Method for implementing anaerobic ammonium oxidation process without nitrite electron acceptor

Abstract

A novel anaerobic ammonium oxidation implementation method implemented by taking a solid as an electron acceptor, relating to the field of sewage treatment. The present invention aims at the restriction problem of anaerobic ammonium oxidation practical application caused by lack of electron acceptor nitrite in practical sewage. A solid electrode and Fe (III) are respectively used as electron acceptors, and when inoculums are anaerobic sludge and aerobic activated sludge, an anaerobic ammonium oxidation process implemented by replacement with the solid electron acceptor can be implemented under an anaerobic condition. The present invention proves that the feasibility of using different inoculums and different electron acceptors to implement anaerobic ammonium oxidation, provides a basis for further performance optimization and reactor design, provides a new idea for implementing anaerobic ammonium oxidation when in lack of nitrite, and has important significance for developing novel domestic sewage denitrification technology.

Description

A method for achieving anaerobic ammonium oxidation without nitrite electron acceptor Technical Field

[0001] The present invention belongs to the field of sewage treatment and relates to an autotrophic denitrification technology; in particular, it relates to a method for realizing an anaerobic ammonium oxidation reaction without the need for a nitrite electron acceptor. Background Art

[0002] Anaerobic ammonium oxidation (ANAMMOX) is a metabolic process in which autotrophic microorganisms oxidize ammonia nitrogen under anaerobic conditions using nitrite as an electron acceptor. Compared with traditional biological denitrification technologies, ANAMMOX technology offers advantages such as requiring no organic carbon source, reducing aeration requirements by 50-60%, generating virtually no N₂O greenhouse gas, and producing minimal sludge. However, the limited availability of nitrite electron acceptors in actual wastewater is a major factor limiting its widespread application.

[0003] In recent years, the electroactivity of anaerobic ammonium oxidizing (ANAMMOX) bacteria has been demonstrated. The process of ANAMMOX coupled with iron reduction is called ferric ammonium oxidation (Feammox), which involves microorganisms oxidizing ammonia nitrogen under anaerobic conditions using Fe(III) as an electron acceptor. Ferric ammonium oxidizers, or electroactive ANAMMOXs, transfer electrons extracellularly to solid electron acceptors such as iron-manganese oxides or electrodes, changing the traditional understanding of the nitrogen cycle. Anammox bacteria are also present in Feammox communities, suggesting the potential for population interactions between the two. ANAMMOX, based on extracellular electron transfer using different electron acceptors, offers a novel approach to addressing the challenge of denitrification in mainstream wastewater lacking NO₂- as an electron acceptor. Ammonia oxidation processes using electrodes as electron acceptors may involve electroactive Anammox bacteria, Feammox bacteria, and other electroactive bacteria. However, significant research remains on functional microorganisms capable of both extracellular electron transfer and ANAMMOX, as well as their population interactions and metabolic regulation mechanisms. Summary of the Invention

[0004] In order to solve the limitation of anaerobic ammonium oxidation reaction caused by the lack of nitrite electron acceptor in the prior art, the present invention aims to explore the possibility of using solid electron acceptors to replace nitrite to realize anaerobic ammonium oxidation reaction, and provide a new anaerobic ammonium oxidation reaction process that does not require nitrite electron acceptors, thereby solving the problem of the practical application of anaerobic ammonium oxidation caused by the lack of electron acceptor nitrite in actual sewage.

[0005] In order to solve the above technical problems, the present invention provides a method for realizing anaerobic ammonium oxidation reaction without the need for nitrite electron acceptor, and the technical solution adopted is as follows:

[0006] The method of the present invention uses electrodes and trivalent iron as solid electron acceptors, uses sludge as inoculum, and realizes an autotrophic anaerobic ammonia oxidation process using solid as electron acceptor without adding an external carbon source.

[0007] A two-chamber anaerobic microbial dye cell was constructed using a reaction system with electrodes as solid electron acceptors. The anode of the microbial fuel cell served as the anaerobic ammonium oxidation (ANAMMOX) component, and the trivalent iron (Fe) served as the chemical cathode. The closed circuit had an external resistance of 100 Ω. The anolyte was ANAMMOX culture medium, and the sludge was inoculated into the anolyte by adding 1 mL of sludge per 100 mL of liquid culture medium. The catholyte was potassium ferricyanide solution, and the cells were incubated under anaerobic conditions at a constant temperature and in the dark. The ANAMMOX culture medium did not contain NO⁻. The reactors were maintained at a constant temperature and in the dark at 35°C.

