JPH04234986A - Microbial adsorbent and its production - Google Patents

Abstract

PURPOSE:To obtain the subject adsorbent having high microbial adsorption efficiency without any clogging by impregnating a hydrophilic porous fiber substrate with a solution containing 4-vinylpyridine and a cross-linking divinyl monomer in a solvent, removing the solvent, copolymerizing the monomers and subsequently carrying out quaternizing treatment. CONSTITUTION:4-Vinylpyridine, a cross-linking divinyl monomer (e.g. divinylbenzene) and a radical polymerization initiator (e.g. azobisisobutyronitrile) are dissolved in a solvent (e.g. ethanol) and a hydrophilic porous fiber substrate (e.g. rayon fiber) is then impregnated with the resultant solution in the solvent, which is then evaporated at ambient temperature to apply the aforementioned monomers to the constituent fiber surfaces of the hydrophilic porous fiber substrate. The obtained fiber substrate is subsequently allowed to stand in a thermostatic chamber at 80 deg.C temperature for 5hr to carry out copolymerization. The formed copolymer is then subjected to quaternizing treatment with a halide (e.g. benzyl bromide) to afford the objective microbial adsorbent in which a water-insoluble or sparingly water-soluble vinylic copolymer having functional groups expressed by the formula (R is benzyl, etc.; X is halogen) and further a cross-linked structure sticks.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microbial adsorbent used as a carrier for holding microorganisms in, for example, a bioreactor or a biosensor, and a method for producing the same. More specifically, the present invention relates to a microbial adsorbent that has a vinyl copolymer with excellent microbial adsorption ability attached to its surface and has dramatically improved microbial adsorption ability, and a method for producing the same.

0002

[Prior Art] Conventionally, microbial adsorbents used as microbial holding carriers in bioreactors, biosensors, etc. have been made of vinyl pyridine and crosslinkable divinyl monomers, and have been quaternized with halides. A method consisting of a combination has been proposed.

[0003] This vinyl copolymer can capture microorganisms in a living state with high efficiency, and is insoluble in water, so it does not pollute the environment and is used as an excellent product. was.

However, this vinyl copolymer has
Since it is insoluble not only in water but also in ordinary organic solvents, it can only be handled as a solid and is difficult to process, so the vinyl copolymer itself has been used as a microbial adsorbent in the form of beads. As a result, the surface area per unit weight becomes small, and the copolymer inside the beads does not contribute to adsorption of microorganisms, resulting in poor adsorption efficiency.

[0005] As a microorganism adsorbent developed to eliminate such problems, there is one described in JP-A-1-207141.

This is a vinyl copolymer made of vinyl pyridine and a crosslinkable divinyl monomer, which is quaternized with a halide and coated on the surface of a granular base material. The surface area per unit weight of the copolymer is increased to improve the adsorption efficiency of microorganisms.

However, when this microbial adsorbent is used in a bioreactor or a biosensor, the microorganisms adsorbed to the microbial adsorbent propagate between each microbial adsorbent, resulting in clogging and blocking of the substrate flow path. The problem was that it was limited.

[0008]

[Problems to be Solved by the Invention] The present invention has been made in view of the above circumstances, and provides a microorganism that has enhanced adsorption efficiency for microorganisms and does not cause clogging due to the proliferation of microorganisms. The object of the present invention is to provide an adsorbent and a method for producing the same.

[0009]

[Means and operations for solving the problem] In order to achieve the above object, the invention according to claim 1 provides the following:

0010

[Case 2]

(However, in the formula, R is a benzyl group, C4-C
A water-insoluble or poorly water-soluble vinyl copolymer having a functional group represented by an alkyl group or a pentafluorophenylmethyl group (X is a halogen atom) and having a crosslinked structure using a crosslinkable divinyl monomer as a crosslinking agent. The gist of the invention is "a microbial adsorbent characterized in that the coalescence is attached to the surface of the constituent fibers of a hydrophilic porous fiber base material."

[0012] In addition, the invention according to claim 2 provides that ``a hydrophilic porous fiber base material is impregnated with a solvent solution containing 4-vinylpyridine and a crosslinkable divinyl monomer, and then the solvent is removed and the 4-vinyl pyridine is The gist of this paper is ``a method for producing a microbial adsorbent, which comprises quaternizing a vinyl copolymer obtained by copolymerizing pyridine and a crosslinkable divinyl monomer with a halide.''

