Method for short-cut nitrification and denitrification high-efficiency biological denitrification of low ammonia-nitrogen sewage in low-temperature environment based on regulation of functional microorganism

Abstract

This invention discloses a method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment through short-cut nitrification and denitrification based on functional microbial regulation. A biofilm carrier attached to *Pseudomonas aeruginosa* is added to an activated sludge reactor, and aeration and stirring continue. The functional signaling substances secreted by the *P. aeruginosa*-attached biofilm carrier can regulate nitrogen metabolism in nitrifying bacteria and inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria. This increases the ammonia oxidation activity of the reactor at 28℃, 18℃, 14℃, and 10℃ by 23.02%, 60.69%, 61.06%, and 88.88%, respectively, and the nitrite accumulation rates are 82.58%, 89.06%, 90.59%, and 83.83%, respectively, thereby achieving short-cut nitrification and denitrification biological nitrogen removal and providing a superior regulation method for improving activated sludge biological nitrogen removal.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a method for efficient biological denitrification of low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation. Background Technology

[0002] China's urban wastewater treatment plants primarily employ traditional nitrification-denitrification biological nitrogen removal technology, which is currently the most economical, efficient, and widely used technology. The nitrogen-converting microorganisms in these wastewater treatment systems are mainly thermophilic, with an optimal growth temperature of 25-30℃. However, in winter, the water temperature in urban wastewater treatment plants in northern China generally drops below 15℃. This low temperature directly restricts the ammonia oxidation process during nitrification and denitrification, leading to the problem of excessive ammonia nitrogen and total nitrogen levels in effluent during winter. This forces these plants to achieve compliant wastewater discharge at the cost of high energy and chemical consumption, creating a stark contradiction with the "dual carbon" (carbon dioxide, carbon sequestration, and carbon emissions) target. Therefore, it is currently necessary to adopt energy-efficient and high-performance operating strategies to ensure stable compliant wastewater discharge.

[0003] Wastewater treatment systems are multi-microbial symbiotic systems. During their growth, microorganisms release signaling substances into the environment and regulate their metabolic functions by sensing changes in these substances' concentrations. These signaling substances are common metabolites in the environment that drive intercellular communication and cooperation; this process, known as intercellular communication, is considered a universal way for microorganisms to establish micro-ecological relationships. Intercellular communication participates in regulating microbial metabolism, aggregation, and movement. In anaerobic ammonia oxidation systems, the functional signaling substances C6-HSL and C8-HSL participate in processes such as the tricarboxylic acid cycle, amino sugar synthesis, and nucleotide sugar metabolism, promoting the active expression of related genes and accelerating the secretion of extracellular substances, leading to an increase in the system's denitrification capacity from 22.2% to 74.6% and 76.2%, respectively. Targeted regulation of intercellular communication by adding exogenous functional signaling substances can effectively control problems such as low biological denitrification efficiency and difficulty in achieving effluent ammonia nitrogen and total nitrogen standards. However, these strategies are costly, and the signaling substances are easily quenched in complex systems, making it difficult to maintain their function sustainably. Therefore, by increasing the abundance of functional signaling producers and promoting the secretion of functional signaling substances in the intercellular communication network of the ammonia oxidation process in the activated sludge system, the ammonia oxidation activity can be enhanced and the biological denitrification efficiency of wastewater can be improved.

