Method for efficiently enriching ammonia-oxidizing archaea in sewage in step-by-step synergistic manner

Abstract

The invention provides a method for efficiently enriching ammonia-oxidizing archaea in sewage in a step-by-step synergistic manner, and belongs to the technical field of microorganism culture and sewage treatment.The method comprises the following steps that S1, a bacterial liquid sample to be enriched is added into an enrichment culture medium containing antibiotics for subculture; and S2, carrying out microporous filtration culture on part of the bacterial liquid obtained in the step S1 to enrich AOA, and then carrying out dilution subculture on the enriched bacterial liquid. According to the AOA enrichment method provided by the invention, by combining antibiotic treatment, multiple filtration and dilution culture strategies, the limitation of the prior art on single-genus AOA enrichment is remarkably broken through, multi-genus AOA can be effectively enriched in a complex sewage environment at a time, and the method has important significance on AOA research and application.

Description

Technical Field

[0001] The invention relates to the technical field of microbial cultivation and sewage treatment, and in particular to a method for step-by-step, synergistic and efficient enrichment of ammonia oxidizing archaea in sewage. Background Art

[0002] Excessive discharge of nitrogen in sewage is not only the main factor causing eutrophication of water bodies, but also produces nitrous oxide (N2O), a potent greenhouse gas, which exacerbates global warming and poses severe challenges to ecosystems and human health. Therefore, in the process of sewage treatment, reducing nitrogen content and effectively reducing N2O emissions are always crucial tasks. Among the existing denitrification methods, biological methods are widely used due to their simple operation, low cost and no secondary pollution. In the traditional biological denitrification process, the reaction is mainly initiated by ammonia oxidizing bacteria (AOB), which is responsible for converting ammonia nitrogen into nitrite, and then the subsequent denitrification process. In this process, not only a large amount of aeration is required to maintain the efficient progress of the reaction, but also N2O is produced, which increases the operating energy consumption of the process and the carbon emissions in the sewage treatment system.

[0003] For a long time, AOB has been considered the main ammonia-oxidizing microorganisms in sewage treatment systems. However, as research deepened, scientists discovered ammonia-oxidizing archaea (AOA) in extreme environments. AOA was subsequently enriched and isolated in a variety of environments such as oceans, soils, and hot springs, further enriching the diversity of ammonia-oxidizing organisms. AOA has a high affinity for substrates and can efficiently oxidize ammonia under extreme conditions. In addition, the N2O produced by AOA during the ammonia oxidation process is much lower than that of AOB, which can significantly reduce the negative impact on the environment.

[0004] In the context of the current global energy crisis and climate change, the development of AOA-based denitrification processes is expected to become one of the important breakthroughs in energy conservation and emission reduction in sewage treatment plants. By culturing more AOA strains from sewage environments, we can deepen our understanding of the physiological characteristics of different AOA lineages and further explore their potential in practical applications. However, the AOA strains or cultures currently available for research are very limited, and there are mainly the following challenges: First, the enrichment method is single, and the types of AOA strains that are successfully enriched are rare, usually limited to the dominant AOA genus in sewage; second, the enrichment process of AOA is difficult and time-consuming, and it usually takes several years of subculture to obtain stable enrichments; third, the enrichment is difficult. As an artificially controlled environment, the sewage treatment system has a small number of AOA, which increases the difficulty of enrichment. At present, the enrichment methods of AOA in sewage mainly rely on antibiotics or dilution culture methods, which are usually time-consuming and have limited enrichment effects. In addition, traditional technologies can usually only enrich AOA of a single genus, and it is difficult to enrich AOA of different genera in a complex sewage environment. These limit the further research and application development of AOA, and it is urgent to develop new methods to overcome these problems. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a step-by-step and coordinated method for efficiently enriching ammonia oxidizing archaea from sewage, aiming to solve the technical problems of existing AOA enrichment methods being single, difficult and time-consuming.

[0006] In order to achieve the above object, the present invention specifically adopts the following technical solutions: A method for efficiently enriching ammonia-oxidizing archaea in sewage in a step-by-step manner comprises the following steps: S1, adding the bacterial liquid sample to be enriched to the enrichment medium containing antibiotics for subculture; S2. Perform microfiltration on part of the bacterial solution obtained in step S1 to screen AOA, and then dilute and subculture the enriched bacterial solution to further enrich AOA and eliminate AOB.