[0013] As the hydrophilic porous fiber base material in the present invention, various forms such as nonwoven fabrics, woven fabrics, and knitted fabrics containing hydrophilic fibers such as rayon, cotton, and glass fibers can be selectively used depending on the purpose. You can. In particular, it is preferable to use a hydrophilic nonwoven fabric having a three-dimensional structure because it has a large surface area and low pressure loss. In addition, since hydrophilic nonwoven fabrics can have vinyl copolymers thinly and uniformly adhered to the surface of their constituent fibers, the surface area per unit weight of vinyl copolymers can be reduced compared to conventional beads or vinyl copolymers. It also has the advantage that it can be made much larger by combining it than by coating the surface of a powder base material. In addition, the hydrophilic porous fiber base material contains constituent fibers such as polyethylene and polypropylene for retaining the shape of the base material in addition to the above-mentioned hydrophilic fibers, and these shape-retaining fibers and the above-mentioned hydrophilic fibers The blending ratio of is appropriately determined in consideration of the use of the microbial adsorbent, the amount of vinyl copolymer attached, etc. In addition, the hydrophilic porous fiber base material is not limited to those containing hydrophilic fibers, but also base materials made of hydrophobic fibers such as polypropylene and polyethylene,
The fiber surface of this base material may be subjected to a hydrophilic treatment such as coating with a hydrophilic resin, sulfonation treatment, fluorination treatment, ultraviolet ray or radiation irradiation, corona discharge treatment, or plasma treatment. The vinyl copolymer is attached to the fiber surface of the hydrophilic porous fiber base material constructed in this way.

The vinyl copolymer in the present invention has 4-
Vinyl pyridine and a crosslinkable divinyl monomer are copolymerized and quaternized with a halide. Examples of crosslinkable divinyl monomers used in this vinyl copolymer include aromatic divinyl compounds such as divinylbenzene, divinyltoluene, and divinylnaphthalene, and aliphatic divinyl compounds such as ethylene glycol diacrylate and ethylene glycol dimethacrylate. It is possible to list the following. These crosslinkable divinyl monomers can be used alone or in combination of two or more.

The crosslinkable divinyl monomer and 4-vinylpyridine are applied to the hydrophilic porous fiber base material in the form of a solution. In other words, the crosslinkable divinyl monomer and 4-vinylpyridine can be combined with alcohols, esters,
By adding it to an organic solvent such as phenol or ether to form a solution, and soaking the hydrophilic porous fiber base material in this solution to impregnate it, or by coating it on the surface of the base material, a predetermined amount of it can be obtained. The crosslinkable divinyl monomer and 4-vinylpyridine are applied in the form of monomers to the surface of the constituent fibers of the base material. Next, part or all of the organic solvent is removed, and the crosslinkable divinyl monomer and 4-vinylpyridine are attached to the surface of the constituent fibers of the hydrophilic porous fiber base material. The proportion of the crosslinkable divinyl monomer attached to the fiber surface of the hydrophilic porous fiber base material is preferably 20 mol% or less based on the total amount of monomer attached. decreases. The amount of vinyl copolymer attached to the hydrophilic porous fiber base material is not particularly limited, but the amount of vinyl copolymer attached to the microorganism adsorbent is 0.01 to 20 w.
t% range has high microbial adsorption ability and is economical. Further, a commonly used radical initiator such as benzoyl peroxide or azobisisobutyronitrile is added to the solution containing each monomer. Furthermore, a reactive vinyl monomer such as styrene or methyl methacrylate may be added to the solution containing each monomer.

The crosslinkable divinyl monomer and 4-vinylpyridine thus attached in the form of monomers to the surface of the constituent fibers of the hydrophilic porous fiber base material are copolymerized under heating. The polymerization temperature is 40 to 140°C under normal pressure or increased pressure. The obtained copolymer is prepared by reacting pyridine with a halide such as alkyl halide, benzyl halide, or pentafluorophenylmethyl halide.
The vinyl copolymer having a crosslinked structure of the present invention is obtained. The structural formula of a vinyl copolymer when divinylbenzene is used as a crosslinking agent is shown below.

