Method for reducing nitrate nitrogen in wastewater into ammonia nitrogen by using double-bacterium inoculant and application of double-bacterium inoculant

Abstract

The invention relates to a method for reducing nitrate nitrogen in wastewater into ammonia nitrogen by using a double-bacterium inoculant and application of the double-bacterium inoculant. The method comprises the following steps: respectively carrying out expanding culture, washing and resuspending on electron donating bacteria and dissimilatory nitrate reduction ammonium-producing bacteria, and then mixing in a co-culture medium for co-culture to form a double-bacteria microbial inoculum; the co-culture medium containing the double-bacterium agent is introduced into wastewater to be treated, and nitrate nitrogen in the wastewater is reduced into ammonia nitrogen in an anaerobic environment. According to the method, nitrate nitrogen in the wastewater can be reduced and converted into energy storage substances with high energy density, the problems of low utilization rate of ammonium nitrogen fertilizer and non-point source pollution can be solved, and the method has important value for nitrogen pollution control and agricultural green sustainable development.

Description

Technical Field

[0001] The present application relates to the technical field of wastewater treatment, and in particular to a method and use of reducing nitrate nitrogen in wastewater to ammonia nitrogen using a dual-bacteria agent. Background Art

[0002] With the development of chip, photovoltaic and other industries, high concentrations of nitrate (NO 3 - ) and nitrite (NO 2 - ) The discharge of wastewater has increased significantly, posing a threat to environmental safety and human health. On the other hand, nitrogen is one of the essential nutrients in the growth and development of crops, and ammonium fertilizer is an important nitrogen source supply. However, the utilization rate of nitrogen fertilizer in my country is only 30%-40% in the season, and ammonium nitrogen fertilizer is easily oxidized to nitrate in the soil through microbial nitrification. Nitrate is easily lost in the soil, which not only causes low nitrogen fertilizer utilization, but also causes non-point source and groundwater pollution.

[0003] Dissimilatory nitrate reduction to ammonium (DNRA) bacteria are widely distributed in ecosystems and soil environments and can reduce nitrate to ammonium (NH 4 + ). However, the low organic matter content in the environment and the insufficient supply of electrons often inhibit the DNRA reaction due to the competition in the denitrification process.

[0004] Therefore, there is a need for an improved method and use for reducing nitrate nitrogen in wastewater to ammonia nitrogen. Summary of the invention

[0005] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0006] On the one hand, the present application provides a method for reducing nitrate nitrogen in wastewater to ammonia nitrogen using a dual-bacteria agent, comprising separately expanding, washing, and resuspending electron-donating bacteria and dissimilatory nitrate-reducing ammonium-producing bacteria, and then mixing them in a co-culture medium to form a dual-bacteria agent; introducing the co-culture medium containing the dual-bacteria agent into the wastewater to be treated to reduce the nitrate nitrogen in the wastewater to ammonia nitrogen under an anaerobic environment.

[0007] As used in this application, the term "electron donating bacteria" refers to bacteria that can use carbon-containing organic matter to donate electrons to their own extracellular environment, including Shewanella oneidensis MR-1, Pseudomonas aeruginosa, Geobacter sulfurreducens, Rhodoferax ferrireducens, Geovibrio ferrireducens, etc.

[0008] As used in this application, the term "dissimilatory nitrate-reducing ammonium-producing bacteria (DNRA bacteria)" refers to bacteria that can use nitrate and nitrite as electron acceptors to reduce them to ammonium and release them outside the body, including Desulfobacter sp., Escherichia coli, Thiobacillus denitrificans, Aeromonas sp., etc.

[0009] As used in this application, the term "wastewater" may also be referred to as sewage, including industrial wastewater, discharged domestic sewage, agricultural wastewater, etc. In this application, sewage may include a carbon source, a nitrogen source, and trace elements, etc., but does not contain highly toxic or highly oxidizing chemicals, such as haloacetic acids, hydrogen peroxide, or its content does not affect the activity of microorganisms. In this application, the initial carbon-nitrogen ratio of the wastewater may be within the range of 0.5-10.0.

[0010] In the present application, the term "carbon source" may include, but is not limited to, sodium acetate, sodium lactate, sodium citrate, glucose and glycerol.

[0011] In this application, the term "nitrogen source" mainly includes nitrate nitrogen, i.e. nitrate (NO 3 - ) (such as sodium nitrate) and nitrite (NO 2 - ).

