CN114686469B - Immobilized bacteria algae microsphere and preparation method thereof - Google Patents
Immobilized bacteria algae microsphere and preparation method thereof Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
- C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
- C02F3/325—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae as symbiotic combination of algae and bacteria
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The disclosure relates to an immobilized bacteria algae microsphere, which contains bacillus subtilis, microalgae, a carrier and a cross-linking agent; the preservation number of the bacillus subtilis is CGMCC No.12317; the carrier is sodium alginate and gelatin, and the cross-linking agent is calcium chloride solution. The algae microsphere can rapidly and efficiently remove the sulfonamide antibiotics in the environment.
Description
Technical Field
The present disclosure relates to the technical field of microbial degradation of antibiotic residues, and in particular, to an immobilized bacteria algae microsphere and a preparation method thereof.
Background
The sulfanilamide antibiotics are antibiotics containing the structure of the sulfanilamide, and have the characteristics of stable chemical property, simple and convenient use, wide antibacterial spectrum and the like, so that the sulfanilamide antibiotics are not only used as human medicines, but also widely applied to the fields of animal husbandry, veterinary clinic and the like. After the medicine is used, residues in foods such as animal meat, milk, eggs and the like are serious and even threaten human health, can cause diseases such as aplastic anemia, granular leucopenia and the like, and low-concentration medicine residues can also induce the drug resistance of pathogenic bacteria.
At present, the use of microbial degradation to remove antibiotics in the environment is becoming a major research hotspot for biological removal methods, with increasing attention of environmental workers. The separation and identification of high-efficiency sulfa antibiotic degrading bacteria are carried out by scientific workers in many countries, and bacillus subtilis, achromobacter xylosoxidans, penicillium oxalicum, penicillium citrinum, aspergillus oryzae and the like are proved to have the function of degrading sulfa antibiotic. However, the biggest disadvantage of the current biological treatment method for degrading the sulfonamide antibiotics is that drug-resistant bacteria and super bacteria are generated, if the bacteria flow into the environment without treatment, the sensitivity of human beings to the antibiotics is reduced or even lost, and the removal of the degradation bacteria becomes a difficult problem.
Therefore, development of a rapid, efficient and environment-friendly sulfanilamide antibiotic degradation method is needed to obtain the maximum sulfanilamide antibiotic degradation effect, and has important scientific significance and application prospect in the research of antibiotic biodegradation theory and method.
Disclosure of Invention
The present disclosure provides an immobilized bacterial algae microsphere and a method for preparing the same, which can rapidly and efficiently remove sulfonamide antibiotics in the environment.
In order to achieve the above object, a first aspect of the present disclosure provides an immobilized bacteria algae microsphere, wherein the microsphere contains bacillus subtilis, microalgae, a carrier and a crosslinking agent; the preservation number of the bacillus subtilis is CGMCC No.12317; the carrier is sodium alginate and gelatin, and the cross-linking agent is calcium chloride solution.
A second aspect of the present disclosure provides a method for preparing the immobilized bacteria-algae microsphere according to the first aspect, wherein the method comprises the following steps:
s1, inoculating bacillus subtilis into a guaiacol solid culture medium, performing plate culture, transferring into an LB solid culture medium, and performing constant-temperature shake culture to a logarithmic phase to obtain bacterial liquid, wherein the preservation number of the bacillus subtilis is CGMCC No.12317;
s2, inoculating microalgae into a TAP culture medium, and culturing to a logarithmic phase to obtain a microalgae culture solution;
s3, mixing sodium alginate, gelatin and water to obtain a carrier solution;
s4, uniformly mixing the bacterial liquid and the microalgae culture liquid to obtain a mixed bacterial algae solution; adding the mixed bacteria algae solution into the carrier solution, uniformly mixing, titrating into a calcium chloride solution for crosslinking, and then cleaning to obtain the immobilized bacteria algae microsphere.
A third aspect of the present disclosure provides use of the immobilized bacterial algae microsphere of the first aspect for removal of sulfonamide antibiotics in an environment.
The fourth aspect of the disclosure provides an application of bacillus subtilis with a preservation number of CGMCC No.12317 in removing sulfonamide antibiotics in the environment.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 shows the mechanism of the algae system against sulfonamide degrading bacteria.
