KR100684603B1 - Novel hybrid immobilization method of microorganisms and bead produced therefrom - Google Patents

Abstract

The present invention relates to a method for producing a microorganism immobilized carrier and to a microorganism immobilized carrier prepared thereby, more specifically,

1) preparing a microorganism immobilization carrier precursor solution in which microorganisms are immobilized to polyvinyl alcohol by mixing and dissolving xanthan gum in an aqueous polyvinyl alcohol solution and adding a microorganism to be fixed and a carbon source;

2) modifying the surface of the carrier into a sphere by dropping the solution of the microorganism immobilized carrier precursor prepared in step 1) into an aqueous solution of boric acid containing a surfactant;

3) The spherical carrier obtained in step 2) is aged in the aqueous solution of boric acid, then added to an aqueous solution of phosphate, aged, filtered, and then aged and washed with water, thereby processing the carrier to facilitate the movement of the substrate and the product. ; And

4) preparing a microorganism immobilized carrier by culturing the carrier processed in the step 3) to facilitate the movement of the substrate and the product in a microbial growth medium, followed by filtration, separation and washing;

It relates to a method for producing a microorganism immobilized carrier comprising a and a microorganism immobilized carrier prepared by the method.

 Microbial Immobilized Carrier, Polyvinyl Alcohol, Xanthan Gum, Surfactant

Description

A method for preparing a microorganism immobilization carrier and a microorganism immobilization carrier produced by the same {NOVEL HYBRID IMMOBILIZATION METHOD OF MICROORGANISMS AND BEAD PRODUCED THEREFROM}

1 is a photograph showing the shape of the microorganism immobilization support of Example 1 (FIG. 1A) and Comparative Example 1 (FIG. 1B).

Figure 2 is a graph comparing the nitrate nitrogen removal ability of the microorganism immobilization support prepared in Example 1 and the microbial immobilization support prepared in Comparative Examples 1 and 2.

The present invention relates to a method for preparing a microorganism immobilization carrier and to a microorganism immobilization carrier produced by the same, and more particularly, to prevent loosening and entanglement of the microorganism immobilization carrier in an aqueous solution, and to immobilize a large amount of microorganisms. A method for producing a microorganism immobilization carrier having a spherical form capable of minimizing the outflow of microorganisms from and a microorganism immobilization carrier produced thereby.

Methods for preparing a microorganism immobilization carrier include entrapment and encapsulation. Among these, the trapping method is a method generally used.

The entrapment method is a method of trapping microorganisms in the inner structure of the matrix form, the matrix is a natural material such as agar, agarose, k-carrageenan, alginate and chitosan, or polyacrylamide, polyurethane, polyethylene glycol and poly It is made of synthetic material such as vinyl alcohol. However, there is a disadvantage that natural materials are not toxic to microorganisms, but weak, and synthetic materials are strong, but they are toxic to some microorganisms and lower their activity.

In the entrapment method, polyvinyl alcohol (PVA) has been commonly used as an immobilizing carrier, and a solution of boron and polyvinyl alcohol is formed by dropping an aqueous polyvinyl alcohol solution into an aqueous solution of boric acid to form a matrix. . The more spherical the immobilized carrier, the higher the immobilization efficiency.

Since the conventional method of preparing an immobilized carrier using polyvinyl alcohol was to prepare a carrier by simply reacting boric acid and polyvinyl alcohol, carriers made during the process were entangled with each other, or the shape was not spherical, and the tail was not formed. There was a problem that formed. In addition, since the produced carriers are carriers prepared using a simple entrapment method, there is a problem that microorganisms immobilized on the carrier easily leak out of the carrier. In addition, in the conventional method of immobilizing microorganisms using polyvinyl alcohol, since the amount of microorganisms is immobilized by the internal matrix is limited, the efficiency of the microorganism immobilization carrier could not be maximized.

Encapsulation is a method of immobilizing microorganisms in a carrier having a thin film. The alginate method is mainly used. The carrier is prepared in a manner opposite to the entrapment method. A method of forming a thin outer film by dropping an aqueous calcium chloride solution mixed with microorganisms into an alginate aqueous solution. However, the carrier prepared by the encapsulation method can immobilize a large amount of microorganisms because there is a lot of space therein, but there is a disadvantage that it is easily destroyed by external shocks and influences because the inside is an empty space.

