A method for preserving anaerobic ammonium oxidation sludge

Abstract

The present invention discloses a method for preserving anaerobic ammonium oxidation (ANAMMOX) sludge, comprising: washing the ANAMMOX sludge multiple times with a preservation solution, placing the ANAMMOX sludge in a preservation container at a preservation temperature of 4°C, and creating an anoxic environment in the preservation container; periodically replacing the preservation solution with artificial simulated wastewater during the preservation period, raising the preservation temperature to a first temperature to enhance the performance of the ANAMMOX sludge; and re-using the preservation solution to replace the artificial simulated wastewater after the reaction is completed, and lowering the preservation temperature to 4°C. By setting parameters such as the preservation temperature, the composition of the preservation solution, and the metabolic enhancement cycle, the present invention preserves ANAMMOX sludge in an economical, convenient, long-term, and effective manner, laying an important foundation for the construction of an ANAMMOX sludge bacterial strain resource library. It also provides bacterial source security for the industrial application of the ANAMMOX process.

Description

Technical Field

[0001] The present invention belongs to the field of environmental protection technology, relates to the preservation of anaerobic ammonium oxidation sludge, and in particular to an economical, convenient, long-term and effective anaerobic ammonium oxidation sludge preservation method. Background Art

[0002] Anaerobic ammonium oxidation (ANAMMOX) is a biological reaction under anaerobic conditions that uses ammonia as an electron donor and nitrite as an electron acceptor to produce nitrogen gas. This reaction can simultaneously remove both nitrogen pollutants. Compared to traditional biological denitrification processes, ANAMMOX offers advantages such as high nitrogen removal efficiency, the absence of an external organic carbon source, and low operating costs, offering promising prospects for industrial application.

[0003] Anaerobic ammonium oxidizing (ANAMMOX) bacteria have a long doubling time, slow growth, and difficulty cultivating pure cultures, making their application in actual wastewater treatment difficult. Enriching and effectively preserving ANAMMOX strains is an effective measure to address this lack of application. Furthermore, seasonal fluctuations in the flow of certain industrial wastewater, rural sewage, and influent from tourist city sewage treatment plants can cause ANAMMOX processes to suspend operation. Preserving ANAMMOX sludge during these operations is an unavoidable technical issue. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides an anaerobic ammonium oxidation sludge preservation method for economically, conveniently, long-term and effectively preserving anaerobic ammonium oxidation bacteria.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for preserving anaerobic ammonium oxidation sludge, which comprises the following steps:

[0007] 1) Wash the anaerobic ammonium oxidation sludge several times with a preservation solution and place it in a preservation container at a storage temperature of 4°C in an anoxic environment;

[0008] 2) During the storage period, the storage solution is periodically replaced with artificial simulated wastewater, and the storage temperature is increased to a first temperature to enhance the performance of the anaerobic ammonium oxidation sludge. After the reaction is completed, the storage solution is used again to replace the artificial simulated wastewater, and the storage temperature is lowered to 4°C;

[0009] The preservation solution is a mixed solution of inorganic salts and organic salts, with a pH of 7.27-7.32.

[0010] The anaerobic ammonium oxidation sludge is non-floc anaerobic ammonium oxidation sludge.

[0011] As a preferred embodiment of the present invention, the inorganic salts are nitrate and ferrous salt, the nitrate concentration is 7-9 mM, and the ferrous salt concentration is 5.2 mM.

[0012] In the present invention, nitrate and ferrous salts are the substrates for the ferrous dissimilatory nitrate reduction to ammonia coupled anaerobic ammonium oxidation (DNRA-Anammox) reaction of anaerobic ammonium oxidizing bacteria. Since nitrite is the main cause of substrate inhibition of anaerobic ammonium oxidizing bacteria, and nitrite is in a production-consumption mode (generated and consumed at the same time) during the reaction, compared with the traditional anaerobic ammonium oxidation reaction with ammonia nitrogen and nitrite as the substrate, the ferrous DNRA-Anammox reaction using nitrate and ferrous salt as the substrate is not prone to substrate inhibition and is particularly suitable for low-temperature storage of anaerobic ammonium oxidizing bacteria. At the same time, ferrous salts also help maintain the morphology of anaerobic ammonium oxidation sludge and avoid the disintegration of biofilm sludge or granular sludge.

[0013] The substrate concentration in the preservation solution is set at 5.2 mM ferrous sulfate and 7-9 mM sodium nitrate, with a relatively excessive amount of sodium nitrate. This is because nitrate is a less susceptible substrate to inhibition than ferrous sulfate. Once the ferrous sulfate in the habitat is depleted, anaerobic ammonium-oxidizing bacteria can still utilize their extracellular polymeric substances (EPS) to initiate the DNRA-Anammox reaction, using organic matter as an electron donor, further enhancing their viability and preservation effectiveness.

