JPH0462717B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention provides a carrier for immobilizing microorganisms such as yeast and bacterial cells,
In particular, it relates to a carrier made of ceramics. (Prior Art) In recent years, immobilized enzymes have been put to practical use as catalysts for production and analysis of sugars, amino acids, etc., and their development and use are rapidly progressing. On the other hand, in order to avoid the troublesome work of isolating, purifying, and immobilizing enzymes produced by microorganisms, research is being conducted on microorganism immobilization technology that uses microorganisms such as enzyme-producing bacteria by immobilizing them on carriers. For example, Japanese Patent Application Publication No. 1983-
No. 11288, No. 59-39290, No. 59-198976, No. 60
-160885 each bulletin suggests fixing microbial cells on anion exchange fibers, polyamine compounds, acetalized polyvinyl alcohol fibers, alginate metal salt gels, etc., and also proposes fixing microorganisms on anion exchange fibers, polyamine compounds, acetalized polyvinyl alcohol fibers, alginate metal salt gels, etc., and also uses polyacrylamide, agar, κ-carrageenan, etc. , etc., are known, and some methods have already been put to practical use.
However, such organic carriers have the disadvantage that they are easily destroyed by mechanical stimulation and have low contact efficiency with oxygen and substrates. Compared to such organic carriers, inorganic carriers such as glass and ceramics have greater dimensional stability, can be reused by burning off degraded or dead microorganisms, and are less susceptible to PH changes and chemical attack of bacteria and solutions. Because of its advantages, such as its resistance to However, since microorganisms are immobilized on inorganic carriers by physical adsorption, the contact efficiency with oxygen is good, and while preparation is easy and inexpensive, the binding force between microorganisms and carriers is relatively small, and microorganisms are easily washed away. There is a serious drawback. (Problems to be Solved by the Invention) In order to solve the above-mentioned problems, the present invention was developed as a result of repeated research on the physical adsorption behavior of enzyme-producing microorganisms to ceramics, which is an inorganic carrier. The present invention was achieved based on the finding that the compatibility between the two is closely related. An object of the present invention is to provide microorganism-immobilized ceramics that can maintain high catalytic efficiency over a long period of time. Another object of the present invention is to expand the range of applicable types of ceramic carriers for immobilizing microorganisms, and also to expand the range of applicable types of microorganisms. (Means for Solving the Problems) The carrier for immobilizing microorganisms of the present invention to achieve the above-mentioned object is characterized by being made of ceramics whose interfacial potential has been modified by chemical modification with organosilane. It is. The above modification is when the charge sign of the electrokinetic potential is reversed,
This includes both cases where they are increased within the same sign. In a preferred embodiment of the carrier for immobilizing microorganisms of the present invention, the organosilane is an organosilylamine, and the amine is added to the ceramic surface via the silane. In another preferred embodiment, the organosilane is an organosilycarboxylic acid,
It is made by adding carboxyl groups to the surface of ceramics via silane. Furthermore, the absolute value of the altered electrokinetic potential is preferably at least 40 mV, more preferably at least 50 mV, most preferably at least 100 mV.
V, especially at least 140 mV. Ceramics applied to the present invention can include silicates, metal oxides, and mixtures thereof, as well as carbides such as carbon, boron, and silicon, and selenides, such as silica; kaolinite, pyrophyllite, Alumina silicate such as sillimanite and mullite; Alkali silicon alumina such as feldspar, mica, montmorillonite, and liusite; Magnesium silicate such as cordierite, steatite, talc, and forsterite; Magnesium aluminate with spinel structure; corundum, saphire, bauxite, gibbsite, etc. alumina; other apatite, titanium white, porcelain, brick, etc.
Among these, cordierite, mullite, alumina and zirconia are preferred. These are used in the form of powders, fine particles, sintered bodies, porous bodies, and molded bodies having a penetrating multi-cell structure such as honeycomb. In the present invention, first, the surface of the ceramic is chemically modified with organosilane to modify the interfacial potential of the ceramic. Here, the interfacial potential is also called ζ-potential, and is the potential difference between colloidal particles in an electrolyte solution and a solution. value. This interfacial potential depends on the type, concentration and electrolyte.
The pH varies depending on the pH, and as shown in Table 1, for ceramics, some materials have a positive charge, such as zirconium oxide and alumina, but many others, such as quartz, cordierite, and mullite, have a negative charge. be. On the other hand, useful microorganisms are not constant in their charge polarity or amount; for example, the same species of yeast, Saccharomyces cerevisiae, as shown in Table 2.
As a result of actual measurements, it was found that only the subspecies (Cerevisiae) showed various signs and values depending on the subspecies.

