a suitable PVA concentration being 10-20% by weight. The gelling treatment time with boric acid is preferably about 15 to 30 minutes when a saturated aqueous boric acid solution is used.

The preferred concentration of the phosphoric acid or phosphate aqueous solution is 5 to 15% by weight, and an immersion time of about 1 to 2 hours gives an ideal product. Sodium phosphate, sodium dihydrogen phosphate, potassium phosphate, ammonium phosphate or the like can be used for the aqueous phosphate solution.

Another preferred method for carrying out the method of the present invention is to mix boric acid with phosphoric acid or an aqueous solution of phosphoric acid, that is, the aqueous boric acid solution contains 3 to 20% by weight of phosphoric acid or phosphoric acid. This is a method in which an aqueous solution of boric acid is used and gelation and curing are simultaneously performed. Preferably, the aqueous boric acid solution contains saturated boric acid and 5 to 15% by weight of phosphoric acid or phosphate. The gelling and curing treatment time is 30 minutes to 3 hours, preferably 1 to 2
It's time.

The mixing weight ratio of the polyvinyl alcohol aqueous solution used in the present invention and the microorganism or enzyme aqueous solution is preferably 1: 2 to 2: 1.

The feature of the method of the present invention is to first build a gel basic structure in an aqueous boric acid solution in a short time, and since the contact time is short at this time, the damage of boric acid to microorganisms or enzymes can be reduced to the minimum. Yes (Japanese Patent Laid-Open No. 61-
According to Hashimoto's boric acid method in 100193, a contact time of 15 to 24 hours was required). further,
In the gel hardening process, it is to use a phosphoric acid or phosphate aqueous solution having a buffering action.Phosphorus element is not only harmless to microorganisms because it is also an essential element for energy metabolism of microorganisms, it also activates the microorganisms. Have the effect of

The PVA gel produced according to the present invention can be applied to the entrapping immobilization technology for enzymes, industrial microorganisms, wastewater treatment microorganisms, animal and plant cells and the like. Examples of the enzyme include amylase, cellulase, protease, glucosidase and the like. As the microorganism, bacteria, fungi, algae, protozoa or a mixture thereof can be used,
Examples of microorganisms include alcohol-fermenting yeast, nitrifying bacteria, denitrifying bacteria and activated sludge, anaerobic digestion sludge, methanated sludge, and denitrification sludge. Specifically as activated sludge microorganisms, activated sludge microorganisms was conditioned with agricultural or industrial waste water and the like, and further, as is the specific organism, brewer's yeast (Saccharomyces cerevisiae (Saccharomyces cere
visiae )), steroid-converting bacteria (Arthrobacter)
Simplex ( Arthrobacter simplex ) and the like. All of the above are applicable objects of the PVA gel method of the present invention.

Example 1 20 g of a 15 wt% PVA (saponification degree of 99% or more, polymerization degree of 2000) aqueous solution and a concentrated solution of denitrification sludge (denitrification sludge microorganisms were obtained from a denitrification tank in a biological denitrification process in a laboratory). The sludge concentration is
20 g (which is 50 g / l) are mixed and this PVA-sludge mixture is added dropwise to a gently stirred saturated aqueous boric acid solution to form a spherical gel with a diameter of about 3 mm and left in the solution for 20 minutes. Then, it was taken out and immersed in an 8 wt% sodium dihydrogen phosphate solution for 40 minutes, and finally the gel granules were taken out and washed with water. 20 g of the obtained immobilized microbial carrier was mixed with 80 ml of artificial wastewater containing potassium nitrate and methanol as main components (concentration of nitrogen nitrate 100 ppm, methanol 350 ppm), put in a 125 ml serum bottle, and batched under anoxic conditions. Performed denitrification. After culturing for 2 hours, the concentration of nitrogen nitrate in the artificial waste water dropped to 32 ppm.

