A method for quickly domesticating anammox bacteria

Abstract

The application provides a method for quickly domesticating anaerobic ammonia oxidation bacteria, relates to the field of novel and efficient nitrogen removal technology, and comprises the following steps: constructing a coupled biological system, then adding simulated wastewater containing ammonium chloride and sodium nitrite into the coupled biological system, adding trace element nutrient solution after adjusting the pH to 7-8 by using anhydrous sodium carbonate, aerating the coupled system by using high-purity nitrogen gas to remove the residual air in the system, regularly sampling and monitoring the sludge and effluent in the reactor; after the sludge in the reactor appears obvious red-brown color and the ammonia nitrogen and nitrite nitrogen in the effluent are significantly removed, the domestication of the anaerobic ammonia oxidation bacteria is completed. The application utilizes anaerobic activated sludge, utilizes the principle of traditional microbial degradation of nitrogen-containing pollutants, introduces iron oxide composite material as an electron carrier to catalyze the degradation process of nitrogen-containing pollutants, improves the degradation efficiency of the traditional biological method, explores a new way to strengthen the degradation of nitrogen-containing pollutants, and reduces the cost.

Description

Technical Field

[0001] This invention relates to the field of novel and efficient denitrification technology, and in particular to a method for rapidly acclimatizing anaerobic ammonia-oxidizing bacteria. Background Technology

[0002] With the development of industries such as petrochemicals, food processing, and pharmaceuticals, and the continuous improvement of people's living standards, the amount of nitrogen compounds in industrial wastewater and domestic sewage has increased dramatically, putting enormous pressure on the environment. Currently, the control of nitrogen-containing wastewater has become a hot topic in the wastewater treatment field. Biological denitrification has received widespread attention due to its economic efficiency, ease of operation, and lack of secondary pollution. However, traditional biological denitrification technologies suffer from drawbacks such as long process flows, large footprints, frequent need for external carbon sources, high energy consumption, and high costs, which limit their further application. In recent years, anaerobic ammonium oxidation (Anammox) technology has attracted widespread attention because it can simultaneously remove ammonia, nitrates, and nitrites. Anammox uses ammonia as an electron donor and nitrates or nitrites as electron acceptors, directly converting nitrogenous substances into nitrogen gas (N2). Compared with traditional biological denitrification, anaerobic ammonium oxidation technology can save 40% of aeration volume and requires no organic carbon source, significantly reducing the cost of nitrogen-containing wastewater treatment, thus showing high application prospects. However, anaerobic ammonia oxidizing bacteria grow slowly, have a long start-up process, are easily inhibited in activity, and are difficult to cultivate, which limits their application in the process. Summary of the Invention

[0003] The purpose of this invention is to solve the technical problems of slow growth, long start-up process, easily inhibited activity, and difficulty in cultivation of anaerobic ammonia oxidizing bacteria in the prior art.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for rapidly acclimatizing anaerobic ammonia-oxidizing bacteria includes the following steps: constructing a coupled biological system, then adding simulated wastewater containing ammonium chloride and sodium nitrite into the coupled biological system, adjusting the pH to 7-8 with anhydrous sodium carbonate, adding trace element nutrient solution, aerating the coupled system with high-purity nitrogen to remove any residual air, and periodically sampling and monitoring the sludge and effluent in the reactor; the acclimatization of anaerobic ammonia-oxidizing bacteria is complete when the sludge in the reactor turns a distinct reddish-brown color and ammonia nitrogen and nitrite nitrogen in the effluent are significantly removed.

[0005] Preferably, constructing a coupled biological system includes the following steps: taking an appropriate amount of nitrified sludge from the denitrification process section of a municipal wastewater treatment plant, cleaning it, and adding it into a reactor, while simultaneously adding an appropriate amount of iron oxide composite material into the reactor to construct a coupled biological system.

[0006] Preferably, the iron oxide composite material is Fe3O4@MIL-101 (Fe), and the dosage is 1 g / L.

[0007] Preferably, the simulated wastewater contains 50 mg / L ammonium chloride, 85.1 mg / L sodium nitrite, and SL-4 trace element nutrient solution at a concentration of 10 mL / L.

