JPH11225740A - High layer type interfacial bioreactor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an interfacial bioreactor having good efficiency, in which defects which conventional various interfacial bioreactors have are dissolved. SOLUTION: This bioreactor is obtained by superposing plural flat plate type interfacial bioreactors in high layers, connecting organic solvent layers of these interfacial bioreactors to each other and circulating the solvent. The flat plate type interfacial bioreactor is obtained by locating a hydrophilic immobilization plate-like carrier in which nutrient and water are contained and a proliferated microorganism film is attached and fixed on the surface on the bottom of the bioreactor and locating the proliferated microorganism film thereon, providing an organic solvent layer containing a hydrophobic substrate thereon, bringing a proliferated microorganism film into contact with the organic solvent layer to convert the hydrophobic substrate by a microorganism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for centrally controlling the entire organic solvent layer by stacking a plurality of plate-type interfacial bioreactors in a vertical direction and interconnecting organic solvent layers containing reaction substrates. The present invention relates to a high-rise interfacial bioreactor capable of performing microbial conversion.

[0002]

2. Description of the Related Art In recent years, research and development of bioconversion using biocatalysts in organic synthetic chemistry have been conducted around the world in the background of rising global environmental problems, resource saving and energy saving problems, and expanding demand for optically active compounds. [Ota Hiromichi, Yuka, 41, 1018 (19
83); Masami Shimizu and Hideaki Yamada, 41, 1064 (198)
3)]. The bioconversion is roughly classified into an enzymatic method using a purified enzyme and a microbial conversion method using a microbial cell itself as a biocatalyst. The latter is an inexpensive biocatalyst and a coenzyme-requiring reaction. It has the advantage of being able to be used at low cost for redox reactions, and many applications have been reported [Ohta, H., et al., Agric.
Biol. Chem., 46, 579 (1982); Oda, S., e.
t al., Biosci. Biotech. Biochem., 56, 1216.
(1992)].

[0003] On the other hand, bioconversion from the viewpoint of the solubility of a substrate in a reaction solvent can be divided into the conversion of a water-soluble substrate and the conversion of a water-insoluble (poorly) -soluble substrate. For the latter, various measures have been reported to enhance the solubility of the substrate. For example, a method of adding a surfactant [Nakahara, T., et al., J. Ferment. Technol., 5
9, 415 (1981)], a method of adding a water-miscible organic solvent [Freeman, A. and Lilly, MD, Appl. Microb.
Biotechnol., 25, 495 (1987)], a method of adding a water-immiscible organic solvent or a two-phase aqueous-organic solvent reaction method [Hocknull, MD and Lilly, MD, App.
l. Microbiol. Biotechnol., 33, 148 (199
0)], a crystal fermentation method [Kondo, E. and Masuo, E., J. Ge
n. Appl. Microbiol., 7, 113 (1961)] and the like. However, microbial conversion methods using microbial cells as biocatalysts have various practical limitations because microorganisms generally die or cannot grow in organic solvents. The inventor of the present invention has previously proposed an interfacial bioreactor as a technique for overcoming the above-mentioned weak point of microbial conversion of a water-insoluble (poorly soluble) substrate [Patent No. 2542766; Shinobu Oda and Hiromichi Ota; 70, 538 (1997); Oda, S. and Ohta,
H., Biosci. Biotech. Biochem., 56, 2041 (1
992)]. The interface bioreactor attaches microorganisms to the surface of a hydrophilic immobilization carrier containing a nutrient source and water,
The microorganisms are brought into contact with the substrate on the surface of the carrier, and the microorganisms are converted in that state.The microorganisms can proliferate vigorously even when they come into contact with hydrophobic organic solvents such as paraffins. This system is applicable to various microorganism conversion reactions. Furthermore, they discovered that the microorganism used as a biocatalyst in the interfacial bioreactor actively maintained metabolic activity, and used acetyl coenzyme A derived from glucose metabolism as an acetyl donor, and a primary alcohol added to this in an organic solvent. (Metabolism-microbial conversion fusion method, coupling system) that acetylates acetyl by using alcohol acetyltransferase present in yeast cells as a catalyst [Od
a, S., et al., Appl. Environ. Microbiol., 62, 2
216 (1996); Oda, S. and Ohta, H., J. Ferme.
nt. Bioeng., 83, 243 (1997)].

