Heterotrophic nitrification-aerobic denitrification bacterial agent as well as preparation method and application thereof in water pollution remediation

Abstract

The invention relates to a preparation method of a heterotrophic nitrification-aerobic denitrification bacterial agent and an obtained HNAD bacterial agent. The preparation method comprises the following steps: obtaining a heterotrophic nitrification denitrification strain; mixing the obtained heterotrophic nitrification nitrogen removal strain with an aerobic denitrification bacterium solution to obtain a compound HNAD (Heterotrophic Nicotinamide Adenine Dinucleotide) flora; and inoculating the obtained compound HNAD flora into a bio-membrane reactor, constructing a stable biofilm culturing HNAD bio-membrane reactor, and domesticating and enriching to obtain the HNAD microbial inoculum. The invention also provides an application of the HNAD microbial inoculum in nitrogen-polluted rare earth mine groundwater remediation. The HNAD microbial agent disclosed by the invention has a remarkable denitrification effect and a wide application prospect on nitrogen-polluted rare earth mine underground water, and has important significance on improving the ecological environment, and the HNAD microbial agent and the remediation method disclosed by the invention have important significance on protecting underground water resources.

Description

Technical Field

[0001] The invention belongs to the field of microbial environment, and specifically relates to a heterotrophic nitrification-aerobic denitrification bacterial agent with high-efficiency denitrification, a preparation method thereof, and application in the remediation of nitrogen pollution in groundwater of rare earth mines. Background Art

[0002] Due to their unique physical and chemical properties, rare earth elements play an irreplaceable role in modern industry and are widely used in electronics, energy, chemical industry, military and other fields. However, the mining and refining process of rare earths is often accompanied by environmental pollution problems, among which groundwater nitrogen pollution is particularly serious. Its water chemical characteristics are mainly characterized by strong acidity, high concentrations of ammonia nitrogen and nitrate nitrogen, and rich rare earth elements and heavy metals. Therefore, how to solve the groundwater pollution problem left by in-situ leaching of rare earth mines is a key issue that urgently needs to be solved for the green and sustainable development of the current rare earth industry.

[0003] At present, the wastewater treatment plants in rare earth mining areas mainly use the traditional nitrification and denitrification process. To ensure efficient denitrification, it is usually necessary to pre-treat the wastewater (such as pH adjustment), and set up aerobic tanks (nitrification stage) and anaerobic tanks (denitrification stage) respectively. The separation of these two processes increases the complexity of the system. At the same time, in order to make the biological concentration in the reaction system higher, it is often necessary to return the sludge. Once the parameter setting is wrong, it is very easy to cause the sludge to float, resulting in a decrease in denitrification effect or even system collapse. In addition, the traditional nitrification and denitrification process is sensitive to environmental conditions such as pH, temperature, heavy metals, etc., and the wastewater needs to be pre-treated, which increases its process flow and operating costs to a certain extent.

[0004] In response to the above problems, scientists have discovered a new type of microorganism in recent years - heterotrophic nitrification-aerobic denitrification (HNAD). This type of microorganism can simultaneously carry out nitrification and denitrification under aerobic conditions, that is, simultaneous nitrification and denitrification. This characteristic has attracted widespread attention in the field of water treatment. HNAD bacteria have the advantages of high denitrification rate, strong resistance to heavy metals, reduced energy consumption and carbon emissions, and strong environmental adaptability. Moreover, the bacteria can effectively treat high-nitrogen wastewater without nitrite and nitrate accumulation, thus avoiding secondary pollution in the traditional denitrification process. However, despite the many advantages of HNAD bacteria, most of the research work still remains on the study of simulated wastewater, and there are still some challenges in practical applications. For example, how to efficiently screen HNAD strains with strong denitrification ability from complex environmental samples, and how to improve the environmental adaptability and denitrification efficiency of these strains through domestication and enrichment. In addition, how to effectively apply HNAD strains screened and domesticated under laboratory conditions to large-scale groundwater remediation projects is also an urgent problem to be solved. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a heterotrophic nitrification-aerobic denitrification bacterial agent and a preparation method thereof, as well as an application thereof in the remediation of nitrogen pollution in groundwater of rare earth mines. The remediation method of the heterotrophic nitrification-aerobic denitrification bacterial agent described in the present invention has the advantages of high efficiency, low cost and environmental friendliness, and can achieve efficient removal of nitrogen compounds in groundwater of rare earth mines.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing a heterotrophic nitrification-aerobic denitrification bacterial agent, comprising the following steps: S1: extracting indigenous microbial sludge from the environment of a sewage treatment plant around a rare earth mining area, inoculating the indigenous microbial sludge into a heterotrophic nitrification enrichment medium for cultivation for 7-10 days, and completing screening to obtain heterotrophic nitrification and denitrification bacterial strains;

[0008] S2. The obtained heterotrophic nitrification and denitrification bacteria with an MLVSS of 1.4-1.6 mg / L and an aerobic denitrification bacteria solution with a concentration of 5-6 g / L are mixed at an inoculation ratio of (2-5):1, and then inoculated into a heterotrophic nitrification-aerobic denitrification enrichment medium to obtain a composite HNAD bacterial flora;

[0009] S3. The obtained composite HNAD bacterial community is inoculated into a biofilm reactor to construct a stable biofilm HNAD biofilm reactor, and then the HNAD biofilm reactor is injected into the rare earth mine groundwater from the source area of ​​the indigenous microbial sludge to domesticate the HNAD bacterial community for no less than 7 days;

[0010] S4. Enrichment to obtain HNAD bacterial agent.

[0011] In some of the embodiments, in steps S1 and S2, the C / N ratio of the culture solution in the culture medium is 4-5, and the pH value is controlled at 7-8.

