JPH10150982A - Support for bioreactor and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inexpensive support high in mechanical strength, rich in microorganisms immobilization, and useful e.g. in continuous fermentation tanks for brewing Japanese wine, by heat treatment of zeolite at a specified temperature followed by grinding and classifying the resultant porous body. SOLUTION: This support for bioreactors is obtained by heat treatment of zeolite (e.g. natural zeolite) at 1200-1400 deg.C followed by grinding and classifying the resultant porous body. This support has an average granular size of 0.15-3.0 (pref. 0.5-3.0)mm, bulk density of 0.7-1.0g/ml and fine pores open on the surface each 5-400μm in average size, being inexpensive, high in mechanical strength, also rich in microorganisms immobilization, thus useful e.g. in continuous fermentation tanks for brewing Japanese wine (alcohol).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bioreactor used in a continuous fermenter for sake (alcohol) brewing using microorganisms, and more particularly to a carrier for immobilizing microorganisms in a bioreactor.

[0002]

2. Description of the Related Art In recent years, as a technology utilizing the activity of microorganisms, such as alcohol fermentation of sake or brewing of soy sauce,
The technology related to a bioreactor, which is a continuous fermenter using microorganisms, has been actively researched and put into practical use. Here, a bioreactor generally refers to immobilizing so-called biocatalysts such as microorganisms such as yeasts and bacteria on various carriers filled in the apparatus, and using the biochemical reactions catalyzed by the catalysts to efficiently produce substances. Refers to the reactor to be performed.

[0003] There are various carriers for immobilizing such microorganisms. For example, FC Rep.
ort 12 (1994) No. 8 Ceramics carrier for bioreactor Mitsuo Kawase, Gypsum & Lime No. 251 (1994) Alcohol fermentation by immobilized yeast using porcelain apatite sintered body as a carrier Toru Ikegami, Shunji Koumoto, etc. Inorganic carriers such as ceramic beads, glass beads, and the like described in Jpn. However, such an inorganic carrier is, for example, a carrier using silicon dioxide and has a bulk density of 0.2 to 0.5 g / cm.
2. The compressive breaking strength is 20 to 80 kg / cm2, and the mechanical strength is brittle and there is a drawback that it is weak in friction. Moreover, it is difficult to use it on a practical scale because it is expensive.

[0004] In addition, Iwate prefecture brewed food test report 25 (1991)
Development of new product (sake sake) using bioreactor
Katsuo Omori describes alginic acid beads and xanthone beads as a polymer-immobilized carrier. However, such a polymer-immobilized carrier has a large amount of yeast immobilized because it is in a gel form, but is weaker than an inorganic carrier and is very difficult to use on a practical scale.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a bioreactor carrier having high mechanical strength, being inexpensive, and having a large amount of immobilized microorganisms, and a method for producing the same.

[0006]

The above object is achieved by the following constitution. (1) Porous particles obtained by heat treatment of zeolite, having an average particle diameter of 0.15 to 3.0 mm, a bulk density of 0.7 to 1.0 g / ml, and pores open to the surface A carrier for a bioreactor, comprising: (2) The bioreactor carrier according to (1), wherein the average pore diameter of the pores is 5 to 400 μm. (3) The bioreactor carrier according to (1) or (2), wherein the zeolite is a natural zeolite. (4) The zeolite is subjected to a heat treatment at 1200 to 1400 ° C., and the obtained porous body is pulverized and classified to obtain the above (1).
A method for producing a bioreactor carrier for obtaining the bioreactor carriers of (3). (5) The zeolite has an average particle size of 0.5 to 3.0.
(4) The method for producing a carrier for a bioreactor according to the above (4). (6) The method for producing a bioreactor carrier according to the above (4) or (5), wherein the zeolite is a natural zeolite.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The bioreactor carrier of the present invention is preferably porous particles obtained by heat-treating natural zeolite, and has an average particle diameter of 0.15 to 3.0 mm.
It has an open pore having a bulk density of 0.7 to 1.0 g / ml and a number average pore diameter of preferably 5 to 400 μm on the surface. Here, natural zeolite is clinoptilite,
It is called modenite and is a compound composed of aluminosilicate and has various components in addition to silicon and aluminum as main components.

