JPH08163989A - Interfacial bioreactor system - Google Patents

Abstract

PURPOSE: To provide an efficient interfacial bioreactor easy to scale up, designed to subject a water-insoluble or sparingly water-soluble stock to bioconversion. CONSTITUTION: This interfacial bioreactor system is made up of a reaction column 1 packed at a rate of 10-70% with a granular hydrophilic immobilizing carrier 3 having a granular diameter of 5-100mm, immobilized with microorganisms in proliferable or viable state and containing a liquid medium composed of water and nutrients necessary for the microorganisms proliferation or living and an aeration column 5 connected to the reaction column 1, designed to circulate a reaction solvent 6 containing a virtually water-insoluble or sparingly water-soluble reaction substrate from the lower part to upper part of the reaction column at a rate of 10-10000ml/min and capable of feeding and mixing the reaction solvent with oxygen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an interfacial bioreactor system for microbial conversion of a water-insoluble or sparingly soluble raw material.

[0002]

2. Description of the Related Art Conventionally, a method of converting a water-soluble and inexpensive raw material into a useful substance by using a microorganism has been used particularly in the production of an optically active substance represented by a raw material such as pharmaceuticals, agricultural chemicals and electronic materials. [Ota Hiromichi, ification, 14 volumes, 823
(1983); Yu Fujisawa et al., Yuka, 44, 519 (1).
986); Hirota Ota, Bioscience and Industry, 44, 823 (1991)]. On the other hand, as the microbial conversion of the raw material that is not soluble in water, in order to compensate for the low solubility and dispersibility of the raw material, an emulsion method or a surfactant addition method [T. Nakaha
ra, et al . , J. et al. Ferment. Techo
l. , 59, 415 (1981)], a method for adding a water-miscible organic solvent [A. Freeman and M.D. D. Li
lly, Appl. Microbiol. Biotec
hnol. 25, 495 (1987)], water-immiscible organic solvent addition or water / organic solvent two-phase reaction method [M. D. Hocknull and M.D. D. Lil
ly, Appl. Microbiol. Biotech
nol. , 33, 148 (1990)] have been proposed, but these methods cannot avoid the toxicity of raw materials and additives and produce a target substance at a high concentration. Is difficult.

On the other hand, an interfacial bioreactor using a microorganism that grows at the interface between a hydrophilic carrier and a hydrophobic organic solvent as a biocatalyst is also known. The interfacial bioreactor has excellent advantages such as improved solubility of raw materials and products, avoidance of toxicity thereof, and abundant oxygen supply from organic solvent, and can be used for almost all microorganisms. And further microbial redox, hydrolysis,
It has the advantage that many reactions such as esterification can be performed with good results [S. Oda, et al . ,
Biosci. Biotech. Biochem. , 5
Vol. 6, 2041 (1992); JP-A-5-91878, JP-A-5-344896, JP-A-6-88.
JP-A-6-90, JP-A-6-95, JP-A-6-197773, and JP-A-6-197.
777].

[0004]

The above-mentioned interfacial bioreactor often uses an agar plate as a hydrophilic carrier, and there is a report on the premise of increasing the scale [S. Oda, et al . , J. et al. Ferment. B
ioeng. , 78, 149 (1994)]. It is still insufficient for practical use, and has the following problems. In the interfacial bioreactor, due to the high oxygen-dissolving power of the hydrophobic organic solvent that is the reaction solvent, the microbial reaction proceeds sufficiently with a significantly smaller aeration amount than the aqueous reaction, but the reaction tower becomes larger and its height increases. In this case, it is necessary to incorporate a ventilation line. In this case, if air is directly ventilated into the reaction column filled with the particulate carrier, an air trap will be generated immediately, causing a serious problem. further,
In a particle-filled bioreactor, mechanical agitation is practically impossible, and it has a great influence not only on oxygen supply but also on material supply to microorganisms and release of products outside the cells.
In the reaction tower filled with the particulate carrier, the larger the scale of the reaction tower is, the larger the packing amount of particles becomes, and the particles in the lower part of the reaction tower are compressed (consolidated) by their own weight, and the particles themselves may collapse. This compaction and particle collapse cause a blockage of the liquid delivery system and cause an increase in pressure, which is a major problem in terms of maintaining pump operation and safety.

