Novel enrichment and separation method applied to nitrous oxide reducing bacteria

Abstract

The invention discloses a novel enrichment and separation method applied to nitrous oxide reducing bacteria. The enrichment and separation method comprises the following steps: adding activated sludge into an enrichment reactor for enrichment and domestication, adding enrichment nutrient substances once a week, automatically supplying N2O to realize that the concentration of the N2O in the enrichment reactor is 0.1-2.0% of the volume of the enrichment reactor in real time, maintaining an anoxic environment, and performing domestication at intervals in the domestication process. The method comprises the following steps: performing sample collection and DNA extraction on a microorganism sample in a reactor, performing abundance quantification on a nosZ gene for coding N2O reductase NOS through a real-time fluorescent quantitative polymerase chain reaction, and judging whether N2O reducing bacteria in an enrichment reactor are successfully enriched or not by judging whether the abundance of the nosZ gene meets the requirement or not and evaluating the N2O reducing capacity and / or N2O reducing potential of the nosZ gene. According to the method provided by the invention, the enrichment efficiency of N2O reducing bacteria can be improved, and an accurate N2O reduction process identification method is established.

Description

Technical Field:

[0001] The present invention relates to the field of environmental microbiology technology, and particularly relates to a new enrichment and separation method applied to nitrous oxide-reducing bacteria. Background Art:

[0002] As a strong greenhouse gas, the greenhouse effect potential of N2O is nearly 300 times that of CO2. The emission of N2O caused by biological nitrogen removal in sewage treatment plants is greater than the initial expectation, which has attracted people's attention. N2O is a by-product of nitrification mediated by ammonia-oxidizing autotrophic microorganisms and also an intermediate product of denitrification mediated by heterotrophic denitrifying bacteria.

[0003] Currently, most of the research on N2O-reducing microorganisms in the environment still stays on the research of the community structure and biodiversity of environmental samples. The enrichment and research of pure bacteria with specific functions can better reflect the environmental value of microorganisms. Enriching activated sludge with N2O emission reduction through continuous cultivation is of great significance for applying N2O-reducing bacteria to emission reduction in the sewage treatment industry. Summary of the Invention:

[0004] The present invention solves the problems existing in the prior art and provides a new enrichment and separation method applied to nitrous oxide-reducing bacteria. The method proposed by the present invention can improve the enrichment efficiency of N2O-reducing bacteria, especially the enrichment of nosZ cladeII-type reducing bacteria with higher affinity for N2O, and establish an accurate method for identifying the N2O reduction process.

[0005] The object of the present invention is to provide a new enrichment and separation method applied to nitrous oxide-reducing bacteria, including the following steps: adding activated sludge into an enrichment reactor for enrichment and domestication, adding enrichment nutrients once a week, automatically supplying to make the N2O concentration in the enrichment reactor reach 0.1%-2.0% of the volume of the enrichment reactor in real time, and maintaining an anoxic environment. During the domestication process, samples of the internal microorganisms of the enrichment reactor are collected and DNA is extracted at regular intervals. The abundance of the nosZ gene encoding nitrous oxide reductase NOS is quantitatively analyzed by real-time fluorescence quantitative polymerase chain reaction (qPCR). Whether the enrichment of N2O-reducing bacteria in the enrichment reactor is successful is determined by evaluating its N2O reduction ability and / or N2O reduction potential based on whether the abundance of the nosZ gene meets the requirements.

[0006] Preferably, the composition ratio of the enriched nutrients is as follows: KH2PO4 1.0064 mg / L, NaOH 0.02 mg / L, MgSO4·7H2O 0.5177 mg / L, CH3COONa·3H2O 6.1608 mg / L, NH4Cl 0.7116 mg / L, Yeast extract (yeast extract) 2 mg / L, trace elements 2 mL / L, and the balance is water; the composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

[0007] Preferably, the mass ratio of the enriched nutrients added each time to the mass of the activated sludge is ≥1.5. Further preferably, the mass ratio of the enriched nutrients added each time to the mass of the activated sludge is 2:1.

[0008] Preferably, the N2O concentration in the enrichment reactor reaches 1.0% of the volume of the enrichment reactor.