[0008] This application also provides a method for investigating the denitrification performance of anaerobic ammonia-oxidizing bacteria under complex environments, comprising the following steps: S1: A quantitative concentration of NH4Cl solution was prepared and added to the acclimated anaerobic ammonia-oxidizing bacteria. Then, different concentrations of NaNO2 solution were prepared and added to the sludge. A certain amount of SL-4 solution was added, and the pH was adjusted with Na2CO3. The iron oxide composite material coupled with the anaerobic biological system was then sealed. Samples were taken and analyzed at specific time intervals, and the concentrations of ammonia nitrogen and nitrite nitrogen were determined using a UV spectrophotometer. S2: In an anaerobic biological system coupled with iron oxide composite materials, samples are taken at specific times for analysis to evaluate and analyze the long-term stable operation performance of the anaerobic ammonia oxidation process under complex environmental conditions.

[0009] Preferably, the concentration of the NH4Cl solution in S1 is 13.08 mg / L; the different concentrations of NaNO2 are 12.7 mg / L, 17.26 mg / L, and 21.82 mg / L, respectively.

[0010] Preferably, the determination time in S2 is 81, 84, 87, 90, 93, or 96 days.

[0011] Preferably, the investigation and analysis method involves measuring the concentrations of ammonia nitrogen and nitrite nitrogen in the reactor to determine the long-term stable operation of the reactor.

[0012] This invention discloses a method for rapidly acclimating anaerobic ammonia-oxidizing bacteria and exploring its denitrification performance under complex environments. According to this invention, no complex reaction requirements and conditions are needed; simply adding a small amount of iron oxide composite material can enhance the degradation efficiency of anaerobic organisms for nitrogenous pollutants and strengthen the acclimation of anaerobic ammonia-oxidizing sludge. Furthermore, the iron oxide composite material can be recovered under an external magnetic field and reused multiple times, further reducing wastewater treatment costs. This invention utilizes anaerobic activated sludge and the traditional principle of microbial degradation of nitrogenous pollutants, introducing iron oxide composite material as an electron carrier to catalyze the degradation process of nitrogenous pollutants. This improves the degradation efficiency of traditional biological methods, explores a new pathway to enhance the degradation of nitrogenous pollutants, reduces costs, and has significant practical application value. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the acclimatization of anaerobic ammonia oxidation sludge in one embodiment of the present invention. Figure 2 This is a schematic diagram illustrating the effect of concentration on the denitrification performance of anaerobic ammonia-oxidizing bacteria in one embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the long-term stability assessment of an anaerobic ammonia oxidation system according to one embodiment of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0015] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0016] Related studies have shown that iron is one of the essential elements for microbial growth, accounting for approximately 0.02% of cell dry weight, and is crucial for the metabolism of anaerobic ammonia oxidizing bacteria. The iron content in anaerobic ammonia oxidizing bacteria is much higher than in general microorganisms. This is because the physiological activities of anaerobic ammonia oxidizing bacteria are highly dependent on iron-binding proteins, and changes in the iron valence state within these proteins play a key role in electron transfer during the anaerobic ammonia oxidation reaction. Furthermore, iron is also an important component of key enzymes in nitrite reduction and ammonia oxidation processes. In summary, a stable supply of iron is essential for maintaining the high activity of anaerobic ammonia oxidizing bacteria. Among numerous iron-based nanomaterials, magnetite nanoparticles (Fe3O4) have outstanding application value in catalysts, magnetohydrodynamics, targeted drug delivery, and disease diagnosis and treatment due to their low toxicity, inherent biocompatibility, high saturation magnetization, and room-temperature superparamagnetism at the critical size. Fe3O4 nanoparticles can ionize into ferrous ions (Fe2+). 2+ ) and ferric ions (Fe 3+ Improving the culture conditions in the anaerobic ammonia oxidation reactor, while Fe 2+ and Fe 3+ The presence of Fe3O4 nanoparticles maintains a high redox potential environment, enhancing anaerobic ammonia oxidation activity and creating a suitable environment for the growth and spread of anaerobic ammonia-oxidizing bacteria. Furthermore, Fe3O4 nanoparticles possess excellent semi-conductivity, significantly promoting electron transfer rates between anaerobic ammonia-oxidizing microorganisms, thus facilitating efficient nitrogen removal within anaerobic systems. However, Fe3O4 nanoparticles exhibit poor dispersibility in aqueous solutions and are prone to sedimentation and large-scale aggregation, severely limiting their application in environmental remediation.