[0017]

[Chemical formula 3]

[0018] In this vinyl copolymer,

[C4]

It is thought that the functional group represented by the following functions to adsorb microorganisms while keeping them in an active state. Since this functional group is positively charged and the cell surface of microorganisms is generally negatively charged, it is presumed that electrostatic interaction is one important factor. The amount of the halide to be used is appropriately determined depending on the content of 4-vinylpyridine units in the copolymer.

The quaternized vinyl copolymer is substantially insoluble or poorly soluble in water and organic solvents, and exhibits properties similar to those of the vinyl copolymer shown in the conventional example. Become something. In addition, the vinyl copolymer of the present invention is attached as a monomer to the surface of the constituent fibers of the hydrophilic porous fiber base material and copolymerized on the base material. The surface area per unit weight of is increased, and its microbial adsorption efficiency is dramatically improved.

Furthermore, as described above, the hydrophilic porous fiber base material of the present invention contains hydrophilic fibers or has been subjected to a hydrophilic treatment. Furthermore, the vinyl copolymer of the present invention is applied to the surface of the constituent fibers of the hydrophilic porous fiber base material in the form of a solution in which 4-vinylpyridine and a crosslinkable divinyl monomer are dissolved in an organic solvent such as an alcohol. and copolymerized on the substrate. Therefore, the affinity between the hydrophilic porous fiber base material and the solution containing 4-vinylpyridine and the crosslinkable divinyl monomer is good, and the solution will spread to every corner of the base material. In addition, the 4-vinylpyridine and crosslinkable divinyl monomer applied to the hydrophilic porous fiber base material move to the surface of the base material as the organic solvent dries.In other words, the applied monomer moves in the direction of drying. This also prevents the so-called monomer migration phenomenon. Furthermore, the affinity between the hydrophilic porous fiber base material and the vinyl pyridine units constituting the vinyl copolymer is good, and the adhesion strength of the vinyl copolymer to the base material is also improved.

[0022] The microorganisms to be adsorbed by the microorganism adsorbent of the present invention include bacteria, fungi, algae, viruses, and the like.

[0023] Through experiments conducted by the present inventors, it has become clear that the vinyl copolymer of the present invention requires moisture in order to fully exhibit its characteristic microbial adsorption ability. . Therefore, when the microbial adsorbent of the present invention is used in an air layer, it is necessary to use alkaline earth metal salts such as calcium chloride, magnesium chloride, lithium chloride, potassium metasilicate, titanium sulfate, etc. together with the vinyl copolymer. Hydrophilic porous fibers contain deliquescent substances, water-soluble polymers such as polyvinyl alcohol, polyacrylates, vinyl pyrrolidone, and humectants such as silica gel, zeolite, collagen, diphosphorous pentoxide, magnesium oxide, and ethylene glycol. It is attached to the surface of the constituent fibers of the base material. As a result, the microbial adsorbent exhibits excellent microbial adsorption ability as in the case of a water-based adsorbent.

[0024] When the microbial adsorbent of the present invention is used only for the purpose of removing microorganisms, a bactericidal agent is attached to the surface of the constituent fibers of the hydrophilic porous fiber base together with the vinyl copolymer of the present invention. By doing so, it is possible to obtain a microbial adsorbent that has not only microbial adsorption ability but also sterilization performance. These disinfectants include, for example, antibiotic polymyxins, positive surfactants such as quaternary ammonium salts, amphoteric surfactants such as alkylaminoethylglycine, piganides such as chlorhexidine and polyhexamethylene piguanidine, and high-grade surfactants such as undecylenic acid. Fatty acids, metals and metal ions, phenols, etc. can be used.

In particular, when it is desired that the disinfectant be water-insoluble, use antibacterial zeolite in which zeolite is substituted with a metal ion having bactericidal properties, or piganide or nitride added to the polymer chain of polyvinyl, polyacrylate, polyester, etc. Polymer-type immobilized bactericides with immobilized tetraammonium salts, silicone-type immobilized bactericides such as 3-(trimethoxysilyl)-propyltrimethyloctadecyl ammonium chloride, and the like can be suitably used.