[0009] After adding N2O, the N2O concentration in the enrichment reactor is adjusted to 0.1%-2.0% of the volume of the enrichment reactor. The N2O concentrations are 0, 0.1%, 0.2%, 0.5%, 1.0%, and 2.0% respectively. Maintaining a low N2O concentration is beneficial to the enrichment of nosZ Clade II bacteria, and the enrichment reactor is kept in an anoxic state.

[0010] The enrichment reactor is set up as six serum bottles with different headspace N2O concentration gradients, namely 0, 0.1, 0.2, 0.5, 1.0, and 2.0%. Maintaining a low N2O concentration is beneficial to the enrichment of nosZ Clade II bacteria. The serum bottles need to be kept in an anoxic state. Nitrogen gas is aerated into the bottles for 10 minutes every month, and then sealed with a rubber stopper. N2O and enriched nutrients are injected into the headspace through a syringe.

[0011] Preferably, the specific steps for determining whether the N2O-reducing bacteria in the enrichment reactor are successfully enriched are as follows: The nosZ gene encoding the NOS enzyme and the nir gene are measured by qPCR. The primers used are the nosZ gene measurement primers and the nirS and nirK gene measurement primers. Observe the gene copy number and ratio at the pre-culture and post-culture stages of qPCR. If the copy number after enrichment culture is more than twice that at the initial stage, it indicates that the N2O-reducing bacteria are successfully enriched. If the ratio of (nirS + nirK) / (nosZcladeI + nosZcladeII) decreases, it indicates that the reduction potential of the N2O-reducing bacteria after enrichment has increased.

[0012] Further preferably, the specific steps for evaluating the N2O reduction potential are as follows: First, DNA is extracted, and the nosZ gene encoding the NOS enzyme is measured by qPCR. The primers used are the nosZ gene measurement primers. By measuring the activated sludge at different cultivation stages, if the nosZ Clade I and II types increase, it indicates an increase in the N2O reduction potential. The increase in the nosZ Clade II type, which has a higher affinity for N2O, can better illustrate the success of enrichment.

[0013] Preferably, during the domestication process, every other month, microbial samples inside the reactor are collected and DNA is extracted.

[0014] Preferably, the evaluation of the N2O reduction ability is as follows: Every other month, the reduction ability of N2O-reducing bacteria in the activated sludge is characterized by continuously detecting the N2O gas concentration for 24 hours.

[0015] Further preferably, the specific steps for evaluating the N2O reduction ability are as follows: Every other month, after centrifuging the activated sludge, it is washed with PBS buffer solution and then centrifuged again. The lower-layer sludge is taken, and a denitrification medium and a saturated N2O solution are added to the lower-layer sludge. The volume ratio of the activated sludge to the denitrification medium is 1:20, and the volume ratio of the activated sludge to the denitrification medium is 7.5:1. After sealing, the N2O degradation kinetics is measured. If the reduction ability continuously increases or remains stable, it proves the successful enrichment of N2O-reducing bacteria in the enrichment reactor. The concentration of the saturated N2O solution is 150 μmol / L.

[0016] Even further preferably, the composition ratio of the denitrification medium is as follows: KNO3 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.6 g / L, succinic acid 2.8 g / L, Na2HPO4 1.05 g / L, trace elements 2 mL, and the balance is water. After preparation, it is autoclaved at 121 °C; the composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

[0017] The present invention also protects the application of the enrichment and separation method in the enrichment of nitrous oxide-reducing bacteria.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The method proposed by the present invention can effectively enrich N2O-reducing bacteria, improving the enrichment efficiency compared with the existing methods, especially for the enrichment of nosZ Clade II bacteria with higher affinity for N2O, and the N2O reduction ability of the sludge is good.

