CN117487792A - Heterotrophic nitrification-aerobic denitrification microorganism immobilization method - Google Patents
Heterotrophic nitrification-aerobic denitrification microorganism immobilization method Download PDFInfo
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- 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 23
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 23
- 239000000661 sodium alginate Substances 0.000 claims abstract description 23
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 23
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 21
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 21
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 9
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 8
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- 239000001110 calcium chloride Substances 0.000 claims abstract description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 33
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
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- 239000001963 growth medium Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000008367 deionised water Substances 0.000 claims description 13
- 229910021641 deionized water Inorganic materials 0.000 claims description 13
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- 238000010438 heat treatment Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
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- UKVHQGHRJIEIML-UHFFFAOYSA-L calcium boric acid dichloride Chemical class [Cl-].[Cl-].[Ca+2].OB(O)O UKVHQGHRJIEIML-UHFFFAOYSA-L 0.000 description 1
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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
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- 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
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Abstract
The invention relates to an immobilization method of heterotrophic nitrification aerobic denitrification microorganisms, belonging to the technical field of immobilization microorganisms; the technical method adopts polyvinyl alcohol and sodium alginate as immobilized carriers, adopts saturated boric acid and calcium chloride as cross-linking agents, immobilizes heterotrophic nitrification aerobic denitrifying bacteria into spheres, and forms the immobilized carriers after washing and air-drying the immobilized bacteria with normal saline; the heterotrophic nitrification aerobic denitrifying bacteria immobilized carrier can simultaneously perform nitrification and denitrification in the same reactor, overcomes the defects of the traditional process, reduces the capital construction and operation cost in the sewage treatment process, and has good application prospect.
Description
Technical Field
The invention relates to a heterotrophic nitrification-aerobic denitrification microorganism immobilization method, in particular to a method for immobilizing thalli by using polyvinyl alcohol (PVA), sodium Alginate (SA), biochar (BC) and the like as composite carriers, and belongs to the technical field of immobilized microorganisms.
Background
The control stability of the biological denitrification process is to be improved, and the current biological method is not ideal in solving the problems of slow bacterial growth and loss, which limits the effect of the biochemical process. In order to improve the biological process, it is necessary to increase the efficiency of the microorganisms and to control the reaction process while maintaining the activity and concentration of bacterial colonies. Immobilized microbial technologies have been developed for further research and development of microbial processes.
Common microorganism immobilization techniques can be divided into two major categories, physical and chemical. Physical methods mainly include adsorption and embedding methods, while chemical methods mainly employ crosslinking methods. The physical adsorption method uses substances with strong adsorption capacity (such as activated carbon, silica gel, etc.) to fix microorganisms on a carrier. The method has the advantages of simple operation, low cost and less loss of microbial activity. However, due to the limited surface area of the support, the immobilized amount of microorganisms is limited. In addition, the acting force between the microorganism and the carrier is smaller and is easy to be dispersed by water flow, thereby reducing the recycling rate and the treatment efficiency. Another embedding method is the most commonly used method for immobilizing microorganisms. The structure of the embedding coefficient can be classified into gel embedding and microcapsule methods. Gel entrapment is the encapsulation of cells within a tiny lattice of gel, or within an ultrafiltration membrane of a semipermeable membrane polymer. The method has low cost and simple operation, but has large mass transfer resistance, and limits the catalytic activity of cells, thereby affecting the action of microorganisms. Chemical crosslinking is typically accomplished by mixing the cells with various hydrogels and encapsulating the cells in a carrier using glutaraldehyde crosslinking agents. However, this approach has some drawbacks. First, organic reagents are required to be used in the preparation process, and the reagents are toxic and have certain harm to the environment and workers. Secondly, large-scale production and preparation cannot be realized. In addition, the embedding of the hydrogel can influence the contact of the thalli and the substrate, thereby influencing the activity utilization rate of the thalli.
