Method for rapidly starting PD / A system and improving denitrification performance based on PPGF enrichment anaerobic ammonium oxidation bacteria

Abstract

The invention belongs to the technical field of sewage treatment, and provides a method for quickly starting a PD / A system and improving denitrification performance based on PPGF-enriched anaerobic ammonium oxidation bacteria. The method comprises the following steps: adding 300ml of inoculated sludge into a reactor by using an up-flow anaerobic bio-membrane reactor; pPGF with a filling ratio of 30% is added in 11d; the inlet water is artificially distributed water, the pH value of the inlet water is adjusted to 7.8-9.1 by using NaHCO3, nitrogen is added for air stripping, so that the dissolved oxygen concentration of the inlet water is less than 0.3 mg.L <-1 >, and an anaerobic environment is kept; placing the reactor at 31 + / -1 DEG C, refluxing once every 30 minutes, continuously operating for 150 days at a refluxing ratio of 2: 1, and analyzing the performance of the reactor during the period; the time required for starting common activated sludge PD / A is shortened, the denitrification efficiency of the system is improved, the common activated sludge is used for enriching anaerobic ammonia oxidation functional bacteria, and a new way is provided for stable operation of anaerobic ammonia oxidation of inoculated common activated sludge.

Description

Technical Field

[0001] The present invention belongs to the technical field of sewage treatment, and particularly relates to a method for rapidly starting a PD / A system and improving nitrogen removal performance by enriching anaerobic ammonium oxidation bacteria based on polyurethane porous gel filler (PPGF). Background Technique

[0002] In the treatment of municipal wastewater, the main challenge faced by the anaerobic ammonium oxidation (Anammox) technology is the stable acquisition of the substrate NO2 - -N. Recently, the PD / A process combining partial denitrification (PD) with Anammox technology has stable and efficient performance. The PD / A process can not only provide a stable NO2 - -N substrate for Anammox bacteria, but also effectively remove organic matter in municipal wastewater, achieving simultaneous deep nitrogen and carbon removal. However, the application of Anammox technology in municipal sewage treatment still faces challenges, which mainly stem from the slow growth, long doubling time, and difficulty in enrichment of Anammox bacteria, resulting in a time-consuming start-up process of the bioreactor. To solve this problem, biomass immobilization technology, especially the granular sludge system and the biofilm system, is considered an effective solution due to its high biomass concentration and tolerance to environmental fluctuations. However, due to the wastewater volume and other engineering problems, the granular system may not be suitable for domestic sewage treatment in municipal sewage treatment. On the contrary, the biofilm system, due to its long sludge retention time and high aggregation ability of anaerobic ammonium oxidation bacteria (AnAOB), is considered an effective way for AnAOB enrichment.

[0003] In the research and application of Anammox biofilm technology, the selection of suspended carriers plays a crucial role in the attachment of microorganisms and the development of biofilms. Traditional carriers, such as spherical polypropylene or tubular polyethylene (PE) materials, although excellent in durability, have deficiencies in microbial attachment ability and fluidization performance, limiting the overall performance of the biofilm reactor. In recent years, sponge carriers made of polyurethane have been favored by researchers due to their large specific surface area and characteristics that promote microbial attachment. However, due to their low density, although these carriers are easy to suspend in the reactor, their fluidization performance is not ideal, which may affect the mass transfer efficiency and the uniform distribution of biofilms.

[0004] Polyurethane porous gel filler (PPGF), which is a new type of porous gel filler with unique physical and chemical properties. From the perspective of structural design, PPGF adopts a wall-like structure. Compared with the ribbed structure of traditional polyurethane sponges, its specific surface area is significantly increased, which provides a larger contact interface for the mass transfer process, thereby improving the mass transfer efficiency.

[0005] In terms of pore structure design, PPGF adopts a macropore-micropore composite structure system. Among them, the macropore structure provides good contact conditions for the gas-liquid-solid three-phase, ensuring the high efficiency of mass transfer; the micropore structure is rich in hydrophilic active groups such as amino, carboxyl, and epoxy groups. The presence of these functional groups endows the material with excellent water absorption performance. It should be noted that PPGF forms a gel state after water absorption. At this time, its hydrophilicity is significantly enhanced, and it can quickly adsorb and fix microorganisms and bioenzymes. This characteristic not only significantly improves the attachment efficiency of microorganisms but also makes the filler quickly settle in the water body by changing the material density to be greater than that of water. From the perspective of material properties, the porous structure of PPGF ensures a high porosity while optimizing the transfer of substances inside and outside the carrier. Its unique elastic characteristics further promote the attachment of microorganisms and the formation kinetics of biofilms. These comprehensive characteristics enable PPGF to exhibit excellent performance in the anaerobic ammonium oxidation (Anammox) process, specifically reflected in significantly shortening the process startup time and improving the nitrogen removal efficiency of the system. Experimental studies have shown that the application of PPGF can effectively promote the enrichment and activity improvement of Anammox bacteria, providing a new technical approach for biological nitrogen removal from wastewater. Summary of the Invention

