CN110452900B - Preparation method of PVA-SA composite immobilized carrier for embedding degradation microorganisms - Google Patents

Preparation method of PVA-SA composite immobilized carrier for embedding degradation microorganisms Download PDF

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CN110452900B
CN110452900B CN201910788354.XA CN201910788354A CN110452900B CN 110452900 B CN110452900 B CN 110452900B CN 201910788354 A CN201910788354 A CN 201910788354A CN 110452900 B CN110452900 B CN 110452900B
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杨宗政
王菊
吴志国
许文帅
赵晓宇
吴芮
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Tianjin University of Science and Technology
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Abstract

A preparation method of a PVA-SA composite immobilized carrier for embedding degradation microorganisms comprises the following steps: the method is characterized by comprising the following steps: a) under the aseptic condition, mixing a bacterial suspension for degrading microorganisms with the sterilized composite material solution; the composite material solution comprises 95-105 g/L polyvinyl alcohol, 9-11 g/L sodium alginate and 28-32 g/L SiO24.5-5.5 g/L CaCO35-7 g/L of active carbon and the balance of deionized water; b) dropwise adding the composite material solution uniformly mixed with the bacterial suspension obtained in the step 1) into a cross-linking agent in a sterile environment, and hardening to obtain the immobilized carrier, wherein the cross-linking agent is a calcium chloride solid with the mass fraction of 1.8-2.2% dissolved in a saturated boric acid solution, and the pH value of the calcium chloride solid is adjusted to 6.1-6.3 by using a sodium hydroxide solution.

Description

Preparation method of PVA-SA composite immobilized carrier for embedding degradation microorganisms
Technical Field
The invention relates to an embedded microorganism immobilized carrier for biological treatment of coking wastewater and a preparation method thereof.
Background
Coking wastewater is wastewater generated in the processes of coke refining, gas purification and coking recovery, contains a plurality of complex organic pollutants including various organic matters such as quinolines, pyridines, phenols, benzenes and the like, and is listed as sewage difficult to treat. Due to the addition (Additive), synergy (synergy), Antagonism (Antagonism) and other effects among various pollutants, when the conventional biological method is adopted for wastewater treatment, the coking wastewater has high toxicity to the growth of microorganisms, so that when the biological method is adopted for treating the coking wastewater, the problems of low tolerable pollutant concentration, poor degradation efficiency, easy inactivation of activated sludge and the like exist, and the application of the measure of treating the composite polluted coking wastewater by the biological method is seriously influenced.
The technology of embedding and immobilizing microorganisms is the key research point in the field of water treatment at present, and the immobilized carrier for embedding and degrading microorganisms is adopted, so that the treatment effect of treating the coking wastewater by utilizing the microorganisms can be improved, the defects that microbial cells are too small, the separation from a water solution is difficult, secondary pollution is easily caused and the like are overcome, and an enzyme system in the cells can be completely stored and can react more fully. The proper and fine immobilized micro-scaffold can endow immobilized cells with certain characteristics, and the immobilized carrier microspheres with adsorption and biodegradation characteristics can enable target pollutants to be adsorbed on the surface of a carrier, so that the degradation strain is promoted to fully contact the pollutants, and the degradation process is accelerated. However, in the research, it is found that when the degrading microorganisms are specific screened complex flora, the degradation effect is adversely affected by the conventional ways of embedding the microorganisms and preparing the immobilized carriers, so that the problem to be solved in the prior art is to be solved by providing a method for preparing the PVA-SA complex immobilized carriers for embedding the degrading microorganisms, which can fully exert the effect of degrading the microorganisms to treat the coking wastewater.
Disclosure of Invention
In order to solve the problems, the invention obtains the preparation method of the immobilized carrier embedded with the degradation microorganisms, which can fully exert the capacity of degrading the microorganisms to treat the coking wastewater, and the immobilized carrier prepared by the method by optimizing the preparation process of the immobilized carrier on the basis of screening a high-efficiency degradation flora of phenol, pyridine and quinoline.