The interfacial bioreactors used in these reports include an agar plate type reactor using an agar plate as a carrier, an agar-coated filter plate and a plate filled with a plate-like carrier based on polyvinyl alcohol in a vertical or horizontal arrangement. The dominant carrier-filled reactor was the dominant one. However, in the former flat plate reactor, the reaction field is limited to a two-dimensional plane, and the amount of an organic solvent layer containing a substrate that can be processed at one time is limited, and thus it is not suitable for scale-up.

On the other hand, in a plate-shaped carrier-filled reactor, the amount of the organic solvent per unit area of the carrier, in other words, the base mass per unit amount of the catalyst is more than twice as large as that of the plate-type reactor, and as a result, the reaction speed is increased. Is inevitable. Furthermore, in a granular carrier-filled interfacial bioreactor filled with a granular carrier, the flow of a hydrophilic granular carrier in a hydrophobic organic solvent is practically impossible. Blockage of the particle gap due to its own weight and accompanying inhibition of liquid flow are recognized, and scale-up is difficult. Accordingly, there is a strong demand for the development of a more practical system for an interfacial bioreactor that exerts its power in the microbial conversion of a water-insoluble (poorly soluble) substrate in view of the scale-up.

[0006]

The present inventors have conducted intensive studies on an interfacial bioreactor which has overcome the above-mentioned problems of the conventional flat plate type, plate-like carrier-filled type or granular carrier-filled type reactors. Combines the advantages of an organic solvent layer amount per unit surface area of a carrier, in other words, a flat plate reactor in which the base mass per unit catalyst amount can be set arbitrarily and appropriately, and a plate-shaped carrier-filled reactor that can be easily scaled up. In other words, the conventional interfacial bioreactor is constructed by stacking a plurality of flat-plate reactors in the up-down direction and constructing a high-layer reactor in which the organic solvent layers are connected so that the organic solvent layers can be centrally controlled. Succeeded in solving the disadvantages of the present invention, and completed the present invention.

Thus, according to the present invention, a nutrient source and water are contained therein, and a growing microbial membrane adheres to the surface.
The immobilized hydrophilic immobilized plate-shaped carrier is positioned at the bottom with the microbial membrane facing upward, and an organic solvent layer containing a hydrophobic substrate is provided thereon to separate the growing microbial membrane and the organic solvent layer. A plurality of plate-type interfacial bioreactors that are brought into contact to convert the hydrophobic substrate into microorganisms are stacked in a high layer, and the organic solvent layers of each interfacial bioreactor are interconnected and circulated. Is provided.

Hereinafter, the high-rise interfacial bioreactor of the present invention will be described in more detail with reference to FIG.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As shown in FIG. 1, a high-layer interfacial bioreactor according to the present invention comprises a plate-type interfacial bioreactor (2) in which a hydrophilic immobilized carrier (1) represented by an agar plate is positioned at the bottom. ) Is defined as one independent section (unit), and a microorganism film (3) is formed on the upper surface of the hydrophilic immobilized carrier by growing microorganisms which are biocatalysts.
An organic solvent layer (4) in which a substrate is dissolved is added on the microbial membrane (3) in a layered manner to complete a reactor unit. A plurality of the reactor units are sequentially stacked in the vertical direction, and the outlet of the organic solvent overflow nozzle (5) installed in the reactor of each unit is sequentially opened on the organic solvent layer of the reactor unit immediately below the reactor unit. The organic solvent phase overflowing from is naturally spouted through the nozzle (5) so as to be sequentially sent to the reactor unit immediately below. The organic solvent layer which has overflowed in this way reaches the lowermost reactor unit, and the organic solvent layer flowing out therefrom is sent to the uppermost portion by the liquid sending pump (6). Thereby, the organic solvent layers of all the reactor units are interconnected and circulated. An outlet for collecting and collecting the organic solvent layer can be provided in this liquid sending line. Thus, the organic solvent layers of a number of reactor units can all be centrally controlled as one continuous layer.

However, the first advantage of the interfacial bioreactor of the present invention is that it has both advantages of a plate-type interfacial bioreactor and a plate-shaped carrier-filled interfacial bioreactor. That is, in the high-layer interface bioreactor according to the present invention, each unit reactor (compartment) has a configuration similar to the flat interface bioreactor.
Aseptic operations such as reactor preparation and carrier inoculation can be easily performed for each individual unit reactor, and the amount of the organic solvent layer per unit surface area of the carrier can be set arbitrarily and low. In addition, the interface bioreactor of the present invention is a high-rise type in which unit reactors are vertically stacked, and the organic solvent layers are connected to each other and can be centrally controlled. It is possible to expand the reactor in a three-dimensional direction.

