JPH0951794A - Porous carrier for bioreactor - Google Patents

Abstract

(57) Abstract: The present invention relates to a porous carrier for a bioreactor. (1) A porous carrier for a bioreactor, comprising a water-swellable polyurethane gel having continuous vents. (2) The porous carrier for a bioreactor according to (1), wherein the polyurethane gel has a volume swelling ratio of 150% to 1000% obtained by reacting a polyol compound, an isocyanate compound and water.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bioreactor (immobilized biocatalyst) by binding and immobilizing animal and plant cells and microorganisms.
As a porous carrier.

[0002]

2. Description of the Related Art Various carriers are used in bioreactors. If these are used, a water-containing gel carrier (non-porous) such as a polyacrylamide gel carrier, a polyethylene glycol gel carrier, a polyvinyl alcohol gel carrier, and an alginate gel carrier is used by immobilizing animal and plant cells and microorganisms in the gel. Type, polyurethane carrier, cellulose carrier, polypropylene carrier, polyvinyl formal carrier, ceramics carrier, and other porous carriers (not hydrous gels), surface-bound type carriers that adsorb and immobilize animal and plant cells or microorganisms on the porous surface, etc. Is mentioned.

Since the hydrous gel carrier uses a carrier that encloses animal and plant cells and microorganisms in advance, it has the advantage that the treatment capacity rises quickly during the initial operation of the bioreactor, but since it contains a high degree of water, it has a physical strength. Inferior to. There is a great risk of abrasion and disintegration during use in the reaction tank.

Further, since the animal and plant cells and microorganisms are encapsulated in the hydrous gel, the aged cells and microorganisms cannot be easily removed and the transport of the substrate and the product becomes slow.

On the other hand, since porous carriers such as polyurethane and polypropylene carriers are hydrophobic, animal and plant cells and microorganisms are difficult to adsorb.

Cellulose carriers are eroded by microorganisms and have a low service life. The polyvinyl formal carrier has drawbacks such as that an inexpensive industrial production method has not been established.

[0007]

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide a porous carrier for a bioreactor which is highly water-containing, has excellent physical strength and is not eroded by microorganisms.

[0008]

Means for Solving the Problems The porous carrier for bioreactor of the present invention for solving the above problems is a porous carrier having continuous pores made of water-swellable polyurethane gel.

Further, the polyurethane gel has a volume swelling ratio of 150% to 1000% obtained by reacting a polyol compound, an isocyanate compound and water.

When this polyurethane gel is used as a carrier for a bioreactor, the stirring efficiency in the tank and the density of animal and plant cells and the density of microorganisms can be increased to realize a high processing capacity.

[0011]

In the present invention, the porous carrier having water-swelling polyurethane gel and having continuous pores (hereinafter referred to as polyurethane gel porous body) is made of polyurethane, which is extremely hydrophilic, and has a large amount of water content. It has the property of being stored in the material and has excellent affinity for plant and animal cells and microorganisms.

Further, since it is a water-containing gel, it has a porous structure with continuous pores, so that animal and plant cells and microorganisms easily enter the pores of the carrier, and animal and plant cells and microorganisms existing in the culture solution or treated water are outside the carrier. It is efficiently attached, bonded and fixed to the surface or the surface of pores.

Also, due to the large surface area of the carrier due to the existence of pores communicating from the surface to the inside of the carrier, a large amount of animal and plant cells and microorganisms in the culture solution or the water to be treated are attached to and bonded to the carrier, and the pores are Animal and plant cells and microorganisms fixed to the part do not easily peel off when flowing.

Further, unlike conventional hydrous gels, it has high shear resistance, and therefore, in the state where a large amount of animal and plant cells or microorganisms treated as a biocatalyst are immobilized on the outer surface and the inner surface of the pores of the carrier in a high density. It enables efficient stirring.

The polyurethane gel porous body is obtained by reacting a polyol compound, an isocyanate compound and water. Polyurethane gel porous material has an average pore size of 10μ
m to 5 mm, preferably 100 μm to 3 mm, having a porous structure with continuous pores.