In order to solve the problems, the technical scheme provided by the invention is as follows:
a preparation method of a PVA-SA composite immobilized carrier for embedding degradation microorganisms comprises the following steps: the method is characterized by comprising the following steps:
a) under the aseptic condition, mixing a bacterial suspension for degrading microorganisms with the sterilized composite material solution; the composite material solution is prepared from 95-105 g/L of polyvinyl alcohol, 9-11 g/L of sodium alginate, 28-32 g/L of silicon dioxide, 4.5-5.5 g/L of calcium carbonate, 5-7 g/L of activated carbon and the balance of deionized water.
b) Dropwise adding the composite material solution uniformly mixed with the bacterial suspension obtained in the step 1) into a cross-linking agent in a sterile environment, and hardening to obtain an immobilized carrier, wherein the cross-linking agent is a calcium chloride solid with the mass fraction of 1.8-2.2% dissolved in a saturated boric acid solution, and the pH value of the calcium chloride solid is adjusted to 6.1-6.3 by using a sodium hydroxide solution;
the degrading microorganism is screened by the following method
1) Sampling: collecting a wastewater sample with sludge from a coking wastewater treatment device, wherein the wastewater sample comprises effluent of an aerobic treatment device and effluent of an anaerobic treatment device;
2) culturing and transferring: adding a sample into a liquid inorganic salt culture medium containing phenol, pyridine and quinoline, culturing at 27-33 ℃, detecting the degradation effect of the sample by ultraviolet scanning, taking the sample as a culture end point when a scanning curve is flattened, performing multiple transfer under the same culture condition, taking the supernatant after the multiple transfer as an inoculation bacterium of a screened high-efficiency degradation flora, wherein the liquid inorganic salt culture medium comprises the following components in percentage by weight: NaCl 0.9-1.1 g/L, NH4NO3 0.9～1.1g/L，K2HPO41.20～1.8g/L，KH2PO4 0.4～0.6g/L，MgSO4·7H20.15-0.25 g/L of O, 7.0-7.2 of pH, 10-100 mg/L of phenol, 10-100 mg/L of pyridine and 10-100 mg/L of quinoline, and the balance of distilled water, wherein the phenol, the pyridine and the quinoline are used as the only carbon nitrogen source;
3) separation and purification: and (3) coating and separating the degradation flora obtained in the step 3) by adopting an LB solid culture medium, repeatedly purifying for many times, and preserving the strain.
The preparation method of the PVA-SA composite immobilized carrier for embedding the degrading microorganisms is characterized in that the proportion of the liquid inorganic salt culture medium is preferably 1.0g/L NaCl and NH4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO40.5g/L，MgSO4·7H20.2g/L of O, 40-60 mg/L of phenol, 40-60 mg/L of pyridine and 40-60 mg/L of quinoline, wherein the culture medium is placed in a triangular flask and cultured under the conditions of 30 ℃ and 180 rpm.
The preparation method of the PVA-SA composite immobilized carrier for embedding the degrading microorganisms is characterized in that the flora mainly comprises the following components in the number ratio of 95-105: 25-30: 210-230: 55-65: 90-100 of Rhodococcus ruber, Clostridium botulinum for pharmaceutical use, Paracoccus acridicola, Microbacterium keratidea and Rhodococcus qingshengii.
The preparation method of the PVA-SA composite immobilized carrier for embedding the degradation microorganisms is characterized in that the preparation method of the bacterial suspension comprises the following steps: transferring the inoculated strain into LB liquid culture medium, culturing at 30 deg.C and 180rmp for 48h to obtain seed solution, inoculating 1mL of the seed solution into 100mL of LB liquid culture medium, culturing at 30 deg.C and 180r/min for 24h, centrifuging at 6000r/min and 4 deg.C for 10min, discarding supernatant, blowing with sterile water for resuspension, centrifuging, discarding supernatant, repeating the above steps for 3 times, and preparing the obtained thallus into OD with inorganic salt solution6002.0 of bacterial suspension, wherein the inorganic salt content of the inorganic salt solution is 1.0g/L of NaCl and NH4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO4 0.5g/L，MgSO4·7H2O0.2 g/L, 1mL of microelement stock solution/L, wherein the components of the microelement stock solution are KI 0.005g and MnSO4·4H2O 0.2g，CuSO4·2H2O 0.02g，ZnSO4·7H2O 0.2g，Na2MoO4·2H2O 0.25g，H3BO3 0.008g，FeCL36H2O 0.1.1 g, adding water to 100 mL; the composite material solution comprises 100g/L polyvinyl alcohol, 10g/L sodium alginate and 30g/L SiO25g/L CaCO3The cross-linking agent is prepared by dissolving 2% calcium chloride solid in a saturated boric acid solution and adjusting the pH value to 6.22 by using 0.5mol/L sodium hydroxide, the hardening treatment condition is that the cross-linking treatment is carried out for 12-24 hours at 4 ℃, and the volume ratio of the bacterial suspension to the composite material solution is 5-30: 100, preferably 18-22: 100.
