JPH0528107B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to immobilized bacterial cells or enzymes and methods for producing the same. [Prior art] Equipment that immobilizes highly concentrated bacterial cells or enzymes on carriers and performs various reactions has been used in fields such as the pharmaceutical and food industries, and in recent years has been particularly used in wastewater treatment using living organisms, energy It is also beginning to be adopted in the field of production. As methods for immobilizing bacterial cells or enzymes, carrier-binding methods, cross-linking methods, entrapment methods, etc. have been known. Among these, the entrapping method is a method that reduces the leakage of bacterial cells and allows the carrier and the bacterial cells or enzymes themselves to be bonded together. Since there is no chemical bonding with other substances, it has the advantage that there is little decrease in the activity of bacterial cells or enzymes due to immobilization, and that it can be immobilized simultaneously with various substances.
Widely used. Carriers used for entrapping immobilization include alginic acid, carrageenan, agar,
Natural polymers such as albumin, synthetic polymers such as acrylamide, photocrosslinkable resins, polyvinyl alcohol, and polystyrene are known. [Problems to be solved by the invention] Among these, the immobilization method using alginic acid is widely used because it can be immobilized by simple operations and there is little decrease in activity, but other natural polymers are also used. Including, the strength is low and the durability is poor,
There are problems such as decomposition by microorganisms. On the other hand, a method is known in which microalginate gel is added to silica sol to gel the silica to obtain immobilized bacterial cells or enzymes with a silica gel-alginate double structure (Japanese Patent Laid-Open No. 60-221089). .
However, this method has the disadvantage that the manufacturing method is complicated and the silica gel part is not suitable for the growth of microorganisms, and the advantage of immobilized bacterial cells is that microorganisms grow inside the carrier and the activity of the gel increases. few. In addition, since microscopic alginate gel is separately prepared, recovered, and added to the silica sol, the operation is complicated in this respect as well. On the other hand, synthetic polymers have the advantages of high strength, good durability, and resistance to decomposition by microorganisms.
In particular, acrylamide is widely used. However, in immobilization with acrylamide, the activity of bacterial cells and enzymes often decreases due to the toxic effects of polymer monomers, crosslinking agents, polymerization agents, etc., or due to heat during polymerization reactions. A known method for minimizing the decrease in activity when immobilizing with acrylamide is to add a flocculant to the suspension of bacterial cells to be immobilized, form microflocs, and then entrapment immobilize with acrylamide. Publication No. 61-68196). However, flocculation using a flocculant differs depending on the type of microorganisms and enzymes, and it is difficult to form a uniform floc of various types of microbial cells and enzymes. Also, if the flocs are too large, the strength of the gel will be reduced. Therefore, it is desirable that the size of the flocs be several hundred microns, but when creating flocs of this size using a flocculant, bacteria or enzymes that do not become flocs and remain suspended. In many cases, these bacterial cells or enzymes are deactivated upon immobilization with acrylamide. The present invention has been made to solve the above-mentioned conventional problems, and provides an immobilized bacterial cell or enzyme that has high physical strength, good durability, and a small decrease in activity during immobilization, and a method for producing the same. The purpose is to [Means for Solving the Problems] That is, the present invention is an immobilized microbial cell or enzyme characterized by dispersing and retaining minute alginate-immobilized microbial cells or an enzyme in an acrylamide gel matrix, and The manufacturing method includes a step of mixing bacterial cells or an enzyme with an alginic acid aqueous solution to form a mixed sol, and a step of spraying this sol into a mixed solution of an acrylamide monomer and an alginate gelling agent to form a fine alginate gel.
The method is characterized in that it includes a step of gelling a mixed liquid in which the minute alginate gel is dispersed. The bacterial cells to be immobilized in the present invention may be any strain cultured in pure water, or may be mixed bacterial cells such as activated sludge. In addition to purified enzymes, crude enzymes include crushed bacterial cells, animal and plant cells,
It may also be a cell organ or the like. Moreover, the concentration can be adjusted arbitrarily. On the other hand, an alginic acid aqueous solution is prepared by dissolving sodium salt, potassium salt, or ammonium salt of alginic acid in water. When the concentration is high, the viscosity is high, making it inconvenient to handle and difficult to spray. If the concentration is low, when it comes into contact with the gelling agent and gels, it becomes a thin film and becomes entangled with each other, so that it may not form a microgel. Therefore, the concentration of alginic acid should be such that it forms micro-clumps when gelatinized, and the viscosity is not too high;
1% is desirable. In addition, acrylamide, which becomes the polymer matrix that further encloses this microalginate gel,
alginate gelling agent and optionally crosslinking agent,
It is mixed with a polymerization initiator and stored as a monomer mixture. Calcium chloride is often used as a gelling agent for alginic acid, and calcium acetate, aluminum sulfate, aluminum chloride, etc. are also used. The concentration of these gelling agents is usually about 1 to 10% (w/v) in the monomer mixture. The concentration of the acrylamide monomer is preferably 10 to 35%, and N,N'-methylenebisacrylamide, N,N'-propylenebisacrylamide, etc. can be used as the crosslinking agent, and the concentration in the monomer mixture is 0.5 to 35%. 6% is desirable. As the polymerization initiator, ammonium persulfate, potassium persulfate, etc. are used. Next, the aqueous alginic acid solution containing the bacterial cells or enzymes described above is sprayed into the monomer mixture. and,
