A method for repairing nitrogen-polluted groundwater by coupling biological repair with a groundwater circulation well technology

Abstract

The present application relates to a kind of biological repair and groundwater circulation well technology coupling repair nitrogen contaminated groundwater method, belong to groundwater pollution site control and repair technical field.In the nitrogen pollution area groundwater is set with the circulation well being connected with surface, circulation well is sequentially provided with upper screen section, packer, lower screen section and wellhead is provided with cover and is equipped with aeration hole etc..After fixing heterotrophic nitrification-aerobic denitrification bacteria group S60 on vinylon silk carrier, it is fixed in groundwater circulation well, to establish coupling repair system and complete repair work.In which, by the continuous oxygen supply of surface air pump, the driving effect of heterotrophic nitrification-aerobic denitrification bacteria group S60 in circulation well to groundwater, can expand its scope of action, and improve denitrification treatment efficiency, shorten repair cycle, while reducing or avoiding further pollution to the environment, effectively realize the repair of nitrogen pollutants in groundwater.

Description

Technical Field

[0001] The present invention relates to the technical field of groundwater contaminated site control and remediation, and in particular to a method for remediating nitrogen-contaminated groundwater by coupling bioremediation with groundwater circulation well technology. Background Art

[0002] At present, traditional technologies for treating organic pollutants in groundwater, such as landfill excavation, pumping and heat treatment, are widely used, but they are costly and may cause significant disturbance to the environment. In contrast, groundwater in situ remediation technology has the advantages of high pollutant removal efficiency, short remediation cycle, and easy control of secondary pollution. It does not require groundwater extraction and treatment, and can significantly reduce the disturbance of the remediation process to the environment. In particular, groundwater circulation well (GCW) technology drives water circulation through steam stripping, continuously injects air into the aquifer, increases the dissolved oxygen content, and provides a superior growth environment for aerobic microorganisms. However, due to the complex and changeable hydrochemistry, hydrogeology and environmental conditions of groundwater, traditional nitrification and denitrification processes and new anaerobic ammonia oxidation, short-range nitrification and denitrification, simultaneous nitrification and denitrification methods are difficult to effectively control their denitrification effects in underground environments, and their oxygen supply conditions need to be adjusted at any time to achieve ideal denitrification performance.

[0003] To address these challenges, heterotrophic nitrification-aerobic denitrification (HN-AD) technology, an emerging biological denitrification technology, shows great potential in underground environments due to its sustainable oxygen supply and easy control. This technology demonstrates efficient denitrification performance, adaptability to extreme environments, the ability to utilize organic substrates, and the ability to simultaneously nitrify and denitrify. To further improve the denitrification efficiency and application scope of HN-AD technology, this paper proposes a new in situ groundwater remediation method that combines HN-AD technology with GCW technology. This method aims to optimize the denitrification effect and range of action of the HN-AD strain by providing the required oxygen through GCW technology. Summary of the Invention

[0004] 1. Technical problem to be solved by the invention:

[0005] In light of the numerous limitations and shortcomings of existing technologies, the present invention aims to provide a coupled process combining groundwater circulation wells with heterotrophic nitrification and aerobic denitrification. This process, primarily used for remediating nitrogen-contaminated groundwater, addresses the low denitrification efficiency, long remediation cycles, and susceptibility to secondary pollution associated with traditional methods. Implementation of the present invention can effectively improve the treatment efficiency of nitrogen-contaminated groundwater, shorten the remediation cycle, and reduce or avoid further environmental pollution.

[0006] 2. Technical solution:

[0007] A first aspect of the present invention provides a method for remediating nitrogen-contaminated groundwater by coupling bioremediation with groundwater circulation well technology, comprising the following steps:

[0008] Step 1, preparing vinylon fibers loaded with heterotrophic nitrification-aerobic denitrification bacteria: placing the vinylon fibers into a sequencing batch bioreactor containing heterotrophic nitrification-aerobic denitrification bacteria, aerobic aeration to maintain dissolved oxygen at 3.0-5.0 mg / L, and culturing at 20-30° C. for 1-2 days;

[0009] Step 2: Set up one or more circulation wells in the area where groundwater nitrogen contamination remediation is required;

[0010] Step 3, fixing the vinylon yarn loaded with heterotrophic nitrification-aerobic denitrification bacteria in step 1 inside the groundwater circulation well;

[0011] Step 4: Adjust the height of the circulation well relative to the groundwater level and the aeration flow rate in the circulation well to form a three-dimensional water circulation system, so that the groundwater to be repaired flows through the vinylon fiber loaded with heterotrophic nitrification-aerobic denitrification bacteria to establish a repair system for the repair of nitrogen-contaminated groundwater.

