Methods and applications of using dual-bacterial inoculants to reduce nitrate nitrogen to ammonia nitrogen in wastewater.

Abstract

This application relates to a method and its application for reducing nitrate nitrogen to ammonia nitrogen in wastewater using a dual-bacterial inoculant. The method involves separately expanding, washing, and resuspending electron-donating bacteria and dissimilatory nitrate-reducing ammonium-producing bacteria, then mixing them in a co-culture medium to form a dual-bacterial inoculant; introducing the co-culture medium containing the dual-bacterial inoculant into the wastewater to be treated, and reducing nitrate nitrogen to ammonia nitrogen in the wastewater under anaerobic conditions. This method can reduce nitrate nitrogen in wastewater into a high-energy-density energy storage substance, and can also alleviate the problems of low utilization rate of ammonium nitrogen fertilizer and non-point source pollution, which is of great value for nitrogen pollution control and green sustainable agricultural development.

Description

Technical Field

[0001] This application relates to the field of wastewater treatment technology, and more particularly to a method and application for reducing nitrate nitrogen in wastewater to ammonia nitrogen using a dual-bacterial agent. Background Technology

[0002] With the development of industries such as chips and photovoltaics, it contains high concentrations of nitrates (NO3). - ) and nitrite (NO2) - The significant increase in wastewater discharge poses a threat to environmental safety and human health. On the other hand, nitrogen is an essential nutrient for crop growth and development, and ammonium fertilizers are a crucial nitrogen source. However, the utilization rate of nitrogen fertilizer in my country during the current season is only 30%-40%. Ammonium nitrogen fertilizers are easily oxidized into nitrates in the soil through microbial nitrification. Nitrates are easily lost from the soil, leading not only to low nitrogen fertilizer utilization but also to non-point source and groundwater pollution.

[0003] Dissimilatory nitrate-reducing ammonium (DNRA) bacteria are widely distributed in ecosystems and soil environments, capable of reducing nitrates to ammonium (NH4+) through respiration. + However, the low organic matter content and insufficient electron supply in the environment often inhibit the DNRA reaction due to the competitive effect of the denitrification process.

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

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

[0006] In one aspect, this application provides a method for reducing nitrate nitrogen in wastewater to ammonia nitrogen using a dual-bacterial 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-bacterial agent; introducing the co-culture medium containing the dual-bacterial agent into the wastewater to be treated, so as to reduce nitrate nitrogen in the wastewater to ammonia nitrogen under anaerobic conditions.

[0007] As used in this application, the term "electron-donating bacteria" refers to bacteria capable of donating electrons to their extracellular environment using carbon-containing organic matter, including Shewanella MR-1 ( Shewanella oneidensis MR-1), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), sulfur-reducing bacteria ( Geobacter sulfurreducens Iron-reducing red bacteria () Rhodoferax ferrireducens Iron-reducing Vibrio ( Geovibrio ferrireducens )wait.

[0008] As used in this application, the term "dissimilatory nitrate-reducing ammonium-producing bacteria (DNRA bacteria)" refers to bacteria capable of reducing nitrates and nitrites to ammonium using nitrates and nitrites as electron acceptors and releasing them outside the body, including desulfurobacteria (…). Desulfobacter sp. ), Escherichia coli ( Escherichia coli ), denitrifying thiobacillus ( Thiobacillus denitrificans Aeromonas ( Aeromonas sp. )wait.

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

[0010] In this 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" primarily includes nitrate nitrogen, i.e., nitrate (NO3). - (such as sodium nitrate) and nitrite (NO2) - ).

[0012] In this application, the term "trace element" may include, but is not limited to, at least one of MgSO4, Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, and ZnCl2.

[0013] In one exemplary embodiment, Shewanella MR-1 is expanded in TSA medium; Pseudomonas aeruginosa is expanded in NB medium; Thiobacillus thioreductoides is expanded in DSMZ Medium 826 medium; Rhodotorula ferricyanipeptides is expanded in ATCC Medium 1768 medium; and Vibrio ferricyanipeptides is expanded in DSMZ Medium 826 medium.

[0014] In one exemplary embodiment, *Dethiobacillus* is cultured in DSMZ Medium 63 medium; *Escherichia coli* is cultured in LB medium; *Denitrifying Thiobacillus* is cultured in DSMZ Medium 113 medium; and *Aeromonas* is cultured in LB medium.