FIG. 1 shows an electron micrograph of the porous structure of the surface of the polyurethane gel porous body usable in the present invention.

The polyurethane gel porous material usable in the present invention preferably has a volume swelling degree in water defined by the formula (1) in the range of 150% to 1,000%.

[0018]

[Equation 1]

The point at which there is no weight loss after drying at 100 ° C. is absolutely dried. It is immersed in pure water at 25 ° C., and the point at which the volume change disappears is defined as the volume at the time of complete swelling.

The volume is obtained by measuring the length of each side of the rectangular parallelepiped or cubic carrier. With a volume swelling degree of less than 150%, it is not different from a general polyurethane porous body and it cannot be said to be a hydrogel. A volume swelling degree of more than 1000% is not practical because the strength is too low.

The polyurethane gel porous material usable in the present invention preferably has an air permeability of 10 to 300 cm ³ / cm ² · S, more preferably 50 to 200 cm ³ / cm ² · S.

The air permeability is measured according to the air permeability test of JIS L1096 general woven fabric test method using a polyurethane gel porous body having a thickness of 10 mm.

When the air permeability is 10 cm ³ / cm ² · S or less,
Since the flowability of the porous body in the polyurethane gel reaction tank in the processing tank becomes poor, there are problems such as a decrease in processing capacity and an increase in energy cost required for flowing the carrier, which is not preferable. When it is 300 cm ³ / cm ² · S or more, the surface area of the porous body becomes small and animal and plant cells and microorganisms cannot be immobilized at high density.

Furthermore, when the polyurethane gel porous body is added to the treatment tank, air with a low air permeability remains in the pores, so that it does not easily settle in a short period of time and remains in a floating state near the culture solution or the treated water surface. , Does not serve as a carrier.

The above-mentioned polyurethane gel porous body is generally synthesized by the following method. For example, water-soluble ethylene oxide having at least two terminal hydroxyl groups in one molecule /
Propylene oxide copolymerized polyether-based polyol is reacted with an isocyanate compound to produce at least 2 per molecule.
A mixture containing a water-soluble ethylene oxide / propylene oxide copolymerized polyether-based polyurethane prepolymer having one terminal isocyanate group and free polyisocyanate is synthesized.

Next, by reacting this mixture with water, a polyurethane gel porous body can be produced.

To describe the method for producing a polyurethane gel porous body in more detail, first, (ethylene / propylene) glycol, which is a copolymer of ethylene oxide and propylene oxide, and ethylene oxide / propylene oxide of at least a trivalent polyhydric alcohol. A polyether-based polyol selected from copolymerized polyether-based polyols and the like, which has a hydroxyl equivalent of at least 500 and is water-soluble, and an amount of about 1 to 4 mol per one hydroxyl group of the polyether polyol (2 ~ 8 equivalents) of a diisocyanate compound are reacted to produce a reaction mixture.

The diisocyanate in the reaction mixture is at least one selected from the group consisting of tolylene diisocyanate, xylylene diisocyanate, naphthylene diisocyanate, diphenylmethane diisocyanate, biphenylene diisocyanate, diphenyl ether diisocyanate, tolidine diisocyanate, hexamethylene diisocyanate and isophorone diisocyanate. A seed of diisocyanate is fisted.

A polyurethane gel porous body can be produced by reacting water with a reaction mixture consisting of the above-mentioned water-soluble polyurethane prepolymer and free polyisocyanate.

When the amount of water is an excess of less than 300 equivalents of hydroxyl groups with respect to 1 equivalent of isocyanate groups in the reaction mixture, a polyurethane gel porous body suitable for the present invention can be obtained.

A typical method for producing a polyurethane gel porous body has been illustrated. The volume swelling ratio in water is 150% to 10%.
A polyurethane gel porous body manufactured by a manufacturing method other than this method can be used in the present invention as long as it satisfies the condition of 00%.

