Method for promoting rapid biofilm formation of anammox under non-deoxidized conditions

Abstract

This invention discloses a method for promoting rapid biofilm formation in anaerobic ammonia oxidation under non-deaeration conditions, comprising the following steps: S1. Adding polyethylene packing material to a reactor and inoculating it with anaerobic ammonia oxidation sludge; S2. Introducing water into the reactor, wherein the total nitrogen concentration in the influent is 200–600 mg·L⁻¹. ‑1 The mass concentration ratio of ammonia nitrogen to nitrite nitrogen is 1:1 to 1.32, and the influent is not deoxygenated. The COD / TN ratio in the influent is controlled to be 0.3 by adding an organic carbon source. S3. The reactor is operated for expansion cultivation, achieving a biomass of 15-16 mg VSS / carrier per unit packing material and increasing EPS content to 50-60 mg / g VSS, thereby achieving rapid biofilm formation in the anaerobic ammonia oxidation process. Compared to traditional biofilm cultivation methods, the method of this invention can effectively shorten the biofilm start-up time, improve the operating efficiency of the anaerobic ammonia oxidation reactor, and enhance the system's antioxidant capacity, enabling it to maintain high-efficiency operation even without deoxygenation.

Description

Technical Field

[0001] This invention relates to the field of wastewater biological treatment technology, specifically to a method for promoting rapid biofilm formation in anaerobic ammonia oxidation under non-deaeration conditions. Background Technology

[0002] With rapid industrialization and urbanization, wastewater discharge is constantly increasing, and nitrogen pollution has become a significant challenge for water environment management. Traditional biological nitrogen removal processes mainly rely on nitrification-denitrification, which requires an external organic carbon source and has high operating costs. In contrast, anaerobic ammonium oxidation (Anammox) technology has received widespread attention in recent years due to its advantages such as no need for an external carbon source, low sludge production, low energy consumption, and high nitrogen removal efficiency, and has shown promising application prospects in wastewater treatment. This process relies on anaerobic ammonium-oxidizing bacteria (AnAOB) to directly convert ammonium and nitrite into nitrogen gas under anaerobic or low-oxygen conditions, achieving highly efficient nitrogen removal. However, due to the slow growth of AnAOB and its high sensitivity to environmental conditions, its enrichment and stable operation in wastewater treatment systems still face many technical challenges.

[0003] Biofilm technology has proven to be an effective means of improving the stability of anammox processes. By introducing a biofilm carrier, AnAOB can attach and grow on the carrier surface, forming a stable biofilm structure, thereby extending sludge retention time, reducing microbial loss, and enhancing the system's resistance to environmental disturbances. However, the natural formation of biofilms typically takes a long time, and in engineering applications, the slow biofilm formation is a significant factor limiting the widespread adoption of anammox processes. Therefore, accelerating biofilm formation, improving AnAOB enrichment efficiency, and enhancing biofilm stability are important directions for current research. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide a method for rapidly promoting biofilm formation in anaerobic ammonia oxidation (ANAO) reactors by optimizing the addition of organic carbon sources such as glucose under anaerobic conditions. Compared to traditional biofilm cultivation methods, this invention effectively shortens the biofilm start-up time, improves the operating efficiency of the ANAO reactor, and enhances the system's antioxidant capacity, enabling it to maintain high-efficiency operation even under ANA conditions.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0006] This invention provides a method for promoting rapid biofilm formation in anaerobic ammonia oxidation biofilms under non-deoxygenation conditions, comprising the following steps:

[0007] S1. Add polyethylene packing material to the reactor and inoculate it with anaerobic ammonia oxidation sludge;

[0008] S2. Water is introduced into the reactor, wherein the total nitrogen concentration in the water is 200–600 mg·L⁻¹. -1 The mass concentration ratio of ammonia nitrogen to nitrite nitrogen is 1:1 to 1.32, and the influent does not undergo deoxygenation treatment; the COD / TN ratio in the influent is controlled to be 0.3 by adding an organic carbon source to the influent.

[0009] S3. Operate the reactor to achieve a biomass of 15-16 mg VSS / carrier per unit packing material and an EPS content of 50-60 mg / g VSS, thereby achieving rapid biofilm formation of the anaerobic ammonia oxidation biofilm.

