Functional microorganism regulation-based short-cut nitrification and denitrification efficient biological denitrification method for low-temperature-environment low-ammonia-nitrogen sewage

Abstract

The invention discloses a functional microorganism regulation based short-cut nitrification and denitrification high-efficiency biological denitrification method for low-temperature environment low-ammonia nitrogen sewage, which comprises the following steps: adding a biofilm carrier attached with pseudomonas aeruginosa into an activated sludge reactor, and aerating and stirring to continuously operate; functional signal substances capable of being secreted by the biofilm carrier attached with the pseudomonas aeruginosa can regulate nitrogen metabolism of nitrifying bacteria and inhibit expression of nitrite oxidoreductase of nitrite oxidizing bacteria, the ammonia oxidation activity of a reactor operating at 28 DEG C, 18 DEG C, 14 DEG C and 10 DEG C is improved by 23.02%, 60.69%, 61.06% and 88.88% respectively, the nitrite accumulation rates are 82.58%, 89.06%, 90.59% and 83.83% respectively, and the ammonia oxidation activity of the reactor operating at 28 DEG C, 18 DEG C, 14 DEG C and 10 DEG C is improved by 23.52%, 60.69%, 61.06% and 88.88% respectively. Therefore, short-cut nitrification and denitrification biological nitrogen removal is realized, and a better regulation and control method is provided for improving activated sludge biological nitrogen removal.

Description

Technical Field

[0001] The present invention belongs to the field of wastewater treatment, and in particular relates to a method for efficient biological denitrification of low-ammonia nitrogen sewage in a low-temperature environment by short-range nitrification and denitrification based on the regulation of functional microorganisms. Background Art

[0002] China's urban wastewater treatment plants primarily utilize traditional biological denitrification technology based on nitrification and denitrification, currently the most cost-effective, efficient, and widely used technology. Nitrogen-transforming microorganisms in wastewater treatment systems are primarily mesophilic, with an optimal growth temperature of 25-30°C. However, winter water temperatures in urban wastewater treatment plants in northern China typically drop below 15°C. These low temperatures directly restrict the ammonia oxidation process during nitrification and denitrification, resulting in excessive levels of ammonia nitrogen and total nitrogen in effluent from these plants in winter. Consequently, these plants are forced to achieve compliance with wastewater discharge standards at the expense of high energy and chemical consumption, creating a stark conflict with the "dual carbon" goals. Therefore, energy-efficient and efficient operation strategies are currently needed to ensure consistent and standard wastewater discharge.

[0003] Wastewater treatment systems are multi-microbial symbiotic systems. Microorganisms release signaling substances into the environment during their growth and regulate their metabolic functions by sensing changes in their concentrations. Signaling substances are common metabolites in the environment that drive intercellular communication and collaboration. This process, known as intercellular communication, is considered a universal mechanism for establishing microbial relationships. Intercellular communication regulates microbial metabolism, aggregation, motility, and other biological processes. In anaerobic ammonium oxidation (ANAMMOX) 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 expression of related genes and accelerating the secretion of extracellular substances, resulting in an increase in the system's denitrification capacity from 22.2% to 74.6% and 76.2%, respectively. Targeted regulation of intercellular communication by exogenously adding functional signaling substances can effectively address issues such as low biological denitrification efficiency and difficulties in meeting effluent ammonia and total nitrogen standards. However, these strategies are costly to implement, and signaling substances are susceptible to quenching in complex systems, making them difficult to sustain. Therefore, targeting the intercellular communication network of the ammonia oxidation process in the activated sludge system, the abundance of functional signal substance producers is increased and the secretion of functional signal substances is promoted, thereby enhancing the ammonia oxidation activity and improving the biological denitrification efficiency of sewage.

