A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide

Abstract

The present invention relates to a method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide, belonging to the technical field of water environment treatment. In the present invention, loofah sponge, an agricultural waste rich in cellulose, is used as a raw material, and a slow-release carbon source material is prepared by freeze-thaw alkali treatment, and then an anaerobic-aerobic composite denitrifying bactericide is constructed using it as a carrier. In the present invention, the slow-release carbon source material serves as a carrier to provide necessary carbon sources and living spaces for denitrifying strains. Aerobic denitrifying strains are enriched and grown on the periphery of the carrier, and anaerobic denitrifying strains are enriched and grown in the center of the carrier. By means of the consumption and utilization of dissolved oxygen by aerobic denitrifying strains on the periphery of the carrier during the denitrification process, an anoxic environment is created for the center of the carrier to avoid the inhibitory effect of excessive dissolved oxygen concentration on anaerobic denitrification. The present invention comprehensively utilizes the synergistic effect of two denitrifying strains and can obtain a significant denitrification and nitrogen removal effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of water environment treatment, and particularly relates to a method for constructing a long-term anaerobic-aerobic composite denitrifying bactericide. Background Art

[0002] With the rapid advancement of industrialization, urbanization, and modern agriculture, a large amount of nitrogen-rich wastewater is discharged into water bodies, causing eutrophication problems in water bodies, seriously affecting the normal physiological functions of aquatic organisms, and even further threatening the drinking water safety of humans. Therefore, how to effectively reduce nitrogen pollution in wastewater has become an urgent task for the country's green and healthy development.

[0003] Biological nitrogen removal technology is currently the most economical, efficient, green, and environmentally friendly method for treating nitrogen pollution and has been widely applied. Its principle is mainly to use nitrifying strains to convert NH4 + into NO3 - and NO2 - , and then use denitrifying strains to gradually reduce NO3 - and NO2 - to N2O, NO, and N2 under the action of a series of biological enzymes. Denitrification is of great significance in the process of wastewater nitrogen removal. According to the different requirements for dissolved oxygen in the reaction process, denitrifying strains can be divided into anaerobic denitrifying strains and aerobic denitrifying strains. The initially isolated denitrifying strains were all anaerobic denitrifying strains, so scholars all believed that the denitrification reaction could only occur under anaerobic conditions. With the continuous in-depth study of denitrifying strains, Dutch scholars Professor Robertson and Kuenen first discovered and proposed aerobic denitrifying strains in 1983. Subsequently, aerobic denitrifying strains of different genera were successively isolated and identified from different environments.

[0004] However, most denitrifying strains in nature belong to anaerobic bacteria, and aerobic denitrifying strains are not dominant species in the environment, so it is difficult to screen and isolate them. In addition, compared with anaerobic denitrifying strains, although aerobic denitrifying strains can carry out denitrification under aerobic conditions, the aerobic denitrification rate is relatively slow, and the initial concentration of nitrogen in the wastewater is required not to be too high. In addition, generally, the denitrification of aerobic denitrifying strains cannot completely convert the nitrogen in the wastewater into N2, and its main product is the greenhouse gas N2O. While the denitrification of anaerobic denitrifying strains mainly produces N2, only a small amount of N2O and NO will be produced.

[0005] Currently, the research on the application of aerobic denitrification nitrogen removal technology mainly focuses on the laboratory bench-scale stage, and there are few reports on the pilot-scale and plant-scale tests. Most of the research uses biological enhancement methods, that is, adding aerobic denitrifying bacteria (groups) to the reactor in the form of bactericide in order to improve the nitrogen removal effect of the reactor. Summary of the Invention

[0006] The object of the present invention is to provide a method for constructing a long - acting anaerobic - aerobic composite denitrifying bactericide. This method combines anaerobic denitrifying strains and aerobic denitrifying strains, uses a slow - release carbon source material as a biological carrier to construct a long - acting anaerobic - aerobic composite denitrifying bactericide, and applies it to the denitrifying system to improve the denitrifying performance of the wastewater treatment system.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A method for constructing a long - acting anaerobic - aerobic composite denitrifying bactericide, comprising the following steps:

