A method for rapidly expanding anaerobic ammonia oxidation sludge by adding organic carbon source and antibiotics

Abstract

The application provides a method for rapidly expanding anaerobic ammonia oxidation sludge by adding organic carbon source and antibiotics, and belongs to the technical field of sludge expansion. The application adopts a continuous stirring reactor, takes mature anaerobic ammonia oxidation sludge as an inoculation source, takes simulated wastewater containing ammonia nitrogen and nitrite nitrogen as influent, adds anhydrous glucose with a COD / TN of 0.4 as an organic carbon source, cooperates with the strategy of dynamically adding antibiotic enrofloxacin, and realizes that the sludge biomass is expanded to more than five times of the initial value under the condition of the micro-oxygen condition of the influent and high activity is maintained. The method of the application relieves the problem of slow growth of anaerobic ammonia oxidation bacteria, and provides technical support for process engineering application.

Description

Technical Field

[0001] This invention relates to the field of sludge expansion technology, and in particular to a method for rapidly expanding anaerobic ammonium oxidation sludge by adding organic carbon sources and antibiotics in synergistic effect. Background Technology

[0002] With the increasing global demand for wastewater treatment, efficient and low-energy biological nitrogen removal technologies have become a research hotspot. Anaerobic ammonia oxidation (AAO) technology, due to its low operating costs and high nitrogen removal efficiency, is widely considered one of the most promising wastewater treatment technologies. AAO can convert electron donor ammonium and electron acceptor nitrite into nitrogen gas under anaerobic or anoxic conditions, achieving highly efficient nitrogen removal without the need for an external carbon source. However, the extremely slow growth rate of AAO bacteria and their high sensitivity to environmental changes limit the widespread application of AAO in practical wastewater treatment.

[0003] Currently, the expansion methods for anammox sludge typically rely on inorganic carbon sources. While this can increase sludge volume to some extent, the expansion cycle is long, inefficient, and the activity is unstable, making it difficult to meet practical engineering needs. In recent years, studies have shown that anammox bacteria possess the potential to metabolize organic carbon sources. Our research group has found that anammox bacteria (such as *Candidatus Kuenenia*) can metabolize simple organic carbon sources like glucose for growth. However, the addition of organic carbon sources may lead to the excessive proliferation of non-target bacteria (such as denitrifying bacteria), competing with anammox bacteria and even eliminating the target bacteria. This makes rapid expansion difficult to achieve solely by adding organic carbon sources.

[0004] In existing technologies, antibiotics are primarily added to sludge cultivation systems to suppress resistant or competing bacteria in the sludge, selectively enriching specific functional strains to improve the sludge's treatment efficiency for target pollutants. Therefore, this technology mainly focuses on the selective enrichment of specific functional strains, rather than the core measure of maintaining stable microbial community structure.

[0005] Therefore, there is an urgent need to provide a propagation method that can both maintain the relative abundance of anaerobic ammonia-oxidizing bacteria and stabilize the bacterial community structure. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for rapidly expanding anaerobic ammonia oxidation sludge by synergistically adding an organic carbon source and antibiotics. This invention adds anhydrous glucose as an organic carbon source to promote rapid biomass growth within the reactor, and simultaneously adds the antibiotic enrofloxacin as a stabilizer to maintain the relative abundance of anaerobic ammonia oxidation bacteria within the reactor, thus stabilizing the bacterial community structure. This results in the successful expansion of anaerobic ammonia oxidation sludge by more than five times within 40 days, exhibiting high abundance and activity. This provides technical guidance and process parameters for broadening the application of engineered anaerobic ammonia oxidation processes.

[0007] The purpose of this invention is to provide a method for rapidly expanding anaerobic ammonia oxidation sludge by adding organic carbon sources and antibiotics in a synergistic manner, comprising the following steps:

[0008] Mature anaerobic ammonia oxidation sludge was inoculated into the reactor, and glucose was used as an organic carbon source in conjunction with antibiotics. Under microaerobic conditions in the influent, the anaerobic ammonia oxidation sludge was expanded to achieve the expansion of sludge biomass.