Therefore, in order to further improve the biological denitrification process, optimization and innovation are required on the method for immobilizing microorganisms to improve the efficiency and stability of microorganism immobilization.
The invention aims to provide a heterotrophic nitrification-aerobic denitrification microorganism immobilization method, wherein one of the methods is a microorganism immobilization method taking polyvinyl alcohol (PVA) and Sodium Alginate (SA) as carriers. The preparation method has the advantages of low preparation condition requirement, simple equipment, low energy consumption, no toxic pollutant in the preparation process, safety to the environment and staff, and realization of large-scale preparation and production; the finally prepared immobilized microorganism has low cost and high quality stability.
It is still another object of the present invention to provide a method for sample scanning detection of immobilized heterotrophic nitrification-aerobic denitrification bacteria.
The technical scheme of the invention is as follows:
a heterotrophic nitrification-aerobic denitrification microorganism immobilization method, comprising the following steps:
1) Preparing an immobilization carrier: preparing the immobilized carrier raw material and ammonia-free deionized water into a solution by using an electromagnetic heating furnace;
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium, and carrying out centrifugal resuspension;
3) Preparing a carrier embedding complex: sterilizing the immobilized carrier material prepared in the step 1), cooling to room temperature, and mixing and stirring with the bacterial suspension obtained in the step 2);
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the immobilized microorganism carrier embedding complex obtained in the step 3), adding the mixture into a crosslinking agent to crosslink and activate the complex, standing and solidifying at room temperature, and finally obtaining the crosslinked-embedded microorganism carrier complex.
The heterotrophic nitrification-aerobic denitrification microorganism immobilization method comprises the following steps:
1) Preparing an immobilization carrier: sequentially adding Biochar (BC) and silicon dioxide into ammonia-free deionized water by taking polyvinyl alcohol (PVA) and Sodium Alginate (SA) as substrate raw materials, stirring and heating by using an electromagnetic heating furnace, and preparing a solution serving as an immobilization carrier PVA-SA-BC;
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium for 20-30 hours, and carrying out centrifugal resuspension;
3) Preparing a carrier embedding complex: selecting heterotrophic nitrification-aerobic denitrification bacterial suspension growing in logarithmic growth phase, and regulating bacterial suspension OD by using PBS solution 600 Mixing and fully stirring the immobilized carrier PVA-SA-BC after sterilization and cooling to room temperature and the heterotrophic nitrification-aerobic denitrification bacteria suspension according to the volume ratio of (10-30): 1, wherein the volume ratio is 0.2-2.0;
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the obtained carrier embedding complex of immobilized microorganism, and carrying out crosslinking activation on the carrier embedding complex by using saturated boric acid and calcium chloride to obtain the crosslinked-embedded microorganism carrier complex after crosslinking activation.
The heterotrophic nitrification-aerobic denitrification microorganism immobilization method comprises the steps that the immobilized heterotrophic nitrification-aerobic denitrification bacteria are heterotrophic nitrification-aerobic denitrification bacteria QD-21 #paracoccus spQD-21), which was preserved in China Center for Type Culture Collection (CCTCC) at 11 and 14 days of 2022, and which was assigned the eight 299 th channel of the armed division of the chinese city, the hubei province, and the strain was assigned the CCTCC NO: m20221801, heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21) its morphological and physiochemical characteristics include:
1) Gram-negative bacteria;
2) The bacterial form is microscopic detected as short rod-shaped bacteria;
3) On the culture medium, the colony forms a milky white smooth round shape with a raised middle part, and the colony edges are irregular.
The heterotrophic nitrification-aerobic denitrification microorganism immobilization method comprises the following steps of: washing the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 sample with deionized water for three times, respectively dehydrating with 30%, 50%, 70% and 90% ethanol, drying in a pre-cooled vacuum freeze dryer at-40 ℃ for 20-24 h to form freeze-drying, adhering the sample to a sample table by using a conductive adhesive tape, placing in a vacuum coating machine, spraying gold on the surface of the sample, taking out the sample, and observing the carrier embedding complex structure of immobilized microorganisms of the carrier PVA-SA-BC immobilized bacteria QD-21 by using a scanning electron microscope after the sample preparation.
