Domestication and enrichment method of anaerobic ammonia oxidation coupled arsenic reduction functional flora
Through a multi-stage culture process, the anaerobic ammonia oxidation coupled arsenic reduction functional flora was successfully domesticated and enriched, which solved the problem of lack of functional microorganisms in this field in the prior art, achieved the coupling of ammonia oxidation and arsenic reduction under anaerobic conditions, and significantly improved the arsenic reduction rate.
Patent Information
- Application Number
- CN202510150884.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-11
- Publication Date
- 2025-04-25
AI Technical Summary
There are no reports on functional microorganisms in the prior art for coupling arsenic reduction process, which has led to the infancy of research in this field and it is difficult to effectively deal with the problem of environmental nitrogen and arsenic pollution.
Through a multi-stage culture process, including light-shading culture and enrichment culture, the anaerobic ammonia oxidation coupled arsenic reduction functional flora are successfully acclimated and enriched. The specific steps include: the first stage is to use water containing ammonia nitrogen and nitrosity nitrogen for light-shading culture, the second stage is to add arsenate for domestication and culture, and the third stage is to further adjust the arsenate concentration for functional flora to enrich the functional flora.
The coupling of ammonia oxidation and arsenic reduction process under anaerobic conditions was achieved, and the functional flora of anaerobic ammonia oxidation coupled arsenic reduction was successfully identified and enriched. In particular, the abundance of Ca.Kuenenia was significantly improved, and it had complete ammonia oxidation and arsenic respiratory reduction genes, with a high arsenic reduction rate.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of environmental microorganisms, and in particular relates to a method for domesticating and enriching an anaerobic ammonium oxidation coupled arsenic reduction functional bacterial community. Background Art
[0002] Nitrogen in the natural environment is a key element in maintaining ecological balance and nutrient cycles, and its circulation process has an important impact on the ecosystem. However, due to human activities, especially the excessive use of nitrogen fertilizers and long-term mining and metal refining activities, nitrogen and arsenic coexist and form pollution in certain environments. This nitrogen-arsenic coexistence pollution problem is particularly prominent in ecosystems such as wetland sediments, paddy field soils, agricultural drainage, tailings liquid and aquifers, posing a serious threat to water quality and ecosystems.
[0003] Microorganisms, as an important part of the ecosystem, participate in the physical, chemical, biochemical and geochemical processes in the environment, thereby affecting the migration and transformation of various metal and non-metal elements. As one of the core processes of the element cycle in the ecosystem, the nitrogen cycle is closely related to the arsenic form transformation process. In recent years, a large number of studies have shown that the microbial transformation of arsenic is closely linked to the cycle of environmental nitrogen. For example, nitrification and denitrification are conducive to the adsorption and fixation of arsenic; denitrification can also couple the arsenic demethylation process, thereby increasing arsenic toxicity; iron ammonia oxidation can affect the reduction and release of arsenic, etc.
[0004] Anaerobic ammonium oxidation (anammox) is a new type of carbon-neutral biological denitrification technology that has attracted much attention in recent years and has been widely used in the treatment of various types of nitrogen-containing wastewater. A large number of studies have shown that anaerobic ammonium oxidation can be coupled with the reduction of other electron acceptors such as Fe(Ⅲ), Mn(IV) and sulfate to drive the biogeochemical cycles of multiple elements. Functional microorganisms involved in these processes are enriched in a variety of habitats, including paddy soils, wetlands and wastewater treatment systems. The latest research has found that the anaerobic ammonium oxidation process can be coupled with the As(Ⅴ) reduction process. Similar to iron-ammonia oxidation, related microorganisms use ammonia as an electron donor to generate NO2 under anaerobic conditions. - , N2, and simultaneously reduce As(V) to As(III).
