A method for culturing anaerobic ammonia oxidation granular sludge resistant to high concentration of nano Fe3O4
By using gradient acclimatization and pH-controlled culture methods to stimulate heme oxygenase secretion, the inhibition problem of Anammox bacteria in a high-concentration nano-Fe3O4 environment was solved, achieving the stability and high-efficiency denitrification performance of the Anammox process and expanding its application range.
Patent Information
- Application Number
- CN202410408601.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-07
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2044-04-07
AI Technical Summary
Anammox bacteria are easily inhibited in high-concentration nano-Fe3O4 environments, resulting in unstable denitrification performance and making them difficult to apply in actual wastewater treatment.
By employing a gradient acclimatization method combined with controlled final concentration and pH adjustment, the secretion of heme oxygenase was directionally stimulated to cultivate anaerobic ammonia oxidation granular sludge tolerant to high concentrations of nano-Fe3O4. Through heme degradation metabolism mediated by heme oxygenase, the environmental adaptability of Anammox bacteria was improved.
The Anammox process was stabilized in a high-concentration nano-Fe3O4 environment, enhancing the oxidative stress resistance and sedimentation performance of Anammox bacteria, broadening its application range, and ensuring efficient denitrification.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of sewage treatment, and particularly relates to a culture method of anaerobic ammonia oxidation granular sludge resistant to high-concentration nano Fe3O4. BACKGROUND
[0002] At present, energy and cost efficiency and sustainable development gradually evolve into the benchmark of the sewage treatment industry. The autotrophic anaerobic ammonia oxidation (Anammox) process greatly breaks through the traditional biological denitrification method in terms of concept and technology, and has the advantages of high denitrification efficiency, low operation cost, small land occupation and the like. However, the Anammox bacteria are easily affected by various emerging pollutants such as nano materials, plastics and antibiotics in wastewater, and often face great challenges in actual wastewater treatment.
[0003] Nano ferroferric oxide (Fe3O4) is one of the most active and high-surface-area iron compounds, and plays an important role in environmental remediation and wastewater treatment. For example, the magnetism and thermodynamic stability of Fe3O4 make it suitable for use as an adsorbent in environmental remediation processes, and it is often used for wastewater treatment, in-situ groundwater remediation and recovery of valuable metals from mine water; Fe3O4 is also used to remove recalcitrant organic pollutants in soil and water due to its high catalytic efficiency. Therefore, a large amount of Fe3O4 can be used in sewage treatment plants to remove inorganic compounds, organic compounds, pesticides, viruses and other pathogens.
[0004] It is reported that the concentration of nanoparticles (NPs) in wastewater ranges from μg / L to mg / L, while previous studies can reach 1000 mg / L and above. However, due to the easy agglomeration of Fe3O4 to form large aggregates, its mobility in the environment is quite limited, resulting in accumulation. Fe3O4 in wastewater accumulates in sludge due to biological effects, and the concentration of FeNPs in the form of Fe3O4 phase in sludge has been detected to be 2.20 x 10 10 Fe3O4 has antibacterial properties and can induce ROS production and oxidative stress inside and outside the cell, ultimately leading to damage to microbial cell membrane integrity, proteases or DNA. Therefore, Fe3O4 in wastewater will have a negative impact on Anammox bacteria, resulting in unstable denitrification performance. Based on this, it is of great significance to develop a culture method of Anammox granular sludge resistant to high-concentration Fe3O4 for the industrial application and promotion of the Anammox process. SUMMARY
[0005] In view of the deficiencies of the prior art, the application provides a culture method of anaerobic ammonia oxidation granular sludge resistant to high-concentration nano Fe3O4, which successfully cultures the Anammox granular sludge resistant to high-concentration nano Fe3O4 by adopting gradient acclimation + control of final concentration + pH down-regulation to directionally stimulate the secretion of hematin oxygenase, improves the environmental adaptability of the Anammox bacteria by using the hematin degradation metabolism mediated by hematin oxygenase, realizes the stable performance of the Anammox process, is beneficial to the application of the Anammox process in actual wastewater treatment, and solves the problem of long-term growth inhibition of high-concentration nano Fe3O4 on the Anammox bacteria, thus creating the possibility for the practical application and popularization of the Anammox system in the treatment of wastewater containing nano materials.
[0006] To achieve the above object, the application is implemented by the following technical scheme:
[0007] The application discloses a culture method of anaerobic ammonia oxidation granular sludge resistant to high-concentration nano Fe3O4, and the steps are as follows:
[0008] (1) an upflow anaerobic sludge bed reactor is adopted to inoculate Anammox granular sludge;
[0009] (2) wastewater containing ammonia nitrogen, nitrite nitrogen, inorganic salt and trace elements is used as influent, and the reactor is cultured under anaerobic conditions until the denitrification effect of the reactor is in a stable state;
[0010] (3) Fe3O4 is added to the reactor in a gradient mode, the final concentration of Fe3O4 in the reactor is controlled to be 1800-2000 mg / L, the pH value of the influent of the reactor is adjusted to be 6.8-6.9, the hematin oxygenase gene of the anaerobic ammonia oxidation bacteria group in step (2) is directionally stimulated, and the anaerobic ammonia oxidation granular sludge resistant to high-concentration nano Fe3O4 is cultured.