[0007] In the above method, the bacterial solution samples to be enriched can be pre-screened according to the actual situation to obtain bacterial solution samples with richer initial AOA for enrichment. For example, in some embodiments of the present invention, by extracting sample DNA, and then comparing the AOA contained in different samples amoA The copy number of the gene was determined by selecting samples with a higher copy number (i.e., a richer AOA number) for processing to obtain the bacterial solution to be enriched.

[0008] In the above method, the antibiotic used is at least one of ampicillin, carbenicillin, streptomycin, kanamycin and tetracycline. Adding appropriate antibiotics to the enrichment medium can effectively inhibit bacterial growth without affecting archaea, thereby achieving rapid enrichment of AOA. In some embodiments of the present invention, the five antibiotics of ampicillin, carbenicillin, streptomycin, kanamycin and tetracycline are used to jointly inhibit the miscellaneous bacteria in the culture system. Preferably, the concentration of each antibiotic is 45-55 mg / L.

[0009] In the above method, the enriched medium is used to provide a suitable growth environment for AOA. In some embodiments of the present invention, the enrichment medium includes 0.9-1.1 g / L NaCl, 0.36-0.44 g / L MgCl2·6H2O, 0.45-0.55 g / L KCl, 0.09-0.11 g / L CaCl2 and 0.18-0.22 g / L KH2PO4, and each liter of enrichment medium also includes 1 mL 1 MNH4Cl solution, 2 mL 1 M NaHCO3 solution, 1 mL 0.5 M sodium pyruvate solution, 1 mL Fe-NaEDTA solution and 1 mL of trace element solution; wherein the Fe-NaEDTA solution includes 196.2-239.8 g / L EDTA-Na2 and 47.25-57.75 g / L FeSO4·6H2O, and the trace element solution includes 1.35-1.65 g / L FeCl2·4H2O, 0.09-0.11 g / L CaCl2 and 0.18-0.22 g / L KH2PO4. MnCl2·4H2O, 0.17~0.21 g / L CoCl2·6H2O, 0.022~0.026 g / L NiCl2·6H2O, 0.015~0.019 g / LCuCl2·2H2O, 0.032~0.040 g / L NaMoO4·2H2O, 0.063~0.077 g / L ZnCl2, 0.056~0.068 g / LHBO3.

[0010] In the above method, the method of subculture in step S1 is preferably: monitoring the ammonia nitrogen concentration in the enrichment medium, when the ammonia nitrogen consumption exceeds 80%~90%, replacing 70%~80% of the upper clear liquid with a fresh enrichment medium containing antibiotics, thus completing one subculture, and enrichment is performed after multiple subcultures.

[0011] In the prior art, the enrichment culture of AOA usually adopts the dilution subculture method, but due to the slow growth of AOA and the long generation time, the dilution subculture method is difficult to retain the AOA of the previous culture cycle. As the number of subcultures increases, the ammonia oxidation activity gradually disappears, resulting in the inability to continue the enrichment. The solution of the present invention adopts a culture method of replacing the supernatant, which can effectively retain the AOA of the previous culture cycle and avoid loss.

[0012] In the above method, step S2 includes the following two stages: S21. Screening AOA by at least one microfiltration using the size difference between AOA and other bacteria; S22. The bacterial solution obtained by enrichment in step S21 is diluted and subcultured by gradually reducing the inoculation ratio to further enrich AOA and eliminate AOB.

[0013] In view of the difference between the size of AOA and the size of general bacteria, such as the cell size of AOA is usually in the range of about 0.2-1 μm, and the cell size of AOB is usually in the range of about 0.5-1.5 μm, the present invention attempts to use microporous filtration to treat the bacterial solution after enrichment culture, so as to enrich and obtain more types of AOA. Microporous filtration can be specifically performed through a filter membrane with a suitable pore size. It can be understood that in order to achieve selective filtration, the pore size of the filter membrane should be able to effectively separate AOA from general bacteria. In some embodiments of the present invention, step S21 uses a 0.45 μm sterile needle filter for microporous filtration. Under this condition, part of the AOA can be allowed to pass, while larger particles and bacteria are retained and removed.