The batch denitrification test was repeated using the same immobilized microbial carrier. As a result of measuring the denitrification rate of immobilized microorganisms by exchanging artificial wastewater of the same component every day, on the 7th day
After reaching 0.65 mg N / g-gel / h, all of the continuous operations up to the 30th day maintained this rate, and it was found that the biochemical activity was fairly stable. On the other hand, immobilized microbial cells having the same sludge content were produced by the method of Hashimoto Sho (JP-A-61-100193), and the same experiment as this example was conducted under the same conditions. As a result, after culturing for 2 hours in batch denitrification, the concentration of nitrogen nitrate in the artificial waste water was 82 ppm. When batch denitrification was repeated, finally on the 15th day,
A denitrification rate of 0.55 mg N / g-gel / h was reached.

Example 2 A 20% by weight aqueous solution of PVA (saponification degree: 99% or more, polymerization degree: 2000) and a denitrifying sludge concentrate (sludge concentration 30 g / L) were mixed at a ratio of 1: 1.
Were uniformly mixed (denitrification sludge microorganisms were obtained from the nitrification tank of the biological denitrification process in the laboratory). This PVA-sludge was subjected to the microorganism immobilization step as in Example 1.

Using the thus obtained immobilized microbial carrier (particle size: 3 mm), an artificial wastewater containing 200 ppm of ammonia nitrogen was continuously introduced into a bioreactor having an operating volume of 10 L (inflow amount: 30 L /
As a result of continuous operation for 10 days at a carrier filling rate of 25% and an air flow rate of 20 L / min, the concentration of ammonia nitrogen in the effluent was 9 ppm, and 92% of ammonia nitrogen was converted to nitrogen nitrate.

Example 3 Pig farm wastewater (COD concentration 1500-2000 ppm, total nitrogen concentration
Concentrated sludge solution obtained by centrifuging activated sludge acclimatized at 200-300ppm for 1 month and 18 wt% PVA (saponification degree 99
%, The degree of polymerization is 2000) The aqueous solution is uniformly mixed at a weight ratio of 1: 1 and added dropwise to a mixed solution containing 5% by weight boric acid and 10% by weight sodium dihydrogen phosphate and immersed for 1 hour. Spherical gel granules with a diameter of 3 mm were formed. The obtained immobilized microbial carrier was treated in the same bioreactor as in Example 2 to treat swine farm wastewater. The operating conditions of the reactor were the same as in Example 2. After continuous operation for 20 days, the COD concentration of the effluent is 200
〜300ppm and total nitrogen concentration also dropped to 120〜180ppm.

Example 4 10 g of a 20 wt% PVA (saponification degree 99% or more, polymerization degree 2000) aqueous solution and brewer's yeast ( Saccharomyces cerevisiae )
10 g of the centrifugally concentrated solution (cell concentration: 30 g / l) was thoroughly stirred and mixed, and subjected to the same microorganism immobilization step as in Example 1. 3% by weight of 15 g of the obtained immobilized microbial carrier (particle diameter 2 mm)
As a result of mixing with 150 ml of a glucose-containing medium, transferred to a flask, and cultured by shaking at 30 ° C. for 8 hours, the alcohol production concentration in the culture was 10.2 g / l. On the other hand, when the free cells were cultured for 8 hours under the same cell amount and the same culture condition, the alcohol production concentration in the culture solution was 10.6 g / l.

Example 5 10 g of a 20% by weight aqueous solution of PVA (saponification degree: 99% or more, polymerization degree: 2000) and steroid-converting bacteria ( Arthrobacter simpl)
ex ) 10 g of the concentrated solution (cell concentration: 20 g / l) was thoroughly mixed with stirring and subjected to the same microorganism immobilization step as in Example 3. 15 g of the obtained immobilized microbial carrier (particle size: 2 mm) was mixed with 150 ml of a medium containing 0.2% by weight of hydrocortisone, and the volume was increased.
Transferred to a 500 ml flask and shake-cultured for Δ ¹ -dehydrogenation biochemical reaction of the steroid, and cultured for 5 hours. As a result, 90% of the substrate was converted to prednisolone.