[0004] The application of sequencing batch biofilm reactors (SBBRs) in low-temperature denitrification has been studied, but it usually requires optimization of packing size and filling rate or combination with other technologies (such as magnetic fields). Existing low-temperature denitrification technologies mainly include traditional nitrification and denitrification, short-cut nitrification and denitrification (PND), anaerobic ammonia oxidation (Anammox), etc. However, these methods have problems such as reduced efficiency, unstable operation, high energy consumption or the need for exogenous carbon sources at low temperatures. For example, the activity of ammonia-oxidizing bacteria in traditional methods is inhibited at low temperatures, resulting in substandard effluent; Anammox is difficult to start at low temperatures and has poor effect in treating low-concentration wastewater. In addition, in existing technologies, although the exogenous addition of signal substances (such as AHLs) can improve denitrification efficiency, the cost is high and the signal substances are easily degraded. Pseudomonas aeruginosa, as a common bacterial species in the activated sludge microecological system, is the core bacterial species in the intercellular communication network of the ammonia oxidation process. It can secrete a variety of functional signal substances such as C6-HSL, C8-HSL, C10-HSL and pyocyanin. Related studies have confirmed that C6-HSL and C8-HSL can significantly improve the efficiency of biological denitrification. Through the analysis of winter activated sludge samples from 23 sewage treatment plants, it was found that the microorganisms related to the ammonia oxidation process in the activated sludge system are mainly Nitrososphaera and Nitrosomonas is the dominant population, and Nitrosomonas europaea It can utilize signal molecules such as C6-HSL, C8-HSL, and C10-HSL. Pyocyanin is a biologically active phenazine pigment and a nitric oxide antagonist that can eliminate nitric oxide produced during the metabolism of ammonia-oxidizing bacteria. Nitric oxide has the potential to inhibit the expression of related enzymes in ammonia-oxidizing bacteria, resulting in a decrease in the abundance of ammonia-oxidizing bacteria. At the same time, pyocyanin can inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria, leading to the accumulation of nitrite nitrogen. Therefore, by adding biofilm carriers attached to Pseudomonas aeruginosa to the activated sludge system, the abundance of functional signal substance producers in the activated sludge system is increased, the intercellular communication behavior of the ammonia oxidation process in the activated sludge system is regulated, the ammonia oxidation activity of the activated sludge is enhanced, the nitrite accumulation rate is increased, and short-range nitrification and denitrification are achieved, thereby improving the efficiency of biological denitrification. Summary of the Invention

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

[0006] The purpose of the present invention is achieved through the following technical solutions: A method for efficient biological denitrification of low-ammonia nitrogen wastewater by short-range nitrification and denitrification in a low-temperature environment based on functional microbial regulation, comprising the following steps: The carrier material is grown as a biofilm in a pure culture system of Pseudomonas aeruginosa to form a carrier attached with Pseudomonas aeruginosa; the attached carrier is added to an activated sludge reactor, with its volume accounting for 12.8%-16.0% of the effective volume of the reactor; a sequencing batch operation mode is adopted, including an aerobic nitrification section and an anoxic denitrification section, and the functional signal substances secreted by Pseudomonas aeruginosa are used to enhance ammonia oxidation activity and inhibit nitrite oxidation, thereby achieving short-term nitrification and denitrification.

[0007] As a more preferred technical solution of the present invention, the carrier material is formed into a biofilm in a pure culture system of Pseudomonas aeruginosa as follows: The freeze-dried powder of Pseudomonas aeruginosa was activated in NB medium and then transferred to LB medium. After culturing for 24-48 hours, it was used as a concentrated bacterial liquid. The concentrated bacterial liquid was inoculated into LB broth medium and cultured for 24-48 hours. The carrier materials were added in groups to the LB broth culture to form biofilms for 2-3 weeks. After the biofilm culture was completed, the surface of the carrier was light green as a whole, with pink dot-like colonies growing.

[0008] As a more preferred technical solution of the present invention, the carrier material is non-woven fabric.

[0009] As a more optimal technical solution of the present invention, the volume of the attached carrier accounts for 14.4% of the effective volume of the reactor.

[0010] As a more optimal technical solution of the present invention, the dissolved oxygen concentration of the influent of the activated sludge reactor is above 2.00 mg / L.