The heterotrophic nitrification-aerobic denitrification microorganism immobilization method comprises the step of preparing the inorganic salt liquid culture medium C 4 H 4 Na 2 O 4 3.375 g/L、NH 4 Cl 0.382 g/L、K 2 HPO 4 0.5 g/L、KH 2 PO 4 1 g/L、MgSO 4 ·7H 2 O0.1 g/L, trace element solution 2 mL, distilled water to constant volume to L L, pH 6.0-8.0; the PBS buffer has a pH of 7-7.8.
The heterotrophic nitrification-aerobic denitrification microorganism immobilization method comprises the step 3) of using PBS buffer solution to resuspend the bacterial solution, wherein the concentration OD 600 0.2-2.0, and the temperature of the immobilized carrier after sterilization and cooling is 20-30 ℃; the concentration (v/v) of the saturated boric acid and the calcium chloride solution added in the step 4) are respectively 2-3% and 1-3%, the crosslinking temperature is 10-30 ℃, and the crosslinking time is 6-12 h.
The invention has the remarkable effects that:
the microorganism immobilization method is simple and convenient, low in preparation condition requirement, simple in equipment and low in energy consumption, toxic pollutants are not generated in the preparation process, the safety of the environment and workers can be ensured, and the method is also suitable for large-scale production and can realize large-scale preparation; the immobilized microorganism prepared by the invention has lower cost and high quality stability. The preparation process has mild condition, can keep the biological activity of the immobilized thalli, and compared with the traditional microorganism immobilization method, the preparation method has the advantages of better molding, high stability, high strength and small external environment interference resistance by adding different carrier materials to make the thalli more stable and increasing the mass transfer efficiency; the heterotrophic nitrification aerobic denitrifying bacteria immobilized carrier can simultaneously perform nitrification and denitrification in the same reactor, overcomes the defects of the traditional process, reduces the capital construction and operation cost in the sewage treatment process, and has good application prospect.
Drawings
FIG. 1 is a scanning electron microscope image of the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 of the optimal PVA-SA-BC carrier obtained under a 1000-fold scanning electron microscope. Wherein A is the outer surface of the 10% PVA-1% SA-BC carrier and B is the cross section of the 10% PVA-1% SA-BC carrier.
As can be seen from the analysis of FIG. 1, the immobilized bacteria carrier shown in FIG. 1 (10% PVA-1% SA-BC) is the optimal immobilized bacteria carrier, and the carrier has the characteristics of inner elasticity and outer elasticity, thereby being more beneficial to protecting microorganisms and preventing the loss of microorganisms. Integrates environmental protection, reactivity and hydrophilicity, and has the characteristics of high water impact resistance, high mass transfer performance, high microorganism loading capacity, high wastewater treatment effect and the like.
Description of the embodiments
The invention is further illustrated by the following examples.
The invention relates to a novel microorganism immobilization method, which comprises the following steps:
1) Preparing an immobilization carrier: preparing the immobilized carrier raw material and ammonia-free deionized water into a solution by using an electromagnetic heating furnace.
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium, and carrying out centrifugal resuspension.
3) Preparing a carrier embedding complex: sterilizing the immobilized carrier material prepared in the step 1), cooling to room temperature, and mixing and stirring with the bacterial suspension obtained in the step 2).
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the immobilized microorganism carrier embedding complex obtained in the step 3), adding the mixture into a crosslinking agent to crosslink and activate the complex, standing and solidifying at room temperature, and finally obtaining the crosslinked-embedded microorganism carrier complex.