[0011] Preferably, in step (3), the initial concentration of Fe3O4 added is 200 mg / L, the total nitrogen removal rate of the reactor is greater than 80% after each concentration is operated, and the maintenance time is greater than or equal to 3-5 days; Fe3O4 is continuously added, and the addition concentration is 2-3 times the addition amount of the last time.
[0012] Preferably, the Fe3O4 is in a granular form, and the diameter is 20-50 nm.
[0013] Preferably, the Fe3O4 is treated by an ultrasonic device at a power of 0.3-0.5 W / mL for 5-10 min before being added.
[0014] Preferably, the average diameter of the Anammox granular sludge is 367.5±150.5 mu m; and the dominant genus of the Anammox bacteria is Candidatus Brocadia.
[0015] Preferably, the suspended solid concentration of the inoculation mixture of the Anammox granular sludge is 3500-4000 mg / L, and the inoculation volume accounts for 30-35% of the total volume of the upflow anaerobic sludge bed reactor.
[0016] Preferably, the condition for judging whether the anaerobic ammonia oxidation granules are resistant to high-concentration nano-Fe3O4 in step (3) is that the total nitrogen removal rate is maintained at more than 80%, and the hematin oxygenase gene abundance in the reactor is more than 2 times the hematin oxygenase gene abundance of the inoculated anaerobic ammonia oxidation granular sludge.
[0017] Preferably, the wastewater comprises NH4 + -N, NO2 - -N, KHCO3, KH2PO4, EDTA, trace element solution I and trace element solution II.
[0018] Preferably, the NH4 + -N and NO2-N have a mass concentration ratio of 1:1.
[0019] Preferably, the trace element solution I comprises NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O; and / or, the trace element solution II comprises CuSO4·5H2O, ZnSO4·7H2O, CoCl2·6H2O, MnCl2·4H2O, NaMoO4·2H2O, NiCl2·6H2O, NaSeO4 and H3BO3.
[0020] The present application has the following beneficial effects:
[0021] 1. The sensitivity of Anammox bacteria in a high-concentration nano-Fe3O4 environment is improved, which greatly promotes the process operation stability and increases the application range. The Anammox granular sludge resistant to high-concentration Fe3O4 is cultured by adopting the gradient domestication + control of final concentration + pH down-regulation mode, which can effectively improve the high resistance and self-recovery ability of the Anammox system disturbed by Fe3O4, achieve the purpose of efficient and stable denitrification, broaden the types of wastewater treated by the anaerobic ammonia oxidation process, and promote the engineering development.
[0022] 2. The Anammox granular sludge that tolerates high concentrations of Fe3O4 cultured by the present invention greatly improves the heme degradation metabolism of functional bacteria, enhances the ability to resist oxidative stress, improves the sensitivity of functional bacteria to the environment, and broadens the application range of the Anammox process. There are a variety of factors that stress Anammox bacteria in biological treatment systems, including oxygen exposure, low temperature, plastics, heavy metals and metal (oxide) NPs. These factors can induce the production of intracellular ROS, resulting in a negative impact on denitrification performance. The heme oxygenase gene of the Anammox granular sludge that tolerates high concentrations of Fe3O4 cultured by the present invention is highly secreted and performs the function of degrading heme, which is beneficial for the Anammox bacteria to alleviate oxidative damage and repair cellular metabolism disturbed by ROS. Anammox bacteria are rich in heme, which provides favorable conditions for the activation of the heme oxygenase gene. In addition, the free iron of heme degradation metabolites can promote the synthesis of ferritin and hemoglobin of Anammox bacteria, which is beneficial to the recovery of Anammox bacteria activity and repair of the denitrification performance of the damaged system.
[0023] 3. The Anammox granular sludge cultured in the present invention that tolerates high concentrations of Fe3O4 has large particle size and high sedimentation performance. The surface of Anammox bacteria is negatively charged, while Fe3O4 is positively charged. This reverse charge can generate electrostatic force to make Fe3O4 adhere to the surface of Anammox bacteria to form larger particles, thereby enhancing the stability of the sludge itself. In addition to Fe3O4 itself, the Fe3O4 released by it is also 2 + Can react with PO4 in the influent matrix KH2PO4 3- The combination promotes the formation of blue iron ore on the surface of Anammox bacteria and improves the granulation of Anammox sludge. Furthermore, Fe₃O₄ can stimulate Anammox bacteria to secrete more extracellular polymeric substances (EPS). These EPS contain a rich variety of functional groups, including hydroxyl and carboxyl groups, which can serve as adsorption sites for Fe₃O₄, amplifying its role as a cell nucleus. At the same time, the increase in EPS content also facilitates the adhesion of microbial subunits, thereby forming larger particles. This improves the settling performance of Anammox granular sludge, effectively prevents sludge loss, and achieves large-scale enrichment of anaerobic ammonium oxidation sludge, thereby achieving long-term, stable, and efficient denitrification performance in the anaerobic ammonium oxidation process.