Detailed Description
Specific embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
In one aspect, the present disclosure provides an immobilized bacterial algae microsphere comprising bacillus subtilis, microalgae, a carrier, and a cross-linking agent; the preservation number of the bacillus subtilis is CGMCC No.12317; the carrier is sodium alginate and gelatin, and the cross-linking agent is calcium chloride solution.
According to the present disclosureThe content of the bacillus subtilis is 10 based on the total mass of the microsphere 8 -10 9 CFU/g, wherein the content of microalgae is 1.2-1.5 wt%, the content of sodium alginate is 3-4 wt%, the content of gelatin is 1-1.2 wt%, and the content of calcium chloride is 0.9-2 wt%.
According to the disclosure, wherein the biomass ratio of the microalgae to the bacillus subtilis is 1:1.5-2.5.
The relative biomass of the microalgae and the bacillus subtilis is expressed as an OD value, which is calculated according to a formula (10 4 /mL）=1557OD 680 34.176 the OD of the Bacillus subtilis was calculated according to the formula (10 9 /mL)=34.041OD 600 -0.1227。
According to the present disclosure, wherein the microalgae is Chlorella (GY-H4 Chlorella sp.), oenothera biennis (GY-D33,Selenastrum capricornutum) And scenedesmus obliquus (GY-D13,Scendesmus obliquus) At least one of them.
In another aspect, the present disclosure provides a method for preparing the immobilized bacterial algae microsphere of the first aspect, wherein the method comprises the steps of:
s1, inoculating bacillus subtilis into a guaiacol solid culture medium, performing plate culture, transferring into an LB solid culture medium, and performing constant-temperature shake culture to a logarithmic phase to obtain bacterial liquid, wherein the preservation number of the bacillus subtilis is CGMCC No.12317;
s2, inoculating microalgae into a TAP culture medium, and culturing to a logarithmic phase to obtain a microalgae culture solution;
s3, mixing sodium alginate, gelatin and water to obtain a carrier solution;
s4, uniformly mixing the bacterial liquid and the microalgae culture liquid to obtain a mixed bacterial algae solution; adding the mixed bacteria algae solution into the carrier solution, uniformly mixing, titrating into a calcium chloride solution for crosslinking, and then cleaning to obtain the immobilized bacteria algae microsphere.
According to the present disclosure, the concentration of the bacterial liquid is 1.0X10 8 -1.0×10 9 CFU/mL; the saidThe concentration of the microalgae culture solution is 6.0X10 7 -8.0×10 7 CFU/mL; the concentration of sodium alginate in the carrier solution is 0.03-0.05g/mL, and the concentration of gelatin is 0.01-0.012g/mL; the concentration of the calcium chloride solution is 0.009-0.02g/mL.
According to the present disclosure, the guaiacol culture medium comprises the following components: beef extract, peptone, naCl and CuSO 4 Guaiacol and agar; the LB solid culture medium comprises the following components: tryptone, yeast powder, naCl and agar; the TAP culture medium comprises H 2 NC(CH 2 OH) 3 、NH 4 Cl、MgSO 4 、CaCl 2 、K 2 HPO 4 、KH 2 PO 4 、Na 2 EDTA、ZnSO 4 、H 3 BO 3 、MnCl 2 、FeSO 4 、CoCl 2 、CuSO 4 、(NH 4 ) 6 Mo 7 O 24 And CH (CH) 3 COOH。
According to the disclosure, the concentration of beef extract in the guaiacol solid culture medium is 4-6g/L, the concentration of peptone is 9-11 g/L, the concentration of NaCl is 4-6g/L, and the concentration of CuSO 4 The concentration of guaiacol is 0.030-0.034 g/L, the concentration of guaiacol is 0.3-0.5 g/L, and the concentration of agar is 19-21g/L; the concentration of tryptone in the LB solid culture medium is 9-11 g/L, the concentration of yeast powder is 4-6g/L, the concentration of NaCl is 9-11 g/L, and the concentration of agar is 19-21g/L; the TAP culture medium has a concentration of H 2 NC(CH 2 OH) 3 Is 2.0X10 g -2 mol/L，NH 4 Cl concentration was 7.0X10 -3 mol/L，MgSO 4 Is 8.30X10 g -4 mol/L，CaCl 2 Is 4.50X10 g -4 mol/L，K 2 HPO 4 Is 1.65X10 g -3 mol/L，KH 2 PO 4 Is 1.05X10 g -3 mol/L，Na 2 EDTA concentration of 1.34×10 -4 mol/L，ZnSO 4 Is 1.36×10 in concentration -4 mol/L，H 3 BO 3 Is 1.84×10 in concentration -4 mol/L，MnCl 2 Is 4.00×10 in concentration -5 mol/L，FeSO 4 Is 3.29×10 in concentration -5 mol/L，CoCl 2 Is 1.23×10 in concentration -5 mol/L，CuSO 4 Is 1.00×10 in concentration -5 mol/L，(NH 4 ) 6 Mo 7 O 24 Is 9.28X10 g -7 mol/L，CH 3 COOH concentration was 1.67X 10 -2 mol/L。
In yet another aspect, the present disclosure provides the use of the immobilized bacterial algae microsphere of the first aspect for removal of sulfonamide antibiotics in an environment.