[0012] In some embodiments, the composition of the culture medium in the heterotrophic nitrification enrichment medium in S1 includes: 0.90-0.95 g / L NaHCO3, 0.34-0.38 g / L KH2PO4, 0.34-0.38 g / L NH4Cl, 0.27-0.31 g / L MgSO4·7H2O and 0.0010-0.0012 g / L CaCl2, 0.75-0.8 g / L CH3COONa, and the pH value is controlled at 7-8; more preferably, the composition of the culture medium includes: 0.92 g / L NaHCO3, 0.36 g / L KH2PO4, 0.36 g / L NH4Cl, 0.29 g / L MgSO4·7H2O and 0.0011 g / L CaCl2, 0.80 g / L CH3COONa.

[0013] In some embodiments, the culture conditions in S1 and S2 are: temperature of 25-30° C., dissolved oxygen (DO) of 3-5 mg / L.

[0014] In some embodiments, the composition of the culture medium in the heterotrophic nitrification-aerobic denitrification enrichment medium in S2 includes: 0.90-0.95 g / L NaHCO3, 0.41-0.45 g / L KNO3, 0.34-0.38 g / L KH2PO4, 0.45-0.50 g / L NH4Cl, 0.27-0.31 g / L MgSO4·7H2O and 0.0010-0.0012 g / L C aCl2, 1.18-1.22 g / L CH3COONa, pH 7-8, more preferably, the composition of the culture solution includes: 0.92 g / L NaHCO3, 0.43 g / L KNO3, 0.36 g / L KH2PO4, 0.47 g / L NH4Cl, 0.29 g / L MgSO4·7H2O and 0.0011 g / L CaCl2, 1.20 g / L CH3COONa.

[0015] In some of the embodiments, the rare earth mining area surrounding sewage treatment plant is a local sewage treatment plant in Longnan Zudong rare earth mining area, Ganzhou City, Jiangxi Province.

[0016] In some of the embodiments, the aerobic denitrifying bacteria is Qiangwei 99.

[0017] In some of the embodiments, the water chemical characteristics of S3 are: ammonia nitrogen: 91.72±2.55 mg / L, nitrate nitrogen: 83.81±4.33 mg / L, nitrous acid: 0.24±0.34 mg / L, total nitrogen: 186.84±10.84 mg / L, chemical oxygen demand is almost 0, and it is strongly acidic (pH is 4.95±0.22).

[0018] The injected rare earth mine groundwater was set to a C / N ratio of 4-5 by adding sodium acetate.

[0019] In some of the embodiments, when constructing the HNAD biofilm reactor in S3: the biological fixed filler is put into the plexiglass cylindrical reactor at a filling rate of 60-70%, the aeration rate is set to 0.15L / (L·min), the temperature is 25-30°C, and the HRT is 6-8h; the HNAD bacterial community is stably formed by exposure for 1-3 days.

[0020] In some embodiments, the volume ratio of the heterotrophic nitrifying and denitrifying bacteria to the aerobic denitrifying bacteria liquid is (2-3.5):1.

[0021] The amount of the composite HNAD bacterial community inoculated into the biofilm reactor is: the heterotrophic nitrification-aerobic denitrification bacteria agent is inoculated into the biofilm reactor at a volume ratio of 1%-3% of the volume of the biofilm reactor.

[0022] The second aspect of the present invention is to provide a heterotrophic nitrification-aerobic denitrification bacterial agent (HNAD bacterial agent) obtained according to any one of the above preparations.

[0023] In some of the embodiments, the dominant genera of the heterotrophic nitrification-aerobic denitrification bacteria include Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira, and it is further preferred that Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira account for more than 20% of the bacteria in the heterotrophic nitrification-aerobic denitrification bacteria.

[0024] In some embodiments, the heterotrophic nitrifying-aerobic denitrifying bacteria grow with ammonia nitrogen, nitrate or nitrite as a nitrogen source.

[0025] The third aspect of the present invention is to provide the use of the heterotrophic nitrification-aerobic denitrification bacterial agent in the remediation of nitrogen-contaminated rare earth mine groundwater.

[0026] A fourth aspect of the present invention is to provide a method for repairing nitrogen-contaminated rare earth mine groundwater, comprising the following steps:

[0027] The heterotrophic nitrification-aerobic denitrification bacteria are inoculated into a biofilm reactor to construct a HNAD biofilm reactor with stable biofilm.

[0028] Then the HNAD biofilm reactor is put into the nitrogen-contaminated rare earth mine groundwater that needs to be repaired for operation;

[0029] Or the repair method includes the following steps: adding the above-mentioned heterotrophic nitrification-aerobic denitrification bacterial agent into the nitrogen-contaminated rare earth mine groundwater that needs to be repaired.

[0030] In some of the embodiments, the heterotrophic nitrification-aerobic denitrification bacteria are inoculated into the biofilm reactor at a ratio of 1%-3%, further 2%-3% of the volume of the biofilm reactor.

[0031] In some of the embodiments, when constructing the HNAD biofilm reactor in S4: the biological fixed filler is put into the plexiglass cylindrical reactor at a filling rate of 60-70%, the aeration rate is set to 0.12-0.18L / (L·min), the C / N ratio is set to 4-5, the temperature is 25-30°C, and the HRT is 6-8h; the HNAD bacteria are stably formed by exposure for 1-3 days.

[0032] The present invention obtains the HNAD bacterial agent of microbial flora with high-efficiency denitrification ability through a specific screening, compounding and domestication process. Compared with the prior art, the advantages of the HNAD bacterial agent are mainly reflected in the following aspects: (1) Synchronous nitrification and denitrification: The HNAD bacterial agent can simultaneously perform nitrification and denitrification under aerobic conditions, and the denitrification rate is extremely fast; (2) Strong environmental adaptability: The domesticated HNAD bacterial flora can adapt to the special environment of rare earth mine groundwater, including high ammonia nitrogen, high COD, strong acidity and other characteristics; (3) Simple process flow and low cost: Compared with the traditional nitrification-denitrification process, the remediation method of the present invention simplifies the operation steps, reduces infrastructure and operation costs, and has better economy; (4) It has less impact on the environment and is a green and sustainable groundwater remediation method. In summary, the present invention provides an HNAD bacterial agent with excellent high-efficiency remediation effect for nitrogen pollution in groundwater of rare earth mines and an innovative remediation technology for nitrogen pollution in groundwater of rare earth mines, which has significant denitrification effect and broad application prospects, and is of great significance to improving the ecological environment and protecting groundwater resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Denitrification performance of dominant denitrifying nitrifying bacteria during the domestication process.

