CN114958817A - Binuclear shell loaded microbial material and preparation method and application thereof - Google Patents
Binuclear shell loaded microbial material and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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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
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
Abstract
The invention provides a binuclear shell load microbial material, a preparation method and application thereof, which are used for mineralizing heavy metals in polluted water; the preparation method of the binuclear shell supported microbial material comprises the following steps: firstly, embedding a microbial liquid in an agar solution to obtain an agar-supported microbial inner shell; then, placing the agar-loaded microorganism inner shell in calcium chloride solution to enable the surface of the agar-loaded microorganism inner shell to cover a calcium chloride layer; and finally, embedding the agar-supported microorganism inner shell covered with the calcium chloride layer in a sodium alginate solution mixed with urea to obtain the double-nucleocapsid-supported microorganism material. The invention adopts the binuclear shell load microbial material to repair heavy metal pollution, and can effectively solve the problem that urease-producing microorganisms cannot tolerate high-concentration heavy metals; the urea and the microorganisms can be embedded separately, so that the problems of additional urea source and advanced reaction are solved. Has wide industrialization prospect and market value.
Description
Technical Field
The invention relates to the technical field of heavy metal polluted water body remediation, in particular to a binuclear shell load microbial material and a preparation method and application thereof.
Background
Heavy metals are stable and not easily eliminated, and often enter the human body through the food chain, causing damage to the brain, kidney, bones, lungs, liver, reproductive system, and the like of the human. The traditional cadmium pollution treatment method mainly comprises physical, chemical and biological methods. The physical and chemical treatment method has the characteristics of high treatment efficiency and simple process. However, the equipment cost and the operation cost are expensive, and secondary pollution is easily caused. Thus, more environmentally friendly and inexpensive bioremediation methods are receiving increasing attention.
The biological adsorption method has many advantages, the raw material source is rich, the variety is many, and the cost is low; and the adsorption speed is high, the adsorption capacity is large, the selectivity is good, and the method is particularly effective in treating low-concentration wastewater. Chinese invention patent (CN109364887A) discloses a porous calcium alginate adsorbent, the fiber molecular chain of which contains a large amount of hydroxyl and carboxyl, has good adsorption capacity to cadmium ions, and can be used for treating heavy metal sewage. However, alginate gel beads are dissolved under the influence of pH increase, so that the heavy metal treatment effect cannot be exerted for a long time.
Biomineralization is a research hotspot for removing heavy metals, and the most common and effective biomineralization process is caused by urease hydrolysis of urea. Urea is decomposed by urease to form ammonium salt which is alkaline, and the combination and existence of calcium and carbonate are facilitated. The principle of biologically inducing carbonate precipitation to repair heavy metals comprises the following steps: firstly, urease is produced by urease-producing bacteria in the metabolic process; secondly, urease hydrolyzes urea to generate ammonium ions and carbonate ions; finally, the generated carbonate ions react with the heavy metal ions to form carbonate precipitates, so that the heavy metals can be quickly mineralized, and the harm of the heavy metals is reduced. In addition, calcium alginate and agar are excellent carriers, and can adsorb cadmium ions and protect urease-producing microorganisms from cadmium toxicity when being used for coating urease bacteria.
CN113373139A an immobilized microorganism material for treating wastewater containing heavy metal ions and a preparation method thereof, the immobilized microorganism material comprises an embedding framework and urease-producing microorganisms, urea and a buffer agent embedded in the embedding framework, the embedding framework is formed by cross-linking sodium alginate and calcium salt, and the patent solves the problems that urea in the heavy metal ion wastewater is easy to hydrolyze and the MICP process is difficult to smoothly carry out without urea in the prior art. However, the urea and the urease bacteria are simultaneously wrapped in the embedding framework, so that the urea can be decomposed into ammonium ions and calcium carbonate ions in advance, the PH in the wastewater is not favorably improved, and the heavy metals cannot be effectively removed.