[0005]

[Means for Solving the Problems] The present inventors have conducted extensive studies for the purpose of developing an interfacial bioreactor that does not have the above-mentioned problems and is efficient and easy to scale up. An external liquid in which a particulate hydrophilic immobilization carrier adhered / immobilized in a proliferative or viable state is filled in the reaction tower and an aeration tower is connected to the reaction tower to enable oxygen supply and mixing of the content liquid. It was found that the above object can be achieved by incorporating a circulation line, and the present invention has been completed.

Thus, according to the present invention, a particle in which a microorganism is adhered / immobilized on the surface in a state where it can grow or survive and which contains a liquid medium consisting of a nutrient source and water necessary for the growth or survival of the microorganism. A reaction column filled with a particulate hydrophilic immobilization carrier having a diameter of 5 to 100 mm at a filling rate of 10 to 70%, and a reaction including a substantially water-insoluble or sparingly soluble reaction substrate connected to the reaction column Provided is an interface bioreactor system comprising a vent column capable of circulating a solvent from a lower part of the reaction column to an upper part thereof at a circulation rate of 10 to 10,000 ml / min and supplying and mixing oxygen to the reaction solvent. To be done.

According to the present invention, a particulate hydrophilic immobilization carrier (hereinafter, also referred to as "particulate carrier") is adhered and immobilized on the surface in a state where microorganisms can grow or survive.
Into the reaction tower filled with, the reaction solvent containing the water-insoluble or sparingly soluble reaction substrate is injected from the aerating and stirring tower by a liquid-sending pump, and circulated, so that in the reaction tower filled with the particulate carrier. A sufficient stirring and mixing effect is produced, supply of oxygen and a reaction substrate to the microorganism and promotion of release of the product from the microorganism by the microorganism can be promoted to efficiently carry out the microorganism conversion reaction.

Therefore, the first feature or advantage of the present invention is to use an external liquid circulation line incorporating an aeration and stirring tower,
The point is that the reaction solvent in the reaction tower is circulated at a high speed, whereby a sufficient stirring and mixing effect of the reaction solvent can be obtained while the particulate carrier in the reaction tower is fixed as a reaction bed. Moreover, by supplying oxygen to the aeration and stirring tower, the uptake of oxygen into the reaction solvent is significantly improved, and the effect of supplying oxygen and reaction substrates to the cells and the effect of releasing the product to the outside of the cells are significantly improved. To do.

The second feature or advantage of the present invention resides in that the liquid is fed into the reaction column through the external liquid circulation line from the lower part of the reaction column, whereby the whole particle mass (reaction bed) is obtained. Tends to be lifted upward in the liquid flow, and the own weight of the reaction bed (particle lump) of the particulate carrier is canceled or reduced, so that even if the reaction tower becomes large,
The effect that the compaction and the collapse of the particles can be easily avoided is obtained.

A third feature or advantage of the present invention is that, when the activity of the microorganism is reduced after the reaction, the particulate carrier having the low activity microorganism adhered to the surface is recovered by steam sterilization, and the microorganism film is washed by surface cleaning. It is possible to drastically reduce the cost of the immobilized carrier because it can be removed and then immersed in a liquid medium to replace the aqueous layer inside the carrier with a fresh liquid medium and repeatedly used.

The fourth feature or advantage of the present invention is that the removal of microbial cells after the reaction is very easy. In the emulsion method, which is a normal microbial conversion method for water-insoluble raw materials, the microbial cells may be separated due to a small difference in specific gravity between the microorganism and the reaction substrate or product, or because the reaction substrate or product adheres to the cell surface. Although it is very difficult to separate the product from the product, in the present invention, since the microorganisms are adhered and immobilized on the surface of the particulate carrier, the removal of the particulate carrier is carried out by passing the reaction solvent containing the reaction substrate through the reaction tower and aeration. It can be carried out simply by extracting it from the tower and the circulation line.