[0024] 4. The method for cultivating Anammox granular sludge that tolerates high concentrations of Fe3O4 provided by the present invention can avoid the problem of failure to stimulate the heme oxygenase gene and irreversible inhibition of anaerobic ammonia-oxidizing bacteria activity caused by too little or too much Fe3O4 addition by specifically limiting the final concentration of Fe3O4 and the addition method, thereby achieving the ability to tolerate high concentrations of Fe3O4.
[0025] 5. The application provides a method for culturing Anammox granular sludge resistant to high concentration of Fe3O4, wherein the initial concentration of Fe3O4 is 200-300 mg / L, the total nitrogen removal rate of the reactor is above 80% under each concentration, and the maintenance time is 3-5 days. Then, Fe3O4 is continuously added, and the concentration of Fe3O4 is 1-2 times of the last addition. In this way, the concentration of Fe3O4 is continuously increased without affecting the recovery of the total nitrogen removal rate to above 80%, so as to ensure that the anaerobic ammonia oxidation bacteria have a certain adaptation time to Fe3O4. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 Figure 1 is a structural schematic diagram of an upflow anaerobic sludge bed reactor, wherein 1 is a water inlet bucket, 2 is a water inlet pump, 3 is a water inlet pipe, 4 is an electromagnetic valve, 5 is a constant temperature water bath device, 6 is a water outlet bucket, 7 is a multi-parameter instrument, 8 is a pH probe, 9 is a DO probe, and 10 is a UASB reactor.
[0027] Figure 2 Figure 3 is a diagram of nitrogen concentration and denitrification performance during the operation of the reactor, wherein TNRE is the nitrogen removal efficiency. DETAILED DESCRIPTION
[0028] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application. If not specifically indicated, the technical means used in the embodiments are conventional means known to those skilled in the art.
[0029] The upflow anaerobic sludge bed reactor used in the application is shown in Figure 1. Figure 1 The bottom of the UASB reactor cylinder 1 is connected with the water inlet pipe 3 through the electromagnetic valve 4, the water inlet pipe 3 is provided with the water inlet pump 2, and the other end of the water inlet pipe 3 is connected with the water inlet bucket 1. The pH probe 8 and the DO probe 9 are arranged in the UASB reactor cylinder 1, the pH probe 8 and the DO probe 9 are connected with the multi-parameter instrument 7, the water outlet bucket 6 is connected with the UASB reactor cylinder 1 through a pipeline near the top of the UASB reactor cylinder 1, and the constant temperature water bath device 5 is connected with the UASB reactor cylinder 1 through a pipeline near the top and the bottom of the side wall of the UASB reactor cylinder 1.
[0030] The application discloses a method for culturing anaerobic ammonia oxidation granular sludge resistant to high concentration of nano-Fe3O4, and the steps are as follows:
[0031] (1) using an upflow anaerobic sludge bed reactor, inoculating Anammox granular sludge; the average diameter of the Anammox granular sludge is 367.5±150.5 μm; the dominant genus of the Anammox granular sludge is Candidatus Brocadia. The suspended solid concentration of the inoculation mixed liquor of the Anammox granular sludge is 3500-4000 mg / L, and the inoculation volume accounts for 30-35% of the total volume of the upflow anaerobic sludge bed reactor.
[0032] (2) using wastewater containing ammonia nitrogen, nitrite nitrogen, inorganic salts and trace elements as influent, under anaerobic conditions with a temperature of 35±1℃, influent pH of 7.5±0.1 and hydraulic retention time set to 1-3 h, the total nitrogen removal rate of the reactor reaches more than 80% after enrichment culture for ≥3-5 days, i.e. the denitrification effect of the reactor is in a stable state;
[0033] (3) adding Fe3O4 to the reactor in a gradient manner, controlling the final concentration of Fe3O4 in the reactor to be 1800-2000 mg / L, and simultaneously adjusting the pH value of the influent of the reactor to be 6.8-6.9, to direct stimulation of the heme oxygenase gene of the anaerobic ammonia oxidation bacteria group in step (2), and to culture anaerobic ammonia oxidation granular sludge resistant to high-concentration nano-Fe3O4. The initial concentration of Fe3O4 added is 200 mg / L, and each concentration is run until the total nitrogen removal rate of the reactor is more than 80% for ≥3-5 days; continue to add Fe3O4, and the addition concentration is 2-3 times the addition amount of the last time. The Fe3O4 is in a granular form with a diameter of 20-50 nm. The Fe3O4 is treated by an ultrasonic device at a power of 0.3-0.5 W / mL for 5-10 min before addition.
[0034] Further, the condition for judging whether the anaerobic ammonia oxidation granular matter is resistant to high-concentration nano-Fe3O4 in step (3) is that the total nitrogen removal rate is kept at more than 80%, and the heme oxygenase gene abundance in the reactor is more than 2 times the heme oxygenase gene abundance of the inoculated anaerobic ammonia oxidation granular sludge.
[0035] Further, the wastewater includes NH4 + -N, NO2 - -N, KHCO3, KH2PO4, EDTA, trace element solution I and trace element solution II. The NH4 +The mass concentration ratio of -N and NO2-N is 1:1. The trace element solution I includes NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O; and / or, the trace element solution II includes CuSO4·5H2O, ZnSO4·7H2O, CoCl2·6H2O, MnCl2·4H2O, NaMoO4·2H2O, NiCl2·6H2O, NaSeO4 and H3BO3.
