JPS61100193A - Preparation of immobilized enzyme, immobilized microorganism and group of immobilized microorganism - Google Patents

Abstract

PURPOSE:To produce an immobilized product resistant to the attack of microorganisms, having high gel strength, and formable to an arbitrary form, at a low cost, by gelatinizing a mixture of an aqueous solution of polyvinyl alcohol and an enzyme or microorganism, etc. in a saturated aqueous solution of boric acid. CONSTITUTION:One or more kinds of enzymes, microorganisms or microbial groups are mixed with an aqueous solution of polyvinyl alcohol, and the mixture is gelatinized in saturated aqueous solution of boric acid to obtain the enzymes, etc. included and immobilized in the monodeol lattice of polyvinyl alcohol-H3 BO3. The polyvinyl alcohol is preferably a partially saponified polyvinyl alcohol having a polymerization degree of 1,000-2,000 and a saponification degree of 87-89%, or a completely saponified polyvinyl alcohol having a saponification degree of >=98%.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for producing an immobilized enzyme, an immobilized microorganism, and an immobilized microorganism group, and in particular, a method for producing an immobilized enzyme, an immobilized microorganism, and an immobilized microorganism group. The present invention relates to a method of immobilization in a type of polymer lattice.

(Conventional technology) Since the early 1950s, enzymes have been bonded to carriers and cross-linked.

There has been a lot of research into fixing it using comprehensive methods, and some of it has already been put into practical use. Since 1975, so-called "%1hole Ce1l Entrapme" has been developed, which entraps microorganisms themselves instead of enzymes into polymeric materials.
nt” (hereinafter abbreviated as WCE) has started to attract attention.

WCE using various polymer materials has been reported.

Typical polymeric materials that have been reported so far that can be used for WCE include acrylamide, carrageenan, sodium alginate, and agar.

Acrylamide has been featured in many studies due to its low cost compared to other polymeric materials. However, it has disadvantages such as not being able to be formed into a spherical shape which is advantageous for continuous processing, and requiring rapid immobilization because the acrylamide monomer is toxic to microorganisms. -Carrageenan is used as a polymeric material for immobilizing yeast in experimental plants for ethanol fermentation because it has low toxicity to microorganisms and can be formed into spherical shapes.

However, natural carrageenan has a λ−
Since it contains carrageenan, it must be separated, which has the disadvantage of increasing costs.

Sodium alginate has the advantage of being cheap and can be formed into a spherical shape, but it has the advantage of being cheap and can be formed into a spherical shape, but it
+ There is a drawback that the gel strength becomes unstable in the presence of cations essential for microorganisms such as K+. Furthermore, agar is 1
Although it can be molded into a spherical shape, it has the disadvantage of weak gel strength.

In this way, the WC that has been developed and reported so far
All of the polymer materials for E use have fatal drawbacks, such as low price, * This is why the development of polymer materials that are less toxic to living things and have strong gel strength is awaited.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and aims to be inexpensive, non-toxic to microorganisms, and capable of being molded into any shape. An object of the present invention is to provide a high polymer material for WCE, an immobilized enzyme, an immobilized microorganism, and a method for producing an immobilized microorganism group. Another object of the invention is to
The purpose of the present invention is to provide a method for easily immobilizing enzymes, microorganisms, and microorganism groups under mild conditions using a high-profile polymer material for WCE that has strong gel strength (and can achieve rapid immobilization).

(Means for Solving the Problems) The method for producing an immobilized enzyme, an immobilized microorganism, and an immobilized group of microorganisms of the present invention includes mixing at least one of the enzyme, the microorganism, and the group of microorganisms with an aqueous polyvinyl alcohol solution. By gelling in a saturated boric acid aqueous solution,
The above object is achieved by entrapping and immobilizing at least one of an enzyme, a microorganism, and a group of microorganisms in the motupur lattice of polyvinyl alcohol-H5BOs.