[0004] The application of sequencing batch biofilm reactors (SBBRs) in low-temperature nitrogen removal has been studied, but it usually requires optimization of packing material size and filling ratio, or the combination of other technologies (such as magnetic fields). Existing low-temperature nitrogen removal technologies mainly include conventional nitrification-denitrification, short-cut nitrification-denitrification (PND), and anaerobic ammonia oxidation (Anammox). However, these methods suffer from reduced efficiency, unstable operation, and the need for high energy consumption or exogenous carbon sources at low temperatures. For example, in conventional methods, the activity of ammonia-oxidizing bacteria is inhibited at low temperatures, resulting in substandard effluent; Anammox is difficult to start up at low temperatures and has poor treatment effect on low-concentration wastewater. In addition, in existing technologies, although the exogenous addition of signaling substances (such as AHLs) can improve nitrogen removal efficiency, it is costly and these signaling substances are easily degraded. *Pseudomonas aeruginosa*, a common microbial species in activated sludge microecology, is a core species in the intercellular communication network of the ammonia oxidation process. It can secrete various functional signaling substances such as C6-HSL, C8-HSL, C10-HSL, and pyocyanin. Related studies have confirmed that C6-HSL and C8-HSL can significantly enhance biological nitrogen removal efficiency. Analysis of activated sludge samples from 23 wastewater treatment plants during winter revealed that the microorganisms related to the ammonia oxidation process in the activated sludge system are mainly... Nitrososphaera and Nitrosomonas As the dominant species, and Nitrosomonas europaea It can utilize signaling molecules such as C6-HSL, C8-HSL, and C10-HSL. Pseudomonas aeruginosa is a bioactive phenazine pigment and a nitric oxide antagonist, capable of eliminating nitric oxide produced during the metabolism of ammonia-oxidizing bacteria. Nitric oxide can potentially inhibit the expression of intracellular enzymes related to ammonia-oxidizing bacteria, leading to a decrease in the abundance of ammonia-oxidizing bacteria. Simultaneously, pseudomonas aeruginosa can inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria, leading to nitrite nitrogen accumulation. Therefore, by adding a biofilm carrier attached to Pseudomonas aeruginosa to the activated sludge system, the abundance of functional signaling molecule producers in the activated sludge system can be increased, the intercellular communication behavior of the ammonia oxidation process in the activated sludge system can be regulated, the ammonia oxidation activity of the activated sludge can be enhanced, the nitrite accumulation rate can be increased, short-cut nitrification and denitrification can be achieved, and thus the biological nitrogen removal efficiency can be improved. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient biological nitrogen removal method for low-ammonia nitrogen wastewater under low-temperature environment short-cut nitrification and denitrification based on functional microbial regulation. It was found that adding a biofilm carrier with attached Pseudomonas aeruginosa can improve the ammonia nitrogen removal rate and nitrite accumulation rate of activated sludge. Pseudomonas aeruginosa can secrete functional signaling substances to regulate the nitrogen metabolism of ammonia-oxidizing bacteria and can eliminate nitric oxide produced during the metabolism of ammonia-oxidizing bacteria, thus avoiding the inhibition of ammonia-oxidizing bacteria activity by nitric oxide. At the same time, it can also inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A highly efficient biological nitrogen removal method for low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation includes the following steps:

[0008] The carrier material is attached to a Pseudomonas aeruginosa pure culture system to form a carrier on which Pseudomonas aeruginosa is attached. The attached carrier is then added to the activated sludge reactor, with its volume accounting for 12.8%-16.0% of the effective volume of the reactor. The sequencing batch reactor is operated in a sequencing batch reactor mode, which includes an aerobic nitrification section and an anoxic denitrification section. The functional signaling substances secreted by Pseudomonas aeruginosa enhance the ammonia oxidation activity and inhibit the nitrite oxidation, thereby achieving short-cut nitrification and denitrification.

[0009] As a preferred technical solution of the present invention, the carrier material is attached to a membrane in a pure culture system of Pseudomonas aeruginosa as follows:

[0010] After activating the lyophilized Pseudomonas aeruginosa powder in NB medium, it was transferred to LB medium and cultured for 24-48 hours before being used as a concentrated bacterial solution. The concentrated bacterial solution was inoculated into LB broth medium and cultured for 24-48 hours. The carrier materials were then added in groups to LB broth medium for biofilm culture for 2-3 weeks. After the biofilm culture was completed, the surface of the carrier was light green with pink dot-like colonies growing on it.

[0011] As a preferred technical solution of the present invention, the carrier material is non-woven fabric.

[0012] As a more preferred technical solution of the present invention, the volume of the attached carrier accounts for 14.4% of the effective volume of the reactor.

[0013] As a preferred technical solution of the present invention, the dissolved oxygen concentration in the influent of the activated sludge reactor is above 2.00 mg / L.