It reacts with the gelling agent in the monomer mixture to form a fine alginic acid immobilized gel. As spray devices, pressurized spray nozzles, gas atomization nozzles, and the like are known. Since these nozzles usually spray the alginic acid aqueous solution at a pressure of about 1 to 120 kg/cm ³ , a relatively large volume of the gelling agent aqueous solution in the saucer is required. Therefore, in order to separate and recover the generated fine alginate gel,
It takes time and effort to recover the material by centrifugation. Therefore, in the production method of the present invention, a gelling agent is added to the monomer mixture, and an alginic acid aqueous solution containing bacterial cells or enzymes is sprayed onto the monomer mixture itself. This method is simpler than the method of collecting and adding. The particle size of the micro alginate gel thus created ranges from several tens of microns to several hundreds of microns, but considering the strength of the double-structured gel obtained by entrapping and fixing this micro alginate gel with acrylamide, the micro alginate gel is The particle size is preferably 500 microns or less. Next, the monomer mixture containing the fine alginic acid gel obtained as described above is polymerized to form a matrix that holds the fine alginic acid gel dispersed therein. In the polymerization method of acrylamide, it is necessary to further add a polymerization accelerator. As the polymerization accelerator, tetramethylethylenediamine, β-dimethylamide propionitrile, etc. can be used. The acrylamide gel containing fine alginate gel obtained in this manner is used in a shape such as spherical, membrane, or fiber, depending on the purpose. In this case, the fine alginate gel has a size of several tens of microns to several hundred microns, and does not separate from the acrylamide gel that forms the matrix. [Examples] Next, the present invention will be explained based on Examples.
The present invention is not limited to this. FIG. 1 is a block diagram showing an example of an apparatus for obtaining immobilized bacterial cells or oxygen of the present invention. The alginic acid aqueous solution of a predetermined concentration stored in the alginic acid aqueous solution tank 1 is mixed with the bacterial cells or enzymes stored in the concentrated bacterial cell tank 2 in the mixing tank 3.
On the other hand, acrylamide monomer is stored in a monomer tank 4, and together with the alginate gelling agent and other additives in an additive tank 5, a monomer mixing tank 6
mixed in. This monomer mixture is sent to the spray chamber 7, where the bacterial cells or enzyme-containing aqueous alginic acid solution is sprayed toward the monomer mixture. The monomer mixture containing the fine alginate gel thus obtained is sent to the immobilization device 8 and polymerized. As a method for polymerizing acrylamide, there is a method in which a monomer mixture is dropped into an organic solvent circulating in a tube. According to this method, a spherical gel with arbitrary grains can be obtained. In this case, a polymerization accelerator may be added to the organic solvent. Alternatively, there is a method in which a polymerization accelerator is added to the monomer mixture and then poured into a mold to form a gel. Next, an example of a method for immobilizing bacterial cells using this device will be described. Example 1 The denitrifying bacterium Pseudomonas denitrificans was treated with potassium nitrate.
A broth medium containing 0.2 g/ml was used, and static culture was performed at 30°C. The cultured cells were collected and dispersed in water to obtain a concentrated bacterial solution (wet weight of cells: 0.2 g/ml). Mix 4 ml of this concentrated bacterial solution with 8 ml of 0.5% sodium alginate aqueous solution. The concentration of sodium alginate was approximately 30%. Meanwhile, 12ml of 20% acrylamide monomer aqueous solution
, 6 ml of 2% N,N'-methylenebisacrylamide, and 2 ml of 1M calcium chloride aqueous solution,
Create a monomer mixture and add 160μ of ammonium persulfate aqueous solution (0.4g/ml) as a polymerization initiator.
Added. This monomer mixture was introduced into a spray chamber, where the alginic acid aqueous solution containing the bacterial cells was sprayed through a self-made gas atomization nozzle. As a result, fine alginate gel with a particle size of several tens of microns to several hundred microns was produced in the monomer mixture. Following this step, the monomer mixture containing the fine alginate gel is
3.14mm Tygon tube (Norton,
A 20μ droplet was added to the soybean oil circulating in the TYGON TUBING. Tetramethylethylenediamine is added to soybean oil as a polymerization accelerator in an amount of 1% based on the soybean oil. The tube length was 2 m, and the soybean oil circulation speed was 100 mm/min. The dropped monomer mixture containing fine alginic acid gel circulates through the tube while being sandwiched between soybean oil, and the polymerization reaction proceeds. The immobilized bacterial cells obtained as follows have a diameter of approximately 3
mm, it was a strong and elastic double-structured gel that contained fine alginate gels. Subsequently, after washing the obtained solidified bacterial cells, potassium nitrate was added.
It was inoculated into 400 ml of synthetic wastewater containing 0.2 g/ml, and stirred at 30°C to perform denitrification treatment. When the change in nitrogen concentration (mg/) with respect to treatment time (hr) was measured, the results were as shown in FIG. 2A. For comparison, 0.5
% sodium alginate was mixed with 8 ml of water to prepare a gel in which bacterial cells were directly entrapping immobilized on acrylamide, and the same denitrification treatment as above was performed.
The results are shown in Figure 2B. Example 2 A double-structured immobilized bacterial cell prepared in the same manner as in Example 1 was packed into a column with an inner diameter of 30 mm and a length of 150 mm.
The same synthetic wastewater as in Example 1 was flowed in at a rate of 1/day, and denitrification treatment was performed at 30°C. For the first few days, the denitrification rate was low, but for about a month thereafter, it showed a high denitrification rate. The results are shown in Table 1.