[0006] The present invention aims to solve the problems of slow startup and low nitrogen removal performance of ordinary activated sludge in anaerobic ammonium oxidation, and provides a method for rapidly starting up the PD / A system and improving nitrogen removal performance based on PPGF to enrich anaerobic ammonium oxidation bacteria. The present invention proposes to shorten the startup time of anaerobic ammonium oxidation for treating wastewater with a low carbon-nitrogen ratio by inoculating ordinary activated sludge and improve the nitrogen removal performance by adding PPGF to the reactor, and explore its mechanism of action. The purpose of the present invention is to reveal the application of PPGF in anaerobic ammonium oxidation of ordinary activated sludge, in order to provide a new idea for the rapid startup and stable operation of anaerobic ammonium oxidation of ordinary activated sludge.

[0007] The present invention is realized by the following technical solutions: A method for rapidly starting up the PD / A system and improving nitrogen removal performance based on PPGF to enrich anaerobic ammonium oxidation bacteria, using an upflow anaerobic biofilm reactor, adding 300 ml of inoculated sludge to the reactor; adding PPGF with a filling ratio of 30% at 11 d; the influent is artificial wastewater, adjusting the pH of the influent to 7.8 - 9.1 with NaHCO3, and blowing with nitrogen to make the dissolved oxygen concentration of the influent less than 0.3 mg·L -1 , maintaining an anaerobic environment; the reactor is placed at 31 ± 1 °C, refluxing once every 30 minutes, and continuously operating for 150 d with a reflux ratio of 2:1. During this period, the performance of the reactor is analyzed; Among them: the specific surface area and porosity of PPGF are: S > 4000 m 2 / m 3 ; Ψ = 98%; The influent contains 40 - 67 mg / L NH4Cl, 33.9 - 83.26 mg / L Na2NO3, 420 mg / L NaHCO3, 300 mg / L CaCl 2· 2H2O, 200 mg / L MgSO4·7H2O, 25 mg / L KH2PO4. In addition, 1 ml / L of trace element solution I and trace element solution II are added. The components of the trace element solutions are as follows: Trace element solution I (g / L): 6.37 g of EDTA·2Na and 9.14 g of FeSO4·7H2O; Trace element solution II (g / L): 19.11 g of EDTA·2Na, 0.24 g of CoCl2·6H2O, 0.25 g of CuSO4·5H2O, 0.22 g of NaMoO4·2H2O, 0.19 g of NiCl2·7H2O, 0.014 g of H3BO4, 0.43 g of ZnSO4·7H2O, and 0.99 g of MnCl2·4H2O.

[0008] Specifically, it includes the following steps: (1) Inoculate the returned sludge and let it stand for 24 h. Discard the supernatant. The measured MLSS is 5.9 g / L and the MLVSS is 4.0 g / L. Take 300 mL of sludge and inoculate it into the reactor. (2) Start the partial denitrification and anaerobic ammonium oxidation coupling reactor, i.e., operate the PD / A system: 2 L of wastewater enters from the bottom, and the influent time is 25 min; the bottom effluent, and the time is 5 min; the reaction temperature is controlled at 31 ± 1 °C, and the dissolved oxygen (DO) is controlled at 0.30 ± 0.05 mg / L. It is refluxed once every 30 min, and the reflux ratio is 2:1; Intermittent influent is adopted, and the HRT is controlled at 24 h; The reactor operates continuously for 150 d, which is divided into three stages, namely stage I for enriching short - range denitrifying bacteria, stage II for enriching anaerobic ammonium - oxidizing bacteria, and stage III for improving the nitrogen removal performance of the reactor. Stage I is from 1 to 10 d, the influent NO3 - -N concentration is 42 mg·L -1 , and the influent NH4 + -N concentration is 45 mg·L -1 ; Stages II and III are from 11 to 150 d, the influent NO3 - -N concentration is 75 mg·L -1 , and the influent NH4 + -N concentration is 60 mg·L -1 ; At the 11th day of the reactor operation, PPGF is added. (3) Performance analysis: Conduct routine water quality analysis on the reactor effluent every day, including NO2 - -N, NO3 - -N, NH4 +-N, COD, pH. The water sample was filtered through a 0.45 µm water-based filter head, and NO2 - -N was analyzed by N-(1-naphthyl)-ethylenediamine spectrophotometry, and NO3 - -N was analyzed by ultraviolet spectrophotometry, and NH4 + -N was analyzed by Nessler reagent spectrophotometry; COD was analyzed by rapid digestion spectrophotometry; Biofilm sludge samples were taken on the 10th, 75th, and 150th days to analyze their EPS content. The microbial protein content was determined by the Bradford method, and the polysaccharide concentration was determined by the anthrone-sulfuric acid colorimetric method; Sludge samples were taken on the 1st, 75th, and 150th days for microbial sequencing analysis; (4) Correlation network analysis: Calculate the Spearman rank correlation coefficient to illustrate the association between genera related to denitrification in all samples. When the correlation coefficient is greater than 0.5 and the p-value is less than 0.05, it is determined to be significantly correlated; Network visualization uses the Gephi platform.