[0034] Figure 2 Results of microbial diversity analysis of dominant denitrifying and nitrifying bacterial species.

[0035] Figure 3 Effect of the inoculation ratio of heterotrophic nitrification and aerobic denitrification on the performance of HNAD (the inoculation ratios of heterotrophic nitrification and aerobic denitrification were 1:0, 0:1, 1:1, 2:1, 5:1, and 7:1, respectively).

[0036] Figure 4 Denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria during acclimation process.

[0037] Figure 5 Ammonia nitrogen (NH4 + -N) as the only nitrogen source.

[0038] Figure 6 Nitrate (NO3 - -N) as the only nitrogen source.

[0039] Figure 7 Nitrous acid (NO2 - -N) as the only nitrogen source.

[0040] Figure 8 Denitrification performance with ammonia nitrogen + nitrate nitrogen as initial nitrogen source.

[0041] Fig. 9 Denitrification performance with ammonia nitrogen + nitrous acid as initial nitrogen source.

[0042] Fig.10 Denitrification performance with nitrate + nitrous acid as initial nitrogen source.

[0043] Fig.11 Denitrification performance with ammonia nitrogen + nitrate nitrogen + nitrous acid as initial nitrogen source.

[0044] Fig.12 Denitrification performance under different carbon source types.

[0045] Fig.13 Denitrification performance under different carbon sources.

[0046] Fig.14 Denitrification performance at different C / N ratios.

[0047] Fig.15 Denitrification performance at different initial pH values.

[0048] Fig.16 Denitrification performance with different ammonia nitrogen concentrations as the only nitrogen source.

[0049] Fig.17 Denitrification performance under different nitrate nitrogen concentrations as the only nitrogen source.

[0050] Fig.18 Denitrification performance under different aeration conditions during long-term operation.

[0051] Fig.19 Changes in environmental factors under different aeration conditions during long-term operation.

[0052] Fig. 20 Microbial community structure of HNAD at the phylum and genus levels under different aeration conditions.

[0053] Fig.21 Changes of key functional genes of HNAD bacterial agent under different aeration conditions.

[0054] Fig. 22 Possible nitrogen metabolism pathways of HNAD bacterial agents.

[0055] Fig.23 SEM electron microscope image of HNAD bacterial agent sludge particles. DETAILED DESCRIPTION

[0056] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0057] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0058] HNAD: Heterotrophic Nitrification-Aerobic Denitrification.

[0059] MLVSS: mixed liquid volatile suspended solids.

[0060] Hydraulic Retention Time (HRT): The average residence time of the wastewater to be treated in the reactor.

[0061] The present invention is further described in detail below with reference to specific embodiments.

[0062] In some embodiments of the present invention, a heterotrophic nitrification-aerobic denitrification bacterial agent for remediation of nitrogen pollution in rare earth mine groundwater is provided, and a preparation method thereof is also provided. In some embodiments of the present invention, a method for remediation of nitrogen pollution in rare earth mine groundwater using the heterotrophic nitrification-aerobic denitrification bacterial agent is also provided.

[0063] The present invention extracts indigenous microbial sludge from the environment of sewage treatment plants around rare earth mining areas, screens out dominant denitrifying bacteria, and compounding with specific aerobic denitrifying bacteria to form an efficient HNAD bacterial community. Through domestication and enrichment, as well as application tests in actual rare earth mine groundwater environments, the HNAD bacterial agent obtained by the present invention not only has strong adaptability and high denitrification efficiency, but also has a simple process flow, low infrastructure and operating costs, and is environmentally friendly, and is expected to become an effective alternative technology to traditional nitrification and denitrification processes.

[0064] Qiangwei 99: purchased from Yichun Qiangwei Microbiology Technology Co., Ltd., the main ingredients are hyperosmotic aerobic denitrifying bacteria, denitrifying alcaligenes, and carriers, with a total live bacteria content of ≥30x10 8 cfu / g, moisture ≤12%.

[0065] Example 1: Domestication of dominant denitrifying nitrifying bacteria

[0066] Indigenous microbial sludge (MLVSS of 1.4-1.6 mg / L) was extracted from a local sewage treatment plant in Longnan Zudong rare earth mining area, Ganzhou City, Jiangxi Province (Longnan branch of Sanda Membrane Environmental Technology Co., Ltd.), and inoculated into a heterotrophic nitrification enrichment medium at a volume ratio of 5%. The culture speed was 200 r / min, and the C / N ratio in the culture medium was set to 5 (99% pure sodium acetate). The culture medium was replaced every day, and the concentrations of ammonia nitrogen, nitrate nitrogen, and nitrous acid were tested regularly (daily). When the ammonia nitrogen concentration was stable at below 10 mg / L for a long time, the sludge was subjected to microbial detection to ensure that the heterotrophic nitrifying bacteria in the system were the dominant species. The culture medium was completely extracted and the sludge was collected. At this time, the heterotrophic nitrifying and denitrifying bacteria were obtained, and the HNAD bacterial community MLVSS was 2.2-2.5 mg / L.

[0067] like Figure 1 As shown, after one week of continuous acclimatization, NH4 + The removal rate of -N can be stabilized at more than 90%. The domesticated sludge was selected for 16S rRNA gene amplification, and the bacterial primers used for amplification were 338F (5′-ACTCCTACGGGAGGCAGCA-3′, SEQ ID NO: 1) and 806R (5-′GACTACHVGGGTWTCTAAT-3′, SEQ ID NO: 2). Figure 2As shown, the dominant nitrifying bacteria species after domestication were mainly Thauera (22.67%), SM1A02 (1.39%), Nitrosomonas (3.00%) and Nitrospira (14.28%), among which Thauera and SM1A02 played the main role in heterotrophic nitrification-aerobic denitrification.