In yet another aspect, the present disclosure provides the use of bacillus subtilis with a accession number of CGMCC No.12317 for the removal of sulfonamide antibiotics in an environment.
The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.
Example 1
1. Screening of sulfonamide antibiotic degradation strain and enzyme activity research thereof
1.1 screening of strains
1.1.1 Medium composition formulation
Enrichment of medium components: 3g/L of beef extract, 10g/L of peptone, 5g/L of NaCl and hydrochloric acid with different concentrations; LB solid medium composition: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl and 20g/L of agar; 0.04% guaiacol solid medium composition: beef extract 5g/L, peptone 10g/L, naCl 5g/L, cuSO 4 0.032g/L, 0.4g/L of guaiacol and 20g/L of agar; inorganic medium composition: naCl 1.0g/L, K 2 HPO 4 0.5g/L，KH 2 PO 4 0.5g/L，MgSO 4 0.2g/L。
1.1.2 screening of sulfonamide antibiotic-degrading Strain
1mL of bacillus licheniformis of CGMCC No.12316, 1mL of bacillus subtilis of CGMCC No.12317 and 1mL of bacillus subtilis of CGMCC No.12318 are respectively added into a 250mL triangular flask, 100mL of liquid enrichment medium with 200mg/L of sulfonamide antibiotics is added, and the culture is carried out in a shaking table at 30 ℃ and 150r/min for 3 days to obtain a bacterial solution I; taking 0.1mL of bacterial liquid I, coating the bacterial liquid I on an LB plate for culturing for 24 hours, observing colony growth conditions, and determining that bacteria exist in a liquid enrichment culture medium with 200mg/L of sulfonamide antibiotics; observing colony growth on a flat plate, adding 5mL of bacterial liquid I into 100mL of liquid enrichment culture medium with the sulfonamide antibiotics content of 400mg/L, and culturing in the dark at 30 ℃ in a shaking table at 150r/min for 3d to obtain bacterial liquid II; diluting the bacterial liquid II to a proper concentration, plating on a plate for culturing for 24 hours, observing colony growth, and then adding 5mL of the bacterial liquid II into 100mL of a liquid enrichment culture medium with the sulfonamide antibiotics content of 600mg/L, and culturing in a shaking table at the temperature of 30 ℃ for 3 days in the dark at the speed of 150r/min to obtain bacterial liquid III; according to the method, the concentration of the sulfonamide antibiotics is gradually increased to be respectively 200mg/L, 400mg/L, 600mg/L, 800mg/L, 1000mg/L, 1200mg/L, 1400mg/L, 1600mg/L, 1800mg/L and 2000mg/L so as to obtain high-concentration-resistant sulfonamide antibiotic strains; diluting 2000mg/L enrichment medium to a proper concentration, coating 0.1mL of liquid on LB solid medium, picking single colony, and streaking and purifying, which proves that bacillus licheniformis with CGMCC No.12316 can survive under high-concentration sulfonamide antibiotics, while bacillus licheniformis with CGMCC No.12316 and bacillus subtilis with CGMCC No.12318 can not survive under high-concentration sulfonamide antibiotics.