[0008] Using trivalent iron as a solid electron acceptor, the process can be carried out in an anaerobic bottle by adding 1 mL of inoculated sludge to every 100 mL of anaerobic ammonium oxidation culture medium. The culture is cultured under anaerobic conditions at a constant temperature and in the dark with magnetic stirring. The anaerobic ammonium oxidation culture medium does not contain NO2- and iron ions.

[0009] Based on the above scheme, a specific method is to proceed as follows:

[0010] Step 1: After the inoculated sludge is sieved through a mesh, it is repeatedly washed three times with culture medium and then inoculated into the reactor at an inoculum volume of 10% of the reaction volume;

[0011] Step 2: For a system using electrodes as electron acceptors, a microbial fuel cell dual-chamber reactor device was used. The cathode and anode chambers were separated by a proton exchange membrane. The two electrodes were tightly attached to a titanium wire and connected to an external circuit. A 1000 Ω resistor was connected in series to the external circuit and connected to a data acquisition device via a wire for real-time voltage data recording. The anolyte was an anaerobic ammonium oxidation culture solution that removed NO2-, and the cathode was a mixture of 100 mM potassium ferrocyanide and 100 mM PBS. This allowed the anaerobic ammonium oxidation reaction to occur at the anode with the electrodes as electron acceptors.

[0012] The reaction solution was replaced every three days under anaerobic conditions at a volume of 50% of the reaction volume. After the replacement, the nitrogen trioxide concentration and pH value in the reactor were measured.

[0013] Based on the above solution, there is another specific method that follows the following steps:

[0014] Step 1: After the inoculated sludge is sieved through a mesh, it is repeatedly washed three times with culture medium and then inoculated into the reactor at an inoculum volume of 10% of the reaction volume;

[0015] Step 2: For systems using ferric iron as an electron acceptor, use an anaerobic flask as the reaction apparatus, housing a 2 cm diameter rotor. The reaction liquid is an anaerobic ammonium oxidation culture medium that removes NO₂⁻, or iron ions. The ferric iron used in the reaction includes, but is not limited to, ferric chloride, ferric oxide, and ferric hydroxide.

[0016] The reaction solution was replaced every five days under anaerobic conditions at a volume of 50% of the reaction volume. After the replacement, the nitrogen trioxide concentration and pH value in the reactor were measured.

[0017] Furthermore, in a system using an electrode as an electron acceptor, the liquid culture medium components also include: 70 mg / L-120 mg / L ammonium chloride; 2.5 mg / L potassium bicarbonate; 0.06 mg / L sodium dihydrogen phosphate; 0.15 mg / L magnesium sulfate; 0.15 mg / L anhydrous calcium chloride; 1.25 mL / L trace element I and 1.25 mL / L trace element II.

[0018] Furthermore, in a system using trivalent iron as an electron acceptor, the liquid culture medium also includes: 70 mg / L-120 mg / L ammonium chloride, 2.5 mg / L potassium bicarbonate; 0.06 mg / L sodium dihydrogen phosphate; 0.15 mg / L magnesium sulfate; 0.15 mg / L anhydrous calcium chloride; 1.25 mL / L trace element I and 1.25 mL / L trace element II.

[0019] The liquid culture medium is prepared with deionized water.

[0020] Furthermore, the system using the electrode as the electron acceptor is cultured statically in the dark at a constant temperature of 35°C.

[0021] Furthermore, the electrode material used in the reactor using the electrode as the electron acceptor should be pretreated using, but not limited to, the following method: soaking in acetone for 10 h, then rinsing its surface with deionized water, and then heating it at 450° C. for 30 minutes in a muffle furnace.

[0022] Furthermore, the proton exchange membrane used to separate the anode and cathode in a reactor with electrodes as electron acceptors should be pretreated as follows: the proton exchange membrane is sequentially placed in 5% hydrogen peroxide, deionized water, 0.5 mol / L sulfuric acid, and deionized water, and each condition is heated in a 50°C water bath for 1 hour. The treated proton exchange membrane is then immersed in deionized water for storage.

[0023] Furthermore, the system using trivalent iron as an electron acceptor is cultured in the dark using a magnetic stirrer.

[0024] Furthermore, the stirring conditions of the magnetic stirrer are: 35° C. and a rotation speed of 100 rpm.

[0025] A method for achieving anaerobic ammonium oxidation without the need for a nitrite electron acceptor. Based on the above method, the ammonia nitrogen degradation status and product generation in the system are judged by monitoring the changes in the three nitrogens (i.e., NH4+, NO2- and NO3-) in the system.