[0009] The antibiotic administration strategy is to start from day 1, with an initial concentration of 0.2–1 mg / L. -1 Adjustments are made based on the biomass growth of the sludge in the reactor; for every 1–2 g·L increase in biomass... -1 The antibiotic concentration increased by 0.2–1 mg / L. -1 ;

[0010] The COD / TN ratio of the influent is 0.4.

[0011] In some embodiments of the present invention, the antibiotic is enrofloxacin;

[0012] The reactor is a continuous stirred reactor to ensure full contact between the culture medium and the substrate.

[0013] In some embodiments of the present invention, the dissolved oxygen concentration under the micro-oxygen conditions is between 0 and 0.29 mg·L⁻¹. -1 .

[0014] In some embodiments of the present invention, the total nitrogen in the influent is composed of NH4. + -N and NO2 - -N composition; the NH4 + -N and NO2 - The mass concentration ratio of -N was 1:1 to 1.32, and the concentrations were 100 to 300 mg·L⁻¹. -1 Total nitrogen is 200–600 mg·L. -1 ;

[0015] Furthermore, when the COD / TN ratio of the influent is 0.4, the COD concentration is 80–240 mg·L⁻¹. -1 .

[0016] In some embodiments of the present invention, the biomass concentration of the mature anaerobic ammonium oxidation sludge inoculated in the continuous stirred reactor, expressed as volatile solids (VSS), is 1–3 g·L⁻¹. -1 .

[0017] In some embodiments of the present invention, the biomass concentration in the continuous stirred reactor after inoculation with mature anaerobic ammonium oxidation sludge is 5–8 g·L⁻¹, expressed as volatile solids (VSS). -1 .

[0018] In some embodiments of the present invention, the dominant bacterial genus in the anammox sludge is Candidatus Kuenenia. In this invention, the addition of antibiotics helps maintain the relative abundance of anammox bacteria in the sludge, preventing their elimination and thus maintaining the bacterial community structure; the influent contains nutrients necessary for the growth of anammox bacteria, making it suitable for their growth.

[0019] In some embodiments of the present invention, the pH of the influent is 7.6 to 7.8, which is most suitable for the growth of anaerobic ammonia-oxidizing bacteria Candidatus Kuenenia, and the hydraulic retention time is 5 to 8 hours; the culture temperature is 16-30°C.

[0020] In some embodiments of the present invention, the mass concentration of glucose is 85.36–256.08 mg·L. -1 Furthermore, it contains 120 mg / L. -1 The required COD addition is 128.04 mg·L⁻¹. -1 Anhydrous glucose.

[0021] In some embodiments of the present invention, the influent contains NH4Cl, NaNO2, and C6H. 12 O6, CaCl2·2H2O, MgCl2·6H2O, KHCO3, NaH2PO4, Trace Element I stock solution and Trace Element II stock solution

[0022] The trace element I stock solution was prepared from EDTA·2Na and FeSO4·7H2O;

[0023] The trace element II stock solution contains: MnCl2, ZnSO4·7H2O, CuSO4·5H2O, Na2MoO4·4H2O, H3BO3, CoCl2·6H2O, NiCl2·6H2O, and vitamin tablets.

[0024] In some embodiments of the present invention, the propagation time is not less than 40 days.

[0025] The technical solution of the present invention has the following advantages over the prior art:

[0026] This invention utilizes the synergistic effect of adding organic carbon sources and antibiotics to rapidly expand anammox sludge. Within 40 days, the anammox sludge was successfully expanded more than five times and exhibited high activity. Experiments have demonstrated that this method is effective for the expansion of anammox sludge.