And when the composite solution is dropwise added into the cross-linking agent, controlling the size of the liquid drop to ensure that the diameter of the prepared composite immobilized carrier bead is 0.3-0.7 cm, preferably 0.4-0.6 cm.
The invention also provides an immobilized carrier obtained by adopting the preparation method of any one PVA-SA composite immobilized carrier embedded with the degradation microorganisms.
Compared with the prior art, the invention has the beneficial effects that:
1. the preparation method of the PVA-SA composite immobilized carrier for embedding the degradation microorganisms, which is provided by the invention, has the advantages of simple operation, low requirement on equipment, low price and relatively safe preparation process.
2. According to the invention, the raw material proportion and the preparation process of the composite material solution are optimized, the obtained immobilized carrier utilizes the adsorption performance of the active carbon in the raw materials to adsorb pollutants and accelerate biodegradation, and the immobilized carrier beads prepared by the optimized method have better capacity of degrading complex pollutants.
3. The immobilized carrier obtained by the preparation method of the invention provides a wide space for the growth of microorganisms, is beneficial to the growth and reproduction of the microorganisms and the removal of pollutants, and increases the application prospect of biodegradation in the field of environmental remediation.
4. The invention has simple manufacturing principle and reasonable structural design, the diameter of the prepared immobilized microorganism pellet is about 0.5cm, the aperture ratio is high, the mass transfer performance is good, the biological affinity is strong, and the invention can be better applied to the environmental pollution remediation. The method has the advantages of low operation cost, convenient operation, wide application in the field of environmental pollution treatment, high practical application value and easy popularization and application.
Drawings
FIG. 1 is a topographical view of the internal microstructure of beads of an immobilization carrier prepared by the method of example 4;
FIG. 2 is a surface microstructure of beads of the immobilization support prepared by the method of example 4.
Detailed Description
The technical solution of the present invention will be further described with reference to the following embodiments.
Example 1: method for screening phenol, pyridine and quinoline high-efficiency degradation flora
The method comprises the following steps:
1) sampling
Coking wastewater with sludge is collected from a sewage treatment device of Tangshandafeng coking Co., Ltd, and comprises 1mL of raw water (with sludge), 1mL of effluent of an anaerobic pool (with sludge), 1mL of effluent of an aerobic pool (with sludge) and 1mL of effluent of a secondary sedimentation pool (with sludge). A. the
2) Culture and transfer
Adding the coking wastewater collected at each process point collected in the step 1) into 100mL of liquid inorganic salt culture medium, wherein the proportion of NaCl to NH is 1.0g/L4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO4 0.5g/L，MgSO4·7H2O0.2 g/L, phenol 50mg/L, pyridine 50mg/L, quinoline 50 mg/L. Placing in a triangular bottle mouth, sealing with a sealing film (the surface of the sealing film has air holes), and culturing in a shaking table at 30 deg.C and 180 rmp. And (3) detecting the degradation effect by ultraviolet full-wavelength scanning, wherein the maximum absorption peaks of phenol are at 210nm and 270nm, the maximum absorption peaks of quinoline are at 313nm and 225nm, the maximum absorption peak of pyridine is at 256nm, and the scanning curve is flattened after 3 days of culture to show that the phenol, the pyridine and the quinoline are completely degraded, and the result is used as the culture endpoint. And after the culture end point is reached, transferring the supernatant as an inoculation bacterial liquid, and performing multiple transfers under the same culture conditions to obtain the high-efficiency degradation flora.