[0020] 2. The enrichment and separation method proposed by the present invention uses automatic supply of N2O gas and organic nutrients to domesticate activated sludge bacteria, enriching heterotrophic N2O-reducing bacteria that reduce N2O under the conditions of organic matter and N2O supply. The bacteria inside the domesticated activated sludge are separated and purified by gradient dilution and anaerobic method, and finally the nosZ gene encoding N2O reductase NOS in the bacteria is determined by polymerase chain reaction (PCR) and real-time fluorescence quantitative polymerase chain reaction (qPCR) to determine that the obtained bacteria are N2O-reducing bacteria. The present invention is expected to become the technical support for the research of N2O "source" and "sink", and is also one of the key technologies to optimize the N2O reduction process, with great development potential in practical engineering. Description of the Drawings:

[0021] Figure 1 Schematic diagram of the N2O-reducing bacteria enrichment device for the N2O-reducing bacteria enrichment and separation method of the present invention:

[0022] Figure 2 Fitting curve of the N2O reduction ability of sludge at different N2O concentrations in the enrichment bottle in Example 1;

[0023] Figure 3 Michaelis-Menten equation fitting curve of the N2O reduction kinetics of sludge domesticated with different concentrations of N2O in Example 1;

[0024] Figure 4 Change of the nosZ gene abundance per unit volume of sludge and the change of the ratio of (nirS + nirK) / (nosZcladeI + nosZcladeII) during the enrichment process of different concentrations of N2O in Example 1;

[0025] Description of the reference numerals: 1. Syringe; 2. Sealing plug; 3. Activated sludge; 4. N2O gas storage tank; 5. Concentrated nutrient storage tank; 6. Image output device; 7. Micro respiratory system; 8. Magnetic stirrer; 9. Glass tube; 10. N2O probe; 11. Fixed bracket; 12. Incubator shaker. Detailed Embodiments:

[0026] The following examples are further descriptions of the present invention, rather than limitations on the present invention.

[0027] Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents in this article are all commercially available products commonly used in this technical field.

[0028] As Figure 1 shown, the following method for enriching and separating N2O-reducing bacteria is realized by an N2O-reducing bacteria enrichment device. Add activated sludge 3 to the enrichment reactor, seal it with a sealing plug 2, and carry out enrichment and domestication. Add enrichment nutrients through a syringe 1 every week. N2O is supplied by an N2O gas storage tank 4, and N2O is automatically supplied to achieve an N2O concentration of 0.1%-2.0% of the volume of the enrichment reactor in real time. The enrichment nutrients are supplied by a concentrated nutrient storage tank 5, and an anoxic environment is maintained. During the domestication process, samples of the microorganisms inside the reactor are collected and DNA is extracted at regular intervals.

[0029] After domestication for a certain period of time, use a sterilized pipette tip to aspirate 10 mL of microbial liquid from the enrichment reactor, extract DNA and perform qPCR reaction on it using a soil DNA extraction kit, and quantify the abundance of the nosZ functional gene encoding NOS enzyme. After each DNA collection, the sludge needs to be concentrated, that is, the sludge is transferred to a sterile 50 mL centrifuge tube and centrifuged to remove the supernatant, and then the precipitate is transferred to an enrichment bottle and enrichment nutrients are added, and N2 is aerated again to achieve an anoxic state inside the bottle. The device is placed in a culture shaker 12 at 25°C and 180 rpm for cultivation.

[0030] After culturing for a certain period of time, take 0.3 mL of the activated sludge in the enrichment bottle, add 0.7 mL of PBS buffer solution, shake for 30 s, then centrifuge at 6000 rpm for 1 min, remove the supernatant and add PBS buffer solution again, repeat the washing once, and finally take the lower layer of sludge and add it to a 6 mL glass tube 9. Add 6 mL of denitrification medium and 40 μL of saturated N2O solution to the glass tube. The saturated N2O solution is provided by inserting an N2O probe 10 in the micro gas system 7 into the glass tube 9. The N2O probe 10 is fixed by a fixing bracket 11, and a magnetic stirrer 8 is used to mix evenly. After sealing and waiting for the baseline to stabilize, measure the degradation kinetics of N2O three times. Through the image output device 6, a kinetic image can be obtained, so as to determine whether the N2O-reducing bacteria are successfully enriched.

