A method for rapidly starting up a PD / A system and improving denitrification performance based on PPGF enrichment of anaerobic ammonia oxidation bacteria

Abstract

The application belongs to the technical field of sewage treatment, and provides a method for rapidly starting a PD / A system and improving denitrification performance based on PPGF enrichment of anaerobic ammonia oxidation bacteria. An upflow anaerobic biofilm reactor is used, 300ml of inoculated sludge is added to the reactor, PPGF with a filling ratio of 30% is added at 11d, artificial water is used as influent, NaHCO3 is used to adjust the influent pH to 7.8-9.1, nitrogen is used for stripping to make the dissolved oxygen concentration of the influent less than 0.3mg·L ‑1 , an anaerobic environment is maintained, the reactor is placed at 31±1℃, backflow is performed once every 30min, the backflow ratio is 2:1, and the reactor is continuously operated for 150d, during which the performance of the reactor is analyzed, the time required for starting the ordinary activated sludge PD / A is shortened, the denitrification efficiency of the system is improved, ordinary activated sludge is used to enrich anaerobic ammonia oxidation functional bacteria, and a new approach is provided for the stable operation of inoculated ordinary activated sludge anaerobic ammonia oxidation.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a method for rapidly starting up a PD / A system and improving denitrification performance by enriching anaerobic ammonia-oxidizing bacteria based on polyurethane porous gel packing (PPGF). Background Technology

[0002] In the treatment of urban wastewater, the main challenge faced by anammox technology is the substrate NO2. - Stable acquisition of NO2-. Recently, the PD / A process, which combines partial denitrification (PD) with Anammox technology, has shown stable and efficient performance. The PD / A process not only provides a stable supply of NO2 to Anammox bacteria, but also... - -N matrix can effectively remove organic matter from urban wastewater, achieving simultaneous deep denitrification and carbon removal. However, the application of Anammox technology in urban wastewater treatment still faces challenges. These challenges mainly stem from the slow growth and long doubling time of Anammox bacteria, making them difficult to accumulate and resulting in a time-consuming start-up process for bioreactors. To address this issue, biomass immobilization technologies, particularly granular sludge systems and biofilm systems, are considered effective solutions due to their high biomass concentration and tolerance to environmental fluctuations. However, due to wastewater volume and other engineering issues, granular systems may not be suitable for treating domestic wastewater in urban wastewater treatment. Conversely, biofilm systems, due to their longer sludge retention time and high aggregation capacity of anaerobic ammonia-oxidizing bacteria (AnAOB), are considered an effective way to enrich AnAOB.

[0003] In the research and application of Anammox biofilm technology, the selection of suspension carriers plays a crucial role in microbial attachment and biofilm development. Traditional carriers, such as spherical polypropylene or tubular polyethylene (PE), while exhibiting excellent durability, have shortcomings in microbial attachment capacity and fluidization performance, limiting the overall performance of biofilm reactors. In recent years, sponge carriers made of polyurethane have gained favor among researchers due to their large specific surface area and properties that promote microbial attachment. However, these carriers, due to their low density, while easily suspended in the reactor, do not have ideal fluidization performance, which may affect mass transfer efficiency and the uniform distribution of the biofilm.

[0004] Polyurethane porous gel filler (PPGF) is a novel type of porous gel filler with unique physical and chemical properties. From a structural design perspective, PPGF adopts a wall-like structure, which significantly increases its specific surface area compared to the ribbed structure of traditional polyurethane foam. This provides a larger contact interface for mass exchange processes, thereby improving mass transfer efficiency.

[0005] In terms of pore structure design, PPGF adopts a macroporous-microporous composite structure system. The macroporous structure provides excellent contact conditions for the gas-liquid-solid three-phase system, ensuring efficient mass transfer; the microporous structure is rich in hydrophilic active groups such as amino, carboxyl, and epoxy groups, which endow the material with excellent water absorption properties. Notably, PPGF forms a gel state after absorbing water, at which point its hydrophilicity is significantly enhanced, enabling rapid adsorption and immobilization of microorganisms and enzymes. This characteristic not only significantly improves the attachment efficiency of microorganisms but also, by changing the material density to be greater than water, achieves rapid settling of the packing material in water. From a material performance perspective, the porous structure of PPGF, while ensuring high porosity, also optimizes the transfer of matter inside and outside the carrier. Its unique elastic properties further promote the attachment of microorganisms and the kinetics of biofilm formation. These comprehensive characteristics enable PPGF to exhibit excellent performance in the anammox process, specifically in significantly shortening the process start-up time and improving the system's denitrification efficiency. Experimental studies have shown that the application of PPGF can effectively promote the enrichment and activity enhancement of Anammox bacteria, providing a new technical approach for biological denitrification of wastewater. Summary of the Invention

[0006] This invention addresses the problems of slow start-up and low nitrogen removal performance in conventional activated sludge anaerobic ammonia oxidation (AAM) systems by providing a method for rapidly starting up a PD / A system and improving nitrogen removal performance based on PPGF-enriched anaerobic ammonia oxidizing bacteria. This invention proposes to shorten the start-up time and improve nitrogen removal performance of AAM systems treated with low C / N ratio wastewater by adding PPGF to the reactor, and explores its mechanism of action. This invention aims to reveal the application of PPGF in conventional activated sludge AAM oxidation, providing a new approach for rapid start-up and stable operation of conventional activated sludge AAM oxidation.