3) Separation and purification:
and separating the bacterial liquid of the high-efficiency degradation flora (hereinafter referred to as enriched bacterial liquid) obtained after multiple transfer by adopting a dilution plate coating method. 10 tubes of 9mL sterile water are arranged according to the proportion of 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8And numbering in sequence. Under the aseptic operation condition, 1mL of enriched bacterial liquid is sucked and placed in 9mL of aseptic water in a first tube and vibrated to obtain 10-1Bacterial liquid with concentration. The same method is used to sequentially dilute to obtain 10-8Bacterial liquid with concentration. Are respectively from 10-3、10-4、10-5、10-6、10-7、10-8The bacterial liquid of (2) was pipetted into 100. mu.L each on a plate with LB solid medium, and was spread evenly on the plate by a glass coating rod, and 2 plates were prepared for each concentration of bacterial liquid. The plate inoculated with the microorganism was incubated in an incubator at 30 ℃. The LB solid medium comprises 10g of peptone, 10g of sodium chloride, 5g of beef extract, 20.0-25.0 g of agar and 1000ml of distilled water, wherein the pH value is 7.0-7.2. After the bacteria grow on the flat plate, selecting the bacteria with better growth vigor for further purification. One-loop colonies were picked aseptically using an inoculating loop, inoculated onto freshly sterilized plates, and further purified for the selected strains. The strain is purified by adopting a plate marking method, and the specific process is as follows: the inoculating loop with colony is first parallel scribed at 3-4 lines on one side of the new plate, the culture dish is rotated by about 70 degrees, the remainder of the inoculating loop is burnt off, after cooling, the inoculating loop is parallel scribed for the second time through the first scribing part, and then the inoculating loop is scribed for the third time through the second scribing part in the same way without overlapping the front and back lines. The plate inoculated with the microorganism was incubated in an incubator at 30 ℃. And identifying the purified dominant bacterial strain by adopting a PCR (polymerase chain reaction) method, wherein the PCR method comprises the steps of extracting DNA (deoxyribonucleic acid) of the dominant bacterial strain, carrying out PCR amplification on the extracted DNA by adopting a PCR amplification instrument, carrying out agarose gel electrophoresis on the DNA, and finally sequencing and analyzing the genome 16SrDNA to determine the species. The high-efficiency degrading flora obtained by screening mainly comprises Rhodococcus ruber (Rhodococcus ruber), medicinal clostridium botulinum (Clostridium botulinum), Acidococcus acridae (Paracoccus acridae), Microbacterium keratolyticum (Microbacterium keratalyticum) and Rhodococcus qingshengii (Rhodococcus cornus) through identification. The quantity ratio of red Rhodococcus ruber (Rhodococcus ruber), medicinal botulis (botulibacter medicamentivorans), Paracoccus acridicus (Paracoccus acridae), Microbacterium keratolyticum (Microbacterium keratalyticum), and Rhodococcus celebratum (Rhodococcus guinshengii) is 100: 27: 218: 58: 96.
and (3) preservation of strains: mixing 750 μ L of glycerin and 750 μ L of bacteria liquid, and preserving at-20 ℃.
Examples 2 to 5: a preparation method of a PVA-SA composite immobilized carrier for embedding degradation microorganisms.
Preparing a degrading flora seed solution: the enriched bacterial liquid obtained in example 1 is transferred into LB liquid culture medium, and cultured for 48h at 30 ℃ in 180rmp environment, so as to obtain seed liquid.
Preparation of bacterial suspension: inoculating the above 1mL seed solution into 100mL LB culture medium, culturing at 180r/min and 30 deg.C for 24h, centrifuging at 6000r/min and 4 deg.C for 10min, discarding supernatant, blowing with sterile water to resuspend, centrifuging, discarding supernatant, repeating the above steps for 3 times, and using inorganic salt solution (the inorganic salt content of the inorganic salt solution is NaCl 1.0g/L, NH) to obtain thallus4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO4 0.5g/L，MgSO4·7H2O0.2 g/L, 1mL of microelement stock solution/L, wherein the components of the microelement stock solution are KI 0.005g and MnSO4·4H2O 0.2g，CuSO4·2H2O 0.02g，ZnSO4·7H2O 0.2g，Na2MoO4·2H2O 0.25g，H3BO3 0.008g，FeCl36H2O 0.1g, water to 100mL) to OD600The suspension was 2.0.
