CN117965524B - Preparation method and application of immobilized microorganism carrier - Google Patents
Preparation method and application of immobilized microorganism carrier Download PDFInfo
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- CN117965524B CN117965524B CN202410382499.0A CN202410382499A CN117965524B CN 117965524 B CN117965524 B CN 117965524B CN 202410382499 A CN202410382499 A CN 202410382499A CN 117965524 B CN117965524 B CN 117965524B
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- attapulgite
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
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- PLHJDBGFXBMTGZ-WEVVVXLNSA-N furazolidone Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OCC1 PLHJDBGFXBMTGZ-WEVVVXLNSA-N 0.000 claims abstract description 40
- 229960001625 furazolidone Drugs 0.000 claims abstract description 40
- 229960000892 attapulgite Drugs 0.000 claims abstract description 29
- 229910052625 palygorskite Inorganic materials 0.000 claims abstract description 29
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 18
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- 239000000661 sodium alginate Substances 0.000 claims abstract description 12
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000003513 alkali Substances 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 8
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- 239000002245 particle Substances 0.000 claims abstract description 7
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- 238000007710 freezing Methods 0.000 claims abstract description 3
- 230000008014 freezing Effects 0.000 claims abstract description 3
- 241000193755 Bacillus cereus Species 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000009629 microbiological culture Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 239000011574 phosphorus Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims 1
- 238000009360 aquaculture Methods 0.000 abstract description 7
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- 239000005017 polysaccharide Substances 0.000 abstract description 3
- -1 polysaccharide sodium alginate Chemical class 0.000 abstract description 3
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- 239000000969 carrier Substances 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 6
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- 238000002474 experimental method Methods 0.000 description 5
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
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- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241000192710 Microcystis aeruginosa Species 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a preparation method and application of an immobilized microorganism carrier, comprising the following steps: s1, preparing alkalized attapulgite: mixing attapulgite with alkali liquor, oscillating and centrifuging, taking precipitate, washing and drying to obtain alkalized attapulgite; s2, preparing SA-P immobilized particles: adding the alkalized attapulgite into a sodium alginate solution, stirring to fully mix the alkalized attapulgite, adding a microbial inoculum, stirring and mixing, freezing and thawing, soaking the mixture in a CaCl 2 solution, and fully reacting to obtain SA-P immobilized particles. The invention utilizes the high porosity of the attapulgite, uses NaOH to modify, increases the surface negative charge of the microbial carrier to improve the adsorption performance, and finally utilizes natural polysaccharide sodium alginate to crosslink and provide an adhesion matrix for microorganisms, thereby effectively degrading furazolidone, COD, TP and TN in the aquaculture tail water.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method of an immobilized microorganism carrier and application of the immobilized microorganism carrier in cultivation tail water treatment.
Background
The pollutants of the cultivation wastewater mainly comprise organic matters, nitrogen, phosphorus, heavy metals, pathogenic microorganisms and the like. Wherein, the culture wastewater with higher organic matters, nitrogen and phosphorus content is one of the main reasons for causing water eutrophication and water bloom. These eutrophic waters attract a large number of phytoplankton and zooplankton, resulting in lack of oxygen and deterioration of water quality in the water, affecting the growth and reproduction of fish and other aquatic organisms. In addition, pathogenic microorganisms in the aquaculture wastewater can also pose a threat to aquatic organisms and human health. To solve this problem, a series of processing techniques and control measures have been adopted. Including physical treatment methods such as precipitation, filtration, sterilization, evaporation, etc.; chemical treatment methods such as biodegradation, ozone oxidation, hydrogen peroxide catalytic oxidation, and the like; biological treatment methods such as biofilters, biooxidations, biofilm processes, and the like.