[0031] In the following embodiments, a method for enriching and separating nitrous oxide-reducing bacteria includes the following steps:

[0032] (1) Add the secondary sedimentation tank sludge of the sewage treatment plant to the enrichment reactor (enrichment bottle) for enrichment and domestication, and manually transport nutrients through the enrichment device regularly. Inject and add the enrichment nutrients from the top rubber stopper with a syringe once a week, and automatically supply N2O to achieve the N2O concentration in the enrichment reactor to 0.1%-2.0% of the volume of the enrichment reactor in real time; during the domestication process, every 1 month, centrifuge the sediment to remove the excess supernatant, replenish the culture medium to the initial state, and collect the sludge for DNA extraction;

[0033] The composition ratio of the enrichment nutrients is: KH2PO4 1.0064 mg / L, NaOH 0.02 mg / L, MgSO4·7H2O 0.5177 mg / L, CH3COONa·3H2O 6.1608 mg / L, NH4Cl 0.7116 mg / L, Yeast extract (yeast extract) 2 mg / L, and trace elements 2 mL / L. The composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, citric acid 10 g / L;

[0034] (2) Every other month, characterize the reduction ability of N2O-reducing bacteria in the sludge by continuously detecting the N2O gas concentration in the headspace for 24 hours; take the sludge every month to measure the reduction ability of N2O per unit of sludge by microelectrode. After taking 0.3 mL of sludge and centrifuging it at 6000 rpm for 1 minute, add PBS buffer solution to wash and then centrifuge again to remove the supernatant, repeat the washing once, and finally take the lower-layer sludge and add it to a 6 mL glass tube, then add 6 mL of denitrification medium and 40 μL of saturated N2O solution, seal it and measure the N2O degradation kinetics 3 times. If the reduction ability continues to increase or remains stable, it proves that the N2O-reducing bacteria are successfully enriched;

[0035] The composition ratio of the denitrification medium is: KNO3 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.6 g / L, succinic acid 2.8 g / L, Na2HPO4 1.05 g / L, trace elements 2 mL. The composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, citric acid 10 g / L; after preparation, autoclave at 121 °C;

[0036] (3) Evaluate the N2O reduction potential of the cultured sludge: First, extract DNA and measure the nosZ gene encoding the NOS enzyme using qPCR with the primers for nosZ gene determination. By measuring the sludge at different culture stages, if the nosZ Clade I and II types increase, it indicates an increase in the N2O reduction potential. In particular, the increase in the nosZ Clade II type, which has a higher affinity for N2O, can better demonstrate successful enrichment.

[0037] The enrichment bottles are set as serum bottles with 6 different headspace N2O concentration gradients, namely 0, 0.1, 0.2, 0.5, 1.0, and 2.0%, respectively. Maintaining a low N2O concentration is beneficial for the enrichment of nosZ Clade II type bacteria. The serum bottles need to be kept in an anoxic state. Aerate N2 gas into the bottles for 10 minutes every month, then seal with a rubber stopper, and inject N2O and nutrients into the headspace through a syringe.

[0038] When conducting N2O reduction kinetics measurements, after adding nutrients and N2O, collect 1 mL of headspace gas through a syringe at 0, 2 h, 5 h, 12 h, and 24 h, measure the air pressure in the bottle with a barometer, dilute the collected gas to 20 mL with nitrogen, and then measure the N2O concentration with a GC.

[0039] When conducting microelectrode measurements of the N2O reduction kinetics of the sludge, load the sludge washed with PBS and the denitrification medium into a 6 mL glass bottle, then seal with a sealing film, inject a saturated N2O solution with a trace syringe to make the N2O concentration 120 μmol / L. When the probe measures that the dissolved N2O concentration decreases to a lower level and stabilizes, add the saturated N2O solution again until it decreases to a lower level. Add it 3 times in total, and finally obtain the time-concentration curve and get the kinetic parameters by fitting the Michaelis equation.

[0040] The microorganism inoculated in the following examples is the activated sludge from the secondary sedimentation tank of a sewage treatment plant.