[0015] Optionally, the initial inoculation OD of the two microbial agents 600The value is between 0.05 and 0.2; for example, 0.05, 0.1, or 0.2, but it is not limited to the listed values. Other unlisted values ​​within this range also apply.

[0016] Optionally, the temperature for propagating the two microbial agents is 30-37℃; for example, 30℃, 33℃ or 37℃, but not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0017] Optionally, Shewanella MR-1, Pseudomonas aeruginosa, Escherichia coli, Thiobacillus denitrificationus, and Aeromonas are cultured under aerobic conditions; while Iron-reducing Rhodotorula rubrum, Iron-reducing Vibrio dysplasia, Thiobacillus thioreductoids, and Thiobacillus denitrificationus are cultured under anaerobic conditions.

[0018] In an exemplary embodiment, the co-culture medium includes nitrate, buffer solution, and trace elements; optionally, the nitrate is NaNO3; optionally, the buffer solution is selected from one or two 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.

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

[0020] In an exemplary embodiment, the washing of the electron-donating bacteria and DNRA bacteria includes the following steps: centrifuging the cultured bacterial agent and then washing it with a biological buffer solution.

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

[0022] Optionally, the biological buffer solution is selected from at least one of Tris-HCl buffer solution, HEPES buffer solution and 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, but not limited to the listed values, other unlisted values ​​within this 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, etc., such as the pH range of 7.0-7.4, but not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0025] In an exemplary embodiment, when the initial carbon-to-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 in the range of 5:1-10:1, for example, 5:1, 6:1, 8:1 or 10:1, but not limited to the listed values, other unlisted values ​​within this range are also applicable.

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

[0027] In one exemplary embodiment, the addition of the carbon source further includes the addition of a signaling molecule to the wastewater or co-culture medium.

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

[0029] In one exemplary embodiment, a reduction reaction is carried out after deoxygenation using an inert gas aeration system.

[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, but not limited to the listed values. Other unlisted values ​​within this range are also applicable.

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

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

[0033] In one exemplary embodiment, a carbon source and / or signaling molecules are further added to the co-culture medium or the wastewater.

[0034] Quorum sensing (QS) is a communication mechanism among microorganisms that regulates microbial community structure and extracellular electron transport (EET) through the secretion of signaling molecules, playing a crucial role in microbial EET processes. This application utilizes QS regulation to transfer electrons generated by electron-donating bacteria to DNRA bacteria via EET, thereby significantly enhancing NH4 production by DNRA bacteria. + Performance is an effective strategy for controlling nitrate nitrogen pollution and improving soil fertility.

[0035] This application provides a novel method for regulating the reduction of nitrate (or nitrite) to ammonium production by bicellular synergistic interaction based on the principle of enhancing biochemical transformation through extracellular electron transfer. This method can enhance the electron synergistic interaction between electron-donating bacteria and DNRA bacteria, improve electron utilization, and efficiently reduce nitrate nitrogen pollutants into ammonia nitrogen (NH4), which has resource and energy properties. + The efficiency of ammonium synthesis in the dual-strain system is at least three times higher than that of DNRA alone, and the selectivity of nitrate nitrogen reaches more than 97%.

[0036] The method described in this application can reduce nitrate nitrogen produced by the nitration reaction to NH4 in situ without the need for electrode material preparation or external energy input. + .

[0037] The microbial composition used in this application can be applied on a large scale to farmland soil, and it has a wide range of usable raw materials, making it a green and sustainable application.

[0038] The method described in this application is simple and efficient, and has a wide range of applications.

[0039] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the embodiments described in the description and drawings. Attached Figure Description

[0040] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0041] Figure 1 Transmission electron micrographs 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 this application are shown.

[0042] Figure 2The graph shows the changes in the concentrations of nitrate ammonia and ammonia nitrogen over time when nitrate nitrogen in wastewater is reduced to ammonia nitrogen using a single DNRA bacterium (Escherichia coli) in Comparative Example 1 and a dual-bacterial agent used in Example 2 of this application. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application are described in detail below. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

[0044] In the following embodiments and comparative examples, unless otherwise specified, all operations were performed under conventional conditions or conditions recommended by the manufacturer. All raw materials used, unless otherwise specified, are commercially available products.

