JPS63287477A - Fermentation by immobilized microorganism and apparatus therefor - Google Patents

Abstract

PURPOSE:To improve cultivation efficiency, by lifting a liquid culture medium through a vertical through-hole honeycomb cell by the lifting action of bubbles and, at the same time, lowering a part of lifted medium through the outside or inside of the cell, thereby circulating the liquid medium. CONSTITUTION:A culture vessel 3 contains a vertical through-hole honeycomb cell 4 composed of porous ceramics. A liquid medium containing reaction substrate is put into the culture vessel 3, air is introduced into the culture vessel 3 from its bottom and lifted along the vertical hole of the cell and made to contact and react with enzyme-producing microorganisms supported on a wall surface of the cell. In the above process, a part of the medium lifted through the vertical hole of the cell by the floating force of introduced air bubbles is circulated by descending through the outside 7 or inside of the cell. The flow rate of the medium in the cell can be increased by this process to enable the maintenance of the attached amount of the microorganisms constantly at the optimum level, improvement in the contact efficiency between the substrate and the microorganisms, remarkable acceleration of the reaction and stabilization of the reaction over a long period.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a fermentation method using immobilized microorganisms and an apparatus used therein, more specifically, a liquid medium containing a reaction substrate,
A method of passing through the vertical cells of a through-cell type honeycomb made of a porous ceramic material while aerating from the bottom thereof, and causing a reaction by contacting with enzyme-producing microorganisms adsorbed and supported on the ceramic partition walls in the cells, and an apparatus used therein. Regarding.

(Prior art) In recent years, immobilized enzymes have been used as catalysts for the production of sugars, amino acids, etc.
It has been put into practical use as an analytical catalyst, and its development and use are progressing rapidly. On the other hand, in order to purely isolate, purify, and immobilize the enzymes produced by microorganisms, research into microorganism immobilization technology that uses microorganisms such as enzyme-producing bacteria by immobilizing them on carriers is underway to save time and effort. Some methods have already been put into practical use, such as methods for immobilizing bacterial cells by entrapment methods using organic polymer carriers. 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 killed microorganisms, and are less susceptible to pH changes and chemical attack of bacteria and solutions. In addition to having advantages such as being resistant to Therefore, research and development of bioreactors using microorganism-immobilized inorganic carriers is rapidly gaining momentum.

The most typical bioreactor of this type known to date is
As shown in the outline in Fig. 2, microorganisms are immobilized in a cylindrical vertical reaction tank 3 equipped with a reaction substrate-containing liquid medium supply port 1 at the bottom and a reaction product-containing liquid medium outlet 2 at the top. Through-cell honeycomb 4 made of porous ceramic material
are fitted so that the long axis of the cell 5 is substantially vertical.

Since the honeycomb 4 has a size and arrangement that occupies almost the entire horizontal section of the reaction tank 3, the vertical flow of the medium S that is supplied from the lower supply port 1 and overflows and discharged from the upper discharge port is as follows. Flow through the vertical cells of the honeycomb. This device also has an aeration means 6 at the bottom of the tank for supplying and diffusing oxygen to the culture medium.

In such a device, the substrate in the culture medium that is injected through the lower supply port, while rising through the honeycomb cells together with aeration bubbles, comes into contact with the microorganisms adsorbed and supported on the inner walls of the cells, and undergoes a reaction due to the action of enzymes produced by the microorganisms. It is discharged from the upper outlet along with the reaction products. At this time, the flow rate of the medium is maintained at an appropriate value in order to maintain or increase the reaction efficiency, and the amount of oxygen necessary for the metabolism of the microorganisms is supplied by aeration.

(Problems to be Solved by the Invention) In the above-mentioned reaction system, microorganisms that have proliferated over time generally accumulate in the honeycomb, narrowing the cells and eventually blocking them, preventing the passage of aeration bubbles. Air bubbles aggregate and remain at the bottom of the honeycomb, obstructing the smooth flow of the medium, and reducing the amount of oxygen diffused and dissolved into the medium. Therefore, even if the substrate is distributed, the activity of microorganisms, especially those in the inner layer, declines due to lack of oxygen, leading to a rapid decrease in the yield of the desired reaction product. Increasing the flow rate of the medium in order to restore the yield not only reduces the reaction time, but also causes microorganisms to escape and be washed away by the shear force generated by the rapid liquid flow, creating a vicious cycle in which efficiency is further reduced.