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

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

[0011] By analyzing the changes in the effluent nitrogen concentration, nitrogen removal efficiency, COD, and microbial community structure, the present invention proves the feasibility of PPGF in promoting the rapid start-up of PD / A in conventional activated sludge and improving the denitrification performance, providing a new approach for the stable operation of PD / A treating low-carbon nitrogen ratio wastewater with conventional activated sludge as the inoculated sludge.

[0012] The results show that in the PD / A reactor, the rapid start-up of PD / A in conventional activated sludge can be promoted by adding PPGF. When the reactor with PPGF addition was successfully started within 79 days, the NRE was 98% at the same time; Anaerobic ammonium-oxidizing bacteria were effectively enriched in PPGF, Cadidatus The relative abundance of Jettenia reached 22% on the 150th day, and the contribution rate of anaerobic ammonium oxidation to the total nitrogen removal reached 87%.

[0013] By adding PPGF into the reactor, the present invention shortens the start-up time required for the conventional activated sludge PD / A and improves the nitrogen removal efficiency of the system, and explores the changes in the nitrogen concentration, nitrogen removal efficiency and microbial community structure in the effluent during the reaction process. Using conventional activated sludge to enrich anaerobic ammonium oxidation functional bacteria is a necessary condition for the stable operation of improving the nitrogen removal performance of low C / N wastewater, and the present invention provides a new way for this. Description of the Drawings

[0014] Figure 1 Changes in nitrogen removal performance during the start-up process: In the figure: (a) shows the changes in the ammonia nitrogen concentration of the influent and effluent and the ammonia nitrogen removal efficiency; (b) shows the changes in the NO-3-N concentration of the influent and effluent, the effluent NO-2-N concentration and the NO-3-N removal efficiency; (c) shows the changes in the TN concentration of the influent and effluent and the TN removal efficiency; (d) shows the contributions of Anammox and denitrification to the TN removal efficiency; Figure 2 Shows the changes in COD and nitrogen concentration during a typical cycle of the PD / A process on the 145th day; Figure 3 Characteristics of EPS content in biofilms during operation: Concentrations of S-EPS, LB-EPS, TB-EPS, total EPS and the PN / PS ratio; Figure 4 Shows the changes in the composition of the reactor microbial community at the phylum level; Figure 5 Shows the relative abundances of anaerobic ammonium-oxidizing bacteria (AnAOB) and denitrifying bacteria (DNB) in the reactor biofilm and flocculent sludge; In the figure: (a) is for DNB; (b) is for AnAOB; (c) is for the main microorganisms involved in nitrogen removal; Figure 6 Shows the genus-level species correlation network; Figure 7 Shows the relative abundances of key nitrogen metabolism enzymes detected in the reactor biofilm and flocculent sludge on the 150th day; Figure 8 Shows the biofilm nitrogen metabolism pathway (AMX: anammox; DNRD: dissimilatory nitrate reduction; ANR: assimilatory nitrate reduction). Detailed Embodiments

[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, but not all, of the embodiments of the present invention; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope 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 belongs. All materials cited herein and the materials they cite will be incorporated by reference.

[0017] Equivalent technologies of the specific embodiments described that can be learned by those skilled in the art through routine experiments will be included in this application.

[0018] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0019] I. Reaction device and method: An upflow anaerobic biofilm reactor was used in the experiment, with a height of 36 cm, an inner diameter of 12 cm, and an effective volume of 4 L. 2 L of wastewater entered through the bottom by a peristaltic pump, and the influent time was 25 min. The bottom effluent time was 5 min; the reaction temperature was controlled at 31 ± 1 °C, the DO was controlled at (0.30 ± 0.05) mg / L, and it was refluxed once every 30 min with a reflux ratio of 2:1. PPGF (2 cm * 2 cm * 1 cm), and the filling ratio was 30%. The reactor was covered and sealed to maintain an anaerobic environment. The PPGF was purchased from Henan Yishuijing Environmental Protection Technology Co., Ltd.

[0020] 1. Inoculated sludge and simulated wastewater: The reflux sludge was taken from Zhengyang Wastewater Treatment Plant in Jinzhong City, Shanxi Province. 300 mL of inoculated sludge was inoculated into the reactor, and its MLSS was 5.90 g·L -1 , and the MLVSS / MLSS was 0.68.

[0021] Artificial water distribution was used, containing NH4Cl (40 - 67 mg / L), Na2NO3 (33.9 - 83.26 mg / L), NaHCO3 (420 mg / L), CaCl 2·2H2O (200 mg / L), MgSO4·7H2O (200 mg / L), KH2PO4 (25 mg / L), 1 mL / L of trace element I and trace element II. The components of the trace element solutions are as follows. Trace element solution I (g / L) contains 6.37 g of EDTA·2Na and 9.14 g of FeSO4·7H2O, and trace element solution II (g / L) contains 19.11 g of EDTA·2Na, 0.24 g of CoCl2·6H2O, 0.25 g of CuSO4·5H2O, 0.22 g of NaMoO4·2H2O, 0.19 g of NiCl2·7H2O, 0.014 g of H3BO4, 0.43 g of ZnSO4·7H2O, and 0.99 g of MnCl2·4H2O. The pH of the influent is adjusted to 7.8 - 9.1 with 0.1 mol·L -1 NaOH.