1.2 Screening and detection of laccase-producing strains
Preparing a solid culture medium containing 0.04% of guaiacol; inoculating Bacillus licheniformis of CGMCC No.12316, bacillus subtilis of CGMCC No.12317 and Bacillus subtilis of CGMCC No.12318 in 0.04% guaiacol solid culture medium, culturing at 30deg.C for 3d, observing whether the colony has red color development ring, wherein the strain with color development has Bacillus licheniformis of CGMCC No.12316 and Bacillus subtilis of CGMCC No.12317, and the Bacillus subtilis of CGMCC No.12318 has no color development.
Scraping bacillus licheniformis of bacterial CGMCC No.12316, bacillus subtilis of CGMCC No.12317 and bacillus subtilis of CGMCC No.12318 in a solid flat plate for 4d simultaneously into sterile water, adding 0.6mol/L ABTS (2-acrylamido-2-methylpropanesulfonic acid), 0.1mol/L citric acid solution and 0.2mol/L sodium dihydrogen phosphate solution into the sterile water, and carrying out water bath at 36 ℃ for 5min to observe whether color change exists. The guaiacol plate is cultivated, and the developed strains include bacillus licheniformis with CGMCC No.12316 and bacillus subtilis with CGMCC No.12317; and (3) detecting laccase produced by bacteria, wherein the bacillus subtilis of CGMCC No.12317 is a bacterial strain which has the function of degrading sulfonamide antibiotics and can produce laccase at the same time.
2. Correspondence between optical density and algae related index
2.1 Establishing a growth curve of microalgae
And (3) establishing a growth curve of the chlorella, the capricorn moon algae and the scenedesmus obliquus. Three algae growth cycles were determined including adaptation phase, logarithmic growth phase, stabilization phase, decay phase. The algae in logarithmic growth phase has the fastest growth rate, vigorous metabolism, active enzyme system, approximate cell number of living algae and general algae, basically consistent chemical composition, morphological and physical properties, and algae after 20 hr in logarithmic growth phase is selected for preparing algae microsphere.
2.2 Establishing the relation between the optical density and the algae cell concentration
The algae cells cultured to the stable period are diluted to different multiples, and the algae density is counted under a microscope through a blood cell counting plate, and the corresponding algae cell optical density value is measured by adopting an ultraviolet spectrophotometry, so that the linear relation between the algae density and the optical density is established. The wavelength of the optical density value is subjected to spectrum scanning by an ultraviolet spectrophotometer, and the peak position is selected to be the optimal wavelength at the highest. In the visible light range, the absorbance of the three algae has obvious absorption peaks at 675nm, is relatively stable, and finally 675nm is selected as the optimal absorption wavelength for measuring the biomass of the 3 algae. As the algal cells increased, the optical density values became correspondingly larger, and the optical density values of 3 algal cells showed a remarkably positive linear correlation with their biomass (R 2 >0.99 Description that the corresponding algal biomass can be expressed by an optical density value, i.e., (10) 4 /mL）=1557OD 680 -34.176
2.3 Establishment of growth curve of bacillus subtilis of CGMCC No.12317
Inoculating small amount of Bacillus subtilis CGMCC No.12317 into fresh LB liquid culture medium with certain volume, and culturing under proper conditionTime measurement of the amount of bacteria in the culture medium, the optical density value became larger as the concentration of the strain increased, indicating that the corresponding Bacillus subtilis biomass could be expressed by the optical density value, i.e. (10) 9 /mL)=34.041OD 600 -0.1227。
3. Construction of immobilization System
Weighing 4g of sodium alginate and 1g of gelatin, placing in a beaker with 100mL of distilled water, sealing by a sealing film, and sterilizing at 121 ℃ for 30min; crosslinking solution (1% calcium chloride): weighing 5g of calcium chloride, and placing in a beaker with 500mL of distilled water and large capacity at 121 ℃ for 30min; the sodium alginate gelatin solution was placed in a 36 ℃ water bath. Wherein chlorella is added according to the formula calculation (ten thousand/mL) =1557OD 680-34.176; the bacterial liquid is prepared by scraping bacillus subtilis strain into sterile distilled water after plate culture, shaking to uniformity, measuring OD value, and obtaining a solution according to a formula (10 9 /m L) = 34.041OD600-0.1227 additions; in the order of magnitude of 1:2 the ratio of chlorella to bacterial liquid. Stirring uniformly, titrating into the cooled calcium chloride solution, and slightly shaking during the process to avoid the adhesion of the pellets; after the titration was completed, the pellets were fixed for 24 hours and washed with sterile water in an ultra clean bench.
