JPH0468913B2 - - Google Patents

Description

[Detailed description of the invention]

(Technical Field of the Invention) The present invention relates to a bioreactor element in which microorganisms containing enzymes used in biochemical reactions are immobilized on a ceramic honeycomb structure, and a method for producing the same. (Description of Prior Art) In recent years, biochemical reactions using microorganisms containing enzymes have developed rapidly and have come to be widely used in fields such as organic synthesis, food industry, and analytical chemistry. In this case, it is required to separate the microorganisms from the products after the reaction and reuse them, and in response to this, technology for immobilizing microorganisms has been developed. Conventionally, there have been two methods for immobilizing microorganisms: a physical adsorption method in which microorganisms are physically adsorbed onto a carrier, and a covalent bonding method in which microorganisms containing enzymes are commonly bonded to various water-insoluble beads or pellets. A cross-linking method in which microorganisms are cross-linked to a carrier using a cross-linking reagent such as glutaraldehyde or bisdiazobenzidine having a functional group of 3 or more, an ionic bonding method in which microorganisms are bound to a carrier through ionic bonding, or a Encapsulation methods are known in which microorganisms are encapsulated in a polymer gel lattice or covered with a semipermeable polymer film. (Problems with conventional technology) However, the covalent bonding method, crosslinking method, and ionic bonding method have drawbacks such as changes in the properties of microorganisms due to immobilization and a large decrease in activity. The problem was a decrease in the activity of microorganisms. Furthermore, in such comprehensive methods, only the surface of the gel exhibits microbial activity, so there are problems such as low activity per unit amount of microbial cells, increased pressure loss, and blockage of flow channels by solid matter. It was hot. As a technique to solve the problems of such a comprehensive method, the applicant has also conducted an immobilization method using a ceramic honeycomb structure as a carrier (Japanese Patent Application Laid-Open No. 1989-1999-1).
43382), in this method, the opening length of the through holes in the ceramic honeycomb structure had to be increased in order to attach the gel containing microorganisms, and there was a limit to the surface area of the through holes. Furthermore, in the physical adsorption method, due to the shape of conventional carriers, the interaction with the carrier is weak compared to the frictional force between the bacterial cells and the liquid, so there was a problem that microorganisms were easily released during fermentation. Therefore, the purpose of the present invention is to:
In the physical adsorption method, which has a low decrease in microbial activity,
The amount of immobilized microorganisms is larger than that of conventional immobilization, and
It is an object of the present invention to provide a bioreactor element in which the contact area between a substrate and microorganisms is large and the microorganisms are difficult to be released from the carrier, and a method for producing the same. Another object of the present invention is to provide a bioreactor element and a method for producing the same that eliminate problems of pressure loss and blockage of flow paths, and facilitate the release of gas when gas generation is involved. be. (Means for Solving the Problems) As a result of intensive research in order to solve the above problems, the inventors of the present invention found that a ceramic honeycomb structure, which had not yet been used in a physical adsorption method, was used as a carrier. It was discovered that a bioreactor element capable of achieving the above object and a method for producing the same can be obtained by using the present invention, and the present invention has been completed. The present invention provides a ceramic honeycomb structure having countless pores and a plurality of parallel through holes surrounded by partition walls, in which microorganisms containing enzymes are immobilized by physical adsorption. a bioreactor element in which the average pore diameter of the partition walls of the through holes of the ceramic honeycomb structure is 30 μm to 120 μm, the porosity of the ceramic honeycomb structure is 30% to 70%, and the ceramic honeycomb structure The present invention relates to a bioreactor element in which the opening length of the through-hole in the body is 1 mm to 5 mm. The present invention also provides a ceramic honeycomb structure having a plurality of parallel through holes having countless pores and surrounded by partition walls, wherein the average pore diameter of the partition walls of the through holes is 30 μm to 120 μm. Yes, porosity is 30
% to 70% and the opening length of the through hole is 1 mm to 5