Disclosure of Invention
In order to further solve the problem of heavy metal pollution in water, the application provides a binuclear shell load microbial material, a preparation method and application thereof, which are used for mineralizing heavy metal in polluted water; firstly, embedding the microorganism in agar to obtain an agar-loaded microorganism inner shell; and then covering a calcium chloride layer outside the agar-supported microorganism inner shell, and finally embedding the agar-supported microorganism inner shell in a sodium alginate solution mixed with urea to obtain the double-core shell-supported microorganism material, namely the double-core shell-supported microorganism material can tolerate heavy metal with higher concentration and improve the removal rate of the heavy metal.
A preparation method of a binuclear shell-loaded microbial material comprises the following steps:
firstly, embedding a microbial liquid in an agar solution to obtain an agar-supported microbial inner shell;
then, placing the agar-loaded microorganism inner shell in calcium chloride solution to enable the surface of the agar-loaded microorganism inner shell to cover a calcium chloride layer;
finally, embedding the agar-loaded microorganism inner shell covered with the calcium chloride layer in a sodium alginate solution of mixed urea to obtain a double-nucleocapsid loaded microorganism material;
the microorganism is urease bacteria, and the volume dosage ratio of the microorganism liquid to the agar solution is 1 (5-10).
The urease is Bacillus pasteurianus
The concentration of the agar solution is 20-50 g/L; the concentration of calcium chloride was 100-1000 mM.
The pasteuria bacillus liquid is obtained by the following method: inoculating Paenibacillus pasteurii in culture medium, performing shake culture at 28 + -0.5 deg.C for 2 days, and regulating bacterial liquid concentration to OD by centrifugal concentration or adding physiological saline 600 ＝1。
The culture medium comprises 20g of yeast powder and 15g of NH 4 Cl，59mg NiCl 2 Adding water to complement to 1L; the pH of the culture medium is 9.2-9.3.
The agar-supported microorganism inner shell is prepared by the following method: preparing agar solution with concentration of 20-50g/L, shaking in a conical flask, placing in a microwave oven, and heating with strong fire for 2 min until agar is dissolved; when the temperature of the dissolved agar solution is reduced to 50 +/-0.5 ℃ (because the temperature is the freezing point of the agar at 40 ℃, the agar has fluidity at 50 ℃ so as to uniformly mix bacteria), adding the microbial solution (OD600 ═ 1-10) into the agar, shaking uniformly, and standing and cooling in a refrigerator at 4 ℃ until the agar is solidified; the agar was divided into agar blocks of uniform size using a 6x6mm mesh cutter to obtain an inner shell of agar-supported microorganisms.
The concentration of the sodium alginate solution in the mixed urea sodium alginate solution is 5-20g/L, and the concentration of urea is 100 mM.
The preparation method of the binuclear shell supported microbial material specifically comprises the following steps:
placing the agar-loaded microorganism inner shell in 100-; in addition, preparing a mixed solution of sodium alginate and urea, wherein the concentration of a sodium alginate aqueous solution in the sodium alginate solution of the mixed urea is 5-20g/L, and the concentration of the urea is 100 mM; and finally, dripping the mixed solution of sodium alginate and urea onto the agar-supported microorganism inner shell covered with the calcium chloride layer by using a peristaltic pump, and uniformly covering the surface with a calcium alginate gel layer mixed with urea to obtain the double-core-shell supported microorganism material. The binuclear shell loaded microbial material is put into a cadmium-containing heavy metal polluted water solution to fix cadmium in wastewater.
The binuclear shell supported microbial material obtained by the preparation method.
The application of the binuclear shell supported microbial material is characterized in that the binuclear shell supported microbial material is used for treating wastewater containing heavy metal ions, can tolerate heavy metal with higher concentration and improves the removal rate of the heavy metal.
Compared with the prior art, the invention has the following advantages:
(1) the invention has simple operation and low cost. The binuclear shell load microbial material is adopted to repair heavy metal pollution, so that the problem that urease-producing microbes cannot tolerate high-concentration heavy metals can be effectively solved;
(2) the urea and the microorganisms can be embedded separately, so that the problems of additional urea source and advanced reaction are solved. In addition, the slow release of the urea can effectively improve the utilization efficiency of the urea, and has wide industrial prospect and market value.