【table】

【table】

[Interfacial potential of microorganisms]

Target microorganisms are collected by centrifugation from the culture solution. 1.5 to 3.0 x ¹⁰ cells/ml of bacterial cells in a solution made with HCl and KCl to a pH of 3.0 and an electrical conductivity of 0.5 millisiemens.
Suspend to a concentration of . Place the suspension in a Blitz cell and energize it. The electrophoretic speed of microbial particles is measured using a microscope, and the positive or negative charge is determined. The −ζ potential is determined from the measured value of the electrophoresis speed using the following formula. ζ=4πμ/D・A/i・C・u×9×10 ¹⁰ However, ζ: Zeta potential (mV) μ: Viscosity coefficient of liquid (Poise) D: Dielectric constant of liquid (-) A: Potential gradient Cross-sectional area of the cell in the perpendicular direction (cm ² ) i: Current flowing in the cell (mA) C: Electrical conductivity of the suspension (S) u: Migration speed of particles (cm/sec) [Interfacial electrokinetic potential of ceramics] The target ceramic is finely ground and suspended in the same solution as above. Using the supernatant after stirring well and allowing it to stand for a while, the concentration is adjusted to the same range as above and the interfacial potential is measured in the same manner. The adsorption amount of yeast, which was used as a microorganism, was measured as follows. [Adsorption amount] 1 g of ceramics ground to a particle size of 1 to 1.4 mm was added to 10 ml of a yeast suspension with an optical density (OD ₅₇₀ ) of approximately 2 at a wavelength of 570 nm, and after shaking at a temperature of 4°C for 2 hours,
OD ₅₇₀ was measured again. The amount of decrease from the initial OD ₅₇₀ value was defined as the amount of adsorption. Example 1 Cordierite was crushed to give an average particle size of 1 to 1.4 mm.
1 g of the prepared product was placed in a 300 ml flask equipped with a reflux condenser together with 100 ml of toluene and a boiling stone. This was heated to the boiling point of toluene, 105°C, and 10 ml of organosilane modifier was added thereto, followed by heating and refluxing for 5 hours. Next, the toluene was filtered off, and the cordierite was washed with toluene, further washed with acetone, and then air-dried. KBM903 (manufactured by Shin-Etsu Chemical Co., Ltd., γ-aminopropyltriethoxysilane) as the above organosilane
and KBM603 [manufactured by the same company, N-(β
-aminoethyl)-γ-aminopropyltrimethoxysilane] Two types of modified cordierite were prepared. Yeast suspension was carried out using Satucharomyces cerevisiae Z-73 and KSC-44 in Table 2 above, using an aqueous solution containing 3% by weight of ethanol and 3% by weight of glucose and whose pH was adjusted to 3.0 with hydrochloric acid as a dispersion medium. The liquid was prepared and the amount of chemically modified cordierite adsorbed was measured. Comparative Example 1 The amount of yeast adsorption was measured in the same manner as in Example 1, without chemically modifying the cordierite used in Example 1, which was ground to an average particle size of 1 to 1.4 mm. The results obtained in Example 1 and Comparative Example 1 are shown in Table 3.