[0014] As a preferred embodiment of the present invention, the organic salts are betaine and EDTA-2Na, the concentration of betaine is 1-3 mM, and the concentration of EDTA-2Na is 7.8 mM.

[0015] In the present invention, betaine is an essential stress product. Its intracellular accumulation as a compatible solute can protect anaerobic ammonium oxidizing bacteria in adverse habitats from starvation and low-temperature stress. At the same time, betaine can also act as a stress protectant to enhance the growth rate and enzyme activity of anaerobic ammonium oxidizing bacteria facing environmental stress. Therefore, anaerobic ammonium oxidizing bacteria can survive under environmental stress and recover quickly when environmental conditions improve. Experiments have shown that when anaerobic ammonium oxidizing bacteria are in low-temperature, low-substrate environmental conditions, the addition of 1-3mM betaine can quickly restore their initial metabolic rate when habitat conditions improve.

[0016] In the present invention, EDTA-2Na is a chelating agent with a high affinity for alkaline earth and heavy metal ions and the ability to form stable complexes. Due to the instability of ferrous iron and its potential for a series of complex physicochemical reactions, the retention rate of ferrous iron after dissolution fluctuates between 33.43±0.66% and 65.61±0.24%. This is clearly not conducive to efficient nitrate removal, especially when the influent ferrous iron concentration is low, such as below 5.2 mM. Therefore, EDTA-2Na can be used to improve the solubility and bioavailability of ferrous iron, thereby enhancing the ferrous DNRA-Anammox reaction.

[0017] As a preferred embodiment of the present invention, the non-floc anaerobic ammonium oxidation sludge is biofilm sludge or granular sludge.

[0018] As a preferred embodiment of the present invention, the first temperature is 35-37°C.

[0019] As a preferred embodiment of the present invention, the concentration of ammonia nitrogen in the artificial simulated wastewater is 5 mM, the concentration of nitrite is 5-6.6 mM, and the pH is 6.5-8.5.

[0020] As a preferred embodiment of the present invention, the contents of various mineral elements in the artificial simulated wastewater are: MgSO4·7H2O 300 mg / L, NaHCO3 1250 mg / L, KH2PO4 10 mg / L, CaCl2·2H2O 5.6 mg / L, and trace elements I and II 1.25 mL / L respectively, wherein the trace element I is composed of: EDTA 5 g / L and FeSO4 5 g / L; the trace element II is composed of EDTA 15 g / L, H3BO4 0.014 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, ZnSO4·7H2O 0.43 g / L, CoCl2·6H2O 0.24 g / L, NiCl2·6H2O 0.19 g / L, NaMoO4·2H2O 0.22g / L and NaSeO4·10H2O0.21g / L.

[0021] As a preferred embodiment of the present invention, in step 2), the preservation solution is periodically replaced every 14-21 days.

[0022] As a preferred embodiment of the present invention, in step 1), the anoxic environment in the storage container is achieved by flushing nitrogen or argon into the storage container for 2-5 minutes.

[0023] As a preferred embodiment of the present invention, when the preservation solution is replaced with artificial simulated wastewater, the indicator for the end of the anaerobic ammonium oxidation reaction is that the concentration of ammonia nitrogen or nitrite is lower than 10-20 mg-N / L.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The present invention sets parameters such as storage temperature, storage solution composition and metabolic enhancement cycle to economically, conveniently, long-term and effectively preserve anaerobic ammonium oxidation sludge, laying an important foundation for the construction of an anaerobic ammonium oxidation sludge strain resource library, and also provides a bacterial source guarantee for the industrial application of the anaerobic ammonium oxidation process.

[0026] 2) The preservation method of the present invention can preserve anaerobic ammonium oxidation sludge for a long time for 126 days.

[0027] 3) The preservation method of the present invention is economical, convenient, long-term, and effective. When stored at 4°C, the anaerobic ammonia oxidation specific activity (SAA) retention rate of the metabolic enhancement group increased significantly from 47.8% of the long-term starvation group to 93.4%, an increase of nearly 2 times. Obviously, the preservation method provided by the present invention is long-term and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the optimal EDTA-2Na / Fe(II) ratio for the DNRA-Anammox reaction of the present invention.

[0029] Figure 2 It is the optimal pH value for DNRA-Anammox reaction. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] In the present invention, all the raw materials, reagents or equipment used can be purchased from the market.