[0026] Beneficial effects of the present invention:

[0027] The present invention realizes the anaerobic ammonium oxidation reaction without the addition of nitrite, breaks through the traditional barriers, and realizes the anaerobic ammonium oxidation reaction without the need for coupling reaction and the addition of exogenous nitrite and organic matter.

[0028] The present invention achieves excellent ammonia nitrogen removal at concentrations between 70 and 90 mg / L. In particular, when activated sludge is used as the inoculum, excellent denitrification performance is maintained even at concentrations up to 120 mg / L. This excellent tolerance to ammonia nitrogen concentrations makes this novel anaerobic ammonium oxidation reaction promising for practical wastewater denitrification applications.

[0029] In a system using trivalent iron as an electron acceptor, the addition of trivalent iron has little effect on the system's denitrification capacity within a certain concentration range (5-15 mg / L), indicating that anaerobic ammonia oxidation reaction can be achieved using low concentrations of trivalent iron, which has good application advantages.

[0030] The present invention demonstrates the feasibility of achieving anaerobic ammonium oxidation using different inocula and different electron acceptors, provides a basis for further performance optimization and reactor design, and offers new ideas for achieving anaerobic ammonium oxidation in the absence of nitrite, which is of great significance for the development of new domestic sewage denitrification technologies.

[0031] The present invention not only provides a new idea for achieving anaerobic ammonium oxidation in the absence of nitrite, which is of great significance for the development of new domestic sewage denitrification technology, but also breaks the conventional idea of ​​anaerobic ammonium oxidation, that is, the anaerobic ammonium oxidation reaction process can be achieved without the enrichment of anaerobic ammonium oxidizing bacteria in the traditional sense. Implementation Method

[0032] The present invention is further described below with reference to specific examples, but the present invention is not limited to the examples. The technical solution of the present invention is not limited to all the specific embodiments listed below.

[0033] Example 1: This example uses activated sludge as inoculum and electrodes as electron acceptors to achieve anaerobic ammonium oxidation. The specific steps are as follows:

[0034] 1. Construct an H-type microbial fuel cell with an effective anode and cathode volume of 200 mL, constructed from primary borosilicate glass. The anode and cathode compartments are separated by a proton exchange membrane (Nifion 117, Shanghai Hesen). A carbon brush (3 × 3 × 15 cm) serves as the anode, and a graphite felt (4 × 3 cm) serves as the cathode. The anode and cathode are connected by wires, with an external series resistor of 1000 Ω.

[0035] 2. Connect the two ends of the resistor to an electrochemical workstation and record the voltage across it every 10 minutes. The anode solution is the anaerobic ammonium oxidation culture medium that removes NO2-, and the cathode solution is a mixture of 100 mM potassium ferricyanide and 100 mM phosphate buffer.

[0036] 3. The reaction liquid in the anode chamber was aerated with high-purity nitrogen for 15 minutes and then sealed to ensure the anaerobic system. One end of the anode chamber was connected to a sampling needle equipped with a three-way valve, and the other end was connected to an air bag filled with high-purity nitrogen. Anaerobic sampling and liquid replacement were achieved through the three-way valve, and the culture was static and dark at a constant temperature of 35°C.

[0037] The anolyte composition in step 2 is: 70-120 mg / L ammonium chloride, 2.5 mg / L potassium bicarbonate; 0.06 mg / L sodium dihydrogen phosphate; 0.15 mg / L magnesium sulfate; 0.15 mg / L anhydrous calcium chloride; 1.25 mL / L trace element I and 1.25 mL / L trace element II, prepared with deionized water.

[0038] The above-mentioned trace element I is: 5 g / L ethylenediaminetetraacetic acid; 5 g / L ferrous sulfate, prepared with deionized water.

[0039] The above-mentioned trace elements II are: 0.43 g / L zinc sulfate heptahydrate; 0.24 g / L cobalt chloride hexahydrate; 0.99 g / L manganese chloride tetrahydrate; 0.25 g / L copper sulfate pentahydrate; 0.19 g / L nickel chloride hexahydrate; 0.014 g / L boric acid; 0.21 g / L sodium selenate decahydrate; 0.22 g / L sodium molybdate dihydrate; 0.05 g / L sodium tungstate dihydrate; and 15 g / L ethylenediaminetetraacetic acid, prepared with deionized water.

[0040] Pretreatment of the carbon brush and carbon paper in step 1: Soak the carbon brush and carbon paper in acetone for 10 hours, rinse their surfaces with deionized water, and then heat them at 450°C for 30 minutes in a muffle furnace.