[0012] Furthermore, the heterotrophic nitrifying and aerobic denitrifying bacterial community is bacterial community S60, which was deposited on August 12, 2024, at the General Microbiology Center of the China National Committee for the Preservation of Microorganisms and Cultures, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO. 31650. Testing showed that the composite bacterial community S60 was viable. The proposed taxonomic name for the composite bacterial community S60 is Halomonas sp.

[0013] Furthermore, the height of the circulation well relative to the groundwater level is adjusted to 40-50 cm, and the aeration flow rate is controlled at 0.3-0.8 m 3 / h.

[0014] Furthermore, the wellhead of the circulation well is provided with a cover and an aeration hole, the aeration pipe passes through the aeration hole and enters the circulation well, the aeration pipe is connected to an air pump at one end outside the circulation well, and is connected to an aeration head at one end inside the circulation well; wherein, air is introduced into the circulation well by the air pump to keep the dissolved oxygen in the circulation well at 2.0-6.0 mg / L.

[0015] Furthermore, one or more of glycogen, alcohols, and acids are added to the circulation well as a carbon source every 1 to 10 days.

[0016] Preferably, sodium acetate solution is injected into the circulation well every 2 days.

[0017] The second aspect of the present application provides a remediation device coupling a groundwater circulation well with a heterotrophic nitrification-aerobic denitrification process.

[0018] The materials and equipment required by the device also include a simulation tank, a peristaltic pump, an aeration pipe, an in-well aeration head, an air pump, and a gas flow meter. Those skilled in the art can reasonably adjust the device as needed.

[0019] Further, the heterotrophic nitrification-aerobic denitrification bacterial community is bacterial community S60, which needs to be activated and fixed. The specific method is as follows: the vinylon filament is placed in a sequencing batch bioreactor containing bacterial community S60, aerobic aeration is performed to maintain the dissolved oxygen at 3.0-5.0 mg / L, and the culture is performed at 20-30℃ for 1-2 days.

[0020] Further, by adjusting the relative groundwater level of the circulation well to 40-50 cm and controlling the aeration flow rate in the circulation well to 0.3-0.8 m 3 / h, a three-dimensional water circulation system is formed, which makes the groundwater to be remediated flow through the vinylon filament loaded with the heterotrophic nitrification-aerobic denitrification bacterial community.

[0021] The third aspect of the present application provides an application of a groundwater circulation well coupled with a heterotrophic nitrification-aerobic denitrification device in remediation of a nitrogen-polluted groundwater environment.

[0022] The fourth aspect of the present application provides a domestication method of bacterial community S60, which comprises the following steps:

[0023] Step A: inoculate landfill leachate into a culture medium, activate the treatment, and obtain a bacterial solution;

[0024] Step B: transfer the above bacterial solution to a sequencing batch bioreactor containing a culture solution, control the DO concentration to be 3.0-5.0 mg / L by a gas flow meter, domesticate and culture at 20-30℃ until the removal of NH4 + -N by the bacterial community reaches a stable state, collect the bacterial solution, and obtain a composite bacterial community with heterotrophic nitrification-aerobic denitrification function.

[0025] Further, the more detailed preparation method in step B is as follows:

[0026] a. According to the change of NH4 + -N in the domestication process, the domestication process is divided into three stages, i.e., stage I, stage II, and stage III; different concentrations of NH4 + -N and COD are injected in each stage.

[0027] b. The bacteria solution obtained in step 1 is transferred to a sequencing batch bioreactor containing the stage I culture solution, and the DO concentration is controlled to be 3.0-5.0 mg / L by a gas flow meter, and the bacteria are acclimated and cultured at 25°C;

[0028] c. During the acclimation process, 30%-50% of the bacterial suspension is taken out every 24 hours, and an equal amount of fresh culture solution is replaced;

[0029] d. The bacterial solution samples collected periodically are used to determine the concentrations of TN, NH4 + -N and COD;

[0030] e. When the concentrations of TN, NH4 + -N and COD change little, the next stage, i.e., stage I enters stage II, and higher concentrations of NH4 + -N and COD are injected in stage II;

[0031] f. Steps c and d are repeated until the concentrations of TN, NH4 + -N and COD change little, the next stage, i.e., stage II enters stage III, and higher concentrations of NH4 + -N and COD are injected in stage III;

[0032] g. Until the removal of NH4 + -N by the bacterial population reaches a stable state, the bacterial solution is collected, and a bacterial population with HN-AD function is obtained;

[0033] Further, in the above steps, the concentrations of NH4 + -N and COD injected in stage I are 50 mg / L±2.0 mg / L and 500 mg / L±20 mg / L, respectively; the concentrations of NH4 + -N and COD injected in stage II are 100 mg / L±4.0 mg / L and 1000 mg / L±40 mg / L, respectively; and the concentrations of NH4 + -N and COD injected in stage III are 200 mg / L±8.0 mg / L and 2000 mg / L±80 mg / L, respectively.