The present inventor has conducted extensive research in order to break such a vicious cycle and provide a fermentation method and device that utilizes a microorganism-immobilized ceramic honeycomb and that does not cause a decrease in reaction efficiency and yield over time. It has been reached.

(Means for solving problems) The fermentation method using immobilized microorganisms according to the present invention is as follows.

A reaction substrate-containing liquid medium is passed through the vertical cells of a through-cell honeycomb made of a ceramic porous body while being aerated from the bottom, and brought into contact with enzyme-producing microorganisms adsorbed and supported on the ceramic partition walls in the cells. In the reaction method, a portion of the medium rising inside the cell at a flow velocity formed by floating blades of aerated air bubbles directed toward the lower end opening of the cell is transferred from the upper end opening of the cell to the outside or inside of the honeycomb. It is characterized in that the amount of microorganisms adsorbed on the ceramic partition wall is maintained at an appropriate level by refluxing the medium downward through the reflux path and guiding it again to the lower end opening of the cell to form an upward and downward circulating flow of the medium.

In a preferred embodiment of the method of the invention, the linear velocity of the liquid medium rising in the cell is 20-500 mtn/sec, preferably 100-300 mtn/sec.

Further, in the method of the present invention, the bubbles aerated toward the lower end opening of the vertical channel are preferably guided to the opening.

The apparatus for carrying out the method of the present invention is a cylindrical vertical reaction tank equipped with a reaction substrate-containing liquid medium supply port and a reaction product-containing liquid medium outlet, and a through-hole made of a ceramic porous body for immobilizing microorganisms. In an apparatus in which a cell-type honeycomb is loaded so that the long axis of the cells is substantially vertical, and has an aeration means for aerating from below into the honeycomb, a reflux path for descending the culture medium extending vertically is connected to the honeycomb. This is a fermentation device using immobilized microorganisms, characterized in that it is formed on the outside or inside of a.

The honeycomb in the apparatus of the invention preferably has a cross-sectional area of at least about 30% of the cross-sectional area of the reaction vessel, and its preferred height is at least about 30% of the medium head. Further, a suitable cell pitch diameter is about 1.0 to 20 mm, and if it exceeds 20 mm, the surface area becomes too small and a sufficient amount of bacterial cells cannot be supported, which is not preferable.

In a preferred embodiment of the device of the invention, a guide member is provided at the honeycomb bottom for guiding the aerated air to the cell openings at the honeycomb bottom.

The cross-sectional area of the medium descending channel is preferably at least 1/2 of the total cross-sectional area of the cells.

Further, the descending flow path is preferably formed along the inner wall of the reaction tank.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The configuration of the present invention will be described in further detail below with respect to the embodiments shown in the accompanying drawings.

FIG. 1 is a schematic explanatory diagram of a specific example of the apparatus of the present invention.

In the figure, a cylindrical vertical reaction tank 3, such as a cylindrical or rectangular tube shape, is provided with a supply port 1 for a substrate-containing liquid medium and a discharge port 2 for discharging a medium containing a reaction product and an unreacted substrate. A porous ceramic body for immobilizing microorganisms is housed therein, for example, a through-cell type honeycomb 4 made of a sintered body. The honeycomb is loaded so that the long axes of the plurality of parallel open-ended cells 5 are substantially perpendicular. Substantially vertical is understood to mean that a slight inclination is acceptable as long as the medium flowing through the cell flows smoothly. Further, an aeration means 6, such as a perforated aeration plate or an aeration cylinder communicating with a compressed air supply source, is provided at the bottom of the reaction tank 3, and the structure is configured such that aeration can be directed toward the lower end openings of the cells at the bottom of the honeycomb. do.

The most important point of the device of the present invention is that a return flow path 7 is provided vertically outside the honeycomb, through which the culture medium that has flowed through the cells 5 and ascended can descend again.

In the illustrated example, the reflux passage 7 has an annular cross section divided by the outer wall of the honeycomb and the inner wall of the reaction tank, but for example, as shown in FIG. Alternatively, as illustrated in FIG. 4, the gap between the cylindrical reaction tank and the prismatic honeycomb inscribed therein may be used as the reflux path.

In short, the arrangement of the return flow path can be arbitrarily determined as long as it has a cross-sectional area sufficient to reduce fluid resistance so that the culture medium that has flowed upward through the honeycomb can descend in a laminar flow.