[0014] As a preferred embodiment of the present invention, the influent substrate of the activated sludge reactor contains 37.63-29.63 mg / L of NH4. + -N and COD of 107.56-92.19 mg / L, pH 7.5-8.5.

[0015] As a preferred embodiment of the present invention, the sludge concentration in the activated sludge reactor is 1710.06-4175.68 mg / L. MLSS (Mixed Lime Solids Saturation) refers to the suspended solids concentration in the mixed liquor of wastewater and activated sludge.

[0016] As a preferred technical solution of the present invention, the activated sludge reactor is a sequencing batch reactor (SBR).

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] This invention can enhance the ammonia oxidation activity of activated sludge and increase the nitrite accumulation rate under conditions of 28℃, 18℃, 14℃ and 10℃, thereby enabling the reactor to achieve better denitrification effect.

[0019] This invention starts from the intercellular communication mechanism of microorganisms. By increasing the abundance of core species in the intercellular communication network during the ammonia oxidation process, the concentration of functional signaling substances in the reactor is increased, thereby regulating nitrogen conversion-related functional microorganisms and fundamentally improving the efficiency of biological denitrification.

[0020] This invention achieves a nitrite accumulation rate of over 80% while enhancing ammonia oxidation activity, and maintains long-term stable operation at 15°C. This application proposes a highly efficient and stable strategy to enhance ammonia oxidation activity and achieve short-cut nitrification, maintaining long-term stability even at low temperatures of 10-14°C, significantly improving biological nitrogen removal efficiency and providing a valuable reference for enhancing biological nitrogen removal in actual wastewater treatment. The added biofilm carrier attached to *Pseudomonas aeruginosa* can secrete functional signaling substances to regulate nitrogen metabolism and species abundance in ammonia-oxidizing bacteria, while also inhibiting the expression of nitrite oxidase in nitrite-oxidizing bacteria.

[0021] This invention covers a wide temperature range of 10-28℃, making it suitable for wastewater treatment in northern my country during winter (when water temperatures are often below 15℃), and it exhibits strong adaptability to influent COD (100 mg / L) and ammonia nitrogen (30 mg / L). The sequencing batch biofilm reactor (SBBR) has demonstrated stability in practical engineering and is easy to promote. Attached Figure Description

[0022] Figure 1 (a) A graph showing long-term water quality monitoring data for the nitrification stage of a biofilm carrier group with attached Pseudomonas aeruginosa;

[0023] Figure 1 (b) is a graph showing the long-term water quality monitoring data of the nitrification section in the control group;

[0024] Figure 2 (a) A graph showing water quality monitoring data for long-term nitrification and denitrification of a biofilm carrier group with attached Pseudomonas aeruginosa;

[0025] Figure 2 (b) is a graph showing the long-term water quality monitoring data of the control group undergoing nitrification and denitrification.

[0026] Figure 3 Ammonia oxidation activity comparison between the group with biofilm carriers attached to Pseudomonas aeruginosa and the control group;

[0027] Figure 4The graph shows the nitrate oxidation rate of the group with biofilm carriers attached to Pseudomonas aeruginosa and the control group.

[0028] Figure 5 Figure 1 shows the extraction and detection of signal molecules in a pure culture system of Pseudomonas aeruginosa.

[0029] Figure 6 A graph showing the nitrogen conversion activity of Pseudomonas aeruginosa under aerobic conditions;

[0030] Figure 7 This is a graph showing the nitrogen transformation activity of Pseudomonas aeruginosa under anaerobic conditions. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but it is not intended to limit the present invention.

[0032] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0033] The carrier material described in this invention is a medium used in biofilm reactors (such as biofilters, biological contact oxidation tanks, MBBRs, etc.) for microbial attachment and growth. Its core function is to provide a large specific surface area and a suitable environment to promote the formation of a stable biofilm by microorganisms. Common biofilm carrier materials are applicable to this application.