4. Method for degrading sulfonamide antibiotics by using immobilization system
2.5mL of sulfonamide antibiotic solution (1 mg/mL) and immobilized bacteria algae microsphere are added into an inorganic culture medium (47.5 mL); culturing the culture medium at 30deg.C and 180r/min under illumination, sampling 0d,2d,4d,6d, and 8d, and measuring the degradation amount of sulfonamide antibiotics.
Example 2
1. The moon algae (GY-D33,Selenastrum capricornutum) Construction of immobilization System
Weighing 4g of sodium alginate and 1g of gelatin, placing in a beaker with 100mL of distilled water, sealing by a sealing film, and sterilizing at 121 ℃ for 30min; crosslinking solution (1% calcium chloride): weighing 5g of calcium chloride, and placing in a beaker with 500mL of distilled water and large capacity at 121 ℃ for 30min; the sodium alginate gelatin solution was placed in a 36 ℃ water bath. Wherein, the moon algae is calculated according to the formula (ten thousand/mL) =1557OD 680 -34.176 addition; the bacterial liquid is bacillus licheniformis strain which is scraped to be sterile after flat-plate cultureDistilled water was shaken to uniformity, and after the OD value was measured, the mixture was subjected to a test according to the formula (10 9 /mL)=34.041OD 600 -0.1227 addition; in the order of magnitude of 1:2, the ratio of the horn moon algae to the bacterial liquid. Stirring uniformly, titrating into the cooled calcium chloride solution, and slightly shaking during the process to avoid the adhesion of the pellets; after the titration was completed, the pellets were fixed for 24 hours and washed with sterile water in an ultra clean bench.
2. Degradation of sulfonamide antibiotics using an immobilization system
2.5mL of sulfonamide antibiotic solution (1 mg/mL) and immobilized bacteria algae microsphere are added into an inorganic culture medium (47.5 mL); the medium was incubated at 30℃for 180r/min under light conditions, sampled at 0d,2d,4d,6d and 8d, and the sulfonamide antibiotic degradation rate was measured, and the results are shown in Table 1.
Example 3
1. Scenedesmus obliquus (GY-D13,Scendesmus obliquus) Construction of immobilization System
Weighing 4g of sodium alginate and 1g of gelatin, placing in a beaker with 100mL of distilled water, sealing by a sealing film, and sterilizing at 121 ℃ for 30min; crosslinking solution (1% calcium chloride): weighing 5g of calcium chloride, and placing in a beaker with 500mL of distilled water and large capacity at 121 ℃ for 30min; the sodium alginate gelatin solution was placed in a 36 ℃ water bath. Wherein, scenedesmus obliquus calculates (ten thousand/mL) =1557OD according to the formula 680 -34.176 addition; the bacterial liquid is prepared by scraping bacillus licheniformis strain into sterile distilled water after plate culture, shaking to uniformity, measuring OD value, and obtaining the bacterial liquid according to the formula (10 9 /mL)=34.041OD 600 -0.1227 addition; in the order of magnitude of 1:2, the ratio of the scenedesmus obliquus to the bacterial liquid. Stirring uniformly, titrating into the cooled calcium chloride solution, and slightly shaking during the process to avoid the adhesion of the pellets; after the titration was completed, the pellets were fixed for 24 hours and washed with sterile water in an ultra clean bench.
2. Degradation of sulfonamide antibiotics using an immobilization system
2.5mL of sulfonamide antibiotic solution (1 mg/mL) and immobilized bacteria algae microsphere are added into an inorganic culture medium (47.5 mL); the medium was incubated at 30℃for 180r/min under light conditions, sampled at 0d,2d,4d,6d and 8d, and the sulfonamide antibiotic degradation rate was measured, and the results are shown in Table 1.
Comparative example 1
The algae microsphere was prepared according to the method of example 1, the only difference being that the strain used in the experiment was bacillus subtilis, the preservation number of which was CGMCC No.12318. 2.5mL of sulfonamide antibiotic solution (1 mg/mL) and immobilized bacteria algae microsphere are added into an inorganic culture medium (47.5 mL); the culture medium was incubated at 30℃for 180r/min under light conditions, samples were taken at 0d,2d,4d,6d and 8d, and after 10-fold dilution of each sample, the degradation amount of the sulfonamide antibiotic was measured.
