JPS58107182A - Immobilization and proliferation of living microbial cell - Google Patents

Abstract

PURPOSE:To carry out the titled immobilization and proliferation process effectively, by drying a mixture of PVA, living microbial cells and an aqueous suspension of a specific clay mineral, at a low-temperature, adding a medium for the proliferation of microbials to the dried product, and extending the diameter of the proliferated microbial colony to a specific value. CONSTITUTION:An aqueous suspension containing (A) PVA having a saponification degree of >=95mol% and a viscosity-average polymerization degree of >=1,500, (B) a clay mineral having laminar structure and containing a three-layer (2:1-type) composite structure as the basic unit (e.g. bentonite), and (C) living microbial cells, wherein the concentration of the dissolved PVA is 1-15wt%, the concentration of suspended clay mineral is 0.7-15wt%, and the weight ratio of PVA to the clay mineral is 1/5-8/1, is dried at -6-+40 deg.C to a dehyration degree of >=15wt% and a water-content of 20-92wt% (wet basis), to obtain microbial cells embedded in a gel. A medium for the proliferation of microorganisms is added to the gel and the diameter of the formed colony of the proliferated microbial cells is extended to 30-250mum to obtain the titled immobilized and proliferated microbial cells.

Description

[Detailed Description of the Invention] The present invention relates to a method for immobilizing and propagating living microorganisms, and in particular,
By dehydrating an aqueous suspension containing polyvinyl alcohol, clay minerals, and viable microorganisms until a predetermined dehydration rate and water content are reached, the viable microorganisms are encapsulated (embedded) in the resulting gel. After fixation), it relates to a method for effectively propagating the same. The non-invention is to dry a mixed suspension aqueous solution of a specific concentration (composition) containing polyvinyl alcohol, viable microorganisms, and a specific clay mineral under specific conditions. Comprehensive (capture/
By supplying a growth medium thereto, the microbial cell enclosing cavity expands, and the growth medium is supplied so as to keep the size of this cavity within a certain limit (diameter 250 μfrL). This is based on the fact that it has been found that by controlling the amount, effective growth and utilization of immobilized living microorganisms within the gel can be achieved.

That is, the present invention uses polyvinyl alcohol with a saponification degree of 95 mol% or more and a viscosity average degree of polymerization of 1,500 or more, a laminated clay mineral whose basic unit is a three-layer type (2:1 type) composite layer, and microorganisms. Contains three types of living bacterial cells, and has a dissolved concentration of polyvinyl alcohol of 1 to 15 wt%,
The suspension concentration of the clay mineral is 0.7 to 15wtX, and the concentration ratio (weight ratio) of the polyvinyl alcohol and the clay mineral is 115
The suspended aqueous solution adjusted to ~8/1 is dried at -6 to +40°C, with a dehydration rate of 15 wt% or more and a water content of 20 to 92 w.
t% (wet body standard) and include living microorganisms (
Embedded gel) is obtained, a microorganism growth medium is supplied to this gel, and the diameter of the grown microorganism colony formed thereby is expanded by 1 to a range of 30 μm to 250 μm. This provides a method for immobilizing and propagating the body.

Attempts to capture (enclose) viable microorganisms in a gel and then fully proliferate it are already known, but as summarized in (1) to (6) below, each method has its drawbacks, and even better immobilization methods are needed. A chemical propagation method has been desired.

(1) Corynebacterium simplex (Cory
nebα-cterium simplex
er 5irILptez)), Bacillus subtilis, Pseudomona8pxtida
), etc., are immobilized in polyacrylamide gel, then supplied with nutrients (medium) and grown, and their respective activities, such as steroid conversion ability, α-amylase production ability, benzene oxidation decomposition ability, etc. There are known methods for increasing (Nature + 268, Oct.

28.796 (1976), Biotech, Bio
eng, +20.1267 (1978), Ev, ro
p, J, App t, Mi Crab io
l.

Biotechnol, +5.283 (1978),
Higashi, 75 (1977)).

However, in these cases, the rate of increase in activity due to proliferation is only about 5 to 10 times at most, and the raw material monomer (acrylamide) of this immobilization carrier is highly toxic (harmful to microorganisms) (Fermentation and Industry, 85. 92 (1977), Bi
otech.

Bioeng, +20.1267 (1978)),
Additionally, microorganisms (proteins) are damaged by the radical initiators used for immobilization (polymerization of monomers and crosslinking of polymers) (5science+ 142.67
8 (1968), Adv, Bi-ochem, Eng.
+ 5.125 (1977), Agr, 13io1゜Qhem, + 4 2, 688 (1978), Ihi →
Nicho 9, 43.269 (1979)), and the immobilizing gel itself is soft (Kagaku 5-gaku Kogaku, 40.189 (1976)), which is extremely unsatisfactory in practice.

(2) It has been widely practiced since ancient times to immobilize various microorganisms on agar and then grow them.
Although it has poor mechanical strength and can be used in the laboratory, it is difficult to use it industrially as an immobilization carrier.

In addition, it has been pointed out that cracks occur from inside the gel as microorganisms multiply (Biotech, Biogn
g, r 22.681 (1980)).

(3) Leuconostoc mesenteroides (Le
rbconos-toc mesenteroides
) f, polyvinyl a) L/ :I-There is also an attempt to propagate it after it has been incorporated into a boric acid complex gel (Japanese Unexamined Patent Application Publication No. 1983-1979)
185295). However, in this case, the boric acid complex (gel) produced from polyvinyl alcohol with a high degree of saponification is weak and difficult to mold. In this case, this difficulty can be considerably improved by using polyvinyl alcohol with a low degree of saponification, but since the gel has strong stickiness, it is difficult to maintain the shape after molding, making it impractical.

(4) Saccharomyces cerevisiae (ScLCCnα
There is also a proposal to proliferate romycesserevisiae) after immobilizing it on a gelatin membrane, but the activity increases only about 4 times, and the gel molded body is soft and its shape is unstable (Bjot- ech,Bioeng
, , 22-11785 (1980)).

(5) There have been attempts to immobilize Myces cerevisiae on calcium alginate gel and then grow it, but as is often the case with natural polysaccharide gels, it is prone to bacterial contamination, and if you pick it up with your fingertips, it will grow slightly. By applying finger pressure, it immediately loses its shape and becomes like a soft paste11, or the gel structure is destroyed by the phosphates supplied as a nutritional source (Eiotech, B
ioeng, 19.387 (1977)).

(6) Saccharomyces cerevisiae (gacch, ar
omyces ce-revisiae), Satucharomyces carlsbergensis (S, carlsber
gensis), Acetobacter suboxidans (Acetobacter 5ttboxydans)
), Serτatia rn
, arscescens), Nisierisia coli (Es
cherichia coli), Brevibacterium flavum
um), Streptomyces phaeochromogenes C
8treptomycesphaeochromoge
nes ), Brevibacterium ammoniagenes (
Erevibacterium ammoniagen
es) Y, f.

There is also an attempt to propagate these numbers after complete fixation. However, this gel is weak (tensile strength < 1 kg, 4♂)
, and even after curing treatment using potassium chloride, hexamethylene diamine, glutaraldehyde, tannin, etc., the strength is comparable to that of polyacrylamide gel (1 to 1
．． 5 kF! /cmF),
It is extremely weak. Therefore, as microorganisms multiply within the gel, the gel structure cracks and is destroyed (J
, 5solid panase Biochem, 2.22
5 (1977), Enzyme Microb
, Technol., 1.95 (1979), J.
, Ferment, Technol, +58, (4
) 827 (1980), Polymers, 29.288 (198
0)).

As seen in these examples, all of the conventional carriers for immobilizing microorganisms have problems, and even better carriers (gels) are being developed (Kobunshi, 29.288 (1980)).

Unlike these known methods, according to the present invention, a gel is generated in the process of drying a mixed suspension of polyvinyl alcohol, clay minerals, and viable microorganisms, and almost all of the viable microorganisms are - encapsulated in this gel. In the present invention, in this immobilization (inclusion) process, acids, alkalis, radiation, radical generators,
No organic solvents, reaction reagents, etc. are used, and no secondary curing treatment is required. The gel of the present invention is a rubber-like elastic body that is highly water-containing and has excellent permeability to carbon sources, nitrogen sources, oxygen gas, carbon dioxide gas, and other inorganic substances required for the activity of living microorganisms, and has a high tensile strength. At the same time, the carrageenan (〈1klil/clrL”)
Carrageenan with secondary hardening treatment (1-1.5 kl
i'/cm2) 'e surpassing strength (>2 to 9/m") k
have

By supplying a growth medium to this gel, living microorganisms naturally proliferate, but in the gel of the present invention, the microorganism enclosing cavities are expanded as a result of this proliferation.

However, because the gel of the present invention is extremely elastic and has excellent mechanical strength, it has undergone rapid proliferation, with the diameter of the cavity within the gel reaching 8 to 30 times (250 μm) the initial diameter (8 to 80 μm). It has the feature that it can withstand (swelling) and the gel structure is not destroyed.

However, in the gel of the present invention, the diameter of the proliferating colony is about 8
When it reaches 0 μm, some of the proliferating bacterial cells pass through at least a part of the mesh of the cavity wall (gel tissue consisting of a micro-network structure) and are discharged from the cavity, and continue to proliferate at the discharge position (small cavity).・The discharge process is repeated, and finally, it is expelled from the gel through the cavity near the gel surface.

Furthermore, when the diameter of the growing colony exceeds about 250 μm by supplying a growth medium, the amount of bacterial cells shed increases significantly.

Therefore, in order to avoid the loss of proliferating bacterial cells,
It is preferable to keep the diameter of most of the proliferating bacterial colonies to 80 μm or less. By the way, generally, at the same time as living microorganisms multiply, at least a portion of them die. Since it is undesirable for this autolyzed residual content (mainly cell wall constituents) of dead and sterilized bacteria to continue to accumulate in viable bacterial colonies, the gel of the present invention also allows this to be added intermittently or continuously to the gel. must be removed outside. However, since there is still no known method for selectively expelling only dead bacteria (residues from autolysis) out of the gel, it is unavoidable to remove part of the bacterial colony consisting of live and dead bacteria. , intermittent'-1: or continuously must be allowed to flow out of the gel. In this regard, in the present invention, the above object can be easily achieved by achieving the total colony diameter of 80 μm or more. Furthermore, if the colony diameter exceeds 250 μmf, this is not a good idea as it will lead to a large amount of the proliferating microbial cells being washed away.