[0041] Pretreatment of the proton exchange membrane in step 1: The proton exchange membrane was sequentially placed in 5% hydrogen peroxide, deionized water, 0.5 mol / L sulfuric acid, and deionized water. Each condition was heated in a 50°C water bath for 1 hour. The treated proton exchange membrane was then immersed in deionized water for storage.

[0042] In this example, the reactor uses electrodes as electron acceptors. Half of the total volume of the anode and cathode liquids is replaced every three days. The NH4+, NO2-, and NO3- concentrations and pH of the anode liquid are measured, and the voltage data across the MFC is recorded using an electrochemical workstation data logger.

[0043] The system in this example maintained an NH₄⁺-N removal rate of 65±5%, and the system voltage remained stable at 30 mV during the optimal phase of reactor operation. The system also demonstrated sensitivity to high NH₄⁺-N concentrations: increasing the NH₄⁺-N concentration within a certain range enhanced the system's power generation capacity. However, when the NH₄⁺-N concentration exceeded 100 mg / L, both the system's ammonia nitrogen removal rate and voltage were suppressed. However, at an NH₄⁺-N concentration of 30 mg / L, the system's NH₄⁺-N removal rate remained unchanged.

[0044] Example 2: This embodiment uses anaerobic sludge as inoculum and ferric iron as electron acceptor to achieve anaerobic ammonium oxidation. The specific steps are as follows:

[0045] 1. The anaerobic sludge for inoculation was purchased from Jiayi Environmental Protection. The bacterial strain was taken from an anaerobic sequencing batch reactor pilot plant that was operating stably, with an SV30 of approximately 70-80%. Before inoculation, the anaerobic sludge was repeatedly washed three times with culture medium and then inoculated into the reactor at an inoculum rate of 10%.

[0046] 2. The device of this embodiment uses an anaerobic bottle, adds anaerobic ammonia oxidation culture solution that removes nitrite and iron ions, and inoculates anaerobic sludge after nitrogen aeration for 15 minutes and then seals it. It is cultured at a constant temperature of 35°C in the dark with magnetic stirring and a speed of 100 rpm. One end of the reactor is connected to a sampling needle equipped with a three-way valve, and the other end is connected to an air bag equipped with high-purity nitrogen. Anaerobic sampling and liquid replacement are achieved through the three-way valve. Half the volume of the reaction solution is replaced every 5 days, and the concentrations of NH4+, NO2-, NO3-, Fe(II) and pH are measured.

[0047] In this example, ferric chloride was used as the doping substance for the ferric iron electron acceptor, and the effects of different ferric chloride dosages on the denitrification effect of the system were explored.

[0048] The composition of the anaerobic ammonium oxidation culture medium in step 2 is: 70-120 mg / L ammonium chloride, 2.5 mg / L potassium bicarbonate; 0.06 mg / L sodium dihydrogen phosphate; 0.15 mg / L magnesium sulfate; 0.15 mg / L anhydrous calcium chloride; and 1.25 mL / L trace element II.

[0049] The above-mentioned trace elements II include: 0.43 g / L zinc sulfate heptahydrate; 0.24 g / L cobalt chloride hexahydrate; 0.99 g / L manganese chloride tetrahydrate; 0.25 g / L copper sulfate pentahydrate; 0.19 g / L nickel chloride hexahydrate; 0.014 g / L boric acid; 0.21 g / L sodium selenate decahydrate; 0.22 g / L sodium molybdate dihydrate; 0.05 g / L sodium tungstate dihydrate; and 15 g / L ethylenediaminetetraacetic acid.

[0050] During the operation of the reactor of this embodiment, ammonia nitrogen oxidation and Fe(III) reduction were detected, achieving an ammonia nitrogen removal rate of 65-85%.

[0051] When the ammonia nitrogen concentration increased to 90 mg / L, the system lost its denitrification ability and showed strong sensitivity to ammonia nitrogen concentration. This shows that when using the method of this embodiment, the ammonia nitrogen pollutant concentration and fluctuation range of the application scenario should be considered.

[0052] During the operation of this example, it was found that the ammonia nitrogen removal capacity of the system did not change with the increase of ferric iron concentration (5-15 mg / L), indicating that in actual application, it is possible to consider adding ferric iron in small amounts and multiple times or adding ferric iron in large amounts and multiple times to reduce the reaction cost.