Example 6 10 g of a 15% by weight aqueous solution of PVA (saponification degree: 99% or more, polymerization degree: 2000) was thoroughly mixed with 3 g of isoamylase and 2 g of β-amylase by stirring, and the same procedure as in Example 1 was conducted. It was subjected to an immobilization process. Obtained immobilized enzyme carrier (particle size 2 mm) 15
150 g of a substrate solution containing 50 g / l of liquefied amylase
The mixture was mixed with 1 and transferred to a flask having a volume of 500 ml, and the starch was hydrolyzed by shaking and stirring. As a result, 3 hours after the reaction, the maltose concentration was 41 g / l and the substrate conversion rate was 82%.

[0027]

According to the method of the present invention, an immobilized biocatalyst having high water resistance and mechanical strength and excellent biochemical activity can be easily produced in a short time at low cost.

[Brief description of drawings]

FIG. 1 is a production flow chart of a biocatalyst with immobilized microorganisms or enzymes of the present invention.

Claims (16)

[Claims] 1. A living organism in which a microorganism or enzyme is immobilized by polyvinyl alcohol which forms a gel sphere by subjecting a mixture of polyvinyl alcohol and a microorganism or enzyme to gelation treatment with an aqueous boric acid solution having a concentration of 3 wt% to saturated. In the catalyst production method, the gelation time is 10 minutes to 2 minutes.
The method is characterized by immersing the obtained gel sphere in an aqueous solution of 3 to 20% by weight phosphoric acid or a phosphate for 30 minutes or more to cure the gel sphere. 2. A mixture of polyvinyl alcohol and a microorganism or an enzyme is subjected to a gelation treatment with a boric acid solution having a concentration of 3% by weight to a saturated aqueous solution to immobilize the microorganism or the enzyme with polyvinyl alcohol to form gel spheres. In the method for producing a catalyst, an aqueous boric acid solution containing 3 to 20% by weight of phosphoric acid or a phosphoric acid salt is used as an aqueous boric acid solution, and the gelation and curing treatment time is 30 minutes to 3 hours.
A method characterized by simultaneously performing gelation and curing. 3. The method according to claim 1, wherein the boric acid aqueous solution is a saturated boric acid aqueous solution, and the gelling time is 15 minutes to 30 minutes. 4. The concentration of phosphoric acid or phosphate aqueous solution is 5
The method according to claim 1, wherein the time for immersing the gel spheres at -15% by weight is 1-2 hours. 5. The boric acid aqueous solution contains saturated boric acid and 5 to 15 parts.
A method according to claim 2 which contains wt% phosphoric acid or phosphate. 6. The method according to claim 2, wherein the gelling and curing treatment time is 1 to 2 hours. 7. The method according to claim 1 or 2, wherein the phosphoric acid or phosphate is selected from the group consisting of sodium phosphate, sodium dihydrogen phosphate, potassium phosphate, ammonium phosphate and phosphoric acid. . 8. The degree of polymerization of polyvinyl alcohol is from 1000 to
A process according to claim 1 or 2, wherein the saponification degree at 3000 is 70 to 98% or more and the polyvinyl alcohol is used as an aqueous solution of 10 to 20% by weight. 9. The degree of polymerization of polyvinyl alcohol is 1500 to
9. The method of claim 8, wherein the degree of saponification at 2000 is 95-98% or more. 10. The method according to claim 1 or 2, wherein the mixing weight ratio of the polyvinyl alcohol aqueous solution and the microorganism or enzyme aqueous solution is 1: 2 to 2: 1. 11. The method according to claim 1 or 2, wherein the microorganism is a bacterium, a fungus, an alga, a protozoan, or a mixture thereof. 12. The method according to claim 1, wherein the microorganism is an activated sludge microorganism. 13. The method according to claim 12, wherein the activated sludge microorganism is an activated sludge microorganism acclimatized with agricultural or industrial wastewater. 14. The method according to claim 1 or 2, wherein the microorganism is brewer's yeast. 15. The method according to claim 1 or 2, wherein the microorganism is a steroid-converting bacterium. 16. The enzyme according to claim 1, which is amylase, cellulase, protease or glucosidase.
The method described.