Therefore, in the present invention, the diameter of the bacterial colony is set to 250 μm.
m or less, for example, 30 to 250 μm, to avoid useless loss of a large amount of proliferated live bacteria and to effectively proliferate all the immobilized live bacteria, and to utilize this, as well as to remove dead bacteria as necessary. Can be discharged.

The diameter of the bacterial colony in the present invention can be easily controlled, for example, by monitoring the expansion status of the colony and controlling the total amount of growth medium supplied.

The present invention uses polyvinyl alcohol and clay minerals together.

Regarding the combined use of polyvinyl alcohol and clay minerals, certain effects different from the combined effects in the present invention are already well known. In other words, by spraying polyvinyl alcohol (diluted aqueous solution) on the surface soil of the stadium,
There are attempts to improve the soil to make it relatively less stiff. Also, a small amount of polyvinyl alcohol (diluted aqueous solution) to the field.
Attempts to improve soil permeability or water retention by spraying
Furthermore, a technique is also known in which a small amount of polyvinyl alcohol' is added to muddy water (clay suspension water) to promote coagulation and sedimentation of clay (colloidal particles).

In these cases, it has been confirmed that the action of polyvinyl alcohol causes changes in the dispersion of soil particles or in the particle size of the soil.13- However, at least outwardly, it is just soil, and it can be used not only in water but also in dry conditions. Even in powder form, it is extremely brittle and crumbles easily. It is also known that a hard film can be obtained by floating clay in an aqueous polyvinyl alcohol solution and drying it. However, this is a hard film with poor water absorption. The gel of the present invention is completely different from the conventional polyvinyl alcohol/clay composites known as soil conditioners or hard films mentioned above.

It is also well known that clay minerals such as montmorillonite, vermiculite, and synthetic taeniolite adsorb enzymes (or polyvinyl alcohol, etc.). However, it has been proven that this is a phenomenon in which enzymes and the like invade between clay mineral crystal layers, and the distance between the crystal layers is said to be 1.5 to 6 nm at most. Therefore, this adsorption crystal layer spacing is less than -14= the thickness of the cell wall of microorganisms (20 to 80 nm), and it is clear that microorganisms (1 to 10 μm) cannot penetrate into this layer at all. Furthermore, it is already known to use either polyvinyl alcohol or clay mineral as a carrier for immobilizing microorganisms. However, as summarized in (1) to (5) below, all of the conventionally known methods have significant drawbacks.

(1) A microorganism-containing film can be obtained by mixing a polyvinyl alcohol aqueous solution and microorganism cells and then air-drying the mixture.
This membrane is weak, has poor water resistance, and has a low microbial trapping capacity.

Although some proposals have been made to support (reinforce) this membrane with nylon cloth, most of the above-mentioned difficulties still remain. In addition, a characteristic of polyvinyl alcohol film is that it has low permeability to carbon sources, nitrogen sources, inorganic substances, etc. required for microbial activity, reducing the activity of immobilized microorganisms (Japanese Unexamined Patent Application Publication No. 1983-1972).
145592, Special Publication No. 55-35415, Plastic Materials Course 14, p. 185 (Sho 45), Nikkan Kogyo Shimbunsha).

(2) After mixing the polyvinyl alcohol water bath solution and microorganisms or enzymes, remove all oxygen and produce 60 ft of cobalt (gamma rays).
The method of crosslinking and gelling polyvinyl alcohol by irradiation is also well known. However, in this case, even if a radiation protection substance such as glycerin is used in combination, the negative effects on microorganisms cannot be completely avoided, the cost of irradiation increases, and the resulting gel is weak, so secondary treatments using other chemical reagents are often required. Requires curing treatment (Biotech).

Bioeng, + 15-1607 (1978),
Fermentation and Industry, 35.92 (1977)).

(3) It has been well known for a long time that when boric acid or a boric acid aqueous solution is added to an aqueous polyvinyl alcohol solution, it immediately gels, and methods for immobilizing microorganisms that utilize this principle have of course been attempted. However, the gel produced from highly saponified polyvinyl alcohol is weak and difficult to mold. By using polyvinyl alcohol with a low degree of saponification,
This difficulty can be improved slightly, but since it is a sticky gel,
For example, it is difficult for molded products such as cut pieces to maintain their shape (Japanese Patent Application Laid-open No. 185295-1983).

(4) A method for producing a polyvinyl alcohol/silicic acid composite yeast membrane has also been proposed by adding acid to an aqueous suspension containing polyvinyl alcohol, tetraethyl silicate, and microorganisms and drying in air, but this membrane is also weak. In this case, even if the membrane is freeze-dried after adding an acid, the mechanical strength of the resulting membrane is rather reduced, and it is almost impossible to mold it.

In any case, the step of adding acid to the microorganism suspension to adjust the pH to below 8 is included, and the negative effect on the microorganisms cannot be ignored.
80858).

(There have been attempts to completely physically adsorb microorganisms onto bentonite, kaolin, acid clay, activated clay, activated carbon, calcium phosphate, alumina, silica gel, zeolite, pumice, etc., but these have low adsorption capacity and are immobilized. The intense desorption after (adsorption) is often pointed out (Polymer, y 2, 828 (1967), Catalyst, Moon,
71 (1972), Chemical Engineering, 1 Port, 357 (1974)
), Chemical Industry, 1975.1206, Membrane, Mi (5) 8
89 (1978), Fermentation Association Journal, 28, 267 (196
5), Journal of the Organic Synthesis Association, Dandan, 471 (1970)).

These facts support that the present invention provides a technique for immobilizing live microorganisms that is completely different from conventional knowledge regarding the interaction between clay minerals and enzymes or between clay minerals and polyvinyl alcohol.

In the present invention, the degree of saponification is 95 mol% or more, preferably 97
Polyvinyl alcohol with a mole % or more is used. Said soil 18 - saponification degree of 80 to 88 mol%, which is considered preferable for improvement
Even if this polyvinyl alcohol is used in the present invention, only a soft gel similar to mud is produced.

Further, in the present invention, polyvinyl alcohol having a viscosity average degree of polymerization of 1.500 or more, for example 1,600 to 2.800, is used.

As the degree of polymerization of polyvinyl alcohol decreases, the mechanical strength of the resulting gel also decreases.
600) is recommended.

In the present invention, first, 1 to 15 ut of polyvinyl alcohol is used.
% aqueous solution. Although this concentration can be further increased, the viscosity of the aqueous solution at room temperature is 10,000 c.
In addition, it is difficult to handle as it may increase in viscosity or gel during storage. (and is completely different from the gel of the present invention).

The amount of polyvinyl alcohol to be used is 175 to 8 times the amount of clay mineral described later. If the usage ratio (weight ratio) of polyvinyl alcohol and clay mineral is low, for example, 1
/1o, the resulting gel has poor mechanical strength. Although the mixing ratio of polyvinyl alcohol and clay mineral is "/1000-'/100 in the above-mentioned soil improvement, the gel of the present invention can never be obtained under such conditions.

When the ratio of polyvinyl alcohol to clay mineral used is 10, a gel with poor water content is produced, which is not preferable as a carrier for immobilizing a microorganism medium box.

The type 3 clay mineral used in the present invention is bentonite, which is known as a general clay.
However, it is cheap and easy to obtain. This clay is tuff,
It is an aggregate of colloidal particles mainly composed of montmorillonite produced by the weathering of rhyolite, etc., and is found in various places such as Hokkaido, Akita, Yamagata, Niigata, Gunma, and Shimane. Montmorillonite, the main component of bentonite, is smectite (SMLgcti).
te), silica (tetrahedral structure), alumina,
It is a laminated structure whose basic unit is an 8-layer (2:1) composite layer of silica (tetrahedral structure).Moreover, a part of the aluminum that makes up all the composite layers is replaced with magnesium, so that the gaps between the composite layers are smooth. may contain water and cations such as sodium, potassium, calcium, lithium, strontium, barium, aluminum, cesium, magnesium, ammonium or hydrogen. For example, an approximate (representative) structural formula of montmorillonite is (At ygMy 7)
5iho,.

(Q B )2 ・Z H2O(K + Ha + C
c + Hr N & rug r Al + Lx +
Denoted as Cs*Sr,Ba)y. Nontronite (n
otLtronite (iron substitution), 21-hectorite (magnesium substitution), saponite (magnesium substitution), beideLlite (aluminum substitution), sauconite
t) (iron, magnesium, zinc substitution), volkonskite (chromium substitution)
These are also often found in the bentonite, also called montmorillonite group minerals.

Bentonite contains about 50 to 85% of montmorillonite and the above-mentioned montmorillonite group minerals, but also contains quartz, feldspar, zeolite, kaolin, illite (mica,
micα), cristobalite (cristovaLi)
te) 14 children are mixed. Therefore, although the composition (wt%) of bentonite is not constant, it is 5sOz 42~6
8, AJ2QB 14-28, H7011-23, ki
qO1-25, Fe, 0.0-4, NCL200-8.
5, Ca0O~8, KtOO, 1~0.7, Tie,
0-0.7, FeOO-0.3, 22-p2o, o-0.04 are common.

The Japanese Pharmacopoeia Law stipulates tests for bentonite's swelling ability and gel-forming ability (ability to form a mushy magnesium oxide/bentonite composite gel), but commercially available bentonite (reagent) usually does not pass this standard. . However, such bentonite can also be used as the clay mineral of the present invention without any problem. Bentonite also has sodium chloride, sodium hydroxide, sodium carbonate, sodium nitrate, ammonium hydroxide, sodium pyrophosphate, and sodium hexametaphosphate (sodium metaphosphate low weight) to reduce its swelling property, dispersibility, specific surface area, etc. It is often treated with aqueous solutions such as hydrochloric acid, sulfuric acid, and citric acid. In the present invention, it is not necessary to particularly perform such treatment on bentonite, but even if such treatment is performed, there is no problem in carrying out the present invention.