[0068] The condition parameters of the culture medium in the above heterotrophic nitrification enrichment medium are: 0.92g / L NaHCO3, 0.36g / L KH2PO4, 0.36g / L NH4Cl, 0.29g / L MgSO4·7H2O and 0.0011g / L CaCl2, 0.80g / L CH3COONa, and the culture conditions are: pH value is controlled at 7-8, temperature is 25-30℃, and dissolved oxygen (DO) is 3-5mg / L.

[0069] Example 2: Effect of heterotrophic nitrification and aerobic denitrification inoculation ratio on HNAD performance

[0070] The heterotrophic nitrification and denitrification bacteria obtained in Example 1 and the aerobic denitrification bacterial solution (Strong Micro 99, 5-6 grams dissolved in 1L of water, 5.5g dissolved in 1L of water in this embodiment) were mixed and inoculated in a heterotrophic nitrification-aerobic denitrification enrichment medium (inoculated at a volume ratio of 5%) according to an inoculation volume ratio of 1:0, 0:1, 1:1, 2:1, 5:1, and 7:1, the temperature was 25-30°C, the DO was 5-6mg / L, the C / N ratio was set to 5 (99% pure sodium acetate), the speed was 200r / min, and the concentrations of ammonia nitrogen, nitrate, nitrite, pH, and DO were measured once every hour; the experimental data were collected and statistically analyzed, and the culture was cultured for at least 3 days, and then all the culture solution was extracted, the sludge was collected, and the HNAD bacterial community (MLVSS was 2.7-3.2mg / L) with synchronous nitrification-denitrification function under aerobic conditions was obtained.

[0071] The composition of the culture solution of the above heterotrophic nitrification-aerobic denitrification enrichment medium is: 0.92g / L NaHCO3, 0.43g / L KNO3, 0.36g / L KH2PO4, 0.47g / L NH4Cl, 0.29g / L MgSO4·7H2O and 0.0011g / L CaCl2, 1.20g / L CH3COONa, and the pH value is controlled at 7-8.

[0072] like Figure 3As shown in the figure, when heterotrophic nitrifying bacteria are added alone, the removal performance of ammonia nitrogen and nitrate nitrogen is not good; when aerobic denitrifying bacteria are added alone, it is not conducive to the removal of ammonia nitrogen (the removal rate is only about 66%), but the removal performance of nitrate nitrogen can reach 100%. In addition, when the inoculation ratio of heterotrophic nitrification: aerobic denitrification is increased to 2:1, the removal rates of ammonia nitrogen and nitrate nitrogen of HNAD can reach 100%; however, when the ratio is increased to 7:1, the ammonia nitrogen removal rate drops rapidly. This shows that the inoculation ratio of heterotrophic nitrification: aerobic denitrification is (2-5:1). During the experiment, multiple rounds of experiments can be adjusted within the range of (2-5:1) to obtain HNAD bacterial flora with synchronous nitrification-denitrification function under aerobic conditions. Too high or too low will affect the overall denitrification performance.

[0073] Example 3: Acclimation and enrichment of HNAD flora in rare earth mine groundwater

[0074] The HNAD bacterial community prepared in Example 2 was inoculated at a volume ratio of 5% (the volume ratio of the HNAD bacterial community to the water inlet of the reactor) and inoculated into the biofilm reactor. The HNAD biofilm reactor was constructed in a conventional manner (the biological fixed filler-polyurethane sponge, porosity 97%, specific surface area 800m 2 / m 3 , put into the organic glass cylindrical reactor at a filling rate of 60-70%, and the aeration rate is set to 0.15L / (L·min). The reactor needs to be aired for 1-3 days before operation to stabilize the HNAD bacteria to form a biofilm. After the biofilm is stable, the rare earth mine groundwater can be introduced for denitrification). The rare earth mine groundwater is injected into the HNAD biofilm reactor to domesticate and enrich the HNAD flora. The concentrations of ammonia nitrogen, nitrate, nitrite and changes in factors such as pH, DO and ORP in the reactor effluent are regularly tested, as well as the growth of the HNAD flora and microbial diversity are regularly monitored to ensure the high diversity of microorganisms and the dominant position of HNAD bacteria such as Thauera and unclassified_Rhodobacteraceae. Finally, an efficient denitrification HNAD bacterial agent that can adapt to the complex environment of rare earth mine groundwater is obtained.

[0075] In the above acclimation process, the rare earth mine groundwater injected into the HNAD biofilm reactor is set to a C / N ratio of 5 (sodium acetate with 99% purity) by adding sodium acetate, the rotation speed is 200r / min, the aeration rate is set to 0.15L / (L·min), the temperature is 25-30°C, the HRT is set to 6h, and the pH value does not need to be controlled. The water chemical characteristics of the above rare earth mine groundwater (derived from the rare earth mine groundwater of the local sewage treatment plant in the Longnan Zudong rare earth mining area of ​​Ganzhou City, Jiangxi Province, where indigenous microbial sludge is extracted) are: ammonia nitrogen: 91.72±2.55mg / L, nitrate nitrogen: 83.81±4.33mg / L, nitrous acid: 0.24±0.34mg / L, total nitrogen: 186.84±10.84mg / L, chemical oxygen demand is almost 0, strong acidity (pH is 4.95±0.22), rich in rare earth elements and heavy metals.

[0076] The contents of rare earth elements and heavy metals can be found in the following table:

[0077]

[0078] like Figure 4 As shown in the figure, the removal rates of ammonia nitrogen and nitrate nitrogen of HNAD bacterial agent were 27.1% and 50.4% respectively on the first day of the biofilm reactor. With the increase of operation time and the regular replacement of wastewater, HNAD bacterial community gradually formed stable biofilms on the filler. After the third day of continuous operation, the removal rates of ammonia nitrogen and nitrate nitrogen can reach more than 90%, indicating that HAND bacterial community can quickly adapt to the groundwater environment of rare earth mines and achieve efficient denitrification. The training of HAND bacterial community was successful. It is recommended to ensure at least 7 days of acclimatization time during actual operation.