[0005] However, research on the anaerobic ammonium oxidation coupled arsenic reduction process is still in its infancy, and there are no reports on functional microorganisms in this process in the existing technology. Therefore, exploring the functional bacterial flora of anaerobic ammonium oxidation coupled arsenic reduction is of great significance for understanding this new nitrogen-arsenic coupling transformation pathway. This will not only help promote the development of carbon-neutral biotechnology based on anaerobic ammonium oxidation to address environmental nitrogen and arsenic pollution problems, but also provide valuable research ideas for nutritional relationships that may link nitrogen metabolism to a series of terminal electron acceptors. Summary of the invention
[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for domesticating and enriching anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria.
[0007] The technical solution of the present invention is achieved in this way:
[0008] A method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria, specifically comprising the following steps:
[0009] (1) Phase I: The mature anaerobic ammonium oxidation granular sludge in stable operation is inoculated into the reactor and cultured under light-shading conditions using water containing ammonia nitrogen, nitrite nitrogen, inorganic salts and trace elements as the influent matrix; the concentrations of ammonia nitrogen and nitrite nitrogen are both 70-90 mg / L;
[0010] (2) The second stage: After the reactor effluent is stable, water containing ammonia nitrogen, arsenate, inorganic salts and trace elements is used as the inlet matrix for light-shading and acclimatization culture; wherein the concentration of ammonia nitrogen is 30-40 mg / L, and the concentration of arsenate is 1 mg / L;
[0011] (3) The third stage: After the reactor effluent returns to stability, water containing ammonia nitrogen, arsenate, inorganic salts and trace elements is used as the inlet matrix for light-shading culture, thereby enriching the functional bacteria community for anaerobic ammonia oxidation coupled with arsenic reduction; among which, the concentration of ammonia nitrogen is 30-40 mg / L, and the concentration of arsenate is 2 mg / L.
[0012] Furthermore, the reactor is a sequencing batch reactor.
[0013] Furthermore, in step (1), step (2) and step (3), the inorganic salts in the influent matrix include: KHCO3 2.5 g / L, CaCl2·2H2O 0.2 g / L, MgSO4·7H2O 0.2 g / L, and KH2PO4 0.025 g / L.
[0014] Furthermore, in step (1), step (2) and step (3), the trace elements in the influent matrix include: EDTA2Na19.1g / L, ZnSO4 0.43g / L, CoCl2·6H2O 0.24g / L, MnCl2·4H2O 0.99g / L, CuSO4·5H2O 0.25g / L, NaMoO4·2H2O 0.22g / L, NiCl2·2H2O 0.19g / L, NaWO4·2H2O 0.05g / L, and H3BO3 0.014g / L.
[0015] Furthermore, in step (1), step (2) and step (3), the reactor is operated in a constant temperature incubator at a temperature of 34±1° C. and a rotation speed of 120 rpm in the dark.
[0016] Furthermore, in step (1), the hydraulic retention time is 0.8-1 d, and the culture period is 15-20 d.
[0017] Furthermore, in step (2), the hydraulic retention time is 5 days and the culture period is 40-50 days.
[0018] Furthermore, in step (1), step (2) and step (3), nitrogen aeration is used to remove oxygen in the influent substrate before the influent substrate enters the reactor.
[0019] Furthermore, in step (3), the hydraulic retention time is 5 days and the culture period is 80-90 days.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] 1. The present invention gradually improves the ability of microorganisms to simultaneously oxidize ammonia and reduce arsenic through a multi-stage cultivation process, and successfully domesticates and enriches a microbial population that can couple ammonia oxidation and arsenic reduction processes under anaerobic conditions.
[0022] 2. The present invention successfully identified a group of anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria. It is worth noting that the abundance of the dominant species, the anaerobic ammonium oxidizing bacteria genus Ca. Kuenenia, increased significantly to 57.41%, and it carried complete genes encoding the anaerobic ammonium oxidation process and arsenic respiratory reduction process (arrAB).
[0023] 3. The domestication and enrichment method provided by the present invention not only helps to promote the development of carbon-neutral biotechnology based on anaerobic ammonia oxidation to address environmental nitrogen and arsenic pollution problems, but also provides valuable research ideas for nutritional relationships that may link nitrogen metabolism with a series of terminal electron acceptors. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 -Community distribution of anaerobic ammonium-oxidizing granular sludge at the genus level.