[0009] S1. Preparation of slow - release carbon source material: Crush loofah sponge and place it in sodium hydroxide solution. After soaking at room temperature, subject it to repeated freeze - thaw cycles several times, centrifuge to collect the precipitate, wash it thoroughly with tap water until neutral, put it in an oven and dry it at 50 °C to constant weight, and use a 60 - mesh sieve to remove too fine particles. The obtained product is the slow - release carbon source material;

[0010] S2. Compound of denitrifying strains: Inoculate each denitrifying strain into beef extract peptone medium, culture it on a shaker until the bacterial liquid becomes turbid, centrifuge to collect the bacterial cells, and prepare the seed liquid of each denitrifying strain with PBS buffer solution respectively;

[0011] Compound the seed liquids of each anaerobic denitrifying strain according to a ratio, shake and mix them on a shaker to obtain the seed liquid of the compound anaerobic denitrifying strains;

[0012] Compound the seed liquids of each aerobic denitrifying strain according to a ratio, shake and mix them on a shaker to obtain the seed liquid of the compound aerobic denitrifying strains;

[0013] S3. Preparation of anaerobic denitrifying bactericide: Inoculate the seed liquid of the compound anaerobic denitrifying strains into beef extract peptone medium, add sterile slow - release carbon source material, place it in a shaker and culture it at 37 °C and 180 rpm for a fixed time, then centrifuge to remove the supernatant, wash the precipitate by shaking with sterile normal saline in a 60 - mesh nylon net to remove the surface bacteria, and obtain the anaerobic denitrifying bactericide;

[0014] S4. Preparation of anaerobic - aerobic composite denitrifying bactericide: Inoculate the seed liquid of the compound aerobic denitrifying strains into beef extract peptone medium, place it in a shaker and culture it at 28 °C and 180 rpm for 24 h, transfer the anaerobic denitrifying bactericide obtained in step S3 into the culture solution and continue to culture it, so that the aerobic denitrifying strains are loaded on the periphery of the slow - release carbon source material, centrifuge to remove the supernatant, and obtain the anaerobic - aerobic composite denitrifying bactericide;

[0015] S5. Domestication of anaerobic-aerobic composite denitrifying bacteria agent: Transfer the anaerobic-aerobic composite denitrifying bacteria agent obtained in step S4 into a nitrate medium, and place it in a shaker for domestication culture at 30 °C and 100 rpm, so that the aerobic denitrifying strain and the anaerobic denitrifying strain are stably colonized on the periphery and inside of the carrier respectively. Centrifuge to collect the precipitate, and vacuum freeze-dry it with skim milk and glycerol as protective agents to obtain a long-acting anaerobic-aerobic composite denitrifying bacteria agent.

[0016] Furthermore, the solid-liquid ratio of the loofah sponge and the sodium hydroxide solution in step S1 is 1 g: 6 - 8 mL, the concentration of the sodium hydroxide solution is 1 - 3%, the normal-temperature soaking time is 10 - 30 h, the freezing and thawing temperatures are -80 °C and 30 °C respectively, the freezing and thawing times are 4 h and 1 h respectively, and the number of freezing and thawing cycles is 1 - 3 times.

[0017] Furthermore, the denitrifying strains in step S2 include anaerobic denitrifying bacteria (Pseudomonas stutzeri CCTCC HB20082814, Pseudomonas aeruginosa CCTCC AB 93066, Paracoccus yeei CCTCC AB 206665) and aerobic denitrifying bacteria (Pseudomonas sp. CICC 25068, Delftia lacustris CICC 20570). The seed liquid concentration of each denitrifying strain is 10 8 CFU / mL. The compounding volume ratio of the anaerobic denitrifying strain seed liquid is Pseudomonas stutzeri:Pseudomonas aeruginosa:Paracoccus yeei = 3 - 5:3 - 5:5 - 9, and the compounding volume ratio of the aerobic denitrifying strain seed liquid is Pseudomonas sp.:Delftia lacustris = 1 - 3:3 - 7. The seed liquid mixing conditions are a temperature of 30 °C, a rotation speed of 180 rpm, and a time of 15 min. The anaerobic denitrifying bacteria are purchased from the China Center for Type Culture Collection, and the aerobic denitrifying bacteria are purchased from the China National Center for Industrial Culture Collection.