[0034] Further, the compound bacterial population with heterotrophic nitrification-aerobic denitrification function is obtained by acclimating for 60 days in step B of the above method, and is named as bacterial population S60.

[0035] The fifth aspect of the present application provides a method for repairing nitrogen pollutants in groundwater by using the bacterial population S60.

[0036] 3. The present application has the following beneficial effects:

[0037] (1) The present invention couples groundwater circulation well technology with heterotrophic nitrification-aerobic denitrification process to construct a biological denitrification reactor that can be used in underground environment. By continuously supplying oxygen, the necessary growth conditions are provided for the S60 bacterial community, thereby improving its denitrification efficiency and shortening the repair period of groundwater nitrogen pollution; for example, after 28 days, NH4 + -N concentration dropped from 154mg / L to 35.15mg / L; NO3 - -N concentration decreased from 4.57 mg / L to 2.40 mg / L (on the 120th day, NO3 - -N concentration dropped to 0.40 mg / L).

[0038] (2) Promote the circulation of groundwater through circulation well technology, expand the scope of bacterial community action, and effectively achieve the remediation of nitrogen pollutants in groundwater over a larger area.

[0039] (3) The present invention cleverly uses the circulation well technology to continuously supply oxygen to the bacterial colony S60, allowing it to achieve ideal denitrification performance without the need to frequently adjust the oxygen supply conditions, which can greatly reduce the subsequent maintenance costs.

[0040] (4) The present invention uses a vinylon yarn loaded with S60 bacteria to maintain a stable remediation effect over a 120-day groundwater remediation process without the need to replace the vinylon yarn or add S60 bacteria. This significantly reduces maintenance costs and improves the long-term stability of the system.

[0041] (5) The biological denitrification reactor constructed by the present invention is easy to construct in an underground environment and has low cost.

[0042] (6) The technology provided by the present invention avoids the risk of secondary pollution and is an in-situ remediation technology with high efficiency and good safety, which has broad application prospects and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a sequencing batch bioreactor (SBR) device of the S60 bacterial colony of the present invention.

[0044] Figure 2(a)-Figure 2(c) Figure 2(a) shows the changes in nitrogen concentrations and carbon source utilization during the acclimation of denitrification functional bacteria. + -N concentration changes; Figure 2 (b) shows the changes of TN and NO2 during the acclimation of denitrification functional bacteria. - -N and NO3 - -N concentration changes; Figure 2(c) shows the changes in chemical oxygen demand (COD) during the acclimation of denitrifying functional bacteria.

[0045] Figure 3 This is the composition structure of the S60 bacterial community.

[0046] Figure 4 For the experimental device materials of the present invention, Figure 4 a is medium sand (26-40 mesh), Figure 4 b is the actual circulation well and its design drawing. Figure 4 c is a biological carrier (vinylon silk).

[0047] Figure 5 This is a physical diagram of the device for coupling the heterotrophic nitrification-aerobic denitrification process of the groundwater circulation well of the present invention.

[0048] Figure 6 Schematic diagram of the device for coupling the groundwater circulation well with the heterotrophic nitrification-aerobic denitrification process of the present invention.

[0049] Figure 7 This is the changing pattern of TN on days 0, 1, 2 and 4 during the operation of the simulation tank after the implementation of the present invention.

[0050] Figure 8 This is the changing pattern of TN on the 8th, 12th, 16th and 20th days during the operation of the simulated tank after the implementation of the present invention.

[0051] Figure 9 This is the changing pattern of TN on the 28th, 36th, 48th and 60th days during the operation of the simulated tank after the implementation of the present invention.

[0052] Figure 10 This is the changing pattern of TN on the 90th and 120th days during the operation of the simulated tank after the implementation of the present invention.

[0053] Figure 11 The NH4 on the 0th and 1st days during the operation of the simulated tank after the implementation of the present invention + -The changing pattern of N.

[0054] Figure 12 The NH4 values ​​on the 2nd, 4th, 8th and 12th days of the simulated tank operation after the present invention is implemented are shown in Table 1. + -The changing pattern of N.

[0055] Figure 13 The NH4 values ​​on the 16th, 20th, 28th and 36th days of the simulated tank operation after the present invention is implemented are shown in Table 1. + -The changing pattern of N.

[0056] Figure 14 The NH4 values ​​on the 48th, 60th, 90th and 120th days of the simulated tank operation after the implementation of the present invention are shown in the figure. + -The changing pattern of N.