[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 ( Escherichia coli, Purchased from BeiNa Bio (BNCC357962), expanded in LB medium, initial inoculation OD of E. coli. 600 The value was 0.05, and the culture was carried out aerobically at 37℃ until the stationary phase (OD). 600 The value was stabilized at around 1.0); E. coli was washed four times with 30 mM pH 7.4 PBS buffer solution and then resuspended to form an E. coli culture for later use.

[0048] (2) Prepare the culture medium. The culture medium in this example includes 10 mM NaNO3 as a simulated nitrogen source, a buffer solution composed of 20 mM KH2PO4 and 20 mM Na2PO4 (used to adjust the pH of the culture medium to about 7), and trace elements composed of 9.6 μM Na2-EDTA, 9.0 μM FeCl3·6H2O, 0.1 μM MnCl2·4H2O, 1.0 μM Na2MoO4·2H2O, 0.8 μM CuCl2·2H2O, 2.5 μM ZnCl2, 0.5 mM MgSO4, and 0.5 mM CaCl2; inoculate the Escherichia coli culture into the co-culture medium, and inoculate the initial OD. 600 The value was 0.15. Continue culturing to increase the OD value in the culture medium. 600 The value is 0.5 or higher.

[0049] (3) Introduce the culture medium containing Escherichia coli into the wastewater containing sodium nitrate (initial carbon-to-nitrogen ratio less than 5:1), and add glycerol as a carbon source to make the C / N (molar ratio) of carbon source to sodium nitrate 6:1; use argon gas to aerate for 20 min to form an anaerobic environment, and then add NO3 at 30℃. - Biological reduction reaction produces ammonia nitrogen NH4 + .

[0050] Using Nessler's reagent as the colorimetric reagent, the ammonia nitrogen content generated in the water was determined by colorimetry. The concentration of ammonia nitrogen generated within the 11-hour reaction time was 6.2 mg / L.

[0051] Example 1 (without adding signaling 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 bacterium Shewanella ( Shewanella oneidensis MR-1 (purchased from BeiNa Biotechnology BNCC274831) was expanded in TSA medium. The initial inoculation OD of Shewanella was... 600 The value was 0.1, and the culture was carried out aerobically at 30℃ until the stationary phase (OD). 600 The value stabilized at around 1.0; the dissimilatory nitrate-reducing ammonium-producing bacteria *Escherichia coli* (… Escherichia coli, Purchased from BeiNa Bio (BNCC295132), expanded in LB medium, initial inoculation OD of E. coli. 600 The value was 0.05, and the bacteria were cultured aerobically at 37°C until the stationary phase. Shewanella was washed three times with 20 mM pH 7.2 Tris-HCl buffer and then resuspended to form a Shewanella culture for later use. Escherichia coli was washed four times with 30 mM pH 7.4 PBS buffer and then resuspended to form an Escherichia coli culture for later use.

[0054] (2) Prepare the co-culture medium. In this example, the co-culture medium consists of 10 mM NaNO3 as a simulated nitrogen source, a buffer solution composed of 20 mM KH2PO4 and 20 mM Na2PO4 (used to adjust the pH of the medium to about 7), and trace elements composed of 9.6 μM Na2-EDTA, 9.0 μM FeCl3·6H2O, 0.1 μM MnCl2·4H2O, 1.0 μM Na2MoO4·2H2O, 0.8 μM CuCl2·2H2O, 2.5 μM ZnCl2, 0.5 mM MgSO4, and 0.5 mM CaCl2. Inoculate the Shewanella bacterial culture into the co-culture medium and inoculate the initial OD. 600The value was 0.15; E. coli culture was inoculated into the co-culture medium, and the initial OD value was [value missing]. 600 The value was 0.15, indicating the total OD value of the two bacterial agents in the co-culture medium. 600 The value was 0.3, and culturing continued to increase the OD value in the culture medium. 600 The value is 0.5 or higher.

[0055] (3) Introduce the co-culture medium containing two bacterial agents into the wastewater containing sodium nitrate (initial carbon-to-nitrogen ratio less than 5:1), and add glycerol as a carbon source to make the C / N (molar ratio) of carbon source to sodium nitrate 6:1; use argon gas to aerate for 20 min to form an anaerobic environment, and add NO3 at 30℃. - Biological reduction reaction produces ammonia nitrogen NH4 + .