[0017] Metal-organic frameworks (MOFs) are a novel type of porous crystalline inorganic-organic hybrid material composed of metal clusters or metal ions. The porous organic ligands are coupled through strong bonds, and their large surface area, ultra-high porosity, diverse building blocks, and tunability have led to their widespread application in catalysis. Among them, iron-based Lavossell frameworks (MILs) possess unique structural properties, including porous structure, large surface area, and extensive iron site distribution, resulting in their widespread use across various fields. MIL-101(Fe) is the most representative iron-based MIL, characterized by its large pore size, large surface area, and large cell volume. This study combines MIL-101(Fe) with Fe3O4 nanoparticles, which not only overcomes the shortcomings of Fe3O4 nanoparticles such as easy aggregation and poor stability in anaerobic systems, but also leverages the strong magnetic properties of Fe3O4 nanoparticles to achieve the recycling of the composite material, thus contributing to further reductions in wastewater denitrification treatment costs.

[0018] This invention uses common nitrifying bacteria as inoculated sludge, introduces iron oxide composite materials, evaluates their effect on the domestication of anaerobic ammonia-oxidizing bacteria, and explores the denitrification performance of this coupled system under complex conditions.

[0019] A method for rapidly domesticating anaerobic ammonia-oxidizing bacteria includes the following steps: constructing a coupled biological system and domesticating anaerobic ammonia-oxidizing bacteria: Specifically, in one embodiment, constructing a coupled biological system includes the following steps: Take an appropriate amount of nitrified sludge from the denitrification process section of a municipal wastewater treatment plant, clean it thoroughly, and add it to the reactor. Simultaneously, add an appropriate amount of iron oxide composite material to the reactor to construct a coupled biological system. The iron oxide composite material is Fe3O4@MIL-101 (Fe), and the dosage is 1 g / L.

[0020] The steps for acclimating anaerobic ammonia-oxidizing bacteria are as follows: Simulated wastewater containing 50 mg / L ammonium chloride (NH4Cl) and 85.1 mg / L sodium nitrite (NaNO2) is added to the coupled system. After adjusting the pH to 7-8 with anhydrous sodium carbonate (Na2CO3), 10 mL / L of trace element nutrient solution (SL-4) is added. High-purity nitrogen is used to aerate the coupled system to remove any residual air. The sludge and effluent in the reactor are sampled and monitored regularly.

[0021] Once the sludge in the reactor turns a distinct reddish-brown color and ammonia nitrogen and nitrite nitrogen in the effluent are significantly removed, the acclimatization of anaerobic ammonia oxidizing bacteria is complete. This application also provides a method for investigating the denitrification performance of anaerobic ammonia-oxidizing bacteria under complex environments, comprising the following steps: S1: A quantitative concentration of NH4Cl solution (13.08 mg / L) was prepared and added to the acclimated anaerobic ammonia-oxidizing bacteria. Then, different concentrations of NaNO2 solution were prepared and added to the sludge. In one embodiment, the different concentrations of NaNO2 solution were 12.7 mg / L, 17.26 mg / L, and 21.82 mg / L. A certain amount of SL-4 solution was added, and the pH was adjusted with Na2CO3. The iron oxide composite material coupled with the anaerobic biological system was sealed. During the reaction, the temperature was controlled at 33℃, the residence time at 48 h, the rotation speed at 2.4 r / min, the reflux ratio at 10, and the ratios of nitrite nitrogen to ammonia nitrogen at 0.97, 1.32, and 1.67, respectively. Sampling and analysis were performed at specific time points, and the concentrations of ammonia nitrogen and nitrite nitrogen were determined using a UV spectrophotometer. S2: In an anaerobic biological system coupled with iron oxide composite materials, samples are taken at specific times for analysis to evaluate and analyze the long-term stable operation performance of the anaerobic ammonia oxidation process under complex environmental conditions.