The result shows that the immobilized composite system prepared in the disclosure can rapidly reduce the concentration of the sulfonamide antibiotics in the polluted water body, and can reduce the concentration of the sulfonamide antibiotics by more than eighty percent in 8 days, and the composite system can achieve the purpose of rapidly removing the pollution of the sulfonamide antibiotics in the water body.
The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. An immobilized bacteria algae microsphere is characterized in that the microsphere contains bacillus subtilisBacillus(s) (-)Bacillus subtilis) Microalgae, a carrier and a crosslinking agent; the preservation number of the bacillus subtilis is CGMCC No.12317; the carrier is sodium alginate and gelatin, and the cross-linking agent is calcium chloride solution.
2. The algae microsphere according to claim 1, wherein the bacillus subtilis content is 10 based on the total weight of the microsphere 8 -10 9 CFU/g, wherein the content of microalgae is 1.2-1.5 wt%, the content of sodium alginate is 3-4 wt%, the content of gelatin is 1-1.2 wt%, and the content of calcium chloride is 0.9-2 wt%.
3. The algae microsphere of claim 1, wherein the biomass ratio of the microalgae to the bacillus subtilis is 1:1.5-2.5.
4. The bacterial algae microsphere according to claim 1, wherein the microalgae is chlorella vulgarisChlorella sp.) Radix seu herba Heterophyllae SphaerocallisSelenastrum capricornutum) And Scenedesmus obliquus (L.) KummerScendesmus obliquus) At least one of them.
5. The method for preparing immobilized bacteria algae microsphere according to claim 1, wherein the method for preparing comprises the following steps:
s1, inoculating bacillus subtilis into a guaiacol solid culture medium, performing plate culture, transferring into an LB solid culture medium, and performing constant-temperature shake culture to a logarithmic phase to obtain bacterial liquid, wherein the preservation number of the bacillus subtilis is CGMCC No.12317;
s2, inoculating microalgae into a TAP culture medium, and culturing to a logarithmic phase to obtain a microalgae culture solution;
s3, mixing sodium alginate, gelatin and water to obtain a carrier solution;
s4, uniformly mixing the bacterial liquid and the microalgae culture liquid to obtain a mixed bacterial algae solution; adding the mixed bacteria algae solution into the carrier solution, uniformly mixing, titrating into a calcium chloride solution for crosslinking, and then cleaning to obtain the immobilized bacteria algae microsphere.
6. The method according to claim 5, wherein the concentration of the bacterial liquid is 1.0X10 8 -1.0×10 9 CFU/mL; the concentration of the microalgae culture solution is 6.0X10 7 -8.0×10 7 CFU/mL; the concentration of sodium alginate in the carrier solution is 0.03-0.05g/mL, and the concentration of gelatin is 0.01-0.012g/mL; the concentration of the calcium chloride solution is 0.009-0.02g/mL.