The further technical scheme is as follows: the microorganism immobilization method comprises the following steps:
1) Preparing an immobilization carrier: polyvinyl alcohol (PVA) and Sodium Alginate (SA) are used as substrate raw materials, biochar (BC) and silicon dioxide are sequentially added into ammonia-free deionized water, an electromagnetic heating furnace is used for stirring and heating, and the solution is prepared to be used as an immobilization carrier PVA-SA-BC.
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium for 20-30 hours, and carrying out centrifugal resuspension.
3) Preparing a carrier embedding complex: selecting heterotrophic nitrification-aerobic denitrification bacterial suspension growing in logarithmic growth phase, and regulating bacterial suspension OD by using PBS solution 600 Mixing and fully stirring the immobilized carrier PVA-SA-BC after sterilization and cooling to room temperature and the heterotrophic nitrification-aerobic denitrification bacteria suspension according to the volume ratio of (10-30): 1, wherein the volume ratio is 0.2-1.0.
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the obtained carrier embedding complex of immobilized microorganism, and carrying out crosslinking activation on the carrier embedding complex by using saturated boric acid and calcium chloride to obtain the crosslinked-embedded microorganism carrier complex after crosslinking activation.
The microbial immobilization method is characterized in that the thallus for immobilization is selected from one of the following bacteria with a preservation unit of China Center for Type Culture Collection (CCTCC) and an address of eight 299 # in Wuchang district of Wuhan, hubei province: heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus sp.QD-21). The invention relates to a heterotrophic nitrification-aerobic denitrification bacteria immobilization method, which is characterized in that heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21), which was preserved in China Center for Type Culture Collection (CCTCC) at 11 and 14 days of 2022, and which was assigned the eight 299 th channel of the armed division of the chinese city, the hubei province, and the strain was assigned the CCTCC NO: m20221801; the heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21) its morphological and physiochemical characteristics include:
1) Gram-negative bacteria;
2) The bacterial form is microscopic detected as short rod-shaped bacteria;
3) On the culture medium, the colony forms a milky white smooth round shape with a raised middle part, and the colony edges are irregular.
The further technical scheme is as follows: the heterotrophic nitrification-aerobic denitrification bacteria immobilization method comprises the step of preparing the inorganic salt liquid culture medium C 4 H 4 Na 2 O 4 3.375 g/L、NH 4 Cl 0.382 g/L、K 2 HPO 4 0.5 g/L、KH 2 PO 4 1 g/L、MgSO 4 ·7H 2 O0.1 g/L, trace element solution 2 mL, distilled water to constant volume to L L, pH 6.0-8.0; the PBS buffer has a pH of 7-7.8.
In the heterotrophic nitrification-aerobic denitrification bacteria immobilization method, PBS buffer solution is used for resuspension of bacteria liquid in the step 3), and the concentration OD thereof 600 0.2-2.0, and the temperature of the immobilized carrier after sterilization and cooling is 20-30 ℃; the concentration (v/v) of the saturated boric acid and the calcium chloride solution added in the step 4) are respectively 2-3% and 1-3%, the crosslinking temperature is 10-30 ℃, and the crosslinking time is 6-12 h.
The invention discloses a method for scanning and detecting immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 samples, which comprises the following steps: washing the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 sample with deionized water for three times, respectively dehydrating with 30%, 50%, 70% and 90% ethanol, drying in a pre-cooled vacuum freeze dryer at-40 ℃ for 20-24 h to form freeze-drying, adhering the sample to a sample table by using a conductive adhesive tape, placing in a vacuum coating machine, spraying gold on the surface of the sample, taking out the sample, and observing the carrier embedding complex structure of immobilized microorganisms of the carrier PVA-SA-BC immobilized bacteria QD-21 by using a scanning electron microscope after the sample preparation.