[0079] The HAND bacterial community multiplied in large quantities on the filler, and the sludge was collected to obtain the HAND bacterial agent (MLVSS was 2.7-3.2 mg / L), which was stored at minus 80 degrees Celsius for future use.

[0080] The HAND bacterial community enriched by the above method has Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira as the main bacterial genera, see Fig.10 and the table below:

[0081] We took samples from the Longnan branch of Sanda Membrane Environmental Technology Co., Ltd. in March 2022, April 2023, and July 2024, respectively. We took 3-5 samples each time and repeated the above operations. We were able to obtain HAND bacteria that can quickly adapt to the groundwater environment of rare earth mines and achieve efficient denitrification. Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira are the dominant genera, accounting for more than 20%. The microbial community structure can be found in Fig. 20 (b).

[0082] Example 4: Effects of different single nitrogen sources and mixed nitrogen sources on HNAD denitrification performance

[0083] The HNAD bacterial agent obtained after domestication in Example 3 was inoculated at a volume ratio of 5%, and 7 biofilm reactors were constructed as described in Example 3. The C / N ratio was set to 4.5 (99% pure sodium acetate), the aeration rate was set to 0.15L / (L·min), the DO was stabilized at about 5-6mg / L, the temperature was 25-30°C, the HRT was set to 9h, the speed was 200r / min, and the initial pH was set to 7-8. The nitrogen source in each reactor was set to 0.31mg / L NH4 + -N, 0.56mg / L NO3 - -N, 0.38mg / L NO2 - -N, 0.31mg / L NH4 + -N+0.56mg / L NO3 - -N, 0.31mg / L NH4 + -N+0.38mg / L NO2 - -N, 0.56mg / L NO3 - -N+0.38mg / L NO2 - -N, 0.31mg / L NH4 + -N+0.56mg / L NO3 - -N+0.38mg / L NO2 - -N. Regularly test the changes in the concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO, ORP and other factors in the reactor effluent.

[0084] Figure 5 When ammonia nitrogen was the only nitrogen source, the total nitrogen removal rate was 92.3% after the reactor was operated for 8 hours. Figure 6 When nitrate is the only nitrogen source, the total nitrogen removal rate reaches 97.8% after the reactor runs for 8 hours; Figure 7 When nitrite is the only nitrogen source, the total nitrogen removal rate reaches 91.5% after the reactor runs for 8 hours.

[0085] like Figure 8-Figure 11 As shown in the figure, HNAD agent showed good denitrification performance with different mixed nitrogen sources as the initial nitrogen source. The removal rates of total nitrogen (TN) were 84.2% (NH4 + -N+NO3 - -N), 81.6% (NH4 + -N+NO2 - -N)、71.5%(NO3 - -N+NO2 - -N)、92.9%(NH4 + -N+NO3 - -N+NO2 - -N). As can be seen from the above, the HNAD bacterial agent has obvious heterotrophic nitrification and aerobic denitrification capabilities under aerobic conditions.

[0086] Example 5: Effect of Carbon Source Type on HNAD Performance

[0087] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into a biofilm reactor (constructed as described in Example 3) at a volume ratio of 10% and operated for 6 hours, with an initial pH value of 7, an aeration rate of 3 L (L·min), C / N=15, and NH4 + -N:120mg / L,NO3 - -N: 50mg / L, speed 200r / min, temperature 25-30℃. The carbon sources were set as sodium acetate, sodium succinate, sodium citrate, glucose and sucrose. Regular sampling was performed to detect the changes in the concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO and other factors in the reactor effluent.

[0088] like Fig.12 As shown, sodium acetate, sodium succinate, sodium citrate, glucose and sucrose as carbon sources of HNAD bacterial agent all showed excellent denitrification performance, among which sodium acetate, sodium succinate and sodium citrate could reach 100% TN removal rate in 6 hours, and glucose and sucrose could reach more than 90%.

[0089] Example 6: Effects of different carbon source types on the performance of HNAD under long-term operation

[0090] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into the biofilm reactor at a volume ratio of 3%, and the aeration rate was set to 1.5L (L·min), DO was stabilized at 5-6mg / L, C / N=4.5, the rotation speed was 200r / min, the temperature was 25-30°C, and the HRT was 6h. During the operation, groundwater from a rare earth mine was continuously added, and the water chemical characteristics of the groundwater from the rare earth mine were as follows: ammonia nitrogen: 91.72±2.55mg / L, nitrate nitrogen: 83.81±4.33mg / L, nitrous acid: 0.24±0.34mg / L, total nitrogen: 186.84±10.84mg / L, chemical oxygen demand was almost 0, strongly acidic (pH was 4.95±0.22), and rich in rare earth elements and heavy metals.

[0091] The carbon sources are selected in order: sodium acetate, sodium acetate (industrial sodium acetate), composite carbon source, sodium acetate + composite carbon source, sodium acetate (industrial sodium acetate) + composite carbon source. Regular sampling is performed to detect the concentrations of ammonia nitrogen, nitrate, and nitrite in the reactor effluent. Fig.13 As shown in the figure, HNAD bacteria can maintain efficient denitrification performance under long-term operation and can use different carbon source types for denitrification. It can also be found that the composite carbon source mainly affects NO3 - -N affects the removal of TN. It can be seen that the HNAD bacterial agent of the present invention can adapt to different types of carbon sources and can further reduce the denitrification operation cost in practical applications.

[0092] Example 7: Denitrification performance at different C / N ratios

[0093] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into the biofilm reactor at a volume ratio of 3% and operated for 6 h. The initial pH value was 7, the aeration rate was set to 3 L (L·min), C / N=15, and NH4 + -N:120mg / L,NO3 - -N: 50mg / L, speed 200r / min, temperature 25-30℃. C / N ratio was set to 5:1, 10:1, 15:1 and 20:1 respectively, and the carbon source was 99% pure sodium acetate. Regular sampling was performed to detect the changes in the concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO and other factors in the reactor effluent.