The natural zeolite is vitrified and melted by heat treatment at 1000 ° C. or higher. At that time, the structure contains moisture, and the moisture is continuously released during the heating process (before and after vitrification). For this reason, it easily foams and becomes porous in the heating process, and the heating element becomes pumice-like. As such natural zeolites, those from Nitsui-machi, Akita Prefecture and from Fujisato-machi, Akita Prefecture are known in Japan, but those from Futatsui Town (estimated reserve 8.5 million tons) are preferable. As a zeolite from Futatsuimachi, for example, the composition of a natural zeolite (hereinafter sometimes abbreviated as SZ) manufactured by San Zeolite Co., Ltd. is represented by weight percentage, and SiO 2: 6
9.38%, Al2O3: 11.02%, Fe2
O3: 0.92%, MgO: 0.60%, Na2
O: 3.34%, CaO: 1.31, K2O: 3.1
7%, P2O5: 0.04%, water of crystallization (zeolite water): 8.09%, adsorbed water: 2.08%, etc., and a natural zeolite manufactured by Nippon Zeolite (hereinafter sometimes abbreviated as NZ) Has a composition of SiO 2: 69.7%, A
l2O3: 12.2%, Fe2O3: 1.1
%, MgO: 1.6%, TiO2: 0.1%, Na2
O: 3.5%, CaO: 0.7, K2O: 3.7
% Of crystallization water (zeolite water). That is, the weight ratio of silica / alumina is about 5 to 7 and the oxide of alkali metal, alkaline earth metal and phosphorus is contained in a weight percentage of about 6 to 12%.

[0009] As a non-natural zeolite, there is a synthetic zeolite. However, ordinary commercially available zeolites are vitrified by heating, but it is difficult to obtain foaming properties equivalent to those of natural zeolites. However, a synthetic zeolite may be used as long as it has the same composition as the above-mentioned natural zeolite.

The size of the zeolite used as a raw material has an average particle size of 0.5 to 3.0 mm, preferably 1.0 to 2.0 mm. If the average particle size is smaller than 0.5 mm, the surface area of the material increases, the evaporation of water at a temperature lower than 1000 ° C. increases, and when the material is vitrified, the generation of bubbles becomes insufficient and good foaming is obtained. And the ability to immobilize microorganisms is reduced. If the average particle diameter is larger than 3 mm, the surface of the material is vitrified and melted first, the water vapor inside the material is trapped, and many extremely large bubbles are generated.
Therefore, the number of pores having a desired size is reduced, and the ability to immobilize microorganisms is reduced.

The heating temperature of the zeolite is 1200
To 1400 ° C, particularly 1260 to 1300 ° C, preferably 1270 to 1290 ° C, and particularly preferably 1270 to 1280. If the heating temperature is too low, the generated bubbles are too small than the target size. If the heating temperature is too high, the generated bubbles are too large than the target pore size. The heating time may be appropriately determined depending on the particle size of the raw material and the heating temperature, but as a holding time not including the temperature raising time, a range of 2 hours or less is preferable, and a range of 5 to 90 minutes is more preferable. In particular, about 10 minutes is preferable.
The heating time is preferably about 400 ° C./hr, and the heating atmosphere may be air.

After zeolite as a raw material is heated and fired, it is ground / classified to a predetermined size. As a pulverizing method, a small amount can be easily pulverized with a mortar or the like, but on a practical scale, a dry pulverizing device such as a roll crusher is usually used. In addition, as a method of classification, the classification can be easily performed by using a sieve having openings of a predetermined size. By pulverizing the porous body in this way, the glass layer on the surface is removed and the cells become open cells (pores), in which microorganisms can be immobilized. Therefore, the zeolite particles having a desired size, which are separated and heated so that they do not adhere to each other, are turned into spherical particles, are covered with a glass film, do not release bubbles, and have an ability to immobilize microorganisms. Is low.