The second advantage of the interfacial bioreactor of the present invention is that the organic solvent layers of the individual unit reactors (compartments) are interconnected and centrally controlled, thereby reducing the variation of the conversion reaction rate for each unit reactor. It can be eliminated. Since the variation in the conversion speed for each unit reactor means the variation in the degree of progress of the conversion reaction, it is possible to obtain a high-purity product by eliminating the variation. Further, in the interfacial bioreactor of the present invention, since the organic solvent layer can be treated as a continuous layer, quantitative analysis using only one sample is sufficient, and addition of additives, continuous operation, and the like become extremely easy.

Further, a third advantage of the interfacial bioreactor of the present invention is that it does not require a carrier having a high mechanical strength such as a plate-type carrier-filled interfacial bioreactor. In a plate-shaped carrier-filled interfacial bioreactor, the individual carriers must maintain strong strength to avoid bending or breakage. For that purpose, the carrier must have a strong skeleton such as a stainless steel frame inside the carrier, and the carrier material itself must also maintain strong strength.
In the present invention, the support is made of a material having a weak strength for a flat plate reactor, and need not have a strong skeleton inside.

In the present invention, the hydrophilic carrier provided at the bottom of each reactor unit (compartment) includes, for example, a carbon source such as glucose, a nitrogen source such as urea, an inorganic salt such as magnesium sulfate, and a trace amount such as yeast extract. There is no particular limitation as long as it can contain very common nutrients and water, such as nutrients, and those conventionally used for immobilizing microbial cells can also be used. , For example, agar,
Carriers such as natural polymers such as alginic acid, carrageenan, and starch matrix; synthetic polymers such as polyvinyl alcohol and polyacrylamide; and inorganic porous materials such as foam glass can be used (see Japanese Patent No. 2542766). However, since the carrier used in the present invention is installed at the bottom of each reactor and used, it does not need to have exceptionally strong strength, and a low-strength gel such as an agar plate can also be used. Rather, the agar plate does not require replacement of the aqueous phase inside the gel with a liquid medium after preparation, such as a carrier based on polyvinyl alcohol, and allows the nutrient source to be included in the gel at the time of gel preparation. This is advantageous because it can be performed.

[0014] Such a hydrophilic immobilized carrier, for example,
When microbial cells are adhered (inoculated) by a method of applying a cell suspension, a method of dipping a carrier in the cell suspension, or the like, the inoculated microbial cells are Divide and proliferate using nutrient sources, and form a membrane (microbial membrane) on the carrier surface
To form

The microorganisms that can be used in the interface bioreactor of the present invention are not particularly limited, and any of bacteria, actinomycetes, yeasts, molds and the like can be used.

The organic solvent usable for the organic solvent layer of the high-layer interface bioreactor in the present invention is not particularly limited as long as it is a hydrophobic organic solvent which does not exhibit toxicity to microorganisms. Organic solvents such as chain paraffins, long-chain esters and ethers can be used (see Japanese Patent No. 2542766). Regarding the selection of the organic solvent, the solubility of the substrate and the product, physical properties such as boiling point, price and supply stability, toxicity to microorganisms, etc. may be selected as an index. Normal decane is one of the satisfactory organic solvents. Normal decane is safe to handle because of its excellent solubility of many hydrophobic substrates and products and its low boiling point. Further, it is suitable because it can be obtained in large quantities at a low price of about 200 yen per kg and furthermore, it does not exhibit toxicity to almost all bacteria, actinomycetes, yeasts and molds.

The substrate that can be present in the organic solvent layer is not particularly limited either, and can be arbitrarily selected according to the desired bioconversion. Ketones or olefins; lipophilic alcohols, aldehydes or olefins as oxidation substrates; lipophilic esters or epoxides as hydrolysis substrates; lipophilic alcohols or carboxylic acids as esterification substrates; Fat-soluble ketones which are substrates for amination can be exemplified.