[0025] Figure 2 - Community distribution at genus level after the second phase.
[0026] Figure 3 - Community distribution at the genus level after the third phase.
[0027] Figure 4 -Graph of experimental results of isotope activity test.
[0028] Figure 5 - Annotation results of genes related to nitrogen metabolism and arsenic reduction in enrichment cultures. DETAILED DESCRIPTION
[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.
[0030] Example 1
[0031] A method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria, comprising the following steps:
[0032] (1) Preparation of inorganic culture matrix
[0033] The inorganic culture matrix is an aqueous solution containing matrix, inorganic salts and trace elements. The matrix and its concentration are: NH4Cl 70 mg N / L, NaNO2 70 mg N / L; the inorganic salts and their concentrations are: KHCO3 2.5 g / L, CaCl2·2H2O 0.2 g / L, MgSO4·7H2O 0.2 g / L, KH2PO4 0.025 g / L; the trace elements and their concentrations are: EDTA2Na 19.1 g / L, ZnSO4 0.43 g / L, CoCl2·6H2O 0.24 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, NaMoO4·2H2O 0.22 g / L, NiCl2·2H2O 0.19 g / L, NaWO4·2H2O 0.05 g / L, H3BO3 0.014 g / L.
[0034] (2) Phase 1
[0035] Take an appropriate amount of mature anaerobic ammonium oxidation granular sludge in stable operation and wash it three times with 1×PBS. Subsequently, 30g of the washed anaerobic ammonium oxidation granular sludge was inoculated into a sequencing batch reactor with an effective volume of 1.2L, and the remaining samples were placed in a -80℃ refrigerator for high-throughput sequencing to determine the abundance of the uncultured samples. Then add 1L of the prepared inorganic culture matrix to the reactor, aerate with nitrogen until the oxygen is completely removed, and then place it in a constant temperature incubator with a temperature of 34±1℃ and a speed of 120rpm to run continuously in the dark. The culture cycle is 20d and the hydraulic retention time is 1d. The culture cycle can be extended or shortened according to the sludge activity to ensure that the system effluent ammonia nitrogen and nitrite nitrogen indicators are stable and run for more than one week below 5mg / L.
[0036] (3) Second stage
[0037] After the reactor effluent stabilized, the influent matrix was adjusted to 30 mg N / L ammonia nitrogen as an electron donor and 1 mg As / L arsenate instead of nitrite as an electron acceptor for the second stage of acclimatization and enrichment culture. The operation was continued, and the other inorganic salts and trace element solution components remained unchanged. The hydraulic retention time was adjusted to 5 days. The culture time was 45 days, which was the time when the reactor was continuously running and stabilized, that is, the ammonia nitrogen degradation was maintained at a constant value. The culture cycle was appropriately extended or shortened according to the activity of the reactor to ensure stable operation for more than one week. At the same time, sludge samples were collected for high-throughput sequencing and microbial community analysis.
[0038] (4) Stage 3
[0039] The influent arsenate concentration was further adjusted to 2 mg As / L for the enrichment culture of the functional microbial flora in the third stage. The other inorganic salt and trace element solution components remained unchanged to obtain high-abundance functional microorganisms, and the hydraulic retention time was adjusted to 5 days. The culture time lasted for 85 days, the reactor operated continuously and stably, the effluent ammonia nitrogen degradation rate was maintained at a constant value again, and the effluent arsenic reduction rate was above 80%. At this time, the reactor was enriched with flora that could achieve anaerobic ammonia oxidation coupled with arsenate reduction. Similarly, sludge samples at the end of the third stage were collected for sequencing analysis.
[0040] The community distribution of uncultured samples, i.e., anaerobic ammonium-oxidizing granular sludge at the genus level is shown in Figure 2. Figure 1 As shown, the bacterial community was mainly composed of Denitratisoma (20.10%), norank_f__A4b (16.34%), norank_c__SJA-28 (8.02%), unclassified_f__Brocadiaceae (7.73%), Candidatus_Kuenenia (7.72%), and Subgroup_10 (3.94%).