The size of the pulverized porous particles is preferably in a range of an average particle diameter of 0.15 to 3.0 mm, particularly preferably 0.6 to 2 mm. If the particle diameter is less than 0.15, the strength and the ability to immobilize microorganisms are low and not practical. If the particle diameter exceeds 3 mm, the surface area decreases, and the ability to immobilize microorganisms decreases. The bulk density is 0.7 to 1.0 g / ml, preferably 0.75 to 0.9 g / ml, particularly 0.8 to 0.9 g / ml.
ml. When the bulk density is less than 0.7 g / ml, the strength of the carrier is reduced and the number of microorganisms in the entire bioreactor is reduced. When the bulk density exceeds 1.0 g / ml, the liquid passing rate is reduced, the production efficiency is reduced, and clogging or the like is easily caused.

The porous particles have open pores on the surface. By having such open pores, microorganisms can be immobilized in the pores. The number average pore diameter of the opened pores is 5 to 400 μm, more preferably 1 to 400 μm.
It is preferably from 0 to 200 μm. If the diameter of the pores is too large or too small, the ability to immobilize microorganisms is reduced. The diameter of such pores is determined by SEM (scanning electron microscope)
And observe the surface of the pores, and measure the average of the minor axis and the major axis as an average of about 50 pores. Further, the number of pores per predetermined surface area, that is, the pore density, for example, the number of pores per 0.5 × 0.5 mm is preferably 5 to 30, particularly preferably 7 to 20. If the pore density is too low, the ability of immobilizing microorganisms per unit area will decrease, and if the pore density is too high, the diameter of the pores will decrease and the ability to immobilize microorganisms will decrease. Such pores are preferably continuous.

The compressive strength of the porous particles 9Kg / mm ² or more, further 10 Kg / mm ² or more. Compressive strength is 9kg / m
If it is less than m ² , the strength required as a carrier cannot be obtained.
The upper limit of the compressive strength is about 200 kg / mm ² .

Next, a method for producing such porous zeolite particles will be described.

A natural zeolite having an average particle size of 0.5 to 3.0 mm is placed on an alumina setter, and preferably at a temperature rising rate of from room temperature to about 400 ° C./hr, from 1260 to 130 ° C.
Heat to 0 ° C. and hold for 2 hours or less, preferably 5-10 minutes. Next, stop heating, cool naturally to room temperature,
A porous body (foam) is obtained. The resulting porous body is pulverized and classified using a sieve of a desired size. Larger ones are further pulverized and classified, and this is repeated until a desired size is obtained. Next, the obtained porous particles having a predetermined particle size are washed with water to remove fine powder, and the edges are scraped off.
Finally, the carrier is obtained by drying.

As the microorganism to be immobilized, yeast (Sacchromyces cerevisiae, Sa) used for alcohol fermentation, brewing and the like can be used.
ccharomyces uvarum), Escherichia coli,
Lactic acid bacteria (genus Lactobacillus, Streptococcus), acetic acid bacteria (genus Acetobacter), Bacillus subtilis
And the like, and transformants (genetically modified organisms) thereof, among which yeasts for brewing are suitable. Such yeast for brewing may be any as long as it produces ethyl alcohol, carbon dioxide, etc., by metabolizing the brewing raw material liquid. Specifically, Saccharomyces
Sacchromyces cerevisiae, Saccharomyces uvarum and the like. Particularly preferred yeasts are the Sake Yeast Association No. 6, 7, 7, 9 and 10 strains for sake fermentation. , No. 11, No. 14, No. 15, etc., and any of them can be immobilized on the carrier of the present invention.

As a method for immobilizing such a microorganism on a carrier, the carrier and the microorganism are immersed in a predetermined culture solution,
By culturing for a predetermined time, it can be easily immobilized on a carrier. As a culture solution, for example, YEPD liquid medium and yeast medium for yeast, NB medium for bacteria,
Peptone medium and the like, the culture time is the type of microorganism,
The temperature is appropriately determined depending on the cultivation temperature and the type of medium.
It is about 4 to 72 hours.