[0027] The beneficial effects of this invention are mainly reflected in: ① the synergistic cultivation of anammox sludge using organic carbon sources and antibiotics broadens the application field of anammox technology; ② the sludge biomass of the anammox sludge cultivated within 40 days, expressed as volatile solids concentration (VSS), is 7.075 g·L⁻¹. -1 It is more than five times the initial biomass, alleviating the problem of slow growth of anaerobic ammonia-oxidizing bacteria. Detailed Implementation

[0028] To address the technical problems identified in the background section, the present invention is implemented in the following manner:

[0029] This invention innovatively introduces the antibiotic enrofloxacin into the anammox sludge expansion process for the first time, simultaneously adding the organic carbon source glucose. Through the precise regulatory effect of enrofloxacin, the stability of the microbial community structure in the anammox sludge is effectively maintained during rapid expansion. By employing a synergistic addition strategy of glucose and enrofloxacin, this invention achieves rapid expansion of the anammox sludge quantity, effectively alleviating the problem of slow sludge growth and providing a scientific basis and process guarantee for the promotion of anammox technology in engineering applications.

[0030] This invention provides a method for rapidly expanding anaerobic ammonia oxidation sludge by synergistically adding organic carbon sources and antibiotics, comprising the following steps:

[0031] In the reactor, glucose is used as an organic carbon source in conjunction with antibiotics to expand the anaerobic ammonia oxidation sludge under micro-aerobic conditions in the influent, thereby achieving the expansion of sludge biomass.

[0032] The antibiotic administration strategy is to start from day 1, with an initial concentration of 0.2–1 mg / L. -1 Adjustments are made based on the biomass growth of the sludge in the reactor; for every 1–2 g·L increase in biomass... -1 The antibiotic concentration increased by 0.2–1 mg / L. -1 ;

[0033] The COD / TN ratio of the influent is 0.4.

[0034] In a specific embodiment of the present invention, the antibiotic is enrofloxacin; the present invention effectively maintains the stability of the microbial community structure in the rapid expansion process of anammox sludge through the precise regulatory effect of enrofloxacin, avoiding the excessive proliferation of non-target microbial communities (such as denitrifying bacteria), and keeping the relative abundance of anammox bacteria such as Candidatus Kuenenia stable.

[0035] The reactor is a continuous stirred reactor to ensure full contact between the culture medium and the substrate.

[0036] In a specific embodiment of the present invention, the dissolved oxygen concentration under the micro-oxygen conditions is between 0 and 0.29 mg·L⁻¹. -1 For example, it can be 0, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, etc.

[0037] In a specific embodiment of the present invention, the total nitrogen in the influent is composed of NH4. + -N and NO2 - -N composition; the NH4 + -N and NO2 - The mass concentration ratio of -N was 1:1 to 1.32, and the concentrations were 100 to 300 mg·L⁻¹. -1 Total nitrogen is 200–600 mg·L. -1 ;

[0038] Furthermore, when the COD / TN ratio of the influent is 0.4, the COD concentration is 80–240 mg·L⁻¹. -1 For example, the values ​​can be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 mg·L. -1 wait.

[0039] In a specific embodiment of the present invention, the biomass concentration of the mature anaerobic ammonium oxidation sludge inoculated in the continuous stirred reactor, expressed as volatile solids (VSS), is 1–3 g·L⁻¹. -1 For example, the dosage can be 1, 1.5, 2, 2.5, or 3 g·L. -1 .

[0040] In a specific embodiment of the present invention, the biomass concentration in the continuous stirred reactor after inoculation with mature anaerobic ammonium oxidation sludge is 5-8 g·L⁻¹, calculated as volatile solids (VSS). -1 For example, the values ​​can be 5, 5.5, 6, 6.5, 7, 7.5, or 8 g·L.-1 wait.

[0041] In a specific embodiment of the present invention, the dominant bacterial genus in the anammox sludge is Candidatus Kuenenia. In this invention, the addition of antibiotics helps maintain the relative abundance of anammox bacteria in the sludge, preventing their elimination and thus maintaining the bacterial community structure; the influent contains nutrients necessary for the growth of anammox bacteria, making it suitable for their growth.

[0042] In a specific embodiment of the present invention, the pH of the influent is 7.6 to 7.8, which is most suitable for the growth of anaerobic ammonia-oxidizing bacteria Candidatus Kuenenia, and the hydraulic retention time is 5 to 8 hours; the culture temperature is 16-30°C.