[0041] After the second stage, the distribution of communities at the genus level was as follows: Figure 2 As shown, the bacterial community was mainly composed of Denitratisoma (19.09%), Candidatus_Kuenenia (10.07%), norank_f__A4b (7.86%), norank_c__SJA-28 (7.42%), and norank_o__SBR1031 (5.49%).
[0042] After the third stage, the distribution of communities at the genus level is as follows: Figure 3As shown, the bacterial community was mainly composed of Candidatus_Kuenenia (57.41%), unclassified_o__Anaerolineales (2.95%), Lautropia (2.71%), unclassified_c__Ignavibacteriales (2.56%), and Candidatus_Jettenia (1.73%).
[0043] from Figure 1 , Figure 2 and Figure 3 It can be seen that the abundance of anaerobic ammonia oxidizing bacteria (Ca. Kuenenia) increased significantly during the domestication and enrichment culture process, from 7.72% to 57.41%, becoming the dominant genus.
[0044] Example 2
[0045] Stable isotope activity test: the experimental method is to construct a microcosm incubation system to verify the ability of enriched cultured sludge to couple anaerobic ammonia oxidation with arsenic reduction.
[0046] Prepare three 50mL serum bottles and inoculate 3g of 150d enrichment sludge into each of the three serum bottles. Then add 40mL of sterile culture medium: 30mg N / L 15 NH4Cl, KHCO3 2.5g / L, CaCl2·2H2O 0.2g / L, MgSO4·7H2O0.2g / L, KH2PO4 0.025g / L, EDTA2Na 19.1g / L, ZnSO4 0.43g / L, CoCl2·6H2O0.24g / L, MnCl2·4H2O 0.99g / L, CuSO4·5H2O 0.25g / L, NaMoO4·2H2O 0.22g / L, NiCl2·2H2O0.19g / L, NaWO4·2H2O 0.05g / L, H3BO3 0.014g / L. All bottles were degassed with argon (99.99%) for 30 minutes and sealed with butyl rubber stoppers and crimped aluminum caps, respectively. NaH2AsO4 (50 mg As / L) was dissolved in deionized water, sterilized and sealed in a serum bottle as a sterile stock solution for later use.
[0047] Set three processing methods: (1) 15 NH4Cl; (2) 15 NH4Cl+As(V); (3) 15NH4Cl+As(V)+C2H2. In the corresponding groups above, the initial concentration of As(V) was 2 mg As / L. When acetylene (C2H2) was added, 5 mL of headspace was replaced by acetylene to block the N2 generation pathway from N2O to N2 (an important step in denitrification) and the anaerobic ammonium oxidation process. Subsequently, all vials were shaken thoroughly to homogenize the mixture and dissolve the acetylene, and then cultured in a dark shaker at 34°C for 10 days at a speed of 140 rpm. Liquid samples were collected at 0, 2, 5, 8 and 10 days of culture and immediately filtered with a 0.22 μm filter membrane for subsequent measurement of the effluent non-valence arsenic concentration. At the same time, a 1 mL gas sample was collected to analyze whether there was any arsenic in the headspace gas. 15 N-labeled gaseous N2 was then used to assess the anaerobic ammonium oxidation-coupled arsenic reduction capacity of the enrichment culture.
[0048] Figure 4 is the experimental result of isotope activity test, among which, Figure 4 (a) is the concentration of arsenate in the effluent of each experimental group; Figure 4 (b) is the concentration of arsenite in the effluent of each experimental group; Figure 4 (c) is the number of samples in each experimental group. 29 N2 generation; Figure 4 (d) is the number of 30 The generation of N2. As can be seen from the figure, Figure 4 The concentration of arsenate in the effluent of each experimental group in a. Compared with the treatment group with only ammonium chloride added, the treatment with both ammonium chloride and arsenate added had a significant 30 N2 and 29 The generation of N2 directly proves that the domesticated and enriched bacterial community of the invention has the ability to couple the anaerobic ammonium oxidation process with the arsenic reduction process.