In the present invention, in addition to the above-mentioned bentonite, acid clay (thin clay, Kambara earth), activated clay,
Fullers earth, Florida earth, Georgia earth f can be used. These include kaolinite, which does not belong to either montmorillonite or the three-layer clay minerals similar to montmorillonite described below, and allophane (α1loph), which is considered an amorphous clay mineral.
αne) Although it contains a considerable amount of f, the main component is still montmorillonite group minerals.

As the clay mineral in the present invention, in addition to montmorillonite group minerals, three-layer type (2:1 type) clay minerals similar to montmorillonite described below can be used. That is, the pottery stones produced in the Nagasaki system (Goto mine), Okayama system (Tamaishi mine), and Nagano system (Honami mine, Yonago mine) are mainly composed of pyrophyllite. These clays are distinguished from montmorillonite clays in that they have extremely low magnesium content and show almost no swelling property, but they are 8-layered silica-alumina-silica (2:1
It is similar to montmorillonite in that it is a laminated structure based on shaped composite layers.

Talcum (ta) is produced in Hyogo, Okayama, Hiroshima, Yamaguchi, and Nagasaki regions.
lc) differs from montmorillonite in that it has a low aluminum content and a particularly high magnesium content, but it is a laminated structure based on a three-layer (2:1 type) composite layer of silica, alumina, and silica. .

Clay in the Kumamoto and Niigata regions contains many illites. this is,
Depending on the content of iron, fluorine, magnesium, etc.,
hydromica, gloconite (
glowco-nite), muscovite (rmbsc
It is subdivided into ovite, mica, iltite, and others, but all of them have a high potassium content and are distinguished from montmorillonite.

However, these are also three-layered types of silica, alumina, and silica (
2:1 type) clay mineral.

Vermiculite, which is mainly produced in Brazil, the United States (Pennsylvania), and India, has long been attracting attention in Japan as vermiculite and vermiculite, but it differs from montmorillonite in that it has a high magnesium content. However, it is similar to montmorillonite in that it has a laminated structure with three mausoleum-shaped composite layers of silica (tetrahedron)/alumina and magnesia (octahedron)/silica (tetrahedron) as its basic units.

In the present invention, any of the three-layered clay minerals described above can be used, and an artificially synthesized three-layered clay mineral can also be used. For example, taeniolite from southern Greenland contains potassium,
It belongs to illites rich in fluorine and magnesium, and can be obtained by melting a mixture of sodium fluoride, lithium fluoride, magnesium oxide, and silicon dioxide, and may be used in the present invention.

In the present invention, the above 8-layer clay mineral has a particle size of 00-15t.
It is best to use it as a powder with a size of +(100 mesh) or less. The bentonite mentioned above usually contains the majority (50-95%
) is dominated by particles finer than 74 μm (200 mesh) in diameter, especially the coarse clay content (0.2-2 μm) and the fine clay content (0.2-2 μm).
It is advantageous because it is rich in 0.2 μm). Talc (ta)
lc) is also used for cosmetics in the range of 150 to 270 mesh (0
, 1 to 0.05) is commercially available. 30 to 100 meshes of acid clay, activated clay, fuller's earth, pyrophyllite, illite, vermiculite, etc.
If granular products with a diameter of 0.59 to 0.15) are used, the mechanical strength of the resulting gel tends to be non-uniform;
It is best to use it by crushing it to a size of 50 or more.

In the present invention, the clay mineral (containing clay) powder is added to the polyvinyl alcohol aqueous solution and dispersed (suspended), or the clay mineral suspension water is prepared in advance,
This can be mixed into the polyvinyl alcohol aqueous solution.

It is also not advisable to add polyvinyl alcohol to the clay mineral suspension water to dissolve it. In any case, the concentrations of polyvinyl alcohol and clay mineral in the resulting aqueous suspension of polyvinyl alcohol and clay mineral are 1-1, respectively.
5 wtX preferably 2-18 wt% and 0.7-1
The content is 5 wt%, preferably 1 to 18 wt%, and the concentration ratio (weight ratio) of the polyvinyl alcohol clay mineral is 175 to 8/1, preferably 1/4 to 6, as described above. The aqueous suspension solution obtained in this way is not necessarily neutral; for example, in the case of bentonite, it often has a pH of 8 to 10, and in the case of activated clay and acid clay, it has a pH of 8 to 6.
shows. On the other hand, the growth and activity of the living microorganisms immobilized in the present invention each have their own optimal pH ranges. To give some examples, Esc. coli 7~'1
．． 5. Saccharomyces cerevisiae (ScLCC9c
'roTnyegs cg"r-evisiag)
5-7, Ashbyagoss
ypii) 6.8~? , 0, Bacillus subtilis (B
aci-11us swbtilis) 4.8-6.
8. Acetobacter acetosum
er acetoswm) 8.5-6.5, Trametas x-sanguinea (Trametas sangwine)
a) 2-6 etc. Therefore, the pH value of the above-mentioned suspended aqueous solution, the optimum pH value of the viable microorganism to be immobilized.
For the purpose of matching the H value, pH as typified by hydrochloric acid, sulfuric acid, nitric acid, citric acid, sodium hydroxide, and potassium hydroxide.
It is better to use a regulator.

291 In the present invention, all the materials used for preparing the aqueous suspension solution can be sterilized in advance, but it is most convenient to sterilize them all at once after the preparation of the aqueous suspension solution is completed.

Sterilization can be completed at 100°C x 5 min in some cases, but if the suspended aqueous solution is contaminated with heat-resistant bacteria, sterilization may be completed at 120°C x 15 t = tL~6 as necessary.
High pressure/steam sterilization must be performed. UV irradiation (
A sterilization method can also be used, but its effectiveness is limited to the irradiated surface, so it is desirable to use it in combination with the heat sterilization method. Furthermore, in any case, these sterilization treatments do not particularly alter the properties of the materials used in the present invention (polyvinyl alcohol, clay minerals), thereby causing no hindrance to the implementation of the present invention.

The sterilized suspension aqueous solution is mixed with live microorganisms to be immobilized. The non-invention is that acids, alkalis, radiation, radical generators, organic solvents, reaction reagents, etc. are not used at all in the immobilization process, so there is no damage to living microorganisms, and therefore, living microorganisms are It is possible to immobilize all heat-resistant and non-heat-resistant living microorganisms, regardless of their resistance to organic reagents, acid resistance, alkali resistance, and radiation resistance. , Aspergillus spp., Rhizopus (Rui
Molds such as the genus Zopus, Pseudomonas
Domonas genus, Acetobacter
cter), Streptomyces (Strepto-
my c e s ) genus, Escher
Bacteria such as the genus Ichia, Sacc.
haγomyces), Candida (Cαη, di
Examples include yeasts of the genus da). As the viable microbial cells, any of the following can be used: frozen/dried preserved microbial cells, a culture solution for growing live microorganisms, or a suspension of living microorganisms that is concentrated by centrifugation from this culture solution. It is most convenient to mix live microorganisms in a focal chamber at room temperature, but when immobilizing live heat-resistant microorganisms, the operation should be performed at a temperature higher than room temperature, depending on their heat resistance. This is even more preferable from the viewpoint of preventing bacterial contamination. At low temperatures, the viscosity of the suspended aqueous solution becomes high and the mixture of living microorganisms and
The dispersion is relatively slow, but if you keep this in mind, the above operation k.

It may also be carried out at a temperature of ~15°C. In the present invention,
There is no limit to the amount of live microorganisms added (initial immobilized amount),
For example, the amount can be arbitrarily selected within the range of 1/2 o, o00 to 7 times the total amount of polyvinyl alcohol and clay mineral, but from the viewpoint of immobilizing almost all of the living microorganisms, the suspended aqueous solution It is preferable to limit the amount to 7 times or less the total amount of polyvinyl alcohol and clay minerals in the container.In this case, by going through the drying (gelation) process described later, 96 to 98% of the viable microorganisms can be reduced to damage. Capture (inclusion/immobilization)
can.

In the present invention, care is taken not to contaminate the mixed suspension aqueous solution of polyvinyl alcohol, clay minerals, and viable microorganisms obtained in this way, and care is taken not to be directly irradiated with germicidal light (ultraviolet light). At least some water is removed from the aqueous suspension. This dehydration (drying) treatment can be done by leaving it at room temperature in a sterile room, or by ventilating it in a sterile room.
In addition to drying by blowing air, it is also possible to dry under reduced pressure (suction) using a vacuum desiccage goo whose interior is sterilized in advance and sterile filters (filters to prevent germs from entering) are attached to the intake and exhaust ports. In any case, at high temperatures, a hard film is formed on the resulting gel,
Since this is unfavorable for the activity and growth (multiplication) of living microorganisms, the drying temperature is preferably 40°C or lower. In the present invention, we operate at temperatures of -6 to +40°C. In this case, as a container for drying the sample, use a container with a wide evaporation area, which is convenient for uniform and rapid drying, and use a container for drying the sample (suspended water bath liquid), for example, 1.5 to 10
It is best to spread it out to about the same thickness.

33- In the present invention, irrespective of the composition of the aqueous suspension solution, a drying treatment must be performed. In this case, the dehydration rate can be, for example, 10 wtCX, or even 15 wtCA or more. As a result of this treatment, the viscosity of the aqueous suspension solution becomes extremely high, and then the entire aqueous suspension solution is assimilated (jelly-formed, gelled). Excessive dehydration must be avoided, as dehydration progresses the hardening of the gel and reduces its water resistance. He's talented.
A gel with excessive residual water is weak. Therefore, in the present invention, once it is confirmed that the suspended aqueous solution has turned into a gel and that the water content of this wet gel has reached a range of 20 to 92 wt%, the drying process is stopped. When the highly elastic gel obtained in this way is immersed in sterilized water, it gradually absorbs water and reaches a water content of 50 to 95 wt% in half a day, but the elasticity and strength of the gel remain almost unchanged, and
Even if it is stirred or shaken at 40°C, it will not become soft for a long period of time.