[0011] As a more optimal technical solution of the present invention, the influent matrix of the activated sludge reactor contains 37.63-29.63 mg / L NH4 + -N and COD of 107.56-92.19 mg / L, with a pH of 7.5-8.5.

[0012] As a more optimal technical solution of the present invention, the sludge concentration in the activated sludge reactor is 1710.06-4175.68 mg / L. The sludge concentration MLSS refers to the suspended solids concentration of the mixed liquor after the sewage and activated sludge are mixed. As a more optimal technical solution of the present invention, the activated sludge reactor is a sequencing batch biofilm reactor.

[0013] Compared with the prior art, the present invention has the following advantages and effects: The present invention can enhance the ammonia oxidation activity of activated sludge and improve the nitrite accumulation rate under the conditions of 28°C, 18°C, 14°C and 10°C, thereby enabling the reactor to achieve better denitrification effect.

[0014] The present invention starts from the intercellular communication mechanism of microorganisms, increases the abundance of core species in the intercellular communication network during the ammonia oxidation process, increases the concentration of functional signal substances in the reactor, and then regulates the functional microorganisms related to nitrogen conversion, thereby fundamentally achieving an improvement in the biological denitrification efficiency.

[0015] The present invention achieves a nitrite accumulation rate of more than 80% while improving ammonia oxidation activity, and can operate stably for a long time at 15°C. The present application proposes an efficient and stable strategy that can enhance ammonia oxidation activity and achieve short-range nitrification. It can still be stable for a long time under low temperature conditions of 10-14°C, greatly improving the efficiency of biological denitrification, and is more conducive to providing a reference for strengthening the biological denitrification effect of actual sewage treatment. The added biofilm carrier attached to Pseudomonas aeruginosa can secrete functional signal substances to regulate the nitrogen metabolism and species abundance of ammonia oxidizing bacteria, and can also inhibit the expression of nitrite oxidase in nitrite oxidizing bacteria.

[0016] This system covers a wide temperature range of 10-28°C, making it suitable for winter wastewater treatment in northern my country (where water temperatures often fall below 15°C). It also exhibits strong adaptability to influent COD (100 mg / L) and ammonia nitrogen (30 mg / L). The sequencing batch biofilm reactor (SBBR) has proven its stability in practical projects and is readily applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 (a) is the long-term water quality monitoring data of the nitrification section of the biofilm carrier group with attached Pseudomonas aeruginosa; Figure 1 (b) is the long-term water quality monitoring data of the nitrification section of the control group; Figure 2 (a) is the water quality monitoring data of the long-term nitrification and denitrification operation of the biofilm carrier group with attached Pseudomonas aeruginosa; Figure 2 (b) is the water quality monitoring data of the long-term nitrification and denitrification operation of the control group; Figure 3 This is a graph showing the ammonia oxidation activity of the biofilm carrier group with attached Pseudomonas aeruginosa compared to the control group; Figure 4 This is a graph showing the nitrite oxidation rate of the biofilm carrier group with attached Pseudomonas aeruginosa and the control group; Figure 5 This is a diagram of signal molecule extraction and detection in a pure culture system of Pseudomonas aeruginosa; Figure 6This is a graph showing nitrogen conversion activity of Pseudomonas aeruginosa under aerobic conditions; Figure 7 This is a diagram of nitrogen transformation activity of Pseudomonas aeruginosa under anaerobic conditions. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to the embodiments, but is not intended to limit the present invention thereto.

[0019] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0020] The carrier material described in this invention is a medium used in biofilm reactors (such as biofilters, biological contact oxidation tanks, and MBBRs) to support the attachment and growth of microorganisms. Its core function is to provide a large specific surface area and a suitable environment to promote the formation of stable biofilms. Common biofilm-forming carrier materials are suitable for this application.