[0007] This invention is achieved by the following technical solution: a method for rapidly starting up a PD / A system and improving denitrification performance based on PPGF-enriched anaerobic ammonia-oxidizing bacteria. The method utilizes an upflow anaerobic biofilm reactor, adding 300 ml of inoculum sludge to the reactor; adding PPGF at a filling ratio of 30% at day 11; using artificially prepared influent, adjusting the pH of the influent to 7.8–9.1 with NaHCO3, and using nitrogen gas for stripping to ensure the dissolved oxygen concentration in the influent is less than 0.3 mg·L⁻¹. -1 To maintain an anaerobic environment, the reactor was placed at 31±1℃ and refluxed every 30 minutes with a reflux ratio of 2:1 for 150 days. During this period, the reactor performance was analyzed. Among them, the specific surface area and porosity of PPGF are: S > 4000 m² 2 / m 3 Ψ=98%; The influent contains 40–67 mg / L NH₄Cl, 33.9–83.26 mg / L Na₂NO₃, 420 mg / L NaHCO₃, and 300 mg / L CaCl₂. 2· The following solutions were added: 2H₂O, 200 mg / L MgSO₄·7H₂O, 25 mg / L KH₂PO₄, and 1 ml / L trace element solution I and trace element solution II. The components of the trace element solutions are as follows: Trace element solution I g / L: 6.37 g EDTA·2Na and 9.14 g FeSO₄·7H₂O; Trace element solution II g / L: 19.11 g EDTA·2Na, 0.24 g CoCl₂·6H₂O, 0.25 g CuSO₄·5H₂O, 0.22 g NaMoO₄·2H₂O, 0.19 g NiCl₂·7H₂O, 0.014 g H₃BO₄, 0.43 g ZnSO₄·7H₂O and 0.99 g MnCl₂·4H₂O.

[0008] Specifically, the steps include the following: (1) Inoculate the return sludge, let it stand for 24 hours, discard the supernatant, and measure the MLSS as 5.9 g / L and MLVSS as 4.0 g / L; take 300 mL of sludge to inoculate into the reactor; (2) Start-up of the partially denitrified and anaerobic ammonia oxidation coupled reactor, i.e., operation of the PD / A system: 2L of wastewater enters through the bottom, with an influent time of 25min; water exits from the bottom, with a time of 5min; the reaction temperature is controlled at 31±1℃, dissolved oxygen (DO) is controlled at 0.30±0.05mg / L, and reflux is performed every 30min with a reflux ratio of 2:1; intermittent influent is adopted, and the HRT is controlled at 24h; the reactor runs continuously for 150 days, divided into three stages: Stage I enriches short-range denitrifying bacteria, Stage II enriches anaerobic ammonia oxidizing bacteria, and Stage III improves the denitrification performance of the reactor; Phase I lasts 1–10 days, with influent NO3 - -N concentration was 42 mg·L⁻¹ -1 NH4 inlet water + -N concentration is 45 mg·L -1 Stages II and III last for 11–150 days, with influent NO3... - -N concentration is 75 mg·L -1 NH4 inlet water + -N concentration is 60 mg·L -1 PPGF was added after the reactor had been running for 11 days. (3) Performance analysis: Routine water quality analysis, including NO2, is performed on the reactor effluent daily. - -N, NO3 - -N, NH4 +-N, COD, pH, water sample filtered using a 0.45µm water filter, NO2 - -N was analyzed using the N-(1-naphthyl)-ethylenediamine spectrophotometric method. NO3 - -N was analyzed using ultraviolet spectrophotometry, and NH4 was... + -N was analyzed using Nessler's reagent spectrophotometry; COD was analyzed using rapid digestion spectrophotometry; biofilm sludge samples were taken at days 10, 75, and 150 to analyze EPS content, microbial protein content was analyzed using the Bradford method, and polysaccharide concentration was analyzed using the anthrone-sulfuric acid colorimetric method; sludge samples were taken at days 1, 75, and 150 for microbial sequencing analysis. (4) Correlation network analysis: Calculate Spearman's rank correlation coefficient to illustrate the association between all denitrification-related genera in all samples. When the correlation coefficient is greater than 0.5 and the p value is less than 0.05, it is judged as significant correlation. Network visualization is performed using the Gephi platform.

[0009] Furthermore, the effective volume of the reactor is 4L.

[0010] The method for microbial sequencing analysis of sludge samples was to extract sample DNA using a kit, and use 341F: 5'-CCTACGGGAGGCAGCAG-3' and 805R: 5'-GACTACHVGGGTATCTAATCC-3' primers for the V3-V4 region of 16S rRNA, followed by Illumina high-throughput sequencing and metagenomic sequencing.

[0011] This invention demonstrates the feasibility of PPGF in promoting the rapid start-up of conventional activated sludge PD / A and improving its denitrification performance by analyzing changes in effluent nitrogen concentration, nitrogen removal efficiency, COD, and microbial community structure. It provides a new approach for using conventional activated sludge as inoculated sludge to treat low C / N ratio wastewater and ensure stable operation of PD / A systems.