[0094] like Fig.14 As shown in Figure 2, with the increase of C / N ratio, the removal rate of TN showed an overall increasing trend, among which NO3 - -N removal rate is less affected by the C / N ratio. When the C / N ratio is 5:1, after 1 hour of denitrification, NO3 - -N is almost completely removed. NH4 +The removal rate of -N is greatly limited by the C / N ratio, and it is recommended to be set to greater than 5: 1. Nitrite has always been at a low level and is less harmful.

[0095] Example 8: Denitrification performance at different initial pH values

[0096] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into the biofilm reactor at a volume ratio of 2% and operated for 6 h. The aeration rate was set to 3 L (L·min), C / N=15, and NH4 + -N:120mg / L,NO3 - -N: 50mg / L, speed 200r / min, temperature 25-30℃, carbon source is 99% pure sodium acetate. Since the pH value of groundwater in rare earth mines is mostly strongly acidic, the initial pH values ​​are set to 2, 3, 4, 5 and 6 respectively. Regular sampling is performed to detect the changes in the concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO and other factors in the reactor effluent.

[0097] like Fig.15 As shown in the figure, when the reaction was carried out for 6 hours, the HNAD bacteria showed excellent denitrification performance at the initial pH value of 2-6, and the TN removal rate could reach 100%. This shows that HNAD bacteria have extremely strong acid resistance, which is mainly due to the rapid denitrification and alkali production ability of aerobic denitrifying bacteria, which can quickly neutralize the environmental pH value and provide alkalinity for the acid production of nitrification; and nitrite has always been at a low level, with less harm. Therefore, for rare earth mine groundwater with strong acidity and containing ammonia nitrogen and nitric nitrogen, heterotrophic nitrification-aerobic denitrifying bacteria have an absolute advantage over other bacteria.

[0098] Example 9: Denitrification performance with different ammonia nitrogen concentrations as the only nitrogen source

[0099] The HNAD bacteria obtained after domestication in Example 3 were selected and inoculated into the biofilm reactor at a volume ratio of 2% for 6 hours. The aeration rate was set to 3L (L·min), C / N=15, the speed was 200r / min, the temperature was 25-30°C, and the carbon source was 99% pure sodium acetate. + -N、NO3 - -N concentration is affected by seasonality, so the HNAD bacterial agent was used to treat different initial NH4 + -N and NO3 - -N concentration performance. Among them, NH4 + -N is the only nitrogen source when NH4 + The concentrations of -N were set to 110, 130, 150, 170 and 200 mg / L respectively; - -N is the only nitrogen source when NO3 -The concentration of -N was set to 50, 80, 110, 140 and 170 mg / L respectively. The concentration of ammonia nitrogen, nitrate, nitrite, pH, DO and other factors in the reactor effluent were sampled regularly to detect the changes.

[0100] Fig.16 and Fig.17 The following are the denitrification performance diagrams under different ammonia nitrogen concentrations and different nitrate nitrogen concentrations as the only nitrogen source. + When the -N concentration was 110 mg / L, the removal efficiency of HNAD was 99% in 6 h, but with the increase of NH4 + With the increase of -N concentration, the removal rate of HNAD agent gradually decreased, especially when NH4 + When the -N concentration increases from 150mg / L to 170mg / L, NH4 + -N removal rate dropped directly from 90% to 78%. NO3 - -N removal rate was less affected by the initial concentration. At 50, 80, 110, 140 and 170 mg / L, NO3 - The concentration of -N was almost completely removed in 2 hours, and the TN removal rate could reach 99% in 3 hours. The accumulation of nitrite nitrogen was very small, and no secondary pollution was generated.

[0101] Example 10: HNAD bacterial agent used for long-term denitrification experiment of groundwater in rare earth mines

[0102] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into the biofilm reactor at a volume ratio of 2%. The biological fixed filler (polyurethane sponge, porosity 97%, specific surface area 800m 2 / m 3 ) Put into organic glass cylindrical reactor at filling rate of 60-70%, C / N ratio is set to 4.5, HRT=6h, temperature is 25-30℃, speed is 200r / min; the reactor is exposed for 2 days before operation to stabilize HNAD bacterial agent biofilm, and after biofilm is stable, groundwater from Longnan Zudong rare earth mine in Ganzhou City, Jiangxi Province is introduced for denitrification; after biofilm is stable, the effective water storage volume of the reactor is about 1L, and a pump air disc and water outlet pipe are provided at the bottom, and the aeration rate is controlled by a gas flow meter; all devices are connected to a timer switch to realize automatic water inlet and outlet. Run for more than 120 days.

[0103] The hydrochemical characteristics of the groundwater in the above rare earth mines are: ammonia nitrogen: 91.72±2.55mg / L, nitrate nitrogen: 83.81±4.33mg / L, nitrous nitrogen: 0.24±0.34mg / L, total nitrogen: 186.84±10.84mg / L, chemical oxygen demand is almost 0, strongly acidic (pH is 4.95±0.22), rich in rare earth elements and heavy metals;

[0104] Reactor operation settings: After 120 days of operation, the aeration parameters were changed according to the denitrification efficiency and removal rate. The aeration parameters were divided into 6 stages, and only the aeration parameters were changed in each stage, namely: stages I-III were continuous aeration (aeration rates were 0.15, 0.25 and 0.10 L / (L·min)), stages IV-VI were intermittent aeration (aeration rate was 0.10 L / (L·min), intermittent time was 1.5h / 1.5h, 1h / 1h and 0.5h / 0.5h); the influent was groundwater from a rare earth mine, and the concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO, etc. in the reactor effluent were tested every day ( Fig.18 , Fig.19 ).