[0021] The sequencing batch biofilm reactor (SBBR) used in this invention has an effective volume of 5 L. A stirring device is installed on the top of the reactor to ensure uniform mixing of mud and water. An aeration device is set at the bottom to ensure aerobic conditions in the system. The DO concentration in the water at different operating stages is greater than 2 mg / L. The test water is artificial synthetic wastewater, and the influent matrix contains 32.36±1.63 mg / L of NH4 + -N and 100.53±6.01 mg / L COD, with a pH of 7.5-8.5. Operating conditions are: dissolved oxygen concentration ≥ 2.00 mg / L, influent matrix containing 30 mg / L NH⁺-N and 100 mg / L COD, a pH of 7.5-8.5, and a sludge concentration of 1710.06-4175.68 mg / L. The carrier volume accounts for 14.4% of the reactor's effective volume. The activated sludge reactor is a sequencing batch biofilm reactor (SBBR) with an operating cycle of 9-17 hours, consisting of 0.5 hours of water inflow, 5-10 hours of aerobic nitrification, 1-4 hours of anoxic denitrification, 2 hours of sedimentation, and 0.5 hours of drainage. 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. The COD concentration is 100 mg / L.

[0022] Non-woven fabric physical and chemical parameters: Fiber diameter: preferably 10-50 μm (fibers that are too thick (>50 μm) have insufficient specific surface area, while fibers that are too thin (<10 μm) tend to clog pores).

[0023] Porosity: 80-95% (when the porosity is <80%, the mass transfer inside the carrier is limited; when the porosity is >95%, the structural strength is insufficient).

[0024] Specific surface area: ≥500 m² / m³ (determined by BET method, when the specific surface area is <300 m² / m³, the bacterial load is less than 10 8 CFU / cm²).

[0025] Surface hydrophilicity: contact angle ≤ 30° (after plasma hydrophilic modification, the initial attachment rate of Pseudomonas aeruginosa decreased by 42% when the contact angle was > 50°).

[0026] Anti-biodegradation: Mass loss rate ≤ 5% after immersion in LB medium for 30 days (polypropylene / polyester composite non-woven fabric is required).

[0027] Carrier structure parameters: Three-dimensional structure: wavy pleat design (pleat height 0.5-1.0mm, spacing 2-3mm).

[0028] Mechanical properties: tensile strength ≥15 N / cm (avoid fiber breakage caused by aeration scouring).

[0029] Carrier pretreatment method: Hydrophilization treatment: Immerse the non-woven fabric in 0.1 mol / L NaOH solution (60°C) for 2 hours, and rinse with deionized water until neutral. Sterilization: sterilize with high pressure steam at 121℃ for 20 minutes, then dry and set aside.

[0030] Quantitative parameters of biofilm culture: Carrier loading density: Specific surface area per liter of culture medium: 200-300 cm² / L (the total surface area of ​​10 carriers in this example is 2 cm×2 cm×2 sides×10 carriers = 80 cm², corresponding to a specific surface area of ​​800 cm² / L for 100 mL of culture medium. Note that this ratio must not exceed 1000 cm² / L, otherwise, insufficient dissolved oxygen will result).

[0031] Culture medium renewal strategy: Replace 50% of the culture medium every 72 hours (maintain a carbon-nitrogen ratio of C / N = 8-10).

[0032] End point determination criteria: Biofilm thickness: 50-80 μm as measured by laser confocal microscopy.

[0033] Bacterial density: qPCR detection of Pseudomonas aeruginosa copy number ≥ 10 8 copies / cm².

[0034] Color quantification: CIE Lab colorimetric system: L value ≤ 70, a value ≥ -5, b* value ≥ 20 (corresponding to light green).

[0035] Dynamic film formation control: Stage 1 (Days 0-7): Shake at 50 rpm to promote initial attachment.

[0036] Phase 2 (8-14 days): The rotation speed is increased to 120 rpm to enhance biofilm stability.

[0037] Metabolite monitoring: Detect C6-HSL concentration every 48 hours (maintain 50-100 nM. When it is lower than 20 nM, supplement with 10 mM sodium succinate to induce signal molecule synthesis).