[0012] The results showed that adding PPGF could promote rapid start-up of conventional activated sludge PD / A reactors. When the reactor was successfully started up within 79 days after PPGF addition, the NRE (nitrogenous regeneration rate) was 98%; anaerobic ammonia oxidizing bacteria were effectively enriched in PPGF. Cadidatus The relative abundance of Jettenia reached 22% on day 150, and anaerobic ammonia oxidation contributed 87% to the removal of total nitrogen.

[0013] This invention shortens the start-up time of conventional activated sludge PD / A reactor and improves the system's denitrification efficiency by adding PPGF to the reactor. The changes in effluent nitrogen concentration, denitrification efficiency, and microbial community structure during the reaction process were investigated. Enriching anaerobic ammonia-oxidizing bacteria with conventional activated sludge is a necessary condition for improving the stable operation of denitrification performance in low C / N wastewater, and this invention provides a new approach for this purpose. Attached Figure Description

[0014] Figure 1 The changes in nitrogen removal performance during startup are shown in the figure: (a) shows the changes in ammonia nitrogen concentration in the influent and effluent and the ammonia nitrogen removal efficiency; (b) shows the changes in NO₃⁻ N concentration in the influent and effluent, the NO₂⁻ N concentration in the effluent and the NO₃⁻ N removal efficiency; (c) shows the changes in TN concentration in the influent and effluent and the TN removal efficiency; (d) shows the contribution of Anammox and denitrification to TN removal efficiency. Figure 2 The changes in COD and nitrogen concentrations during a typical cycle of the PD / A process on day 145; Figure 3 Characteristics of EPS content in the biofilm during operation: concentrations of S-EPS, LB-EPS, TB-EPS, total EPS, and PN / PS ratio; Figure 4 This refers to changes in the phylum-level composition of the reactor's microbial community. Figure 5 The figures show the relative abundance of anaerobic ammonia-oxidizing bacteria (AnAOB) and denitrifying bacteria (DNB) in the reactor biofilm and flocculent sludge; in the figure: (a) DNB; (b) AnAOB; (c) the main microorganisms involved in denitrification. Figure 6 A genus-level species correlation network; Figure 7 The relative abundance of key nitrogen metabolism enzymes detected in biofilm and flocculent sludge in the reactor on day 150. Figure 8 This refers to the nitrogen metabolism pathway in biological membranes (AMX: anammox; DNRD: dissimilatory nitrate reduction; ANR: assimilatory nitrate reduction). Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0017] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0019] I. Reaction Apparatus and Methods: An upflow anaerobic biofilm reactor was used in the experiment. The reactor was 36 cm high, 12 cm in inner diameter, and had an effective volume of 4 L. 2 L of wastewater was introduced through a peristaltic pump from the bottom over a period of 25 min. Wastewater was discharged from the bottom over a period of 5 min. The reaction temperature was controlled at 31 ± 1℃, and the dissolved oxygen (DO) was controlled at (0.30 ± 0.05) mg / L. Reflux was performed every 30 min at a reflux ratio of 2:1.PPGF (2 cm * 2 cm * 1 cm), with a filling ratio of 30%. The reactor was sealed with a lid to maintain an anaerobic environment. PPGF was purchased from Henan Yishuijing Environmental Protection Technology Co., Ltd.

[0020] 1. Inoculation sludge and simulated wastewater: The return sludge was taken from Zhengyang Wastewater Treatment Plant in Jinzhong City, Shanxi Province. 300 mL of inoculated sludge was used to inoculate the reactor, with a MLSS of 5.90 g·L⁻¹. -1 The MLVSS / MLSS ratio is 0.68.

[0021] Artificially prepared water containing NH4Cl (40–67 mg / L), Na2NO3 (33.9–83.26 mg / L), NaHCO3 (420 mg / L), and CaCl2 2·The solution contained 2H₂O (200 mg / L), MgSO₄·7H₂O (200 mg / L), KH₂PO₄ (25 mg / L), and 1 mL / L of trace element I and trace element II. The trace element solutions were as follows: Trace element solution I (g / L) contained 6.37 g EDTA·2Na and 9.14 g FeSO₄·7H₂O; Trace element solution II (g / L) contained 19.11 g EDTA·2Na, 0.24 g CoCl₂·6H₂O, 0.25 g CuSO₄·5H₂O, 0.22 g NaMoO₄·2H₂O, 0.19 g NiCl₂·7H₂O, 0.014 g H₃BO₄, 0.43 g ZnSO₄·7H₂O, and 0.99 g MnCl₂·4H₂O, using 0.1 mol·L⁻¹. -1 NaOH was used to adjust the pH of the influent to 7.8–9.1.

[0022] 2. Reactor operating parameters: The reactor operated for a total of 150 days, divided into three stages. The time and influent water quality of each stage are shown in Table 1.