The interface bioreactor system of the present invention will be described in more detail below.

The material for the particulate hydrophilic immobilization carrier that can be used in the present invention can contain and retain an aqueous liquid medium containing a nutrient source for microorganisms, and supplies the nutrient source to the microorganisms present on the surface of the carrier. There is no particular limitation as long as it can be used. Specific examples include natural polymers such as alginic acid, carrageenan, starch matrix, agar, and cellulose materials such as filter paper; polyvinyl alcohol, urethane polymer, polyacrylamide, polyacrylamide. Examples thereof include synthetic polymers such as acrylic acid; inorganic substances such as foam glass. However, when the carrier is repeatedly reused, the natural polymer other than the filter paper sphere has a problem that the strength is lowered. Therefore, it is preferable to use a synthetic polymer or an inorganic granular material. It is also possible to use a gelled material of low strength natural polymer or synthetic polymer as a coating agent and coat it on the surface of a strong strength granular material such as an inorganic granular material.

The interfacial bioreactor system of the present invention can be applied to any microorganism, including oxidation, reduction, hydrolysis,
It is possible to carry out various microbial conversion reactions such as ester synthesis reactions. However, the microorganisms that can be immobilized on the carrier as described above may be any microorganisms such as bacteria, molds, yeasts and actinomycetes, and may be either aerobic or anaerobic type. Good. Specifically, for example, Pseudomonas (Pseudomonas) genus Gluconobacter (Gluconobacter) genus Acetobacter (Acetoba
cter ) genus, Ahrithobacter ( Arthrobacter ) genus, Corynebacterium ( Corynebacterium ) genus, Rhodococcus ( Rhodococcus ) genus, Alcaligenes ( Alcaligenes )
Genus Candida (Candida) species, Hansenula (Hansenula)
Genus, mention may be made of Aspergillus (Aspergillus) a microorganism belonging to the genus belonging to such.

The attachment and immobilization of these microorganisms to the carrier is a method known per se, for example, JP-A-5-9187.
It can be carried out according to the method described in the literature such as JP-A-8, etc., and finally it is formed into particles and used.

The inside of these particulate carriers must be used by incorporating a liquid medium for culturing ordinary microorganisms, but the composition of the liquid medium used is not particularly limited, and it depends on the type of microorganisms used. It can be varied over a wide range, but generally one containing a carbon source such as glucose or sucrose, a nitrogen source such as an ammonium salt, an inorganic salt such as a magnesium salt, or a micronutrient source such as a yeast extract is used. be able to. When paraffins are used as the reaction solvent and also serve as the growth substrate, it is not necessary to include a carbon source inside the carrier.

In order to operate the interfacial bioreactor system of the present invention efficiently and in a scaled-up state without causing the above-mentioned problems, the size of the particulate carrier to be used and its packing rate for the reaction tower are appropriately set. The choice is important. Since the surface of the particulate carrier is thickened with the growth of the immobilized microorganisms, the gap between the packed particles is substantially reduced. Furthermore, it is an essential condition for an interfacial bioreactor that an organic solvent phase be present between the particulate carriers having a microbial layer on the surface to form a solid / liquid interface. For that purpose, it is desirable that the particle size of the particulate carrier to be used is large to some extent.
The range of -100 mm, preferably 10-50 mm is suitable. Further, the packing rate of the particulate carrier in the reaction tower is
Considering productivity, generally 10-70%, especially 30-6
It is preferably within the range of 0. Here, the particle size represents the average particle size, and the distribution width of the particle size is approximately in the range of 3 to 150 mm. The particle size can be measured using a caliper or a ruler. The filling rate of the particulate carrier refers to the ratio of the particulate carrier to the working volume (total volume of the particulate carrier and the reaction solvent).