[0056] Using Nessler's reagent as a colorimetric reagent, the ammonia nitrogen content generated in the water was detected by colorimetry. Within a reaction time of 11 hours, the concentration of ammonia nitrogen generated was 24 mg / L. This indicates that the reduction efficiency of the dual-bacterial agent of this application is significantly greater than that of the single dissimilatory nitrate-reducing ammonium-producing bacteria (such as Escherichia coli) used in Comparative Example 1. The dual-bacterial agent of this 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 MR-1 ( Shewanella oneidensis MR-1 (purchased from BeiNa Biotechnology BNCC274831) was expanded in TSA medium. The initial inoculation OD of Shewanella was... 600 The value was 0.1, and the culture was carried out aerobically at 30℃ until the stationary phase (OD). 600 The value stabilized at around 1.0; the dissimilatory nitrate-reducing ammonium-producing bacteria *Escherichia coli* (… Escherichia coli, Purchased from BeiNa Bio (BNCC295132), expanded in LB medium, initial inoculation OD of E. coli. 600 The value was 0.05, and the bacteria were cultured aerobically at 37°C until the stationary phase. Shewanella was washed three times with 20 mM pH 7.2 Tris-HCl buffer and then resuspended to form a Shewanella culture for later use. Escherichia coli was washed four times with 30 mM pH 7.4 PBS buffer and then resuspended to form an Escherichia coli culture for later use.

[0060] (2) Prepare the co-culture medium. In this embodiment, the co-culture medium includes 10 mM NaNO3 as a simulated nitrogen source, a buffer solution composed of 20 mM KH2PO4 and 20 mM Na2PO4 (used to adjust the pH of the medium to about 7), and trace elements composed of 9.6 μM Na2-EDTA, 9.0 μM FeCl3·6H2O, 0.1 μM MnCl2·4H2O, 1.0 μM Na2MoO4·2H2O, 0.8 μM CuCl2·2H2O, 2.5 μM ZnCl2, 0.5 mM MgSO4, and 0.5 mM CaCl2. Inoculate the Shewanella bacterial culture into the co-culture medium and inoculate the initial OD. 600 The value was 0.15; E. coli culture was inoculated into the co-culture medium, and the initial OD value was [value missing]. 600 The value was 0.15, indicating the total OD value of the two bacterial agents in the co-culture medium. 600 The value was 0.3, and culturing continued to increase the OD value in the culture medium. 600 The value is 0.5 or higher.

[0061] (3) Introduce the co-culture medium containing two bacterial agents into the wastewater containing sodium nitrate (initial carbon-to-nitrogen ratio less than 5:1), and add glycerol as a carbon source to make the C / N (molar ratio) of carbon source to sodium nitrate 6:1; continue to add a total concentration of 2 μM of signal molecules composed of C4-AHLs and C6-AHLs (concentration ratio of the two signal molecules 1:1); use argon gas to aerate for 20 min to form an anaerobic environment, and then add NO3 at 30℃. - Biological reduction reaction produces ammonia nitrogen NH4 + .

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

[0063] Depend on Figure 2 It can be seen that the dual-strain agent of this application combines with signaling molecules, enhances the quorum effect, and significantly increases the amount of ammonia nitrogen generated. Within a 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 bacterium is Shewanella MR-1 ( Shewanella oneidensis MR-1, Beina Biotechnology BNCC274831), Pseudomonas aeruginosa ( Pseudomonas aeruginosa, When co-culturing a combination of bacteria (ATCC 9027), Shewanella inoculated with the initial OD 600The value was 0.10, indicating the initial OD value of *Pseudomonas aeruginosa* inoculation. 600 The value was 0.05; the dissimilatory nitrate reducing ammonium-producing bacteria was Escherichia coli, and the other steps were the same as in Example 2.

[0066] Example 4

[0067] In this embodiment, the dissimilatory nitrate-reducing ammonium-producing bacterium is *Escherichia coli* (…). Escherichia coli (BNCC295132) and Denitrifying Thiobacillus ( Thiobacillus denitrificans When co-culturing a combination of bacteria (DSM 12475), Escherichia coli is inoculated with an initial OD of [missing information]. 600 The value was 0.07, indicating an initial OD value of 0.07 for *Thiobacillus denitrificationis* inoculation. 600 The value was 0.08; the electron donor was Shewanella MR-1, and the other steps were the same as in Example 2.