The high-layer interface bioreactor of the present invention comprises:
Although it is a system that can scale up in a narrow space while retaining the advantages of a flat plate type interface bioreactor,
It is not just a matter of stacking up flat reactors in the vertical direction.It can be expanded to a remarkably large scale by stacking multiple stacked high-level interfacial bioreactors and connecting their organic solvent layers further. It is also possible. The overflow of the organic solvent layer from these multiple towers is collected in one place,
It is also possible to send the liquid back to each of the multiple towers, but care must be taken in this case, even if many liquid pumps are used and one liquid pump is used. However, the point is that the liquid sending speed to the multiple towers must be equal. To this end, it is desirable to use as few liquid feeding pumps as possible and to set all liquid feeding lines in parallel. Also, when the overflowed organic solvent layer is collected in one tank and sent by one pump, the pump often involves air. Therefore, as the liquid sending pump, even if air such as a diaphragm pump is used. It is preferable to use a drivable one.

Thus, by multiplying the multi-layered high-layer interface bioreactor, a remarkably large scale-up can be achieved in a narrow space. The number of stages of unit reactors to be stacked depends on the size of the individual unit reactors, especially the height. For example, when using a commercially available stainless steel stacking bat, the height is only 5 cm per stage, so If is fixed firmly, it is about 5 m at 100 steps and about 10 m at 200 steps, and it is possible to remarkably increase the height at a low height. It is sufficient if the height of each unit reactor is about 5 cm, and if it is more than 5 cm, the dead space increases, which is uneconomical.

Furthermore, parallelization depends to some extent on the set number of pumps, but rather on construction space. According to the high-rise interface bioreactor of the present invention, there is an advantage that the number of stacks and the number of towers to be parallelized can be freely determined according to the production plan of the conversion product. By doing so, it is possible to significantly improve the conversion rate even at a low circulation rate of the organic solvent layer.

On the other hand, in the high-layer interface bioreactor of the present invention, a very large number of unit reactors are required for scale-up, but each unit reactor can be aseptically prepared and stored. So, for example,
Even when a nutrient agar plate that is extremely susceptible to germ contamination is used as a carrier, there is an advantage that scale-up is not hindered.

[0022]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to only these examples.

Example 1 A stainless steel stacking vat having a length of 26 cm, a width of 34 cm and a height of 5 cm was used as a reactor unit, and was stacked in 10 stages to assemble a high-layer interfacial bioreactor as shown in FIG. Peptone 5 for each bat
g, yeast extract 3g, malt extract 3g, magnesium sulfate 1g, glucose 10g, agar 20g and a nutrient agar medium consisting of distilled water 1 liter (pH 6.0) 500m
1 was dispensed and cured to obtain a flat plate. Add Hansenula saturnus IFO 080 to the plate medium.
No. 9 was inoculated with 5 ml of a daily culture solution, and allowed to stand still for 1 day to grow a microbial membrane. After connecting the overflow nozzles of each unit with a Viton pipe, a 4% ( RS ) -citronellol / decane solution was injected from the top of the 1.8 liter using a liquid sending pump, and overflowed in order. An organic solvent layer was filled in each reactor.
Thereafter, the organic solvent layer was circulated at a liquid sending rate of 100 ml / min while reciprocatingly shaking the high-layer interfacial bioreactor comprising 10 stages (units) at a shaking number of 40 strokes / min. The sample was sampled every day from a sample collection port installed in the middle of the line from to the liquid sending pump, and the conversion reaction was traced using gas chromatography.

The present strain oxidizes ( S ) -citronellol of racemic citronellol and preferentially accumulates ( S ) -citronellic acid, but the conversion reaction proceeds from the first day and starts on the seventh day. The accumulated concentrations of acid and citronellol reached 19.2 and 18.5 g / l, respectively. After completion of the reaction, the entire decane solution layer was recovered, and 1 kg of silica gel was used.
Was applied to the column. After removing decane using hexane, citronellol and citronellic acid were eluted with hexane-ethyl acetate (8: 2) and ethyl acetate, respectively. After removal of the solvent, citronellol was Jones-oxidized to derive citronellic acid. After both the obtained citronellic acids were derivatized to acid chloride, they were amidated with ( R ) -1- (1-naphthylethyl) amine, and the absolute configuration and the enantiomeric excess were measured using liquid chromatography. As a result, the absolute configurations of the corresponding citronellol and citronellic acid are ( R ) and ( S ), respectively, and the enantiomeric excess is 7%.
2, 81% ee.