[0043] In a specific embodiment of the present invention, the mass concentration of glucose is 85.36–256.08 mg·L. -1 For example, the dosage can be 85.36, 85, 90, 95, 100, 150, 200, 250, or 256.08 mg·L. -1 wait.

[0044] Furthermore, it contains 120 mg·L -1 The required COD addition is 128.04 mg·L⁻¹. -1 Anhydrous glucose.

[0045] In a specific embodiment of the present invention, the influent contains NH4Cl, NaNO2, and C6H. 12 O6, CaCl2·2H2O, MgCl2·6H2O, KHCO3, NaH2PO4, Trace Element I stock solution and Trace Element II stock solution

[0046] The trace element I stock solution was prepared from EDTA·2Na and FeSO4·7H2O;

[0047] The trace element II stock solution contains: MnCl2, ZnSO4·7H2O, CuSO4·5H2O, Na2MoO4·4H2O, H3BO3, CoCl2·6H2O, NiCl2·6H2O, and vitamin tablets.

[0048] In a specific embodiment of the present invention, the propagation time is not less than 40 days.

[0049] 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.

[0050] Example 1:

[0051] This invention provides a method for rapidly expanding anaerobic ammonia oxidation sludge by synergistically adding organic carbon sources and antibiotics. The specific steps are as follows:

[0052] (1) A continuous stirred reactor with an effective volume of 1 L was used, and the biomass, expressed as volatile solids concentration (VSS), was 2.875 g·L⁻¹. -1 Mature anaerobic ammonia oxidation sludge was used as the inoculum source. After inoculation, the biomass in the reactor, expressed as volatile solids concentration (VSS), was 1.217 g·L⁻¹. -1 With a content of 150 mg·L -1 ammonia nitrogen and 150 mg·L -1 Simulated wastewater containing nitrite and without deoxygenation treatment was used as the influent, with a hydraulic retention time of 6 hours. The sludge was cultured under the conditions of 24℃ and influent pH 7.6.

[0053] The composition of other components in the simulated wastewater used is as follows: ammonia nitrogen 150 mg·L⁻¹ -1 Nitrite 150 mg·L -1 KHCO3 1500 mg·L -1 MgCl2·6H2O 1500mg·L -1 56 mg / L CaCl2·2H2O -1 NaH2PO4 10 mg·L -1 The trace element I and trace element II stock solutions were prepared using water as the solvent; the organic carbon source was added at a rate of 128.04 mg·L⁻¹ each time. -1 .

[0054] The composition of the trace element I stock solution is as follows:

[0055] (a) EDTA·2Na 5g·L -1 ,

[0056] (b) FeSO4·7H2O 9.14 g·L -1 .

[0057] The composition of the trace element II stock solution is as follows:

[0058]

[0059] (2) Four continuous stirred reactors, each with an effective volume of 1.0 L, were set up. X1, with a COD / TN ratio of 0.4, served as the experimental group; X2, X3, and X4, with COD / TN ratios of 0, 0.2, and 0.7, served as the control groups. X1, X3, and X4 were supplemented with 128.04 mg·L⁻¹, respectively. -1 64.02 mg·L -1 and 224.07 mg·L -1Anhydrous glucose was used. After 40 days of cultivation, the average nitrogen removal efficiency of X1 was 74.85%, while the average nitrogen removal efficiency of X3 and X4 was around 69%. The average nitrogen removal efficiency of X2 without added organic carbon source was 65.93%. On the 40th day, a 6-hour in-situ batch experiment was conducted in all four reactors to test the anaerobic ammonia oxidation activity. X1 achieved the highest total nitrogen removal rate of 70.46%, X2 achieved 53.47%, while X3 achieved only 24.25% and X4 achieved 46.73%. Combining the average nitrogen removal efficiency and the results of the in-situ batch experiment, it can be demonstrated that when the COD / TN ratio is 0.4, the reactor exhibits the highest nitrogen removal efficiency and sludge activity.