The polyvinyl alcohol (PVA) is a synthetic organic polymer material, has the advantages of high mechanical strength of a carrier, difficult decomposition by microorganisms and the like, has a certain influence on the activity of the microorganisms in the embedding process, and has slightly poor mass transfer capacity; sodium Alginate (SA) is a natural organic polymer material, has the characteristics of easy molding, high mechanical strength, no toxicity or harm and good mass transfer performance, but is easy to decompose by microorganisms, and the mechanical strength of the single sodium alginate polymer material is low, so that the single sodium alginate polymer material cannot meet the practical application; biochar (BC) is a common adsorbent, and is derived from agricultural wastes, plays an important role in reducing greenhouse gas emission, has a rich pore structure and a large surface area, and can adsorb microorganisms due to the addition of the biochar, and the additive material of the biochar has a porous structure, so that the mechanical strength and mass transfer performance of the embedded carrier are improved; the addition of the silicon dioxide improves the mechanical strength and the physical and chemical stability of the embedding carrier; the composite carrier of the synthetic organic polymer carrier combines natural organic polymer materials with synthetic organic polymer materials, a plurality of additives are added into the composite carrier to adjust the carrier performance, and the carrier can combine the respective advantages of the natural organic polymer materials, the synthetic organic polymer carrier and the synthetic organic polymer materials by utilizing different proportions, so that the composite carrier has great advantages in the aspects of hydrophilicity, reproducibility and biocompatibility.
Example 1: is a basic example of the novel microorganism immobilization method. A novel microorganism immobilization method comprises the following steps: 1) Preparing an immobilization carrier: preparing the immobilized carrier raw material and ammonia-free deionized water into a solution by using an electromagnetic heating furnace;
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium, and carrying out centrifugal resuspension;
3) Preparing a carrier embedding complex: sterilizing the immobilized carrier material prepared in the step 1, cooling, and mixing and stirring with the bacterial suspension obtained in the step 2;
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the immobilized microorganism carrier embedding complex obtained in the step 3, adding the mixture into a crosslinking agent to crosslink and activate the complex, standing and solidifying at room temperature, and finally obtaining the crosslinked-embedded microorganism carrier complex.
The sequence of the steps 1, 2, 3 and 4 is as follows: 1. 2, 3 and 4.
The thallus for immobilization is selected from eight-path 299 No. of Wuhan, hubei province, which is a China Center for Type Culture Collection (CCTCC).
Example 2: is the preferred embodiment. In particular to a microorganism immobilization method for preparing spherical beads from polyvinyl alcohol (PVA) and Sodium Alginate (SA) as immobilization carriers, which comprises the following steps:
1) Preparing an immobilization carrier: 10 g polyvinyl alcohol (PVA) and 1 g Sodium Alginate (SA) are added into 100 mL of 1% ammonia-free deionized water solution with v/v, and the mixture is fully stirred and heated to prepare 10% polyvinyl alcohol (PVA) and 1% Sodium Alginate (SA) solution with w/v; 1 g biochar and 0.5 g silica were added to a 10% polyvinyl alcohol (PVA) and 1% Sodium Alginate (SA) solution of 100 mL and fully dissolved to prepare a PVA-SA-BC solution containing 1% biochar and 0.5% silica by w/v.
2) Preparing a bacterial suspension: the method is characterized in that the method is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: heterotrophic nitrification aerobic denitrifying bacteria QD-21 of M20221801paracoccus spQD-21), culturing in inorganic salt liquid culture medium for 24-30 hr, and selecting heterotrophic nitrification-aerobic denitrification bacteria QD-21 with good growth in logarithmic phaseparacoccus spQD-21) bacterial liquid, centrifuging at 5000rpm for 5min, collecting bacterial cells, washing with PBS buffer with pH of 7-7.8 for 2-3 times, and suspending bacterial liquid again with PBS buffer with pH of 7-7.8 to obtain bacterial liquid OD 600 1.0, to obtain a bacterial suspension for immobilization.
3) Preparing a carrier embedding complex: sterilizing and cooling PVA-SA-BC solution with high pressure steam sterilizing pot at 121deg.C for 30 min to obtain immobilized carrier PVA-SA-BC and OD 600 The heterotrophic nitrification-aerobic denitrification bacteria QD-21 bacterial suspension with the volume ratio of 1.0 (10-30) is fully mixed and stirred.