34- The plate-shaped gel thus obtained can be easily cut into disks, square plates, cubes, etc., just like a rubber sheet.

Of course, it is also possible to punch out a large number of holes in a circular plate and use it as a perforated plate (perforated plate). Although these molded products barely settle in water, their apparent specific gravity is about the same as that of water, and after irregularly emitting light to a height of 6 m into a reaction tower filled with sterile water, the substrate ('!!: bamboo nutrients) whole aqueous solution of medium),
Even if it is gradually introduced from the top or the bottom of the column, there will be no problem with the load bearing capacity of the packed material.If a small amount of this gel is poured into boiling water, it immediately turns into muddy water and the gel does not retain its shape at all. Moreover, even if this muddy water is cooled to 16 to +30°C and further dehydrated, a muddy film is often obtained, instead of a soft gel, which is highly elastic, highly absorbent, and relatively strong. In many cases, it is not possible to obtain a suitable gel. As described above, the gel flower of the present invention is a unique physicochemical phenomenon that is not necessarily expected to be reversible, but according to the present invention, this gelation can be reliably achieved through relatively simple physical operations. be able to.

The cubic gel with a diameter of 2 to 12 mm obtained by the present invention is dissolved in water as a substrate having the optimum temperature and pH value for the growth activity of immobilized living microorganisms. (medium) and brought into contact within a flask, an activity of approximately 85 to 97% is observed compared to the original (non-solidified) viable microbial cells. By further increasing the particle size of this gel, the above relative activity ratio tends to decrease, but in such cases, known substrate diffusion penetration enhancers such as lauryl alcohol sulfate and cetylpyridinium chloride may be used as necessary. , cetyltrimethylammonium bromide, and the like can be used without deteriorating the gel's mechanical strength, lifespan, or other performance.

As described above, according to the present invention, all living microorganisms can be captured in a gel with high water content, high elasticity, and excellent mechanical strength. By controlling the growth i1j of the present invention, it is possible to significantly multiply and increase the activity, thereby achieving the immobilization and proliferation of living microorganisms, which is the objective of the present invention.

The fact that living microorganisms multiply when supplied with a growth medium is not in itself a new fact worth noting, and is a very natural principle; however, since the gel of the present invention is highly elastic, The living microorganisms embedded in the microorganisms easily expand their enclosing cavities and multiply, growing into huge colonies.
Moreover, since the gel of the present invention has excellent mechanical strength,
Rupture (cracks) of the gel tissue due to growth of viable microorganisms is avoided. The gel of the present invention has a tensile strength and a compressive strength of 2 kL/cm2 or more, and has a natural polysaccharide (agar, carrageenan, pectin, etc.) with a strength of 1 k17/cTIL or less.
Alternatively, a secondary hardening treatment product of carrageenan (strength 1 to 15
kli'/c1rL2). However, in the unexploded gel, when the diameter of the colony of proliferating bacteria reaches about 80 μm, some of the proliferating bacteria pass through the mesh of at least a part of the cavity wall and are discharged from the cavity, and the discharge position is (small cavities), the entire process of proliferation and excretion is repeated, and finally, they are expelled from the gel from the cavities near the gel surface.

Further, if the growth medium is still supplied, the diameter of the growth colony exceeds about 250 μmf, and as a result, the amount of bacterial cells washed away increases significantly. Therefore, in the present invention, the diameter of the colony is kept at 80 μm or less in order to completely avoid the washout of the proliferating microbial cells.

In order to completely avoid the accumulation of dead bacteria in the colony, it is possible to adopt a method of supplying a large amount of growth medium to reach a colony diameter of 80 μm or more. However, the village diameter is 2
As mentioned above, if it exceeds 50 μmf, a large amount of proliferating bacterial cells will be washed away, which is not favorable. Therefore, in the present invention, the diameter of the bacterial colony is 250 μm or less, for example, 30 μm or less.
250 μm 38 - Preferably maintained at 50 to 200 μm to avoid useless loss of a large amount of proliferating bacterial cells, and by allowing the colony diameter to reach 80 μm to 250 μm intermittently or continuously, the bacterial colony is killed. Some of the bacterial cells, including bacterial cells (residue from autolysis), are removed from the gel.

That is, in the present invention, the diameter of the proliferating bacterial colony is set to 250 μm.
By controlling the supply of the growth medium so as to keep the amount below m, useless loss of a large amount of growing bacterial cells is completely avoided. That is, in the present invention, the entire growth medium is supplied to the immobilized living microorganisms, a very small amount of the gel is intermittently collected, and the growing colonies within the gel are observed using, for example, a scanning electron microscope.
By stopping the supply of growth medium when the diameter reaches 250 μm or less, a large amount of viable bacterial cells can be washed away.
It is possible to get rid of it.

Partly, in order to increase the activity of immobilized live microbial cells, it is necessary to sieve the entire viable cell concentration within the gel. The number of viable bacteria that can be accommodated in a unit volume of gel is, of course, finite, and its limit value (closest photonic concentration) is calculated mathematically based on the shape and size of the viable bacteria. In a spherical or oval shape (averaging about 5 μm), the number is about 109 mosquitoes/ze. In an actual gel, the gel material also occupies some of the space, so it is impossible to completely achieve the above limit value, but in the present invention, the diameter of the growing bacterial colony does not exceed 250 μm. Within this range, it is possible to achieve bacterial cell concentrations very close to the above-mentioned limit values. That is,
For example, when Saccharomyces cerevisiae of '400 (dry basis weight) is separated from the total amount of polyvinyl alcohol and clay minerals, subjected to the comprehensive treatment of the present invention, and supplied with a growth medium for 8 to 4 days. , in the gel, with a diameter of about 2
The colony of 0077-grown bacteria was dense, and the yeast concentration was estimated to be about 109 cells/1 nt-gel. Similarly, when Saccharomyces cerevisiae was added to about 1/4 o to the total -k of polyvinyl alcohol and clay minerals and the growth medium was supplied for 2 days, bacterial colonies with a diameter of about 100 μm formed in the gel. However, the yeast concentration is still about 109
It is estimated that the number of gels per m/'-gel.

The activity of the sessile-propagated bacterial cells obtained in this manner does not drop sharply even after the supply of the growth medium is stopped, and the same activity often persists for 2 to 3 weeks. Originally 1
After ~2 months, the diameter of the bacterial colony decreases by about 20 to 40% and the activity decreases by about 80% in many cases. 1 medium
By supplying 2 to 24 ml, the activity and colony diameter of the solid-footed bacterial cells are restored to their original values.

In the present invention, a large number of bacterial colonies with a diameter of 30 to 250 μm and 50 to 200 μm are generated in the gel, and a growth medium is then continuously supplied thereto intermittently or in small amounts. By this, the initial bacterial cell activity 'f!
: Characterized by maintaining. Even if the above-mentioned operation is repeated and continued for 4 months or more on the gel of the present invention, there is no change in the strength of the gel structure, and no cracking or destruction of the gel structure is observed.

By subjecting the polyvinyl alcohol/clay composite gel of the present invention to a known curing treatment 'fr:'#U for polyvinyl alcohol fibers or polyvinyl alcohol films, the mechanical strength of the gel is further increased slightly. This known hardening (crosslinking) treatment includes, for example, aldehydes, dialdehydes, diisocyanates, phenols, or metal compounds such as titanium, chromium, and zirconium, as well as borax, acrylonitrile, trimethylolmelamine, and shrimp chlorohydrin. , bis-(β-hydroxyethyl) sulfone, polyacrylic acid, dimethylol urea, maleic anhydride, and the like.

However, the gel of the present invention has the strength (load-bearing capacity) as described above, and the above-mentioned auxiliary curing treatment often damages the immobilized living microorganisms, and the manufacturing cost of the gel is high. In consideration of the unnecessarily increased cost, these curing treatments, which are often used for natural polysaccharides, do not need to be carried out in the present invention.

Next, the present invention will be explained with reference to Examples.

Example: Commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average criticality 2,200.4%, viscosity of water bath liquid (20°C) 5
4 cP) powder 852 (water content 6 wt%) was mixed with water 915
1 to obtain an 8.0 wt% aqueous solution (pH 6,9).

Commercially available bentonite (reagent powder, water content 1'1wt%)
1062 in 1,490 f of water (bentonite)
55wt% suspension 7KC pH 10,1>k obtained, here 6N
4 ml of sulfuric acid was added to adjust the pH value of the suspension water to 6.6.

After mixing the above-mentioned polyvinyl alcohol aqueous solution 1801 and bentonite suspension water 2001, they were subjected to pressurized steam sterilization at 120°C for 6 hours, and then left to cool in a sterile room before being transferred to Sacca O'? Ises Serepici-[-(Sacch
aromyces cerevisiae) 420Tn9
Add 20ml of suspension water (tris(hydroxymethyl)aminomethane buffer, pE 8.0) containing f, and add 7m
Stir for n minutes. The polyvinyl alcohol concentration of this suspended aqueous solution is 8.5 wt%, and the bentonite suspended concentration is 3 w.
t%. On the other hand, the analysis results of the above bentonite (powder) (X-ray diffraction analysis, differential thermal analysis, identification by electron microscope, heating dehydration, interlayer expansion with glycerin, cation exchange nia 8 q/100 f, chemical analysis: 5 i (h 6
7.2, At, 0.6.8, FljtOs 4.1, C
aOO,8, MQO1,6, Ti(h O,4, Mn0
0.1, RQsO, 1, Nat Q 8.2, KtOO
, 4wt%), its dry clay mineral composition (wt%) is montmorillonite group 58, illite group 1, talc 1,
Nozoirophyllite 11, vermiculite 1.