Drawings
FIG. 1 is a schematic view of a binuclear shell-supported microbial material;
FIG. 2 is a comparison of microbial tolerance to heavy metal cadmium;
FIG. 3 is a graph of cadmium removal rate;
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The strains and reagents used in the following examples were all conventional reagents and were obtained from commercial sources.
The pasteuria bacteria used in the examples were commercially available pasteuria bacteria ATCC11859, a strain conventional in the art, such as the strain used in the patent CN109295108A examples.
The media formulations in the examples are as follows: per liter of culture medium contains: 20g of yeast powder, 15g of NH4Cl, 59mg of NiCl 2 Adding water to complement to 1L; the pH value is 9.2-9.3.
Inoculating the mother liquid of the pasteurella to the sterilized culture medium, and carrying out shake culture at the temperature of 28 ℃ for two days. Measuring the light absorption value of the bacterial liquid by using a spectrophotometer after the culture is finished, and regulating the concentration of the bacterial liquid to OD600 (OD 600) 10 by using centrifugal concentration and normal saline for later use; preparing cadmium-containing wastewater with the cadmium concentration of 2mM by using pure water and cadmium chloride for later use.
Preparation of the dual core-shell supported microbial materials used in the following examples:
preparing agar solution with the concentration of 25g/L, dissolving at high temperature and cooling to 50 ℃, and then mixing the bacillus pasteurianus bacterial liquid according to the volume ratio of 1: 9 adding agar, shaking, standing in a refrigerator at 4 deg.C, and cooling to solidify; the agar was divided into agar blocks of uniform size using a 6x6mm mesh cutter to obtain an inner shell of agar-supported microorganisms.
Placing the agar-loaded microorganism inner shell in 100mM calcium chloride solution, and filtering out after the surface is fully covered with a layer of calcium chloride; in addition, 100mM urea is added into the prepared 5g/L sodium alginate aqueous solution to obtain a mixed solution of the sodium alginate and the urea; and finally, dripping the mixed solution of sodium alginate and urea onto the agar-supported microorganism inner shell covered with a layer of calcium chloride by using a peristaltic pump, and uniformly covering a layer of calcium alginate gel mixed with urea on the surface to obtain the double-core-shell supported microorganism material.
Example 1: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacterial liquid of the binuclear shell load microbial material and the cadmium-containing wastewater are OD 600 1 and 1 mM. The reaction system was shaken at 28 deg.CReconditioning for 1 day under bed conditions.
Example 2: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacterial liquid of the binuclear shell load microbial material and the cadmium-containing wastewater are OD 600 1 and 2 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day.
Example 3: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacterial liquid of the binuclear shell load microbial material and the cadmium-containing wastewater are OD 600 1 and 5 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day.
Comparative example 1: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacillus pasteurianus bacterial liquid and the cadmium-containing wastewater are OD 600 1 and 1 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day.
Comparative example 2: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacillus pasteurianus bacterial liquid and the cadmium-containing wastewater are OD 600 1 and 2 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day.
Comparative example 3: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacillus pasteurianus bacterial liquid and the cadmium-containing wastewater are OD 600 1 and 5 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day.
Comparative example 4: preparing 10mL of reaction system; in a 10mL reaction system, the final concentrations of the bacterial liquid of the single-core shell loaded with the microbial material and the cadmium-containing wastewater are OD 600 1 and 1 mM. The reaction system was maintained in a shaker at 28 ℃ for 1 day. Wherein, the preparation of the bacterial liquid of the single-core shell load microbial material comprises the following steps: the Paenibacillus pasteurii and sodium alginate solution (5g/L) were mixed and added dropwise to a 100mM calcium chloride solution.
Test example 1: microbiological activity assay
The increased ammonium concentration in the heavy metal cadmium polluted water is measured by a nano reagent spectrophotometry, and the decomposition capacity of the microorganism on urea is obtained, so that the activity of the microorganism is judged.
Three groups of parallel samples are arranged in each of example 1, comparative example 1 and comparative example 4, and the detection results are averaged.