[0023] Table 1: Reactor Operation Stages 3. The water sample was filtered using a 0.45µm water filter to remove NO2. - -N was analyzed using the N-(1-naphthyl)-ethylenediamine spectrophotometric method. NO3 - -N was analyzed using ultraviolet spectrophotometry, and NH4 was... + -N was analyzed using Nessler's reagent spectrophotometry, and COD was analyzed using rapid digestion spectrophotometry. Biofilm sludge samples were collected at days 10, 75, and 150 for EPS content analysis. Microbial protein content was analyzed using the Bradford method, and polysaccharide concentration was analyzed using the anthrone-sulfuric acid colorimetric method. MLSS and MLVSS were analyzed using standard methods, and pH was determined using a pH meter. In addition, sludge samples from different stages (days 1, 75, and 150) were used to extract DNA using a kit. Primers for the V3-V4 region of 16S rRNA were 341F (CCTACGGGAGGCAGCAG) and 805R (GACTACHVGGGTATCTAATCC). High-throughput sequencing was performed using an Illumina system, and metagenomic sequencing was performed on the sludge sample from day 150.

[0024] 4. Calculation method: The specific calculation formulas involved are as follows: PD and PD / A stages The methods for calculating conversion rate (NTR) are shown below: In the formula: Indicates water output Concentration (mg / L); Indicates water output Concentration (mg / L); Indicates water ingress Concentration (mg / L).

[0025] In the formula: Represents time t Concentration (mg / L); This represents the theoretical consumption of the Anammox reaction. The concentration is mg / L; Indicates water ingress Concentration (mg / L); This represents the theoretical production of the Anammox reaction. Concentration (mg / L); Represents time t Concentration (mg / L).

[0026] Complete denitrification and anaerobic ammonium oxidation are the main denitrification pathways in the PD / A stage, ignoring the NH4 consumed by cellular synthesis. + The calculation of the -N, root Anammox reaction formula is shown below: Contribution rate of anaerobic ammonia oxidation to nitrogen removal: .

[0027] Contribution of complete denitrification to nitrogen removal: In the formula: Indicates water ingress Concentration (mg / L); Indicates water output Concentration (mg / L); This indicates the change in concentration of influent and effluent in mg / L.

[0028] II. Experimental Results 1. Reactor start-up process; changes in effluent nitrogen concentration; denitrification performance of the reactor during start-up process, such as... Figure 1 As shown.

[0029] In Phase I (days 1-10), flocculent sludge is inoculated into the reactor and run for 10 days to ensure its stability and adaptation to the new operating conditions. Influent C / NO3 - -N and pH values ​​are 0.5 and 9, respectively. As shown in Figure 1a, the effluent NH4+... + -N, NO2 - -N and NO3 - -N concentrations were 55.66-40.69 mg / L, 3.75-0 mg / L, and 0 mg / L, respectively. NH4 +The removal rate of -N was -24.23% to -5.93%, and the nitrogen removal efficiency (NRE) was 56.84% to 27.97%. NTR PDmax Only 11.20%. This may be due to changes in environmental conditions causing the lysis of unsuitable bacterial cells in the inoculated sludge, resulting in increased NH4 in the effluent. + -N concentration was higher than influent. As the PD / A process proceeded, the activated sludge primarily underwent endogenous denitrification. To reduce effluent TN concentration and efficiently and rapidly establish the PD / A system, PPGF was added to the reactor at a 30% filling rate, and the influent nitrogen load was increased.

[0030] During phase II (days 11–52), NRE remained almost unchanged. Figure 1 c). The effluent TN concentration was approximately 93.39 mg / L, and NRE was 28.68%. In contrast, during the later stages of the second phase (days 53–75), the effluent TN concentration gradually decreased to 68.63 mg / L, indicating that PD and AnAOB adapted to the influent NH4. + -N and NO3 - Increased NO3- concentration significantly improves denitrification performance. Nevertheless, the average effluent NO3... - -N concentration remains as high as 31.11 mg / L ( Figure 1 b、 Figure 1 c). These results indicate that the denitrification performance of the second stage is still somewhat limited, with the reactor's ARE and NRE at approximately 50.14% and 49.60%, respectively. Notably, the influent C / NO3 ratio... - -N may be too low for short-cut denitrification processes, resulting in relatively low denitrification performance. Furthermore, from day 53 to day 75, the on-site ammonia oxidation rate gradually increased, reaching 1.88 mg NH4 on day 75. + -N / L / h ( Figure 1 a).

[0031] To further improve nitrogen removal performance, during Phase III (days 76-150), the influent C / NO3 ratio was adjusted. - -N increased to 3 on day 76. This adjustment significantly increased the nitrogen removal rate to 79.30% and significantly reduced the effluent TN concentration to 29.32 mg / L. Figure 1 c). Meanwhile, ΔNO3 - -N / ΔNH4 + The average ratio of -N is approximately 1.33, slightly higher than 1.32 (theoretical value). This is likely due to the fact that complete denitrification reduces NO3 in the effluent. --N concentration decreased to 10.21 mg N / L. Furthermore, NO2... - -N concentration also reached its minimum (Figure 1b). After 79 days of operation, the reactor achieved efficient nitrogen removal. When the influent C / NO3 ratio was... - When the -N ratio is 3, the NRE reaches 98.22% and remains almost unchanged during days 79-150. Figure 1 c) The effluent TN concentration was as low as 2.52 mg / L. Furthermore, the anammox activity remained stable at 2.45 mg NH4+. + -N / L / h, accounting for 87.10% of NRE. This is because AnAOB has a high NO2 content. - -N is more competitive than denitrifying bacteria, therefore they can effectively eliminate NH4+. + -N and NO2 - -N indicates that a PD / A process can be established in this system.