[0022] 2. Reactor operating parameters: The reactor operated for a total of 150 days and was 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 samples were filtered through a 0.45 µm hydrophilic filter head. NO2 - -N was analyzed by N-(1-naphthyl)-ethylenediamine spectrophotometry, NO3 - -N was analyzed by ultraviolet spectrophotometry, NH4 + -N was analyzed by Nessler's reagent spectrophotometry, and COD was analyzed by rapid digestion spectrophotometry; Biofilm sludge samples were taken on the 10th, 75th, and 150th days to analyze their EPS content. The microbial protein content was determined by the Bradford method, and the polysaccharide concentration was determined by the anthrone-sulfuric acid colorimetric method; MLSS and MLVSS were analyzed by standard methods, and the pH was measured with a pH meter. In addition, sludge samples from different stages (the 1st day, the 75th day, and the 150th day) were taken, and the DNA of the samples was extracted using a kit. The primers for the V3-V4 region of 16S rRNA were 341F (CCTACGGGAGGCAGCAG) and 805R (GACTACHVGGGTATCTAATCC), and high-throughput sequencing was performed using the Illumina system, and metagenomic sequencing was performed on the sludge sample on the 150th day.

[0024] 4. Calculation methods: The specific calculation formulas involved are as follows: PD and PD / A stages The calculation methods for the conversion rate (NTR) are shown below respectively: ; where: represents the effluent Concentration mg / L; Indicates the effluent Concentration mg / L; Indicates the influent Concentration mg / L.

[0025] ; Where: Indicates the concentration at time t mg / L; Indicates the theoretically consumed by the Anammox reaction Concentration mg / L; Indicates the influent Concentration mg / L; Indicates the theoretically produced by the Anammox reaction Concentration mg / L; Indicates the concentration at time t of mg / L.

[0026] Complete denitrification and anaerobic ammonium oxidation are the main nitrogen removal pathways in the PD / A stage. Ignoring the NH4 + -N consumed by cell synthesis, the calculation based on the Anammox reaction formula is as follows: Anaerobic ammonium oxidation nitrogen removal contribution rate: .

[0027] Complete denitrification nitrogen removal contribution rate: ; Where: Indicates the influent Concentration mg / L; Indicates the effluent Concentration mg / L; Indicates the change in concentration of influent and effluent mg / L.

[0028] II. Experimental results 1. Start-up process of the reactor; change in effluent nitrogen concentration; nitrogen removal performance during the start-up process of the reactor is as Figure 1 shown.

[0029] In stage I (days 1 - 10), flocculent sludge was inoculated into the reactor and operated for 10 days to ensure its stability and adaptation to the new operating conditions. The influent C / NO3 - -N and pH value were 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%, the nitrogen removal efficiency (NRE) was 56.84% to 27.97%, and the NTR PDmax was only 11.20%. This may be due to the change of environmental conditions, which led to the lysis of non-adapted bacterial cells in the inoculated sludge, resulting in the effluent NH4 + -N concentration being higher than that of the influent. As the PD / A process proceeded, endogenous denitrification was mainly carried out in the activated sludge. In order to reduce the effluent TN concentration and establish the PD / A system efficiently and rapidly, PPGF was added to the reactor at a filling rate of 30%, and the influent nitrogen load was increased.

[0030] In stage II (days 11 - 52), the NRE remained almost unchanged ( Figure 1 c). The effluent TN concentration was about 93.39 mg / L, and the NRE was 28.68%. In contrast, in the later stage of the second stage (days 53 - 75), the effluent TN concentration gradually decreased to 68.63 mg / L, indicating that as PD and AnAOB adapted to the influent NH4 + -N and NO3 - -N concentrations increased, the denitrification performance was significantly improved. Nevertheless, the average effluent NO3 - -N concentration was still as high as 31.11 mg / L ( Figure 1 b, Figure 1 c). These results show that the denitrification performance in the second stage was still limited to a certain extent, and the ARE and NRE of the reactor were about 50.14% and 49.60% respectively. It should be noted that the influent C / NO3 - -N may be too low for the shortcut denitrification process, resulting in relatively low denitrification performance. In addition, from day 53 to day 75, the in-situ ammonia oxidation reaction rate gradually increased and reached 1.88 mg NH4 + -N / L / h ( Figure 1 a).