[0010] Appropriate amounts of organic carbon sources can promote the formation of anaerobic ammonia oxidation (AnAOB) biofilms, increase the secretion of extracellular polymeric substances (EPS), enhance microbial adhesion, and accelerate biofilm maturation. Furthermore, proper regulation of organic carbon sources can promote synergistic effects between AnAOBs and other microorganisms, optimize microbial community structure, and improve the system's denitrification efficiency. However, excessive addition of organic carbon sources may lead to excessive proliferation of heterotrophic bacteria, affecting AnAOB growth and biofilm stability. Therefore, rationally controlling the type and amount of organic carbon sources is crucial for accelerating biofilm formation.

[0011] Besides the influence of organic carbon sources, dissolved oxygen (DO) also plays a crucial role in the formation and stability of anaerobic ammonia oxidation biofilms. Although AnAOBs possess a certain degree of oxygen tolerance, high DO concentrations may inhibit their activity and affect the anaerobic ammonia oxidation process. The multilayered structure of the biofilm can effectively buffer the effects of oxygen, protecting the deeper AnAOBs from oxygen toxicity.

[0012] In this invention, the inventors achieved rapid promotion of anaerobic ammonia oxidation biofilm formation by optimizing the addition of organic carbon sources such as glucose to the influent. Specifically, the inventors discovered that controlling the COD / TN ratio in the influent to 0.3 by adding organic carbon sources can optimize EPS secretion, enhance the stability of the biofilm structure, accelerate the biofilm maturation process, improve the enrichment efficiency of AnAOB, and enhance the system's oxygen tolerance and denitrification capacity.

[0013] In step S1 of the present invention, the polyethylene filler is a K3 or K5 type polyethylene carrier, and the filling rate of the polyethylene filler is 30-35% (v / v). For example, it can be 30%, 31%, 32%, 33%, 34%, 35% (v / v), etc.

[0014] In step S1 of this invention, the reactor is preferably a continuously stirred reactor to ensure sufficient contact between the culture medium and the substrate, and to maintain a dissolved oxygen (DO) concentration of 0.19 ± 0.03 mg / L within the reactor, thereby maintaining the growth environment for anaerobic ammonia-oxidizing bacteria. The stirring rate of the continuously stirred reactor is preferably 50–200 rpm, and can be, for example, 50, 80, 100, 120, 150, 180, or 200 rpm.

[0015] In step S1 of this invention, the biomass concentration of the anaerobic ammonia oxidation sludge inoculated in the reactor is 2.3–2.5 g·L⁻¹, calculated as volatile suspended solids (VSS). -1 For example, it can be 2.3, 2.4, or 2.5 g·L. -1 Furthermore, the dominant bacterial genus in the anaerobic ammonia oxidation sludge is Candidatus Kuenenia.

[0016] In step S2 of this invention, the pH of the influent is preferably 7.6 to 7.8, and can be, for example, 7.6, 7.7, 7.8, etc. This pH range is most suitable for the growth of anaerobic ammonia-oxidizing bacteria Candidatus Kuenenia.

[0017] In step S2 of this invention, the temperature inside the reactor is preferably 16–30°C, and can be, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C. The hydraulic retention time is preferably 4 hours.

[0018] In step S2 of this invention, the total nitrogen in the influent is composed of NH4+. + -N and NO2 - The composition is -N, with a total nitrogen concentration of 200–600 mg·L⁻¹. -1 For example, the values ​​can be 200, 300, 400, 500, or 600 mg·L. -1 etc.; NH4 + -N and NO2 - The mass concentration ratio of -N is 1:1 to 1.32, and for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.32, etc.

[0019] In step S2 of this invention, the COD / TN ratio of the influent needs to be controlled at 0.3. By precisely controlling the carbon-nitrogen ratio to 0.3, the secretion of extracellular polymers can be promoted, the microbial community structure can be optimized, the enrichment efficiency of anaerobic ammonia-oxidizing bacteria can be improved, and the competition of heterotrophic bacteria can be inhibited, ensuring the long-term stable operation of the system.

[0020] Furthermore, the COD / TN ratio in the influent can be adjusted to 0.3 by adding glucose. Adding appropriate amounts of glucose promotes synergistic effects between denitrifying bacteria and the system, improving nitrogen removal efficiency and maintaining the bacterial community structure. The influent contains nutrients necessary for the growth of anaerobic ammonia-oxidizing bacteria, making it suitable for their growth. In some embodiments, when the TN of the influent is 300 mg / L... -1 At that time, the COD concentration was 90 mg·L⁻¹. -1 At this point, the glucose dosage was 84.375 mg / L.