[0036] Further, the wastewater includes NH4 + -N 90-150mg / L, NO2 - -N 90-150mg / L, KHCO3500-1000mg / L, KH2PO420-40mg / L, EDTA 5-10mg / L, trace element solution I 2mL / L and trace element solution II 1mL / L. The trace element solution I includes NaCl 500mg / L, KCl 700mg / L, CaCl2·2H2O 700mg / L, MgSO4·7H2O 500mg / L; the trace element solution II includes CuSO4·5H2O 0.25mg / L, ZnSO4·7H2O 0.43mg / L, CoCl2·6H2O 0.24mg / L, MnCl2·4H2O 0.99mg / L, NaMoO4·2H2O 0.22mg / L, NiCl2·6H2O 0.19mg / L, NaSeO4 0.1mg / L and H3BO3 0.014mg / L.
[0037] The application will be further described in combination with specific examples.
[0038] Example 1
[0039] The specific steps of the culture method of the anaerobic ammonia oxidation granular sludge resistant to high-concentration Fe3O4 are as follows:
[0040] (1) Start the anaerobic ammonia oxidation reactor, complete inoculation and stable operation, and the specific method is as follows:
[0041] The mature anaerobic ammonia oxidation granular sludge is used as the inoculation sludge, and the Anammox dominant bacteria genus is Candidatus Brocadia. The inoculation sludge is placed into an upflow anaerobic sludge bed reactor (UASB) with an effective volume of 1.5L Figure 1). The average particle size of the inoculated anaerobic ammonia oxidation granular sludge was 367.5±150.5 μm; the volume of the inoculated sludge accounted for 30% of the volume of the UASB reactor; the mixed liquor suspended solids concentration was 3800 mg / L; simulated wastewater was used as the influent, nitrogen was used for aeration to remove dissolved oxygen before the influent, and the reactor was operated under the conditions of anaerobic, light-avoiding, temperature of 35±1 ℃, influent pH of 7.5, and hydraulic retention time (HRT) of 2 h. The composition of the simulated wastewater was as follows: NH4 + -N 130±10 mg / L, NO2 - -N 130±10 mg / L, KHCO31000 mg / L, KH2PO435 mg / L, EDTA 5 mg / L, and trace element solution I 2 mL / L and trace element solution II 1 mL / L. The composition of the trace element solution I was as follows: NaCl 500 mg / L, KCl 700 mg / L, CaCl2·2H2O 700 mg / L, and MgSO4·7H2O 500 mg / L. The composition of the trace element solution II was as follows: CuSO4·5H2O 0.25 mg / L, ZnSO4·7H2O 0.43 mg / L, CoCl2·6H2O 0.24 mg / L, MnCl2·4H2O 0.99 mg / L, NaMoO4·2H2O 0.22 mg / L, NiCl2·6H2O 0.19 mg / L, NaSeO4 0.1 mg / L, and H3BO3 0.014 mg / L.
[0042] According to the above operation mode, the mass concentration ratio of NH4 + -N and NO2 - -N in the influent tank was 1:1, and after 14 days, the total nitrogen removal rate of the reactor reached 80%, the maintenance time was 3 days, and the stable operation of the reactor was completed, as shown in Figure 2 .
[0043] (2) Entering the high-concentration Fe3O4 domestication stage of the anaerobic ammonia oxidation granular sludge, the specific steps were as follows:
[0044] Fe3O4 material was added to the reactor at a dosage of 200 mg / L, that is, 200 mg of nano Fe3O4 (20-50 nm) material was added per liter of reactor volume, and before the addition, the Fe3O4 was treated at a power of 0.5 W / mL for 10 min, and at the same time, the pH in the influent was reduced from 7.5 to 6.8, and then the reactor was operated under the condition of maintaining the pH value.
[0045] As shown in Figure 2As shown, the first addition of Fe3O4 to the reactor will cause a certain impact on the anaerobic ammonia oxidation system, so that the effluent ammonia nitrogen and nitrite accumulate temporarily during the anaerobic ammonia oxidation process, and the total nitrogen removal rate rises on the 24th day and gradually recovers to 80% after 8 days, with a maintenance time of 3 days; continue to add 400 mg / L Fe3O4 to the reactor, and the total nitrogen removal rate recovers to 80% after 3 days of fluctuation, with a maintenance time of 3 days; again add 1200 mg / L Fe3O4 to the reactor, and the total nitrogen removal rate is not affected, and the gene prediction result of 16S rRNA high-throughput sequencing technology shows that the hemin oxygenase gene (ho-1) is increased by 2.3 times compared with the sludge at the start-up success stage. The above proves that the anaerobic ammonia oxidation granular sludge with high concentration of Fe3O4 tolerance is successfully obtained.
[0046] In addition, after 45 days of continuous operation, the particle size of the anaerobic ammonia oxidation granular sludge gradually increases, and the average particle size increases from 367.5±150.5μm to 650.3±150.5μm. The settling performance is significantly improved, and the SVI5 decreases from 63.29±1.5mL / g SS to 28.67±1.5mL / g SS. At the same time, the content of extracellular polymeric substance (EPS) increases significantly, and the EPS increases from 84.6±2.8mg·g·VSS -1 to 165±2.8mg·g·VSS -1 . It is shown that the characteristics of Anammox granular sludge are enhanced under the interference of high concentration of nano-Fe3O4.