The polyvinyl alcohol (PVA) used in the present invention has a degree of polymerization of soo to a, ooo, preferably 1,00.
0-2,000. Partially saponified poval with a saponification degree of 70% or more, preferably 87% to 89% and/or completely saponified poval with a saponification degree of 98% or more. It takes too much time, and if the temperature is too high, the viscosity will increase, causing problems in handling. 3-40% by volume of such PVA, preferably 10
~20% by volume and an appropriate amount of enzymes, microorganisms, and/or microorganisms of 50% by weight or less based on the PVA are uniformly mixed and stirred, and this mixture is shaped into 9 spheres, threads, or plates in a saturated tisaos solution. Gel and shape. By maintaining this gel in a saturated ustsos solution for 1 to 48 hours, the desired water-resistant immobilized enzyme, immobilized microorganism, and/or group of immobilized microorganisms can be obtained. There is no particular restriction on the processing temperature (a temperature that is advantageous for the enzyme, microorganism, and microorganism group is applied. The enzyme, microorganism, and microorganism group need not be particularly special (although any known temperature may be applied depending on the purpose of use). For example, when the system obtained by the method of the present invention is applied to the treatment of wastewater, enzymes or microorganisms that have phenol decomposition activity are used. For example, activated sludge microorganisms, anaerobic digestive bacteria, photosynthetic bacteria, etc. are used.Other enzymes include amylase, β-glucosidase, cellulase, and protease.

Other enzymes, etc., alcohol fermentation yeast, nitrifying bacteria, denitrifying bacteria, etc. as microorganisms, and activated sludge as microorganisms,
A very large number of microbial groups can be used, such as anaerobic methane fermentation, fiber fermentation, water/sewage/industrial wastewater treatment microbial groups, etc.

Gelled PV with H3BOs obtained as above
Polymer A is extremely stable and strong, and even after continuous use for half a year, the activity of immobilized microorganisms, immobilized enzymes, and immobilized microorganism groups does not decrease, and the gel strength does not decrease. If a small amount of casein, aluminum salt, Cu salt, titanium ion, etc. are mixed in, these will cause a bond with PvA molecules, so that a gelled PVA polymer with even higher gel strength can be obtained.

According to the method of the present invention, no particularly harsh treatment conditions are imposed, and therefore most known enzymes can be used.

Microorganisms, aerobic and anaerobic activated sludge, and other microbial groups can be immobilized at low cost with almost no reduction in activity, and the activity can be maintained for a long period of time.

(Example) One embodiment of the present invention will be described below.

Daitome Kanjo PVA (degree of saponification 98-99% 1 degree of polymerization 1700)
(7) 166g of 20% by volume solution, fill and
Thoroughly mix and stir 55 g of a concentrated mixed solution (sludge concentration 10 g/l) of activated sludge obtained by culturing using the draw total oxidation treatment method.

Add approximately 300 to 400 cc of saturated boric acid (83
803) Added solution. In this way, the PVA-activated sludge mixture is gelled. The obtained gel was stretched into a plate shape with a thickness of 2 mm, and this was left in a saturated boric acid solution for 5 hours. The plate-shaped gel was cut into small pieces of 5 tm x 5 dragons x 2 dragons, and then washed with tap water. The PV obtained in this way
Using A-immobilized activated sludge, synthetic sewage mainly containing meat extract and peptone was treated in a sewage treatment device as shown in the figure. In this device, sewage flowing in from above is aerated in an aeration reaction section (capacity 0.61) 1 in which the fixed activated sludge 100 is arranged.

The aerated water passes through a settling tank 2 and flows out of the system from a treated water outlet 3. Aeration is performed by blowing compressed air into the aeration reaction section 1 via a flow meter 11 from an aeration method 10. The operating conditions are shown in Table 1. Table 2 shows the processing results in steady state.

Table 1 TOC (Total Organic Carbon) - Stable TOC removal of approximately 90% could be achieved despite fluctuations in volume loading. Since the sample sludge contained nitrifying bacteria, it was expected that the nitrification reaction would occur to a considerable extent even in highly loaded liquids, but only less than 5■/2 of N03-N was detected in the treated water. .