Furthermore, in the apparatus of the present invention, it is preferable that a guide member 8 for guiding aerated bubbles to the cell openings at the bottom of the honeycomb be provided at an appropriate position in the space between the bottom surface of the honeycomb and the aeration means. A funnel-shaped annular member is preferred.

(Function) The apparatus of the present invention having the above-mentioned configuration first performs the reaction "
A reaction tank is filled with a suspension of cultured microorganisms required for this purpose, and the microorganisms are adsorbed and carried on the honeycomb. Next, supply port 1
When a liquid medium containing a more reactive substrate is supplied and aeration is directed toward the bottom surface of the honeycomb from the aeration means 6 at the bottom, air bubbles rise in the honeycomb cells, and the medium flows through the cells with the flow velocity formed by the floating blades. It flows through and is sent to the upper surface of the honeycomb, descends through the reflux path 7, and reaches the lower end opening of the cell again. In this way, the upward and downward circulation flow shown by the arrow is formed. The bubble guide member 8 is particularly useful for guiding bubbles into the cell at the start of operation and concentrating the bubble floating blades inside the cell to form a circulating flow.

In such a system, the liquid medium passing through the honeycomb cells is pushed up by rising air bubbles, and its flow rate increases accordingly.
Since an appropriate shearing force acts between the cells and the cell walls, it is possible to remove some of the microorganisms that have grown over time and are adsorbed and supported on the partition walls within the cells, thereby adjusting the adsorption amount to an appropriate amount. A suitable flow rate for performing such an adjusting action is a linear velocity of 2 (1 to 500 mm/s, more preferably 1
It is in the range of 00 to 300 mm/sec. If the flow rate is too high and exceeds the above range, the reaction efficiency will be drastically reduced, and if it is too low, the cell will become narrowed due to the proliferation of microorganisms, and in extreme cases, it will become blocked, making it difficult for smooth oxygen and substrate flow. The supply is inhibited and the reaction efficiency is reduced as well.

The above flow rate can be adjusted by increasing or decreasing the amount of aeration. The appropriate amount of aeration depends on the capacity and head of the reaction tank, the supply amount and viscosity of the medium, the honeycomb structure such as cell pitch, etc., but is usually 0.3 to 1.5 VVM.
A degree of QVVM, preferably about 0.5 to 1, is suitable.

Here, VVM represents the ratio of aeration capacity to device volume per unit time.

According to the present invention, excessive growth and adhesion of microorganisms is prevented by selecting an appropriate aeration amount, and air bubbles are maintained in a good state of passage, descending due to circulation flow, and staying in the medium for a long time. The overall oxygen transfer capacity coefficient (KLa value), which is an indicator of the amount of oxygen dissolved in the water, increases significantly, and the activity of microorganisms becomes active.

Furthermore, since the flow rate of the medium passing through the honeycomb increases, the contact efficiency between the substrate and microorganisms also increases. It has been demonstrated that this increase in contact efficiency and the activation of microorganisms due to the increase in KLa value act synergistically, and the yield of the reaction product is approximately three times that of the conventional method.

(Examples) The present invention will be explained below using Examples, but the present invention is not intended to be limited by the Examples.

In the examples, the KLa value was measured by the so-called sodium sulfite oxidation method.

Example 1 Reactor tanks (A and B) shown in FIGS. 1 and 2, each with a capacity of 2.5 liters, and a degas tank (C) of the same structure and capacity as in FIG. 2, except that no honeycomb is loaded. The KLa values due to aeration were compared using the following. Fill each tank with water,
The KLa values after aeration for 1 hour at an aeration rate of 0.5 VVM with the flow stopped were as follows.

A (device of the present invention) 130 B (known device) 78 C (control) 130 That is, in the experiment using the conventionally known device, the bubble movement was limited to the upward direction only, so the bubble residence time was shortened. As a result, the surface area of the bubbles becomes smaller and the amount of dissolved oxygen decreases due to the association of bubbles in the cell.
4m decreased compared to C.

In the device A of the present invention, the formation of a circulating flow causes the bubbles to flow downward, resulting in a longer residence time, and the flow rate in the cell is high, making it difficult for bubbles to aggregate, so that the air bubbles are approximately between the two. KLa values were obtained.

These results fully confirm that the device B of the present invention is advantageous in terms of contact efficiency between the carrier and the substrate.