Furazolidone is an antibacterial drug widely applied to the field of aquaculture, has the functions of sterilization, bacteriostasis and antivirus, and can be used for preventing and treating various infectious diseases of fish and aquatic animals. In the aspect of aquaculture, the use and the dosage of the furazolidone generally depend on specific diseases and infection degrees, and the proper amount of furazolidone can be added to effectively control pathogenic microorganisms in water bodies and protect fish from being affected by diseases. If the culture tail water containing furazolidone is released into the environment, the furazolidone may permeate into soil and water body and pollute the environment, and as furazolidone is a durable chemical substance, the furazolidone stays in the environment for a long time, and may have long-term negative influence on food chains. The biological treatment technology for furazolidone is still freshly researched at present, so the invention provides a preparation method of an immobilized microorganism carrier capable of efficiently degrading furazolidone and main pollutants of culture tail water.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims at providing a preparation method of an immobilized microorganism carrier, which utilizes the high porosity of attapulgite, uses NaOH to modify, increases the surface negative charge of the microorganism carrier to improve the adsorption performance, and finally utilizes natural polysaccharide sodium alginate to crosslink and provide an adhesion matrix for microorganisms, thereby effectively degrading furazolidone, COD, TP and TN in aquaculture tail water.
In order to achieve the above object, the present invention adopts the following technical scheme:
The preparation method of the immobilized microorganism carrier comprises the following steps:
s1, preparing alkalized attapulgite: mixing attapulgite with alkali liquor, oscillating and centrifuging, taking precipitate, washing and drying to obtain alkalized attapulgite;
S2, preparing SA-P immobilized particles: adding the alkalized attapulgite into a sodium alginate solution, stirring to fully mix the alkalized attapulgite, adding a microbial inoculum, stirring and mixing, freezing and thawing, soaking the mixture in a CaCl 2 solution, and fully reacting to obtain SA-P immobilized particles.
Preferably, in the step S1, the alkali solution is a NaOH solution with a concentration of 1-10%.
Preferably, in the step S1, the addition ratio of the attapulgite to the alkali solution is (4-10) g: (70-110) mL.
Preferably, in the step S2, the microbial agent is Bacillus cereus (WR-3) and is deposited in the China general microbiological culture Collection center, which is the collection number: CGMCC No.29355.
Preferably, in the step S2, the addition ratio of the alkalized attapulgite, sodium alginate and the microbial inoculum is (5-10) g: (50-100) mL: (2-3) mL.
Preferably, in the step S2, the concentration of the sodium alginate solution is 1-2wt% and the concentration of the CaCl 2 solution is 2wt%.
The immobilized microorganism carrier is used for degrading furazolidone, COD, TP and TN in the tail water of the cultivation.
Preferably, the temperature of the culture tail water is 25-35 ℃, and the pH value is 6.5-8.0.
Preferably, the concentration of furazolidone in the culture tail water is 100-300 mg/L.
The invention has the advantages that: the invention utilizes the high porosity of the attapulgite, uses NaOH to modify, increases the surface negative charge of the microbial carrier to improve the adsorption performance, and finally utilizes natural polysaccharide sodium alginate to crosslink and provide an adhesion matrix for microorganisms, thus effectively degrading furazolidone, COD, TP and TN in the aquaculture tail water; the SA-P immobilized microorganism carrier has the advantages of low price, strong microorganism growth capability and no pollution to the environment, has wide application range to conditions such as temperature, pH and the like in practical application, is not easy to be impacted by the external environment, and has good application value and wide application prospect.
Drawings
FIG. 1 is a graph showing the effect of SA-P immobilized microorganism carriers on the treatment of aquaculture tail water;
FIG. 2 is a graph showing the degradation effect of SA-P immobilized microorganism carriers at different initial furazolidone concentrations;
FIG. 3 is a graph showing the degradation effect of SA-P immobilized microorganism carriers at different temperatures;
FIG. 4 is a graph showing the degradation effect of SA-P immobilized microorganism carriers at different pH values;
FIG. 5 is a graph showing the comparison of the degradation effects of SA-P immobilized microorganism carriers and solid microbial agents.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
EXAMPLE 1 preparation of SA-P immobilized microorganism vector
S1, preparation of alkalized attapulgite
Taking a proper amount of attapulgite in an conical flask, adding NaOH alkali liquor with the concentration of 1%, wherein the adding ratio of the attapulgite to the alkali liquor is (4-10) g: (70-110) mL, specifically weighing 4.5 g attapulgite and 90mL alkali liquor, oscillating the conical flask at 150 r/min for one hour at 60 ℃, centrifuging, washing the precipitate with deionized water until the pH of the supernatant is about 7.0, drying the precipitate in a 60 ℃ oven for 1 d, and grinding the obtained solid into powder by a mortar to obtain the alkalized attapulgite.