[0041] Example 1

[0042] A method for enriching and separating nitrous oxide-reducing bacteria, comprising the following steps:

[0043] (1) Activated sludge from the secondary sedimentation tank of a sewage treatment plant was used. 10 mL of activated sludge was inoculated into an enrichment bottle at room temperature for acclimation and enrichment. The enrichment bottle was adjusted to an anoxic state by aerating with N2 for 10 min. N2O was added according to the percentage of the enrichment bottle volume, and the culture medium was injected. N2O was automatically supplied to achieve an N2O concentration of 0.1%-2.0% of the enrichment reactor volume in real time (N2O concentrations were 0, 0.1%, 0.2%, 0.5%, 1.0%, 2.0%). The enrichment culture medium was added once a week, and the mass ratio of the enrichment culture medium to the activated sludge was 2:1.

[0044] The formula of the enrichment culture medium was: KH2PO4 1.0064 mg / L, NaOH 0.02 mg / L, MgSO4·7H2O 0.5177 mg / L, CH3COONa·3H2O 6.1608 mg / L, NH4Cl 0.7116 mg / L, Yeast extract 2 mg / L, trace elements 2 mL / L, and the balance was water; the composition ratio of the trace elements was: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

[0045] The purpose of acclimation was to enrich N2O-reducing bacteria adapted to N2O as a nitrogen source, especially the nosZ clade II type of reducing bacteria with higher N2O affinity. After acclimation for one month using the above method, the enrichment of N2O-reducing bacteria could be initially observed. 10 mL of microbial liquid was aspirated from the enrichment bottle using a sterilized pipette tip, and its DNA was extracted and qPCR reaction was carried out using a soil DNA extraction kit. The abundance of the nosZ functional gene encoding the NOS enzyme was quantitatively analyzed. Microbial samples were collected, DNA was extracted, and nosZ functional gene quantification were carried out every month. When it was found that the nosZ functional gene increased by more than 2 times compared with the initial value and was stable, the enrichment of N2O-reducing bacteria was completed. The change in the abundance of the nosZ functional gene was as Figure 4 shown. After each DNA collection, the sludge needed to be concentrated, that is, the sludge was transferred to a sterile 50 mL centrifuge tube and centrifuged to remove the supernatant, and then the precipitate was transferred to the enrichment bottle and nutrient solution was added, and N2 aeration was used again to achieve an anoxic state in the bottle. The device was placed in a shaker at 25 °C and 180 rpm for cultivation.

[0046] (2) The N2O reduction rate of the system was evaluated, that is, by measuring the removal rate of N2O in the headspace of the bottle, and the rate results were as described Figure 3As shown. After aeration with N2, different percentages by volume of N2O were added. Gas samples were collected at 0, 2 h, 4 h, 8 h, 12 h, and 24 h. Before and after each gas sampling, the air pressure was recorded using a barometer. The N2O concentration in the samples was determined by gas chromatography. The N2O reduction rate graph is as shown in Figure 2 shown.

[0047] (3) Take 0.3 mL of the sludge in the enrichment bottle. After adding 0.7 mL of PBS buffer solution, shake for 30 s, then centrifuge at 6000 rpm for 1 min. After removing the supernatant, add PBS buffer solution again and repeat the washing once. Finally, take the lower-layer sludge and add it to a 6-mL glass tube. Add 6 mL of denitrification medium and 40 μL of saturated N2O solution to the glass tube. After sealing and waiting for the baseline to stabilize, measure the degradation kinetics of N2O three times. The degradation kinetics is as shown in the specification Figure 3 shown.

[0048] The composition ratio of the denitrification medium is as follows: KNO3 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.6 g / L, succinic acid 2.8 g / L, Na2HPO4 1.05 g / L, trace elements 2 mL, and the balance is water. After preparation, it is autoclaved at 121 °C; the composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

[0049] (4) Evaluate the N2O reduction potential of the sludge, that is, measure its nosZ clade I, nosZ clade II, nirS, and nirK functional genes by qPCR. The primers used for the measurement are shown in Table 1 below. The qPCR results are as shown in Figure 4 shown.