[0047] Example 2
[0048] The specific steps of the culture method of the anaerobic ammonia oxidation granular sludge with high concentration of Fe3O4 tolerance are as follows:
[0049] (1) The mature anaerobic ammonia oxidation granular sludge is used as the inoculated sludge, and the dominant genus of Anammox is Candidatus Brocadia. The inoculated sludge is placed in an upflow anaerobic sludge bed reactor (UASB) Figure 1 ) with an effective volume of 1.5L. The average particle size of the inoculated anaerobic ammonia oxidation granular sludge is 367.5±150.5μm; the volume of the inoculated sludge accounts for 35% of the volume of the UASB reactor; the mixed liquid suspended solid concentration is 3500mg / L; the simulated wastewater is used as the influent, nitrogen gas is used for aeration before the influent to remove dissolved oxygen, and the reactor is operated under the conditions of anaerobic, light shielding, temperature of 35±1℃, influent pH of 7.5, and hydraulic retention time (HRT) of 2h.
[0050] The composition of the simulated wastewater is as follows: NH4 + -N 130±10mg / L, NO2 -- N 130±10mg / L, KHCO31000mg / L, KH2PO4 35mg / L, EDTA 5mg / L and trace element solution I 2mL / L, trace element solution II 1mL / L. The composition of trace element solution I is: NaCI 500mg / L, KCI 700mg / L, CaCI2·2H2O 700mg / L, MgSO4·7H2O 500mg / L. The composition of trace element solution II is: CuSO4·5H2O 0.25mg / L, ZnSO4·7H2O 0.43mg / L, CoCI2·6H2O 0.24mg / L, MnCI2·4H2O 0.99mg / L, NaMoO4·2H2O 0.22mg / L, NiCI2·6H2O 0.19mg / L, NaSeO4 0.1mg / L, H3BO3 0.014mg / L.
[0051] According to the above operation mode, the mass concentration ratio of NH4 + -N and NO2 - The mass concentration ratio of -N in the influent tank is 1:1, and after 14 days, the total nitrogen removal rate of the reactor reaches 80%, and the maintenance time is 3 days, and the reactor is successfully and stably operated.
[0052] (2) Enter the anaerobic ammonia oxidation granular sludge tolerance high concentration Fe3O4 domestication stage, the specific steps are:
[0053] Fe3O4 material is added to the reactor at a dosage of 200mg / L, that is, 200mg of nano Fe3O4 (30nm) material is added per liter of reactor volume, and before adding, Fe3O4 is treated at a power of 0.5W / mL for 5min, and at the same time, the pH of the influent is reduced from 7.5 to 6.9, and then the reactor is operated under this pH condition. The ammonia nitrogen and nitrite in the reactor effluent temporarily accumulated, the total nitrogen removal rate increased on the 26th day and gradually recovered to 80% after 9 days, and the maintenance time was 3 days; continue to add 600mg / L Fe3O4 to the reactor, and the total nitrogen removal rate recovers to 80% after 2 days of fluctuation, and the maintenance time is 3 days; again add 1200mg / L Fe3O4 to the reactor, and the total nitrogen removal rate is not affected, and the gene prediction result in 16SrRNA high-throughput sequencing technology shows that the heme oxygenase gene increases by 2.8 times compared with the sludge in the start-up success stage. The above proves that the anaerobic ammonia oxidation granular sludge with tolerance to high concentration Fe3O4 is successfully obtained.
[0054] In addition, the particle size of the anammox granular sludge gradually increased after 48 days of continuous operation, with the average particle size increasing from 367.5 ± 150.5 μm to 530.3 ± 150.5 μm. The settling performance was significantly improved, with SVI5 decreasing from 65.29 ± 1.5 mL / g SS to 32.67 ± 1.5 mL / g SS. At the same time, the content of extracellular polymeric substances (EPS) increased significantly, with EPS increasing from 90.6 ± 2.8 mg·g·VSS -1 to 153 ± 2.8 mg·g·VSS -1 .
[0055] Comparative Example 1
[0056] This comparative example was carried out at the same time as Example 1 and had basically the same experimental setup, with the main difference being that the gradient addition of Fe3O4 was not carried out during the anammox bacteria tolerance to high-concentration Fe3O4 acclimation stage. The details are as follows:
[0057] (1) During the reactor start-up stage, the anammox bacteria inoculation in the anammox reactor and the stable operation parameters and operating conditions were consistent with those of Example 1, i.e., the operating conditions were maintained until the 14th day, and the total nitrogen removal rate of the reactor was stably maintained at 80.78%, with a maintenance time of 3 days, completing the stable operation of the reactor.
