JPS606193A - Preparation of membrane of immobilized microorganism - Google Patents

Abstract

PURPOSE:To obtain the titled membrane useful as a BOD sensor element of the oxygen sensor of a BOD analyzer, by impregnating an aqueous solution of a water-soluble monomer containig microorganisms in a porous polymer film, and polymerizing the monomer by plasma irradiation. CONSTITUTION:A porous polymer membrane (e.g. made of cellulose acetate) is immersed in an aqueous solution of a water-soluble monomer (e.g. acrylamide) mixed with microorganisms (e.g. bacteria, yeasts, etc.) to impregnate the microorganisms and the water-soluble monomer in the membrane. The membrane is taken out of the solution, and the undried impregnated membrane is irradiated with a plasma to polymerize the monomer and obtain the objective membrane containing the microorganisms immobilized to the polymer membrane. EFFECT:There is no restriction in the material of the membrane.

Description

[Detailed description of the invention] The present invention relates to a method for producing an immobilized microbial membrane.

Furthermore, the present invention relates to a method for producing an immobilized microbial film that can be effectively used as a BOD sensor element of an oxygen sensor detection section of a y)', L < )l]0D7iIjl constant device.

The following methods are conventionally known as methods for obtaining immobilized microbial membranes, but each has drawbacks and none of them can be said to be fully satisfactory.

(1) Physical adsorption to ceramic primary etc. This method has the disadvantage that the adsorbed Jt is easily detached from the microorganisms.

(2) 1tc 6 in aqueous solution such as polyvinyl alcohol
1 A method in which a film is formed by mixing living organisms and air drying. This membrane has the disadvantage of poor permeability to carbon sources, nitrogen sources, inorganic substances, and the like.

(3) After mixing collagen fibril liquid and microorganisms,
After casting and air drying, a method of obtaining a film by further dipping in a glutaraldehyde aqueous solution (C) and air drying.
Requires step-by-step operations.

(4) polyacrylamide, alginic acid, carrageenan,
A method of immobilizing r in a gel using agar, etc.

In this case, if it is made into a film, it becomes brittle, and for example, taking polyacrylamide as an example, during polymerization, a polymerization initiator,
Requires components other than monomers, such as polymerization accelerators and crosslinking agents.

In view of the drawbacks of the prior art, the present invention has been made to
Di! The present invention provides a method for producing an immobilized r,i biofilm that can be effectively used in the 130D sensor element of the acid-free sensor detection part of the IllI detection device, and 1? II. The production of the stabilized microbial membrane involves immersing the porous polymer 11 in a water-soluble monomer aqueous solution mixed with microorganisms, impregnating the polymer membrane with the microorganisms and the water-soluble monomer, and then This is carried out by irradiating the undried impregnated material 71''j taken out of the aqueous solution with plasma to polymerize the monomer, thereby fixing the microorganisms to the polymer membrane.

(As the microorganism, microorganisms in the logarithmic growth phase or (qt stationary phase) are used, specifically, for example,
- Monas fluorescens, Bacillus subtilis,
Examples include bacteria such as Pseudomonas aeruginosa, filamentous fungi such as Aspergillus niger and Rhizopus hormocensis, actinobacteria such as Streptomyces griseus, yeasts such as Zatucharomyces cerevituse, and Trichosporon cutanoim, and molds.

Examples of water-soluble monomers include acrylamide, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid, methacrylic acid-2-human[1xyJ, and methyl ester. These water-soluble monomers 41
1. Generally about 5 to 80 weight @% 1.! A small amount of N, IJ is prepared as needed.
A crosslinking agent such as '-methylenebisacrylamide is added thereto. And about 0.1 to 10 η2 per ml of such aqueous solution? Some microorganisms are mixed in there.

A t/c porous polymer membrane is immersed in this aqueous solution, and this fJj4 is impregnated with microorganisms and a water-soluble monomer.

Polymer membrane binding 10 is a porous membrane of cellulose acetate (including cellulose triacetate), polyurethane, polysulfone, polyacrylonitrile, polypropylene, y4': polycyclized vinyl, polyamide, polyimide, polycarbonate, etc., generally C A porous membrane having a porosity of about 5% or more and a maximum pore diameter of about 0.8 to 1.2 μm is used. The immersion is performed at a temperature of about 10 to 40°C for about 1 to 120 minutes.

In this way, the porous membrane impregnated with microorganisms and water-soluble monomers is pulled out of the aqueous solution, and before it dries, it is placed on a substrate such as a glass plate and the monomers are polymerized by plasma irradiation. .