[0031] To further improve the denitrification performance, during stage III (days 76 - 150), the influent C / NO3 - -N was increased to 3 on day 76. This adjustment significantly increased the denitrification rate to 79.30% and significantly reduced the effluent TN concentration to 29.32 mg / L ( Figure 1 c). At the same time, the average ratio of ΔNO3 - -N / ΔNH4 + -N was about 1.33, slightly higher than 1.32 (the theoretical value). This may be due to the complete denitrification, which reduced the effluent NO3 --N concentration decreased to 10.21 mg N / L. In addition, NO2 - -N concentration also reached the minimum value (Figure 1b). After 79 days of operation, the reactor achieved efficient nitrogen removal. When the influent C / NO3 - -N ratio was 3, the NRE reached 98.22%, and remained almost unchanged during the period from day 79 to day 150 ( Figure 1 c), and the effluent TN concentration was as low as 2.52 mg / L. In addition, the anammox activity was also stable at 2.45 mg NH4 + -N / L / h, accounting for 87.10% of the NRE. Since AnAOB has a stronger competitive ability for NO2 - -N than denitrifying bacteria, they can effectively remove NH4 + -N and NO2 - -N, indicating that the PD / A process can be established in this system.

[0032] 2. Analysis of nitrogen removal pathways: To clarify the nitrogen removal pathways in the system, the 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 stages were observed, including the PD stage and the anaerobic ammonia oxidation stage. Within 0 - 120 minutes, the substrates NO3 - -N and COD required for partial nitrogen removal were sufficient. Part of the nitrogen removal process converted organic substrates into nitrates, while part of the NH4 + -N and the generated NO2 - -N were also removed by anammox. Within 120 minutes, the NO2 - -N concentration reached the peak value (34.37 ± 0.14 mg / L), indicating that PD was the main process, and the accumulation rate of NO2 - -N was faster than the consumption rate of anammox. Subsequently, NH4 + -N and NO2 - -N were removed by anammox. However, the COD concentration only decreased to 49.49 ± 3.97 mg / L, indicating that during the nitrogen removal process, the role of anammox was greater than that of denitrification. During the cycle, the NTR PD / A reached 94.35%, indicating that anammox could compete with denitrification for NO2 - -N. During the ammonia oxidation stage, the ratio of ΔNO2 - -N / ΔNH4 + -N was 1.24, slightly lower than 1.32 calculated by the ammonia oxidation equation, indicating that part of the NO3 --N is converted to NO2 in the system - -N. This is consistent with the results of the above batch tests. The removal of nitrogen in the later stage of the reaction mainly depends on the anammox pathway, so anammox is the dominant process at this stage. Finally, 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, the removal rate of TN by denitrification is 8.85%, which shows that Anammox is the main nitrogen removal pathway, with a removal rate of TN exceeding 91.15%. In addition, it is worth mentioning that during partial denitrification, partial nitrification also occurred in the system, which can provide additional NO2 - -N to reduce the NH4 + -N concentration, thereby improving the nitrogen removal performance of Anammox.

[0033] 3. Changes in EPS in the reactor: Microbial cells form biofilms by secreting a large amount of EPS, and these EPS bind the sludge together and adsorb on the sticky aggregates on the surface of PPGF. During the entire operation cycle, as Figure 3As shown, the total EPS content shows a gradually increasing trend. With the enrichment of sludge, the concentration of biofilm EPS increases from 88.77 ± 2.58 mg / g-VSS to 160.94 ± 3.78 mg / g-VSS, a significant increase of 81.30%. The increase in EPS content plays a key role in maintaining biofilm formation, keeping the structural integrity, and protecting the microorganisms continuously adsorbed on the surface of PPGF from the external environment and stress. From the stratification results of EPS, the concentrations of S-EPS and LB-EPS fluctuate less, while TB-EPS has the highest concentration among EPS, and its content gradually increases, reaching 132.90 ± 5.01 mg / g-VSS on the 150th day of the mature biofilm. From the results of each component of EPS, the change in PS concentration is not obvious, while PN has the highest content in the two EPS subpopulations. The concentration of PN in the biofilm system increases 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 containing extracellular enzymes in EPS. This means that the increase in PN concentration provides more extracellular enzymes and nutrients for microorganisms, thus enhancing the activity of microorganisms. As the two main components of EPS, PS and PN regulate the characteristics of biofilms, 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, thus promoting the growth metabolism, aggregation, and interaction of bacteria, and further improving the denitrification performance of the anaerobic system. After 140 days of reactor operation, the PN / PS ratio shows an upward trend throughout the operation cycle and remains at about 4.04.