[0058] (2) The anammox granular sludge tolerance to high-concentration Fe3O4 acclimation stage was entered, and the specific steps were as follows:
[0059] Fe3O4 material was directly added to the reactor at a dosage concentration of 1800 mg / L, i.e., 1800 mg of nano-sized Fe3O4 (20-50 nm) material was added per liter of reactor volume, and the Fe3O4 was treated for 10 min at a power of 0.5 W / mL before addition, while the pH in the influent was reduced from 7.5 to 6.8, and the reactor was then operated under this pH condition. The reactor collapsed on the 16th day, with the total nitrogen removal rate continuously decreasing to 65%, and the gene prediction results of the 16S rRNA high-throughput sequencing technology showed that the heme oxygenase gene decreased by 74% compared to the sludge in the start-up success stage. The above proves that an anammox granular sludge with tolerance to high-concentration Fe3O4 cannot be obtained under conditions lacking a gradient Fe3O4 measure.
[0060] Comparative Example 2
[0061] This comparative example was carried out at the same time as Example 1 and had basically the same experimental setup, with the main difference being that the final concentration of Fe3O4 during the anammox bacteria tolerance to high-concentration Fe3O4 stage was greater than 2000 mg / L. The details are as follows:
[0062] (1) In the reactor start-up phase, the anaerobic ammonia oxidation bacteria inoculation in the Anammox reactor and the stable operation parameters and operation conditions are consistent with those of Example 1, that is, the total nitrogen removal rate of the reactor is stably maintained at 80.5% under the operation conditions until the 14th day, the maintenance time is 3 days, and the stable operation of the reactor is completed.
[0063] (2) Enter the high-concentration Fe3O4-tolerant anaerobic ammonia oxidation granular sludge domestication phase, and the specific steps are as follows:
[0064] Fe3O4 material is added to the reactor at a dosage concentration of 200 mg / L, that is, 200 mg of nano Fe3O4 (20-50 nm) material is added per liter of reactor volume, and before adding, the Fe3O4 is treated for 10 min under a power of 0.5 W / mL, and at the same time, the pH of the influent is reduced from 7.5 to 6.8, and then the reactor is operated under this pH condition. The ammonia nitrogen and nitrite in the reactor effluent temporarily accumulate, and the total nitrogen removal rate increases on the 23rd day and gradually recovers to 80% for 6 days, and the maintenance time is 3 days; continue to add 600 mg / L of Fe3O4 to the reactor, and after 3 days of fluctuation, the total nitrogen removal rate recovers to 80%, and the maintenance time is 3 days; again, 1800 mg / L of Fe3O4 is added to the reactor, and the total nitrogen removal rate is severely inhibited, decreasing from 80% to 60%, and the 16S rRNA high-throughput sequencing technology shows that the hemin oxygenase gene decreases by 40% compared with the sludge in the start-up success phase. The above proves that the final concentration of Fe3O4 greater than 2000 mg / L cannot obtain anaerobic ammonia oxidation granular sludge with high Fe3O4 tolerance.
[0065] Comparative Example 3
[0066] This comparative example is carried out at the same time as Example 1 and has basically the same experimental setup, and the main difference is that the final concentration of Fe3O4 in the high-concentration Fe3O4-tolerant anaerobic ammonia oxidation bacteria stage is less than 1800 mg / L. The specific steps are as follows:
[0067] (1) In the reactor start-up phase, the anaerobic ammonia oxidation bacteria inoculation in the Anammox reactor and the stable operation parameters and operation conditions are consistent with those of Example 1, that is, the total nitrogen removal rate of the reactor is stably maintained at 80.5% under the operation conditions until the 14th day, the maintenance time is 3 days, and the stable operation of the reactor is completed.
[0068] (2) Enter the high-concentration Fe3O4-tolerant anaerobic ammonia oxidation granular sludge domestication phase, and the specific steps are as follows:
[0069] Fe3O4 material was added to the reactor at a dosage concentration of 200 mg / L, i.e. 200 mg of nano-sized Fe3O4 (20-50 nm) material was added per liter of reactor volume, and the Fe3O4 was treated for 10 min at a power of 0.5 W / mL before being added, while the pH of the influent was reduced from 7.5 to 6.8, and then the reactor was operated under this pH condition. The ammonia nitrogen and nitrite temporarily accumulated in the reactor effluent, and the total nitrogen removal rate increased on day 23 and gradually recovered to 80% over 6 days, and the maintenance time was 3 days; 400 mg / L of Fe3O4 was continuously added to the reactor, and the total nitrogen removal rate recovered to 80% after 3 days of fluctuation, and the maintenance time was 3 days; 800 mg / L of Fe3O4 was again added to the reactor, and the total nitrogen removal rate recovered to 80% after 6 days of fluctuation, and the 16S rRNA high-throughput sequencing technology showed that the gene prediction result showed that the heme oxygenase gene did not significantly improve compared with the sludge in the start-up success stage. The above proves that the final concentration of Fe3O4 is less than 1800 mg / L, which cannot stimulate the secretion of heme oxygenase, and therefore the anaerobic ammonia oxidation granular sludge with high tolerance to Fe3O4 cannot be successfully obtained.
[0070] Comparative Example 4
[0071] This comparative example was carried out at the same time as Example 1 and the experimental setup was basically the same, the main difference being that the pH value was kept at 7.5 during the stage of anaerobic ammonia oxidation bacteria tolerating high concentration of Fe3O4. The details are as follows:
[0072] (1) During the start-up stage of the reactor, the inoculation of anaerobic ammonia oxidation bacteria in the Anammox reactor and the stable operation parameters and operating conditions were consistent with those of Example 1, i.e. the total nitrogen removal rate of the reactor was stable at 80% for 3 days, and the stable operation of the reactor was completed.