Rather, TN (total nitrogen) was removed with a high efficiency of 43-50%. This is due to the denitrification reaction occurring in the anaerobic region inside the immobilized activated sludge.

It is understood that by immobilizing the activated sludge, the nitrification reaction by the nitrifying bacteria contained therein and the denitrification reaction occurring inside the immobilized activated sludge are balanced, and nitrogen removal can be performed efficiently.

As a result, by entrapping and immobilizing activated sludge in the PVA-H3BO3 monodiol polymer lattice,
Continuous operation in high load range is possible; TOC is 90%
It has become clear that 40 to 50% of TN can be removed; and 2) there are fewer problems in solid-liquid separation in a sedimentation tank.

U-Example - PVA (Saponification degree 98-99% 9 Polymerization degree 1, 700
) and 10 g of a 10% solution of Acinetobacter sp.
/ j! Cell concentration) 3.3g was thoroughly mixed and stirred, and this was added to the slowly stirred saturated HJO: + solution 1!
A spherical gel (3 to 4 cranes in diameter) was obtained. After slowly stirring this spherical gel in saturated H, BOj for 24 hours.

Washed with tap water. The PVA-microorganism gel thus obtained was activated in an inorganic salt medium containing phenol for 5 days, and its ability to decompose phenol was examined.

This immobilized Aclnetobacter was placed in 200 cc of a medium with a phenol concentration of 1000 μ/l, and aeration was performed to perform a phenol decomposition test. 1000■/
l concentration of phenol reduced this immobilized Ac1 in 2.6 h.
netobactersp.

It was found that this phenol decomposition activity increases as the activation time increases. Ac1neto inside PVA gel
bacter sp.

This is for the purpose of growing. Additionally, immobilized Ac1netob
Actersp, and non-immobilized Aclnetobacterium
When the phenol decomposition activity was compared with that of the immobilized Ac1neto-bacter, approximately 50%
was found to work effectively within the immobilization gel.

Cultured cells of Aclnetobacter, a major phenol-degrading bacterium, were crushed in a French press, and a cell extract obtained was concentrated by salting out ammonium sulfate. 3 g of this crude enzyme solution was mixed with 10 g of the same 10% PVA solution as in Example 2, and the same treatment as in Example 2 was carried out to obtain an immobilized enzyme of the phenol-degrading bacterium Aclnetobacter. When this was brought into contact with 200 cc of phenol at a concentration of 1000 .mu./1 at 30.degree. C., the phenol was completely decomposed after 4 hours.

(Effects of the Invention) As described above, the present invention is a method for easily and quickly immobilizing enzymes, microorganisms, and microorganism groups by incorporating them into inexpensive PVA H3BOi motupur-type polymers under mild conditions. , the resulting immobilized enzyme.

Immobilized microorganisms and groups of immobilized microorganisms can maintain desired activities of enzymes and microorganisms for long periods of time.

The immobilizing polymer material is PVAH3BO3.

Moreover, the immobilization conditions are mild and nothing special.

The enzymes, microorganisms, and microorganism groups to be immobilized do not need to be special; they can be any known enzyme.

Microorganisms and groups of microorganisms may be applied.

4. The figure is a schematic diagram showing a sewage treatment experimental apparatus using immobilized activated sludge obtained by the method of the present invention.

1... Aeration reaction section, 2... Sedimentation section, 3... Treated water outlet.

10... Diffusion method, 100... Fixed activated sludge.

that's all

Claims (1)

[Claims] 1. At least one of the enzyme, microorganism, and microorganism group is mixed with a polyvinyl alcohol aqueous solution and gelled in a saturated boric acid aqueous solution, so that at least one of the enzyme, microorganism, and microorganism group is mixed with polyvinyl alcohol. A method for producing an immobilized enzyme, an immobilized microorganism, and a group of immobilized microorganisms, which comprises entrapping immobilization in the monodeol lattice of H_3BO_3.