As an organic solvent for preparing a solution containing a reaction substrate that is insoluble or sparingly soluble in water supplied to a reaction tower according to the present invention, it shows substantial toxicity to immobilized microbial cells. Those that do not exist are preferable.
Normal paraffins or liquid paraffins represented by methane hydrocarbons having 6 to 20 carbon atoms such as hexane, heptane, octane, nonane, and decane; isoparaffins such as isooctane; pentylbenzene, hexylbenzene, heptylbenzene, octylbenzene Normal alkylbenzenes having 5 to 15 carbon atoms in the aliphatic chain; isoalkylbenzenes such as cumene; alicyclic hydrocarbons such as cyclohexane; ethers such as hexyl ether; aromatic esters such as dibutyl phthalate; Examples thereof include aliphatic esters such as ethyl decanoate; silicone oils such as polydimethylsiloxane.

Of the above reaction solvents, it is preferable to select and use a hydrophobic organic solvent that is substantially harmless to the immobilized individual microorganisms.

On the other hand, as the substantially water-insoluble or insoluble organic compound which can be used as a substrate for the microbial conversion reaction, various compounds can be used depending on the conversion ability of the immobilized microorganisms, and are particularly limited. Not, for example, benzene, toluene, xylene, ethylbenzene, styrene,
Aromatic hydrocarbons such as naphthalene and phenanthrene;
Aliphatic hydrocarbons such as tridecane and tetradecane; alicyclic compounds such as cyclohexanol and cyclohexanone; heterocyclic compounds such as methylimidazole, collidine and picoline; lauric acid, palmitic acid, stearic acid, oleic acid, linole Higher fatty acids such as acids; higher alcohols such as octyl alcohol, decyl alcohol, lauryl alcohol, cetyl alcohol, stearyl alcohol; fatty acid esters such as ethyl caprylate and ethyl caproate.

The concentration of the reaction substrate in the organic solvent is not particularly limited and can be determined according to the toxicity to the immobilized cells. For example, when the substrate is a highly toxic aromatic hydrocarbon, it is 5% to normal paraffin which is a methane hydrocarbon having 10 to 15 carbon atoms.
It can be added at a concentration of up to about 30%. Also,
If the substrate is a relatively less toxic fatty acid ester,
50% or more can be added to normal paraffin.

Among the substrates, the higher fatty acid esters, which are particularly less toxic, are used as a conversion substrate and an organic solvent which is brought into contact with the bacterial cell phase on the immobilization carrier in 100% concentration without being mixed with the organic solvent. be able to.

Similar to higher fatty acid esters, aliphatic hydrocarbons are also particularly less toxic when used as substrates, and are themselves used as organic solvents for contacting the bacterial phase on the immobilization carrier and as conversion substrates. be able to.

In the interfacial bioreactor system of the present invention, the reaction solvent containing the above-mentioned reaction substrate is sent from the lower part of the reaction tower to the upper part and circulated. The circulation rate at that time is one important factor for stably and efficiently operating the interfacial bioreactor of the present invention in a state where the particulate carrier is not compacted or collapsed, and the reaction solvent amount and filling rate are referred to. Can be determined individually, but usually 10 to 100 based on the reaction solvent amount of 1 liter.
The reaction solvent may be injected and discharged at a rate of 00 ml / min, preferably in the range of 100 to 1000 ml / min.

The circulation of the reaction solvent into the reaction tower can be carried out by using an aeration tower connected to the reaction tower. The structure of the aeration tower that can be used in the present invention is not particularly limited, and it is possible to efficiently supply air to the reaction solvent containing the reaction substrate and replenish the consumed reaction substrate,
Any material can be used as long as it can be circulated in the reaction tower at the above circulation speed after mixing.

The particulate carrier-filled interfacial bioreactor system of the present invention and its operating method will be further described with reference to the drawings.