[0034] The sequencing batch reactor (SBBR) used in this invention has an effective volume of 5 L. A stirring device is installed at the top of the reactor to ensure uniform mixing of the sludge and water, and an aeration device is installed at the bottom to ensure aerobic conditions in the system. The dissolved oxygen (DO) concentration in the water is greater than 2 mg / L at different operating temperatures. The experiment used synthetic wastewater as the test water, and the influent substrate contained 32.36 ± 1.63 mg / L of NH4+. +The activated sludge reactor has NH4⁺-N and COD of 100.53±6.01 mg / L, with a pH of 7.5-8.5. Operating conditions are: dissolved oxygen concentration ≥2.00 mg / L, influent substrate containing NH4⁺-N 30 mg / L, COD 100 mg / L, pH 7.5-8.5, and sludge concentration 1710.06-4175.68 mg / L; the carrier volume occupies 14.4% of the reactor's effective volume. The activated sludge reactor is a sequencing batch reactor (SBBR), with an operating cycle of 9-17 hours, including 0.5 hours of influent, 5-10 hours of aerobic nitrification, 1-4 hours of anoxic denitrification, 2 hours of sedimentation, and 0.5 hours of effluent discharge. The aeration rate in the aerobic nitrification section is 80-400 mL / min, and sodium acetate is added as a carbon source in the anoxic denitrification section, with a COD concentration of 100 mg / L.

[0035] Nonwoven fabric physical and chemical parameters:

[0036] Fiber diameter: preferably 10-50 μm (fibers that are too coarse (>50μm) have insufficient specific surface area, and fibers that are too fine (<10μm) are prone to clogging pores).

[0037] Porosity: 80-95% (when the porosity is <80%, the mass transfer inside the carrier is limited; when it is >95%, the structural strength is insufficient).

[0038] Specific surface area: ≥500 m² / m³ (determined by BET method; when specific surface area <300 m² / m³, the bacterial load is less than 10). 8 CFU / cm²).

[0039] Surface hydrophilicity: Contact angle ≤30° (after plasma hydrophilic modification treatment, the initial adhesion rate of Pseudomonas aeruginosa decreased by 42% when the contact angle is >50°).

[0040] Biodegradability: The mass loss rate after soaking in LB medium for 30 days is ≤5% (polypropylene / polyester composite nonwoven fabric must be used).

[0041] Carrier structural parameters:

[0042] Three-dimensional structure: wave-shaped pleats (pleat height 0.5-1.0mm, spacing 2-3mm).

[0043] Mechanical properties: Tensile strength ≥15 N / cm (avoid fiber breakage caused by aeration erosion).

[0044] Vector pretreatment methods:

[0045] Hydrophilization treatment: Immerse the nonwoven fabric in 0.1 mol / L NaOH solution (60℃) for 2 hours, then rinse with deionized water until neutral.

[0046] Sterilization treatment: Sterilize with high-pressure steam at 121℃ for 20 minutes, then dry before use.

[0047] Quantitative parameters of biofilm culture:

[0048] Carrier loading density: 200-300 cm² / L of specific surface area per liter of culture medium (in the example, the total surface area of ​​10 carriers is 2cm×2cm×2 surfaces×10 carriers=80 cm², which corresponds to 800 cm² / L of specific surface area per 100mL of culture medium. It should be noted that this ratio should not exceed 1000 cm² / L, otherwise the dissolved oxygen will be insufficient).

[0049] Culture medium replacement strategy: Replace 50% of the culture medium every 72 hours (maintaining a carbon-to-nitrogen ratio of C / N = 8-10).

[0050] Finish line determination criteria:

[0051] Biofilm thickness: 50-80 μm as measured by laser confocal microscopy.

[0052] Bacterial density: ≥10 copies of Pseudomonas aeruginosa detected by qPCR 8 copies / cm².

[0053] Color quantification: CIE Lab colorimetric system measured L value ≤ 70, a value ≥ -5, b* value ≥ 20 (corresponding to light green).

[0054] Dynamic film attachment control:

[0055] Phase 1 (0-7 days): Shake at 50 rpm to promote initial adhesion.

[0056] Phase 2 (8-14 days): Increase the rotation speed to 120 rpm to enhance biofilm stability.