Plasma polymerization is carried out using, for example, an apparatus as schematically shown in FIG. B, connected to vacuum pump 1, leak valve 2 and main valve 3, vacuum gauge 4
The porous polymer membrane 6 is housed in a plasma reactor 5 equipped with a high-frequency generator 1'Z (
13,56M H2)? and Matsucin Dani Knit 8
Using a high frequency source, the effective power is about 50~20
The oscillation coil 9
This is done by irradiating plasma from the source. In addition, as the reactor, either a cube type or a Pelger type can be used, and as the discharge electrode, an external or internal parallel electrode plate can also be used in addition to a coil-shaped one.

The reason why a undried porous polymer membrane is used is that water is involved in the combination of monomers. Furthermore, it is generally appropriate for plasma polymerization to be carried out at around 20° C., and therefore it is desirable to attach a cooling device to the plasma reactor and carry out the polymerization in a state where the film is cooled.

After irradiation, the film is heated at a temperature of about 4 to 30°C for about 1 to 3 hours.
Leave it for about a day for post-polymerization. Through these processes,
The monomer will polymerize on the porous side of the membrane, and at the same time the microorganisms will become immobilized on the membrane.

As described above, according to the present invention, when microorganisms are immobilized on a polymer membrane using a monomer, it is possible to do it only by short-term plasma irradiation without using a polymerization initiator or a polymerization promoter. can. Furthermore, when trying to chemically bond yeast to a polymer membrane using glutaraldehyde, for example, the membrane material must have a group that has binding properties to an aldehyde group, such as an amide 7 group.
Although restrictions would be imposed on the selection of the type of membrane material, there are no such restrictions according to the method of the present invention.

The obtained immobilized microorganisms fv and il2 can be used, for example, as a BOD sensor in an oxygen sensor detection section of a BOD measuring device. When this membrane is used as a BOD sensor in a BOD measurement device and oxygen consumption is measured using an oxygen electrode, the output voltage shows a linear decrease as the concentration of the sample solution increases, and the measured value of the output voltage Since the base voltage and equilibrium voltage exhibit stable values for about two weeks, the immobilized microbial membrane effectively functions as a BOD sensor.

Next, the present invention will be explained with reference to examples.

Example 1 Yeast Trichosporon cutaneum
(IAM 12206) in a liquid medium of pu6.0 in an incubator (Ml-15 (type 1) manufactured by Sanyo'Ichiki) for 30 minutes.
The cells were cultured aerobically for 48 hours at ℃. Medium composition d1, malt extract 0.3%, yeast extract 0.3%, polypeptone 0.
3%, glucose 1% and buffer 98.1%. The culture state was observed by measuring the absorbance at 630 nm using a visible and ultraviolet absorption photometer (UV-190, manufactured by Shimadzu Corporation). After about 25 hours of culture time, the absorbance becomes about 1.2, and thereafter remains at a constant value.

#f The yeast cells after cultivation are separated using a centrifuge (H-2 manufactured by Domestic Centrifuge).
00 type) at 8000 o. After drying this yeast at 105°C for 24 hours, the 2rnyk
It was mixed into 1me of acrylamide aqueous solution.

This acrylamide aqueous solution had a composition of 1% by weight of acrylamide monomer, 0.3% by weight of N,N/-methylenebisacrylamide crosslinking agent, and 79.7% by weight of reverse osmosis water.

Next, the porous polyurethane membrane was immersed in the yeast-containing aqueous solution at room temperature for 5 minutes. The porous polyurethane II φU1, polyethylene adipate, 4.4°-diphenylcyinone anate, and a polyconisple type polyurethane polymerized using a hydrogynone diethylol ether main armor extender was dissolved in dimethylformamide by weight. It is dissolved to a very high degree of consistency, formed into a film on a glass plate, gelled in an aqueous solution, made porous, and then dried.The wet thickness is 0.14 + njn, and the maximum pore diameter is determined by electron microscopy (Hitachi). - Observation with Akashi MSN-7 model) is l, Q l1m.

The immersed porous polyurethane membrane was immersed in glass 1 in an aqueous solution.
It was lifted up and placed on a glass plate before it was sufficiently dried, and then heated to 0.000 m using the plasma irradiation device shown in Fig. 1.
Plasma irradiation was performed for 60 seconds in an empty page state at 1 Torr. ○ - The plasma reactor is divided by the ring part 1o, and after the polymer membrane is taken out from the reactor, it is heated at 25°C for 24 hours.
After leaving for 11 hours, post-polymerization of acrylamide was carried out.