Preparing immobilized microspheres:
in the first step, an immobilization carrier solution for embedding a bacterial population is prepared. 10g of polyvinyl alcohol and 1g of sodium alginate are weighed into a 250mL beaker, added into 100mL of deionized water and stirred uniformly, and soaked for 28 hours at room temperature to be fully softened. Then 3gSiO was added2、0.5gCaCO3And 0.5g of activated carbon, which is stirred rapidly a few times to prevent caking. Dissolving in a heat-collecting constant-temperature heating magnetic stirrer while stirring in a water bath at 90 ℃, and continuously adding water in the water bath process to ensure that the volume of the mixed solution is not changed. And obtaining the composite material solution after the polyvinyl alcohol and the sodium alginate are completely dissolved. Then sterilizing in a high-temperature high-pressure sterilizing pot at 121 ℃ for 30 min. And (4) mixing the bacterial suspension and the composite material solution uniformly under the aseptic condition. The volume ratio of the bacterial suspension to the composite material solution is A: 100.
in the second step, a crosslinking agent is prepared. The saturated boric acid solution is dissolved with calcium chloride solid with the mass fraction of 2 percent and is adjusted with 0.5mol/L sodium hydroxide to have the pH value of 6.22.
And step three, preparing the sodium alginate-polyvinyl alcohol composite microspheres. Under aseptic condition, uniformly mixing the bacterial suspension and the composite carrier solution cooled to room temperature, sucking the composite material solution uniformly mixed with the bacterial suspension by a glass syringe with a glue dispensing head and carrying out sterilization treatment, and dripping the composite material solution into a cross-linking agent, wherein the controlled force is controlled to ensure that the embedding material descends in the form of liquid drops. Crosslinking at 4 ℃ for 12 h. And obtaining immobilized carrier spheres embedding the flora, filtering the spheres after crosslinking, washing with deionized water for 1-3 times until no white solution is generated, and controlling the diameter range of the finished immobilized carrier spheres to be 0.3-0.7 cm.
The ratio of bacterial suspension to composite solution for examples 2-5 is shown in the following table
Numbering Example 2 Example 3 Example 4 Example 5
A 5 10 20 30
Wherein the morphology of the internal microstructure and the morphology of the surface microstructure (electron microscopy) of the immobilized carrier beads obtained by the method of example 4 are respectively shown in fig. 1 and fig. 2.
Application effect analysis of carrier microspheres for immobilized efficient degradation of flora
The immobilized carrier beads embedded with flora prepared in examples 2-5 are respectively added into inorganic salt liquid culture media containing three characteristic pollutants of phenol, pyridine and quinoline. The initial mass concentration of the three characteristic pollutants is 100 mg/L.
The addition amount of the immobilized carrier beads is 30g/100mL
The concentrations of three pollutants of phenol, pyridine and quinoline are measured by sampling at the conditions of T-30 ℃ and r-180 r/min. The time for the immobilized carrier beads of each example to completely degrade the three contaminants was recorded
Numbering Example 2 Example 3 Example 4 Example 5
Complete degradation time/h 24 18 11 15
From the data, it can be seen that the immobilized carrier beads prepared according to different ratios of the bacterial suspension to the composite material solution can completely degrade three characteristic pollutants, namely phenol, pyridine and quinoline, but the immobilized carrier beads prepared in example 4 have the fastest degradation speed and show better treatment effect.