Preparation of S2 and SA-P immobilized particles
Bacillus cereus WR-3 was selected from the group consisting of Bacillus cereus and deposited at the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 12 and 18 days of 2023, with the deposit number: CGMCC No.29355. The preparation of the microbial inoculum comprises the following steps:
(1) Inoculating WR-3 strain on LB solid plate culture medium, wherein the LB solid plate culture medium comprises the following components: 10.0g g of peptone, 5.0 g g of yeast powder and 10.0g of sodium chloride, adding water to 1.0L and pH of 7.0, placing the mixture in a constant temperature incubator, and culturing at 15 ℃ for about 24. 24 h until colonies grow on the medium; then, single colony is selected and inoculated on LB inclined plane culture medium, and the components of the LB inclined plane culture medium are as follows: yeast extract 5 g/L, tryptone 10 g/L and sodium chloride 10 g/L, and incubated at 15deg.C for 24 h.
(2) The strain cultured in the first step is prepared into bacterial suspension of 1X 10 9 CFU/mL by 5 per mill sterilized normal saline, and inoculated into LB liquid culture medium with 3 percent of inoculation amount, wherein the LB liquid culture medium comprises the following components: yeast extract 5 g/L, tryptone 10 g/L and sodium chloride 10 g/L, shaking culture at 15deg.C, 180 rpm for 24 h to obtain seed solution.
(3) Inoculating the seed liquid in the second step into a fermentation culture medium with 3% of inoculation amount, wherein the components of the fermentation culture medium are a carbon source 10 g/L, a nitrogen source 5 g/L, sodium chloride 10 g/L and pure water, carrying out shaking culture at 15 ℃ and 180 rpm until the viable count is 2X 10 9 CFU/mL, and stopping fermentation to obtain the liquid microbial inoculum.
Preparing a sodium alginate solution with the weight percent of 1, adding alkalized attapulgite, stirring 1 h to fully mix, and then adding Bacillus cereus WR-3 microbial inoculum, wherein the adding ratio of the alkalized attapulgite, the sodium alginate and the microbial inoculum is (5-10) g: (50-100) mL: (2-3) mL, in this example, 50-mL parts of sodium alginate solution, 5-g parts of alkalized attapulgite and 2-3 mL of Bacillus cereus (WR-3) microbial inoculum are specifically taken, stirred 3-h to be fully mixed, added into a spherical mold, put into a refrigerator for freeze thawing, 2wt% of CaCl 2 solution is prepared, a sample obtained in the spherical mold is added into CaCl 2 solution for soaking 24-h parts of the CaCl 2 solution for full reaction, and then placed into a baking oven at 35 ℃ for drying for 2 days, and sealed and stored for standby, thus 8.5g of SA-P immobilized microorganism carrier is obtained.
EXAMPLE 2 Effect of SA-P immobilized microorganism vector on treatment of culture Tail Water
Preparing a water sample of 1L, wherein the initial concentration of furazolidone, COD, TP and TN is 200 mg/L, 120 mg/L, 80 mg/L and 50 mg/L respectively, adding 1 g of the SA-P immobilized microorganism carrier prepared in the embodiment 1, shake culturing at 30 ℃ and 160 r/min, sampling every 24h, measuring the content of furazolidone, COD, TP and TN in the water sample, and calculating the degradation rate, wherein the result is shown in figure 1.
As shown in FIG. 1, after the SA-P immobilized microorganism carrier is used for treating 48 h of the culture tail water, the degradation rate of furazolidone can reach more than 90%, the degradation rates of COD, TP and TN can reach more than 85%, and when the SA-P immobilized microorganism carrier is used for treating 72 h, the degradation rate of furazolidone is 98%, the degradation rates of COD, TP and TN can reach more than 90%, and the treatment effect is excellent.