Therefore, the concentration of the ste-type clay mineral in the aqueous suspension solution (wt%) and the concentration ratio between polyvinyl alcohol and the three-layer clay mineral are 2. This suspension aqueous solution 40091e
In a sterile room, the mixture was poured into a container with a bottom surface of 10 cm x 10 cm and left at 40 to 42°C for 2 days to obtain gel 991 (dehydration rate 75 wtcX% water content 70 wt%). This plate-shaped gel (approximately 10 mm thick) was immersed in 100 M of 0.9% saline that had been sterilized in advance, and as a result, the plate-shaped gel absorbed 110 g of water (water content T8wt%).
reached. Moreover, the yeast was not detected in this immersion liquid. Next, this plate-shaped gel was divided into a large number of strips (8 x 6 x
IO) After cutting, pre-sterilized 0.9% saline solution 1001 nI! As a result of washing the yeast with a gel and analyzing the entire washing solution45-, at least 98% of the original yeast remained in the gel (strips).
I learned that it was included.

゛A cylindrical column made of acrylic resin with a diameter of 3 and a height of 60α was coated with the above gel (strips) 1059 irregularly, and an aqueous substrate solution for ethyl alcohol synthesis (33°C) that had been previously sterilized at 120°C for 20 min. , glucose 5
wt%, magnesium sulfate heptahydrate 60 ppm)'
As a result of feeding from the bottom of the column at a flow rate of c4 omt/m, the ethyl alcohol concentration in the effluent was 1 wt% (4% of the theoretical yield).
%).

Next, the growth medium (glucose 6%, peptone 0.5%, yeast extract 0.8%, malt extract 0.8%) is added to the bottom of the tower.
%, potassium chloride 1%, pH 5, 38°C)' (il-4
As a result of feeding at a flow rate of 3 ml/h, the diameter was 70 mm after 27 hours.
It was observed and confirmed using a scanning electron microscope that many micrometer-sized yeast colonies were produced.

Further, in this case, no cracks were observed in the gel. Next, the 46-substrate aqueous solution was again supplied to the bottom of the column at a flow rate of 40 N/h, and as a result, the ethyl alcohol concentration of the effluent after GiL was 2. Shz+t% (86% of theoretical yield) was reached.

The commercially available bentonite powder (2,07) was subjected to a swelling power test according to the Japanese Pharmacopoeia Act. That is, 100 m/'e of water was collected in a graduated cylinder, and 1
Add in 0 batches. However, the next sample is added after almost all of the previously prepared sample has been deposited. When left for 24 hours after adding the entire amount, the apparent volume of the precipitate was only 9 m/cm, far below the prescribed 20 m/min.

Similarly, bentonite powder (6, Of) was subjected to a gel-forming ability test as specified in the Japanese Pharmacopoeia Law. That is,
After mixing the above powder with 0-80 r of magnesium oxide, it was added in several portions to 200 m of water, and after shaking for 1 h, 1001 nl of the resulting suspension was taken and left for 24 h, but a transparent layer separated into an upper layer. The liquid did not stay at the specified 2 ml (or less), but reached 4 ml.

As described above, although the commercially available bentonite used in this example does not satisfy the provisions of the Japanese Pharmacopoeia Law, it is clear that this does not pose any problem to the present invention, as already stated.

Example 2 Viscosity of commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average polymerization degree 1,700.4% aqueous solution (20°C)
26 cP) powder (moisture content '1 wt%) was mixed with 91 g of water.
92 to obtain an 8 wt% aqueous solution (pH 6,9)'#.

Commercially available vermiculite powder (moisture content 9 wt%) 12 (l
r water 1,200? Dispersed in 8wt% suspension water (pH 6
, 6) were obtained.

After mixing each of the polyvinyl alcohol aqueous solution and vermiculite turbid water 19 (1'), they were subjected to pressure steam sterilization at 120° C. 5
1 L of suspension water (phosphate buffer pH 7) containing 22 tn9 was added and stirred for 7 hours.

The concentrations of polyvinyl alcohol and vermiculite in this aqueous suspension are each 8.8 wtXf.

- From the analysis results of vermiculite (powder), its dry clay mineral composition (wt%) is montmorillonite group 4.
, illites 2, talc 3, pyrophyllite 2, vermiculite 85 (5t0242, Alto, 19
, 'T, @Ot 2 * Fthis 8 * FeO
2, Ca11. MaO'L2, KtOl, NatOl
).

Therefore, the three-layer clay mineral concentration of the above suspended aqueous solution is 8
．． The concentration ratio of polyvinyl alcohol and three-layered clay mineral was 1. This suspension aqueous solution 400f,
In a sterile room, pour into a container with a bottom surface of 10 cr IL
mB? ), gel 72 (dehydration rate: 84 wt%, water content: T: 4 wt%) was obtained. This plate-shaped gel (approximately 7 cm thick) was immersed in 100 ml of 49-0.9% saline solution previously sterilized for 6 hours, and as a result, the plate-shaped gel absorbed water and reached 78 f (water content 75 wt%). Moreover, the bacteria were not detected in this immersion liquid. next,
This plate-shaped gel was cut into many pieces (8 x 8 x 8 pieces) and washed with 100M sterilized 0.9% saline solution, and nephelometric analysis of this washing solution revealed that the original bacterial count was 98. It was confirmed that % was incorporated into the gel (strip).

The above gel (shredded piece) 757 was applied to the column of Example 1 with irregular light, and the column was heated at 12 o'cx 20 rnin in advance.
sterilized culture solution (5% soluble starch, 1% peptone, 1% meat extract, 0.1% yeast extract, 0.5% salt)
%, calcium chloride 0.02%, magnesium sulfate heptahydrate m0.01%, pH 7, 30°C) f, 26rnl/h
After 50 hours, a very small amount of gel was collected and observed with a scanning electron microscope.
A dense bacterial colony with 0μ toxicity was observed, but there were cracks in the gel structure.
0- was not observed, and the effluent at the top of the column was only slightly suspended, and the amount of bacterial cells flowing out was only slight (loss amount per hour of bacterial cell amount in Nigel / 1ooo). I learned that. Furthermore, by supplying growth medium + 1 oh,
As a result of monitoring the growth of bacterial colonies and the turbidity of the tower top effluent, we found that the turbidity of the effluent increases significantly when the colony diameter exceeds 250 μm (8 hours). therefore,
It is effective to control the colony diameter due to multiplication so that it always remains at 250 μm.

Example a Commercially available polyvinyl alcohol (saponification degree 98 mol%, viscosity average degree of polymerization 1,700.4%, viscosity of water bath liquid (20°C) 2
6 cP) powder (moisture content 7 wt%) was mixed with 920 g of water.
1 to obtain an 8.0 wt% aqueous solution (pH 7.0).

Commercially available talc (water content 14 wt%) 12 (lk, water 1,1
Dispersed at 90V, talc 8vyt% suspension water CpH 8)
I got jc.

1 each of the above polyvinyl alcohol aqueous solution and talc suspension water
902 After all mixing, sterilize with steam under pressure at 120°C x 20 inches 8! After applying all the ingredients, leave to cool in a sterile room, and then add Saccharo myces cerevisiae (Saccharo myces cerevisiae).
myces cere-visiae)201Jm9
20 ml of suspension water (phosphate buffer, f 7 ) containing in the solution was added and stirred for 7 min. The concentrations of polyvinyl alcohol and talc in this suspended aqueous solution are 3.8w each.
t%. - From the analysis results of talc (powder), its dry clay mineral composition (wt%) is: montmorillonite group 6, illite group 2, talc 87, pyrophyllite 2,
Vermiculite 1 (MgQ32.5zC) 264
, At2Q31 , Ca0O,8r N(L200.7
+F e20s O, 5). Therefore, the three-layered clay mineral in the aqueous suspension solution is 3.7 wt %, and the concentration ratio between polyvinyl alcohol and the three-layered clay mineral is 1.

This suspension aqueous solution 4002 is placed on the bottom surface 10 in a sterile room.
Pour into a 10crn container and dry in a vacuum dryer (0-9℃,
Gel 100F (dehydration rate 73 wt%, water content 65 wt%)
%) was obtained. This plate-shaped gel (approximately 10 mm thick) was immersed in 1001 nm of pre-sterilized 0.9% saline solution for 6 hours, and as a result, the plate-shaped gel absorbed water and reached 110 f! (moisture content'
10 wt%). Further, the yeast was not detected in this soaking solution. Next, this plate-shaped gel is divided into many strips (
After cutting into 8rm x 8H) 7x 10mm), they were sterilized in advance and washed with 0.9% saline solution, and nephelometric analysis of this washing solution revealed that 96% of the original bacteria were included in the gel (strips). I made sure that it was done.

Example 10 A column was filled with 112 f'r of the above gel (strips) and a growth medium (glucose 4%, yeast extract 1%, ammonium chloride 0.2%) that had been sterilized in advance at 120°C x 20 ml tx. , magnesium sulfate heptahydrate 0.01%, 53-calcium chloride 0.006%, pH 5, 4, 30°C Hr
After sending the gel from the bottom of the column at a flow rate of 86 ml/h for 3 days, a small amount of the gel was taken and observed using a scanning electron microscope. As a result, many yeast colonies with a diameter of 190 μm were observed, but no cracks in the enclosing cavity were observed. Ta.

Next, an aqueous substrate solution for ethyl alcohol synthesis (glucose 1Qwt%, magnesium sulfate heptahydrate 100%
ppm.

7) H5, 5, 30℃) Feed at a flow rate of 84 vertical/h in total,
As a result of analyzing the effluent for ethyl alcohol (gas chromad analysis method) and tocurose (dinitrosalicylic acid method), the glucose residual rate was only 0.8%, and ethyl alcohol < 8%, 4.5 wt% (theoretical yield of 88%). %).

After 1 month, the above ethyl alcohol concentration was 8.8wt%
As a result, a small amount of this gel was collected and similarly observed, and it was found that many yeast colonies had all shrunk to a diameter of 120 μm. Next, the medium was again fed into the bottom of the column at a flow rate of 22 ml/liter for 18 hours, and a trace amount of gel was observed in the same manner. Knew. Then, to the bottom of the tower,
The above substrate aqueous solution for ethyl alcohol synthesis was added at 847 nl/
After 26 hours, the concentration of ethyl alcohol in the effluent was 4511 Jt%. Through the above operation, no yeast was detected in the effluent, and it was confirmed that not only the yeast at the time of immobilization but also almost all the yeast that had grown in the column were captured in the gel.