The interfacial bioreactor basically comprises a reaction tower (1) filled with a particulate carrier (3) and a reaction solvent (6) containing a reaction substrate for aeration and mixing. It has a two-column structure of a tower (5), and both towers are connected by a circulation line (8). The upper part inside the reaction tower (1) has a mesh (2) having a pore size that does not allow the particulate carrier to pass, and in some cases, the same mesh (2) can be provided in the lower part of the reaction tower. However, when the circulation velocity is low and the particle agglomerates do not float, the upper and lower meshes (2)
Can be omitted. The reaction solvent (6) is circulated to the reaction tower (1) through a circulation pump (4), a circulation line (8) and an injection port (not shown) at the bottom of the reaction tower. A sparger may be installed in the lower part to feed the liquid, or a stirring blade may be installed in the lower space to further enhance the mixing efficiency.

The aeration tower (5) can have a sparger (9) at the bottom of the tower, from which the air pump (1
0) aerates the contents through the filter (11). This aeration is for the purpose of supplying oxygen to the reaction solvent, and air is usually used, but pure oxygen or oxygen gas diluted with an inert gas may be used as the case may be. Installation of a sparger (9) is not essential, but in order to increase the efficiency of aeration and stirring of the content liquid,
It is preferred to vent through a sparger (9) which produces fine bubbles. It is also possible to install a stirring blade (not shown) in the aeration tower (5) to further increase the stirring efficiency. On the other hand, although the organic solvent which is the reaction solvent (6) is discharged to the outside of the tower with the ventilation, it is preferable to incorporate the oil mist trap (16) in the exhaust line in order to avoid it. Further, the exhaust duct from the aeration tower (5) is provided with a bent portion (7) at one or more downward positions, and an aerosol of a reaction solvent is attached at the bent portion (7).
By collecting the reaction solvent (6), an exhaust port (1
It is preferable to have a structure that does not go out from 5). Since the scattering of the reaction solvent (6) is not evaporation but scattering of the aerosol by aeration foaming, it is not necessary to attach a condenser to the exhaust line. The collected organic solvent can be returned to the aeration tower (5) again via, for example, a circulation pump (16), or can be returned to the reaction tower (1), which is an occupational health and safety hazard. In addition, the reaction solvent can be prevented from decreasing.

In order to keep the temperature of the reaction liquid in the reaction tower (1), the aeration tower (5) and the circulation line (8) constant, for example,
The reaction tower (1) and / or the aeration tower (5) is kept warm by flowing hot water (13) around the reaction tower (1) and / or the aeration tower (5) via a temperature controller (12). Is preferably temperature controlled. Further, it is preferable to install a liquid surface controller (14) in the reaction tower (1) and / or the aeration tower (5) to keep the liquid surface in the reaction tower and / or the liquid surface in the aeration tower constant. The discharge of the reaction solvent (6) from the reaction tower (1) to the aeration tower (5) is
If necessary, a forced discharge to the circulation line (8) by a circulation pump (not shown) via a liquid level controller (not shown) or a natural discharge without a circulation pump is performed.

The equipment required for post-treatment of the downstream (18) containing the desired product from the reaction tower (1), such as distillation concentrating tower, extraction tank, precipitation tank or crystal production apparatus (these are not shown), is usually The specifications of the target product,
It may be appropriately used depending on the reaction substrate, the reaction solvent species and the like.

In the present interface bioreactor, since the content liquid (reaction solvent) is an organic solvent, it is not always necessary to use a stainless steel plate for the liquid contact part. However, when inoculating and growing the microorganisms on the particulate carrier in the interface bioreactor, the reaction tower, the aeration tower, and the liquid contact part of the circulation line come into contact with water. It is preferable to use a material.

[0032]

EXAMPLES Hereinafter, the case where the microbial conversion reaction is carried out using the interfacial bioreactor of the present invention will be described in more detail with reference to the examples.

Example 1 500 ml of decane as a reaction solvent was injected into a reaction tower having an internal volume of 1.3 liters. To this reaction tower, Isachenkia-Sukuchurata bar Sukuchurata (Is on the surface of the particles
satchenkia scutulata var . scutulata ) IFO 1
Polyethylene glycol / 0070 grown for 1 day
Photocurable resin particles (ENTG-3800, manufactured by Kansai Paint Co., Ltd., diameter: 10 to 20 mm), which is a copolymer of polypropylene glycol, were filled (filling ratio 70%). Immobilization of the yeast on the carrier was carried out by shaking the yeast for 1 day in a liquid medium (pH 6.0) consisting of 5 g of peptone, 3 g of malt extract, 3 g of yeast extract, 10 g of glucose and 1 liter of water. The cells were allowed to stand for 10 minutes to inoculate the cells, and then statically cultured on a lifting net for 1 day.