7. The preparation method according to claim 5, wherein the guaiacol solid medium comprises the following components: beef extract, peptone, naCl and CuSO 4 Guaiacol and agar; the LB solid culture medium comprises the following components: tryptone, yeast powder, naCl and agar; the TAP culture medium comprises the following components: h 2 NC(CH 2 OH) 3 、NH 4 Cl、MgSO 4 、CaCl 2 、K 2 HPO 4 、KH 2 PO 4 、Na 2 EDTA、ZnSO 4 、H 3 BO 3 、MnCl 2 、FeSO 4 、CoCl 2 、CuSO 4 、(NH 4 ) 6 Mo 7 O 24 And CH (CH) 3 COOH。
8. The preparation method of claim 7, wherein the concentration of beef extract in the guaiacol solid medium is 4-6g/L, the concentration of peptone is 9-11 g/L, the concentration of NaCl is 4-6g/L, and the concentration of CuSO 4 The concentration of the guaiacol is 0.030-0.034 g/L, the concentration of the guaiacol is 0.3-0.5 g/L, and the concentration of the agar is 19-21g/L; the concentration of tryptone in the LB solid medium is 9-11 g/L, the concentration of yeast powder is 4-6g/L, the concentration of NaCl is 9-11 g/L, and the concentration of agar is 19-21g/L; the TAP culture medium has a concentration of H 2 NC(CH 2 OH) 3 Is 2.0X10 g -2 mol/L，NH 4 Cl concentration was 7.0X10 -3 mol/L，MgSO 4 Is 8.30X10 g -4 mol/L，CaCl 2 Is 4.50X10 g -4 mol/L，K 2 HPO 4 Is 1.65X10 g -3 mol/L，KH 2 PO 4 Is 1.05X10 g -3 mol/L，Na 2 EDTA concentration of 1.34×10 -4 mol/L，ZnSO 4 Is 1.36×10 in concentration -4 mol/L，H 3 BO 3 Is 1.84×10 in concentration -4 mol/L，MnCl 2 Is 4.00×10 in concentration -5 mol/L，FeSO 4 Is 3.29×10 in concentration -5 mol/L，CoCl 2 Is 1.23×10 in concentration -5 mol/L，CuSO 4 Is 1.00×10 in concentration -5 mol/L，(NH 4 ) 6 Mo 7 O 24 Is 9.28X10 g -7 mol/L，CH 3 COOH concentration was 1.67X 10 -2 mol/L。
9. Use of the immobilized bacterial algae microsphere according to any one of claims 1-4 for removing sulfamethoxazole in an environment.
10. The application of bacillus subtilis with the preservation number of CGMCC No.12317 in removing sulfamethoxazole in the environment.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3922343A (en) * | 1972-09-07 | 1975-11-25 | Kyowa Hakko Kogyo Kk | Antibiotic XK-49-1-B-2 and process for production thereof using streptosporangium vlolaceochromogenes |
| CN105950507A (en) * | 2016-05-24 | 2016-09-21 | 中国农业科学院农业环境与可持续发展研究所 | Bacillus subtilis and bactericide as well as application of bactericide in treatment of livestock and poultry breeding waste water and treatment method |
| CN106434430A (en) * | 2016-09-07 | 2017-02-22 | 中山市润泽生物科技有限公司 | Compound microbial agent for degrading antibiotic and pesticide residues as well as preparation and application thereof |
| CN107140746A (en) * | 2017-05-28 | 2017-09-08 | 天津大学 | Method of the reduction containing bio-toxicity in artificial synthesized antimicrobial water body |
| CN110616170A (en) * | 2019-09-20 | 2019-12-27 | 广东省农业科学院农业资源与环境研究所 | Sulfonamide antibiotic polluted soil in-situ microorganism digestion preparation, preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014011564A2 (en) * | 2012-07-09 | 2014-01-16 | Ira Milton Trachtman | Compositions and method for treatment and prophylaxis of inflammatory bowel disease |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3922343A (en) * | 1972-09-07 | 1975-11-25 | Kyowa Hakko Kogyo Kk | Antibiotic XK-49-1-B-2 and process for production thereof using streptosporangium vlolaceochromogenes |
| CN105950507A (en) * | 2016-05-24 | 2016-09-21 | 中国农业科学院农业环境与可持续发展研究所 | Bacillus subtilis and bactericide as well as application of bactericide in treatment of livestock and poultry breeding waste water and treatment method |
| CN106434430A (en) * | 2016-09-07 | 2017-02-22 | 中山市润泽生物科技有限公司 | Compound microbial agent for degrading antibiotic and pesticide residues as well as preparation and application thereof |
| CN107140746A (en) * | 2017-05-28 | 2017-09-08 | 天津大学 | Method of the reduction containing bio-toxicity in artificial synthesized antimicrobial water body |
| CN110616170A (en) * | 2019-09-20 | 2019-12-27 | 广东省农业科学院农业资源与环境研究所 | Sulfonamide antibiotic polluted soil in-situ microorganism digestion preparation, preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| Cloning of the Bacillus subtilis Sulfanilamide Resistance Gene in Bacillus subtilis;KAREN ORZECH McDONALD et al.;JOURNAL OF BACTERIOLOGY,;第149卷(第1期);第391-394页 * |
| 饲料添加剂对感染球虫肉鸡早期肠道健康的影响;刘雪 等;中国农业大学学报;第23卷(第11期);第80-87页 * |
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