[0021] Furthermore, the influent can be simulated wastewater. In some preferred embodiments of the present invention, the influent contains NH4Cl, NaNO2, and C6H4Cl. 12 O6, CaCl2·2H2O, MgCl2·6H2O, KHCO3, NaH2PO4, trace element I stock solution and trace element II stock solution. The trace element I stock solution is prepared from EDTA·2Na and FeSO4·7H2O; the trace element II stock solution contains: MnCl2, ZnSO4·7H2O, CuSO4·5H2O, Na2MoO4·4H2O, H3BO3, CoCl2·6H2O, NiCl2·6H2O, and vitamin tablets.

[0022] In some embodiments of the present invention, the trace element I stock solution has the following composition:

[0023] (a) EDTA·2Na 5g·L -1 ,

[0024] (b) FeSO4·7H2O 9.14 g·L -1 .

[0025] The composition of the trace element II stock solution is as follows:

[0026]

[0027]

[0028] In step S3 of this invention, the expansion culture operation time is no more than 90 days. After expansion culture, rapid biofilm formation of the anaerobic ammonia oxidation biofilm is achieved, enhancing the mechanical strength of the biofilm and improving the system's resistance to erosion.

[0029] Experiments have shown that the system can still maintain high-efficiency denitrification under the condition of DO = 0.19 ± 0.03 mg / L, with a nitrogen removal rate of 88.83% and a COD removal rate of 88.54%.

[0030] Furthermore, the method described in this invention can be applied to wastewater treatment plants, industrial wastewater treatment, and anaerobic ammonia oxidation process optimization to reduce operating costs and improve denitrification efficiency.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] 1. This invention provides a method for rapidly promoting biofilm formation in anaerobic ammonia oxidation (ANAO) under non-deoxygenation conditions by optimizing the addition of organic carbon sources such as glucose. This method optimizes EPS secretion by precisely controlling the amount of organic carbon source added, enhancing the stability of the biofilm structure, accelerating the biofilm maturation process, improving the enrichment efficiency of AnAOB, and enhancing the system's oxygen tolerance and denitrification capacity. Compared to traditional biofilm cultivation methods, this invention effectively shortens the biofilm start-up time, improves the operating efficiency of the ANAO reactor, and provides a scientific basis and technical support for the engineering application of ANAO technology.

[0033] 2. The method of the present invention optimizes the enrichment conditions of anaerobic ammonia oxidizing bacteria, increases the microbial biomass (VSS) per unit packing material, improves the stability and denitrification efficiency of the biofilm, and enhances the system's antioxidant capacity, enabling it to maintain high-efficiency operation even without deoxygenation.

[0034] 3. Experiments have shown that the method of the present invention can achieve a biomass of 15-16 mg VSS / carrier per unit packing material within 90 days and increase the EPS content to 50-60 mg / g VSS, effectively enhancing the mechanical strength and stability of the biofilm and improving the system's resistance to erosion. This proves that the method is effective for biofilm formation in anaerobic ammonia oxidation sludge. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0036] 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. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0037] In the following examples and comparative examples, the specific composition of the simulated wastewater used is as follows: ammonia nitrogen 150 mg·L⁻¹ -1 Nitrite nitrogen 150 mg·L -1 KHCO3 1500mg·L -1 MgCl2·6H2O 1500mg·L-1 CaCl2·2H2O 56mg·L -1 NaH2PO4 10 mg·L -1 Trace element I and trace element II stock solutions were prepared, with water as the solvent. The composition of trace element I stock solution was: EDTA·2Na 5 g·L⁻¹ -1 FeSO4·7H2O 9.14 g·L -1 The composition of the trace element II stock solution is: MnCl2 0.63 g·L⁻¹ -1 ZnSO4·7H2O 0.43g·L -1 CuSO4·5H2O 0.219 g·L -1 Na₂MoO₄·4H₂O 0.184 g·L⁻¹ -1 H3BO3 0.014 g·L -1 CoCl2·6H2O 0.228g·L -1 NiCl2·6H2O 0.195g·L -1 Vitamin tablets 0.317g / L -1 .

[0038] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available.