[0014] Preferably, in step S21, multiple microporous filtrations are performed, and the entire bacterial solution is filtered and cultured each time. The specific operation is: taking part of the bacterial solution obtained in step S1, inoculating it into an enrichment medium without antibiotics after microporous filtration, and culturing it in the dark; when the ammonia nitrogen consumption exceeds 80%, the entire bacterial solution is centrifuged and the supernatant is removed, and the solution is resuspended with a fresh enrichment medium, and then the resuspended solution is microporously filtered, and the filtered bacterial solution is inoculated into an enrichment medium without antibiotics and continued to be cultured in the dark; and the above steps are repeated.

[0015] The experimental data showed that after step S21, AOA genus different from that enriched in step S1 can be enriched, which provides the possibility of enriching AOA of different genera from sewage environmental samples at one time. However, the AOA content in the bacterial solution obtained in step S21 is low, and due to the lack of antibiotic inhibition, miscellaneous bacteria (such as AOB) can grow, which requires further treatment to eliminate AOB in the culture and make AOA occupy an absolute advantage.

[0016] In view of the above problems, the method of the present invention successfully solves the problem by adopting a dilution passage method with a gradually reduced inoculation ratio in step S22; for example, in some embodiments of the present invention, the initial inoculation ratio is 25%, then adjusted to 20%, and finally reduced to 10%. For the cultivation of AOA, the inoculation ratio will have many effects; for example, a higher inoculation ratio means more starting cells, which can make AOA adapt to the new environment faster, help shorten the lag phase, and bring the growth logarithmic phase forward, but if the inoculation ratio is too high, it may intensify the competition of microorganisms for nutrients and growth space, inhibit the growth of AOA, and may also cause the accumulation of metabolites too fast, which is toxic to cells and affects the growth and metabolism of AOA. Therefore, an appropriate inoculation ratio helps to alleviate excessive competition, maintain the balance of nutrition and space, avoid the negative impact of the accumulation of metabolites on the growth of AOA, and ensure the stability of its growth and metabolism. The present invention effectively reduces the rapid loss of AOA during the dilution passage process by dilution passage + gradually reducing the inoculation ratio, and efficiently enriches AOA in a short time, and effectively eliminates AOB in the bacterial solution on the other hand.

[0017] The step-by-step synergistic and efficient enrichment method of AOA provided by the present invention can be used to enrich and obtain AOA from complex sewage environmental samples. For example, in one embodiment of the present invention, AOA of the genus Nitrocosmicus and AOA of the genus Nitrosotenuis were effectively enriched at one time from biofilm microorganisms in an aerated biological filter tank of a sewage treatment plant.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The method of the present invention is suitable for samples with complex conditions and in which the number of AOA is not dominant. By replacing the supernatant in the early stage and filtering the entire bacterial liquid during filtration culture, the number of AOA is retained to the maximum extent, ensuring that it occupies a dominant position in microbial competition, and successfully achieving effective enrichment of AOA.

[0019] (2) The method of the present invention can achieve the enrichment of at least two genera of AOA. By using antibiotics in the early stage, the miscellaneous bacteria are selectively inhibited, and the dominant AOA genus is effectively enriched. Subsequently, other secondary genera of AOA in the sewage are further separated through multiple filtration operations, thereby achieving the enrichment of multiple genera of AOA, overcoming the limitation of traditional methods that only a single genus of AOA can be enriched.