Example 3 Effect of different Furazolidone initial concentrations on the degradation Effect of SA-P immobilized microbial Carriers
Six groups of water samples with 1L are prepared, the initial concentration of furazolidone is 50, 100, 200, 300, 350, 400 mg/L, the pH value is 7.0, SA-P immobilized microorganism carrier of 1 g is added, shake culture is carried out at 30 ℃ at 160 r/min, sampling is carried out after 48 h, and the residual concentration of furazolidone is detected, wherein the result is shown in figure 2.
As shown in FIG. 2, when the initial concentration of furazolidone is 100-300 mg/L, the degradation rate of SA-P immobilized microorganism carrier to furazolidone can reach more than 85%, the concentration is 300 mg/L, and the degradation rate is 95% at most.
EXAMPLE 4 Effect of different temperatures on the degradation Effect of SA-P immobilized microbial Carriers
Six groups of water samples of 1L are prepared, wherein the initial concentration of furazolidone is 200 mg/L respectively, 1g of SA-P immobilized microorganism carrier prepared in the embodiment 1 is added, the water samples are respectively placed at 15 ℃, 20 ℃, 25 ℃,30 ℃, 35 ℃ and 40 ℃ for shake culture, pH is 7.0, 160 r/min is carried out, 48 h is sampled, the content of furazolidone in the water samples is measured, and the degradation rate is calculated, and the result is shown in figure 3.
As shown in FIG. 3, when the temperature of a water sample is 20-35 ℃, the degradation rate of the SA-P immobilized microorganism carrier to furazolidone is highest and can reach more than 90%, and the temperature application range is wide.
Example 5 Effect of different pH on degradation Effect of SA-P immobilized microorganism vector
Eight groups of water samples of 1L are prepared, wherein the initial concentration of furazolidone is 200 mg/L respectively, 1g of SA-P immobilized microorganism carriers prepared in the embodiment 1 are added, the water samples are respectively placed under the conditions of pH 5, 5.5, 6, 6.5, 7, 7.5, 8 and 8.5, shake culture is carried out at 30 ℃ at 160 r/min, 48 h is sampled, the content of furazolidone in the water samples is measured, and the degradation rate is calculated, and the result is shown in figure 4.
As shown in FIG. 4, when the pH of the water sample is between 6.5 and 8, the degradation rate of the SA-P immobilized microbial carrier on furazolidone is highest and can reach more than 90%, and the pH application range is wide.
Example 6, comparative experiment (one)
Two groups of water samples with the initial concentration of furazolidone of 1L are arranged, the pH value is 7.0, one group of the water samples is added with 8.5 g of the SA-P immobilized microorganism carrier prepared in the embodiment 1, the other group of the water samples is added with 2ml of Bacillus cereus WR-3 microbial inoculum, the temperature is 30 ℃, the shaking culture is carried out at 160r/min, the samples are taken every 24h, the furazolidone content in the water samples is measured, and the degradation rate is calculated, and the result is shown in figure 5.
As shown in FIG. 5, the SA-P immobilized microbial carrier has better degradation effect on furazolidone than the case of only adding Bacillus cereus WR-3 microbial agent, and the degradation effect can reach more than 90% at 48 h.
Example 7, comparative experiment (II)
Two groups of C: N: P were set to 100:5:1, 15-L of water sample, arranging filler, adding 8.5-g of SA-P immobilized microorganism carrier into one group, adding 2ml of Bacillus cereus WR-3 microbial inoculum into the other group, respectively adding the two groups of water samples into a portable strain activation device, starting a stirring device for full dissolution, aerating and oxygenating, keeping the temperature at 20-35 ℃ for 2-3 hours, carrying out strain film hanging, measuring COD values of water inlet and outlet every 24-h, and determining the end and time point of an aerobic film hanging stage by taking COD removal rate as a judgment basis after one week.
The results showed that the number of days for film formation was 6 days for the group to which SA-P immobilized microorganism vector was added, and 13 days for the group to which Bacillus cereus WR-3 was added. Experiments show that the film-forming speed of the SA-P carrier is far greater than that of the solid microbial inoculum directly, and the treatment efficiency of practical application is effectively improved.