The present invention is to solve the above problems, the carrier has a spherical shape without being entangled with each other, the efficiency is good, the substrate and the product in the carrier is easy to move, the amount of microorganisms immobilized in the carrier In addition, an object of the present invention is to provide a method for preparing a microorganism immobilized carrier which is not easily broken by external influences and a microorganism immobilized carrier prepared thereby.

The method for preparing a microorganism immobilization carrier of the present invention is characterized by comprising the following steps:

1) preparing a microorganism immobilization carrier precursor solution in which microorganisms are immobilized to polyvinyl alcohol by adding a microorganism and a carbon source to be dissolved by mixing and dissolving xanthan gum in an aqueous polyvinyl alcohol solution;

2) modifying the surface of the carrier into a sphere by dropping the solution of the microorganism immobilized carrier precursor prepared in step 1) into an aqueous solution of boric acid containing a surfactant;

3) The spherical carrier obtained in step 2) is aged in the aqueous solution of boric acid, then added to an aqueous solution of phosphate, aged, filtered, and then aged and washed with water, thereby processing the carrier to facilitate the movement of the substrate and the product. ; And

4) preparing a microorganism immobilization carrier by culturing the carrier processed to facilitate the movement of the substrate and the product in step 3) in a microbial growth medium, followed by filtration, separation and washing.

In the step 1), the polyvinyl alcohol (PVA) has a large surface free energy, and is useful as a carrier in the field of biological wastewater treatment. In the present invention, 5 to 13% by weight of polyvinyl alcohol having a polymerization degree of 1500 to 3000 Is preferably used in an aqueous solution completely dissolved in distilled water. In the immobilization method using polyvinyl alcohol, the weight of the polyvinyl alcohol is not important because the weight of the polymer is different depending on the degree of polymerization, and it is important that the amount of polyvinyl alcohol used is 5 to 13% by weight. If it is less than% by weight, the carrier is not formed, and it is not preferable. If it exceeds 13% by weight, the strength of the carrier is high but it hardens quickly. It is not desirable because it loses.

In step 1), Xanthan gum is a high molecular weight polysaccharide composed of two glucose (D-glucose) and two mannose (D-mannose) mainly composed of hexose units, one It is a structure in which a total of five strains are repeatedly connected by connecting D-glucuronic acid, which is usually used as a stabilizer, suspending agent, and thickener, and is nontoxic and stable in a wide range of pH or temperature range. The amount of xanthan gum added is preferably 0.1 to 0.3 parts by weight based on 100 parts by weight of the polyvinyl alcohol aqueous solution. Less than 0.1 part by weight is not preferable because a spherical carrier is not made, and an amount exceeding 0.3 parts by weight is not preferable because the surface of the carrier becomes too dense to prevent in-carrier movement of the substrate and the product, thereby preventing encapsulation.

In step 1), the heating temperature is preferably 60 ~ 100 ℃. If the heating temperature is below 60 ° C, polyvinyl alcohol and xanthan gum are easily hardened and microorganism immobilization carriers are not formed. Therefore, if the heating temperature is higher than 100 ° C, not only the process risk increases due to the high temperature but also the manufacturing cost increases. It is not preferable because it is.

In the step 1), the microorganism to be fixed is a microorganism or a harmful or environmental pollutant to produce a useful product if it is known that can be used to prepare a microorganism immobilization carrier in the art to which the present invention belongs There are no particular restrictions on the kind of microorganisms such as microorganisms to be converted to the substance, and for example, Ochrobactrum anthropi SY509 having excellent denitrification ability. In particular, the Ocrobactrum entropy SY509 screened microorganisms having excellent denitrification ability from the activated sludge collected from the Gimpo landfill, and exhibits excellent denitrification effect compared to other existing microorganisms.

In step 1), the initial mixing amount of the microorganism to be fixed is preferably 0.8 ~ 1.5 parts by weight based on 100 parts by weight of polyvinyl alcohol aqueous solution on the basis of dry weight. If the mixing amount is less than 0.8 parts by weight, the concentration of the immobilized microorganisms is low and the efficiency as a microorganism immobilization carrier is not preferable. If the amount is more than 1.5 parts by weight, the concentration of the microorganisms immobilized in the polyvinyl alcohol can be fixed. It is not preferable because is so high that the substrate and the product are not easily transported into the microorganism immobilization carrier, thereby lowering the efficiency as the microorganism immobilization carrier.