[0022] Specifically, in one implementation, the specific time period is 81, 84, 87, 90, 93, or 96 days.

[0023] The present application will be described below with reference to specific embodiments: Example 1: A suitable amount of sludge from the denitrification process of a municipal wastewater treatment plant was taken, cleaned, and became anaerobic sludge. Simulated wastewater containing 50 mg / L NH4Cl and 85.1 mg / L NaNO2 was added to the anaerobic sludge, followed by 10 mL of SL-4 (NH4Cl and NaNO2 act as electron donor and acceptor, respectively). The pH was adjusted to 7-8 with Na2CO3. Then, an iron oxide composite material was added to construct an iron oxide composite material coupled anaerobic biological system. This coupled system was then sealed and aerated to complete the sludge acclimatization. The experimental results are as follows: Figure 1 As shown.

[0024] Within the first 15 days of operation, both ammonia nitrogen and nitrite nitrogen showed some removal, but the removal rates were low. This indicates that anaerobic ammonia oxidation (AAO) had not yet become the dominant reaction in the reactor, and the reaction was still in a lag phase. Between days 15 and 30, both ammonia nitrogen and nitrite nitrogen began to be significantly removed, but the removal ratio still lacked a clear pattern, indicating that AAO was not yet the dominant reaction in the reactor; therefore, the reaction was still in a lag phase. After 30 days, both ammonia nitrogen and nitrite nitrogen began to be significantly removed, with a nitrite nitrogen to ammonia nitrogen removal ratio of 1.48. As the reaction time progressed, the removal of both nitrite nitrogen and ammonia nitrogen gradually increased, while the ratio of their removal gradually decreased. At this point, the reaction was in an activity-enhancing phase. By day 42, the nitrite nitrogen removal rate was 39.9%, and the ammonia nitrogen removal rate was 36.5%. Subsequently, the nitrite nitrogen to ammonia nitrogen removal ratio remained relatively stable at 1.38. By day 80, the removal rate of nitrite nitrogen was 99.6%, and the removal rate of ammonia nitrogen was 96.16%. The ratio of nitrite nitrogen to ammonia nitrogen removal was 1.35, which is close to the ideal ratio of 1.32. After 80 days, the removal rates of nitrite nitrogen and ammonia nitrogen, as well as their ratios, basically stabilized. The dominant reaction in the reactor was now anaerobic ammonia oxidation, which had reached a stable activity stage.

[0025] Example 2: In an upflow anaerobic sludge blanket reactor, iron oxide composite materials, sludge, and 13.08 mg / L NH4Cl were added. While maintaining a constant NH4Cl concentration, the NaNO2 concentration was varied to 12.7 mg / L, 17.26 mg / L, and 21.82 mg / L. 10 mL of trace element nutrient solution SL-4 was then added, and the wastewater pH was adjusted to 7-8 using Na2CO3 to construct an iron oxide-coupled anaerobic biological system. The sludge was sealed, and N2 was introduced to purge residual oxygen from the wastewater to an anaerobic state. At this point, the nitrite nitrogen to ammonia nitrogen ratios were 0.97, 1.32, and 1.67, respectively. During the reaction cycle, at designated sampling time points, water samples were filtered through a 0.22 μm filter, and nitrogen content was determined using a UV-Vis spectrophotometer at wavelengths of 420 and 540 nm.