[0012] In this application, the term "trace elements" may include but is not limited to MgSO 4 、Na 2 -EDTA, FeCl 3 6H 2 O、MnCl 2 ·4H 2 O、Na 2 MoO 4 ·2H 2 O, CuCl 2 ·2H 2O、ZnCl 2 At least one of .

[0013] In an exemplary embodiment, Shewanella MR-1 is cultured in TSA medium; Pseudomonas aeruginosa is cultured in NB medium; Geobacter sulfurreducens is cultured in DSMZ Medium 826 medium; Rhodococcus iron-reducing is cultured in ATCC Medium 1768 medium; and Geovibrio iron-reducing is cultured in DSMZ Medium 826 medium.

[0014] In an exemplary embodiment, Desulfurobacterium is cultured in DSMZ Medium 63 medium; Escherichia coli is cultured in LB medium; Denitrificant Thiobacillus is cultured in DSMZ Medium 113 medium; Aeromonas is cultured in LB medium.

[0015] Optionally, the initial inoculation OD of the two inoculants is 600 The value is 0.05-0.2; for example, 0.05, 0.1 or 0.2, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0016] Optionally, the temperature during the expansion of the two bacterial agents is 30-37°C; for example, 30°C, 33°C or 37°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0017] Optionally, the Shewanella MR-1, Pseudomonas aeruginosa, Escherichia coli, Denitrifying Thiobacillus and Aeromonas are cultured under aerobic conditions; and the iron-reducing Rhodococcus, iron-reducing Vibrio, sulfur-reducing Geobacter and Desulfobacillus are cultured under anaerobic conditions.

[0018] In an exemplary embodiment, the co-culture medium includes nitrate, a buffer solution and trace elements; optionally, the nitrate is NaNO 3 ; Optionally, the buffer solution is selected from KH 2 PO 4 、Na 2 PO 4 One or two of; Optionally, the trace element is selected from Na 2 -EDTA, FeCl 3 6H 2 O、MnCl 2 ·4H 2 O、Na 2 MoO 4 ·2H 2 O, CuCl 2 ·2H 2 O、ZnCl 2 MgSO4 and CaCl 2 One or more of .

[0019] In an exemplary embodiment, the initial OD values ​​of the electron-donating bacteria and the DNRA bacteria in the co-culture medium are 600 The value ratio is 1:1-1:3, and the total initial OD 600 The value is 0.2-0.5, for example, 0.2, 0.3, 0.4 or 0.5, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0020] In an exemplary embodiment, the washing of the electron-donating bacteria and the DNRA bacteria comprises the following steps: centrifuging the expanded bacterial agent and then adding a biological buffer solution for washing.

[0021] Optionally, the number of washing times is 3-5 times, for example, 3, 4 or 5, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0022] Optionally, the biological buffer solution is selected from at least one of a Tris-HCl buffer solution, a HEPES buffer solution and a PBS buffer solution.

[0023] Optionally, the concentration of the biological buffer solution is 20-50 mM; for example, 20 mM, 30 mM, or 50 mM, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0024] Optionally, the pH of the biological buffer solution is 7.0-7.6, for example, 7.0, 7.1, 7.2, 7.3, or 7.4, such as the pH range of 7.0-7.4, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0025] In an exemplary embodiment, when the initial carbon-nitrogen ratio of the wastewater is less than 5:1, in order to enhance the reduction reaction of nitrate nitrogen to ammonia nitrogen in the wastewater, a carbon source can be added to the wastewater or co-culture medium so that the C / N ratio (molar ratio) of the carbon source to the nitrogen source (such as sodium nitrate) in the wastewater is greater than or equal to 5:1, such as within the range of 5:1-10:1, for example, 5:1, 6:1, 8:1 or 10:1, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are equally applicable.

[0026] Optionally, the carbon source is selected from one or more of sodium lactate, sodium citrate, glucose and glycerol.

[0027] In an exemplary embodiment, adding the carbon source further comprises adding a signal molecule to the wastewater or the co-culture medium.

[0028] Optionally, the signal molecule is one or more of C4-AHLs, C6-AHLs and C8-AHLs; the total concentration of the added signal molecules is 1 to 5 μM, for example, 1 μM, 3 μM, or 5 μM, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0029] In an exemplary embodiment, the reduction reaction is performed after deoxygenation by aeration using an inert gas.