Claims (3)

1. A preparation method of a PVA-SA composite immobilized carrier for embedding degradation microorganisms comprises the following steps: the method is characterized by comprising the following steps:
a) under the aseptic condition, mixing a bacterial suspension for degrading microorganisms with the sterilized composite material solution; the composite material solution comprises 95-105 g/L polyvinyl alcohol, 9-11 g/L sodium alginate and 28-32 g/L SiO24.5-5.5 g/L CaCO35-7 g/L of activated carbon and the balance of deionized water, wherein the volume ratio of the bacterial suspension to the composite material solution is 18-22: 100;
b) dropwise adding the composite material solution uniformly mixed with the bacterial suspension obtained in the step 1) into a cross-linking agent in a sterile environment, and hardening to obtain an immobilized carrier, wherein the cross-linking agent is a calcium chloride solid with the mass fraction of 1.8-2.2% dissolved in a saturated boric acid solution, and the pH value of the calcium chloride solid is adjusted to 6.1-6.3 by using a sodium hydroxide solution;
the degrading microorganism is screened by adopting the following method:
1) sampling: collecting a wastewater sample with sludge from a coking wastewater treatment device, wherein the wastewater sample comprises effluent of an aerobic treatment device and effluent of an anaerobic treatment device;
2) culturing and transferring: adding a sample into a liquid inorganic salt culture medium containing phenol, pyridine and quinoline, culturing at 27-33 ℃, detecting the degradation effect of the sample by ultraviolet scanning, taking the sample as a culture end point when a scanning curve is flattened, performing multiple transfer under the same culture condition, taking the supernatant after the multiple transfer as an inoculation bacterium of a screened high-efficiency degradation flora, wherein the liquid inorganic salt culture medium comprises the following components in percentage by weight: NaCl 0.9-1.1 g/L, NH4NO3 0.9～1.1g/L，K2HPO4 1.20～1.8g/L，KH2PO4 0.4～0.6g/L，MgSO4·7H2O0.15-0.25 g/L, pH 7.0-7.2, phenol 10-100 mg/L, pyridine 10-100 mg/L, quinoline 10-100 mg/L, and distilled water, phenol, pyridineQuinoline is the only carbon nitrogen source;
3) separation and purification: coating and separating the degrading flora obtained in the step 2) by adopting an LB solid culture medium, performing streak purification for many times, and preserving the seeds, wherein the flora comprises the following components in a number ratio of 95-105: 25-30: 210-230: 55-65: 90-100 of Rhodococcus ruber (Rhodococcus ruber), Clostridium botulinum for pharmaceutical use (Patulibacterium mediastinvorans), Paracoccus acridicola (Paracoccus crassifae), Microbacterium keratolyticum (Microbacterium keratitanium), and Rhodococcus qingshengii (Rhodococcus guingshengii);
the preparation method of the bacterial suspension comprises the following steps: transferring the inoculated strain into LB liquid culture medium, culturing at 30 deg.C and 180rmp for 48h to obtain seed solution, inoculating 1mL of the seed solution into 100mL of LB liquid culture medium, culturing at 30 deg.C and 180r/min for 24h, centrifuging at 6000r/min and 4 deg.C for 10min, discarding supernatant, blowing with sterile water for resuspension, centrifuging, discarding supernatant, repeating the above steps for 3 times, and preparing the obtained thallus into OD with inorganic salt solution6002.0 of bacterial suspension, wherein the inorganic salt content of the inorganic salt solution is 1.0g/L of NaCl and NH4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO4 0.5g/L，MgSO4·7H2O0.2 g/L, 1mL of microelement stock solution/L, wherein the proportion of the microelement stock solution is KI 0.005g and MnSO4·4H2O 0.2g，CuSO4·2H2O 0.02g，ZnSO4·7H2O 0.2g，Na2MoO4·2H2O 0.25g，H3BO3 0.008g，FeCl3·6H20.1g of O, and adding water to 100 mL; the composite material solution is prepared from 100g/L of polyvinyl alcohol, 10g/L of sodium alginate, 30g/L of silicon dioxide, 5g/L of calcium carbonate, 6g/L of activated carbon and the balance of deionized water, the cross-linking agent is obtained by dissolving 2% calcium chloride solid in a saturated boric acid solution and adjusting the pH value to 6.22 by 0.5mol/L of sodium hydroxide, and the hardening treatment condition is that the cross-linking treatment is carried out for 12-24 hours at the temperature of 4 ℃.
2. The method of claim 1, wherein the PVA-SA composite immobilization carrier comprises a biodegradable microorganismThe preparation method is characterized in that the proportion of the liquid inorganic salt culture medium is that NaCl is 1.0g/L and NH4NO3 1.0g/L，K2HPO4 1.5g/L，KH2PO4 0.5g/L，MgSO4·7H20.2g/L of O, 40-60 mg/L of phenol, 40-60 mg/L of pyridine and 40-60 mg/L of quinoline, wherein the culture medium is placed in a triangular flask and cultured under the conditions of 30 ℃ and 180 rpm.
3. The immobilized carrier obtained by the preparation method of the PVA-SA composite immobilized carrier embedded with the degradation microorganisms according to any one of claims 1 to 2.
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