Prepare a ceramic honeycomb structure having a diameter of mm, suspend microorganisms containing enzymes in an aqueous solution containing nutrients, immerse the ceramic honeycomb structure in this aqueous solution, and fill the pores of the ceramic honeycomb structure with the aqueous solution. This relates to a method for producing a bioreactor element in which microorganisms are infiltrated into the pores, and then microorganisms are cultured inside the pores and the microorganisms are immobilized inside the pores by physical adsorption. The ceramic honeycomb structure used in the present invention is made of ceramic material such as alumina, mullite, and cordierite. This is because the ceramic honeycomb structure has the advantage of having the optimum pore size and porosity for immobilizing microorganisms containing enzymes, ensuring a large contact area, and having small pressure loss. The average pore diameter of the partition walls of the through holes in this ceramic honeycomb structure is preferably 30 μm to 100 μm, more preferably 30 μm to 70 μm. The average pore size is
If the diameter is less than 30 μm, it is difficult for microorganisms to enter the pores.
This is because if the diameter exceeds 100 μm, microorganisms will easily flow out from the pores. Further, the opening length of the through holes in the ceramic honeycomb structure is 1 mm to 5 mm, preferably 2 mm to 3 mm. If the opening length of the through hole is less than 1 mm, microorganisms will not be immobilized easily or uniformly, and the flow path may be blocked by air bubbles, etc., resulting in a sudden increase in pressure loss. This is because if the opening length of the pores is greater than 5 mm, a disadvantage arises in that the volume occupied by the ceramic honeycomb structure necessary for the reaction becomes too large. Furthermore, the porosity, which indicates the proportion of voids in the partition walls of the ceramic honeycomb structure, is 30 to 70%, preferably
It is between 40% and 60%. This is because if the porosity is less than 30%, the amount of microorganisms required for the reaction is insufficient, while if it exceeds 70%, the problem arises that the strength of the ceramic honeycomb structure decreases. In the method for producing a bioreactor element according to the present invention, the temperature of the microorganism suspension is set at 25 to 30°C.
℃, and the liquid viscosity is 0.5 to 30 c.p. Also, when infiltrating the inside of the pores of the ceramic honeycomb structure,
By using a vacuum degassing method using an aspirator or the like, air bubbles inside the pores of the ceramic honeycomb structure can be easily removed, and accordingly, microorganisms can be introduced quickly. In this case, the ceramic honeycomb structure is arranged so that the through holes of the ceramic honeycomb structure are oriented in the vertical direction so that the bubbles to be deaerated can easily escape. Next, in culturing the microorganisms, it is preferable to perform shaking culture to grow the microorganisms and fill the inside of the pores with the microorganisms. Note that microorganisms that can be used in the present invention include yeast fungi, bacteria, actinobacteria, molds, and the like, and the enzyme is an enzyme that microorganisms inherently contain. (Example) Hereinafter, the present invention will be explained with reference to Examples. Example 1 Alcohol-fermented yeast Satucharomyces cerevisiae was cultured in a culture solution (yeast extract 0.15%, NH ₄ Cl 0.25%,
_{K2HPO4 0.55} %, _{MgSO47H200.025} %, _NaCl0.1
%, _CaCl2 0.001%, citric acid 0.3%) at pH 5.4
10 ⁵ cells/ml were suspended. The cordierite honeycomb structure was immersed in this liquid, and then the liquid was allowed to penetrate into the pores of the ceramic honeycomb structure while being vacuum degassed using an aspirator for 15 minutes. Next, the yeast was cultured in a constant temperature bath at 30° C. for 3 days to grow and immobilize the yeast. The 21 types of ceramic honeycomb structures used had the opening lengths of through holes, porosity, and average pore diameter listed in Table 1. Next, for comparison, three types of bioreactor elements were manufactured using a conventional comprehensive method. First, two of these are 30% yeast suspension and 30% yeast suspension.
It was manufactured by mixing aqueous solution of sodium alginate (wt%) at a temperature of 40°C to form a gel-like liquid, immersing a ceramic honeycomb structure in this liquid, and then blowing off the excess adhered gel with compressed air. The remaining one uses the same gel mixture,
This was dropped into a 3% by weight aluminum sulfate solution to obtain a bead-shaped immobilized microorganism. For each of the bioreactor elements produced as described above, a reaction tube with a diameter of 5 cm and a length of 30 cm was used, and a 20% by weight aqueous glucose solution was flowed into the reaction tube at a rate of 40 ml/hr from the lower part of the reaction tube, and continuously replaced for 15 days. After that, the ethanol production amount was compared. The results obtained are shown in Table 1.