[0018] Furthermore, the liquid loading amount of the beef extract peptone medium in step S3 is 300 mL / 500 mL conical flask, the inoculation amount of the anaerobic denitrifying compound strain seed liquid is 1 - 5%, the solid-liquid ratio of the slow-release carbon source material and the beef extract peptone medium is 1 g:10 - 20 mL, the fixed culture time is 48 - 96 h, and the oscillation washing time is 20 - 40 s.

[0019] Furthermore, the liquid loading amount of the beef extract peptone medium in step S4 is 300 mL / 500 mL conical flask, the inoculation amount of the aerobic denitrifying compound strain seed liquid is 1 - 5%, and the continued culture time is 6 - 18 h.

[0020] Further, the nitrate medium formula described in step S5 is as follows: glucose 10 g / L, potassium nitrate 2 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, pH 7.2 - 7.5. The loading volume of the nitrate medium is 300 mL / 500 mL conical flask, and the acclimation culture time is 3 - 5 d.

[0021] The present invention uses loofah sponge, an agricultural waste rich in cellulose, as a raw material to prepare a slow-release carbon source material through freeze-thaw alkali treatment, and then uses it as a carrier to construct an anaerobic-aerobic composite denitrifying bactericide, which has the following beneficial effects:

[0022] (1) The present invention prepares a slow-release carbon source material by freeze-thaw alkali treatment of the agricultural waste loofah sponge, and uses it as a carrier to provide a necessary living environment and carbon source for denitrifying strains, which not only reduces the carbon supplementation cost of denitrification and nitrogen removal, but also realizes a long-term denitrification and nitrogen removal effect.

[0023] (2) The present invention constructs an anaerobic-aerobic composite denitrifying bactericide with the slow-release carbon source material as a carrier. Aerobic denitrifying strains grow on the periphery of the carrier, and anaerobic denitrifying strains grow in the center of the carrier. By means of the consumption and utilization of dissolved oxygen by aerobic denitrifying strains on the periphery of the carrier during the denitrification process, an anoxic environment is created for the center of the carrier to avoid the inhibitory effect of too high dissolved oxygen concentration on anaerobic denitrification, and the synergistic effect of the two denitrifying strains is comprehensively utilized to obtain a significant denitrification and nitrogen removal effect. Specific embodiments

[0024] The present invention will be further described in detail below in conjunction with embodiments.

[0025] In the following embodiments, anaerobic denitrifying bacteria (Pseudomonas stutzeri CCTCC HB 20082814, Pseudomonas aeruginosa CCTCC AB 93066, Paracoccus denitrificans CCTCC AB 206665) were purchased from the China Center for Type Culture Collection.

[0026] Aerobic denitrifying bacteria (Pseudomonas sp. CICC 25068, Delftia lacustris CICC 20570) were purchased from the China Center for Industrial Culture Collection Management. Embodiment

[0027] A method for constructing a long-term anaerobic-aerobic composite denitrifying bactericide is prepared according to the following steps:

[0028] S1. Preparation of slow-release carbon source material: Crush 100 g of loofah sponge and place it in 700 mL of sodium hydroxide solution with a mass fraction of 2%. Soak it at room temperature for 20 h, freeze it at -80 °C for 4 h, thaw it at 30 °C for 1 h, repeat the freeze-thaw process 2 times, centrifuge to collect the precipitate, wash it thoroughly with tap water until neutral, put it in an oven and dry it at 50 °C to constant weight, and remove the too-fine particles with a 60-mesh sieve. The obtained product is the slow-release carbon source material.

[0029] S2. Preparation of seed liquid of denitrifying strains: Inoculate each denitrifying strain into the beef extract peptone medium respectively, and culture it in a shaker until the bacterial liquid becomes turbid. Centrifuge to collect the bacteria, and prepare the seed liquid of the strains with a bacterial concentration of 10 8 CFU / mL with PBS buffer saline. Mix the anaerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas stutzeri:Pseudomonas aeruginosa:Paracoccus denitrificans = 4:4:7, and put it in a shaker at 30 °C and 180 rpm for 15 min to oscillate and mix to obtain the anaerobic denitrifying compound strain seed liquid. Mix the aerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas:Delftia lacustris = 2:5, and put it in a shaker at 30 °C and 180 rpm for 15 min to oscillate and mix to obtain the aerobic denitrifying compound strain seed liquid.