[Table] As is clear from the above table, the interfacial potential of cordierite without chemical modification is -32 mV, and the interfacial potential of Satucharomyces cerevisiae Z-73 is +13 mV with the opposite sign, so there is a certain degree of interfacial potential. Although the amount of adsorption was shown, the adhesion strength could not necessarily be said to be large. Furthermore, although Satucharomyces cerevisiae KSC-44, which has an interfacial potential of the same sign, is hardly adsorbed, in the chemically modified product of the present invention, the interfacial potential of cordierite is significantly changed to the positive sign side. Therefore, it showed a large amount of adsorption. Furthermore, compared to cordierite with an interfacial potential of +113 mV, the adsorption amount of cordierite exceeding +140 mV was significantly increased, and these bonds were also extremely strong. When a glucose solution was passed through the cell, yeast growth was observed, but almost no runoff was observed, and good activity could be maintained for a long period of time. Example 2 Alumina was chemically modified in the same manner as in Example 1, except that the alumina shown in Table 1 was used as a ceramic carrier and organosilane KBM903 was used as a modifier to obtain a carrier having amino groups on the surface. Ta. Add azelaic acid to 10ml of water after air drying.
A PH5.0 solution containing 0.4 g of 1-cyclohexyl-3-(morpholinoethyl)carbodiimidemeth-p-toluenesulfonic acid was added thereto as a catalyst, and the mixture was shaken overnight at 60°C. After the reaction was completed, the solution was filtered, washed with water, and then dried. When the interfacial potential of the obtained carrier was measured,
The charge had changed from positive to negative. In addition, Satsukaromyces cerevisiae Z-73 and KSC-
The adsorption amount of each of the 44 substances was measured. Comparative Example 2 The amount of yeast adsorption was measured in the same manner as in Example 2 without chemically modifying the alumina used in Example 2 above. The results of the above Example 2 and Comparative Example 2 are summarized in Table 4.

[Table] As is clear from the table above, since the interfacial potential of alumina is originally positively charged, Z-73, which has the same interfacial potential, shows almost no affinity, and it is difficult to fix it by adsorption. However, the material according to the present invention exhibited stable and high immobilization rate due to its large adsorption power.
It will be appreciated that this significantly widens the range of applications for carrier and microorganism combinations. Example 3 When the alumina used in Example 2 was chemically modified with KBM903 in the same manner as in Example 1, the interfacial potential increased from +28 mV to +142 mV. When Saccharomyces cerevisiae KSC-44 was adsorbed onto this material, it showed an excellent adsorption amount of 1.98. Comparative Example 4 Mullite was surface-treated in the same manner as in Example 1, except that mullite was used as the ceramic and KBC1003 (manufactured by Shin-Etsu Chemical Co., Ltd., trimethoxyvinylsilane) was used as the organosilane modifier. When the amount of yeast adsorbed to mullite before and after the surface treatment was measured in the same manner as in Example 1, there was virtually no significant difference observed before and after the surface treatment for both Satucharomyces cerevisiae Z-73 and KSC-44. (Effects of the Invention) As is clear from the above explanation and Examples, the microorganism immobilization carrier of the present invention has an organic amine or carboxyl group firmly bonded to the surface via silane. Since it is made of ceramics, it exhibits an extremely stable and modified interfacial potential, and in combination with this, microorganisms with an oppositely charged interfacial potential can be absorbed in large quantities with a strong adsorption force that is far superior to conventional simple van der Waals adsorption. Adsorb. Therefore, in combination with the advantage of physical adsorption, which is good contact efficiency between microorganisms and oxygen and substrates, it exhibits extremely excellent action and activity, and because it reduces runoff during operation, it maintains strong activity for a long period of time. can be maintained for a period of time. Furthermore, although conventional ceramic carriers based on physical adsorption can be applied to certain microorganisms, they do not show satisfactory adsorption to other microorganisms, and if the microorganisms are considered living organisms, ceramics There were differences in the difficulty of adsorption, and each had restrictions on the applicable range. The present inventors have elucidated this adsorption mechanism, and the present invention, which was completed based on this, has lifted the above-mentioned restrictions and has been able to arbitrarily expand the range of application of ceramic carriers and the range of application of microorganisms, and is expected to be useful for future microorganisms. It is expected to make a major contribution to the development of immobilization technology and industrialization.

Claims (1)

[Scope of Claims] 1. A carrier for immobilizing microorganisms, characterized in that it is made of ceramics which have been chemically modified with organosilane to change the electrokinetic potential to a value of at least 40 mV in absolute value in a given charge sign. 2 Claim 1, wherein the charge sign of the electrokinetic potential of the ceramics is reversed by the chemical modification.
A carrier for immobilizing microorganisms as described in . 3. The carrier for immobilizing microorganisms according to claim 1, wherein the chemical modification increases the electrokinetic potential of the ceramic at the same charge sign.