200 ml of the reaction solvent was injected into the aeration tower (internal volume: 300 ml), and the mixture was circulated for 1 hour using a circulation pump.
The reaction solvent was circulated between the reaction tower and the aeration tower at a flow rate of 00 ml / min. Then, the conversion substrate 1-decanol 4
0 g was injected into the aeration tower, and the air aeration rate was 100 ml /
Minutes, the reaction solvent was circulated at a liquid circulation rate of 100 ml / min, and sampling was performed daily for 1 to 5 days from the start of the reaction.
The product decanoic acid concentration was quantified using gas chromatography. As a result, accumulation of decanoic acid was recognized from the first day after the reaction was started, and 26 g / l of decanoic acid was accumulated in the reaction for 5 days. After the reaction was completed, the reaction solution was recovered from the reaction tower, the aeration tower and the circulation line, and about 80% of the reaction solvent decane was distilled off by vacuum distillation. Then 100 water
After adding ml, the solution was made alkaline with a 10% sodium hydroxide solution to collect decanoic acid on the aqueous layer side, and the aqueous solution was further acidified with 10% hydrochloric acid, and decanoic acid was extracted with ethyl acetate. The extract was dehydrated with anhydrous magnesium sulfate and then ethyl acetate was removed to give decanoic acid of 23 g /
1 was obtained (gas chromatography purity 97%). On the other hand, after the reaction is completed, the total amount of the particulate carrier is recovered from the reaction tower,
High-pressure steam sterilization was performed using an autoclave. After sterilization, the microbial layer on the particle surface was removed by high-speed stirring and washing with ion-exchanged water mixed with an alkaline detergent, and further, high-speed stirring and washing with ion-exchanged water three times. After that, the water inside the carrier was replaced with the liquid medium by immersing it in the above-mentioned liquid medium overnight, and after inoculating and proliferating in the same manner as in the above-mentioned first time, in the same manner as in the above-mentioned first time test. A decanoic acid synthesis test was conducted. The resulting decanoic acid was 17 g /
It was l.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of an interfacial bioreactor system of the present invention.

[Explanation of symbols]

 1 Reaction Tower 2 Mesh 3 Particulate Carrier 4 Circulation Pump 5 Aeration Tower 6 Reaction Solvent 7 Exhaust Duct Bend 8 Exhaust Port 9 Sparger 10 Air Pump 11 Filter 12 Temperature Controller 13 Hot Water 14 Liquid Level Controller 15 Exhaust Port 16 Oil Mist Trap 17 Circulation pump 18 downstream

Claims (4)

[Claims] 1. A microparticle having a particle size of 5 to 100 mm, which has a microorganism attached to and immobilized on its surface in a state where it can grow or survive and which contains a liquid medium consisting of water and a nutrient source necessary for the growth or survival of the microorganism. A reaction tower filled with the particulate hydrophilic immobilization carrier at a packing rate of 10 to 70%, and a reaction solvent connected to the reaction tower and containing a substantially water-insoluble or sparingly soluble reaction substrate are contained in the reaction tower. An interfacial bioreactor system comprising an aeration tower that can be circulated from the lower part to the upper part at a circulation rate of 10 to 10000 ml / min and can supply and mix oxygen to the reaction solvent. 2. The interfacial bioreactor system according to claim 1, wherein the particulate hydrophilic immobilization carrier is a synthetic polymer granule. 3. The interfacial bioreactor according to claim 1, wherein the reaction solvent is a hydrophobic organic solvent which is substantially harmless to microorganisms. 4. The interfacial bioreactor system according to claim 1, which is repeatedly reused by washing the particulate hydrophilic immobilization carrier after use and replacing the inside with a liquid medium.