[0030] S3. Preparation of anaerobic denitrifying agent: Inoculate the anaerobic denitrifying compound strain seed liquid into 300 mL of beef extract peptone medium at a ratio of 3%, add 20 g of sterile slow-release carbon source material, put it in a shaker at 37 °C and 180 rpm for fixed culture for 72 h, then centrifuge to remove the supernatant, and wash the precipitate in a 60-mesh nylon net with sterile normal saline for 30 s to remove the bacteria on its surface to obtain the anaerobic denitrifying agent.

[0031] S4. Preparation of anaerobic-aerobic composite denitrifying agent: Inoculate the aerobic denitrifying compound strain seed liquid into 300 mL of beef extract peptone medium at a ratio of 3%, and culture it in a shaker at 28 °C and 180 rpm for 24 h. Transfer the anaerobic denitrifying agent obtained in step S3 into the culture solution and continue to culture for 12 h to make the aerobic denitrifying strain load on the periphery of the slow-release carbon source material. Centrifuge to remove the supernatant to obtain the anaerobic-aerobic composite denitrifying agent.

[0032] S5. Domestication of anaerobic-aerobic composite denitrifying agent: Transfer the anaerobic-aerobic composite denitrifying agent obtained in step S4 into 300 mL of nitrate medium, and culture it in a shaker at 30 °C and 100 rpm for 4 d to make the aerobic denitrifying strain and anaerobic denitrifying strain stably colonize on the periphery and inside of the carrier respectively. Centrifuge to collect the precipitate, and vacuum freeze-dry it with skim milk and glycerol as protective agents to obtain the long-acting anaerobic-aerobic composite denitrifying and nitrogen-removing agent. Example

[0033] A method for constructing a long - acting anaerobic - aerobic composite denitrifying bactericide is prepared according to the following steps:

[0034] S1. Preparation of slow - release carbon source material: Crush 100 g of loofah sponge and place it in 600 mL of sodium hydroxide solution with a mass fraction of 1%. Soak it at room temperature for 10 h, freeze it at - 80 °C for 4 h, thaw it at 30 °C for 1 h, centrifuge to collect the precipitate, wash it thoroughly with tap water until neutral, put it in an oven and dry it at 50 °C to constant weight, and remove the too - fine particles with a 60 - mesh sieve. The obtained product is the slow - release carbon source material.

[0035] S2. Preparation of denitrifying strain seed liquid: Inoculate each denitrifying strain into the beef extract - peptone medium respectively, and culture it in a shaker until the bacterial liquid becomes turbid. Centrifuge to collect the bacteria, and prepare the bacterial concentration into 10 8 CFU / mL strain seed liquid with PBS buffer salt solution. Mix the anaerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas stutzeri:Pseudomonas aeruginosa:Paracoccus denitrificans = 3:3:5, put it in a shaker and oscillate and mix at 30 °C and 180 rpm for 15 min to obtain the anaerobic denitrifying compound strain seed liquid. Mix the aerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas:Delftia lacustris = 1:3, put it in a shaker and oscillate and mix at 30 °C and 180 rpm for 15 min to obtain the aerobic denitrifying compound strain seed liquid.

[0036] S3. Preparation of anaerobic denitrifying bactericide: Inoculate the anaerobic denitrifying compound strain seed liquid into 300 mL of beef extract - peptone medium at a ratio of 1%, add 30 g of sterile slow - release carbon source material, put it in a shaker and fix - culture at 37 °C and 180 rpm for 48 h, then centrifuge to remove the supernatant, and wash the precipitate in a 60 - mesh nylon net with sterile normal saline by oscillating for 20 s to remove the bacteria on its surface to obtain the anaerobic denitrifying bactericide.

[0037] S4. Preparation of anaerobic - aerobic composite denitrifying bactericide: Inoculate the aerobic denitrifying compound strain seed liquid into 300 mL of beef extract - peptone medium at a ratio of 1%, culture it in a shaker at 28 °C and 180 rpm for 24 h, transfer the anaerobic denitrifying bactericide obtained in step S3 into the culture solution and continue to culture for 6 h, so that the aerobic denitrifying strain is loaded on the periphery of the slow - release carbon source material, and then centrifuge to remove the supernatant to obtain the anaerobic - aerobic composite denitrifying bactericide.