[0060] (3) On day 40, the sludge biomass of each reactor in (2) was measured. The biomass in reactor X1, with a COD / TN ratio of 0.4, expressed as volatile solids concentration (VSS), was 6.425 g·L⁻¹. -1 Under the same conditions, the biomass expressed as volatile solids concentration (VSS) in X2, X3, and X4 was 4.426 g·L⁻¹. -1 5.478 g·L -1 and 5.971 g·L -1 The biomass in X1 was 31.1%, 14.7%, and 7.1% higher than that in X2, X3, and X4, respectively. Through COD / TN optimization experiments, it was demonstrated that the sludge biomass expansion efficiency was highest when the COD / TN ratio was 0.4.

[0061] Example 2:

[0062] (1) Based on a COD / TN ratio of 0.4, four continuous stirred reactors with an effective volume of 1.0 L were set up, with an anaerobic ammonia-oxidizing bacterium genus Candidatus Kuenenia having a relative abundance of 16.82% and a biomass expressed as volatile solids concentration (VSS) of 2.875 g·L⁻¹. -1 Mature anaerobic ammonia oxidation sludge was used as the inoculum source. After inoculation, the biomass in the reactor, expressed as volatile solids concentration (VSS), was 1.352 g·L⁻¹. -1 With a content of 150 mg·L -1 ammonia nitrogen and 150 mg·L -1 The simulated wastewater containing nitrite but without deoxygenation treatment was used as the influent, with a hydraulic retention time of 6 hours. The sludge was cultured at 24°C and an influent pH of 7.6. The composition of other components in the simulated wastewater used was as described in Example 1.

[0063] (2) Initial enrofloxacin dosage: 0.5 mg / L -1 For every 2 g·L increase in biomass within the reactor, expressed as volatile solids (VSS),... -1 Enrofloxacin concentration increased by 0.5 mg / L-1 Z1 was the experimental group, Z2 was the group without enrofloxacin, and the concentration of enrofloxacin was fixed at 0.5 mg / L. -1 and 1 mg·L -1 Z3 and Z4 served as the control group. The four reactors operated for a total of 40 days, with enrofloxacin added to Z1, Z3, and Z4 starting from day 1. The average denitrification efficiency of Z1 was 76.22%, while the average denitrification efficiencies of Z3 and Z4 were both around 74%. The average denitrification efficiency of Z2, without enrofloxacin, was 77.41%. The denitrification performance of the four reactors was roughly the same, indicating that the addition of enrofloxacin did not significantly affect the denitrification performance of the anaerobic ammonia oxidation process.

[0064] (3) On day 40, the sludge biomass and relative abundance of each reactor in (2) were measured. The biomass in reactor Z1, which was dynamically dosed with enrofloxacin, expressed as volatile solids concentration (VSS), was 7.075 g·L⁻¹. -1 Under the same conditions, the biomass expressed as volatile solids concentration (VSS) in Z2, Z3, and Z4 was 4.028 g·L⁻¹. -1 6.300 g·L -1 and 5.975 g·L -1 The biomass in Z1 was 43.1%, 10.9%, and 15.5% higher than that in Z2, Z3, and Z4, respectively. The relative abundance of *Candidatus Kuenenia* in reactor Z1 with dynamic enrofloxacin dosing was 17.85%, while under the same conditions, the relative abundances in Z2, Z3, and Z4 were 3.36%, 9.54%, and 11.93%, respectively. This indicates that dynamic enrofloxacin dosing can stabilize the relative abundance of anaerobic ammonia oxidizing bacteria in the anaerobic ammonia oxidation system, preventing them from being eliminated due to the proliferation of competing bacteria caused by glucose addition, thus maintaining the stability of the bacterial community structure and successfully expanding the anaerobic ammonia oxidation sludge.