[0039] Example 1

[0040] This embodiment provides a method for promoting rapid biofilm formation in anaerobic ammonia oxidation biofilms under non-deoxygenation conditions. The specific steps are as follows:

[0041] A continuous stirred-tank reactor (CSTR) with an effective volume of 11L was used, with K3 type polyethylene packing material added as a microbial carrier at a filling rate of 30% (v / v) to increase the attachment area for microorganisms and enhance the enrichment capacity of anaerobic ammonia oxidizing bacteria (AnAOB). Anaerobic ammonia oxidation sludge was used as the inoculum, with an initial sludge concentration (VSS) of 2.35–2.37 g / L, derived from a long-term stable-operation anaerobic ammonia oxidation reactor. Simulated wastewater was used as the influent, containing ammonia nitrogen (NH4+). +The concentrations of nitrite nitrogen (NO₂-N) and total nitrogen (TN) were both 150 mg / L, with a total nitrogen concentration of 300 mg / L. The hydraulic retention time (HRT) was set to 4 h. The reactor temperature was controlled at 30 °C, the pH was maintained at 7.6 using a phosphate buffer solution, and the dissolved oxygen (DO) was controlled at 0.19 mg / L to maintain the activity of anaerobic ammonia-oxidizing bacteria. In this embodiment, glucose was used as the organic carbon source, with the COD / TN ratio precisely controlled at 0.3 and the dosage at 84.375 mg / L, corresponding to a COD concentration of 90 mg / L, to optimize biofilm formation and denitrification performance.

[0042] After 90 days of operation, a complete brick-red sludge layer formed on the surface of the packing material, with a biofilm thickness of 800 μm and a unit packing biomass (VSS) of 15.65 mg VSS / carrier. Under the condition of COD / TN = 0.3, the extracellular polymeric substance (EPS) content reached 57.92 mg / g VSS, promoting microbial attachment and improving biofilm stability.

[0043] Microbial community analysis showed that the anaerobic ammonia-oxidizing bacterium *Candidatus Kuenenia* had the highest relative abundance, reaching 27.90%. Simultaneously, the synergistic effect of denitrifying bacteria enhanced nitrogen removal efficiency, ultimately achieving a nitrogen removal rate of 88.83% and a COD removal rate of 88.54%. Furthermore, the system maintained high-efficiency nitrogen removal even at DO = 0.19 ± 0.03 mg / L, indicating that the optimized biofilm structure enhanced the system's oxygen tolerance.

[0044] Example 2

[0045] In this embodiment, three reactors were set up with different COD / TN ratios of 0.1, 0.3 and 0.5, respectively, to explore the effects of different organic carbon dosages on biofilm formation and denitrification performance.

[0046] The reactor is a 1L continuous stirred reactor (CSTR) filled with K5 type polyethylene packing material at a filling rate of 30% (v / v). The inoculum sludge is anaerobic ammonia oxidation sludge with an initial sludge concentration (VSS) of 2.3 g / L. The influent ammonia nitrogen (NH4+)... + -N) and nitrite nitrogen (NO2) - The concentrations of nitrogen (N) were all 150 mg / L, the total nitrogen concentration was 300 mg / L, the hydraulic retention time (HRT) was set to 4 h, the experimental temperature was controlled at 30 ℃, the pH was maintained at 7.6~7.8, and the dissolved oxygen (DO) was controlled at 0.19±0.03 mg / L to ensure a micro-aerobic environment.

[0047] In the COD / TN ratio group of 0.1, the glucose dosage was 28.125 mg / L, corresponding to a COD concentration of 30 mg / L. After 90 days of operation, biofilm formation on the packing surface was slow, with a unit packing biomass (VSS) of 10.84 mg VSS / carrier and EPS content of 42.15 mg / g VSS. Microbial community analysis showed that the abundance of anaerobic ammonia-oxidizing bacteria Candidatus Kuenenia was only 19.43%, with a final nitrogen removal rate of 72.31% and a COD removal rate of 75.26%. This indicates that under low COD / TN ratio conditions, biofilm adhesion is weak and nitrogen removal efficiency is poor.

[0048] In the COD / TN = 0.3 group, the glucose dosage was 84.375 mg / L, corresponding to a COD concentration of 90 mg / L. After 90 days of operation, a complete brick-red sludge layer formed on the surface of the packing material, with a biofilm thickness of 800 μm. The unit packing biomass (VSS) reached 15.74 mg VSS / carrier, and the EPS content reached 57.92 mg / g VSS. Microbial community analysis showed that the anaerobic ammonia oxidizing bacteria Candidatus Kuenenia had the highest abundance, reaching 27.90%, with a final nitrogen removal rate of 88.83% and a COD removal rate of 88.54%. This indicates that an appropriate amount of organic carbon source can promote the enrichment of anaerobic ammonia oxidizing bacteria and improve the system's denitrification capacity.

