Strain for efficiently removing nitrite nitrogen and construction method and application of couplant of strain
By screening and fixing the functional strain Bacillus megaterium LZW-3, the strain coupling was formed, which solved the problems of low nitrosity nitrogen removal efficiency and insufficient environmental adaptability in the prior art, and achieved high efficiency in complex water environments , stabilize the degradation effect of nitrosity nitrogen to achieve the goal of water purification.
Patent Information
- Application Number
- CN202411453776.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-17
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art has problems of low efficiency and insufficient environmental adaptability in removing nitrosity nitrogen in water bodies, especially in complex water environments, which are difficult to effectively degrade for a long time.
Screened out the functional strain Bacillus megaterium LZW-3, which efficiently transforms nitrogen pollutants, and form strain couplings to improve the stability and impact resistance of microorganisms and enhance the nitrosome. Nitrogen removal effect.
It significantly improves the degradation efficiency and stability of nitrosity nitrogen, so that it can efficiently remove nitrosity nitrogen in water under different pH and temperature conditions, reduces nitrogen pollution in water, and achieves water purification.
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Figure CN119162048A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a strain capable of efficiently removing nitrite nitrogen and a construction method and application of a coupled product thereof. Background Art
[0002] Water is a necessary consumable in human production and life, but the freshwater resources that humans can use are only 1% of the total global water resources. With the continuous acceleration of industrial and agricultural development, nitrogen-containing pollutants are discharged into water bodies, causing eutrophication of water bodies. Therefore, finding an efficient and environmentally friendly method to remove nitrite nitrogen from water bodies has become a research hotspot in the current environmental field. At present, traditional methods for removing nitrite nitrogen mainly include biological methods, chemical methods and physical methods. Among them, biological methods have attracted much attention due to their environmental protection, economy and high efficiency. As a new type of biological method, carrier-immobilized microbial technology has unique advantages in removing nitrite nitrogen from water bodies. Compared with traditional biological methods, fixed microbial technology can better protect microbial species and improve degradation rate. At the same time, it can effectively reduce the number of microbial species released and reduce secondary pollution to water bodies, and has better development and application prospects. Summary of the invention
[0003] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for constructing and applying a strain for efficiently removing nitrite nitrogen and its coupled products, by screening functional bacteria that efficiently convert nitrogen pollutants and utilizing immobilization to enhance microbial activity, thereby reducing the concentration of nitrite nitrogen in water bodies and reducing nitrogen pollution in water bodies, thereby achieving the goal of purifying water bodies.
[0004] Technical solution: In the first aspect, the present invention provides a strain for efficiently removing nitrite nitrogen, wherein the strain is Bacillus megaterium Bacillus megaterium LZW-3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 7, 2024, with the deposit number CGMCC NO.31636.
[0005] Furthermore, the specific culture method of the strain is: inoculating and culturing the strain with Luria-Bertani medium; wherein the inoculation amount is 0.2~2%, the initial pH value of the Luria-Bertani medium is 5~9, the culture temperature is 15~35°C, the rotation speed is 0~250r / min, and the culture time is 16~24h.
[0006] In a second aspect, the present invention provides an application of a strain for efficiently removing nitrite nitrogen as described in any of the above items in nitrogen-containing wastewater, wherein when the strain degrades nitrite nitrogen in nitrogen-containing wastewater, the input amount of the strain is 0.2~1%; the degradation pH is 6-8, and the degradation temperature is 20-35°C.
[0007] In a third aspect, the present invention provides a method for constructing a strain coupling product for efficiently removing nitrite nitrogen, comprising the following steps: S1. The concave soil is sterilized and dried to obtain pretreated concave soil; S2. The strain having high efficiency in removing nitrite nitrogen described in any one of the above items is constructed into a bacterial suspension using PBS buffer; S3. The bacterial suspension is mixed with the pretreated attapulgite (the amount of the pretreated attapulgite is 1-5%), and the mixture is shaken and adsorbed at 100-190 r / min and 15-45° C., centrifuged, and freeze-dried to obtain a strain coupling product.
[0008] Furthermore, in S3, the mass of the mixed liquid is 500-3000 g; and the amount of pretreated attapulgite added to the mixed liquid is 1-5%.
[0009] Furthermore, in S3, the time of oscillation adsorption is 4-16h.