[0034] 4. At the phylum level, such as Figure 4As shown, the microbial community of the biofilm exhibited significant dynamic changes. Bacterial phyla with a relative abundance greater than 1% were considered the main bacterial phyla. On the 75th day, 8 main bacterial phyla were found in the biofilm, including Chlorobi (14.97%), Proteobacteria (13.73%), Bacteroidetes (6.71%), Planctomycetacia (5.21%), Acidobacteria (4.12%), Armantimonadetes (3.73%), Gemmatimonadota (2.81%), and Actinobacteriota (1.03%). Notably, the phylum Planctomycetota, including AnAOB, reached 2.81%. During the entire stable operation period (days 80 - 150), the abundances of Proteobacteria and Actinobacteriota in the biofilm increased significantly, reaching 29.91% and 23.94% respectively, indicating that they had become the dominant phyla. Meanwhile, the abundance of the Bacteroidetes group increased to 8.86%. However, the relative abundances of Chlorobaculum, Limnohabitans, Acidobacteria, and Armantimonadetes decreased significantly, accounting for 11.51%, 4.42%, 1.63%, and 1.51% respectively, suggesting that these microbial phyla might have played important roles in the initial stage of biofilm formation. The abundance of Armillaria, a common phylum in the ammonia oxidation system, decreased slightly during the steady-state operation. It has been reported that Armatimonadota can cause excessive consumption of NH4 + -N in the ammonia oxidation system, which might be the reason for the ΔNO2 - -N / ΔNH4 + -N ratio being less than the theoretical value here. These results indicate that functional microorganisms exhibit different succession patterns during biofilm formation.

[0035] 5. At the genus level, as Figure 5 shown, in addition to AnAOB, bacterial genera capable of PD were also enriched in the biofilm, thus ensuring the NO2 - -N supply for AnAOB. On the 75th day, the abundance of denitrifying bacteria (including Thauera, Pseudomonas, Denitratisoma, and unclassified Comamonadaceae) reached 3.92% (Figure 5a). During days 80 - 150, the biofilm gradually matured, and AnAOB became the dominant bacterium, with its abundance increasing from 0.19% to 22.00%; the main 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 and gradually became the dominant genus in AnAOB later. Its relative abundance on day 150 was as high as 21.73%, higher than that reported in other anaerobic systems. The second dominant bacteria in the biofilm were denitrifying bacteria (21.14%), and the main genera were Thauera and Denitratisoma ( Figure 5 c). When the C / NO3 - -N ratio was 3, the organic matter could be used as an electron donor to convert nitrate into N2, thus reducing the accumulation of nitrate in the system. In this case, the denitrifying bacteria became active, and the growth environment of AnAOB was improved, thereby increasing the nitrogen removal efficiency. Therefore, when the C / NO3 - -N ratio was 3, AnAOB showed stronger ability in substrate competition compared with heterotrophic bacteria. In addition, with the increase in relative abundance, AnAOB even competed with heterotrophic denitrifying bacteria for nitrite, thus increasing the contribution of Anammox to nitrogen removal in the system. Therefore, these results indicate that AnAOB and heterotrophic bacteria can coexist harmoniously in the reactor. On the contrary, on day 150, denitrifying bacteria dominated in the flocculent sludge (30.43%), and the main genera included Thauera, Denitratisoma and Pseudomonas. AnAOB became the second dominant bacteria (15.66%). These results indicate that the biofilm is more conducive to the enrichment of AnAOB. In addition, Candidatus the abundance of _Jettenia in the biofilm was as high as 21.73%, while it was only 15.55% in the flocculent sludge, indicating the success of the cultivation of anaerobic ammonium oxidation sludge.

[0036] 6. Microbial correlation network analysis: As Figure 6 shown (Spearman correlation coefficient greater than 0.5 or less than -0.5, significance level p < 0.05) was used to identify possible relationships between key microorganisms. There were obvious positive correlation relationships among several specific subsets of denitrifying bacteria in the biofilm. For example, Candidatus _Jettenia and Thauera, Thauera and Pseudomonas, Comamonas, Candidatus_Competibacter and Hyphomicrobium, indicating a synergistic relationship between denitrifying bacteria and AnAOB. In other words, Thauera and Pseudomonas with partial denitrifying ability can produce NO2 - -N for AnAOB by reducing NO3 - -N. These results indicate that the synergistic application of denitrification and Anammox processes can achieve efficient and stable nitrogen removal.

[0037] 7. Metabolic mechanism analysis: To clarify the metabolic mechanism of the highly active biofilm, the sequencing results were compared with the KEGG database to obtain the relevant pathways of nitrogen metabolism in the reactor and the relative abundances of the functional genes involved as Figure 7 shown. Considering the synergistic process of denitrification and anaerobic ammonium oxidation, the key enzymes of functional bacteria in the biofilm sludge and flocculent sludge in the reactor were further studied.