[0020] (3) The method of the present invention effectively improves the enrichment efficiency. In traditional methods, the dilution culture method is effective, but it is time-consuming and inefficient, especially when the initial AOA abundance is low. The present invention optimizes the enrichment process, shortens the culture time, and improves the enrichment efficiency by combining antibiotic enrichment, multiple filtration, and finally using the dilution culture method, so that AOA of different genera can be stably enriched in a relatively short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is a physical picture of the culture vessel used in the AOA enrichment process of the embodiment of the present invention; Figure 2 This is a graph showing changes in nitrogen concentration during the antibiotic selective enrichment culture of AOA in an embodiment of the present invention; Figure 3 This is a graph showing changes in nitrogen concentration during microporous filtration combined with dilution subculture of AOA in an embodiment of the present invention; Figure 4 Archaea and bacteria in the enrichment culture process of the present invention amo A gene PCR amplification pattern changes, where lane 1 is the sample obtained after 6 micropore filtration cultures, lane 2 is the sample obtained after 4 dilution subcultures, and lane 3 is the archaea amo A(arch- amo A) Positive control, lane 4 is bacteria amo A(bac- amo A) Positive control, lane M is DNA marker; Figure 5 The figure is a trend chart of the change of the number of AOA and AOB in different samples obtained in the embodiment of the present invention, wherein A0 is the inoculation source, A1 is the sample obtained after enrichment and subculture under antibiotic conditions, and A2 is the sample obtained after microporous filtration combined with dilution and subculture; Figure 6 Graph showing the community diversity analysis results of different cultures obtained in the examples of the present invention, wherein A0 is the inoculum source, A1 is the sample obtained after enrichment and subculture under antibiotic conditions, and A2 is the sample obtained after microporous filtration combined with dilution and subculture. DETAILED DESCRIPTION

[0023] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" in the description and claims of the present invention and any variations thereof are intended to cover non-exclusive inclusions.

[0025] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0026] Existing methods for enriching AOA from sewage systems have high selectivity for specific genera, and have problems such as single method, difficulty in enrichment, and long time consumption, which limit the in-depth study of AOA diversity and ecological functions. In order to solve this problem, the present invention provides an optimized step-by-step collaborative method for enriching different AOA from sewage environments. In the scheme of the present invention, AOA of a specific genus is first successfully enriched through antibiotic treatment combined with a subculture method of replacing the supernatant; then, a filter membrane with a specific pore size is used to filter and culture part of the enrichment, and the entire bacterial solution is filtered and inoculated during each subculture to screen out AOA of another genus; finally, combined with the dilution culture method, AOB in the culture is effectively eliminated, achieving stable enrichment of AOA as the only ammonia oxidizing microorganism.

[0027] The scheme of the present invention effectively breaks through the limitations of the existing technology by optimizing the subculture process and flexibly combining antibiotic treatment, multiple filtration and dilution culture strategies, and provides a method for quickly and efficiently enriching AOA. The AOA enriched by the method of the present invention includes two genera, Nitrocosmicus and Nitrosotenuis, while in the existing AOA enrichment methods, usually only AOA of one genus can be enriched.

[0028] A specific embodiment is listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0029] Example 1 This example provides a step-by-step method for synergistically and efficiently enriching ammonia-oxidizing archaea in sewage, comprising the following steps: (1) Selection and pretreatment of activated sludge.

[0030] Sludge samples were collected from a domestic sewage treatment plant in Wuhan, specifically anoxic activated sludge from the anoxic tank of the sewage treatment plant and biofilm samples from the aerated biological filter. The same volume of sludge samples was taken for DNA extraction, and the AOA contained in each milliliter of sample was compared using a fluorescent quantitative method. amoA The copy number of the gene showed that the copy number of AOA in the biofilm sample of the aerated biological filter was one order of magnitude higher than that in the activated sludge of the anoxic tank. Therefore, in this case, the biofilm sample of the aerated biological filter with a richer number of AOA was selected as the inoculated sludge.

[0031] Pretreatment of inoculated sludge: In a clean bench, place the filter media of the aerated biological filter in a 50 mL centrifuge tube, add an appropriate amount of sterile water, and shake on a vortex mixer for 15 minutes to remove the biofilm on the filter media. Repeat this step to finally prepare a sufficient amount of bacterial suspension for subsequent inoculation culture.

[0032] (2) Antibiotics selectively enrich AOA.

[0033] In a clean bench, add 5 mL of uniform bacterial suspension to 50 mL of enrichment medium containing antibiotics, and culture in a 37°C constant temperature incubator away from light. Regularly monitor the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the culture medium. When the ammonia nitrogen in the culture medium is consumed by more than 80%, replace 75% of the supernatant and replenish it with fresh medium containing antibiotics to complete one subculture. Repeat the above steps for multiple subcultures.

[0034] (3) Microfiltration combined with dilution subculture to synergistically screen AOA.