[0038] S5. Domestication of anaerobic-aerobic composite denitrifying bacteria agent: Transfer the anaerobic-aerobic composite denitrifying bacteria agent obtained in step S4 into 300 mL of nitrate medium, place it in a shaker and domestication culture at 30 °C and 100 rpm for 3 d, so that the aerobic denitrifying strain and the anaerobic denitrifying strain are stably colonized on the periphery and inside of the carrier respectively. Centrifuge to collect the precipitate, and vacuum freeze-dry it with skim milk and glycerol as protective agents to obtain a long-acting anaerobic-aerobic composite denitrifying bacteria agent. Example

[0039] A method for constructing a long-acting anaerobic-aerobic composite denitrifying bacteria agent is prepared according to the following steps:

[0040] S1. Preparation of slow-release carbon source material: Crush 100 g of loofah sponge and place it in 800 mL of sodium hydroxide solution with a mass fraction of 3%. Soak it at room temperature for 30 h, freeze it at -80 °C for 4 h, thaw it at 30 °C for 1 h, repeat the freeze-thaw cycle 3 times, centrifuge to collect the precipitate, wash it thoroughly with tap water until neutral, place it in an oven and dry it at 50 °C to constant weight, and remove the too-fine particles with a 60-mesh sieve. The obtained product is the slow-release carbon source material.

[0041] S2. Preparation of denitrifying strain seed liquid: Inoculate each denitrifying strain into beef extract peptone medium respectively, and culture it in a shaker until the bacterial liquid becomes turbid. Centrifuge to collect the bacteria, and prepare the bacteria concentration to be 10 8 CFU / mL of strain seed liquid with PBS buffer salt solution. Mix the anaerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas stutzeri:Pseudomonas aeruginosa:Paracoccus yeei = 5:5:9, place it in a shaker and oscillate and mix at 30 °C and 180 rpm for 15 min to obtain an anaerobic denitrifying compound strain seed liquid. Mix the aerobic denitrifying strain seed liquid according to the volume ratio of Pseudomonas:Delftia lacustris = 3:7, place it in a shaker and oscillate and mix at 30 °C and 180 rpm for 15 min to obtain an aerobic denitrifying compound strain seed liquid.

[0042] S3. Preparation of anaerobic denitrifying bacteria agent: Inoculate the anaerobic denitrifying compound strain seed liquid at a ratio of 5% into 300 mL of beef extract peptone medium, add 15 g of sterile slow-release carbon source material, place it in a shaker and fixedly culture at 37 °C and 180 rpm for 96 h, then centrifuge to remove the supernatant, and oscillate and wash the precipitate in a 60-mesh nylon net with sterile normal saline for 40 s to remove the bacteria on its surface to obtain the anaerobic denitrifying bacteria agent.

[0043] S4. Preparation of anaerobic-aerobic composite denitrifying bacterium agent: Inoculate the seed solution of aerobic denitrifying compound strains into 300 mL of beef extract peptone medium at a ratio of 5%, place it in a shaker and culture at 28 °C and 180 rpm for 24 h. Transfer the anaerobic denitrifying bacterium agent obtained in step S3 into the culture solution and continue to culture for 18 h, so that the aerobic denitrifying strains are loaded on the periphery of the slow-release carbon source material. Centrifuge to remove the supernatant to obtain the anaerobic-aerobic composite denitrifying bacterium agent.

[0044] S5. Domestication of anaerobic-aerobic composite denitrifying bacterium agent: Transfer the anaerobic-aerobic composite denitrifying bacterium agent obtained in step S4 into 300 mL of nitrate medium, place it in a shaker and domesticate and culture at 30 °C and 100 rpm for 5 d, so that the aerobic denitrifying strains and anaerobic denitrifying strains are stably colonized on the periphery and inside of the carrier respectively. Centrifuge to collect the precipitate, and vacuum freeze-dry it with skim milk and glycerol as protectants to obtain a long-acting anaerobic-aerobic composite denitrifying bacterium agent.