In step 1), the carbon source is a substrate of the microorganism to be fixed, and serves to activate the microorganism. There is no particular limitation on the carbon source that can be used in the present invention, and preferably at least one selected from the group consisting of glucose, methanol, ethanol and acetate can be used. The amount of carbon source added in step 1) is preferably 0.2 to 0.5% by weight based on the weight of the polyvinyl alcohol aqueous solution. If the carbon source is less than 0.2% by weight, the activity of the microorganisms may be lost during the process of making the microorganism immobilization carrier, and if the carbon source exceeds 0.5% by weight, a high concentration of substrate is added to the microorganisms that are inhibited during the carrier production process. It is undesirable because it has the effect of reducing the activity of microorganisms and is expensive.

In the step 2), the boric acid aqueous solution, it is preferable to use a saturated solution of 10 ~ 15% concentration for the complete reaction of boric acid and PVA. If the concentration of the aqueous solution of boric acid is less than 10%, the concentration of boron which can be combined with polyvinyl alcohol is low, and it is not preferable because a compact matrix cannot be formed, the strength of the carrier is weakened, and the perfect sphere cannot be formed. If it exceeds, it is not preferable because the effect of increasing the boric acid concentration is not obtained, which is inefficient.

In the step 2), the surfactant has a hydrophilic group and a lipophilic group at the same time to adsorb to the interface in a dilute solution to reduce the surface tension, and to penetrate, disperse, emulsify, Means a substance that acts as bubbles. The surfactant usable in the present invention is preferably a nonionic surfactant having no group dissociated into ions in an aqueous solution, and more preferably a TWEEN-based nonionic surfactant. The addition amount is preferably 0.1 to 0.5% by volume based on the volume of the aqueous solution of boric acid at a concentration of 10 to 15%. If it is less than 0.1 vol%, it is not preferable because a spherical carrier is not formed. If it exceeds 0.5 vol%, it is not preferable because surfactants react.

When the present invention reacts and deforms the surface of the polyvinyl alcohol carrier to which microorganisms are immobilized with the xanthan gum and the surfactant described above, the present invention not only forms a perfect spherical carrier that does not get entangled with each other, but also by using these materials It has been found that it can have both characteristics of encapsulation and of encapsulation.

In step 3), the aging in the boric acid aqueous solution is preferably made for 1 to 3 hours at 20 ~ 40 ℃. It is not preferable that the aging temperature is less than 20 ° C. because it takes a long time to harden the carrier, and if it exceeds 40 ° C., it is also not preferable because it takes a long time to harden the carrier. In addition, since the aging time is less than 1 hour, polyvinyl alcohol and boric acid are not sufficiently bonded, and it is not preferable, since it may affect the activity of the immobilized microorganisms.

In step 3), the carrier aged in the boric acid solution is filtered and separated, and then, in an aqueous solution of 6-8% phosphate, such as sodium orthophosphate, at 20-40 ° C. for 1 to 2 hours. Mature. If the concentration of the aqueous solution of phosphate is less than 6%, the saponification reaction by phosphate does not occur, and thus the polyvinyl alcohol and boric acid are released, which is not preferable because the immobilized microorganisms flow out of the carrier. It is not preferable because it rises and the carrier becomes hard and the pores of the carrier become dense and the movement of the substrate and the product is not easy. In addition, if the aging temperature in the aqueous solution of phosphate is less than 20 ° C, it is not preferable because the carrier hardens for a long time, and if it exceeds 40 ° C, it is not preferable because the carrier takes too long. If the aging time is less than 1 hour, the saponification reaction is not preferable because time is insufficient, and if it exceeds 2 hours, the saponification reaction occurs too much, which is not preferable because the pores of the carrier become dense.

In step 3), the carrier matured in an aqueous solution of phosphate is filtered and separated, and then aged in 20-40 ° C. for 1 to 2 hours in water, followed by washing. This increases the size of the carrier before the carrier is used for the reaction, thereby increasing the voids in the carrier matrix so that no microorganisms are released, and the mass transfer and transport is easy. This is to prevent backpressure. It is not preferable that the aging temperature in water is less than 20 ° C. because it takes a long time to harden the carrier, and if it exceeds 40 ° C., it is also not preferable because it takes a long time for the carrier to harden. If the maturation time is less than 1 hour, there is insufficient time to wash away the residual boric acid and phosphate by water, and the volume of the carrier is completely increased. This is not preferable because the carrier volume is sufficiently increased after 2 hours and the carrier pore is sufficiently increased.