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the obtained carrier embedding complex, sucking the carrier embedding complex of 10 mL bacteria-containing PVA-SA-BC solution by using a medical disposable 10 mL syringe with the needle head model of 27G, and titrating the PVA-SA-BC solution into a saturated boric acid-calcium chloride solution through the needle head of the syringe to obtain a PVA-SA-BC carrier crosslinking-embedding complex; repeatedly washing with deionized water until no saturated boric acid and calcium chloride remain, eluting free bacteria, and standing at 2-8deg.C for use.
The heterotrophic nitrification-aerobic denitrification bacteria are heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21) was preserved in China Center for Type Culture Collection (CCTCC) at 11 and 14 days of 2022, and the address is 299, and the preservation number is CCTCC NO: m20221801, heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21) its morphology and physiologyThe biochemical features include:
1) Gram-negative bacteria;
2) The bacterial form is microscopic detected as short rod-shaped bacteria;
3) On the culture medium, the colony forms a milky white smooth round shape with a raised middle part, and the colony edges are irregular.
The inorganic salt liquid culture medium is C 4 H 4 Na 2 O 4 3.375 g/L、NH 4 Cl 0.382 g/L、K 2 HPO 4 0.5 g/L、KH 2 PO 4 1 g/L、MgSO 4 ·7H 2 O0.1 g/L, trace element solution 2 mL, distilled water to constant volume to L L, pH 7.2; the pH of the PBS buffer solution is 7-7.8. In step 3, the bacterial suspension was resuspended in PBS buffer at a concentration OD 600 1.0-2.0, and the temperature of the immobilized carrier after sterilization and cooling is 25 ℃; the concentration (v/v) of the saturated boric acid and the calcium chloride solution added in the step 4 are 2.5% and 3%, respectively, the crosslinking temperature is 20 ℃, and the crosslinking time is 12 h.
The immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 are placed in an environment of 4 ℃ for preservation.
Example 3: is a preferred embodiment of a method for scanning and detecting the immobilized heterotrophic nitrification-aerobic denitrification bacteria. The sample scanning detection method comprises the following steps: washing the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 sample with deionized water for three times, respectively dehydrating with 30%, 50%, 70% and 90% ethanol, drying in a pre-cooled vacuum freeze dryer at-40 ℃ for 24 h to form freeze-drying, adhering the sample to a sample table by using a conductive adhesive tape, placing in a vacuum coating machine, spraying gold on the surface of the sample, taking out the sample, and observing the carrier embedding complex structure of immobilized microorganisms of the carrier PVA-SA-BC immobilized bacteria QD-21 by using a scanning electron microscope after the sample preparation.
The scope of the invention is not limited to the embodiments described above.
Claims (6)
1. A heterotrophic nitrification-aerobic denitrification microorganism immobilization method, which is characterized by comprising the following steps:
1) Preparing an immobilization carrier: preparing the immobilized carrier raw material and ammonia-free deionized water into a solution by using an electromagnetic heating furnace;
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium, and carrying out centrifugal resuspension;
3) Preparing a carrier embedding complex: sterilizing the immobilized carrier material prepared in the step 1), cooling to room temperature, and mixing and stirring with the bacterial suspension obtained in the step 2);
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the immobilized microorganism carrier embedding complex obtained in the step 3), adding the mixture into a crosslinking agent to crosslink and activate the complex, standing and solidifying at room temperature, and finally obtaining the crosslinked-embedded microorganism carrier complex.