[0038] The key genes nitrate reductase Nar and periplasmic dissimilatory nitrate reductase nap for the conversion of nitrate to nitrite were observed in both the biofilm sludge and flocculent sludge in the reactor, indicating that the conversion of nitrate to nitrite is a potential pathway for the enrichment of anaerobic ammonium oxidizing bacteria. Specifically, the relative abundances of Nar in the biofilm and flocculent sludge were 0.46% and 0.42% respectively. Nar was mainly distributed in Thauera in the biofilm and flocculent sludge, with relative abundances of 0.12% and 0.14% respectively, followed by Candidatus_ Jettenia, with relative abundances of 0.05% and 0.02% respectively; another Nap was also mainly distributed in Thauera in the biofilm and flocculent sludge, with relative abundances of 0.13% and 0.14% respectively; indicating a relatively high expression level of genes related to short-cut denitrification, and Thauera is the main force in reducing nitrate to nitrite, which is consistent with previous studies. Copper nitrite reductase (NirK) and cytochrome cd1 nitrite reductase (NirS) are processes that can reduce nitrite to nitric oxide during the denitrification process, and it is also an important process for the occurrence of anaerobic ammonium oxidation reaction. For NirS, the relative abundances in the biofilm and flocculent sludge were 0.13% and 0.11% respectively; interestingly, the relative abundances of NirK in the biofilm and flocculent sludge were very low, only 0.0015% and 0.0010% respectively. And nitric oxide reductase NorB / C and nitrous oxide reductase NosZ related to complete denitrification could also be detected. The relative abundances of NorB / C in the biofilm and flocculent sludge were both 0.15%, and the relative abundances of NosZ in the biofilm and flocculent sludge were 0.17% and 0.15% respectively, indicating that the denitrification process also contributed to the nitrogen removal process in the reactor. Hydrazine dehydrogenase (Hdh) and hydrazine synthase (Hzs), the two most important enzymes in the anaerobic ammonium oxidation process, could be detected in both the 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, and the relative abundances of Hdh in biofilm and flocculent sludge were 0.29% and 0.10%, respectively. The relative abundances of these two enzymes in biofilm sludge were higher than those in flocculent sludge. The higher the relative abundances of Hzs and Hdh, the higher the relative abundance of AnAOB bacteria in the biofilm. These results were consistent with the results of microbial community analysis. At the same time, the genes related to nitrification, AmoA / B / C, were all at very low levels, which was the same as the results of microbial community detection, indicating that ammonia nitrogen was mainly removed through the anaerobic ammonium oxidation reaction. Therefore, the Nar and Nap genes dominated the nitrogen transformation in the PD process. The significant prevalence of the Nar gene, together with the limited presence of the NorB / C and NosZ genes related to complete denitrification and the low expression of the genes related to nitrification, AmoA / B / C, was consistent with the significant nitrogen removal contribution of anaerobic ammonium oxidation in the anaerobic biofilm PD / A system. In addition, other nitrogen metabolic pathways, including the functional gene expressions of the complete dissimilatory nitrate reduction process and the assimilatory nitrate reduction process, also existed in the reactor biofilm and flocculent sludge. Ten functional genes were detected in total in the reactor biofilm and flocculent sludge for the dissimilatory nitrate reduction process. Among them, the functional genes Nar and Nap involved in the process of nitrate conversion to nitrite were consistent with the process of denitrifying nitrate conversion to nitrite, and the functional genes involved were NarG / H / I / J and NapA / B; the functional genes NirB / D and NrfA / H involved in the process of nitrite conversion to ammonia nitrogen. The relative abundances of NirB / D in biofilm and flocculent sludge were 0.14% and 0.16%, respectively, and the relative abundances of NrfA / H in biofilm and flocculent sludge were 0.011% and 0.0048%, respectively. The higher expression of NirB / D compared to NrfA / H also indirectly indicated the presence of a higher concentration of nitrite during the nitrogen removal process in the reactor, further demonstrating the excellent PD performance in the reactor. Two functional genes were detected in total in the biofilm sludge and flocculent sludge for the assimilatory nitrate reduction process, namely the functional gene NasA for nitrate conversion to nitrite, and the relative abundances of NasA in biofilm and flocculent sludge were 0.34% and 0.37%, respectively; the functional gene NirA involved in the process of nitrite conversion to ammonia nitrogen, and the relative abundances of NirA in biofilm and flocculent sludge were 0.00048% and 0.00030%, respectively. Based on the above results, a biofilm nitrogen metabolic pathway based on metagenomics was proposed ( Figure 8 ).

[0039] This invention has verified that: the PD / A system started with the self-enrichment of anaerobic ammonium-oxidizing bacteria operates stably in mainstream sewage, achieving deep nitrogen removal, and the enriched anaerobic ammonium-oxidizing bacteria play a dominant role in the nitrogen removal process. Meanwhile, a powerful anaerobic biofilm PD / A system was rapidly started within 79 days. When the influent C / NO3 - -N was 3, the effluent TN concentration was lower than 3 mg / L, the NRE reached 98%, and the anammox removal rate of TN exceeded 87%. The start-up time of PD / A was effectively shortened without inoculating anaerobic ammonium-oxidizing sludge. The gradual implementation of multi-faceted regulation strategies fully integrated and mobilized the advantages of each stage of biofilm development. The strategy of gradually increasing the wastewater feed ratio not only stably established the mainstream environment but also considered the adaptive domestication of the PD / A microbiota to its habitat, effectively promoting the succession of the microbial community. In addition, during the entire operation process, sufficient nitrite was produced while consuming organic matter, eliminating the adverse effects of organic matter on anammox, laying the foundation and providing the necessary support for the final configuration of the core bacterial community in the PD / A biofilm system. In addition, a spatio-temporal structure was created for anammox with PPGF, and the abundance of AnAOB in the biofilm reached 22% on the 150th day. Candidatus _Jettenia is the dominant genus of anaerobic ammonium-oxidation. The in-situ enrichment of anaerobic ammonium-oxidation for treating municipal sewage provides a reliable experimental basis and is expected to be further verified in future long-term laboratory-scale and pilot-scale tests.