[0035] In the clean bench, take 15 mL of bacterial liquid that has been enriched with antibiotics for about 300 days, filter it with a 0.45 μm sterile syringe filter, inoculate it into 135 mL enrichment medium (without antibiotics), and culture it in a 37°C constant temperature incubator in the dark. When the ammonia nitrogen consumption in the culture solution exceeds 80%, centrifuge all the bacterial liquid and discard the supernatant, resuspend it with 15 mL of fresh enrichment medium to prepare a bacterial suspension, filter the bacterial suspension again with a 0.45 μm sterile syringe filter, and inoculate it into 135 mL enrichment medium for further culture. Repeat the above steps 6 times.

[0036] The enriched medium (without antibiotics) was used to continue culturing the bacterial solution obtained by filtration culture by dilution subculture method. The culture device was a 1L sterile conical flask with a culture volume of 800 mL. This step performed a total of 4 dilution subcultures. During the subculture dilution process, the inoculation ratio was gradually reduced in this example. Specifically, the initial inoculation ratio was 25%, then adjusted to 20%, and finally reduced to 10%.

[0037] The preparation method of the enrichment medium used in this example is as follows: prepare a liquid culture medium containing 1 g / L NaCl, 0.4 g / L MgCl2·6H2O, 0.5 g / L KCl, 0.1 g / L CaCl2 and 0.2 g / L KH2PO4, adjust the pH to 7.0 with 0.5 M HCl and 1 M NaOH, sterilize at high temperature, and then add the following filter-sterilized stock solutions to each liter of liquid culture medium: 1 mL NH4Cl solution (1 M), 2 mL NaHCO3 solution (1 M), 1 mL sodium pyruvate solution (0.5 M), 1 mL Fe-NaEDTA solution and 1 mL trace element solution. Among them, the Fe-NaEDTA solution includes 218 g / L EDTA-Na2 and 52.5 g / L FeSO4·6H2O, and the trace element solution contains 1.5 g / L FeCl2·4H2O, 0.1 g / L MnCl2·4H2O, 0.19 g / L CoCl2·6H2O, 0.024 g / L NiCl2·6H2O, 0.017 g / L CuCl2·2H2O, 0.036 g / L NaMoO4·2H2O, 0.07 g / L ZnCl2, and 0.062 g / L HBO3.

[0038] The enrichment medium containing antibiotics refers to the above-mentioned enrichment medium to which five antibiotics, namely, ampicillin, carbenicillin, streptomycin, kanamycin and tetracycline, are added, and the use concentration of each antibiotic is 50 mg / L.

[0039] The following tests were performed during the above enrichment process: ① Collect 3 mL samples from the bottles every 6 days, filter through a 0.22 μm filter membrane, and analyze the concentrations of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the samples to monitor the ammonia oxidation activity of the culture.

[0040] ② At the end of each enrichment stage, the bacteria were collected and DNA was extracted. AOA was qualitatively and quantitatively detected by polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (qPCR) to determine the presence and quantitative changes of AOA in the enrichment. At the same time, the main types and enrichment levels of AOA in the enrichment were determined by 16S rRNA detection.

[0041] The relevant test results and analysis are as follows: Figure 2 and Figure 3The results of monitoring the ammonia oxidation activity of the culture were presented. The results showed that during the entire enrichment culture process, nitrite accumulation showed a stable trend, indicating that the culture had good ammonia oxidation activity; in the early stage of antibiotic treatment, the ammonia oxidation process was relatively slow, with an average passage time of 62 days; as the enrichment progressed, the passage time gradually shortened to 29 days; subsequently, during the microporous filtration and dilution culture process, due to the removal of antibiotic selection pressure, the ammonia oxidation rate was significantly accelerated, and the average passage time was shortened to 16 days.

[0042] Figure 4 Archaea and bacteria in culture amo PCR amplification results of gene A. The results show that after microporous filtration and dilution culture, the PCR amplification map always shows archaea amo A clear band of gene A, indicating that AOA enrichment is highly stable; however, after 6 filtration cultures, PCR amplified bacteria amo The A gene band indicates that some small-sized AOB can pass through the filtration; after further dilution and culture, PCR failed to amplify the bacteria amo The A gene band indicated that the only ammonia oxidizing microorganism in the culture at this time was AOA.