[0032] 2. Denitrification Pathway Analysis: To elucidate the denitrification pathway in the system, changes in COD and nitrogen concentrations during the PD / A process on day 145 were measured under steady-state conditions. As shown in Figure 2, two phases were observed: the PD phase and the anaerobic ammonia oxidation phase. Within 0–120 minutes, the substrate NO3 required for partial denitrification was partially removed. - -N and COD are sufficient. Partial denitrification converts organic substrates to nitrates, while some NH4+ is used. + -N and the generated NO2 - -N is also removed by anammox. Within 120 minutes, NO2... - -N concentration reached a peak (34.37±0.14 mg / L), indicating that PD is the main process, and NO2 - -N accumulates faster than anammox is consumed. Subsequently, NH4 + -N and NO2 - -N was expelled via anammox. However, the COD concentration only decreased to 49.49 ± 3.97 mg / L, indicating that the role of anammox in nitrogen removal was greater than that of denitrification. During the cycle, NTR... PD / A Reaching 94.35% indicates that anammox can compete with denitrification for NO2. - -N. During the ammonia oxidation stage, ΔNO2 - -N / ΔNH4 + The -N ratio is 1.24, slightly lower than the 1.32 calculated by the ammonia oxidation equation, indicating that some NO3 is produced during ammonia oxidation. --N is converted to NO2 in the system. - -N. This is consistent with the results of the batch trials mentioned above. Nitrogen removal in the later stages of the reaction mainly relied on the anammox pathway, thus anammox was the dominant process in this phase. Ultimately, the COD concentration decreased from 200.94±9.56 mg / L to 60.94±0.16 mg / L, and the TN concentration decreased from 126.63±0.43 mg N / L to 4.23±0.11 mg N / L, indicating that nitrogen can be removed through anammox and denitrification. According to stoichiometric analysis, denitrification achieved a TN removal rate of 8.85%, indicating that Anammox is the main nitrogen removal pathway, with a TN removal rate exceeding 91.15%. Furthermore, it is worth noting that partial nitrification also occurred in the system concurrently with partial denitrification, which could provide additional NO2 to AnAOB. - -N, reduces NH4+ + -N concentration, thereby improving the denitrification performance of Anammox.

[0033] 3. Changes in EPS within the reactor: Microbial cells secrete large amounts of EPS to form a biofilm. These EPS bind the sludge together, adsorbing onto the sticky aggregates on the surface of PPGF. Throughout the entire operating cycle, such as... Figure 3As shown, the total EPS content gradually increased. With the enrichment of sludge, the EPS concentration in the biofilm increased significantly from 88.77 ± 2.58 mg / g-VSS to 160.94 ± 3.78 mg / g-VSS, an increase of 81.30%. The increase in EPS content plays a crucial role in maintaining biofilm formation, preserving structural integrity, and protecting microorganisms continuously adsorbed on the PPGF surface from external environmental and stress factors. From the EPS stratification results, the concentrations of S-EPS and LB-EPS fluctuated less, while TB-EPS had the highest concentration among EPS, and its content gradually increased, reaching 132.90 ± 5.01 mg / g-VSS on day 150 of mature biofilm formation. The results for each component of EPS showed that PS concentration did not change significantly, while PN had the highest content in both EPS subgroups. The concentration of PN in the biofilm system increased significantly from 66.92 ± 2.21 mg / g-VSS (day 10) to 135.03 ± 4.27 mg / g-VSS (day 150). PN is the main component of EPS containing extracellular enzymes. This means that the increase in PN concentration provides microorganisms with more extracellular enzymes and nutrients, thereby enhancing microbial activity. PS and PN, as two major components of EPS, regulate biofilm properties such as surface charge, adhesion, and hydrophobicity. Previous studies have shown that a higher PN / PS ratio can enhance the hydrophobic interaction between microorganisms and aqueous solutions, promote electron transfer between sludge particles, thereby promoting bacterial growth, metabolism, aggregation, and interaction, and thus improving the denitrification performance of the anaerobic system. After 140 days of reactor operation, the PN / PS ratio showed an upward trend throughout the entire operating cycle and remained at around 4.04.