[0049] In the COD / TN = 0.5 group, the glucose dosage was 140.625 mg / L, corresponding to a COD concentration of 150 mg / L. After 90 days of operation, a relatively thick sludge layer formed on the surface of the packing material, but the biofilm structure was relatively loose. The unit packing biomass (VSS) was 12.91 mg VSS / carrier, and the EPS content was 48.36 mg / g VSS. Microbial community analysis showed that the abundance of anaerobic ammonia oxidizing bacteria Candidatus Kuenenia decreased to 21.75%, with a final nitrogen removal rate of 81.02% and a COD removal rate of 82.41%. This indicates that excessive organic carbon sources may enhance competition from heterotrophic bacteria, affecting the stable growth of anaerobic ammonia oxidizing bacteria.

[0050] In summary, the method provided by this invention, under non-deoxygenation conditions, optimizes EPS secretion, enhances biofilm structural stability, accelerates biofilm maturation, improves AnAOB enrichment efficiency, and enhances the system's oxygen tolerance and denitrification capacity by precisely controlling the amount of organic carbon source added to achieve a COD / TN ratio of 0.3. Compared to traditional biofilm cultivation methods, this invention effectively shortens the biofilm start-up time, improves the operating efficiency of the anaerobic ammonia oxidation reactor, and enhances the system's oxygen resistance, enabling it to maintain high-efficiency operation even under non-deoxygenation conditions.

[0051] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for promoting rapid biofilm formation in anaerobic ammonia oxidation biofilms under non-deoxygenated conditions, characterized in that, Includes the following steps: S1. Add polyethylene packing material to the reactor and inoculate with anaerobic ammonia oxidation sludge; S2. Water is introduced into the reactor, wherein the total nitrogen concentration in the influent is 200–600 mg·L⁻¹. -1 The mass concentration ratio of ammonia nitrogen to nitrite nitrogen is 1:1 to 1.32, and the influent does not undergo deoxygenation treatment; the COD / TN ratio in the influent is controlled to be 0.3 by adding an organic carbon source; the dissolved oxygen concentration in the reactor is 0.19 ± 0.03 mg / L; the pH of the influent is 7.6 to 7.8, the hydraulic retention time is 4 h, and the expansion temperature is 16 to 30℃; the added organic carbon source is glucose. S3. Run the reactor for expansion culture to achieve a biomass of 15-16 mg VSS / carrier per unit packing material and an EPS content of 50-60 mg / g VSS, thereby achieving rapid biofilm formation of the anaerobic ammonia oxidation biofilm; the expansion culture operation time shall not exceed 90 days.

2. The method for promoting rapid biofilm formation under non-deoxygenation conditions according to claim 1, characterized in that, In step S1, the polyethylene filler is a K3 or K5 type polyethylene carrier; and / or, The polyethylene filler has a filling rate of 30-35% (v / v).

3. The method for promoting rapid biofilm formation in anaerobic ammonia oxidation biofilms under non-deoxygenation conditions according to claim 1, characterized in that, In step S1, the reactor is a continuous stirred reactor with a stirring rate of 50~200 rpm.

4. The method for promoting rapid biofilm formation under non-deoxygenation conditions according to claim 1, characterized in that, In step S1, the biomass concentration of the anaerobic ammonia oxidation sludge inoculated into the reactor is 2.3–2.5 g·L⁻¹, calculated as volatile suspended solids (VSS). -1 ; and / or, The dominant bacterial genera in the anammox sludge are: Candidatus Kuenenia .

5. The method for promoting rapid biofilm formation in anaerobic ammonia oxidation biofilms under non-deoxygenation conditions according to claim 1, characterized in that, In step S2, the influent contains NH4Cl, NaNO2, and C6H. 12 O6, CaCl2·2H2O, MgCl2·6H2O, KHCO3, NaH2PO4, and trace element I and trace element II stock solutions; The trace element I stock solution is prepared from EDTA·2Na and FeSO4·7H2O; the trace element II stock solution contains MnCl2, ZnSO4·7H2O, CuSO4·5H2O, Na2MoO4·4H2O, H3BO3, CoCl2·6H2O, NiCl2·6H2O and vitamin tablets.

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