[0105] The microbial diversity of the reactor sludge was tested by sampling at each stage ( Fig. 20 Specifically, the domesticated sludge was selected for 16S rRNA gene amplification, and the bacterial primers used for amplification were 338F (5′-ACTCCTACGGGAGGCAGCA-3′, SEQ ID NO: 1) and 806R (5-′GACTACHVGGGTWTCTAAT-3′, SEQ ID NO: 2).

[0106] At each stage, samples were taken to detect the key functional genes of the reactor sludge ( Fig.21 ). The relative activities of four key functional genes (ammonia monooxygenase (amo), nitrite oxidoreductase (nxr), nitrate reductase gene (napA) and nitrite reductase (nirS)) were measured using a fluorescent quantitative PCR instrument (LightCycler480). The primer sequences of the relevant genes are shown in Table 10.1. Specifically, the DNA was first extracted according to the instructions of the bacterial DNA extraction kit, where the DNA elution volume was 100 μL, and then the concentration and purity of the extracted DNA were detected using a small molecule nucleic acid analyzer. The OD260 / OD280 value was in the range of 1.8-2.0, indicating that the DNA purity was high. Then, the qPCR reaction system was configured with DNA as a template, mixed and added to a 384-well Roche fluorescent quantitative PCR plate, and finally placed in a fluorescent quantitative PCR instrument for reaction. qPCR was run under the following conditions: first, an initial denaturation at 95°C for 2 min, then amplification using 40 cycles of the program (denaturation at 95°C for 5 s, annealing at 60°C for 30-34 s), and finally fluorescence collection on a PCR instrument. Each sample was replicated in triplicate, and the relative expression level of the corresponding sample was calculated using the 2^(-△△CT) method.

[0107] Table 10.1 Primer sequences of key functional genes

[0108]

[0109]

[0110] Reactor performance evaluation: Figure 18-20 It can be seen that the reactor maintains a high removal efficiency after continuous operation for 122 days, and the total nitrogen removal rate maintains a high removal rate under different parameters, up to 97.35%. The reactor can realize automatic pH adjustment, the original pH value is 4.95±0.2, and the effluent pH value is 8.59±0.09; among the domesticated HNAD bacterial agents, Thauera is the main HNAD genus (accounting for 49.62%-66.24%), ensuring a high diversity of microorganisms is conducive to the reactor to achieve optimal denitrification.

[0111] Study on the denitrification mechanism of HNAD bacteria: Fig.21 As shown in the figure, genes such as amo, nxr, napA, and nirS were detected. These genes are typical heterotrophic nitrification-aerobic denitrification functional genes, which further proves the occurrence of the HNAD process. At the same time, combined with the above case studies and previous studies, the denitrification pathway of the HNAD bacterial agent in the present invention was drawn ( Fig. 22 ):NH4 + -N→NH2OH→NO2 - -N→NO3 - -N→NO2 - -N→NO→N2 is the main process, and H also has ammonia nitrogen assimilation and DNRA effects.

[0112] Fig.23 This is a SEM electron microscope image of the HNAD bacterial agent sludge particles in Example 10. Fig.23 It can be seen that the HNAD bacterial agent is mainly composed of Brevibacterium, the bacteria grow well, the number is large, and there are flocs for them to attach to, which makes HNAD have extremely high denitrification performance.

[0113] Example 11: Study on the stress resistance of HNAD bacterial agent to rare earth and metal elements when used for denitrification of groundwater in rare earth mines

[0114] The HNAD bacterial agent obtained after domestication in Example 3 was selected and inoculated into the biofilm reactor at a volume ratio of 3% for 6 hours. The aeration rate was set to 1.5L (L·min), C / N=4.5, the rotation speed was 200r / min, the temperature was 25-30°C, the carbon source was 99% pure sodium acetate, and the wastewater introduced was groundwater from the Longnan Zudong rare earth mine in Ganzhou City, Jiangxi Province. The concentrations of ammonia nitrogen, nitrate, nitrite, pH, DO and other factors of the reactor effluent were sampled regularly to detect changes. After the denitrification performance was stabilized, the metals and rare earth elements in the effluent and biofilm were detected. The metal concentration was determined by inductively coupled plasma emission spectrometry (ICP-MS: Agulient 7800, USA). The detection indicators included 24 items: Y, Dy, Nd, Gd, Er, La, Sm, Yb, Ho, Ce, Pr, Tb, Tm, Lu, Eu, Ca, K, Mg, Mn, Se, Sr, Zn, Pb, and Al.

[0115] The hydrochemical characteristics of the groundwater in the above rare earth mines are: ammonia nitrogen: 91.72±2.55mg / L, nitrate nitrogen: 83.81±4.33mg / L, nitrous nitrogen: 0.24±0.34mg / L, total nitrogen: 186.84±10.84mg / L, chemical oxygen demand is almost 0, strongly acidic (pH is 4.95±0.22), rich in rare earth elements and heavy metals;

[0116] As shown in Table 11.1, after the experiment, the concentrations of all rare earth elements and some toxic metal elements decreased significantly, and the removal rates were basically above 90%. In addition, the rare earth elements on the biofilm were significantly enriched (Table 11.2), among which the enrichment concentration of Y was as high as 1244.09 mg / L. This shows that the HNAD bacterial agent of the present invention has a strong ability to resist rare earth elements and metal stress, which is also one of the important reasons why the HNAD bacterial agent does not need pretreatment in treating rare earth mine groundwater, and has an absolute advantage over traditional microbial denitrification technology.

[0117] Table 11.1 Changes in concentrations of metals and rare earth elements in groundwater of rare earth mines before and after HNAD bacterial agent treatment

[0118]

[0119] Table 11.2 Concentrations of rare earth elements and metals on HNAD biofilm after the experiment

[0120]

[0121] The above examples demonstrate that the HNAD bacterial agent has obvious advantages in underground denitrification in rare earth mines. The present invention provides a systematic method for remediating nitrogen pollution in groundwater of rare earth mines. From the screening, compounding, domestication to practical application of the HNAD bacterial community, each step has been carefully designed and optimized to ensure the efficiency and stability of the remediation process. At the same time, the present invention also takes into account the long-term stability of the HNAD bacterial community, providing a reliable technical guarantee for practical engineering applications.