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

Claims

1. A method for rapidly starting a PD / A system and improving denitrification performance based on PPGF enrichment of anaerobic ammonia-oxidizing bacteria, characterized in that: An upflow anaerobic biofilm reactor was used, and 300 ml of inoculated sludge was added to the reactor. PPGF with a filling ratio of 30% was added on day 11. The influent was artificially distributed water, and the pH of the influent was adjusted to 7.8-9.1 with NaHCO3. Nitrogen was used for stripping to make the dissolved oxygen concentration of the influent less than 0.3 mg·L -1 , maintaining an anaerobic environment; the reactor was placed at 31±1℃, refluxed once every 30 minutes, and the reflux ratio was 2:1 for 150 days. During this period, the performance of the reactor was analyzed; Among them: The specific surface area and porosity of PPGF are: S>4000m 2 / m 3 ;Ψ=98%; The influent contains 40-67 mg / L NH4Cl, 33.9-83.26 mg / L Na2NO3, 420 mg / L NaHCO3, 300 mg / L CaCl 2· 2H2O, 200mg / L MgSO4·7H2O, 25mg / L KH2PO4, in addition, 1ml / L trace element solution I and trace element solution II were added; the components of the trace element solution were as follows: trace element solution I g / L: 6.37g EDTA·2Na and 9.14g FeSO4·7H2O; trace element solution II g / L: 19.11g EDTA·2Na, 0.24g CoCl2·6H2O, 0.25g CuSO4·5H2O, 0.22g NaMoO4·2H2O, 0.19g NiCl2·7H2O, 0.014g H3BO4, 0.43g ZnSO4·7H2O and 0.99g MnCl2·4H2O.

2. The method according to claim 1, characterized in that: The specific steps include: (1) Inoculate the return sludge, let it stand for 24 hours, discard the supernatant, and measure the MLSS to be 5.9 g / L and the MLVSS to be 4.0 g / L; take 300 mL of sludge and inoculate it in the reactor; (2) Start-up of the partial denitrification and anaerobic ammonium oxidation coupled reactor, i.e., the PD / A system operation: 2L of wastewater enters through the bottom, the water inlet time is 25min; the water is discharged from the bottom, the time is 5min; the reaction temperature is controlled at 31±1℃, the dissolved oxygen (DO) is controlled at 0.30±0.05mg / L, reflux is performed every 30min, and the reflux ratio is 2:1; intermittent water inlet is used, and the HRT is controlled at 24h; The reactor was operated continuously for 150 days and was divided into three stages: stage I to enrich short-range denitrifying bacteria, stage II to enrich anaerobic ammonia-oxidizing bacteria, and stage III to improve the denitrification performance of the reactor; Phase I is 1 to 10 days, with influent NO3 - -N concentration is 42 mg·L -1 , influent NH4 + -N concentration is 45 mg·L -1 ; Stage II and Stage III are 11 to 150 days, and the influent is NO3 - -N concentration is 75 mg·L -1 , influent NH4 + -N concentration is 60 mg·L -1 ; PPGF was added when the reactor was running for 11 days; (3) Performance analysis: Routine water quality analysis is performed on the reactor effluent every day, including NO2 - -N、NO3 - -N、NH4 + -N, COD, pH, water samples were filtered with a 0.45µm water filter, NO2 - -N was analyzed by N-(1-naphthyl)-ethylenediamine spectrophotometry, NO3 - -N was analyzed by UV spectrophotometry, NH4 + -N was analyzed by Nessler's reagent spectrophotometry; COD was analyzed by rapid digestion spectrophotometry; Biofilm sludge samples were taken on the 10th, 75th, and 150th days to analyze their EPS content, microbial protein content using the Bradford method, and polysaccharide concentration using the anthrone-sulfuric acid colorimetric method; sludge was taken on the 1st, 75th, and 150th days for microbial sequencing analysis; (4) Correlation network analysis: Spearman rank correlation coefficient was calculated to illustrate the correlation between all sample denitrification-related genera. When the correlation coefficient was greater than 0.5 and the p value was less than 0.05, it was considered to be significantly correlated. The network visualization uses the Gephi platform.

3. The method according to claim 2, characterized in that: The effective volume of the reactor is 4L. The method according to claim 2, characterized in that: The method for microbial sequencing analysis of sludge samples was to extract sample DNA using a kit, and the primers for the V3-V4 region of 16SrRNA were 341F: 5'-CCTACGGGAGGCAGCAG-3' and 805R: 5'-GACTACHVGGGTATCTAATCC-3', and Illumina high-throughput sequencing and metagenomic sequencing were performed.

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