[0038] Example 1 Commercially available Pseudomonas aeruginosa PAO1 lyophilized powder (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number BMZ114547, all Pseudomonas aeruginosa mentioned below are products purchased from the same company) was activated in 5 mL of NB medium and transferred to 50 mL of LB medium. After culturing for 24-48 hours, it was used as a concentrated bacterial solution. Prepare 100 mL of LB broth medium, inoculate 10% of the Pseudomonas aeruginosa concentrated bacterial solution, and culture at 37°C on a shaker set at 150 rpm for 24-48 hours. After that, the OD value was measured. 600 = 2 or above. Group 10 2 cm x 2 cm x 1 cm carrier materials. Add each group of 10 carrier materials to 100 mL of culture medium and culture for biofilm formation for 2-3 weeks, changing the medium every 3 days. The carrier material is white non-woven fabric. After biofilm formation, the carrier surface will be light green with pink, dotted colonies. Each carrier material for Pseudomonas aeruginosa PAO1 is 2 cm x 2 cm x 1 cm. Ten carriers are hung on each thread, allowing 16-20 threads to fit in a 5-L reactor.

[0039] In the following application examples, there are 18 threads, that is, the volume ratio of the biofilm carriers attached with Pseudomonas aeruginosa to the volume in the activated sludge reactor is 14.4%.

[0040] Application Example 1 The efficient biological denitrification method for sewage of this embodiment comprises the following steps: When operated at 28℃, the MLSS in the biofilm carrier reactor with attached Pseudomonas aeruginosa was 4152.24±20.42 mg / L and the VSS was 1382.09±3.69 mg / L; the MLSS in the control reactor was 4175.68±20.74 mg / L and the VSS was 1575.68±8.92 mg / L. +The SBBR reactor operated for 9 hours per cycle, with a 0.5-hour water inlet and a 5-hour aerobic phase. Aeration was then turned off, followed by a COD of 104.92±6.37 mg / L (sodium acetate as a carbon source) for denitrification. The reactor was anoxic and stirred for 1 hour, followed by settling for 2 hours, and then drained for 0.5 hours. The drainage ratio was 50%. The reactor containing the biofilm carriers containing Pseudomonas aeruginosa exhibited a 23.02% increase in ammonia oxidation activity compared to the control reactor. The reactor containing the biofilm carriers also achieved a nitrite accumulation rate of 82.58%, successfully enhancing the ammonia oxidation activity of the activated sludge and achieving a high nitrite accumulation rate.

[0041] Application Example 2 The efficient biological denitrification method for sewage of this embodiment comprises the following steps: When operated at 18℃, the MLSS in the biofilm carrier reactor with attached Pseudomonas aeruginosa was 4152.24±20.42 mg / L and the VSS was 1382.09±3.69 mg / L; the MLSS in the control reactor was 4175.68±20.74 mg / L and the VSS was 1575.68±8.92 mg / L. + The SBBR reactor operated at a rate of 400 mL / min during the aerobic phase. Each cycle consisted of 11 hours of water inlet, 7 hours of aerobic operation, and aeration. Denitrification was supplemented with a COD of 94.47 ± 5.21 mg / L (sodium acetate as a carbon source). The reactor was anoxic and stirred for 1 hour, allowed to settle for 2 hours, and then drained for 0.5 hours, with a drainage ratio of 50%. Compared to the control reactor, the reactor containing the biofilm carriers containing Pseudomonas aeruginosa exhibited a 60.69% increase in ammonia oxidation activity. The reactor containing the biofilm carriers also achieved a nitrite accumulation rate of 89.06%, successfully enhancing the ammonia oxidation activity of the activated sludge and achieving a high nitrite accumulation rate.