[0010] In a fourth aspect, the present invention provides an application of a strain couple for efficiently removing nitrite nitrogen constructed by any of the methods described above in nitrogen-containing wastewater. When the strain couple degrades nitrite nitrogen in nitrogen-containing wastewater, the input amount of the strain couple is 1-5%; the degradation temperature is 15-35°C.
[0011] Beneficial effects: The innovation of the present invention is that, based on the screening of microorganisms with the ability to efficiently degrade nitrite nitrogen, porous materials are used as carriers to adsorb bacteria and fix the functional bacteria on the carriers. The carriers have rich mineral components and chemical compositions, and can provide a stable microenvironment, so that the functional bacteria can play a more stable role in a specific environment, thereby improving the stability of the microorganisms. At the same time, this combination also improves its adaptability and resistance to environmental changes. The structure of the carrier can provide certain physical protection for the functional bacteria, reduce the impact of adverse factors in the environment on the bacteria, improve the impact resistance of the microorganisms and the conversion efficiency of nitrogen pollutants, enhance the effect of functional bacteria in removing nitrite nitrogen from water bodies, and enable them to effectively remove nitrite nitrogen for a long time in complex water environments. The present invention is a technology that can effectively remove nitrite nitrogen, reduce the concentration of nitrite nitrogen in water bodies, and achieve the goal of purifying water bodies. The present invention can be used in the fields of purification of nitrogen-containing sewage and has a broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Bacillus megaterium strain screened for efficient removal of nitrite nitrogen by the present invention Bacillus megaterium Schematic diagram of the growth of LZW-3 on solid Luria-Bertani medium (left) and Gram staining of the strain (right); Figure 2 Bacillus megaterium at different pH Bacillus megaterium Schematic diagram of the growth and nitrite nitrogen degradation changes of LZW-3; Figure 3 Bacillus megaterium at different temperatures Bacillus megaterium Schematic diagram of the growth and nitrite nitrogen degradation changes of LZW-3; Figure 4 Bacillus megaterium Bacillus megaterium Schematic diagram of the coupling time curve between LZW-3 and the fixed carrier; Figure 5 This is a schematic diagram of the degradation of nitrite nitrogen by the strain coupling; Figure 6 This is a schematic diagram of the degradation of nitrite nitrogen by the strain coupling at different pH; Figure 7 This is a schematic diagram of the degradation of nitrite nitrogen by the strain coupling at different temperatures. DETAILED DESCRIPTION
[0013] The present invention is described in detail below in conjunction with the embodiments.
[0014] Implementation 1: This embodiment provides a method for screening a strain that can efficiently remove nitrite nitrogen. The specific steps are as follows: (1) Enrichment culture: Sludge collected from the Southeast White Shrimp Farming Base in Nantong City, Jiangsu Province was added to a flask containing 200 ml of sodium nitrite and cultured at 30°C for 4 days to obtain enrichment culture medium; (2) Isolation and purification: Use sterile water to dilute the above enriched culture solution to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 There are 6 concentration gradients in total. Use a pipette to take 200 μL of samples from each concentration gradient, spread them on LB medium plates, place them in a constant temperature incubator at 30°C, pick single colonies from the plates, transfer them to LB solid medium, and separate and screen 3 dominant strains. Place them in a constant temperature incubator at 30°C and culture them away from light. Cultivate in a 30°C incubator until single colonies grow. After repeated streaking, select and purify single colonies on the workbench, inoculate them in LB liquid medium and culture them for 24 hours, then prepare sterilized 60% glycerol, store them in a sterile tube in a clean bench, and store them in a -80°C refrigerator; (3) Screening: The strain isolated, purified and preserved in step (2) was inoculated into LB medium and cultured at 30°C and 160 rpm for 24 h. Then, 2.5 ml of the cultured bacterial solution was added to simulated sewage containing glucose and sodium nitrite, and cultured at 30°C and 160 rpm for 48 h. The degradation efficiency of nitrite nitrogen was tested and the best strain was screened, i.e., the strain Bacillus megaterium that efficiently removed nitrite nitrogen. Bacillus megaterium LZW-3, this strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on August 7, 2024, with the deposit number CGMCC NO.31636.
[0015] Implementation 2: This embodiment provides a method for constructing a strain coupling substance that can efficiently remove nitrite nitrogen, as follows: (1) sterilizing the attapulgite at 121°C for 20 min and drying it to obtain pretreated attapulgite; (2) The strain with high efficiency in removing nitrite nitrogen obtained by screening in Implementation Method 1 is used to construct a bacterial suspension using PBS buffer; (3) Mix the bacterial suspension with the pretreated kaolin (the amount of pretreated kaolin is 1-5%) to obtain 500-3000 g of a mixed solution, shake the mixed solution at 100-190 r / min and 15-45°C for adsorption, centrifuge, and freeze-dry to obtain a strain coupling product.