[0034] 4. At the gate level, such as Figure 4As shown, the microbial community of the biofilm exhibited significant dynamic changes. Phylums with a relative abundance greater than 1% were considered dominant. On day 75, eight major bacterial phyla were identified in the biofilm, including *Aerobacterium* (14.97%), *Proteus* (13.73%), *Bacteroides* (6.71%), *Plasmodium* (5.21%), *Acidobacterium* (4.12%), *Brevibacteria* (3.73%), *Plasmodium* (2.81%), and *Actinomyces* (1.03%). Notably, *Planctomycetota*, including *AnAOB*, reached 2.81%. Throughout the stable operation period (days 80–150), the abundance of *Proteus* and *Actinomyces* in the biofilm increased significantly, reaching 29.91% and 23.94%, respectively, indicating that they had become dominant phyla. Simultaneously, the abundance of *Bacteroides* increased to 8.86%. However, the relative abundances of *Aerobacterium*, *Ctenobacter*, *Acidobacter*, and *Armibacter* decreased significantly, accounting for 11.51%, 4.42%, 1.63%, and 1.51%, respectively, suggesting that these microbial phyla may play an important role in the initial stages of biofilm formation. The abundance of *Armillaria*, a common phylum in ammonia oxidation systems, decreased slightly during steady-state operation. *Armatimonadota* has been reported to cause NH4+ oxidase degradation in ammonia oxidation systems. + Excessive consumption of -N may be the cause of ΔNO2 here. - -N / ΔNH4 + The reason why the -N ratio is less than the theoretical value. These results indicate that functional microorganisms exhibit different succession patterns during biofilm formation.

[0035] 5. At the fungal level, such as Figure 5 As shown, in addition to AnAOB, the biofilm also contains bacterial genera capable of PD, thus ensuring the NO2 content of AnAOB. - -N supply. On day 75, the abundance of denitrifying bacteria (including Thaurea, Pseudomonas, Denitrosoma, and unclassified Comamonadaceae) reached 3.92% (Fig. 5a). Between days 80 and 150, the biofilm matured, with AnAOB becoming the dominant bacterium, its abundance increasing from 0.19% to 22.00%; the major bacterial genera included Candidatus Jettenia (21.73%) Candidatus _Brocadia (0.0052%) and Candidatus _Anammoximicrobium (0.26%) (Figure 5b). Among them, Candidatus_Jettenia_ was initially absent in the sludge inoculated on day 0, but gradually became the dominant genus in AnAOB, reaching a relative abundance of 21.73% on day 150, higher than reported in other anaerobic systems. The second most dominant bacteria in the biofilm were denitrifying bacteria (21.14%), mainly Thaurea and Denitrazoma. Figure 5 c). When C / NO3 - When the -N ratio is 3, organic matter can act as an electron donor to convert nitrate into N2, thereby reducing nitrate accumulation in the system. Under these conditions, denitrifying bacteria become active, the growth environment for AnAOB is improved, and thus nitrogen removal efficiency is increased. Therefore, in the C / NO3 ratio... - At a -N ratio of 3, AnAOB exhibited a stronger ability to compete for substrates compared to heterotrophic bacteria. Furthermore, with increasing relative abundance, AnAOB even competed with heterotrophic denitrifying bacteria for nitrite, thus increasing Anammox's contribution to nitrogen removal in the system. Therefore, these results suggest that AnAOB and heterotrophic bacteria can coexist harmoniously in the reactor. Conversely, at day 150, denitrifying bacteria dominated the flocculent sludge (30.43%), with the main genera including Thaurea, Denitrosoma, and Pseudomonas. AnAOB became the second most dominant bacteria (15.66%). These results indicate that biofilms are more conducive to the enrichment of AnAOB. ​​Furthermore, Candidatus The abundance of _Jettenia was as high as 21.73% in the biofilm, while it was only 15.55% in the flocculent sludge, indicating that the cultivation of anaerobic ammonia oxidation sludge was successful.

[0036] 6. Microbial network analysis: such as Figure 6 The values ​​shown (Spearman correlation coefficients greater than 0.5 or less than -0.5, significance level p<0.05) are used to identify potential relationships between key microorganisms. Significant positive correlations exist between several specific subsets of denitrifying bacteria in biofilms, for example... Candidatus The presence of *Jettenia* with *Thauera*, and *Thauera* with *Pseudomonas*, *Comamonas*, *Candidatus* *Competibacter*, and *Hyphomicrobium* indicates a synergistic relationship between denitrifying bacteria and AnAOB. ​​In other words, *Thauera* and *Pseudomonas*, which possess partial denitrification capabilities, can reduce NO3-. - -N generates NO2 for AnAOB. - These results demonstrate that the synergistic application of denitrification and the Anammox process can achieve efficient and stable nitrogen removal.

[0037] 7. Metabolic Mechanism Analysis: To elucidate the metabolic mechanisms of the highly active biofilm, sequencing results were compared with the KEGG database to determine the relevant pathways of nitrogen metabolism in the reactor and the relative abundance of the functional genes involved, as follows: Figure 7 As shown, considering the synergistic process of denitrification and anaerobic ammonia oxidation, the key enzymes of functional bacteria in biofilm sludge and flocculated sludge in the reactor were further studied.