[0073] (2) Enter the stage of domesticating anaerobic ammonia oxidation granular sludge to tolerate high concentration of Fe3O4, the specific steps are as follows:
[0074] Fe3O4 material was added to the reactor at a dosage concentration of 200 mg / L, i.e. 200 mg of nano-sized Fe3O4 (20-50 nm) material was added per liter of reactor volume, and the Fe3O4 was treated at a power of 0.5 W / mL for 10 min before being added. The ammonia nitrogen and nitrite accumulation in the effluent of the anaerobic ammonia oxidation reactor, and the total nitrogen removal rate, increased on day 29 and gradually recovered to 80% over 15 days, and was maintained for 3 days; the reactor was continuously dosed with 600 mg / L of Fe3O4, and the total nitrogen removal rate did not recover to 80% after fluctuating for 25 days, and was 75%; the reactor was again dosed with 1200 mg / L of Fe3O4, and the reactor performance began to collapse on day 27, the total nitrogen removal rate continued to decrease to 62%, and the gene prediction results of the 16S rRNA high-throughput sequencing technology showed that the heme oxygenase gene decreased by 70% compared to the sludge at the start of the successful stage. The above proves that the Anammox bacteria failed to be domesticated at high concentrations of Fe3O4 in the absence of a down-regulated pH of 6.8-6.9, and they could not be stably operated in a high-concentration Fe3O4 environment.
[0075] Comparative Example 5
[0076] This comparative example was carried out at the same time as the experiment of Example 1 and the experimental setup was basically the same, the main difference being that the pH value during the stage of domesticating the anaerobic ammonia oxidation bacteria to tolerate high concentrations of Fe3O4 was lower than 6.8. Details are as follows:
[0077] (1) During the start-up stage of the reactor, the inoculation of anaerobic ammonia oxidation bacteria in the Anammox reactor and the stable operation parameters and operating conditions were consistent with those of Example 1, i.e. the operating conditions were maintained until day 14, the total nitrogen removal rate of the reactor was stably maintained at 82%, the maintenance time was 3 days, and the stable operation of the reactor was completed.
[0078] (2) The specific steps for entering the stage of domesticating the anaerobic ammonia oxidation granular sludge to tolerate high concentrations of Fe3O4 were as follows:
[0079] Fe3O4 material was added to the reactor at a dosage concentration of 200 mg / L, i.e. 200 mg of nano-sized Fe3O4 (20-50 nm) material was added per liter of reactor volume, and the Fe3O4 was treated for 10 min at a power of 0.5 W / mL before being added, while the pH of the influent was reduced from 7.5 to 6.5, and then the reactor was operated under this pH condition. The ammonia nitrogen and nitrite accumulation in the effluent of the anaerobic ammonia oxidation reactor, and the total nitrogen removal rate increased on the 30th day but eventually did not recover to 80%, and was 73%; 600 mg / L of Fe3O4 was continuously added to the reactor, and the total nitrogen removal rate stabilized at 69% after fluctuation; 1200 mg / L of Fe3O4 was again added to the reactor, and the performance of the reactor began to collapse on the 47th day, and the total nitrogen removal rate continued to decrease to 59%, and the gene prediction results of the 16S rRNA high-throughput sequencing technology showed that the heme oxygenase gene decreased by 90% compared with the sludge in the start-up success stage. The above proves that the Anammox bacteria cannot be domesticated to tolerate high concentrations of Fe3O4 under the condition that the pH is lower than 6.8, and it cannot be stably operated in a high-concentration Fe3O4 environment.
[0080] Finally, based on the gene prediction function, the present application provides a domestication method of Anammox granular sludge that tolerates high concentrations of Fe3O4, to support the Anammox bacteria group to achieve stable and efficient nitrogen removal in a high-concentration nano-Fe3O4 environment.
[0081] The study shows that the stress behavior of Anammox bacteria under the stress of NPs is reversible, and the denitrification capacity of Anammox bacteria can recover to the initial level within 70 days under the stress of CuNPs. NPs can produce ROS-induced oxidative stress due to their unique physical and chemical properties (high surface area / volume and surface charge), which is the fundamental reason for their toxicity to bacteria. Therefore, the key to improving the tolerance of bacteria is to remove ROS to alleviate oxidative stress. It is well known that the functional protease of microorganisms regulates its metabolic function. For this, microorganisms have a strong enzyme antioxidant defense system, and common antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), in addition to which thioredoxin reductase (TrxR) and heme oxygenase (HO) also belong to antioxidant enzymes. Among them, heme oxygenase participates in the degradation metabolism of heme and is the key rate-limiting enzyme of the metabolism, and the products are free iron, biliverdin and carbon monoxide, and biliverdin is further reduced to bilirubin by biliverdin reductase (BvdR). HO, biliverdin and bilirubin are effective antioxidants; free iron can be detoxified by extracellular efflux or chelation to ferritin, which is an intracellular iron storage molecule with potential cell protection function. Compared with other microorganisms, Anammox bacteria are rich in a large amount of heme, and some functional enzymes with heme are very important coenzymes such as hydroxylamine oxidoreductase (HAO), which participates in the anaerobic ammonia oxidation reaction and performs the denitrification function. Iron is essential for Anammox bacteria and can directly participate in major metabolic processes such as electron transfer and tricarboxylic acid cycle. Appropriate amount of iron can enhance the activity of Anammox bacteria. Therefore, heme oxygenase is the preferred ROS removal in the enzyme antioxidant system to improve the environmental adaptability of bacteria in Anammox bacteria. In addition, the heme oxygenase gene has been identified in anaerobic bacteria (such as Escherichia coli). Based on this, the increase of heme oxygenase gene in Anammox bacteria can make it a powerful "organ" for intracellular ROS removal, so that Anammox bacteria can tolerate high concentration of nano Fe3O4. The present application increases the abundance of heme oxygenase gene in Anammox bacteria by adopting gradient domestication + control of final concentration + pH down-regulation mode, effectively alleviates the sensitivity of Anammox bacteria to high concentration of nano Fe3O4 and maintains high-efficiency denitrification performance in high concentration of nano Fe3O4 environment.