[0026] The results are as follows Figure 2As shown, in the iron oxide composite material coupling system, the removal effects of ammonia nitrogen and nitrite nitrogen are relatively stable. The effluent concentration of ammonia nitrogen gradually decreases, while the removal rate increases, with the ammonia nitrogen removal rate rising from 83.34% to 95.99%. For nitrite nitrogen, the removal rate reaches its highest level of 98.70% when the ammonia nitrogen to nitrite nitrogen ratio is 1.32, the lowest level of 85.61% when the ratio is 1.67, and the best removal effect of 91.64% when the ratio is 0.97. Anaerobic ammonia oxidizing bacteria exhibit strong denitrification performance within the influent nitrite nitrogen to ammonia nitrogen ratio range of 0.97-1.32. The denitrification performance of anaerobic ammonia oxidizing bacteria is relatively ideal when the influent nitrite nitrogen to ammonia nitrogen concentration ratio is around 1.32.

[0027] The ratio of nitrite nitrogen to ammonia nitrogen in the influent can significantly affect the nitrogen removal performance of an anammox reactor. When the ratio is below the stoichiometric ratio of 1.32 for anammox, the reaction rate does not decrease significantly and the reactor functions normally. However, when the ratio exceeds 1.32, it will lead to a significant excess of nitrite nitrogen, a significant decrease in the reaction rate, and reactor malfunction.

[0028] The principle is that conductive minerals play a similar role in extracellular electron transport in microorganisms as cytochromes outside the membrane, promoting the transfer of electrons from microorganisms to extracellular electron acceptors. Under anaerobic conditions, microorganisms can utilize Fe3O4@MIL-101 (Fe) as an electron carrier to promote electron transfer between cells, thereby improving degradation efficiency.

[0029] Example 3: Simulated wastewater containing 50 mg / L NH4Cl and 85.1 mg / L NaNO2 was added to anaerobic sludge, followed by 10 mL of SL-4 (NH4Cl and NaNO2 acting as electron donor and acceptor, respectively). The pH was adjusted with Na2CO3, and then an iron oxide composite material was added to construct an iron oxide composite-coupled anaerobic biological system. This coupled system was then sealed and aerated, and placed in an anaerobic sludge bed reactor for reaction to complete the acclimation of the anaerobic sludge. The reactor operating conditions were controlled as follows: temperature 33℃, residence time 48 h, rotation speed 2.4 r / min, and reflux ratio 10. Results are as follows: Figure 3 As shown, after 80 days of acclimatization, the anaerobic ammonia-oxidizing bacteria achieved a removal rate of 85.26% for ammonia nitrogen and 98.35% for nitrite nitrogen, demonstrating good denitrification performance. After 80 days, the removal rates of nitrite nitrogen and ammonia nitrogen, as well as their removal ratios, stabilized. The nitrite nitrogen removal rate remained above 97%, and the ammonia nitrogen removal rate reached above 95%, with a removal ratio of approximately 1.32, maintaining good denitrification performance.

[0030] This invention uses common nitrifying bacteria as inoculated sludge, introduces iron oxide composite materials, evaluates their effect on the domestication of anaerobic ammonia-oxidizing bacteria, and explores the denitrification performance of this coupled system under complex conditions.

[0031] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for rapidly acclimatizing anaerobic ammonia-oxidizing bacteria, characterized in that: Includes the following steps: Take an appropriate amount of nitrified sludge from the denitrification process section of a municipal wastewater treatment plant, clean it, and add it to the reactor. At the same time, add an appropriate amount of iron oxide composite material, namely Fe3O4@MIL-101(Fe), to the reactor at a dosage of 1 g / L; construct a coupled biological system. Then, simulated wastewater containing ammonium chloride and sodium nitrite was added to the coupled biological system. After adjusting the pH to 7-8 with anhydrous sodium carbonate, trace element nutrient solution was added. High-purity nitrogen was used to aerate the coupled system to remove any air that might remain in the system. The sludge and effluent in the reactor were sampled and monitored regularly. Once the sludge in the reactor turns a distinct reddish-brown color and ammonia nitrogen and nitrite nitrogen in the effluent are significantly removed, the acclimatization of anaerobic ammonia oxidizing bacteria is complete.

2. The method for rapidly acclimatizing anaerobic ammonia-oxidizing bacteria according to claim 1, characterized in that: The simulated wastewater contained 50 mg / L ammonium chloride, 85.1 mg / L sodium nitrite, and SL-4 trace element nutrient solution at a concentration of 10 mL / L.

Images