[0016] The conditions and effects of degrading nitrite nitrogen in water by the strains screened in Implementation Example 1 and the strains coupled with the strains constructed in Implementation Example 2 were tested. The test steps and results are as follows: Among them, the strain with high efficiency in removing nitrite nitrogen screened in Implementation Method 1 and the strain with high efficiency in removing nitrite nitrogen constructed in Implementation Method 2 are used for the degradation of nitrite nitrogen in water, and the detection steps of the degradation efficiency are as follows: (1) Prepare simulated sewage (components: Vickers salt: 40 ml / L-80 ml / L, sodium nitrite: 30 mg / L-100 mg / L, sucrose: 0.2 g / L-1 g / L); (2) adding the strain with high efficiency in removing nitrite nitrogen screened in Implementation Method 1 and the strain coupled with high efficiency in removing nitrite nitrogen constructed in Implementation Method 2 to the simulated sewage respectively to degrade nitrite nitrogen; (3) Collect degraded water samples using centrifuge tubes; (4) Use a centrifuge to centrifuge the collected degraded water samples to remove impurities; (5) Take the supernatant and use the α-naphthylamine spectrophotometric method to determine the nitrite nitrogen content in the degraded water sample: take 1.00 mL of the supernatant and add it to a 10 mL volumetric flask. Add water to make the volume up to 10 mL, then add 1 mL of p-aminobenzenesulfonic acid solution, mix well and let stand for 3 min, then add 1 mL of sodium acetate buffer and 1 mL of α-naphthylamine solution in turn, mix well and let stand for 30 min, and measure the absorbance at 520 nm using a UV spectrophotometer.
[0017] Figure 2 Bacillus megaterium strains that efficiently remove nitrite nitrogen at different pH values Bacillus megaterium The growth of LZW-3 and the degradation of nitrite nitrogen show the experimental verification effect of strain growth and nitrite nitrogen degradation ability under different pH. The growth and metabolism of the strain are inhibited under acidic conditions, and the degradation efficiency of nitrite nitrogen is not high, less than 40%. In a neutral to alkaline environment, the strain grows well and the nitrite nitrogen degradation efficiency is also high. At a pH of 8, the degradation efficiency of nitrite nitrogen is the highest, reaching 94.52%. It can be seen that the strain exhibits efficient nitrite nitrogen degradation ability under neutral and weakly alkaline conditions with a pH value of 7-9.
[0018] Figure 3 Bacillus megaterium at different temperatures Bacillus megaterium Schematic diagram of the growth and nitrite degradation of LZW-3, showing the experimental verification results of the strain growth and nitrite degradation ability at different temperatures. It can be seen that the strain has good growth in a wide temperature range and has the ability to efficiently degrade nitrite. At a temperature of 15°C, the growth and metabolism of the strain are inhibited, and OD 600 The value was 0.53, and the degradation rate of nitrite nitrogen was 4.445%. However, when the external temperature rose from 15℃ to 20℃, 25℃, 30℃, and 35℃, the degradation rate of nitrite nitrogen by the strain was greatly improved, reaching 100%. Between 20℃ and 35℃, the degradation rate of nitrite nitrogen was stable, all at 100%. At the same time, between 20℃ and 30℃, the growth trend of the strain was good, and OD 600 The value increased from 0.731 to 0.949, and decreased to 0.669 at 35°C.
[0019] Figure 4 Bacillus megaterium Bacillus megateriumThe schematic diagram of the coupling time curve between LZW-3 and the fixed carrier shows the coupling between the strain and the fixed carrier at different times. It can be seen that as time goes by, more and more strains are adsorbed on the carrier. In 0-4h, the adsorption situation changes the most, and the adsorption rate changes from 0% to 72.43%; at 16h, the coupling between the strain and the fixed carrier is the best, and most of the strains are adsorbed on the carrier, with an adsorption rate of 96.86%. At 24h, the adsorption rate decreases, which may be due to the long oscillation time, and the adsorbed strains fall off the carrier.