Viscosity of commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average degree of polymerization 1,700.4% aqueous solution (20'C))
Powder 1044 (water content 8 wt%) of 27 fake 11 mouth CP) was mixed with water 801
2 to obtain a 10.6 wt% aqueous solution (pH 6,8). Add commercially available activated clay (water content 11 wt%) to this aqueous solution.
After addition of 681, 26 m/2N sodium hydroxide was added to the suspension aqueous solution (pH 3.5) obtained by stirring for 25 minutes to adjust the pH to 6.8.

Next, it was subjected to pressure steam sterilization treatment at 120°C x 80 min, left to cool in a sterile room, and then the 2002 Kluyveromyces marxianus (Kluyveromyces marxianus)
myces marxi-anus) 9 om9' was poured into 20 l of suspension water (phosphate buffer) and stirred for 7 tmrL. The polyvinyl alcohol concentration of this suspension aqueous solution is 8.5 wt%, and the activated clay suspension concentration is 5.5 wt%.
It is. - From the analysis results of the activated clay, its dry clay mineral composition (wt%) is 48 montmorillonite group, 7 illite, 4 talc, and no? Ilofilite 14, Vermiculite 2, Kaolinite + Halloysite 17 (5i
O274+ A120318, Fe20311 M
gO2'l Ca, 041 Na2O+201). Therefore, the concentration of the three-layer clay mineral in the aqueous suspension solution is 4 wt %, and the concentration ratio of the polyvinyl alcohol to the eight-layer clay mineral is 2.

This suspension aqueous solution 2211 is placed on the bottom surface 10 in a sterile room.
Pour into a CrrLX10crrL container and place in a vacuum dryer (
-1 to +8°C, 340 to 560 mmHf) for one day, Gel 751 (dehydration rate 66 vt%, water content 52 wt%) was obtained. This plate-shaped gel (about 7 dragons thick)
As a result of 6 hours of immersion in 80M of 0.9% saline that had been sterilized in advance, the plate-shaped gel absorbed water and became 801 (water content: 56 wt%).
) reached. Moreover, the yeast was not detected in this immersion liquid. Next, this plate-shaped gel was divided into a large number of strips (8 mm
After cutting into 8 + u +
, 9% saline 80WLe, and nephelometric analysis of this wash confirmed that at least 98% of the yeast was captured in the gel.

The above-mentioned cut gel 78F was applied to the column of Example 1 with irregular light coating, and a growth medium (inulin 1%, malt extract 0.3%) that had been previously sterilized at 120°C
, yeast extract 0.3%, pH 6,5, 27℃)k14r
It was fed from the bottom of the column at a flow rate of ul/h. As a result of observing the gel after 46 hours using a scanning electron microscope, colonies with a diameter of 200 μm were observed within the gel, but no fissures were observed in the gel structure. After feeding the culture medium for another 18 hours, similar observation revealed that the colony diameter exceeded 250 μm, and although no cracks were observed in the gel structure, yeast were completely observed suspended in the top effluent. Therefore, it was recognized that it was necessary to control the colony diameter so as not to exceed 250 μmf.

Example & According to Example 1, a polyvinyl alcohol 8.0w 6% aqueous solution is prepared.

Bentonite powder 3 of Example 1 (1'k, sodium pyrophosphate water bath g (Na4P 2Q? 10 Hto
10.7 wt%) 56058-7 to obtain a 4.5 wt% suspended aqueous solution <pH 10,6), and add 9 m/s of 6N sulfuric acid to adjust the pH value of the suspended aqueous solution to 6. Adjusted to .9.

After mixing the above polyvinyl alcohol water bath solution 1102 and 60 g of bentonite suspension water, sterilization was performed with fluoro-region steam at 120°C x 6 ml, and then left to cool in a sterile room.
Acter simp-1gz) (also known as Corynebacterium simplex, Corynebacterium
m simplex)0-029'fc-containing suspension (tris(hydroxymethyl)aminomethane/hydrochloric acid buffer,
pli 7.5.2 (lk pour, 7 tin spacing 1
Zeta. The polyvinyl alcohol concentration of this suspended aqueous solution is 4
5wt (X% bentonite suspension concentration is 15ut%. Also, in this case, the suspension concentration of three-layered clay mineral is 1,1
The concentration ratio of w t'As polyvinyl alcohol and three-layered clay mineral is 4.

This suspension aqueous solution (1909) was placed in a sterile room, and the bottom surface 1
Gel 421 (water content: 50 wt%) was obtained by pouring into a 0cIIL x 10 cr/L container and leaving it at room temperature for 2 days. By immersing this plate-shaped gel (approximately 4 mm in thickness) in 6 Qm of previously sterilized 0.9% saline for 6 hours, the gel absorbed water and reached 47 f (water content 55 wt%). After cutting all of this plate-shaped gel into small pieces (8 birds x 8 cities x 4 pieces), they were washed with 69 m of sterilized 0.9% saline solution, and their nephelometric analysis revealed that at least 9 of the original bacteria were present.
It was confirmed that 6% was captured in the gel.

The above-mentioned cut gel 451 was placed on the column of Example 1, and the growth medium (peptone 0.5%, cortisol 1
mM, methyl alcohol 4%, 31°C)k, 1ag/h
The gel was introduced from the bottom of the column at a flow rate of
A total number of bacterial colonies of 0 μm were observed.

Next, prednisolone y (prerionisolon), which had been sterilized at 120°C
e+11β+ 17 α+21-trihydroxy-1,
Substrate solution for synthesis of 4-pregnagene-3゜20-dione (cortisol 311β, 17α, 2
1-) Dihydroxy-4-pregnene-3,20-dione 0. IM, 4% methyl alcohol, 'pli 7.
85°C) was fed to the bottom of the column at a flow rate of 17 m1/h.
After that, the effluent was subjected to ultraviolet absorption spectroscopy (285 nm
As a result of analysis by thin layer chromatography (silica gel-propylene glycol-chloroform) and high performance liquid chromatography (chloroform extraction), the prednisolone concentration was 18 wt% (yield 42 mol%).

Example 0 61- Commercially available polyvinyl alcohol (saponification degree 99.7 mol%,
Viscosity average degree of polymerization 2,600.4% aqueous solution (20℃
) 64 cP) powder (moisture content 9 wt%) was mixed with 1 t of water.
．． Dissolved in 180 r, 6 wt% aqueous solution (pH 6,9)
I got it.

Commercially available acid clay (moisture content 12wt%) 127g! water 1,
Dispersed at 280V, acid clay 8.2wt% suspended water (p
H6) was obtained.

After mixing 40 f of the above polyvinyl alcohol aqueous solution and 120 f of acidic clay suspension water, the mixture was subjected to pressurized steam sterilization at 120°C for 30 min, and then left to cool in a sterile room.
To this, suspension water (phosphate buffer) containing Satucharomyces cerevisiae 22 was stirred between 2Of' and Trnitr.The polyvinyl alcohol concentration of this suspension aqueous solution was 1.5 wt%, and the acid clay suspension Concentration is 5..5wt
%. - From the analysis results of the acid clay mentioned above, the dry clay mineral composition (wt%) is 46% of the montmorillonite group.
, illites 6, talc 1, pyrophyllite 14, 6
2- Vermiculite 4, Kaolinite Hallosite 23 (
S'LQz 69 + A120318 + CaO8
+ Mg01.5, A7+zzQ0.1 r &OO
, 1+Fe20s 2 ). Therefore, the concentration of the three-layered clay mineral in the aqueous suspension solution is 4 wt%, and the concentration ratio of polyvinyl alcohol and the eight-layered clay mineral is 1/8.
It is.

This suspension aqueous solution 18 (1?), in a sterile room, the bottom surface 10
Pour into a 10crIL container and dry in a vacuum dryer (-2 to +
By standing at 7° C. and 100 to 500 wHf for 1 day, 24 tons of yellow gel (water content 88 wt%) was obtained.

This gel plate (approximately 3 mm thick) was sterilized in advance.
As a result of immersion in 40 layers of 9% saline for 6 times, the plate-shaped gel absorbed water and reached 292 (water content 45 wt%). Moreover, the yeast was not detected in this immersion liquid. Next, the entire plate-shaped gel was divided into many pieces (2crrLX 1cr/LX3
After cutting into pieces (rlrn), they were washed with 40% sterilized 0.9% saline solution, and based on the results of nephelometric analysis of this washing solution, the mechanical strength of the gel cut pieces was measured. Strength: 2kg/I2, compressive strength: 8k
g/crn2 or more. Furthermore, even when pinched and pressed with a fingertip, it did not lose its shape and returned to its original shape.

Example 7 Commercially available polyvinyl alcohol (saponification degree 98.4 mol%,
Viscosity average degree of polymerization 1,800.4% aqueous solution (20℃
)29.5cP) powder 84v (water content 5wt%)
8. Dissolve in 916 g of water. Q wt% aqueous solution <73H
6,9) -! I got r.

Commercially available bentonite (reagent powder, water content 12wt%) 1
001 in 1.50 liters of water (dispersed in bentonite) 5
5 wt % Q turbid water (pH 10.4) was obtained, and 4 ml of 6N sulfuric acid was added thereto to adjust the pH value of the suspended water to 7.0.

After mixing the above polyvinyl alcohol paddy '1ff16 (l) and bentonite suspension water 1001, it was subjected to pressure steam sterilization at 120°C x 6 hours, and then left to cool in a sterile room, and then Acetobacter suboxidans (Acet
obacter 5ub-oxyd, ans) 40m
202 of the suspension water (phosphate buffer, pH 7) contained in No. 9 was poured into the flask and stirred for 7-7 L. The polvinyl alcohol concentration of this suspended aqueous solution is 2. 'Iwt%, the turbidity concentration of 14g of bento tea is 8.1wt%. −10,000, said bentonite (
Powder) analysis results (X-ray diffraction analysis, differential thermal analysis, identification by electron microscope, heating dehydration, interlayer expansion with glycerin, cation exchange: 187 q/10 (1, chemical analysis:
SiO2'l1rAl, Os 9 + Fetus 8
+ CaO0,5r MgO8+ Tie20.5
.