[0053] It is hereby stated that experiments have verified that only anaerobic sludge or anaerobic ammonium oxidation sludge can realize the anaerobic ammonium oxidation process with trivalent iron as the electron acceptor, while the anaerobic ammonium oxidation process with electrodes as the electron acceptor is not limited to activated sludge and anaerobic sludge.

[0054] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for achieving the anaerobic ammonium oxidation process without a nitrite electron acceptor, characterized in that, It was carried out in a two-chamber reactor of a microbial fuel cell, with the electrode as a solid electron acceptor, sludge as the inoculum, no additional carbon source added, the anolyte being an anaerobic ammonium oxidation culture medium, the sludge being inoculated into the anolyte, inoculation being carried out by adding 1 mL of inoculated sludge to every 100 mL of liquid culture medium, the catholyte being a potassium ferricyanide solution, and culturing being carried out under anaerobic conditions at a constant temperature in the dark; among them, the anaerobic ammonium oxidation culture medium does not contain NO 2 - 。 2. A method for achieving the anaerobic ammonium oxidation process without a nitrite electron acceptor, characterized in that, Using ferric iron as a solid electron acceptor, sludge as an inoculum, without additional carbon source added, inoculating according to adding 1 mL of inoculated sludge into every 100 mL of anaerobic ammonium oxidation culture solution, culturing under anaerobic conditions with constant temperature, avoiding light and magnetic stirring, wherein the anaerobic ammonium oxidation culture solution does not contain NO 2 - and ferric ions.

3. The method according to claim 1 or 2, characterized in that, The anolyte composition is: 70 - 120 mg / L ammonium chloride, 2.5 mg / L potassium bicarbonate, 0.06 mg / L sodium dihydrogen phosphate, 0.15 mg / L magnesium sulfate, 0.15 mg / L anhydrous calcium chloride, and trace elements; The trace elements are 1.25 mL / L trace element I and / or 1.25 mL / L trace element II; Trace element I includes: 5 g / L ethylenediaminetetraacetic acid, and 5 g / L ferrous sulfate; Trace element II includes: 0.43 g / L zinc sulfate heptahydrate, 0.24 g / L cobalt chloride hexahydrate, 0.99 g / L manganese chloride tetrahydrate, 0.25 g / L copper sulfate pentahydrate, 0.19 g / L nickel chloride hexahydrate, 0.014 g / L boric acid, 0.21 g / L sodium selenite decahydrate, 0.22 g / L sodium molybdate dihydrate, 0.05 g / L sodium tungstate dihydrate, and 15 g / L ethylenediaminetetraacetic acid.

4. The method according to claim 1, characterized in that, For the anaerobic ammonium oxidation reaction with the electrode as the electron acceptor, the inoculum is one or both of anaerobic sludge and aerobic activated sludge; static light - avoiding culture is adopted, and the temperature is kept constant at 35°C; the reaction solution is replaced every three days, and the replacement volume is 50% of the reaction volume.

5. The method according to claim 2, characterized in that, Fe 3+ has a concentration of 5 mg / L - 15 mg / L, and the Fe 3+ is provided by ferric chloride, iron oxide, and iron hydroxide.

6. The method according to claim 1 or 2, characterized in that, The culture solution is aerated for more than 15 min to create an anaerobic environment, and the aeration gas is an inert gas, and the inert gas is one of nitrogen and argon.

7. The method according to claim 1, characterized in that, The pretreatment method of the electrode material used in the reactor with the electrode as the electron acceptor: soak in acetone for 10 h, then rinse its surface with deionized water, and then heat it in a muffle furnace at 450°C for 30 minutes.

8. The method according to claim 1, characterized in that, The pretreatment method of the proton exchange membrane used to separate the anode and cathode in the reactor with the electrode as the electron acceptor should be as follows: sequentially place the proton exchange membrane in hydrogen peroxide with a concentration of 5%, deionized water, sulfuric acid with a concentration of 0.5 mol / L, and deionized water, and heat each condition in a water bath at 50°C for 1 hour, and then soak the treated proton exchange membrane in deionized water for storage.

9. The method according to claim 1, characterized in that, For the anaerobic ammonium oxidation reaction with ferric iron as the electron acceptor, the inoculum is anaerobic sludge or anaerobic ammonium oxidation activated sludge; magnetic stirrer light - avoiding culture is adopted, at 35°C, with a rotation speed of 100 rpm; the reaction solution is replaced every five days, and the replacement volume is 50% of the reaction volume.

10. The method according to claim 1 or 2, characterized in that, By monitoring the NH in the system 4 + , NO 2 - and NO 3 - concentration changes to judge the ammonia nitrogen degradation situation in the system and the generation of products.