[0043] Figure 5 The trend of the number of AOA and AOB in the culture is shown. Whether it is selective enrichment by antibiotics or microfiltration and dilution culture, AOA becomes the dominant ammonia oxidizing microorganism in the culture system, and its number remains at 4.84×10 5 to 4.97×10 5 copies / mL. In contrast, the number of AOBs increased from the initial 6.50×10 5 The copies / mL gradually decreased until they disappeared completely, which was consistent with the above PCR test results.

[0044] Figure 6 The results of community diversity analysis of enriched AOA showed that AOA of the genus Nitrocosmicus was mainly enriched by antibiotic selection, with an abundance of 50.23%, while AOA enriched by microfiltration mainly belonged to the genus Nitrosotenuis, with an abundance of 40.77%.

[0045] In summary, the present invention breaks through the limitation of the existing technology for enriching a single genus of AOA by combining antibiotic treatment, multiple filtration and dilution culture strategies, and successfully achieves the goal of enriching multiple genera of AOA from a complex sewage environment.

[0046] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for efficiently enriching ammonia-oxidizing archaea in sewage in a step-by-step manner, characterized in that: The following steps are involved: S1, adding the bacterial liquid sample to be enriched to the enrichment medium containing antibiotics for subculture; S2. Perform microporous filtration culture on part of the bacterial solution obtained in step S1 to enrich AOA, and then dilute and subculture the enriched bacterial solution.

2. The method according to claim 1, characterized in that The antibiotic is at least one of ampicillin, carbenicillin, streptomycin, kanamycin and tetracycline.

3. The method according to claim 2, characterized in that The antibiotics consist of ampicillin, carbenicillin, streptomycin, kanamycin and tetracycline, and the use concentration of each antibiotic is 45-55 mg / L.

4. The method according to claim 1, characterized in that The enrichment medium includes 0.9-1.1 g / L NaCl, 0.36-0.44 g / L MgCl2·6H2O, 0.45-0.55 g / L KCl, 0.09-0.11 g / L CaCl2 and 0.18-0.22 g / L KH2PO4, and each liter of the enrichment medium is further added with 1 mL 1 M NH4Cl solution, 2 mL 1 M NaHCO3 solution, 1 mL 0.5M sodium pyruvate solution, 1 mL Fe-NaEDTA solution and 1 mL of trace element solution; wherein the Fe-NaEDTA solution includes 196.2-239.8 g / L EDTA-Na2 and 47.25-57.75 g / L FeSO4·6H2O, the trace element solution includes 1.35-1.65 g / L FeCl2·4H2O, 0.09-0.11 g / L CaCl2 and 0.18-0.22 g / L KH2PO4. MnCl2·4H2O, 0.17~0.21 g / L CoCl2·6H2O, 0.022~0.026 g / L NiCl2·6H2O, 0.015~0.019 g / L CuCl2·2H2O, 0.032~0.040 g / LNaMoO4·2H2O, 0.063~0.077 g / L ZnCl2, 0.056~0.068 g / L HBO3.

5. The method according to claim 1, characterized in that The method of subculturing in step S1 is: monitoring the ammonia nitrogen concentration in the enrichment medium, and when the ammonia nitrogen consumption exceeds 80% to 90%, replacing 70% to 80% of the supernatant with fresh enrichment medium containing antibiotics.

6. The method according to claim 1, characterized in that Step S2 includes the following: S21, utilizing the size difference between AOA and other bacteria to enrich AOA by at least one microfiltration; S22, diluting and subculturing the bacterial solution obtained by enrichment in step S21 by gradually reducing the inoculation ratio.

7. The method according to claim 6, characterized in that In step S21, a 0.45 μm sterile syringe filter is used to perform microfiltration on the bacterial solution.

8. The method according to claim 1, characterized in that By detecting AOA in samples amoA The copy number of the gene was determined, and samples with a larger number of AOA were screened for enrichment.

9. Use of the method according to any one of claims 1 to 8 in enriching ammonia oxidizing archaea.

10. The use according to claim 9, characterized in that: The ammonia oxidizing archaea include Nitrocosmicus AOA and / or Nitrosotenuis AOA.

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