Nα2 Q 4 * K200.5 r MnOO,O
r P20* 0.0 + 1120518°C%), its dry clay mineral composition (wt%) is 66 montmorillonites, 1 illite, 1 talc, 14 pyrophyllite, and 1 vermiculite. Therefore, the concentration of the three-layered clay mineral in the suspension water 65-solution is 2.6 wt%, and the concentration ratio of the polyvinyl alcohol to the three-layered clay mineral is 1.

This suspension aqueous solution 180 r'r in the bottom 10
The mixture was poured into a cIn x 10 cIIL container and left at room temperature for 2 days to obtain 55 g of gel (dehydration rate 70 wt%, water content T5 wt%). This plate-shaped gel (thickness approximately 6mγI
)'k, As a result of 6 hours of immersion in 80 m/s of pre-sterilized 0.9% saline, the plate-like gel absorbed water and reached 592 (water content 75'wt%). The bacteria were not detected in the soaking solution of the shredded octopus. Next, this plate-shaped gel was divided into many strips (
After cutting into pieces (8 mm x 8 mm x 6 mm), they were washed with 80 ml of previously sterilized 0.9% saline, and when the washing solution was observed under an optical microscope, a small number of the bacteria were found, but based on the turbidity of the washing solution, This was fully quantified and found to ensure that at least 97% of the original bacteria were encapsulated in the gel (strip). 120℃×20 in advance
Min. sterilized culture medium (D-sorbitol 5%, corn steep liquor 0.1%, ammonium fumarate 0.5%, pli 6.5.30°C
) After collecting 40 pieces into a Sakaro flask (500m/), 1
Sterilize at 20°C for 20 min, leave to cool in a sterile room, then put the above-mentioned cutting gel (fixed north side @) 62 there, attach a cotton plug and place in a constant temperature room at 27-32°C. After 40 hours, a very small amount of the gel was taken and observed using a scanning electron microscope. As a result, many bacterial colonies with a diameter of 190 μm were observed within the gel. At this time, no fissures were observed in the village-encompassing cavity.

Next, the gel was separated from the growth medium and added to an aqueous substrate solution for L-sorbose synthesis (D-sorbitol 5%, pH
6,5) Sakaro flask (5'oo) from which 40mlfe was collected
The L-sorbose in the substrate aqueous solution was 3.4 wt% (yield 70 mol%). Ta.

Commercially available bentonite powder (2, Of) used in this example
Therefore, the above swelling test was conducted according to the Japanese Pharmacopoeia Law.
The apparent volume of the precipitate was only 8 ml, which was far below the specified 2 Qm/(or more). A gel forming ability test was also carried out according to the Japanese Pharmacopoeia Law, but the transparent liquid that separated into the upper layer did not stay at the specified 2 (or less), but reached 12.

As described above, although the commercially available bentonite used in this example does not meet the provisions of the Japanese Pharmacopoeia Law, it is clear that it can be a useful carrier material for bacterial immobilization and proliferation, as already stated.

Examples & Commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average polymerization degree 1,700.4% Viscosity of aqueous solution (20°C)
26 cP) powder 862 (moisture content 7 wt%) total, water 91
4v, 8.0 wt% aqueous solution CpH6,8)'
I got ft.

Commercially available bentonite (reagent powder, water content 15wt%) 1
Bentonite 5 was prepared by dispersing 042 in water at 1,500 F.
．． 5 wt% suspended water (pIi 10.7'Dr: obtained,
Add 4ml of 6N sulfuric acid to this to adjust the pH value of the suspended water to 6.8.
Adjusted to.

After mixing 50 f of the above polyvinyl alcohol aqueous solution and 120 ml of bentonite suspension water, sterilize the mixture with pressurized steam at 120°C x 6 m, then leave it to cool in a sterile room, and then transfer it here.
Seratia mar
20 f of suspension water (phosphate buffer) containing 88Tn9 was poured into the solution and stirred for 7-1 minutes. this)
The polyvinyl alcohol concentration of the turbid water solution is 2wt%,
Bentonite suspension at 0 degrees is 3.5 wt%. Ten thousand,
Analysis results of the bentonite (powder) (X-ray diffraction analysis,
Differential thermal analysis, identification using deuteron microscopy, heating dehydration, interlayer expansion with glycerin-69, cation exchange: 89 Q/100
t, chemical analysis: Si0,6 a7 + At, Os
8.7 + Fe, 0.3.1 + Ca0OJ 2
Mg01.6, NatO8,2, K2O0,8, Ti
1t O, O.

MnOO,0+ P2O5O,0+ K7O15wt%
), its dry clay mineral composition (wt%) is 64 montmorillonites, 1 illite, 8 talc, 18 pyrophyllite, and 1 vermiculite. therefore,
The concentration of the three-layered clay mineral in the suspended water bath was 8 wt %, and the concentration ratio of polyvinyl alcohol to the three-layered clay mineral was 2/8.

This suspension aqueous solution 19 (1'k, in a sterile room, the bottom surface 1
Pour into a 10cm x 0cm container and dry in a vacuum dryer (-6 to +
3℃, 200-400vrrxH? ), Gel 322 (dehydration rate: 88 wt%, water content: 55 wt%) was obtained. This plate-shaped gel (approximately 8 mm thick)
) k, pre-sterilized 0.9% saline 80 crab 6
As a result of immersion, the plate-shaped gel absorbed water and reached a water content of 3670-1 (water content 6 (1 wt%). In addition, no bacteria were detected in this immersion solution. After cutting into small pieces (8 mπ x 8 x 3 pieces), they were washed with 80 ml of sterilized 9.9% saline solution and the washing solution was observed under an optical microscope. We quantified this based on turbidity and found that at least 98% of the original bacteria were entrapped in the gel.

The above-mentioned cut gel 37f was placed irregularly in a cylindrical column made of acrylic resin with a diameter of 3CIn1 and a crystal size of 60CrIL, and a growth medium (glucose 0.4%, yeast extract 1%) that had been sterilized at 120°C for 20 min was added. , peptone 1%, meat extract 0.4%, sodium chloride 0.4
%, pH 7, 30°C) from the bottom of the column at a flow rate of 7rnl/kl'7rnl/. After 38 hours, a very small amount of the gel was taken and observed under a scanning electron microscope. As a result, a bacterial colony with a diameter of 170 μm was observed, but there were no cracks in the enclosing cavity. Next, a substrate for L-inleucine synthesis (glucose 3%,
D-threonine 1.5%, magnesium sulfate heptahydrate 0
．． 07%, phosphate buffer 0.06M, pH 7, 30°C
) As a result of feeding from the bottom of the column at a flow rate of k28 wre/h, 3
After 8 h, the concentration of L-4 soleucine (L-α-aminocaproic acid) in the effluent reached 0.5 wt% (31× the theoretical yield).

Commercially available bentonite powder (2, Or) used in this example
The above swelling test stipulated by the Japanese Pharmacopoeia Law was conducted for
The apparent volume of the precipitate was only 9 ml, which was far below the specified volume of 20 TdIC (more than 20 TdIC), and passed the gel-forming ability test according to the Japanese Pharmacopoeia Law. ) was barely a skeleton.

As described above, none of the three types of commercially available bentonites used in Example 1.7.8 satisfy the provisions of the Japanese Pharmacopoeia Law, but as already mentioned, it is clear that they can be useful microorganism immobilization materials in non-invention. It is.

Example a Commercially available polyvinyl alcohol (saponification degree 97 mol%, viscosity average polymerization degree 2,200.4% aqueous solution viscosity (20°C) 5
4cP) powder (85f (water content 6wt%)) was mixed with 9141g of water.
Dissolve in 8. Owt% aqueous solution CpH6,8)? Obtained.

Illite (produced in Ota City, Shimane Prefecture, moisture content 8wt%) 98 f
i, and dispersed in 1.200 t of water to obtain an 8 wt % illite suspension in water (pH 7.4).

190g of the above polyvinyl alcohol aqueous solution and 195L of illite suspension? After mixing, they were treated with pressurized steam at 120°C for 2 hours several times, and then left to cool in a sterile room, where Acetobacter suboxidans (Acetobacter suboxidans) was added.
Cter 5ub-ozydans) was mixed with suspension water (phosphate buffer pH 7) contained in 40 ml of water and stirred for 7 ml of 73-20 ml of water. The concentrations of polyvinyl alcohol and illite in this 1" water bath solution are 4 wt% each. From the analysis results of illite (powder), its dry clay mineral composition (wt%) is montmorillonite group 1, 87 illites, 1 talc, 6 pyrophyllite, 1 vermiculite (5zO252
+ At20z 24, Fe2us 8 + FeO
3+AigO4, CaO1, Kto 7, N(L20
1. TiO21+ M? L2Q32). Therefore, the 8-layer ψ clay mineral concentration of the above suspended aqueous solution is 8.
8wt%, also polyvinyl alcohol 8F'; 4fJ
The 4 degree ratio with clay minerals is 1.

Store this turbid water bath solution 405 f in a sterile room at 1°C on the bottom.
Pour into a 0αX 10cm container and dry in a vacuum dryer (2 to 1 liters).
Gel 792 (dehydration rate 80'u) t%,
A moisture content of 55 wt%) was obtained. This plate-shaped gel (approximately 8 m thick)
m) was immersed in pre-sterilized 0.9% saline solution = 74-100 m/m for 6 hours, and as a result, the plate-shaped gel absorbed water and
902 (moisture content 6 wt%). Also, the bacteria were not detected after this immersion. Next, this plate-shaped gel was divided into a large number of strips (8mm x 8m*X 9+nyy).
After cutting, the pieces were washed with 100 ml of sterilized 0.9% saline solution, and nephelometric analysis of this washing solution confirmed that 97% of the original bacteria were encapsulated in the gel (strips).