[0042] Application Example 3 The efficient biological denitrification method for sewage of this embodiment comprises the following steps: When operated at 14°C, the MLSS in the biofilm carrier reactor with attached Pseudomonas aeruginosa was 2943.83±29.25 mg / L and the VSS was 2813.62±65.54 mg / L; the MLSS in the control reactor was 3851.89±51.89 mg / L and the VSS was 3081.43±52.25 mg / L. + The SBBR reactor operated for 15 hours per cycle, with a 0.5-hour water inlet and a 10-hour aerobic phase. Aeration was then shut off, and a COD of 90.87±0.66 mg / L (sodium acetate as a carbon source) was added for denitrification. The reactor was anoxic and stirred for 2 hours, allowed to settle for 2 hours, and then drained for 0.5 hours, with a drainage ratio of 50%. Compared to the control reactor, the reactor containing the biofilm carriers containing Pseudomonas aeruginosa exhibited a 61.06% increase in ammonia oxidation activity. The reactor containing the biofilm carriers also achieved a 90.59% nitrite accumulation rate, successfully enhancing the ammonia oxidation activity of the activated sludge and achieving a high nitrite accumulation rate.

[0043] Application Example 4 The efficient biological denitrification method for sewage of this embodiment comprises the following steps: When operated at 10℃, the MLSS in the biofilm carrier reactor with attached Pseudomonas aeruginosa was 1710.06±6.61 mg / L and the VSS was 1601.50±67.02 mg / L; the MLSS in the control reactor was 3250.59±35.77 mg / L and the VSS was 2489.82±10.19 mg / L. + The SBBR reactor operated for 17 hours per cycle, with a 0.5-hour water inlet and a 10-hour aerobic phase. Aeration was then shut off. Denitrification was supplemented with a COD of 102.10±3.54 mg / L (sodium acetate as a carbon source). The reactor was anoxic and stirred for 4 hours, followed by a 2-hour sedimentation period and a 0.5-hour drainage period. The drainage ratio was 50%. The reactor containing the biofilm carriers containing Pseudomonas aeruginosa exhibited an 88.88% increase in ammonia oxidation activity compared to the control reactor. The reactor containing the biofilm carriers also achieved a nitrite accumulation rate of 83.83%, successfully enhancing the ammonia oxidation activity of the activated sludge and achieving a high nitrite accumulation rate.

[0044] The present invention is a microbial regulation based on the intercellular communication mechanism. By adding biofilm carriers attached with Pseudomonas aeruginosa, the acyl homoserine lactones (AHLs, such as C6-HSL, C8-HSL) and pyocyanin secreted by them are used to regulate the activities of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). The AHLs secreted by Pseudomonas aeruginosa have been shown to promote the metabolic functions of ammonia oxidizing bacteria, such as the tricarboxylic acid cycle, amino sugar synthesis, etc., thereby improving the denitrification efficiency. For example, the ammonia oxidation activity of the experimental group increased by 88.88% at 10°C. Pyocyanin, as a nitric oxide (NO) antagonist, can eliminate the toxicity of NO produced by AOB metabolism to itself, while inhibiting the nitrite oxidase activity of NOB, so that the nitrite accumulation rate is stable at more than 80% for a long time. Compared with the traditional strategy of exogenous addition of AHLs, which has the problems of high cost and easy degradation (repeated addition is required), the present application achieves continuous and stable regulation through the endogenous secretion of signal molecules by biofilm carriers. By Figure 1 、 Figure 2 、 Figure 3 as well as Figure 5 As shown, the present invention can eliminate nitric oxide produced during the metabolism of ammonia oxidizing bacteria by adding biofilm carriers attached with Pseudomonas aeruginosa, thus preventing nitric oxide from damaging ammonia oxidizing bacteria and causing a decrease in their abundance. At the same time, the biofilm carriers attached with Pseudomonas aeruginosa can secrete functional signal substances to regulate the expression of nitrogen metabolism-related genes of ammonia oxidizing microorganisms, thereby improving the specific ammonia oxidation activity of the reactor. Figure 1 and Figure 4 As shown, the present invention can inhibit the expression of nitrite oxidoreductase in nitrite-oxidizing bacteria in the reactor by adding a biofilm carrier attached with Pseudomonas aeruginosa, resulting in the accumulation of nitrite nitrogen, thereby achieving short-range nitrification and denitrification and improving the biological denitrification efficiency of the reactor. Figure 6 and Figure 7 As shown, the added biofilm carrier attached with Pseudomonas aeruginosa had no nitrogen conversion activity under aerobic and anoxic conditions. Therefore, it can be proved that the regulatory strategy proposed in the present invention starts from the intercellular communication behavior between microorganisms, and indirectly increases the concentration of functional signal substances by increasing the abundance of core bacterial species, thereby regulating the species abundance and related gene expression of nitrogen conversion-related microorganisms, solving the problems of difficult denitrification and low efficiency faced by urban sewage treatment plants, and improving the efficiency of biological denitrification treatment of sewage.