[0065] This invention, through COD / TN optimization experiments and antibiotic dosing strategy optimization experiments, demonstrates that when the COD / TN ratio is 0.4, enrofloxacin exhibits the highest expansion efficiency when dynamically added based on biomass. Using anhydrous glucose as the organic carbon source and enrofloxacin as the antibiotic, under the condition of a COD / TN ratio of 0.4, the dosing strategy is to start from day 1 with an initial concentration of 0.5 mg·L⁻¹. -1 The adjustment is made based on the biomass growth of the sludge in the reactor, expressed as VSS (volatile solids concentration). For every 2 g·L increase in biomass... -1 Enrofloxacin concentration increased by 0.5 mg / L -1 The biomass of the sludge increased to 7.075 g·L⁻¹. -1 Compared with the initial biomass concentration (1.352 g·L⁻¹),-1 The nitrogen removal efficiency increased more than fivefold, reaching an average of 76.22%. The relative abundance of anaerobic ammonia-oxidizing bacteria (Candidatus Kuenenia) in the sludge remained stable at 17.85%, basically consistent with the initial relative abundance (16.82%). In the control group, sludge activity, nitrogen removal efficiency, and relative abundance of anaerobic ammonia-oxidizing bacteria were all lower than in the experimental group, further verifying the effectiveness of COD / TN optimization and antibiotic dosing strategy optimization.

[0066] This embodiment successfully expanded the biomass of anammox sludge more than fivefold within 40 days by adding glucose (COD / TN ratio of 0.4) as an organic carbon source and dynamically adding enrofloxacin. Simultaneously, the relative abundance of anammox bacteria in the expanded sludge remained relatively consistent and exhibited good nitrogen removal efficiency. This invention provides a new perspective and data support for the expansion of anammox sludge in practical applications, and also offers new ideas and process optimization guidance for the treatment of wastewater containing organic matter using anammox technology.

[0067] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for rapidly expanding anaerobic ammonia oxidation sludge by synergistically adding organic carbon sources and antibiotics, characterized in that, Includes the following steps: Mature anaerobic ammonia oxidation sludge was inoculated into the reactor, and glucose was used as an organic carbon source in conjunction with antibiotics. Under microaerobic conditions in the influent, the anaerobic ammonia oxidation sludge was expanded to achieve the expansion of sludge biomass. The antibiotic administration strategy is to start from day 1, with an initial concentration of 0.2–1 mg / L. -1 Adjustments are made based on the biomass growth of the sludge in the reactor; for every 1–2 g·L increase in biomass... -1 The antibiotic concentration increased by 0.2–1 mg / L. -1 ; The COD / TN ratio of the influent is 0.4; The antibiotic in question is enrofloxacin; The dominant bacterial genera in the anammox sludge are: Candidatus Kuenenia.

2. The method according to claim 1, characterized in that, The reactor is a continuous stirred reactor to ensure full contact between the culture medium and the substrate.

3. The method according to claim 1, characterized in that, The dissolved oxygen concentration under the micro-oxygen conditions is 0-0.29 mg·L⁻¹. -1 .

4. The method according to claim 1, characterized in that, Total nitrogen in the influent is from NH4 + -N and NO2 - -N composition; the NH4 + -N and NO2 - The mass concentration ratio of -N is 1:1 to 1.32, wherein the NH4 + -N and NO2 - The mass concentration of -N is 100–300 mg·L⁻¹. -1 Total nitrogen is 200–600 mg·L. -1 .

5. The method according to claim 1, characterized in that, The biomass concentration of the mature anaerobic ammonium oxidation sludge inoculated in the reactor, expressed as volatile solids (VSS), was 1–3 g·L⁻¹. -1 .

6. The method according to claim 1, characterized in that, After inoculation with mature anaerobic ammonium oxidation sludge and expanded culture, the biomass concentration in the reactor, expressed as volatile solids (VSS), was 5–8 g·L⁻¹. -1 .

7. The method according to claim 1, characterized in that, The pH of the influent is 7.6–7.8, and the hydraulic retention time is 5–8 hours.

8. The method according to claim 1, characterized in that, The mass concentration of the organic carbon source is 85.36–256.08 mg·L⁻¹. -1 .

9. The method according to claim 1, characterized in that, The propagation process shall last for no less than 40 days.

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