[0030] Optionally, the inert gas is one of helium, argon or nitrogen, and the aeration time is 5-20 min, for example, 5 min, 10 min or 20 min, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0031] In an exemplary embodiment, the temperature of the reduction reaction is 25-37°C; for example, 25°C, 30°C, or 37°C, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0032] On the other hand, the present application also provides the use of a dual-bacteria agent in reducing nitrate nitrogen in wastewater to ammonia nitrogen. The dual-bacteria agent is formed by separately expanding, washing, and resuspending electron-donating bacteria and dissimilatory nitrate-reducing ammonium-producing bacteria and then mixing them in a co-culture medium for co-cultivation.

[0033] In an exemplary embodiment, a carbon source and / or a signal molecule is further added to the co-culture medium or the wastewater.

[0034] Quorum sensing (QS) is a communication mechanism between microorganisms. It regulates the microbial community structure and extracellular electron transfer (EET) by secreting signal molecules, and plays an important role in the EET process of microorganisms. In this application, the electrons produced by electron-donating bacteria are transferred to DNRA bacteria through EET through QS regulation, which can significantly increase the NH production of DNRA bacteria. 4 + Performance is an effective strategy to control nitrate nitrogen pollution and improve soil fertility.

[0035] The method of the present application is based on the principle of extracellular electron transfer to enhance biochemical transformation, and provides a new method for regulating the mutual reduction of nitrate (or nitrite) to produce ammonium by double bacteria through quorum sensing effect. The method of the present application can enhance the electron mutualism between electron-donating bacteria and DNRA bacteria, improve the electron utilization rate, and efficiently reduce nitrate nitrogen pollutants into ammonia nitrogen NH with resource and energy properties. 4 + The efficiency of ammonium synthesis in the dual-bacteria system is at least 3 times higher than that of DNRA alone, and the highest nitrate nitrogen selectivity reaches more than 97%.

[0036] The method of the present application can reduce the nitrate nitrogen produced by the nitration reaction to NH in situ without the need to prepare electrode materials and external energy input. 4 + .

[0037] The bacterial composition used in the method of the present application can be applied to farmland soil on a large scale, can utilize a wide range of raw materials, and has the characteristics of green and sustainable application.

[0038] The method of the present application is simple and efficient, and has a wide range of application scenarios.

[0039] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0041] Figure 1 The transmission electron microscope images of the electron-donating bacteria (Shewanella) and DNRA bacteria (Escherichia coli) used in the method for reducing nitrate nitrogen in wastewater to ammonia nitrogen provided in Example 2 of the present application are shown;

[0042] Figure 2 A graph showing the changes in the concentrations of nitrate ammonia and ammonia nitrogen over time when nitrate nitrogen in wastewater is reduced to ammonia nitrogen by using a single DNRA bacterium (Escherichia coli) in Comparative Example 1 and the dual-bacteria bacterial agent used in Example 2 of the present application. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present application more clear, the embodiments of the present application are described in detail below. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other arbitrarily without conflict.

[0044] In the following examples and comparative examples, the operations involved were carried out under conventional conditions or conditions recommended by the manufacturer if the conditions were not specified. The raw materials used were commercially available if the manufacturers and specifications were not specified.

[0045] Comparative Example 1 (only dissimilatory nitrate-reducing ammonium-producing bacteria, i.e., only Escherichia coli)

[0046] The method for reducing nitrate nitrogen in wastewater to ammonia nitrogen provided in this embodiment may include the following steps:

[0047] (1) The dissimilatory nitrate-reducing ammonium-producing bacteria Escherichia coli (purchased from Beina Biotech BNCC357962) were cultured in LB medium. The initial inoculation OD of Escherichia coli was 600 The value was 0.05, and the cells were cultured aerobically at 37°C until the stable phase (OD 600 The value is stable at about 1.0); the Escherichia coli was washed 4 times with 30 mM PBS buffer solution at pH 7.4 and then resuspended to form an Escherichia coli bacterial solution to be used.

[0048] (2) Preparation of culture medium. The composition of the culture medium in this example includes 10 mM NaNO as a simulated nitrogen source. 3 , 20 mM KH 2 PO 4 and 20 mM Na 2 PO 4 A buffer solution composed of 9.6 μM Na 2 -EDTA, 9.0 μM FeCl 3 6H 2 O, 0.1 μM MnCl 2 ·4H 2 O, 1.0 μM Na 2 MoO 4 ·2H 2 O, 0.8 μM CuCl 2 ·2H 2 O, 2.5 μM ZnCl 2 , 0.5 mM MgSO 4 and 0.5 mM CaCl 2 The trace elements composed of E. coli were inoculated into the co-culture medium, and the initial OD 600 The value is 0.15, and the culture is continued until the OD 600 The value is 0.5 or more.