[0057] Metabolite monitoring: C6-HSL concentration was measured every 48 hours (it needs to be maintained at 50-100 nM; if it is below 20 nM, 10 mM sodium succinate should be added to induce the synthesis of signal molecules).

[0058] Example 1

[0059] Commercially available Pseudomonas aeruginosa PAO1 lyophilized powder (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number BMZ114547; all Pseudomonas aeruginosa mentioned below are products with the same product number purchased from this company) was activated in 5 mL of NB medium, then transferred to 50 mL of LB medium and cultured for 24-48 h to be used as a concentrated bacterial suspension. 100 mL of LB broth medium was prepared, inoculated with 10% concentrated Pseudomonas aeruginosa bacterial suspension, and cultured at 37°C on a shaker at 150 rpm for 24-48 h. OD was then measured. 600For samples with a density of 2 or higher, divide 10 pieces of 2 cm x 2 cm x 1 cm carrier material into groups. Add each group of carrier material to 100 mL of culture medium and incubate for 2-3 weeks, changing the culture medium every 3 days. The carrier material is white non-woven fabric. After the incubation period, the surface of the carrier is light green with pink dot-like colonies. For carrier materials attached to Pseudomonas aeruginosa PAO1, each piece is 2 cm x 2 cm x 1 cm, with 10 pieces attached to each thread. A 5 L reactor can hold 16-20 threads.

[0060] In the following application examples, there are 18 lines, which means that the volume ratio of the biofilm carrier with attached Pseudomonas aeruginosa to the activated sludge reactor is 14.4%.

[0061] Application Example 1

[0062] The efficient biological nitrogen removal method for wastewater in this embodiment includes the following steps:

[0063] Operating at 28℃, the MLSS in the reactor with the added biofilm carrier containing *Pseudomonas aeruginosa* was 4152.24 ± 20.42 mg / L, and the VSS was 1382.09 ± 3.69 mg / L; in the control reactor, the MLSS was 4175.68 ± 20.74 mg / L, and the VSS was 1575.68 ± 8.92 mg / L. Influent NH4... + -N=34.01±1.98 mg / L, COD=94.66±3.31 mg / L (LB medium 35 mL), aeration rate of 400 mL / min in the aerobic stage. The SBBR reactor operated for 9 hours per cycle, with 0.5 hours of influent, 5 hours of aerobic operation, aeration shut off, COD=104.92±6.37 mg / L (sodium acetate as carbon source) added for denitrification, 1 hour of anoxic stirring, 2 hours of sedimentation, and 0.5 hours of effluent discharge, with an effluent ratio of 50%. Compared to the control reactor, the reactor with the biofilm carrier attached to Pseudomonas aeruginosa showed a 23.02% increase in ammonia oxidation activity and achieved an 82.58% nitrite accumulation rate, successfully improving the ammonia oxidation activity of activated sludge and achieving a high nitrite accumulation rate.

[0064] Application Example 2

[0065] The efficient biological nitrogen removal method for wastewater in this embodiment includes the following steps:

[0066] Operating at 18℃, the MLSS in the reactor with the added biofilm carrier containing *Pseudomonas aeruginosa* was 4152.24 ± 20.42 mg / L, and the VSS was 1382.09 ± 3.69 mg / L; in the control reactor, the MLSS was 4175.68 ± 20.74 mg / L, and the VSS was 1575.68 ± 8.92 mg / L. Influent NH4... + -N=32.86±1.86 mg / L, COD=104.89±6.51 mg / L (LB medium 35 mL), aeration rate of 400 mL / min in the aerobic stage. The SBBR reactor operated for 11 hours per cycle: 0.5 hours of influent, 7 hours of aerobic operation, aeration shut off, COD=94.47±5.21 mg / L (sodium acetate as carbon source) added for denitrification, 1 hour of anoxic stirring, 2 hours of sedimentation, and 0.5 hours of effluent discharge (effluent ratio 50%). Compared to the control group, the reactor with the biofilm carrier attached to Pseudomonas aeruginosa showed a 60.69% increase in ammonia oxidation activity and achieved an 89.06% nitrite accumulation rate, successfully improving the ammonia oxidation activity of activated sludge and achieving a high nitrite accumulation rate.