In this way, acrylamide is extremely porous; 6-sided polymerization and cross-linking occur, and simultaneous V-fermentation 1':f bacteria also occur.
- ηf is fixed, but it is certain that it is fixed - as follows 1 (
It was done. This polymer was immersed in a phosphate buffer solution with a pH of 7.Q for 24 hours.
) The wavelengths of rl and m were determined. As a result of the X-j, yeast decompression F phenomenon 1: lij could not be eaten.

Example 2 Using the yeast immobilized porous polyurethane produced in Example 1, a BOD measurement device rfi was manufactured.

BOD (biological oxygen demand) is an indicator of the concentration of organic substances that are pollutants in wastewater, and it takes at least 5 days to measure the value, and it is a personal Therefore, it has been desired to develop a mounting method that can quickly determine iq+u by reducing the number of steps.

FIG. 2 is a schematic diagram showing a BOD measuring device, which is equipped with a ftff1 body for dissolved oxygen analysis (Beckman Co., Ltd. 1!).
40260 type) li′fK: used, output m from the electrode
The pressure is a recorder (Yokogawa '11 v: 1066 type) 12
It was recorded using Polyurethane membrane 13 on which yeast yeast was immobilized 2) A pH 7.0 phosphate buffer solution 17 in a beaker 16 of IQO+vj! equipped with a W pusher 15 Remove the lower part of the IJ14 made of tetrafluoroethylene resin, which is the end face of the immersed side of the oxygen electrode 18 that is half immersed in the beaker, so that it is in close contact with the lower part of the IJ14. It was maintained at a constant temperature in a constant temperature bath 19 at 19° C. Under this condition, the output voltage U reached a stable base voltage of 5.75 my.

In this state, 50 pn, 100 pn, and 200 pn of Glucose (Kanto Kagaku 17! product) were applied over 1fiO.
+n included]) H7,0 phosphorus 1-i'l km'
FJJ f(1,,LOo 1.71! Zutsu Kano・,kawa(
i ＠ (／Put it in the beaker and add the above-mentioned electric yuzu (
7. Dip it in and measure its output voltage.

ri'i' iiJ: In the presence of bacteria, 01-order thick r reaction is completed, glucose +02-→Co2-1)120
Used for +ΔE reduction, 11-7; 1 provisional consumption) 11
．． Total 1'H (1 drop of oil with 2 element electrodes) 'L'? Output '1]: If i4 is lower than , then output '7i'j pressure (base') when glucose concentration Oppm is
Ifi Ijmo) 5.75 mV to 200 pp
Output when m? 4T, I'l': up to 12 n#,
It was found that as the concentration increased, the output power decreased significantly. Na j5, this γ1 j11 u f11 day and,
6) N law results for equilibrium voltage at p1 degree.

From this result, it was found that yeast 1J immobilized in the polyurethane membrane
It was found that the bacteria converted gelose and consumed dissolved oxygen in the process, and as a whole functioned as a B(')]) sensor. Base voltage and 50 ppm
As a result of measuring the stability of glucose equilibrium-flj and pressure, it was confirmed that both were stable for about 2 hours.

4 I'i in the drawing? FIG. 1 is a schematic diagram showing one embodiment of a plasma coupling device used in the present invention. Fig. 2 shows the fixed f1) microorganisms H of the oxygen sensor detection section Bob) and i! according to the present invention. 7 is a schematic diagram of the BOD measurement device used for the -r use.

(Min. No. 11) a) I......Vacuum pump 4...
・・・・・・Plasma reactor 6・・・・・・・・・・・・
・Porous polymer membrane 7・・・・・・・・・・・・）%MJ
Wave generating device 8・・・・・・・・・・・・Matching unit 9・・・・・・・・・・・・fl 4 dragon coils 11・
......Dissolved fluorine analysis 112...
......Output possible, pressure recorder 13...
......Microorganism immobilization membrane 18...
...Oxygen electrode proxy valve j) 71st Yoshi IJJ Toshio

Claims (1)

[Claims] 1. A porous polymer sample is immersed in an aqueous solution of a water-soluble monomer mixed with microorganisms, and after impregnating the polymer membrane with the microorganisms and the water-soluble monomer, it is removed from the aqueous solution. 1. A method for producing an immobilized microbial membrane, which comprises irradiating the pulled undried impregnated membrane with plasma to polymerize monomers, thereby immobilizing microorganisms on the polymer membrane. 2. A method for producing an immobilized microbial membrane according to claim 1, which is used for an EOD sensor element of an oxygen sensor detection section of a BOD measuring device.