[0032] The present invention sets parameters such as preservation temperature, preservation solution composition and metabolic enhancement cycle to economically, conveniently, long-term and effectively preserve anaerobic ammonium oxidation sludge, laying an important foundation for the construction of an anaerobic ammonium oxidation sludge strain resource library, and also provides bacterial source guarantee for the industrial application of the anaerobic ammonium oxidation process.

[0033] Example 1

[0034] This embodiment provides a method for preserving anaerobic ammonium oxidation sludge, comprising the following steps:

[0035] 1) Anaerobic ammonium oxidation sludge uses biofilm sludge or granular sludge and is washed three times with a preservation solution before storage. The preservation solution is a mixed solution of inorganic and organic salts, wherein the inorganic salts are nitrates and ferrous salts, and the organic salts are betaine and EDTA-2Na. In this embodiment, sodium nitrate is used as the nitrate, and ferrous sulfate is used as the ferrous salt.

[0036] 2) During the storage period, the storage solution was periodically replaced with artificial simulated wastewater, and the storage temperature was increased to 35-37°C to enhance the performance of the anaerobic ammonia oxidation sludge. After the reaction was completed, the storage solution was used again to replace the artificial simulated wastewater, and the storage temperature was lowered to 4°C.

[0037] The artificial simulated wastewater used has an ammonia nitrogen concentration of 5 mM, a nitrite concentration of 5-6.6 mM, and a pH of 6.5-8.5. The contents of mineral elements in the artificial simulated wastewater used are: MgSO4·7H2O 300 mg / L, NaHCO3 1250 mg / L, KH2PO4 10 mg / L, CaCl2·2H2O 5.6 mg / L, and trace elements I and II 1.25 mL / L each. Among them, the composition of trace elements I is: EDTA 5 g / L, FeSO4 5 g / L; the composition of trace elements II is EDTA 15 g / L, H3BO4 0.014 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, ZnSO4·7H2O 0.43 g / L, CoCl2·6H2O 0.24 g / L, NiCl2·6H2O 0.19 g / L, NaMoO4·2H2O 0.22g / L, NaSeO4·10H2O 0.21g / L.

[0038] like Figure 1 As shown in the figure, the addition of EDTA-2Na has a significant effect on the ferrous DNRA-Anammox process of anaerobic ammonium-oxidizing bacteria. The enhancement effect is best when the EDTA-2Na / Fe(II) is 1.5, and a better nitrate removal rate is achieved. At this time, the ferrous concentration is 5.2mM and the EDTA-2Na concentration is 7.8mM.

[0039] Bell-shaped functions (A) and (B) are often used to simulate the dependence of anaerobic ammonium-oxidizing bacteria on pH, and thus the optimal pH (pH) of anaerobic ammonium-oxidizing metabolism under certain conditions can be obtained. opt ).like Figure 2 As shown, Figure 2 (a) and Figure 2 (b) in the figure are the bell-shaped function (A) curve and the bell-shaped function (B) curve of anaerobic ammonium-oxidizing bacteria using inorganic reducing agents, Figure 2 (c) and Figure 2 Figures (d) and (e) show the bell-shaped function (A) and bell-shaped function (B) curves of anaerobic ammonium oxidizing bacteria utilizing organic reducing agents, respectively. The pH-corrected kinetic fitting revealed that the optimal pH range for the DNRA-Anammox reaction of anaerobic ammonium oxidizing bacteria is 7.27-7.32.

[0040] Bell-shaped function (A):

[0041]

[0042] Bell-shaped function (B):

[0043]

[0044] The storage container is an oxygen-deficient environment, which is achieved by flushing nitrogen or argon into the storage container for 2-5 minutes.

[0045] Comparative Example 1 is different from Example 1 only in that step 2) is not adopted and the product is directly stored at 4°C.

[0046] Comparative Example 2 is different from Example 1 only in that the storage temperature is 20°C.

[0047] The only difference between Comparative Example 3 and Comparative Example 2 is that step 2) is not adopted and the product is directly stored at 20°C.

[0048] After 126 days of storage, the SAA retention rate of the anaerobic ammonium oxidation sludge was tested, and the results are shown in Table 1.