[0026]

EXAMPLES Example 1 An ethanol solution containing 4-vinylpyridine, divinylbenzene and azobisisobutyronitrile in a molar ratio of 99:1:1 is prepared. On the other hand, a fibrous web made of rayon fibers (fineness: 1.5 denier) is hydroentangled to produce a hydrophilic nonwoven fabric having a basis weight of 90 g/m2. After immersing this hydrophilic nonwoven fabric in the ethanol solution,
Ethanol is evaporated at room temperature to adhere 4-vinylpyridine and divinylbenzene to the surface of the constituent fibers of the hydrophilic nonwoven fabric. Note that the amount of adhesion is adjusted to be 2% by weight based on the weight of the hydrophilic nonwoven fabric. Next, the 4-vinylpyridine and divinylbenzene are copolymerized on the hydrophilic nonwoven fabric by leaving it in a thermostat at a temperature of 80° C. for 5 hours. After this, the hydrophilic nonwoven fabric with the copolymer attached was placed in a reaction container, and benzyl bromide and ethanol were added to the same container and reacted at 80°C for 6 hours to perform quaternization treatment. A microbial adsorbent was obtained in which a vinyl copolymer having a pyridinium group and a crosslinked structure was attached to the surface of the constituent fibers.

[0027] The obtained microorganism adsorbent was
Cut into spiral shapes and add E. coli to 2.2 x 1
E. coli is immersed in 200 ml of 0.85% by weight sterile physiological saline containing 0.07 cells/ml and stirred with a magnetic stirrer to adsorb E. coli onto the microbial adsorbent, and the remaining viable bacteria in the solution are removed at regular intervals. The number was measured using the agar plate pouring method, and the eradication rate was determined and shown in Table 1.

Examples 2 to 4 The molar ratio of 4-vinylpyridine and divinylbenzene contained in the ethanol solution used was 95:5 (Example 2
), 90:10 (Example 3), 85:15 (Example 4)
A microbial adsorbent was obtained in the same manner as in Example 1, except that the following was changed.

Using the obtained microbial adsorbent, Example 1
An experiment was conducted in the same manner as above, and the number of viable bacteria remaining in the liquid was measured at regular intervals, and the eradication rate was determined and shown in Tables 1 and 2. As is clear from Tables 1 and 2, the higher the proportion of 4-vinylpyridine, the higher the ability to adsorb microorganisms.

[0030]

[Table 1]

[0031]

[Table 2]

Comparative Example 1 An experiment was conducted in the same manner as in Example 1 using only the hydrophilic nonwoven fabric used in Example 1, and the number of viable bacteria remaining in the solution was measured at regular intervals to determine the sterilization rate. The results are shown in Table 3.

Comparative Example 2 The nonwoven fabric used was made of polypropylene fibers with an average fiber type of 2 μm produced by the melt-blowing method, and had a basis weight of 85 g.
A microbial adsorbent was obtained in the same manner as in Example 1 except that a hydrophobic nonwoven fabric having a thickness of 0.9 mm and a thickness of 0.9 mm was used.

[0034] Using the obtained microbial adsorbent, Example 1
An experiment was conducted in the same manner as above, and the number of viable bacteria remaining in the liquid was measured at regular intervals, and the sterilization rate was determined and shown in Table 4. Although this microorganism adsorbent used a nonwoven fabric with a smaller fiber diameter and a larger surface area than the nonwoven fabric used in Example 1, its ability to adsorb microorganisms was significantly inferior to that of Example 1. This is thought to be because the nonwoven fabric produced by the above melt blowing method is hydrophobic, and therefore the vinyl copolymer cannot be uniformly adhered to the fiber surface.

Comparative Example 3 Using 2 g of insoluble polymer beads made of cross-linked polyvinylpyridinium halide as a microorganism adsorbent, 2 g of Escherichia coli were collected.
．． 200 ml containing 0.0.
It is placed in 85% by weight sterile physiological saline and stirred with a magnetic stirrer to adsorb E. coli to the microbial adsorbent, and the number of viable bacteria remaining in the solution is measured at regular intervals using the agar plate pouring method. The eradication rate was calculated and shown in Table 4.

[0036]

[Table 3]

[0037]

[Table 4]

Example 5 The microbial adsorbent of Example 1 was cut into 35 cm x 6 cm and processed into a spiral shape, and a 200 m sample containing Staphylococcus aureus at a rate of 1.5 x 107 cells/ml was prepared.
E. coli was immersed in 0.85% by weight of sterile physiological saline and stirred with a magnetic stirrer to adsorb E. coli to the microbial adsorbent, and the number of viable bacteria remaining in the solution was measured at regular intervals using the agar plate pour method. The sterilization rate was determined and shown in Table 5.