[0038] Key genes for nitrate-to-nitrite conversion, nitrate reductase Nar and periplasmic dissimilatory nitrate reductase nap, were observed in both biofilm sludge and flocculent sludge in the reactor, indicating that nitrate-to-nitrite conversion is a potential pathway for the enrichment of anaerobic ammonia-oxidizing bacteria. Specifically, the relative abundance of Nar in biofilm and flocculent sludge was 0.46% and 0.42%, respectively. Nar was mainly distributed in Thaurera in both biofilm and flocculent sludge, with relative abundances of 0.12% and 0.14%, respectively, followed by... Candidatus Jettenia was found in 0.05% and 0.02% of the samples, respectively. Another Nap-like substance, Thaurera, was also mainly distributed in biofilms and flocculent sludge, with relative abundances of 0.13% and 0.14%, respectively. This indicates that genes related to short-cut denitrification are highly expressed, and Thaurera is the main agent for reducing nitrate to nitrite, consistent with previous studies. Nitrite reductase (NirK) and cytochrome cd1 were also involved. Nitrite reductase (NirS) is a process in denitrification that reduces nitrite to nitric oxide, and it is also an important process in anaerobic ammonia oxidation. The relative abundances of NirS in biofilms and flocculent sludge were 0.13% and 0.11%, respectively. Interestingly, the relative abundances of NirK in both biofilms and flocculent sludge were very low, at only 0.0015% and 0.0010%, respectively. Both nitric oxide reductase NorB / C and nitrous oxide reductase NosZ, which are related to complete denitrification, were detected. The relative abundance of NorB / C in both biofilm and flocculent sludge was 0.15%, while the relative abundance of NosZ in biofilm and flocculent sludge were 0.17% and 0.15%, respectively, indicating that the denitrification process also contributes to nitrogen removal in the reactor. Hydrazine dehydrogenase (Hdh) and hydrazine synthase (Hzs), the two most important enzymes in anaerobic ammonia oxidation, were detected in both biofilm sludge and flocculent sludge, and were only distributed in... CandidatusIn Jettenia, the relative abundances of Hzs in biofilm and flocculent sludge were 0.10% and 0.038%, respectively, while the relative abundances of Hdh in biofilm and flocculent sludge were 0.29% and 0.10%, respectively. The relative abundance of both enzymes was higher in biofilm sludge than in flocculent sludge. Higher relative abundances of Hzs and Hdh were associated with higher relative abundances of AnAOB bacteria in the biofilm, consistent with the microbial community analysis results. Meanwhile, nitrification-related genes AmoA / B / C were at very low levels, consistent with the microbial community analysis, indicating that ammonia nitrogen was primarily removed via anaerobic ammonia oxidation. Therefore, Nar and Nap genes dominate nitrogen transformation in the PD process. The significant prevalence of Nar genes, coupled with the limited presence of NorB / C and NosZ genes associated with complete denitrification and the low expression of nitrification-related genes AmoA / B / C, is consistent with the significant nitrogen removal contribution of anaerobic ammonia oxidation in the anaerobic biofilm PD / A system. In addition, functional gene expression was observed in the reactor biofilm and flocculated sludge for other nitrogen metabolism pathways, including the complete dissimilatory nitrate reduction (DNT) and assimilatory nitrate reduction (ANT) processes. Ten functional genes were detected in the reactor biofilm and flocculated sludge for the DNT process. Among them, the functional genes Nar and Nap involved in the nitrate-to-nitrite conversion process were consistent with those involved in the denitrification nitrate-to-nitrite conversion process, namely NarG / H / I / J and NapA / B. The functional genes NirB / D and NrfA / H involved in the nitrite-to-ammonia conversion process were also present. The relative abundances of NirB / D in the biofilm and flocculated sludge were 0.14% and 0.16%, respectively, while those of NrfA / H were 0.011% and 0.0048%, respectively. The higher expression of NirB / D compared to NrfA / H indirectly indicates the presence of a higher concentration of nitrite during the denitrification process, further demonstrating the excellent PD performance of the reactor. Two functional genes were detected in both biofilm sludge and flocculent sludge during the nitrate reduction process: NasA, a functional gene involved in the conversion of nitrate to nitrite, with relative abundances of 0.34% and 0.37% in biofilm and flocculent sludge, respectively; and NirA, a functional gene involved in the conversion of nitrite to ammonia nitrogen, with relative abundances of 0.00048% and 0.00030% in biofilm and flocculent sludge, respectively. Based on these results, a metagenomics-based biofilm nitrogen metabolism pathway was proposed. Figure 8 ).