[0049] (3) The culture medium containing Escherichia coli was introduced into the wastewater containing sodium nitrate (the initial carbon-nitrogen ratio was less than 5:1), and glycerol was added as a carbon source so that the C / N (molar ratio) of the carbon source to sodium nitrate was 6:1; argon was used for aeration for 20 min to form an anaerobic environment, and NO 3 - Bioreduction reaction produces ammonia nitrogen NH 4 + .

[0050] The ammonia nitrogen content generated in the water was detected by colorimetry using Nessler's reagent as a colorimetric method. Within a reaction time of 11 hours, the ammonia nitrogen concentration generated was 6.2 mg / L.

[0051] Example 1 (without adding signal molecules)

[0052] The method for reducing nitrate nitrogen in wastewater to ammonia nitrogen provided in this embodiment may include the following steps:

[0053] (1) The electron-donating bacteria Shewanella oneidensis MR-1 (purchased from Beina Biotech BNCC274831) was cultured in TSA medium. The initial inoculation OD of Shewanella oneidensis was 600 The value was 0.1, and the cells were cultured aerobically at 30°C until the stable phase (OD 600 The value was stable at about 1.0); the dissimilatory nitrate-reducing ammonium-producing bacteria Escherichia coli (Escherichiacoli, purchased from Beina Biotech BNCC295132) was cultured in LB medium, and the initial inoculation OD of Escherichia coli was 600 The value was 0.05, and the cells were aerobically cultured at 37°C until the stable phase; Shewanella was washed 3 times with 20 mM Tris-HCl buffer solution at pH 7.2 and then resuspended to form a Shewanella bacterial solution for use; Escherichia coli was washed 4 times with 30 mM PBS buffer solution at pH 7.4 and then resuspended to form an Escherichia coli bacterial solution for use.

[0054] (2) Prepare a co-culture medium. The co-culture medium in this example includes 10 mM NaNO as a simulated nitrogen source. 3 , 20 mM KH 2 PO 4 and 20 mM Na 2 PO 4 A buffer solution composed of 9.6 μM Na 2 -EDTA, 9.0 μM FeCl 3 6H 2 O, 0.1 μM MnCl 2 ·4H 2 O, 1.0 μM Na 2 MoO 4 ·2H 2 O, 0.8 μM CuCl 2 ·2H 2 O, 2.5 μM ZnCl 2 , 0.5 mM MgSO 4 and 0.5 mM CaCl 2 The trace elements of Shewanella were inoculated into the co-culture medium, and the initial OD 600 The value is 0.15; the E. coli liquid is inoculated into the co-culture medium, and the initial OD 600The value is 0.15, and the total OD of the dual bacterial inoculum in the co-culture medium 600 The value is 0.3, and the culture is continued until the OD 600 The value is 0.5 or more.

[0055] (3) The co-culture medium containing the dual bacterial inoculant was introduced into the wastewater containing sodium nitrate (the initial carbon-nitrogen ratio was less than 5:1), and glycerol was added as a carbon source so that the C / N (molar ratio) of the carbon source to sodium nitrate was 6:1; argon was used for aeration for 20 min to form an anaerobic environment, and NO 3 - Bioreduction reaction produces ammonia nitrogen NH 4 + .

[0056] Using Nessler's reagent as a colorimetric agent, the ammonia nitrogen content generated in the water was detected by colorimetry. Within a reaction time of 11 hours, the ammonia nitrogen concentration generated was 24 mg / L, which indicates that the reduction efficiency of the dual-bacteria agent of the present application is significantly greater than the reduction efficiency of the single dissimilatory nitrate-reducing ammonium-producing bacteria (such as Escherichia coli) in Comparative Example 1. The dual-bacteria agent of the present application is more conducive to reducing nitrate ammonia to ammonia nitrogen.