[0067] Application Example 3

[0068] The efficient biological nitrogen removal method for wastewater in this embodiment includes the following steps:

[0069] Operating at 14℃, the MLSS in the reactor with the added biofilm carrier containing *Pseudomonas aeruginosa* was 2943.83±29.25 mg / L, and the VSS was 2813.62±65.54 mg / L; in the control reactor, the MLSS was 3851.89±51.89 mg / L, and the VSS was 3081.43±52.25 mg / L. Influent NH4... + -N=31.41±1.30 mg / L, COD=92.19±4.62 mg / L (LB medium 35 mL), aeration rate of 160 mL / min in the aerobic stage. The SBBR reactor operated for 15 h per cycle, with 0.5 h of influent, 10 h of aerobic operation, aeration shut off, COD=90.87±0.66 mg / L (sodium acetate as carbon source) added for denitrification, 2 h of anoxic stirring, 2 h of sedimentation, and 0.5 h of effluent discharge (effluent ratio 50%). Compared with the control reactor, the reactor with the biofilm carrier attached to Pseudomonas aeruginosa showed a 61.06% increase in ammonia oxidation activity and achieved a 90.59% nitrite accumulation rate, successfully improving the ammonia oxidation activity of activated sludge and achieving a high nitrite accumulation rate.

[0070] Application Example 4

[0071] The efficient biological nitrogen removal method for wastewater in this embodiment includes the following steps:

[0072] Operating at 10℃, the MLSS in the reactor with the added biofilm carrier containing *Pseudomonas aeruginosa* was 1710.06±6.61 mg / L, and the VSS was 1601.50±67.02 mg / L; in the control reactor, the MLSS was 3250.59±35.77 mg / L, and the VSS was 2489.82±10.19 mg / L. Influent NH4... + -N=32.59±1.28 mg / L, COD=105.46±2.10 mg / L (LB medium 35 mL), aeration rate of 80 mL / min in the aerobic stage. The SBBR reactor operated for 17 h per cycle, with 0.5 h of influent, 10 h of aerobic operation, aeration shut off, COD=102.10±3.54 mg / L (sodium acetate as carbon source) added for denitrification, 4 h of anoxic stirring, 2 h of sedimentation, and 0.5 h of effluent discharge, with an effluent ratio of 50%. Compared to the control reactor, the reactor with the biofilm carrier attached to Pseudomonas aeruginosa showed an 88.88% increase in ammonia oxidation activity and an 83.83% nitrite accumulation rate, successfully improving the ammonia oxidation activity of activated sludge and achieving a high nitrite accumulation rate.

[0073] This invention relates to microbial regulation based on intercellular communication mechanisms. By adding a biofilm carrier attached to *Pseudomonas aeruginosa*, the activity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) is regulated using acylhomoserine lactones (AHLs, such as C6-HSL and C8-HSL) and pyocyanin secreted by the bacteria. AHLs secreted by *P. aeruginosa* have been shown to promote the metabolic functions of ammonia-oxidizing bacteria, such as the tricarboxylic acid cycle and amino sugar synthesis, thereby improving denitrification efficiency. For example, the experimental group showed an 88.88% increase in ammonia oxidation activity at 10°C. Pyocyanin, as a nitric oxide (NO) antagonist, can eliminate the self-toxicity of NO produced by AOB metabolism, while simultaneously inhibiting the nitrite oxidase activity of NOB, keeping the nitrite accumulation rate stable above 80% over a long period. Compared to traditional strategies that rely on exogenous AHLs, which suffer from high cost and easy degradation (requiring repeated additions), this application achieves continuous and stable regulation through the endogenous secretion of signaling molecules by the biofilm carrier. Figure 1 , Figure 2 , Figure 3 as well as Figure 5As shown, this invention, by adding a biofilm carrier attached to *Pseudomonas aeruginosa*, can eliminate nitric oxide produced during the metabolism of ammonia-oxidizing bacteria, avoiding damage to ammonia-oxidizing bacteria and subsequent decrease in their abundance caused by nitric oxide. Simultaneously, the biofilm carrier attached to *Pseudomonas aeruginosa* can secrete functional signaling substances to regulate the expression of genes related to nitrogen metabolism in ammonia-oxidizing microorganisms, thereby improving the specific ammonia oxidation activity of the reactor. Figure 1 and Figure 4 As shown, this invention, by adding a biofilm carrier attached to *Pseudomonas aeruginosa*, can inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria in the reactor, leading to nitrite nitrogen accumulation, thereby achieving short-cut nitrification and denitrification and improving the reactor's biological nitrogen removal efficiency. Figure 6 and Figure 7 As shown, the added biofilm carriers with attached Pseudomonas aeruginosa did not exhibit nitrogen conversion activity under both aerobic and hypoxic conditions. This demonstrates that the regulatory strategy proposed in this invention addresses intercellular communication behavior among microorganisms, indirectly increasing the concentration of functional signaling substances by enhancing the abundance of core bacterial species. Consequently, it regulates the species abundance and gene expression of nitrogen conversion-related microorganisms, thereby solving the problems of difficult and inefficient nitrogen removal faced by urban wastewater treatment plants and improving the efficiency of biological nitrogen removal treatment of wastewater.