The polyurethane gel porous body of the present invention has a porous structure even though it is a hydrous gel, and its strength is not different from that of a normal polyurethane porous body. The wear strength of the carrier is significantly higher than that of the gel type carrier mentioned above.

It should be noted that the polyurethane gel porous material of the present invention is capable of entrapping immobilization of animal and plant cells and microorganisms.

When the reaction mixture consisting of the water-soluble polyurethane prepolymer and the free polyisocyanate is reacted with water to produce a polyurethane gel porous body,
As water to be used, water containing plant and animal cells or microorganisms (for example, a suspension culture solution or an activated sludge suspension aqueous solution having an arbitrary cell density and microorganism concentration) can be used.

Since the reaction temperature can be controlled to about 35 ° C., it has little effect on living organisms, and animal and plant cells and microorganisms are dispersed and entrapped and immobilized in the gel matrix of the polyurethane gel porous body.

If necessary, this method can be used to improve the initial startup of the bioreactor.

Details will be shown below with reference to examples of the present invention, but the present invention is not limited to the examples.

[0038]

Example 1 Production of Porous Polyurethane Gel 100 g of a reaction mixture (Sanyo Kasei Co., Ltd., Sanprene WE-104, NCO 3.8%) consisting of a water-soluble polyurethane prepolymer and free polyisocyanate and 100 g of water (123 equivalents of OH to NCO) were mixed and vigorously stirred to foam.

4 mm of the obtained polyurethane gel porous body
It was cut into square dice to obtain a carrier for immobilization. This polyurethane gel porous body has a volume swelling degree in water of 700.
%, The air permeability was 52 cm ³ / cm ² · S.

[0040]

Example 2 67 g of water (82% OH for NCO).
A polyurethane gel porous body was obtained by the method of Example 1 using 3 equivalents). Volume swelling is 990%, air permeability is 87 cm
^{It was 3} / cm ² · S.

[0041]

Example 3 Production of Polyurethane Gel Porous Body Entrapping Immobilized Animal and Plant Cells and Microorganisms 100 g of a reaction mixture consisting of a water-soluble polyurethane prepolymer and free polyisocyanate (Sanprene WE-104 manufactured by Sanyo Kasei Co., Ltd.) Filamentous fungus (Asper
100 g of spore suspension (gillus niger JCM5548) (123 equivalents of OH with respect to NCO) was mixed and vigorously stirred to foam.

4 mm of the obtained polyurethane gel porous body
It was cut into square dice to obtain a filamentous fungus-immobilized carrier. The immobilization carrier had a volume swelling degree in water of 700% and an air permeability of 52 cm ³ / cm ² · S.

[0043]

Example 4 The spore suspension of Example 3 was treated with spinach (S
pinia oleracea L. cv. Okam
e) The same procedure as in Example 3 was carried out except that a 20% (w / w) suspension of cultured cells (OH was 98.4 equivalents relative to NCO) was used to obtain a plant-cultured cell-immobilized carrier. . This immobilization carrier has a volume swelling degree in water of 700% and an air permeability of 1
It was 26 cm ³ / cm ² · S.

[0044]

Example 5 Production of Polyurethane Gel Porous Body Incorporating and Immobilizing Microorganisms that Decompose and Remove Organic Pollutants A reaction mixture consisting of a water-soluble polyurethane prepolymer and free polyisocyanate (Sanprene, Sanyo Kasei Co., Ltd.) 100 g of WE-104) and 100 g of an activated sludge suspension having an activated sludge concentration of 9500 mg / L (122 equivalents of OH to NCO) were mixed and vigorously stirred to foam.

4 mm of the obtained polyurethane gel porous body
It was cut into square dice and used in a water treatment device. The volume swelling degree of this polyurethane gel porous body in water is 70.
The air permeability was 0% and the air permeability was 50 cm ³ / cm ² · S.