[0057] Example 2

[0058] The method for reducing nitrate nitrogen in wastewater to ammonia nitrogen provided in this embodiment may include the following steps:

[0059] (1) The electron-donating bacteria Shewanella oneidensis MR-1 (purchased from Beina Biotech BNCC274831) was cultured in TSA medium. The initial inoculation OD of Shewanella oneidensis was 600 The value was 0.1, and the cells were cultured aerobically at 30°C until the stable phase (OD 600 The value was stable at about 1.0); the dissimilatory nitrate-reducing ammonium-producing bacteria Escherichia coli (Escherichiacoli, purchased from Beina Biotech BNCC295132) was cultured in LB medium, and the initial inoculation OD of Escherichia coli was 600 The value was 0.05, and the cells were aerobically cultured at 37°C until the stable phase; Shewanella was washed 3 times with 20 mM Tris-HCl buffer solution at pH 7.2 and then resuspended to form a Shewanella bacterial solution for use; Escherichia coli was washed 4 times with 30 mM PBS buffer solution at pH 7.4 and then resuspended to form an Escherichia coli bacterial solution for use.

[0060] (2) Prepare a co-culture medium. The co-culture medium in this example includes 10 mM NaNO as a simulated nitrogen source. 3 , 20 mM KH 2 PO 4 and 20 mM Na2 PO 4 A buffer solution composed of 9.6 μM Na 2 -EDTA, 9.0 μM FeCl 3 6H 2 O, 0.1 μM MnCl 2 ·4H 2 O, 1.0 μM Na 2 MoO 4 ·2H 2 O, 0.8 μM CuCl 2 ·2H 2 O, 2.5 μM ZnCl 2 , 0.5 mM MgSO 4 and 0.5 mM CaCl 2 The trace elements of Shewanella were inoculated into the co-culture medium, and the initial OD 600 The value is 0.15; the E. coli liquid is inoculated into the co-culture medium, and the initial OD 600 The value is 0.15, and the total OD of the dual bacterial inoculum in the co-culture medium 600 The value is 0.3, and the culture is continued until the OD 600 The value is 0.5 or more.

[0061] (3) The co-culture medium containing the dual bacterial inoculant was introduced into the wastewater containing sodium nitrate (the initial carbon-nitrogen ratio was less than 5:1), and glycerol was added as a carbon source to make the C / N (molar ratio) of the carbon source to sodium nitrate 6:1; signal molecules composed of C4-AHLs and C6-AHLs were added at a total concentration of 2 μM (the concentration ratio of the two signal molecules was 1:1); argon was used for aeration for 20 min to form an anaerobic environment, and NO was reduced to 0.1% at 30°C. 3 - Bioreduction reaction produces ammonia nitrogen NH 4 + .

[0062] Figure 2 A graph is given showing the changes in the concentrations of nitrate ammonia and ammonia nitrogen over time when nitrate nitrogen in wastewater is reduced to ammonia nitrogen by using a single DNRA bacterium (Escherichia coli) in Comparative Example 1 and the dual-bacteria bacterial agent used in this example.

[0063] Depend on Figure 2 It can be seen that the dual-bacteria agent of the present application is combined with the signal molecule to enhance the quorum sensing effect, and the generation of ammonia nitrogen is significantly increased. Within the reaction time of 11 hours, the concentration of ammonia nitrogen generated reaches 36 mg / L.

[0064] Example 3

[0065] In this embodiment, the electron-donating bacteria is a combination of Shewanella oneidensis MR-1 (Shewanella oneidensis MR-1, Beina Bio BNCC274831) and Pseudomonas aeruginosa (Pseudomonas aeruginosa, ATCC 9027). When co-cultured, the initial OD of Shewanella inoculation is 600 The value is 0.10, and the initial OD of Pseudomonas aeruginosa inoculation 600 The value is 0.05; the dissimilatory nitrate-reducing ammonium-producing bacteria is Escherichia coli, and the other steps are the same as those in Example 2.

[0066] Example 4

[0067] In this embodiment, the dissimilatory nitrate-reducing ammonium-producing bacteria is a combination of Escherichia coli (BNCC295132, Bio-Tech BNCC295132) and Thiobacillus denitrificans (DSM 12475). When co-cultured, the initial OD of Escherichia coli inoculated was 600 The value is 0.07, and the initial OD of Thiobacillus denitrificans inoculation is 600 The value is 0.08; the electron donating bacteria is Shewanella MR-1, and the other steps are the same as in Example 2.

[0068] For Example 3 and Example 4, the concentrations of ammonia nitrogen generated within a reaction time of 11 hours were measured by colorimetry and were 34 mg / L and 40 mg / L, respectively, indicating that electron-donating bacteria or dissimilatory nitrate-reducing ammonium-producing bacteria can have an enhanced effect of reducing nitrate nitrogen to ammonia nitrogen when a combination of multiple bacterial agents is used.