[0045] This application achieves efficient nitrogen removal at low temperatures of 10-28°C, breaking through the bottleneck of traditional nitrification and denitrification processes, which experience a sharp drop in efficiency at low temperatures (<15°C). The reactor was operated for a certain period at 28°C, 18°C, 14°C, and 10°C. The experimental groups with biofilm carriers containing Pseudomonas aeruginosa showed increases in ammonia oxidation activity of 23.02%, 60.69%, 61.06%, and 88.88%, respectively, compared to the control group, and the cumulative nitrite rates were 82.58%, 89.06%, 90.59%, and 83.83%, respectively.

[0046] At 10°C, the experimental group achieved an 88.88% increase in ammonia oxidation activity and an 83.83% nitrite accumulation rate, significantly outperforming conventional short-range nitrification and denitrification (PND) and anaerobic ammonium oxidation (Anammox) at low temperatures (for example, the CANON process achieved a total nitrogen removal rate of only 16.87% at 10°C). Unlike low-temperature enhancement techniques (such as magnetic field enhancement and gradual cooling), which require additional equipment or complex operations, the present invention simplifies this process through microbial regulation.

[0047] Within the temperature range of 10-28°C, the experimental group of this invention saw an increase in ammonia oxidation activity by 23.02%-88.88%, and a nitrite accumulation rate of 82.58%-90.59%. Furthermore, the system maintained stable operation for extended periods at 15°C. In contrast, the ammonia nitrogen removal rate of conventional activated sludge processes often falls below 50% at low temperatures. The sequencing batch biofilm reactor (SBBR), combined with a 14.4% carrier fill rate, optimizes biofilm attachment and mass transfer efficiency. For example, at 10°C, the median suspended sludge concentration (MLSS) ranged from 1710.06 to 4175.68 mg / L, ensuring the abundance of functional bacteria.

[0048] The present invention uses short-term nitrification and denitrification (only ammonia needs to be oxidized to nitrite and then directly reduced to nitrogen gas), saving approximately 25% of oxygen demand and 40% of carbon source consumption, meeting the "dual carbon" goals. Compared with electrode ammonia oxidation or Anammox processes, it does not require an external power supply or strict anaerobic conditions, reducing energy consumption and operational complexity. In existing studies, directly adding AHLs is costly (for example, 1000-nM AHL causes a 7% decrease in COD removal rate) and is easily degraded by quenching bacteria. The present invention achieves endogenous continuous secretion by adding signal substance-producing bacteria (Pseudomonas aeruginosa), which is more cost-effective and has a more stable effect.

[0049] Traditional low-temperature denitrification relies on screening for psychrotrophic bacteria or gradually acclimating them through cooling (which takes several months). This new method, however, uses signaling substances to regulate microbial communities, rapidly boosting AOB activity (for example, ammonia oxidation activity increased by 60.69% at 18°C). Pyocyanin's inhibitory effect on NOB remains effective at low temperatures, whereas conventional methods (such as DO regulation) struggle to inhibit NOB proliferation at low temperatures.