[0122] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a heterotrophic nitrification-aerobic denitrification bacterial agent, characterized in that: The following steps are involved: S1. Indigenous microbial sludge will be extracted from the environment of the sewage treatment plant around the rare earth mining area, and the indigenous microbial sludge will be inoculated into a heterotrophic nitrification enrichment medium for cultivation for 7-10 days, and the screening is completed to obtain heterotrophic nitrification and denitrification bacteria; S2. The obtained heterotrophic nitrification and denitrification bacteria with an MLVSS of 1.4-1.6 mg / L and an aerobic denitrification bacteria solution with a concentration of 5-6 g / L are mixed at an inoculation volume ratio of (2-5):1, and then inoculated into a heterotrophic nitrification-aerobic denitrification enrichment medium to obtain a composite HNAD bacterial flora; S3. The obtained composite HNAD bacterial community is inoculated into a biofilm reactor to construct a HNAD biofilm reactor with stable biofilm, and then the rare earth mine groundwater from the source area of ​​the indigenous microbial sludge is injected into the HNAD biofilm reactor to domesticate the HNAD bacterial community for no less than 3 days; S4. Collect and obtain HNAD bacterial agent.

2. The preparation method according to claim 1, characterized in that: In steps S1 and S2, the C / N ratio of the culture solution in the culture medium is 4-5, and the pH value is controlled at 7-8.

3. The preparation method according to claim 1, characterized in that: The culture conditions in S1 and S2 were: temperature of 25-30°C and dissolved oxygen of 3-5 mg / L.

4. The preparation method according to claim 1, characterized in that: The composition of the culture solution in the heterotrophic nitrification enrichment medium in S1 includes: 0.90-0.95 g / L NaHCO3, 0.34-0.38 g / L KH2PO4, 0.34-0.38 g / L NH4Cl, 0.27-0.31 g / L MgSO4·7H2O and 0.0010-0.0012 g / L CaCl2, 0.75-0.8 g / L CH3COONa, and the pH value is 7-8; and / or The composition of the culture medium in the heterotrophic nitrification-aerobic denitrification enrichment medium in S2 includes: 0.90-0.95 g / L NaHCO3, 0.41-0.45 g / L KNO3, 0.34-0.38 g / L KH2PO4, 0.45-0.50 g / L NH4Cl, 0.27-0.31 g / L MgSO4·7H2O and 0.0010-0.0012 g / L CaCl2, 1.18-1.22 g / L CH3COONa, and the pH is 7-8.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The rare earth mining area surrounding sewage treatment plant is a local sewage treatment plant in Longnan Zudong rare earth mining area, Ganzhou City, Jiangxi Province; and / or the aerobic denitrifying bacteria is Qiangwei 99.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The chemical characteristics of the rare earth mine groundwater described in S3 are: ammonia nitrogen: 91.72±2.55 mg / L, nitrate nitrogen: 83.81±4.33 mg / L, nitrous acid: 0.24±0.34 mg / L, total nitrogen: 186.84±10.84 mg / L, chemical oxygen demand is 0, pH is 4.95±0.22, and it is rich in rare earth elements and heavy metals.

7. The preparation method according to any one of claims 1 to 4, characterized in that: When constructing the HNAD biofilm reactor in S3: the biological fixed filler is put into the organic glass cylindrical reactor at a filling rate of 60-70%, the aeration rate is set to 0.12-0.18L / (L·min), the C / N ratio is set to 4-5, the temperature is 25-30℃, and the HRT is 6-8h; the HNAD bacterial community is stably formed by ventilating for 1-3 days.

8. The heterotrophic nitrification-aerobic denitrification bacterial agent obtained according to the preparation method according to any one of claims 1 to 7.

9. The heterotrophic nitrification-aerobic denitrification bacterial agent according to claim 8, characterized in that: The dominant genera of the heterotrophic nitrification-aerobic denitrification agent include Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira. Preferably, Thauera, SM1A02, unclassified_Rhodobacteraceae, Nitrosomonas and Nitrospira account for more than 20% of the bacteria in the heterotrophic nitrification-aerobic denitrification agent.

10. The heterotrophic nitrification-aerobic denitrification bacterial agent according to claim 8, characterized in that: The heterotrophic nitrification-aerobic denitrification bacteria agent grows with ammonia nitrogen, nitrate or nitrite as a nitrogen source.

11. Use of the heterotrophic nitrification-aerobic denitrification bacterial agent according to any one of claims 8 to 10 in the remediation of nitrogen-contaminated rare earth mine groundwater.

12. A method for remediating nitrogen-contaminated rare earth mine groundwater, characterized in that: The following steps are involved: Inoculating the heterotrophic nitrification-aerobic denitrification bacterial agent according to any one of claims 8 to 10 into a biofilm reactor to construct a HNAD biofilm reactor with stable biofilm; Then the nitrogen-contaminated rare earth mine groundwater to be remediated is injected into the HNAD biofilm reactor for operation; Or the remediation method comprises the following steps: introducing the heterotrophic nitrification-aerobic denitrification bacterial agent described in any one of claims 8 to 10 into the nitrogen-contaminated rare earth mine groundwater that needs to be remediated.

13. The repair method according to claim 12, characterized in that: Inoculating the heterotrophic nitrification-aerobic denitrification bacteria into the biofilm reactor at a volume ratio of 1% to 3% of the volume of the biofilm reactor; and / or When constructing the HNAD biofilm reactor, the biological fixed filler is put into the organic glass cylindrical reactor at a filling rate of 60-70%, the aeration rate is set to 0.12-0.18L / (L·min), the C / N ratio is set to 4-5, the temperature is 25-30°C, and the HRT is 6-8h; the HNAD bacteria are stably formed by stoic exposure for 1-3 days.

Images