[0020] Figure 5 This is a schematic diagram of the degradation of nitrite nitrogen by the strain coupling, showing the experimental verification effect of the strain coupling's ability to remove nitrite nitrogen. The strain coupling has a high efficiency in degrading nitrite nitrogen, and the degradation rate reaches 100% at 16 hours, indicating that the strain coupling has a high efficiency in degrading nitrite nitrogen.
[0021] Figure 6 This is a schematic diagram of the degradation of nitrite nitrogen by strain coupling at different pH values, showing the growth of strain coupling and the degradation of nitrite nitrogen at different pH values. It can be seen that compared with individual strains, strain coupling has higher stability and impact resistance. When the pH value is in the range of 5-9, the degradation rate of strain coupling is basically the same, indicating that strain coupling can degrade nitrite nitrogen in acidic, neutral and alkaline environments, and has a high degradation ability.
[0022] Figure 7 This is a schematic diagram of the degradation of nitrite nitrogen by strain couplings at different temperatures, showing the experimental verification effect of the growth of strain couplings and the degradation ability of nitrite nitrogen at different temperatures. It can be seen that the strain couplings also have the ability to efficiently degrade nitrite nitrogen in a wide temperature range. Under the condition of a temperature of 15°C, the growth and metabolism of the strains are inhibited, but the strain couplings are different. The strain couplings have a high nitrite nitrogen degradation ability at 15°C, with a degradation rate of 98.7%. Between 20°C and 35°C, the degradation rate of nitrite nitrogen is stable, all 100%. However, at 45°C, the nitrite nitrogen degradation ability of the strain couplings dropped to 2.13%, indicating that under higher temperature conditions, the ability of the strain couplings to degrade nitrite nitrogen was inhibited, and the reason may be that the strains died under higher temperature conditions.
[0023] In summary, the present invention screened out the functional bacteria Bacillus megaterium that efficiently transforms nitrogen pollutants. Bacillus megaterium LZW-3 has the ability to effectively degrade nitrite nitrogen in sewage and reduce nitrogen pollution in water bodies. At the same time, the present invention further introduces Bacillus megaterium Bacillus megateriumLZW-3 is fixed on the kaolin carrier, which improves the stability, activity and impact resistance of the microorganisms, thereby enhancing their effect in removing nitrite-nitrogen from water bodies, enabling them to effectively remove nitrite-nitrogen in a complex water environment for a long time.
[0024] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.
Claims
1. A strain for efficiently removing nitrite nitrogen, characterized in that: Bacillus megaterium Bacillus megaterium LZW-3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 7, 2024, with the deposit number CGMCC NO.31636.
2. The strain for efficiently removing nitrite-nitrogen according to claim 1, characterized in that: The specific culture method of the strain is: inoculating and culturing the strain using Luria-Bertani medium; wherein the inoculation amount is 0.2-2%, the initial pH value of the Luria-Bertani medium is 5-9, the culture temperature is 15-35°C, the rotation speed is 0-250r / min, and the culture time is 16-24h.
3. Use of a strain for efficiently removing nitrite-nitrogen as claimed in any one of claims 1 to 2 in nitrogen-containing wastewater, characterized in that: When the strain degrades nitrite nitrogen in nitrogen-containing sewage, the input amount of the strain is 0.2-1%, the degradation pH is 6-8, and the degradation temperature is 20-35°C.
4. A method for constructing a strain coupling product for efficiently removing nitrite nitrogen, characterized in that: The following steps are involved: S1. The concave soil is sterilized and dried to obtain pretreated concave soil; S2. The strain with high efficiency in removing nitrite-nitrogen according to any one of claims 1 to 2 is used to construct a bacterial suspension using PBS buffer; S3. The bacterial suspension is mixed with the pretreated attapulgite (the amount of the pretreated attapulgite is 1-5%), and the mixture is shaken and adsorbed at 100-190 r / min and 15-45° C., centrifuged, and freeze-dried to obtain a strain coupling product.
5. The method for constructing a strain coupling substance according to claim 4, characterized in that: In S3, the amount of pretreated attapulgite added to the mixed solution is 1-5%.
6. The method for constructing a strain coupling substance according to claim 4, characterized in that: In S3, the time of oscillation adsorption is 4-16h.
7. An application of a strain coupling product for efficiently removing nitrite nitrogen constructed by the method according to any one of claims 4 to 6 in nitrogen-containing wastewater, characterized in that: When the strain coupling substance degrades nitrite nitrogen in nitrogen-containing sewage, the input amount of the strain coupling substance is 1-5%; and the degradation temperature is 15-35°C.