[0082] The pH down-regulation plays a crucial role in the heme degradation metabolism mediated by heme oxygenase. The catalysis of heme oxygenase, i.e. the process of degrading heme, requires the participation of redox partner protein (such as iron-sulfur cluster protein), because the heme oxygenase lacking the coenzyme-binding domain cannot directly use the electrons and reducing power in NADPH. Iron is an important coenzyme factor of iron-sulfur cluster protein, when it is loaded onto the subunit, the iron-sulfur cluster protein can transfer the electrons from NADPH to the heme molecule through conformational rearrangement. The present application realizes the loading of iron in the slightly acidic environment, because Fe3O4 releases Fe(II) and Fe(III) under acidic conditions, wherein Fe(II) can be effectively absorbed and utilized by Anammox bacteria, thereby realizing the loading of iron. Therefore, the present application controls the pH to be slightly acidic (6.8-6.9), utilizes the characteristics of Fe3O4 itself releasing Fe(II) and Fe(III), stimulates the iron-sulfur cluster protein as the partner protein of heme oxygenase, and realizes the function of heme oxygenase in degrading heme.
[0083] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.
Claims
1. A method for culturing anaerobic ammonia oxidation granular sludge resistant to high concentration of nano-Fe304, characterized in that: The steps are as follows: (1) using an upflow anaerobic sludge bed reactor, inoculating Anammox granular sludge; (2) using wastewater containing ammonia nitrogen, nitrite nitrogen, inorganic salts and trace elements as influent, enriching and culturing under anaerobic conditions until the denitrification effect of the reactor is in a stable state; (3) adding Fe3O4 to the reactor in a gradient, controlling the final concentration of Fe3O4 in the reactor to be 1800-2000 mg / L, and simultaneously adjusting the pH value of the influent of the reactor to be 6.8-6.9, to direct stimulation of the heme oxygenase gene of the anaerobic ammonia oxidation bacteria group in step (2), and to culture anaerobic ammonia oxidation granular sludge resistant to high-concentration nano-Fe3O4.
2. The culturing method according to claim 1, characterized by: In step (3), the initial concentration of Fe3O4 added is 200 mg / L, and each concentration is run until the total nitrogen removal rate of the reactor is more than 80%, and the maintenance time is ≥3-5 days; continue to add Fe3O4, and the addition concentration is 2-3 times the amount of the last addition.
3. The culturing method according to claim 1, characterized by: The Fe3O4 is in a granular form, with a diameter of 20-50 nm.
4. The culturing method according to claim 3, characterized by: The Fe3O4 is treated by an ultrasonic device at a power of 0.3-0.5 W / mL for 5-10 min before addition.
5. The culturing method of claim 1, wherein: The average diameter of the Anammox granular sludge is 367.5±150.5 μm; and the dominant genus of the Anammox bacteria is Candidatus Brocadia.
6. The culturing method of claim 1, wherein: The suspended solid concentration of the inoculation mixed solution of the Anammox granular sludge is 3500-4000 mg / L, and the inoculation volume accounts for 30-35% of the total volume of the upflow anaerobic sludge bed reactor.
7. The culturing method of claim 1, wherein: In step (3), the condition for judging whether the anaerobic ammonia oxidation granules are resistant to high-concentration nano-Fe3O4 is that the total nitrogen removal rate is maintained at more than 80%, and the abundance of the heme oxygenase gene in the reactor is more than 2 times the abundance of the heme oxygenase gene of the inoculated anaerobic ammonia oxidation granular sludge.
8. The culturing method of claim 1, wherein: The wastewater comprises NH4 + -N, NO2 - -N, KHCO3, KH2PO4, EDTA, trace element solution I and trace element solution II.
9. The culturing method of claim 8, wherein: The NH4 + The mass concentration ratio of -N and NO2-N is 1:
1.
10. The culturing method of claim 8, wherein: The trace element solution I includes NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O; and / or, the trace element solution II includes CuSO4·5H2O, ZnSO4·7H2O, CoCl2·6H2O, MnCl2·4H2O, NaMoO4·2H2O, NiCl2·6H2O, NaSeO4, H3BO3.