The reactor vessel (fermenter) may be appropriately determined depending on the purpose of use, the production amount, the type of microorganism used, and the like. For example, a column type reactor, a fluidized bed type reactor, a stirred tank type reactor, an air lift type reactor Reactors, etc .;
(1991) Development of a new product (sake sake) using a bioreactor The fermenter described in Katsuo Omori can also be used.

[0021]

The present invention will be described more specifically with reference to the following examples.

<Examples 1 and 2> [Preparation of support] Nippon Zeolite No. 6 (abbreviated as NZ6 in the table): A particle size of 0.5 to 1.5 mm was placed on an alumina setter, It was heated and maintained at 1270 ° C. for 10 minutes. Pulverize the resulting porous material, 0.6-
The particles were classified into sizes of 1.0 mm (Example 1) and more than 1.0 to 2.0 mm (Example 2). The bulk density and compressive breaking strength of the obtained porous particle carrier were measured. Here, the compressive fracture strength was calculated by taking out one carrier and measuring it with a granule strength tester, and dividing it by the apparent cross-sectional area obtained by converting the major axis of the carrier into a circle. The number average pore diameter and pore density were calculated from the SEM photograph of the surface. Here, the pore density is
The number of pores present in a 0.5 × 0.5 mm section was measured. Table 1 shows the obtained results.
An example of the M photograph is shown in FIG. The porous body having a particle diameter of less than 0.6 mm was omitted because the compressive breaking strength could not be measured due to the small particle diameter. [Immobilization of yeast] 100 ml of YEPD medium (2% glucose, 1% yeast extract, 2% polypeptone) and 10 g of the carrier obtained above were placed in a 500 ml Erlenmeyer flask,
Sterilization was performed at 21 ° C. for 15 minutes. Thereafter, one platinum loop of Sake Yeast Association No. 7 was inoculated and shake-cultured at 28 ° C. for 2 days to immobilize the yeast on a carrier. [Measurement of Immobilized Yeast] The carrier after yeast immobilization was crushed in sterile water to release yeast, and the number of free yeast in the suspension was measured using a Toma hemocytometer. The number of free yeasts was calculated by dividing the apparent cross-sectional area of the carrier by the apparent volume obtained. Table 1 shows the obtained results. <Examples 3 and 4> Example 1 was repeated except that the following carriers were used.
Yeast was immobilized in the same manner as in 2, and the number of yeasts was counted. Table 1 shows the results.

Sanzeolite 8-20 # (SZ in the table)
8-20 #): Heat and pulverize a particle diameter of 0.8-3.0 mm in the same manner as in Examples 1 and 2 to obtain a particle diameter of 0.6-1.0 mm.
(Example 3) Classified into sizes of more than 1.0 to 2.0 mm (Example 4) to obtain carriers. Table 1 shows the carrier characteristics measured in the same manner as in Examples 1 and 2. <Examples 5 and 6> Except for using the following carriers,
Yeast was immobilized in the same manner as in Example 4, and the number of yeasts was counted. Table 1 shows the results. The porous body having a particle diameter of less than 0.6 mm was omitted because the compressive breaking strength could not be measured due to the small particle diameter.

In Examples 3 and 4, the heating temperature was set to 130
Except that the temperature was 0 ° C., heating and classification were performed in the same manner as in Examples 3 and 4, and 0.6 to 1.0 mm (Example 5), more than 1.0 to 2.0
Each of the particles was classified into porous particles having a size of mm (Example 6) to obtain a carrier. Table 1 shows the characteristics of this carrier measured in the same manner as in Examples 1 and 2. In addition, particle diameter 0.6
Porous materials of less than mm were omitted because their compressive fracture strength could not be measured due to their small particle size.

Comparative Example 1 Commercially available glass beads as a carrier: Except for using a particle diameter of 2 to 3 mm, yeast was immobilized in the same manner as in Examples 1 and 2, and the number of yeasts was counted. The result is shown in Table 1, and an example of the SEM photograph of the surface is shown in FIG.