[0046]

Comparative Example 1 Triol polyalkylene glycol,
Unilube 50TG32U (manufactured by NOF Corporation) 100
g and a catalyst, DABCO33LV (Toyo Soda Co., Ltd.)
3.0g, Toraysilicon SH-19 as foam stabilizer
0 (manufactured by Toray Industries, Inc.) 2.5 g, water 7.0 g as a foaming agent
(OH is 0.93 equivalent to NCO) is mixed well, and Coronate T-80 is used as an isocyanate compound.
It was reacted with 84.1 g (manufactured by Nippon Polyurethane Co., Ltd.). The volume swelling degree of the obtained polyurethane porous body is 130.
%, The air permeability was 108 cm ³ / cm ² · S. Even when placed in water, it did not absorb water and did not settle.

The following evaluations were carried out on the porous bodies obtained in the above examples.

(1) Comparison of abrasion strength of carrier 4 mm was put on a container in which a water resistant sandpaper (No. 100) was stuck on the inner surface of a glass bottle (diameter 40 mm, length 200 mm).
30 ml of corner carrier (measured using a 100 ml graduated cylinder) and 120 ml of water were added and the stopper was added.

This container was stroked at 70 mm and the number of revolutions was 1
It was shaken back and forth at 50 rpm for 20 hours. Thereafter, the carrier inside was taken out and passed through a sieve having a spread of 1 mm.

The volume of carrier remaining in the sieve was measured using a 100 ml graduated cylinder.

[0051]

[Equation 2]

(2) Result

[0053]

[Table 1]

(3) Immobilization test of filamentous fungus 200 ml of culture solution (a) in a 500 ml Erlenmeyer flask
Then, 50 ml of each carrier of Example 1 and Comparative Example 1 having a size of 4 mm was put and autoclaved at 121 ° C. for 20 minutes to obtain a filamentous fungus (Asp
S. ergillus niger JCM5548) was inoculated with one platinum loop.

Using a rotary shaker, this Erlenmeyer flask was
The cells were cultured at 25 ° C. for 4 days with shaking at 140 rpm to obtain immobilized cells.

150 ml of the culture solution (b) was placed in another 500 ml Erlenmeyer flask and sterilized under pressure at 121 ° C. for 20 minutes, and the above-mentioned immobilized cells were aseptically filtered and added. The mixture was shaken at 30 ° C. and 180 rpm as it was, and the same operation was repeated every 6 days to perform subculture and citrate fermentation.

The citric acid concentration of the culture broth after culturing for 6 days was
It was measured using HPLC.

(4) Result

[0059]

[Table 2]

(5) Immobilization test of plant culture cells In a 200 ml Erlenmeyer flask, 40 ml of the culture solution (c),
10 ml of each carrier of Example 1 and Comparative Example 1 having a size of 4 mm was placed, and autoclaved at 121 ° C. for 20 minutes.

In this Erlenmeyer flask, spinach (Spi
nacia oleracea L. cv. Okam
1 g of the suspension-cultured cells of e) was added, and shake culture was performed at 25 ° C. and 100 rpm using a rotary shaker under the light of 8000 lux.

TTC (2,3,5-tri) was used as the viability of spinach suspension culture cells after 12 days of culture.
phenyltetrazolium chloroid
e) The reducing power was measured.

The TTC reducing power was measured without adding a carrier.
Using the spinach suspension culture cells that had been cultured for a day as a control, the following method was performed.

The contents of the Erlenmeyer flask after 12 days of culturing were
Aseptically transfer to a test tube with a 00 ml screw cap and centrifuge at 2000 rpm for 10 minutes to remove the supernatant.

Pre-sterilized 0.05M KH ₂ P
O ₄ -K ₂ HPO ₄ buffer (pH 7.4), 0.05M
Aseptically add 30 ml of TTC and incubate at 30 ° C. for 10 hours in the dark.

After centrifugation at 2000 rpm for 10 minutes, the supernatant is removed and 50 ml of 95% ethanol is added to the precipitate. After boiling for 10 minutes at 100 ° C., the mixture is cooled and the extract is made up to 100 ml with 95% ethanol.