[0045] A method for constructing an anaerobic-aerobic composite denitrifying bacterium agent, which is different from Example 1 in that the loofah sponge is not treated by freeze-thaw and alkali treatment.

[0046] A method for constructing an anaerobic-aerobic composite denitrifying bacterium agent, which is different from Example 1 in that the raw material of the slow-release carbon source is corncob.

[0047] A method for constructing an anaerobic composite denitrifying bacterium agent, which is different from Example 1 in that the composite denitrifying bacterium agent does not contain aerobic denitrifying strains.

[0048] Control Example 4

[0049] A method for constructing an aerobic composite denitrifying bacterium agent, which is different from Example 1 in that the composite denitrifying bacterium agent does not contain anaerobic denitrifying strains.

[0050] Using the composite denitrifying bacterium agents prepared in Examples 1 to 3 and Control Examples 1 to 4, a denitrification experiment was carried out. Prepare a low-carbon wastewater with a total nitrogen concentration of 500 mg / L using potassium nitrate as the nitrogen source. Add each bacterium agent to the wastewater respectively, and calculate the denitrification efficiency of each denitrifying bacterium agent by detecting the total nitrogen content in the wastewater after 96 h. The determination of the total nitrogen content refers to HJ 636-2012 "Determination of Total Nitrogen in Water - Alkaline Potassium Persulfate Digestion UV Spectrophotometer", and the results of the denitrification experiment are shown in Table 1.

[0051] Table 1 Test results of total nitrogen content in wastewater

[0052] Test Items Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Total Nitrogen Content (Unit: mg / L) 3.5 24.5 15 125.5 54.5 113 194 Denitrification Efficiency (Unit: %) 99.3 95.1 97.0 74.9 89.1 77.4 61.2

[0053] It can be seen from the above data that when the composite denitrifying bacteria agents prepared in Examples 1 to 3 are used for wastewater denitrification, the total nitrogen content in the wastewater is reduced from 500 mg / L to 3.5 mg / L, 24.5 mg / L, and 15 mg / L respectively. When the composite denitrifying bacteria agents prepared in Comparative Examples 1 to 4 are used for wastewater denitrification, the total nitrogen content in the wastewater is reduced to 125.5 mg / L, 54.5 mg / L, 113 mg / L, and 194 mg / L respectively. When the composite denitrifying bacteria agents prepared in Examples 1 to 3 are used for wastewater denitrification, their denitrification effects are all better than those of the composite denitrifying bacteria agents prepared in Comparative Examples 1 to 4. This shows that loofah sponge is a high-quality slow-release carbon source raw material, and freeze-thaw alkali treatment can improve the carbon supply performance of loofah sponge. At the same time, using the freeze-thaw alkali-treated loofah sponge as a slow-release carbon source material to construct a long-term anaerobic-aerobic composite denitrifying bacteria agent and comprehensively utilizing the synergistic effect of two denitrifying bacteria strains can achieve a significant denitrification effect.

[0054] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide, characterized in that: It includes the following steps: S1. Preparation of slow-release carbon source material: Using loofah sponge as raw material, after alkali treatment, freeze-thaw treatment and drying, the slow-release carbon source material is obtained; S2. Recombination of denitrifying strains: Inoculate each anaerobic denitrifying strain and each aerobic denitrifying strain into the beef extract peptone medium respectively, culture in a shaker until the bacterial liquid becomes turbid, centrifuge to collect the bacterial cells, and prepare the seed liquid of each denitrifying strain with PBS buffer solution respectively; Recombine the seed liquids of each anaerobic denitrifying strain according to a certain proportion, mix them by shaking in a shaker to obtain the seed liquid of the recombined anaerobic denitrifying strains; Recombine the seed liquids of each aerobic denitrifying strain according to a certain proportion, mix them by shaking in a shaker to obtain the seed liquid of the recombined aerobic denitrifying strains; S3. Preparation of anaerobic denitrifying agent: Inoculate the seed liquid of the recombined anaerobic denitrifying strains into the beef extract peptone medium, add sterile slow-release carbon source material, place it in a shaker for fixed culture at 37 °C and 180 rpm, centrifuge to remove the supernatant, wash the precipitate by shaking with sterile normal saline in a nylon mesh to remove the bacterial cells on its surface, and obtain the anaerobic denitrifying agent; S4. Preparation of anaerobic-aerobic composite denitrifying agent: Inoculate the seed liquid of the recombined aerobic denitrifying strains into the beef extract peptone medium, place it in a shaker for culture at 28 °C and 180 rpm for 24 h, transfer the anaerobic denitrifying agent obtained in step S3 into the culture solution for continuous culture, so that the aerobic denitrifying strains are loaded on the periphery of the slow-release carbon source material, centrifuge to remove the supernatant, and obtain the anaerobic-aerobic composite denitrifying agent; S5. Domestication of anaerobic-aerobic composite denitrifying agent: Transfer the anaerobic-aerobic composite denitrifying agent obtained in step S4 into the nitrate medium, place it in a shaker for domestication culture at 30 °C and 100 rpm, so that the aerobic denitrifying strains and anaerobic denitrifying strains are respectively stably colonized on the periphery and inside of the carrier, centrifuge to collect the precipitate, and obtain the long-acting anaerobic-aerobic composite denitrifying and nitrogen-removing agent after vacuum freeze-drying.