[0049] Table 1 Effects of storage temperature and metabolic enhancement cycle on storage effect

[0050] Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Storage temperature 4℃ 4℃ 20℃ 20℃ Metabolic Enhancement Cycle 14-21 days No reinforcement 14-21 days No reinforcement SAA retention rate after 126 days 93.4％ 47.8％ 47.5％ 36.2％

[0051] As shown in Table 1, 1) compared with storage at a moderate temperature of 20°C, storage at a low temperature of 4°C can better maintain the biological activity of ANAMMOX sludge. When ANAMMOX sludge was stored for 126 days without special treatment, the SAA retention rate at a low temperature of 4°C (Comparative Example 1) was 47.8%, while the SAA retention rate at a moderate temperature of 20°C (Comparative Example 3) was only 36.2%. 2) ANAMMOX biofilm sludge or granular sludge will gradually disintegrate into flocculent sludge after long-term starvation. Therefore, during the long-term storage of ANAMMOX sludge, ANAMMOX metabolism should be periodically enhanced. The specific anaerobic ammonia oxidation metabolism enhancement method is as follows: the anaerobic ammonia oxidation sludge is preserved for a long time for 126 days, and the preservation solution is periodically replaced with artificial simulated wastewater during the preservation period. The period of periodic replacement of the preservation solution is 14-21 days, and the preservation temperature is raised to 35-37 ° C. When the ammonia nitrogen or nitrite concentration is lower than 10-20 mg-N / L, the metabolic enhancement is deemed to be completed, the preservation solution is used again to replace the artificial simulated wastewater, and the preservation temperature is lowered to 4 ° C. Compared with long-term preservation without strengthening anaerobic ammonia oxidation metabolism, strengthening anaerobic ammonia oxidation metabolism with a period of 14-21 days can better maintain the biological activity of anaerobic ammonia oxidation sludge: when preserved at a low temperature of 4 ° C, the SAA retention rate of the metabolic enhancement group (Example 1) is significantly increased from 47.8% of the long-term starvation group (Comparative Example 1) to 93.4%, an increase of nearly 2 times. Obviously, the preservation method provided by the present invention is long-term and effective.

[0052] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preserving anaerobic ammonium oxidation sludge, characterized in that: The preservation method comprises the following steps: 1) Washing the anaerobic ammonium oxidation sludge multiple times with a preservation solution and placing it in a preservation container at a storage temperature of 4°C in an anoxic environment; 2) During the storage period, the storage solution is periodically replaced with artificial simulated wastewater, and the storage temperature is increased to 35-37°C to enhance the performance of anaerobic ammonia oxidation sludge. After the reaction is completed, the storage solution is used again to replace the artificial simulated wastewater, and the storage temperature is lowered to 4°C. The periodic replacement of the storage solution is 14-21 days; The preservation solution is a mixed solution of inorganic salts and organic salts, with a pH of 7.27-7.32, the inorganic salts are nitrates and ferrous salts, the nitrate concentration is 7-9 mM, the ferrous salt concentration is 5.2 mM, the organic salts are betaine and EDTA-2Na, the betaine concentration is 1-3 mM, and the EDTA-2Na concentration is 7.8 mM; The anaerobic ammonium oxidation sludge is non-floc anaerobic ammonium oxidation sludge.

2. The method for preserving anaerobic ammonium oxidation sludge according to claim 1, wherein: The non-floc anaerobic ammonium oxidation sludge is anaerobic ammonium oxidation biofilm sludge or anaerobic ammonium oxidation granular sludge.

3. The method for preserving anaerobic ammonium oxidation sludge according to claim 1, wherein: The artificial simulated wastewater has an ammonia nitrogen concentration of 5 mM, a nitrite concentration of 5-6.6 mM, and a pH of 6.5-8.

5.

4. The method for preserving anaerobic ammonium oxidation sludge according to claim 1 or 3, wherein: The contents of various elements in artificial simulated wastewater are: MgSO4·7H2O 300 mg / L, NaHCO3 1250 mg / L, KH2PO4 10 mg / L, CaCl2·2H2O 5.6 mg / L, and trace elements I and II 1.25 mL / L respectively. Among them, the composition of trace elements I is: EDTA 5 g / L and FeSO4 5 g / L; the composition of trace elements II is EDTA 15 g / L, H3BO4 0.014 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, ZnSO4·7H2O 0.43 g / L, CoCl2·6H2O 0.24 g / L, NiCl2·6H2O 0.19g / L, NaMoO4·2H2O0.22g / L and NaSeO4·10H2O 0.21g / L.

5. The method for preserving anaerobic ammonium oxidation sludge according to claim 1, wherein: In step 1), the anoxic environment in the storage container is achieved by flushing nitrogen or argon into the storage container for 2-5 minutes.

6. The method for preserving anaerobic ammonium oxidation sludge according to claim 1, wherein: When the preservation solution was replaced with artificial simulated wastewater, the indicator for the end of the anaerobic ammonium oxidation reaction was that the ammonia nitrogen or nitrite concentration was lower than 20 mgN / L.

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