The absorbance of this solution at 520 nm was measured, and the value of the cultured cells without addition of carrier was expressed as 100%.

(6) Result

[0069]

[Table 3]

Culture liquid composition Culture liquid (a)

[0071]

[Table 4]

Culture liquid (b)

[0073]

[Table 5]

Culture solution (c)

[0075]

[Table 6]

[0076]

[Sixth Embodiment] A water treatment apparatus using the porous carrier of the present invention will be described below with reference to the drawings.

In FIG. 2, 1 is a first settling tank, 2 is a biological reaction tank, and 3 is a final settling tank. The treated water 4 initially supplied from the settling tank is biologically treated in the biological reaction tank, and the treated water 5 is designed to remove the precipitate in the final settling tank and discharge the supernatant water. .

The biological reaction tank 2 is provided with an air diffuser 6 for aeration for supplying oxygen or air containing oxygen. Air containing oxygen is sent from 6 to a blower motor 7.

Further, the polyurethane gel porous body 8 of the present invention is put into the biological reaction tank 2. Further, if necessary, a polyurethane gel porous body 8b in which microorganisms that decompose and remove organic pollutants are entrapped and fixed is added.

In the reaction tank 2, while the treated water 4 is introduced and the treated water 5 in the tank is sent to the final settling tank 3, air containing oxygen is blown out from the air diffuser 6, and the mixed liquid in the tank is discharged. 9
Is supplied with oxygen.

At this time, an ascending bubble flow occurs, convection of the mixed liquid occurs, and the polyurethane gel porous body floats and circulates in the reaction tank.

Microorganisms that decompose and remove organic pollutants present in the mixed liquid 9 are attached to, bonded to and immobilized on the polyurethane gel porous body 8.

At this time, the polyurethane gel porous body 8 has an extremely high water content and a high affinity for microorganisms. Furthermore, since it has an infinite number of open-pore porous structures, it has a large surface area as a result.

For this reason, the mixed liquid 9 in the tank easily enters the pores communicating from the surface to the inside of the carrier, and the mixed liquid 9
Microorganisms that decompose and remove organic pollutants contained in are efficiently adhered to and bound to the outer surface of the carrier 8 or the surface of the pores, and the microorganisms immobilized on the surface of the pores when the porous body 8 flows. Also hard to peel off.

When the polyurethane gel porous body 8b entrapping and fixing microorganisms which decomposes and removes organic pollutants is used, the entrapping and fixing microorganisms and the microorganisms which are subsequently bound and fixed in the mixed solution 9 are used. Since both decompose and remove organic pollutants, it is possible to enhance the ability of the water treatment device especially at the time of startup.

In any case, the organic pollutant in the mixed solution 9 and the microorganism that decomposes and removes the organic pollutant should be in sufficient contact with each other in a state where the microorganism is maintained at a high concentration. Thus, organic pollutants are biologically treated very efficiently and at high speed.

The size and shape of the polyurethane gel porous material usable in the water treatment apparatus of the present invention are not particularly limited.

However, in order to maximize the outer surface area, a dice shape or a cylindrical shape is preferable. For example, 1
A carrier having a side shape of 2 to 8 mm, a dice shape, or a cylindrical shape having a diameter of 5 mm and a length of 5 mm is suitable.

Further, the polyurethane gel porous material can flow uniformly in the reaction tank when the specific gravity when the adhesion and bond immobilization of microorganisms in the tank reaches a steady state is 1.000 to 1.250. Therefore, it is preferable to control the specific gravity of the porous body within this range.

The results of actual batch type waste water treatment will be described.

The first settling tank 1 in FIG. 2 has an effective volume of 50 l,
The biological reaction tank 2 was made to have an effective volume of 30 l, and the final sedimentation tank 3 was made to have an effective volume of 50 l. The wastewater of the Nisshinbo Tokyo factory was first put in the sedimentation tank 1 to sediment and remove the sediment. The supernatant was transferred to Biological Reactor 2. The biological reaction tank was operated under the following three conditions.