After 1 day of restoration, the detection result is shown in fig. 2, and the test results of example 1 and comparative example 1 show that for the removal of the microbial mineralization of the heavy metal cadmium polluted water, the decomposition of urea is improved by nearly 33 times under the condition of double-nucleocapsid loaded with the microbial material, namely, the activity of urease bacteria is improved by nearly 33 times. The test results of example 1 and comparative example 4 show that the dual nucleocapsid structure of the present invention has better microbial activity than the single nucleocapsid structure of the prior art.
Test example 2: test of ability to remove heavy metal ions
And testing the residual cadmium concentration in the heavy metal cadmium polluted water by an ICP-OES instrument, and then comparing the cadmium concentrations before and after the polluted water is treated to judge the capacity of removing heavy metal cadmium ions.
Three sets of parallel samples were set for each of the examples and comparative examples, and the results were averaged.
After 1 day of restoration, the detection result is shown in fig. 3, for the removal of the microorganisms mineralized in the water polluted by the heavy metal cadmium, under the condition that the binuclear shell carries the microbial material, the removal rate is improved by one third when 2mM cadmium is treated, and is improved from 60% to 80%; the removal rate of 5mM cadmium is improved by more than two times, and is improved from 19% to 63%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A preparation method of a binuclear shell supported microbial material is characterized by comprising the following steps:
firstly, embedding a microbial liquid in an agar solution to obtain an agar-supported microbial inner shell;
then, placing the agar-loaded microorganism inner shell in calcium chloride solution to enable the surface of the agar-loaded microorganism inner shell to cover a calcium chloride layer;
finally, embedding the agar-loaded microorganism inner shell covered with the calcium chloride layer in a sodium alginate solution of mixed urea to obtain a double-nucleocapsid loaded microorganism material;
the microorganism is urease bacteria, and the volume usage ratio of the microorganism liquid to the agar solution is 1 (5-10).
2. The method for preparing a binuclear shell supported microbial material according to claim 1, characterized in that: the urease bacteria are bacillus pasteurii.
3. The method for preparing a binuclear shell supported microbial material according to claim 1, characterized in that: the concentration of the agar solution is 20-50 g/L.
4. The method for preparing a binuclear shell supported microbial material according to claim 1, characterized in that: the calcium chloride concentration was 100-1000 mM.
5. The method for preparing a binuclear shell supported microbial material according to claim 2, characterized in that: the bacillus pasteurii bacterial liquid is obtained by the following method: inoculating Paenibacillus pasteurii in culture medium, performing shake culture at 28 + -0.5 deg.C for 2 days, and regulating bacterial liquid concentration to OD by centrifugal concentration or adding physiological saline 600 ＝1。
6. The method for preparing a binuclear shell supported microbial material according to claim 5, characterized in that: the culture medium comprises 20g of yeast powder and 15g of NH 4 Cl，59mg NiCl 2 Adding water to complement to 1L; the pH value of the culture medium is 9.2-9.3.
7. The method for preparing a binuclear shell supported microbial material according to claim 1, characterized in that: the agar-supported microorganism inner shell is prepared by the following method: heating the agar solution until the agar is dissolved; the temperature of the agar solution to be dissolved is reduced to 50 DEGAfter 0.5 ℃ plus or minus, the microbial inoculum (OD degree. C. +, C.) is added 600 1-10) adding the mixture into an agar solution, shaking up, and cooling to solidify; and (3) cutting the solidified agar into agar blocks with uniform sizes by using a 6x6mm mesh cutter to obtain the agar-loaded microorganism inner shell.
8. The method for preparing a binuclear shell supported microbial material according to claim 1, characterized in that: the concentration of the sodium alginate aqueous solution in the sodium alginate solution of the mixed urea is 5-20g/L, and the concentration of the urea is 100 mM.
9. The binuclear shell-supported microbial material obtained by the production method according to any one of claims 1 to 8.
10. The use of a binuclear shell supported microbial material as claimed in claim 9, wherein: the double-nucleocapsid loaded microorganism material is used for treating wastewater containing heavy metal ions.
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