【table】

[Table] Shows the amount.
In Table 1, the average pore diameter of the partition walls of the through holes of the ceramic honeycomb structure is 30 μm to 120 μm, the porosity is 30% to 70%, and the opening length of the through holes is 1 mm to 5 mm. , sample numbers 4, 5, 7
~20, and sample numbers 1, 2, 3, 6, and 21 are outside the scope of the present invention. As can be seen from the results in Table 1, sample numbers 1 to 21, in which the physical adsorption method was applied to the ceramic honeycomb structure, had higher ethanol production than the comparative products using the inclusion method. Moreover, among sample numbers 1 to 21, sample numbers 4, 5, 7 to 20, which are within the scope of the present invention, have higher ethanol production than sample numbers 1, 2, 3, 6, and 21. ing. This is because the product of the present invention is directly adsorbed onto the carrier, so it can be immobilized on a ceramic honeycomb structure with a finer opening length of through holes than conventional products, and the effective surface area per unit volume is high. The porosity of the body is large and a large amount of yeast can be immobilized, the average pore diameter of the ceramic honeycomb structure is optimal for yeast immobilization, and the ceramic honeycomb structure has a shape with parallel through holes. This is because there is less yeast release due to fluid friction, and gas bubbles that are generated are easier to rise compared to beads, so back mixing is less likely to occur. Example 2 Using a ceramic honeycomb structure with a through-hole opening length of 5 mm, a porosity of 50%, and an average pore diameter of 50 μm, the physical adsorption method of the present invention and the comprehensive method of the conventional method were used in the same manner as in Example 1. Bioreactor elements were manufactured respectively. Furthermore, bead-shaped bioreactor elements having an average bead diameter of 3 mm were produced in the same manner as in Example 1. These bioreactor elements were subjected to ethanol fermentation in the same manner as in Example 1, and in order to observe changes over time, the ethanol production amount was measured when continuous fermentation was performed for 100 days. The results obtained are shown in FIG. 1 for comparison. As is clear from the results shown in Figure 1, the bioreactor element manufactured by the present invention entered the stable production range in a shorter period of time than the conventional method, and continued to produce alcohol at a high concentration for a long period of time. I know that there is. This is because the ceramic honeycomb structure has a high porosity, and especially in the product of the present invention, the pore size is optimal for immobilizing yeast, and since the yeast is in direct contact with the glucose solution, it is thought that yeast multiplication is rapid. . In addition, in the product of the present invention,
Since the yeast is immobilized within the pores of the ceramic honeycomb structure, it is difficult for it to flow out due to the glucose solution, and since the ceramic honeycomb structure has strong mechanical strength, it is thought that the activity will not decrease. Example 3 Preculture medium A for acetic acid bacteria Acetobacter aceti
(In 1, glucose 10g, polypeptone 10g, yeast extract 10g, ethanol 20ml, glacial acetic acid 10g)
10 ⁶ cells/ml were suspended. A cordierite ceramic honeycomb structure with a through-hole opening length of 1 mm, a porosity of 50%, and an average pore diameter of 50 μm was immersed in this solution, and was statically cultured at 30° C. and pH = 3.3 for 4 days. After that, the ceramic honeycomb structure was grown in preculture medium B (1
medium (glucose 10 g, polypeptone 10 g, yeast extract 10 g, ethanol 40 ml, glacial acetic acid 10 g) and cultured with shaking at 30°C and pH=3.3 for 36 hours. The bioreactor element obtained in this way was placed in a tubular reaction tube with a diameter of 5 cm and a length of 20 cm, and a solution containing preculture medium B was added at 50 c.c./hr from the bottom of the reaction tube.
At the same time, air was introduced at a rate of 250 c.c./min. Acetic acid fermentation was carried out using acetic acid bacteria while maintaining the temperature at 30°C and pH = 3.3, and the acetic acid concentration at the reactor outlet was measured. As a comparative product, acetic acid bacteria were immobilized entrapping in 3 mm beads with 3% by weight sodium alginate, and acetic acid fermentation was performed under the same conditions as in this example. These results are shown in Table 2 for comparison.