Conditions Nisshinbo Tokyo factory wastewater treatment equipment activated sludge (sludge concentration 1500 mg / l) Conditions Nisshinbo Tokyo factory wastewater treatment equipment activated sludge (sludge concentration 1500 mg / l) and the polyurethane gel porous body prepared in Example 1 ( 3 l) Conditions Activated sludge (sludge concentration 1500 mg / l) in the wastewater treatment equipment of Nisshinbo Tokyo factory and the polyurethane porous body (3 l) produced in Comparative Example 1

The volume 3 l of the porous material is 30 l of the water to be treated.
Equivalent to 10% of The porous body of Example 1 is a wet porous body 1.
The dry weight per liter is 35 g. Therefore 1 dry weight
05 g becomes a porous body for 3 l.

The porous body of Comparative Example has a dry weight of 15 g per liter of wet porous body. Therefore, 45 g dry weight is 3
It becomes a 1-minute porous body.

As described above, the volume of the porous body was determined by measuring the specific gravity of the porous body, and the volume of 3 l was used exactly from the weight of the porous body. Each 3 liters of the porous body was retained in the Nisshinbo Tokyo factory wastewater treatment aeration tank in advance, and the microorganisms were acclimated for 30 days before being used for the experiment.

The biological reactor was aerated with 4 l / min of air. Aeration start time is set to 0 minutes, and thereafter 60, 120,
Was measured with the NO _X nitrogen (NO _X -N) the total Kjeldahl nitrogen (TKN) when 180,240 minutes.

For the measurement, an ion chromatography and a Kjeldahl method total nitrogen measuring instrument were used, respectively. NO _X -N and TKN were plotted against time, the slope was calculated by the least squares method, and the NO _X production rate and the nitrogen consumption rate were determined.

The conditions show the wastewater treatment capacity when no porous material is added. Conditions indicate the wastewater treatment capacity of activated sludge + porous body. Therefore, conditions, more determined NO _X when the generation rate and nitrogen consumption rate by subtracting the NO _X generation rate and the nitrogen consumption rate conditions of the porous body alone NO _X
The production rate and nitrogen consumption rate are determined. The measurement results are summarized in the table.

[0099]

[Table 7]

From the above results, it was clarified that the use of the polyurethane gel porous body of the present invention enables the ammoniacal nitrogen in the waste water to be decomposed extremely efficiently as compared with the case of using the activated sludge alone.

[0101]

INDUSTRIAL APPLICABILITY The polyurethane gel porous material of the present invention has a high abrasion strength, despite having a high water content, and since it is hydrophilic, animal and plant cells and microorganisms are adsorbed without lowering its physiological activity, Less susceptible to microbial erosion.

[Brief description of drawings]

FIG. 1 is an electron micrograph showing the porous structure of the surface of a polyurethane gel porous body that can be used in the present invention.

FIG. 2 is an explanatory view of a water treatment device using the porous carrier of the present invention.

[Explanation of symbols]

 1 First settling tank 2 Biological reaction tank 3 Final settling tank 4 Treated water 5 Treated water 6 Air diffuser 7 Blower motor 8 Polyurethane gel porous body 8b Polyurethane gel porous body 9 Mixed liquid

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // C12P 7/48 C12P 7/48 (C12P 7/48 C12R 1: 685) C08L 75:04

Claims (3)

[Claims] 1. A porous carrier for a bioreactor, which is made of a polyurethane gel which is water-swellable and has continuous ventilation holes. 2. The porous carrier for a bioreactor according to claim 1, wherein the polyurethane gel has a volume swelling ratio of 150% to 1000% obtained by reacting a polyol compound, an isocyanate compound and water. 3. The air permeability is 10 to 300 cm ³ / cm ² · S.
The porous carrier for a bioreactor according to claim 1 or 2.