[Table] Furthermore, even after about one month, the strength of the immobilized bacterial cells remained high and the elasticity did not change. For comparison, when a similar column was filled with alginate spherical gel containing the same amount of bacterial cells and a similar treatment experiment was performed, the gel dissolved and disappeared within a few days. [Effects of the Invention] As detailed above, according to the present invention, it is possible to obtain immobilized microbial cells or enzymes that exhibit little decrease in activity during immobilization, have high strength, and are durable. In addition, the manufacturing method has the advantage that the process is simple because a fine gel is created by directly spraying an aqueous alginic acid solution containing bacterial cells or enzymes onto the monomer mixture.

[Brief explanation of the drawing]

Fig. 1 is a block diagram showing an example of an apparatus for carrying out the method according to the present invention, and Fig. 2 shows treatment time and nitrogen concentration when denitrification treatment is performed using the immobilized bacterial cells of the present invention. FIG. 1... Alginic acid aqueous solution tank, 2... Concentrated bacterial cell tank, 3... Mixing tank, 4... Monomer tank, 5... Additive tank, 6... Monomer mixing tank, 7... Spray room, 8... Immobilization device.

Claims (1)

[Scope of Claims] 1. An immobilized microbial cell or enzyme, characterized in that microscopic alginate-immobilized microbial cells or an enzyme are dispersed and held in an acrylamide gel matrix. 2. A step of mixing bacterial cells or an enzyme with an alginic acid aqueous solution to form a mixed sol, a step of spraying this mixed sol into a mixed solution of an acrylamide monomer and an alginic acid gelling agent to form a fine alginic acid gel, and a step of forming a fine alginic acid gel. 1. A method for producing immobilized bacterial cells or enzymes, comprising the step of gelling a mixed liquid in which a gel is dispersed.