[0039] This invention verifies that a PD / A system employing anaerobic ammonia oxidizing bacteria self-enrichment startup can stably operate in mainstream wastewater, achieving deep denitrification and ensuring that the enriched anaerobic ammonia oxidizing bacteria dominate the denitrification process. Simultaneously, a powerful anaerobic biofilm PD / A system rapidly starts up within 79 days, achieving a C / NO3 ratio in the influent. - When -N is 3, the effluent TN concentration is below 3 mg / L, NRE reaches 98%, and anammox removes over 87% of TN. This effectively shortens the PD / A start-up time without inoculating anammox sludge. The gradual implementation of multi-faceted control strategies fully integrates and mobilizes the advantages of each stage of biofilm development. The strategy of gradually increasing the wastewater feed ratio not only stably establishes the mainstream environment but also effectively promotes the succession of the microbial community by considering the adaptive acclimatization of the PD / A microbial community to its habitat. Furthermore, the sufficient production of nitrite during the entire operation process simultaneously consumes organic matter, eliminating the adverse effects of organic matter on anammox and laying the foundation and providing necessary support for the final configuration of the core bacterial community in the PD / A biofilm system. In addition, PPGF creates a spatiotemporal structure for anammox, and the abundance of AnAOB in the biofilm reaches 22% on day 150. Candidatus _Jettenia_ is the dominant genus of bacteria in anaerobic ammonia oxidation. The in-situ enrichment treatment of urban wastewater using anaerobic ammonia oxidation provides reliable experimental evidence and is expected to be further validated in future long-term laboratory-scale and pilot-scale trials.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for rapidly starting up a PD / A system and improving denitrification performance based on PPGF-enriched anaerobic ammonia-oxidizing bacteria, characterized in that: An upflow anaerobic biofilm reactor was used, with 300 ml of inoculum sludge added to the reactor. PPGF with a filling ratio of 30% was added at day 11. The influent was artificially prepared, and the pH was adjusted to 7.8–9.1 using NaHCO3. Nitrogen gas was used for stripping to ensure the dissolved oxygen concentration in the influent was less than 0.3 mg·L⁻¹. -1 To maintain an anaerobic environment, the reactor was placed at 31±1℃ and refluxed every 30 minutes with a reflux ratio of 2:1 for 150 days. During this period, the reactor performance was analyzed. Among them, the specific surface area and porosity of PPGF are: S > 4000 m² 2 / m 3 Ψ=98%; The influent contains 40–67 mg / L NH₄Cl, 33.9–83.26 mg / L Na₂NO₃, 420 mg / L NaHCO₃, and 300 mg / L CaCl₂. 2· The following solutions were added: 2H₂O, 200 mg / L MgSO₄·7H₂O, 25 mg / L KH₂PO₄, and 1 ml / L trace element solution I and trace element solution II. The components of the trace element solutions are as follows: Trace element solution I g / L: 6.37 g EDTA·2Na and 9.14 g FeSO₄·7H₂O; Trace element solution II g / L: 19.11 g EDTA·2Na, 0.24 g CoCl₂·6H₂O, 0.25 g CuSO₄·5H₂O, 0.22 g NaMoO₄·2H₂O, 0.19 g NiCl₂·7H₂O, 0.014 g H₃BO₄, 0.43 g ZnSO₄·7H₂O and 0.99 g MnCl₂·4H₂O. Specifically, the steps include the following: (1) Inoculate the return sludge, let it stand for 24 hours, discard the supernatant, and measure the MLSS as 5.9 g / L and MLVSS as 4.0 g / L; take 300 mL of sludge to inoculate into the reactor; (2) Start-up of the partially denitrified and anaerobic ammonia oxidation coupled reactor, i.e., operation of the PD / A system: 2L of wastewater enters through the bottom, with an influent time of 25min; water exits from the bottom, with a time of 5min; the reaction temperature is controlled at 31±1℃, dissolved oxygen (DO) is controlled at 0.30±0.05mg / L, and reflux is performed once every 30min, with a reflux ratio of 2:1; intermittent influent is adopted, and the HRT is controlled at 24h; the reactor runs continuously for 150d, divided into three stages: stage I enriches short-range denitrifying bacteria, stage II enriches anaerobic ammonia oxidizing bacteria, and stage III improves the denitrification performance of the reactor; Phase I lasts 1–10 days, with influent NO3 - -N concentration was 42 mg·L⁻¹ -1 NH4 inlet water + -N concentration is 45 mg·L -1 Stages II and III last for 11–150 days, with influent NO3... - -N concentration is 75 mg·L -1 NH4 inlet water + -N concentration is 60 mg·L -1 PPGF was added after the reactor had been running for 11 days. (3) Performance analysis: Routine water quality analysis, including NO2, is performed on the reactor effluent daily. - -N, NO3 - -N, NH4 + -N, COD, pH, water sample filtered using a 0.45µm water filter, NO2 - -N was analyzed using the N-(1-naphthyl)-ethylenediamine spectrophotometric method. NO3 - -N was analyzed using ultraviolet spectrophotometry, and NH4 was... + -N was analyzed using Nessler's reagent spectrophotometry; COD was analyzed using rapid digestion spectrophotometry; biofilm sludge samples were taken at days 10, 75, and 150 to analyze EPS content, microbial protein content was analyzed using the Bradford method, and polysaccharide concentration was analyzed using the anthrone-sulfuric acid colorimetric method; sludge samples were taken at days 1, 75, and 150 for microbial sequencing analysis. (4) Correlation network analysis: Calculate Spearman's rank correlation coefficient to illustrate the association between all denitrification-related genera in all samples. When the correlation coefficient is greater than 0.5 and the p value is less than 0.05, it is judged as significant correlation. Network visualization is performed using the Gephi platform.

2. The method according to claim 1, characterized in that: The effective volume of the reactor is 4L.

3. The method according to claim 1, characterized in that: The method for microbial sequencing analysis of sludge samples was to extract sample DNA using a kit, and use 341F: 5'-CCTACGGGAGGCAGCAG-3' and 805R: 5'-GACTACHVGGGTATCTAATCC-3' primers for the V3-V4 region of 16S rRNA, followed by Illumina high-throughput sequencing and metagenomic sequencing.

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