[0050] The biofilm carriers attached to Pseudomonas aeruginosa in the present invention have a synergistic effect with the carrier. Pseudomonas aeruginosa is added after biofilm formation in a pure culture system, ensuring the density and activity of the functional bacteria in the biofilm carriers and avoiding competition from other bacteria. Traditionally, mixed bacterial communities have been used or carrier attachment conditions have not been optimized. The 14.4% carrier filling rate provided in the examples of the present invention has been experimentally verified to be the optimal ratio, balancing the biofilm attachment area and the mass transfer efficiency of the reactor (excessive filling rates may cause collision interference). This invention combines short-term nitrification and denitrification with signaling molecule regulation to form a synergistic pathway: enhancing AOB activity, inhibiting NOB activity, and promoting nitrite accumulation. Existing technologies often focus on a single mechanism (e.g., inhibiting NOB alone or optimizing AOB alone). Pseudomonas aeruginosa is a common strain in activated sludge, requiring no additional culture costs, and the biofilm carrier is reusable. Compared to chemical methods (such as the addition of nitrite oxidation inhibitors), this method eliminates the risk of secondary contamination.

[0051] This invention utilizes the innovative application of Pseudomonas aeruginosa biofilm carriers, combined with intercellular communication regulation and the inhibitory effect of pyocyanin, to achieve efficient and stable short-range nitrification and denitrification under low-temperature conditions. 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 fill rate). This significantly surpasses the limitations of existing technologies in terms of reliance on exogenous signaling substances, low-temperature adaptability, and operating costs, demonstrating high practical application value and potential for widespread adoption.

[0052] In the present invention, sludge concentration refers to the suspended solids content of the mixed liquor after sewage and activated sludge are mixed.

[0053] The embodiments of the present invention are not limited to the above-described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention shall be considered equivalent replacement methods and shall be included within the scope of protection of the present invention. Although the embodiments of the present invention have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above-described embodiments within the scope of the present invention.

Claims

1. A method for efficient biological denitrification of low-ammonia nitrogen wastewater in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation, characterized in that: The steps include: The carrier material is grown in a pure culture system of Pseudomonas aeruginosa to form a carrier attached with Pseudomonas aeruginosa; The attached carrier is added to the activated sludge reactor, and its volume accounts for 12.8%-16.0% of the effective volume of the reactor; The sequencing batch operation mode includes an aerobic nitrification section and an anoxic denitrification section. The functional signal substances secreted by Pseudomonas aeruginosa enhance ammonia oxidation activity and inhibit nitrite oxidation, thereby achieving short-term nitrification and denitrification.

2. The method for efficient biological denitrification of low-ammonia nitrogen sewage in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The specific method of forming a biofilm on the carrier material in the pure culture system of Pseudomonas aeruginosa is as follows: The freeze-dried powder of Pseudomonas aeruginosa was activated in NB medium and then transferred to LB medium. After culturing for 24-48 hours, it was used as a concentrated bacterial liquid. The concentrated bacterial liquid was inoculated into LB broth medium and cultured for 24-48 hours. The carrier materials were added in groups to the LB broth culture to form biofilms for 2-3 weeks. After the biofilm culture was completed, the surface of the carrier was light green as a whole, with pink dot-like colonies growing.

3. The method for efficient biological denitrification of low-ammonia nitrogen sewage in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The carrier material is non-woven fabric.

4. The method for efficient biological denitrification of low-ammonia nitrogen sewage in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, 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 denitrification of low-ammonia nitrogen sewage in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The dissolved oxygen concentration of the influent of the activated sludge reactor is above 2.00 mg / L.

6. The method for efficient biological denitrification of low-ammonia nitrogen wastewater in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The influent matrix of the activated sludge reactor contains 37.63-29.63 mg / L of NH4 + -N and COD of 107.56-92.19 mg / L, with a pH of 7.5-8.

5.

7. The method for efficient biological denitrification of low-ammonia nitrogen wastewater in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The sludge concentration in the activated sludge reactor is 1710.06-4175.68 mg / L.

8. The method for efficient biological denitrification of low-ammonia nitrogen wastewater in a low-temperature environment by short-cut nitrification and denitrification based on functional microbial regulation according to claim 1, characterized in that: The activated sludge reactor is a sequencing batch biofilm reactor.

9. The efficient biological denitrification method for sewage according to claim 1, characterized in that: The inlet water temperature of the activated sludge reactor is selected from 10-28°C.

Images