[0074] This application achieves highly efficient nitrogen removal under low-temperature conditions of 10-28℃, overcoming the bottleneck of the sharp drop in efficiency of traditional nitrification-denitrification processes at low temperatures (<15℃). The reactor was operated for certain periods at 28℃, 18℃, 14℃, and 10℃. Compared to the control group, the experimental groups with added biofilm carriers attached to *Pseudomonas aeruginosa* showed increased ammonia oxidation activity of 23.02%, 60.69%, 61.06%, and 88.88%, respectively, and nitrite accumulation rates of 82.58%, 89.06%, 90.59%, and 83.83%, respectively.

[0075] The experimental group showed an 88.88% increase in ammonia oxidation activity and a nitrite accumulation rate of 83.83% at 10℃, significantly outperforming conventional short-cut nitrification-denitrification (PND) and anaerobic ammonia oxidation (Anammox) at low temperatures (e.g., the CANON process only achieved a total nitrogen removal rate of 16.87% at 10℃). While low-temperature enhancement technologies (such as magnetic field enhancement and gradual cooling methods) require additional equipment or complex operations, this invention simplifies the process through microbial regulation.

[0076] Within the temperature range of 10-28℃, this invention improved ammonia oxidation activity by 23.02%-88.88% and nitrite accumulation rate by 82.58%-90.59% in the experimental group, and it can operate stably for a long time at 15℃. In contrast, the traditional activated sludge process often has an ammonia nitrogen removal rate of less than 50% at low temperatures. The sequencing batch biofilm reactor (SBBR) combined with a carrier filling rate of 14.4% optimizes biofilm attachment and mass transfer efficiency, for example, the suspended sludge concentration (MLSS) at 10℃ is 1710.06-4175.68 mg / L, ensuring the abundance of functional bacteria.

[0077] This invention utilizes short-cut nitrification-denitrification (requiring only the oxidation of ammonia to nitrite, followed by direct reduction to nitrogen), saving approximately 25% of oxygen demand and 40% of carbon source consumption, thus meeting the "dual carbon" target. Compared to electrode ammonia oxidation or Anammox processes, it eliminates the need for an external power source or strictly anaerobic conditions, reducing energy consumption and operational complexity. Existing studies using direct addition of AHLs are costly (e.g., 1000-nM AHL reduces COD removal rate by 7%) and are easily degraded by quenching bacteria. This invention achieves endogenous continuous secretion through the addition of signaling molecule-producing bacteria (Pseudomonas aeruginosa), resulting in lower costs and more stable effects.

[0078] Traditional low-temperature denitrification relies on screening for psychrophilic bacteria or gradual cooling and acclimatization (which takes several months). This invention, however, rapidly enhances AOB activity (e.g., a 60.69% increase in ammonia oxidation activity at 18°C) by regulating the microbial community through signaling substances. Pseudomonas aeruginosa's inhibitory effect on NOB remains effective at low temperatures, while conventional methods (such as DO regulation) struggle to inhibit NOB proliferation at low temperatures.