2. The method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: The specific preparation method of the slow-release carbon source material described in step S1 is as follows: Crush the loofah sponge and place it in sodium hydroxide solution, soak it at room temperature, perform freeze-thaw treatment several times, centrifuge to collect the precipitate, wash it thoroughly with water until neutral, dry it to constant weight, and remove the too fine particles with a 60-mesh sieve. The obtained product is the slow-release carbon source material.

3. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 2, characterized in that: The solid-liquid ratio of the loofah sponge to the sodium hydroxide solution is 1 g:6 - 8 mL, the concentration of the sodium hydroxide solution is 1 - 3%, the soaking time at room temperature is 10 - 30 h, the freeze-thaw temperatures are -80 °C and 30 °C respectively, the freeze-thaw times are 4 h and 1 h respectively, and the number of freeze-thaw cycles is 1 - 3 times.

4. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: The anaerobic denitrifying bacteria described in step S2 are Pseudomonas stutzeri CCTCC HB 20082814, Pseudomonas aeruginosa CCTCC AB 93066, and Paracoccus denitrificans CCTCC AB 206665. The aerobic denitrifying bacteria are Pseudomonas sp. CICC25068 and Delftia lacustris CICC 20570. The seed liquid concentration of each denitrifying strain is 10 8 CFU / mL. The compounding volume ratio of the anaerobic denitrifying strain seed liquid is Pseudomonas stutzeri CCTCC HB 20082814:Pseudomonas aeruginosa CCTCC AB93066:Paracoccus denitrificans CCTCC AB 206665 = 3-5:3-5:5-9. The compounding volume ratio of the aerobic denitrifying strain seed liquid is Pseudomonas sp. CICC 25068:Delftia lacustris CICC 20570 = 1-3:3-7. The seed liquid mixing conditions are a temperature of 30°C, a rotation speed of 180 rpm, and a time of 15 min.

5. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: The liquid loading amount of the beef extract peptone medium described in step S3 is 300 mL / 500 mL conical flask, the inoculation amount of the seed liquid of the recombined anaerobic denitrifying strains is 1 - 5%, the solid-liquid ratio of the slow-release carbon source material to the beef extract peptone medium is 1 g:10 - 20 mL, the fixed culture time is 48 - 96 h, and the shaking washing time is 20 - 40 s.

6. The method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: The liquid loading amount of the beef extract peptone medium described in step S4 is 300 mL / 500 mL conical flask, the inoculation amount of the seed liquid of the recombined aerobic denitrifying strains is 1 - 5%, and the continuous culture time is 6 - 18 h.

7. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: The nitrate medium formulation described in step S5 is as follows: glucose 10 g / L, potassium nitrate 2 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, pH 7.2 - 7.

5. The liquid loading of the nitrate medium is 300 mL / 500 mL conical flask, and the acclimation culture time is 3 - 5 d.

8. A method for constructing a long-acting anaerobic-aerobic composite denitrifying bactericide according to claim 1, characterized in that: In the vacuum freeze-drying process described in step S5, skim milk and glycerol are used as protectants.