In step 4), the carrier processed in step 3) is incubated for 20 to 40 ℃, 24 to 36 hours in a microbial growth medium. This is to enhance the activity of the microorganism and also to increase the concentration of the microorganism in the carrier. If the incubation time is less than 24 hours, the active recovery of the microorganisms in the carrier is all performed, which is not preferable because the time is insufficient, and if it exceeds 36 hours, the activity of the microorganisms has already been restored in the carrier, and the amount of the microorganisms is increased enough. There is no change, which is undesirable in terms of carrier production efficiency. The cultured microorganism immobilization carrier can be separated by filtration, washed with water, and used as the next filler.

In the microorganism immobilization carrier of the present invention prepared by the above production method, a fixed amount of microorganism is 30-70% based on the total weight of the carrier, and a large amount of microorganisms can be immobilized, and the carrier is entangled in aqueous solution and It is a spherical carrier which can prevent loosening and has little microbial outflow from the carrier.

The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

Example 1

Step 1: Preparation of Microbial Immobilized Carrier Precursor Solution

5 g of polyvinyl alcohol (polymerization degree 1500) was mixed with 30 ml of distilled water and completely dissolved while heating the temperature to 80 ° C. Then, 0.07 g of xanthan gum was added thereto, stirred, and cooled to room temperature. After stirring, 0.54 g (dry weight) of Ocrobactrum entropy SY509 was added thereto, followed by stirring with addition of 0.1 g of glucose as a carbon source. 50 ml of the precursor solution was prepared by mixing.

Step 2: Preparation of Spherical Activated Microbial Immobilization Carrier

A spherical activated microorganism immobilization carrier was prepared by dropping the solution into the microorganism immobilization carrier precursor solution prepared in step 1 in 300 ml of an aqueous solution of 11% boric acid containing 0.3 volume% of Tween 20 and 0.6 g / l of nitrate-N. (See FIG. 1A).

Step 3: Aging, Filtration and Washing

The aqueous boric acid solution containing the spherical activated microorganism immobilization carrier prepared in step 2 was aged with stirring at 25 ° C. for 2 hours, filtered off, and then aged for 1 hour in 6% sodium orthophosphate solution. It was filtered off. Then, aging and washing were performed for 1 hour in distilled water.

Step 4: incubate, filter and wash

The microorganism immobilization carrier prepared in step 3 was transferred to a microorganism culture medium containing glucose and nitrate nitrogen, and then cultured in a 30 ° C. incubator for 24 hours, and then separated by filtration and washed five times with distilled water to immobilize the microorganism of the present invention. The carrier was prepared.

The prepared microorganism immobilization carrier had a concentration of 35.5% of microorganisms immobilized on polyvinyl alcohol.

Comparative Example 1

Prepared in the same manner as described in Example 1, but was added to the microbial immobilized carrier without the xanthan gum and Tween 20 (see Figure 1b).

Comparative Example 2

The microorganism immobilization support was prepared in the same manner as in Comparative Example 1, except that calcium alginate was added to the polyvinyl aqueous solution in an amount of 2% by weight based on polyvinyl alcohol.

[Comparison of Morphology of Microorganism Immobilized Carriers]

The microbial immobilization carriers prepared in Example 1 and Comparative Example 1 are shown in FIGS. 1A and 1B. The microorganism immobilization support of Example 1 prepared by the method for preparing the microorganism immobilization support of the present invention was in a perfect spherical form as shown in FIG. However, the microorganism immobilization carriers prepared by Comparative Example 1 were in the form of being entangled with each other or having a tail as shown in FIG. 1B.

[Microbial fixation and runoff experiment of microorganism immobilization carrier]

For the microorganism immobilization carrier prepared in Example 1, Comparative Examples 1 and 2, the results of comparing the microorganism immobilization and microbial outflow are shown in Table 1 below.

In Table 1 below, the concentration of microorganism immobilization was measured for each of the carrier precursor prepared in step 1 of Example 1 and the final carrier prepared in step 4, and the effluent concentration of the microorganisms was reacted in a reactor for 7 days, followed by a batch It was measured separately in the batch type reactor and packed bed type reactor.

division Microbial Immobilization Concentration (mg / carrier ^* ) Microbial Outflow Concentration (% / carrier) Carrier precursor Final Batch Reactor Packed Reactor Comparative Example 1 0.4 0.57 15 3 Comparative Example 2 0.4 0.74 8 2.5 Example 1 0.4 1.1 1.8 0.5

* Average weight of carrier: 3mg

As can be seen in Table 1, the microorganism immobilization carrier of the present invention prepared in Example 1 had a very high concentration of microorganism immobilization of the final carrier compared to the microorganism immobilization carrier prepared in Comparative Examples 1 and 2, The concentration of microbial outflow from the microbial immobilization carrier was significantly lower in both packed reactors. Therefore, according to the present invention, the carrier is prevented from being entangled and entangled in an aqueous solution, so that a stable carrier can be obtained, and a large amount of microorganisms can be immobilized, and a perfect spherical microorganism immobilization can minimize the outflow of microorganisms from the carrier. It can be seen that a carrier is provided.