[0079] In this invention, the biofilm carrier for attaching *Pseudomonas aeruginosa* exhibits a synergistic effect with the carrier itself. *Pseudomonas aeruginosa* is added after attachment in a pure culture system, ensuring the functional bacterial density and activity of the biofilm carrier and avoiding competition from other bacteria. Traditionally, mixed bacterial communities or unoptimized carrier attachment conditions are often used. The 14.4% carrier filling rate provided in the examples of this invention has been experimentally verified as the optimal ratio, balancing the biofilm attachment area with the reactor's mass transfer efficiency (excessive filling rate may lead to collision interference).

[0080] This invention combines short-cut nitrification and denitrification with signaling molecule regulation to form a synergistic pathway of "enhancing AOB activity - inhibiting NOB activity - promoting nitrite accumulation." Existing technologies mostly focus on a single mechanism (such as only inhibiting NOB or only optimizing AOB). *Pseudomonas aeruginosa* is a common species in activated sludge, requiring no additional cultivation costs, and the biofilm carrier can be reused. Compared to chemical methods (such as adding nitrite oxidation inhibitors), there is no risk of secondary pollution.

[0081]

[0082] This invention achieves efficient and stable short-range nitrification-denitrification nitrogen removal under low-temperature conditions through the innovative application of *Pseudomonas aeruginosa* biofilm carriers, combined with intercellular communication regulation and the inhibitory effect of pyocyanin. Its innovation lies not only in the breakthrough in microbial regulation mechanisms but also in the optimization of engineering parameters (such as a 14.4% carrier filling rate), significantly surpassing the limitations of existing technologies in terms of exogenous signaling substance dependence, low-temperature adaptability, and operating costs. It possesses high practical application value and widespread potential.

[0083] In this invention, sludge concentration refers to the suspended solids content of the mixture after wastewater and activated sludge are mixed.

[0084] The embodiments of the present invention are not limited to the above-described examples. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, characterized in that, Includes the following steps: The carrier material was attached to a membrane in a pure culture system of Pseudomonas aeruginosa to form a carrier for Pseudomonas aeruginosa. The attached carrier is added to the sequencing batch reactor (SBR) with a volume of 12.8%-16.0% of the effective volume of the reactor; the influent temperature of the SBR is selected from 10-14℃. The sequencing batch reactor (SBR) operation mode includes an aerobic nitrification section and an anoxic denitrification section. It enhances ammonia oxidation activity and inhibits nitrite oxidation by using functional signaling substances secreted by Pseudomonas aeruginosa, and can also eliminate nitric oxide produced during the metabolism of ammonia-oxidizing bacteria, thus achieving short-cut nitrification and denitrification.

2. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The carrier material was implanted into a pure culture system of Pseudomonas aeruginosa as follows: After activating the lyophilized Pseudomonas aeruginosa powder in NB medium, it was transferred to LB medium and cultured for 24-48 hours before being used as a concentrated bacterial solution. The concentrated bacterial solution was inoculated into LB broth medium and cultured for 24-48 hours. The carrier materials were then added in groups to LB broth medium for biofilm culture for 2-3 weeks. After the biofilm culture was completed, the surface of the carrier was light green with pink dot-like colonies growing on it.

3. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The carrier material is non-woven fabric.

4. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The volume of the attached carrier accounts for 14.4% of the effective volume of the reactor.

5. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The dissolved oxygen concentration in the influent of the sequencing batch biofilm reactor is above 2.00 mg / L.

6. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The influent substrate of the sequencing batch reactor contains 37.63-29.63 mg / L of NH4. + -N and COD of 107.56-92.19 mg / L, pH 7.5-8.

5.

7. The method for efficient biological nitrogen removal from low-ammonia nitrogen wastewater in a low-temperature environment based on functional microbial regulation, as described in claim 1, is characterized in that... The sludge concentration in the sequencing batch reactor is 1710.06-4175.68 mg / L.

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