Example 6 The microbial adsorbent of Example 1 was cut to 35 cm x 6 cm and processed into a spiral shape, and 200 ml containing Pseudomonas aeruginosa at a ratio of 1.1 x 107 cells/ml was prepared.
The sample was immersed in 0.85% by weight sterile physiological saline and stirred with a magnetic stirrer to adsorb E. coli to the microbial adsorbent, and the number of viable bacteria remaining in the solution was measured at regular intervals using the agar plate pouring method. The sterilization rate was determined and shown in Table 5. As is clear from Table 5, it can be seen that the microbial adsorbent of the present invention exhibits good adsorption power even when the types of bacteria are different.

[0040]

[Table 5]

Example 7 Made of ultra-fine glass fiber, area weight 75 g/m2, thickness 0
．． A microbial adsorbent was obtained in the same manner as in Example 1, except that a 35 mm hydrophilic glass fiber filter paper (GF-75, manufactured by Toyo Roshi Co., Ltd.) was used. After filling a column with the obtained microbial adsorbent, a liquid in which T-4 bacterial phages were suspended was passed through the column, and most of the T-4 bacterial phages could be collected. In addition, when a similar experiment was conducted using only the above glass fiber filter paper,
Almost no T-4 bacterial phage could be collected.

[Effect of the invention] By having the above configuration, claim 1
In the microbial adsorbent described above, the vinyl copolymer having excellent microbial adsorption ability is attached to the surface of the constituent fibers of the hydrophilic porous fiber base material. Compared to microbial adsorbents made of copolymers, the surface area per unit weight of vinyl copolymers is dramatically increased.
Extremely high microbial adsorption efficiency will be ensured. In addition, in the microbial adsorbent according to claim 1, since the vinyl copolymer is attached to the surface of the constituent fibers of the hydrophilic porous fiber base material, clogging occurs due to the proliferation of microorganisms. The flow path of the substrate is not limited.

Furthermore, since the microbial adsorbent according to claim 1 uses a hydrophilic porous fiber base material, it has good affinity with the vinyl pyridine units constituting the vinyl copolymer. , the adhesion strength of the vinyl copolymer is increased, and a decrease in the microbial adsorption ability due to shedding of the vinyl copolymer can be suppressed.

According to the method for producing a microbial adsorbent according to claim 2, the vinyl copolymer is hydrophilic in the form of a solution in which 4-vinylpyridine and a crosslinkable divinyl monomer are dissolved in an organic solvent such as alcohol. It is applied to the surface of the constituent fibers of the porous fiber base material and copolymerized on the base material. Therefore, compared to conventional microbial adsorbents made of bead-shaped vinyl copolymers, the surface area per unit weight of vinyl copolymers can be increased, dramatically improving microbial adsorption efficiency. I can do it.

Further, according to the method for producing a microbial adsorbent according to claim 2, since the hydrophilic porous fiber base material is impregnated with a solution dissolved in an organic solvent such as alcohol, vinyl-based The polymer can be spread throughout the substrate. Further, the 4-vinylpyridine and crosslinkable divinyl monomer applied to the hydrophilic porous fiber base material can prevent the migration of monomers to the surface portion of the base material as the organic solvent dries.

Claims (1)

[Scope of Claims] [Claim 1] [Formula 1] (wherein, R is a benzyl group, a C4-C16 alkyl group, or a pentafluorophenylmethyl group, and X is a halogen atom). A water-insoluble or poorly water-soluble vinyl copolymer having a crosslinked structure using a crosslinkable divinyl monomer as a crosslinking agent is attached to the surface of the constituent fibers of the hydrophilic porous fiber base material. Microbial adsorbent. Claim 2: A hydrophilic porous fiber base material is impregnated with a solvent solution containing 4-vinylpyridine and a crosslinkable divinyl monomer, and then the solvent is removed to combine the 4-vinylpyridine and the crosslinkable divinyl monomer. A method for producing a microbial adsorbent, which comprises quaternizing a vinyl copolymer obtained by polymerization with a halide.