Comparative Example 2 Yeast was immobilized in the same manner as in Examples 1 and 2, except that the following carriers were used, and the number of yeasts was counted. Table 1 shows the results.

No. 8 (NZ8) manufactured by Nippon Zeolite Co., Ltd .: A particle size of 0.1 to 0.5 mm was heated and classified in the same manner as in Examples 1 and 2 to give a particle size of more than 1.0 to 2.0 mm. The particles were classified into porous particles to obtain carriers. Table 1 shows the characteristics of the carrier measured in the same manner as in Examples 1 and 2, and FIG. 3 shows an example of an SEM photograph of the surface.

Comparative Example 3 Yeast was immobilized in the same manner as in Examples 1 and 2, except that the following carriers were used and the heating temperature was set at 1310 ° C., and the number of yeasts was counted. Table 1 shows the results.

KS Mining Co., Ltd .: A material having a particle size of 20 to 40 mm is heated and classified in the same manner as in Examples 1 and 2, and
The particles were classified into porous particles having a size of 2.0 mm to obtain a carrier. Table 1 shows the characteristics of the carrier measured in the same manner as in Examples 1 and 2, and FIG. 4 shows an example of an SEM photograph of the surface.

Comparative Example 4 Yeast was immobilized in the same manner as in Examples 1 and 2, except that the following carriers were used, and the number of yeasts was counted. Table 1 shows the results.

Sanzeolite 8-20 # (SZ8-
20 #): A particle diameter of 1 to 2 mm is placed on an alumina setter with one grain and one grain separated from each other to prevent particles from adhering to each other.
It was heated at 300 ° C. and used as a carrier without pulverization and classification. Table 1 shows the characteristics of the carrier measured in the same manner as in Examples 1 and 2, and FIG. 5 shows an example of an SEM photograph of the surface.

[0032]

[Table 1]

As is clear from Table 1, the carrier for a bioreactor of the present invention has an immobilizing capacity for yeast that is equal to or about twice that of a conventional glass carrier, and has excellent compressive breaking strength of twice or more. Is shown.

[0034]

According to the present invention, it is possible to provide a bioreactor carrier having high mechanical strength, being inexpensive, and having a large amount of immobilized microorganisms, and a method for producing the same.

[Brief description of the drawings]

FIG. 1 is a drawing-substitute photograph showing an example of the surface of a porous particle obtained according to Examples 1 and 2 of the present invention.

FIG. 2 is an SEM of a glass bead surface of Comparative Example 1 of the present invention.
Drawing substitute photograph which is an example of photograph.

FIG. 3 is a drawing-substitute photograph showing an example of the surface of a porous particle obtained in Comparative Example 2 of the present invention.

FIG. 4 is a drawing-substitute photograph showing an example of the surface of a porous particle obtained in Comparative Example 3 of the present invention.

FIG. 5 is a drawing-substitute photograph showing an example of the surface of a porous particle obtained in Comparative Example 4 of the present invention.

Claims (6)

[Claims] 1. Porous particles obtained by heat-treating zeolite, having an average particle diameter of 0.15 to 3.0 mm, a bulk density of 0.7 to 1.0 g / ml, and being open to the surface. A bioreactor carrier having pores. 2. An average pore diameter of the pores is 5 to 400 μm.
The carrier for a bioreactor according to claim 1, which is: 3. The bioreactor carrier according to claim 1, wherein the zeolite is a natural zeolite. 4. A method for producing a bioreactor carrier, wherein the zeolite is heat-treated at 1200 to 1400 ° C., and the obtained porous body is pulverized and classified to obtain the bioreactor carrier according to claim 1. 5. The zeolite having an average particle size of 0.5 to 5.
5. The method for producing a bioreactor carrier according to claim 4, which is 3.0 mm. 6. The method for producing a bioreactor carrier according to claim 4, wherein the zeolite is a natural zeolite.