[Table] As is clear from the results in Table 2, the product of the present invention had a higher acetic acid concentration than the comparative product. This is because in the case of beads, the reaction liquid is disturbed by the air sent into the reaction tube, causing back mixing, and also because the opening length of the ceramic honeycomb structure is short, and the acetic acid bacteria involved in the reaction have a large adhesion area. This is thought to be due to the high (Effects of the Invention) Compared to conventional bioreactor elements, the bioreactor element according to the present invention has the following effects as a result of adsorbing microorganisms onto the partition walls of the through holes of the ceramic honeycomb structure. (1) Since a ceramic honeycomb structure with short through holes is used as the immobilization carrier, the contact area between the microorganisms and the solution per unit volume is large, resulting in high reaction efficiency. (2) Since the ceramic honeycomb structure has parallel through holes and the microorganisms are immobilized on the partition walls of the through holes of the ceramic honeycomb structure, there is little release of microorganisms due to friction of the flow of the solution. (3) Since microorganisms are adsorbed on the partition walls of the through-holes of the ceramic honeycomb structure, the reaction starts quickly, and the mechanical strength is strong, so the activity does not decrease over a long period of time. (4) Since the ceramic honeycomb structure has parallel through-holes, the gas generated in the gas-generating fermentation system easily rises and dissipates through the through-holes, resulting in reaction disturbance and back-mixing. There is little occurrence of generation, and there is less clogging of the flow path by solid matter. As a result, pressure loss is also small. Therefore, the present invention can be effectively used in various bioreactor elements, particularly in alcoholic fermentation that involves gas generation during the reaction, acetic acid fermentation that requires air, and starch saccharification reactions where the substrate is highly viscous. It is valid.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the number of days and the amount of ethanol production.

Claims (1)

[Claims] 1. Microorganisms containing enzymes are absorbed into the pores of a ceramic honeycomb structure having countless pores and a plurality of parallel through-holes surrounded by partition walls by physical adsorption. The bioreactor element is a fixed bioreactor element, wherein the average pore diameter of the partition walls of the through holes of the ceramic honeycomb structure is 30 μm to 120 μm, and the porosity of the ceramic honeycomb structure is 30% to 70%. A bioreactor element in which the opening length of the through holes of the ceramic honeycomb structure is 1 mm to 5 mm. 2 A ceramic honeycomb structure having countless pores and a plurality of parallel through holes surrounded by partition walls, wherein the average pore diameter of the partition walls of the through holes is
A ceramic honeycomb structure having a diameter of 30 μm to 120 μm, a porosity of 30% to 70%, and an opening length of through holes of 1 mm to 5 mm is prepared, and microorganisms containing enzymes are suspended in an aqueous solution containing nutrients. The ceramic honeycomb structure is immersed in this aqueous solution to allow the aqueous solution to penetrate into the pores of the ceramic honeycomb structure, and then microorganisms are cultured inside the pores and the microorganisms are absorbed into the pores by physical adsorption. A method for producing bioreactor elements that are fixed inside pores.