Example 10 The above gel (1 piece) was irregularly filled with 89P'i in a column, and heated at 120°C x 20 tnin in advance.
A sterilized culture solution (D-sorbitol 5%, corn steep liquor 〇, 2y, ammonium fumarate 1%, pH 6.5) was fed from the bottom of the column at a flow rate of 451 nl/h, After JQA, a small amount of the gel was taken and observed using a scanning electron microscope, and the diameter was 490 μm.
However, cracks in the gel structure were not observed. Next, dihydroxyacetone synthesis substrate aqueous solution (2.5% glycerin, 0.1% corn steep liquor)
, ammonium fumarate 1%, pH 65, 30°C)e
As a result of feeding from the bottom of the tower at a flow rate of 47 m//m, the flow rate was 36 h.
The dihydroxyacetone concentration of the subsequent effluent was 1.1 wt.
% (yield 46 mol%).

Example 1α Commercially available polyvinyl alcohol (saponification degree 99.7 mol%,
Viscosity average degree of polymerization 2,600.4% Viscosity of water bath liquid (20℃
) 64 cP) powder (water content 8 wt%) was mixed with 1 ml of water.
．． 00 OS' and make an 8 wt% aqueous solution (pH 6,8
) was obtained.

Pyrophyllite (produced in Mitsuishi, Okayama Prefecture, water content 6wt%) 7
81 was dispersed in 1.00 Of water to make a 7 wt% suspension of water (
pH 6.5) was obtained.

After mixing the above-mentioned polyvinyl alcohol aqueous solution W 40 f and pyrophyllite suspension water 502, they were subjected to pressurized steam sterilization at 120° C. for 5 hours, and then left to cool in a sterile room. - Buoxidans (G
hwconobactersu, boxyd, ans)
20 f' of suspension water (phosphate buffer) contained in 44 m9 was poured and stirred for 7 min. The polyvinyl alcohol concentration of this suspension aqueous solution is 1 tut%, and the pyrophyllite suspension concentration is d w 1%. -10,000, From the analysis results of the pyrophyllite, its dry clay mineral, river bottom (wt%
) is montmorillonite group 4, illite 0, talc 0,
pyrophyllite 83, vermiculite c) (5z
Qt 66 r AX 20329 +Ti120.0
4. , Pet's O,2, FeOO,1, M'g
OO,5, Ca0O,4, No, 00-03, P,
(1.20 in 10 marks). Therefore, the concentration of the three-layered clay mineral in the aqueous suspension solution is 2.8 wt%, and the degree ratio of polyvinyl alcohol to the three-layered clay mineral is 173.

This suspended aqueous i110? Place the bottom surface 10 in a sterile room.
Pour into a cIn-1×10α container and dry in a vacuum dryer (-8 to +6℃, 2
8 to 470 mvtlH) for half a day.
Gel 301 (dehydration rate 73wt%, water content '10wt%)
I got it. This gel plate (about 3 mm thick) was immersed in 49 i/ml of 0.9% saline that had been sterilized in advance for 6 hours, and as a result, the gel plate absorbed water and reached a water content of 361 (water content: 75 wt%). In addition, the bacteria were not detected in this immersion solution.Next, this plate-shaped gel was divided into many pieces (4 mm x 4 cities x 3
After cutting into pieces, add 40% sterilized saline solution.
It was confirmed from the nephelometric analysis of this washing solution that 98% of the original bacteria were captured in the gel.

Pre-sterilized medium (D-arabitol 5%, corn steep liquor 0.1%, yeast extract 0.1%, potassium phosphate buffer 0.06M, pH 5.35 at 120°C x 20-nm) °C) 501nl'k, Sakaro Frass: + (500m/) taken, 12 o'cx 20
The gel was sterilized with tnin, left to cool in a quiet room for 78 hours, and 8.7 fk of the above-mentioned cut gel was added thereto, a cotton plug was attached, and the mixture was shaken in a constant temperature room at 28 to 32°C.

After 24 hours, a very small amount of the gel was taken and observed using a scanning electron microscope. As a result, bacterial colonies with a diameter of 160 μm were observed, but no cracks were observed in the gel structure. Next, the gel was separated from the growth medium and pre-sterilized D
- Substrate aqueous solution for xylose synthesis (D~arabitol 5%
, Magnesium sulfate Nanamikuchimono 0.02%, Rinj! Potassium buffer 0.06M.

The mixture was poured into 50 ml of pH 5 (35° C.) and similarly shaken for 24 hours. After 32 days, 3.4 wt% of D was added to the substrate aqueous solution.
-xylose (yield 39 mol%) was observed.

As shown in Examples 1 to 10 above, the three-layer type (2:1
It is clear that all clay minerals are useful as carriers for immobilizing microorganisms.

Example 1 was prepared using commercially available polyvinyl alcohol (degree of saponification 97 mol%, viscosity average degree of polymerization L 700.4% water, viscosity of liquid (20°C) 2
7cP) powder 1047 (water content 8wt%) was mixed with 80% water.
07 to make a 106 zat% aqueous solution CpH 6,8)
I got it.

After mixing silicic anhydride 607, magnesium oxide 409, sodium fluoride 42v1 and lithium fluoride 261, the mixture was transferred to a platinum crucible, sealed tightly, and heated at 1885"C for 6 hours using an electric furnace.
h After melting, cool to 810℃1 at a cooling rate of 2.5℃/mix.
The solid material obtained by slow cooling is further cooled at room temperature 1, and then pulverized to a powder finer than 100 meshes (147 μm), and classified to a powder of 200 meshes (147 μm).
81 grams of fine powder (dry powder at 110° C.) with a diameter of 74 μm or less was obtained. The chemical analysis results (wt%) of this powder are: Na5.
6, Mg 12.8, Li 1.7, Si
28.9, 041.2, F9.8, sodium teniolite N'aMQ2 L! (SL< Q+
o) Empirical formula 8 formula that almost matches F2 % In the diffraction analysis results, no crystals other than sodium duolite (fluorine tetrasilicon mica) were observed, but the crystal structure became amorphous during the above grinding process. With this, the content of illite (three-layered clay mineral) is 6.
It was 3%.

Water 1,170 to the above sodium teniolite 809
g'', synthetic illite (mica) 6.4?at%
A water suspension (pH 7) was obtained.

After mixing each of the above polyvinyl alcohol water bath solution and synthetic illite e suspension 1901, they were subjected to pressure steam sterilization at 120°C x 20 mn, and then left to cool in a sterile room.
Here, Acetobacter aceti (Acetobacter aceti)
ter aceti) was added and stirred for Tmltr. The concentration of polyvinyl 81-alcohol in this suspension water bath is 5 wt%, which is the concentration of synthetic illite! The turbidity concentration is 3 wt%, the concentration of the three-layer clay mineral in the suspended aqueous solution is 2 wt%, and the concentration ratio between polyvinyl alcohol and the three-layer clay mineral is 2.5.

Place this suspended aqueous solution 4001-1 in a sterile room, and place the bottom lO
Pour into an α x 10 α container and vacuum dryer (-5 to +15
Gel 90f (dehydration rate 77 wtX% water content 60) Dt
%) was obtained. This plate-like gel (approximately 9 yr thick)
As a result of 6 hours of immersion in 1007 m of 0.9% saline that had been sterilized in advance, the gel plate absorbed water and reached 1142 m (moisture content: 68 t%). Moreover, the bacteria were not detected in this immersion liquid. Next, this plate-shaped gel was cut into many pieces (4 mm x 4 mm x 9 mm) and washed with 100 m of sterilized 0.9% saline solution. It was confirmed that 98% of the bacteria were encapsulated in the gel (strips).

Glucose 10%, corn staple lJ car 0.
1%, yeast extract 0.1%, phosphate buffer 0.06MC
PH7) culture medium (59m) was taken into a Sakaro flask (500g), sterilized at 120°C x 20-n, left to cool in a sterile room, and the above-mentioned cut gel 101 was put into it, and a cotton plug was attached. It was then shaken in a constant temperature room at 28-32°C. 2
After 4 hours, a small amount of the gel was sampled and observed under a scanning electron microscope. As a result, bacterial colonies with a diameter of 100 μm were observed inside the gel. After further shaking for 20 hours, many colonies with a diameter of 170 μm were observed, but no cracks were observed in the gel structure.

Next, a substrate aqueous solution for gluconic acid synthesis (glucose 1O%
, Corn Stape Liquor 0.1%, Phosphate Buffer 0.06M% pH 7, 32°C) 50 d in a Sakalo flask (
5001n, sterilized at 120°C x 20 mtx, left to cool in a sterile room, transferred the long-time shaking gel to this, and shaken for 24 hours in a constant temperature room at 30-33°C. As a result, the gluconic acid concentration of the substrate aqueous solution was 9.2
wtX (yield 85 mol%). In this case, no deformation of the molded gel was observed even though it was shaken for a long time.

Like the natural three-layer clay minerals of Examples 1 to 10, the synthetic three-layer clay minerals are also useful in the present invention.

Patent applicant: Nippon Oil Co., Ltd. -1.

Procedural amendment May 14, 1981 Commissioner of the Patent Office Shima 1) Itsuki Haru 1.Display of the incident 1981 Patent Application No. 206080 2. Name of the invention Bipedalization and multiplication method of living microorganisms 3. Person who makes corrections Relationship to the case Patent applicant Name (444) Nippon Oil Co., Ltd. 4, Agent 1- 6. Contents of amendment (1) All parts of the second part of Book A will be revised.

2-

Claims (1)

[Claims] The degree of saponification is 95 mol% or more, and the viscosity average degree of polymerization is 1.
500 or more polyvinyl alcohol and 8N type (2:1
Type a) Contains a layered structure type clay mineral with laminated layers as the basic unit and viable microorganisms, and has a dissolved concentration of polyvinyl alcohol of 1 to 15 wtX and a suspended concentration of clay mineral of 0.7 ~15wtX, a suspended aqueous solution in which the concentration ratio (weight ratio) of the polyvinyl alcohol and the clay mineral was adjusted to 115 to 8/1 was dried at -6 to +40°C to obtain a dehydration rate of 16wt% or more and a water content of 20. ~92wt% (wet body basis) to obtain a gel that encloses (embeds) living microorganisms, supplies a microorganism growth medium to this gel, and the diameter of the grown microorganism colony formed thereby. 80μm~2
A method for immobilizing and propagating living microorganisms, which is characterized by expanding them to a range of 50μ.