2. A class of heterotrophic nitrification-aerobic denitrification microbial immobilization methods as claimed in claim 1, wherein said method comprises the steps of:
1) Preparing an immobilization carrier: sequentially adding Biochar (BC) and silicon dioxide into ammonia-free deionized water by taking polyvinyl alcohol (PVA) and Sodium Alginate (SA) as substrate raw materials, stirring and heating by using an electromagnetic heating furnace, and preparing a solution serving as an immobilization carrier PVA-SA-BC;
2) Preparing a bacterial suspension: culturing a heterotrophic nitrification aerobic denitrifying bacterium in an inorganic salt liquid culture medium for 20-30 hours, and carrying out centrifugal resuspension;
3) Preparing a carrier embedding complex: selecting heterotrophic nitrification-aerobic denitrification bacterial suspension growing in logarithmic growth phase, and regulating bacterial suspension OD by using PBS solution 600 Mixing and fully stirring the immobilized carrier PVA-SA-BC after sterilization and cooling to room temperature and the heterotrophic nitrification-aerobic denitrification bacteria suspension according to the volume ratio of (10-30): 1, wherein the volume ratio is 0.2-2.0;
4) Immobilization of the activated crosslink-entrapping complexes: fully mixing the obtained carrier embedding complex of immobilized microorganism, and carrying out crosslinking activation on the carrier embedding complex by using saturated boric acid and calcium chloride to obtain the crosslinked-embedded microorganism carrier complex after crosslinking activation.
3. The method for immobilizing heterotrophic nitrification-aerobic denitrification microorganisms according to claim 1 or 2, wherein the immobilized heterotrophic nitrification-aerobic denitrification bacteria are heterotrophic nitrification-aerobic denitrification bacteria QD-21%paracoccus spQD-21), which was preserved in China Center for Type Culture Collection (CCTCC) at 11 and 14 days of 2022, and which was assigned the eight 299 th channel of the armed division of the chinese city, the hubei province, and the strain was assigned the CCTCC NO: m20221801 the morphology and physiological and biochemical characteristics of the heterotrophic nitrification-aerobic denitrification bacteria QD-21 comprise:
1) Gram-negative bacteria;
2) The bacterial form is microscopic detected as short rod-shaped bacteria;
3) On the culture medium, the colony forms a milky white smooth round shape with a raised middle part, and the colony edges are irregular.
4. The heterotrophic nitrification-aerobic denitrification microbial immobilization method as claimed in claim 1 or 2, wherein said method for scanning and detecting the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 bacteria sample comprises the steps of: washing the immobilized heterotrophic nitrification-aerobic denitrification bacteria QD-21 sample with deionized water for three times, respectively dehydrating with 30%, 50%, 70% and 90% ethanol, drying in a pre-cooled vacuum freeze dryer at-40 ℃ for 20-24 h to form freeze-drying, adhering the sample to a sample table by using a conductive adhesive tape, placing in a vacuum coating machine, spraying gold on the surface of the sample, taking out the sample, and observing the carrier embedding complex structure of immobilized microorganisms of the carrier PVA-SA-BC immobilized bacteria QD-21 by using a scanning electron microscope after the sample preparation.
5. A heterotrophic nitrification-aerobic denitrification microorganism immobilization process according to claim 1 or 2, wherein said inorganic salt liquid medium is C 4 H 4 Na 2 O 4 3.375 g/L、NH 4 Cl 0.382 g/L、K 2 HPO 4 0.5 g/L、KH 2 PO 4 1 g/L、MgSO 4 ·7H 2 O0.1 g/L, trace element solution 2 mL, distilled water to constant volume to L L, pH 6.0-8.0; the PBS buffer has a pH of 7-7.8.
6. The method for immobilizing heterotrophic nitrification-aerobic denitrification microorganisms according to claim 2, wherein in said step 3), PBS buffer is used to resuspend the bacterial solution, and the concentration OD thereof 600 0.2-2.0, and the temperature of the immobilized carrier after sterilization and cooling is 20-30 ℃; the concentration (v/v) of the saturated boric acid and the calcium chloride solution added in the step 4) are respectively 2-3% and 1-3%, the crosslinking temperature is 10-30 ℃, and the crosslinking time is 6-12 h.
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