[Nitrate Nitrogen Removal Capability Test]

The results of comparing the nitrate-nitrogen removal ability by measuring the concentration of nitrate-N in the effluent with time for the microbial immobilization carriers prepared in Examples 1 and Comparative Examples 1 and 2 are shown in FIG. 2.

As shown in Figure 2, the microorganism immobilization support of the present invention prepared in Example 1, compared with the conventional microorganism immobilization support prepared in Comparative Examples 1 and 2, the nitrate nitrogen is removed in a short time, and thus the nitrate It can be seen that the nitrogen removal ability is very excellent.

According to the present invention, by using xanthan gum and a surfactant to prepare a microorganism immobilization carrier using polyvinyl alcohol, it is possible to prevent loosening and entanglement of the microorganism immobilization carrier in an aqueous solution and to immobilize a large amount of microorganisms. Complete spherical microbial immobilization carriers are provided that minimize outflow.

Claims (11)

A method for preparing a microorganism immobilization carrier comprising the following steps: 1) preparing a microorganism immobilization carrier precursor solution in which microorganisms are immobilized to polyvinyl alcohol by mixing and dissolving xanthan gum in an aqueous polyvinyl alcohol solution and adding a microorganism to be fixed and a carbon source; 2) modifying the surface of the carrier into a sphere by dropping the solution of the microorganism immobilized carrier precursor prepared in step 1) into an aqueous solution of boric acid containing a surfactant; 3) The spherical carrier obtained in step 2) is aged in the aqueous solution of boric acid, then added to an aqueous solution of phosphate, aged, filtered, and then aged and washed with water, thereby processing the carrier to facilitate the movement of the substrate and the product. ; And 4) preparing a microorganism immobilization carrier by culturing the carrier processed to facilitate the movement of the substrate and the product in step 3) in a microbial growth medium, followed by filtration, separation and washing. The method of claim 1, wherein the polyvinyl alcohol aqueous solution in step 1) is obtained by completely dissolving 5 to 13% by weight of polyvinyl alcohol having a polymerization degree of 1500 to 3000 in distilled water. The method of claim 1, wherein the amount of xanthan gum added in step 1) is 0.1 to 0.3 parts by weight based on 100 parts by weight of an aqueous polyvinyl alcohol solution. The method of claim 1, wherein the temperature in the process of dissolving xanthan gum in the polyvinyl alcohol aqueous solution in step 1) to dissolve is 60 ~ 100 ℃. The method of claim 1, wherein the amount of microorganisms added in step 1) is 0.8 to 1.5 parts by weight based on 100 parts by weight of an aqueous polyvinyl alcohol solution. The method of claim 1, wherein in step 1), the carbon source is added in an amount of 0.2 to 0.5% by weight based on the weight of the aqueous polyvinyl alcohol solution. The method of claim 1, wherein in the step 2), the surfactant is added in an amount of 0.1 to 0.5% by volume based on the volume of the aqueous solution of boric acid at a concentration of 10 to 15%. The method of claim 1, wherein the aging of the boric acid solution in step 3) is carried out at 20 to 40 ° C for 1 to 3 hours. The method of claim 1, wherein in step 3), the phosphate aqueous solution and the aging in water are each performed at 20 to 40 ° C. for 1 to 2 hours, and the phosphate aqueous solution is an aqueous sodium orthophosphate solution at a concentration of 6 to 8%. Method for producing a microorganism immobilized carrier. The method for preparing a microorganism immobilization carrier according to claim 1, wherein the culture in the microbial growth medium in step 4) is carried out at 20 to 40 ° C for 24 to 36 hours. A microorganism immobilized carrier prepared by the method of any one of claims 1 to 10, wherein the concentration of the microorganism immobilized on the polyvinyl alcohol is 30 to 70 wt% based on the total weight of the carrier.

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