[0049] Figure 5 The results of annotation of genes related to nitrogen metabolism and arsenic reduction in enrichment culture. The anaerobic ammonium oxidation coupled with arsenic reduction process may be similar to the iron-ammonia oxidation reported in the literature, and is completed by a consortium of multiple microorganisms, including anaerobic ammonium oxidizing bacteria and other microorganisms carrying N / As metabolic genes. The results of high-throughput sequencing and metagenomic sorting showed that the anaerobic ammonium oxidizing bacteria Candidatus_Kuenenia, as the main dominant species, carries genes encoding ammonia oxidation (hao, nirKS, hzsABC, hdh) and arsenic reduction (arrAB), and may actively participate in the reduction of As(V) and oxidation of ammonia as a potential functional driver. The reactions that occur are shown in formulas (1)(2)(3).
[0050]
[0051] Finally, it should be noted that the above embodiments of the present invention are only examples for illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. It is impossible to list all implementation methods here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.
Claims
1. A method for acclimating and enriching anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria, characterized in that: The specific steps include: (1) Phase I: The mature anaerobic ammonium oxidation granular sludge in stable operation is inoculated into the reactor and cultured under light-shading conditions using water containing ammonia nitrogen, nitrite nitrogen, inorganic salts and trace elements as the influent matrix; the concentrations of ammonia nitrogen and nitrite nitrogen are both 70-90 mg / L; (2) The second stage: After the effluent from the reactor is stable, water containing ammonia nitrogen, arsenate, inorganic salts and trace elements is used as the inlet matrix for light-shading and acclimatization culture; the concentration of ammonia nitrogen is 30-40 mg / L, and the concentration of arsenate is 1 mg / L; (3) The third stage: After the reactor effluent returns to stability, water containing ammonia nitrogen, arsenate, inorganic salts and trace elements is used as the inlet matrix for light-shading culture, thereby enriching the functional bacteria community for anaerobic ammonia oxidation coupled with arsenic reduction; among which, the concentration of ammonia nitrogen is 30-40 mg / L, and the concentration of arsenate is 2 mg / L.
2. The method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria according to claim 1, characterized in that: The reactor is a sequencing batch reactor.
3. The method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria according to claim 1, characterized in that: In step (1), step (2) and step (3), the inorganic salts in the influent matrix include: KHCO3 2.5 g / L, CaCl2·2H2O 0.2 g / L, MgSO4·7H2O 0.2 g / L, and KH2PO4 0.025 g / L.
4. The method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria according to claim 1, characterized in that: In step (1), step (2) and step (3), the trace elements in the influent matrix include: EDTA2Na 19.1 g / L, ZnSO4 0.43 g / L, CoCl2·6H2O 0.24 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, NaMoO4·2H2O 0.22 g / L, NiCl2·2H2O 0.19 g / L, NaWO4·2H2O 0.05 g / L, and H3BO3 0.014 g / L.
5. The method for acclimating and enriching anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria according to claim 1, characterized in that: In step (1), step (2) and step (3), the reactor is operated in a constant temperature incubator at a temperature of 34±1°C and a rotation speed of 120 rpm in the dark.
6. The method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria according to claim 1, characterized in that: In step (1), the hydraulic retention time is 0.8-1d and the culture period is 15-20d.
7. The method for acclimating and enriching anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria according to claim 1, characterized in that: In step (2), the hydraulic retention time is 5 days and the culture period is 40-50 days.
8. The method for acclimating and enriching anaerobic ammonium oxidation coupled with arsenic reduction functional bacteria according to claim 1, characterized in that: In step (1), step (2) and step (3), nitrogen aeration is used to remove oxygen in the influent substrate before the influent substrate enters the reactor.
9. The method for acclimating and enriching anaerobic ammonium oxidation coupled arsenic reduction functional bacteria according to claim 1, characterized in that: In step (3), the hydraulic retention time is 5 days and the culture period is 80-90 days.