[0068] For Examples 3 and 4, the concentrations of ammonia nitrogen generated within a reaction time of 11 hours were measured by colorimetry to be 34 mg / L and 40 mg / L, respectively. This indicates that when multiple bacterial agents are combined, either electron-donating bacteria or dissimilatory nitrate-reducing ammonium-producing bacteria can enhance the reduction of nitrate nitrogen to ammonia nitrogen.

[0069] In addition, when ammonia nitrogen NH4 + After accumulating to a certain concentration in water, the pH of the aqueous solution can be adjusted, and 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 wastewater.

[0070] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for reducing nitrate nitrogen to ammonia nitrogen in wastewater using a dual-bacterial inoculant, characterized in that, The process involves separately expanding, washing, and resuspending electron-donating bacteria and dissimilatory nitrate-reducing ammonium-producing bacteria, then mixing them in a co-culture medium to form a dual-bacterial agent; introducing the co-culture medium containing the dual-bacterial agent into wastewater with an initial carbon-to-nitrogen ratio of less than 5:1 to reduce nitrate nitrogen in the wastewater to ammonia nitrogen under anaerobic conditions. The method further includes adding a carbon source to the co-culture medium or the wastewater to be treated, such that the molar ratio of carbon to nitrogen in the wastewater is greater than or equal to 5:1; after adding the carbon source, a signal molecule is also added to the co-culture medium or the wastewater to be treated. The electron-donating bacteria mentioned therein are selected from one or more of Shewanella oneidensis MR-1, Pseudomonas aeruginosa, Geobacter sulfurreducens, Rhodoferax ferrireducens, and Geovibrio ferrireducens; The dissimilar nitrate-reducing ammonium-producing bacteria are selected from one or more of Desulfobacter sp., Escherichia coli, Thiobacillus denitrificans, and Aeromonas sp.; The signaling molecules are selected from one or more of C4-AHLs, C6-AHLs, and C8-AHLs.

2. The method according to claim 1, wherein the initial OD of the electron-donating bacteria and the dissimilar nitrate-reducing ammonium-producing bacteria in the co-culture medium 600 The ratio of values ​​is 1:1 to 1:3; and / or, The total initial OD of the two bacterial agents 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 *Shewanella MR-1* is expanded in TSA medium; *Pseudomonas aeruginosa* is expanded in NB medium; *Thioreductive *Geobacterium thiocyanate* is expanded in DSMZ Medium 826 medium; *Iron-reducing *Rhodotorula rubrum* is expanded in ATCC Medium 1768 medium; *Iron-reducing *Vibrio geysersis* is expanded in DSMZ Medium 826 medium; and the initial inoculation OD of the electron-donating bacteria... 600 When the value is in the range of 0.05-0.2, the expansion temperature is 30-37℃; Dethiobacillus was expanded in DSMZ Medium 63 medium; Escherichia coli was expanded in LB medium; Denitrifying Thiobacillus was expanded in DSMZ Medium 113 medium; Aeromonas was expanded in LB medium; the initial inoculation OD of the dissimilar 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℃.

4. The method according to claim 1, wherein the carbon source is selected from one or more of sodium lactate, sodium citrate, glucose, and glycerol.

5. The method according to claim 1, wherein the total concentration of the added signaling molecule is 1-5 μM.

6. The method according to any one of claims 1-5, wherein the co-culture medium comprises nitrate, buffer solution, and trace elements.

7. The method according to claim 6, wherein the nitrate is NaNO3; the buffer solution is selected from one or two of KH2PO4 and Na2PO4; and the trace element is selected from one or more of Na2-EDTA, FeCl3·6H2O, MnCl2·4H2O, Na2MoO4·2H2O, CuCl2·2H2O, ZnCl2, MgSO4, and CaCl2.

8. 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 with a biological buffer solution.

9. The method according to claim 8, wherein the biological buffer solution is selected from at least one of Tris-HCl buffer solution, HEPES buffer solution and PBS buffer solution.

10. The method according to claim 8, wherein the concentration of the biological buffer solution is 20-100 mM; and the pH of the biological buffer solution is 7.0-7.6.

Images