[0050]

[0051] The description of the above embodiments is only used to help understand the technical solution and its core idea of the present invention. It should be noted that for those skilled in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A new enrichment and isolation method applied to nitrous oxide-reducing bacteria, characterized in that, It includes the following steps: adding activated sludge into an enrichment reactor for enrichment and domestication, adding enrichment nutrients once a week, automatically supplying N2O in real time to achieve an N2O concentration in the enrichment reactor of 0.1%-2.0% of the volume of the enrichment reactor, and maintaining an anoxic environment. During the domestication process, at regular intervals, collect microbial samples inside the enrichment reactor and extract DNA. Quantify the abundance of the nosZ gene encoding N2O reductase NOS therein by real-time fluorescence quantitative polymerase chain reaction. Determine whether the N2O-reducing bacteria in the enrichment reactor are successfully enriched by evaluating their N2O reduction ability and / or N2O reduction potential based on whether the nosZ gene abundance meets the requirements.

2. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 1, characterized in that, The composition ratio of the enrichment nutrients is as follows: KH2PO4 1.0064 mg / L, NaOH 0.02 mg / L, MgSO4·7H2O 0.5177 mg / L, CH3COONa·3H2O 6.1608 mg / L, NH4Cl 0.7116 mg / L, Yeast extract 2 mg / L, trace elements 2 mL / L, and the balance is water; the composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

3. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 1 or 2, characterized in that, The N2O concentration in the enrichment reactor reaches 1.0% of the volume of the enrichment reactor.

4. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 1, characterized in that, The specific steps for determining whether the N2O-reducing bacteria in the enrichment reactor are successfully enriched are as follows: use qPCR to measure the nosZ gene encoding NOS enzyme and the nir gene, and the primers used are the nosZ gene measurement primer and the nirS and nirK gene measurement primers respectively. Observe the gene copy number and ratio in the pre-culture and post-culture stages of qPCR. If the copy number after enrichment culture is more than 2 times that of the initial stage, it indicates that the N2O-reducing bacteria are successfully enriched. If the ratio of (nirS + nirK) / (nosZcladeI + nosZcladeII) decreases, it indicates that the reduction potential of the enriched N2O-reducing bacteria has increased.

5. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 4, wherein, The specific steps for evaluating the N2O reduction potential are as follows: First, extract DNA, measure the nosZ gene encoding NOS enzyme by qPCR, and the primer used is the nosZ gene measurement primer. By measuring the activated sludge at different stages of culture, if nosZ CladeI and II types increase, it indicates that the N2O reduction potential increases. The increase of nosZ CladeII type with higher affinity for N2O further indicates successful enrichment.

6. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 1, characterized in that, During the domestication process, every month, collect microbial samples inside the reactor and extract DNA.

7. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 1, characterized in that, The evaluation of the N2O reduction ability is: every month, characterize the reduction ability of the N2O-reducing bacteria in the activated sludge by continuously detecting the N2O gas concentration for 24 hours.

8. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 7, characterized in that, The specific steps for evaluating the N2O reduction ability are as follows: Every other month, take the activated sludge, centrifuge it, add PBS buffer solution to wash it and then centrifuge again. Take the lower-layer sludge, add denitrification medium and saturated N2O solution to the lower-layer sludge. The volume ratio of the activated sludge to the denitrification medium is 1:20, and the volume ratio of the activated sludge to the denitrification medium is 7.5:

1. After sealing, measure the N2O degradation kinetics. If the reduction ability continues to increase or remains stable, it proves that the N2O-reducing bacteria in the enrichment reactor are successfully enriched.

9. The novel enrichment and isolation method for nitrous oxide-reducing bacteria according to claim 8, characterized in that, The composition ratio of the denitrification medium is as follows: KNO3 1 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.6 g / L, succinic acid 2.8 g / L, Na2HPO4 1.05 g / L, trace elements 2 mL, and the balance is water. After preparation, it is sterilized under high pressure at 121 °C; the composition ratio of the trace elements is: FeSO4·7H2O 10 g / L, ZnSO4·7H2O 2 g / L, CuSO4·7H2O 4 g / L, NaMoO4·2H2O 0.5 g / L, MnCl2·4H2O 0.1 g / L, H3BO4 0.1 g / L, Na2SeO3 0.3 g / L, and citric acid 10 g / L.

10. Use of the enrichment and separation method according to claim 1 in the enrichment of nitrous oxide-reducing bacteria.

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