EXAMPLE 8 treatment of actual cultivation Tail Water by SA-P immobilized microorganism vector (I)
SA-P immobilized microbial carriers are prepared according to the method of the embodiment 1, the source of treated water is a culture tail water treatment system, a laboratory simulates aeration treatment of a biochemical system, the treatment capacity is 5L, 5g of SA-P immobilized microbial carriers are added, the aeration treatment enables dissolved oxygen to be more than 2 mg/L, the pH of sewage is 6.0-8.0, the initial furazolidone concentration is 100 mg/L,48 h is 1 d for water inlet and outlet once, each time is 1L for water inlet and outlet, each time is 100 mg/L for furazolidone concentration in a water inlet and maintaining system, the content of furazolidone is detected by sampling, and the tracking experiment result is shown in the table 1.
Table 1 furazolidone content in water sample
EXAMPLE 9 treatment of actual cultivation Tail Water by SA-P immobilized microorganism vector (II)
The SA-P immobilized microorganism carrier is prepared according to the method of the embodiment 1, the source of the treated water is a pond culture tail water treatment system, a laboratory simulates aeration treatment of a biochemical system, the treatment capacity is 5L, the SA-P carrier of 5g is added, the aeration treatment ensures that dissolved oxygen is more than 2 mg/L, the pH of sewage is 7.5-8.5, the COD is about 120 mg/L,48 h is 1 d for water inlet and outlet once, the water inlet and outlet is 1L each time, the content of COD, TP and TN in the water is sampled and detected, and the degradation rate of COD, TP and TN is obtained to reach more than 80%.
EXAMPLE 10 SA-P immobilized microbial Carrier sustained Release test
18 Immobilized microorganism carrier materials were prepared as in example 1 and packed into three carrier spheres, each of which was packed with 6 immobilized materials, and ropes and suspension spheres were tethered to the carrier spheres. And selecting one section of the river channel for testing, respectively placing 1 carrier ball in the water inlet area, the treatment area and the ecological purification area, and fixing the carrier balls in the river channel by using the suspension balls, and taking every 24 h to record the morphology and the slow release condition of the material. And when the water flow speed is high, the water flow is immersed into the river bottom for experiments.
The appearance of the material is observed, when the material is put into water for 5 days, the immobilized carrier is not obviously softened, is in a solid state, is not obviously changed in size, is harder in hardness, and after 8 days, the immobilized carrier is obviously softened, the volume is obviously reduced, and the carrier is almost completely dispersed in water within 1 month. In conclusion, the slow release time of the alkalized attapulgite immobilized microorganism carrier is 1 month.
In conclusion, the SA-P immobilized microorganism carrier provided by the invention is added into a culture tail water treatment system, can effectively remove furazolidone, COD, TP and TN, and has a wide application prospect.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.
Claims (6)
1. The preparation method of the immobilized microorganism carrier is characterized by comprising the following steps:
s1, preparing alkalized attapulgite: mixing attapulgite with alkali liquor, oscillating and centrifuging, taking precipitate, washing and drying to obtain alkalized attapulgite;
S2, preparing SA-P immobilized particles: adding the alkalized attapulgite into a sodium alginate solution, stirring to fully mix the alkalized attapulgite, adding a microbial inoculum, stirring and mixing, freezing and thawing, soaking the mixture in a CaCl 2 solution, and fully reacting to obtain SA-P immobilized particles;
The microbial agent is selected from Bacillus cereus (WR-3) and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 12 months and 18 days in 2023, and the preservation number is: CGMCC No.29355.
2. The method for preparing an immobilized microorganism carrier according to claim 1, wherein in the step S1, the alkali solution is 1-10% NaOH solution.
3. The method for preparing an immobilized microorganism carrier according to claim 1, wherein in the step S2, the concentration of the sodium alginate solution is 1-2wt% and the concentration of the CaCl 2 solution is 2wt%.
4. The use of the immobilized microorganism carrier prepared by the preparation method of claim 1, which is used for degrading furazolidone, chemical oxygen demand, phosphorus and nitrogen in the tail water of cultivation.
5. The use according to claim 4, wherein the temperature of the cultivation tail water is 25-35 ℃ and the pH value is 6.5-8.0.
6. The use according to claim 4, wherein the concentration of furazolidone in the cultivation tail water is 100-300 mg/L.
Priority Applications (1)
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