[0069] In addition, when ammonia nitrogen NH 4 + After being enriched to a certain concentration in water, the pH of the aqueous solution can be adjusted, and the ammonia nitrogen can be collected in the form of ammonia gas through a stripping process, thereby achieving the removal and resource utilization of nitrates and nitrites in the wastewater.

[0070] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Those skilled in the art can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for reducing nitrate nitrogen in wastewater to ammonia nitrogen using a double bacterial agent, characterized in that: The method comprises the steps of respectively expanding, washing and resuspending electron-donating bacteria and dissimilatory nitrate-reducing ammonium-producing bacteria, and then mixing them in a co-culture medium and culturing them together to form a dual-bacteria inoculant; and introducing the co-culture medium containing the dual-bacteria inoculant into wastewater to be treated, so as to reduce nitrate nitrogen in the wastewater to ammonia nitrogen under an anaerobic environment.

2. The method according to claim 1, wherein the initial OD of the electron donating bacteria and the dissimilatory nitrate reducing ammonium producing bacteria in the co-culture medium is 600 The ratio of the values ​​is 1:1-1:3; and / or, The total initial OD of the dual bacterial inoculum in the co-culture medium 600 The value is in the range of 0.2-0.

5.

3. The method according to claim 1, wherein the electron-donating bacteria is selected from one or more of Shewanella oneidensis MR-1, Pseudomonas aeruginosa, Geobacter sulfurreducens, Rhodoferax ferrireducens and Geovibrio ferrireducens; and / or, The dissimilatory nitrate-reducing ammonium-producing bacteria are selected from one or more of Desulfobacter sp., Escherichia coli, Thiobacillus denitrificans and Aeromonas sp.

4. The method according to claim 3, wherein Shewanella MR-1 is cultured in TSA medium; Pseudomonas aeruginosa is cultured in NB medium; Geobacter sulfurreducens is cultured in DSMZ Medium 826 medium; Rhodococcus iron-reducing is cultured in ATCC Medium 1768 medium; and Vibrio iron-reducing is cultured in DSMZ Medium 826 medium; the initial inoculation OD of the electron-giving bacteria is 600 The value is in the range of 0.05-0.2, and the expansion temperature is 30-37℃; Desulfobacillus was cultured in DSMZ Medium 63 medium; Escherichia coli was cultured in LB medium; Denitrifying Thiobacillus was cultured in DSMZ Medium 113 medium; Aeromonas was cultured in LB medium; the initial inoculation OD of the dissimilatory nitrate-reducing ammonium-producing bacteria was 600 The value is in the range of 0.05-0.2, and the expansion temperature is 30-37℃.

5. The method according to claim 1, wherein the co-culture medium comprises nitrate, a buffer solution and trace elements; Optionally, the nitrate is NaNO3; Optionally, the buffer solution is selected from one or both of KH2PO4 and Na2PO4; Optionally, the trace elements are selected from one or more of Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, ZnCl2, MgSO4 and CaCl2.

6. The method according to claim 1, wherein the washing comprises washing the electron donating bacteria and the dissimilatory nitrate-reducing ammonium-producing bacteria 3-5 times respectively using a biological buffer solution; Optionally, the biological buffer solution is selected from at least one of a Tris-HCl buffer solution, a HEPES buffer solution and a PBS buffer solution; Optionally, the concentration of the biological buffer solution is 20-100 mM; Optionally, the pH of the biological buffer solution is 7.0-7.

6.

7. The method according to any one of claims 1 to 6, wherein the method further comprises adding a carbon source to the co-culture medium or the wastewater to be treated so that the molar ratio of carbon to nitrogen in the wastewater is greater than or equal to 5:1; Optionally, the carbon source is selected from one or more of sodium lactate, sodium citrate, glucose and glycerol.

8. The method according to claim 7, wherein after adding the carbon source, it further comprises adding a signal molecule to the co-culture medium or the wastewater to be treated; Optionally, the signal molecule is selected from one or more of C4-AHLs, C6-AHLs and C8-AHLs; Optionally, the signal molecule is added at a total concentration of 1-5 μM.

9. Use of a double bacterial agent in reducing nitrate nitrogen in wastewater to ammonia nitrogen, characterized in that: The dual-bacteria inoculant is formed by respectively expanding, washing, and resuspending the electron-donating bacteria and the dissimilatory nitrate-reducing ammonium-producing bacteria, and then mixing and culturing them together in a co-culture medium.

10. The use according to claim 9, wherein a carbon source and / or a signal molecule is further added to the co-culture medium or the wastewater.

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