Comparative Example 1 The same conversion reaction was carried out independently on each of the 10 reactor units without circulating the organic solvent layer of the high-layer interface bioreactor of Example 1. 4% ( RS ) -citronellol /
Decane solution is 180 ml per reactor,
Shaking was set at 40 strokes / min. Samples were sampled individually in 10 stages, and the conversion reaction was followed by gas chromatography. As a result, the oxidation rates in the 10 stages were varied from the first day of the reaction, and were not more than 7 days after the start of the reaction.
On the day, the accumulated concentrations of citronellic acid from the first stage (bottom) to the tenth stage (top) are 16.2 and 15.
4, 19.9, 22.3, 14.8, 23.6, 21.7,
18.2, 19.9 and 25.1 g / l.

Thereafter, the entire organic solvent layer was recovered, and citronellol and citronellic acid were separated in the same manner as in Example 1.
After purification, the absolute configuration and enantiomeric excess were determined. As a result, the absolute configurations of citronellol and citronellic acid were (R) and (S), respectively, and the enantiomeric excess was 66 and 72% ee, respectively. Met.

Example 2 A polyvinyl alcohol-glutaraldehyde-calcium alginate composite crosslinked gel was used as a carrier.
In the same manner as in the above, a 30-stage high-layer interface bioreactor was assembled. 10% complex crosslinked gel for each reactor unit
Polyvinyl alcohol-10% glutaraldehyde-5
% Of sodium alginate (100: 1: 10), cross-linked polyvinyl alcohol and glutaraldehyde for 2 days at room temperature, and then contacted the composite cross-linked gel with 0.1 M calcium chloride solution at room temperature for 2 days. And calcium ions were salt-crosslinked to completely gel. After washing with water, the surface of the composite cross-linked gel plate was sterilized by ultraviolet irradiation, and then 2 liters of a liquid medium of the medium composition (excluding agar) used in Example 1 was dispensed per unit, and the gel was kept at a low temperature for 3 days. The aqueous phase inside the plate was replaced with the liquid medium. Issatchenkia scutulata var. On the surface of this gel plate. scutulata
A microbial membrane was grown by inoculating 5 ml of a daily culture solution of IFO 10070 and statically culturing for 1 day.

The reactor units thus obtained are stacked in 30 stages, and the overflow nozzle is connected with a Viton pipe as shown in FIG. 1, and 3% 1-decanol /
A 4.5 liter decane solution was sent and overflowed sequentially to the lower reactor unit to connect the organic solvent layers. The solution sending speed of the diaphragm pump in the organic solvent layer circulation line was set to 500 ml / min, and the conversion test was performed at 30 ° C. under static conditions. In the conversion reaction from 1-decanol to decanoic acid, the decane layer was analyzed and followed by gas chromatography. As a result, the oxidative conversion reaction was observed from the first day of the reaction, and the accumulated concentration of decanoic acid reached 29.2 g / l on the seventh day of the reaction.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view of an example of a high-layer interface bioreactor of the present invention.

Claims (3)

[Claims] 1. A hydrophilic immobilized plate-like carrier containing a nutrient source and water therein and having a growth microbial membrane adhered and immobilized on the surface thereof is positioned at the bottom with the microbial membrane facing up,
An organic solvent layer containing a hydrophobic substrate is provided thereon, and a plurality of plate-type interfacial bioreactors, which are configured to contact the growing microbial membrane and the organic solvent layer to convert the hydrophobic substrate into microorganisms, are formed in a multi-layered form. Characterized in that the organic solvent layers of each interfacial bioreactor are interconnected and circulated. 2. The method according to claim 1, wherein each of the plate-type interface bioreactors constitutes an independent section, and these sections are vertically stacked, and then the organic solvent layers of adjacent plate-type interface bioreactors are interconnected. Item 1. The bioreactor according to Item 1. 3. The connection of the organic solvent layer of the plate-type interfacial bioreactor follows the sending of the organic solvent layer to the uppermost plate-type interfacial bioreactor, and then the organic solvent layer to the lower plate-type interfacial bioreactor. 2. The bioreactor according to claim 1, wherein the bioreactor is formed by overflowing the organic solvent layer and recirculating the organic solvent layer flowing out from the lowermost portion to the uppermost planar interfacial bioreactor.