Example 2 A 500 rd volume reaction tank of the type shown in FIGS. 1 and 2 with an inner diameter of 52 mm and a head up to the liquid level of 235 mm was filled with a honeycomb made of a ceramic porous body. For the reaction tank shown in FIG. 1, a honeycomb carrier made of fired mullite with a cell pitch of 7.4 m+n, 23 mm opening x 130 mmH, and a net solid capacity of 16 ml was used. This reaction vessel was called the first reactor.

The reaction tank shown in Fig. 2 as the second reactor is made of fired cordierite with a cell pitch of 2.5 man.
mmφX150 mmH, solid net capacity 45m7! A honeycomb carrier was used.

Yeast, Aureobasidium, sp, 5N were added to the honeycombs of both reactors, respectively.
-642 is adsorbed and supported, and an aqueous liquid medium having a composition of 33.5% glucose substrate, 1.1% yeast extract, and pH 5.0 is supplied; 1. Ellis IJ) by degrading glucose while performing OVVM aeration)
- Produced 100ml. At this time, the fluid linear velocity within the honeycomb cells was 200 mm/sec in the first reactor and 50 mm/sec in the second reactor.

In the honeycomb of the second reactor, cell narrowing became noticeable two weeks after the start of fermentation, and the mouth was completely blocked for three weeks. on the other hand,
The honeycomb of the first reactor was able to operate stably and continuously for one month without any blockages. The results of fermentation in both reactors are shown in the table below.

As is clear from the above table of hydraulic retention times, the production rate of the method of the present invention is approximately three times that of the conventional method, which is due to the high contact efficiency between the substrate and yeast. Moreover, the amount of dissolved oxygen is large, indicating that the metabolism of the yeast cells is active.

(Effects of the Invention) As can be fully understood from the above explanation, according to the method and apparatus of the present invention, the formation of a circulating flow increases the flow rate in the cells of the honeycomb carrier of the bioreactor, thereby increasing the adsorption of microorganisms.
is always maintained at an appropriate value, a sufficient amount of oxygen necessary for the metabolism of microorganisms is supplied and diffused into the culture medium through aeration, and the activity of microorganisms is maintained well. The synergistic effect of increasing contact efficiency between the substrate and microorganisms dramatically increases production volume, and enables stable continuous operation over long periods of time, resulting in extremely high productivity and great utility in the biochemical industry. It is possible to provide a bioreactor with the following characteristics.

[Brief explanation of the drawing]

Fig. 1 is a schematic explanatory diagram showing aspects of the apparatus of the present invention, Fig. 2 is a schematic explanatory diagram showing a conventionally known fermentation apparatus, and Figs. It is a schematic plan view showing an aspect. 1...Medium supply port 2...Medium discharge port 3...
・Reaction tank 4...Honeycomb death...Cell
6...Aeration means 7...Recirculation path
Deceased...Guide member Fig. 2 Fig. 3 Fig. 4

Claims (1)

[Claims] 1. A reaction substrate-containing liquid medium is passed through the vertical cells of a through-cell honeycomb made of a ceramic porous body while being aerated from the bottom, and is adsorbed and supported on the ceramic partition walls in the cells. In this method, a portion of the medium rising in the cell at a flow rate formed by floating blades of aerated air bubbles directed toward the lower end opening of the cell is directed toward the lower end opening of the cell and brought into contact with the enzyme-producing microorganism. The amount of microorganisms adsorbed on the ceramic partition walls is maintained at an appropriate level by allowing the culture medium to flow downward through the openings through the reflux channels outside or inside the honeycomb and then guided back to the lower end openings of the cells to form an upward and downward circulating flow of the medium. A fermentation method using immobilized microorganisms characterized by the following. 2. The linear velocity of the medium rising in the cell is 20 to 500
A fermentation method using an immobilized microorganism according to claim 1, wherein the fermentation rate is mm/sec. 3. In a cylindrical vertical reaction tank equipped with a reaction substrate-containing liquid medium supply port and a reaction product-containing medium discharge port, a through-cell type honeycomb made of a ceramic porous body for immobilizing microorganisms is placed so that the long axis of the cells is In an apparatus that is loaded substantially vertically and has an aeration means for aerating from below into the honeycomb, a culture medium descending flow path extending vertically is formed outside or inside the honeycomb. Fermentation equipment using characteristic immobilized microorganisms. 4. A fermentation device using immobilized microorganisms according to claim 3, wherein a guide member is provided at the bottom of the honeycomb for guiding the aerated air to the cell openings at the bottom of the honeycomb.