[0057] Figure 15 The NO3 values ​​on the 0th, 2nd, 8th and 16th days of the simulation tank operation after the present invention is implemented - -The changing pattern of N.

[0058] Figure 16 The change rule of NO3 - -N in the simulation tank running process after the implementation of the application on the 28th, 48th, 90th and 120th day.

[0059] Figure 17 The change rule of NO2 - -N in the simulation tank running process after the implementation of the application on the 0th, 2nd, 8th and 16th day.

[0060] Figure 18 The change rule of NO2 - -N in the simulation tank running process after the implementation of the application on the 28th, 48th, 90th and 120th day. DETAILED DESCRIPTION

[0061] The application provides a coupling process combining a groundwater circulation well and heterotrophic nitrification-aerobic denitrification, taking a composite bacterial group S60 as a representative. The composite bacterial group S60 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO. 31650.

[0062] In practical application, the S60 bacterial group needs to be fixed on a carrier material and placed in the circulation well, and its growth and metabolic activity in the complex groundwater environment will directly affect the remediation effect of the contaminated groundwater layer. Before the actual site remediation application, it is necessary to carry out indoor simulation experiments based on the data of batch experiments for groundwater remediation. That is, the simulation experiments carried out in the laboratory are an important step to understand and optimize the field application technology.

[0063] In the preliminary pollution site investigation, it was found that a groundwater monitoring well located in the Guozhai Domestic Waste Simple Landfill Site in Leizhou City, Zhanjiang City, Guangdong Province was polluted by NH4 + -N and NO3 - -N, with concentrations as high as 154 mg / L and 4.57 mg / L respectively, belonging to Class V water standard. Considering comprehensively, in order to effectively remediate this pollution, the application adopts the coupling method of HN-AD process and groundwater circulation well technology. By injecting air into the circulation well, groundwater circulation is driven to provide the required oxygen for the S60 bacterial group, and promote its growth and reproduction. At the same time, the circulation well is filled with vinylon silk, which has the characteristics of large specific surface area, no biological toxicity, flexibility and loose structure, etc., which is beneficial to the construction of a biofilm reactor. In addition, the application provides the operation process parameters of the groundwater circulation well, and provides the influence of groundwater level and aeration flow on the circulation effect. Then, the change rule of TN, NH4+ -N, NO3 - -N and NO2 - These analyses help verify the effectiveness of the coupling technology disclosed in the present invention in remediating groundwater contaminated by mixed nitrogen sources.

[0064] The following is the experimental setup constructed by the present invention:

[0065] 1. Construction of the simulation tank: The simulation tank is 1.57m×0.1m×0.60m in size and is equipped with an inlet and outlet area. Add 26-40 mesh medium sand to the simulation tank until the height reaches 55cm and tamp it during the filling process to form a simulated aquifer. The groundwater level is controlled at 45cm and the flow rate is 0.6m / d. The air pump is connected to the gas flow meter at 0.7m 3 A circulation well is operated with an aeration flow rate of / h to ensure continuous water circulation. The peristaltic pump transports wastewater to the simulation tank and controls the groundwater flow rate. The air pump introduces air into the circulation well, continuously providing oxygen to the S60 and powering the three-dimensional circulation of groundwater within the aquifer.

[0066] 2. Circulation Well Setup: Two gauze-wrapped circulation wells connected to the surface were installed at one-third and two-thirds of the aquifer. Each well consisted of two nested well pipes, one inside and one outside, equipped with an upper screen section, a packer, and a lower screen section. Perforated floral pipes were installed within the well to facilitate water flow. The upper and lower screen sections were separated by a packer into upper and lower sections. The wellhead was capped and equipped with aeration holes. Aeration pipes entered the circulation well from here and were connected to an air pump and aeration head through the pipes to introduce air to promote water circulation, thus forming a three-dimensional water circulation system underground.

[0067] Figure 4 b provides the size and proportion of the circulation well. Those skilled in the art should know that the size of the circulation well can be adjusted according to the actual situation on site. During the adjustment process, it is necessary to ensure that the inner well of the circulation well is higher than the holes around the outer pipe.

[0068] 3. Construction of biological denitrification reactor: The vinylon yarn was placed in a sequencing batch bioreactor containing 1 L of simulated tank experimental activation medium, inoculated with S60 bacterial solution at a 4% inoculum volume, and the DO concentration was controlled at 4.0 mg / L by bottom aeration. The culture was carried out at 25°C for 24 h, and the OD 600 When the pH reaches approximately 1.0, the bacterial community is immobilized on the carrier. Subsequently, the vinylon yarn is wrapped around the aeration pipe and fixed in the circulation well to construct a biological denitrification reactor suitable for underground environments.

[0069] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and process, which is intended to be used to explain the present invention, but the implementation does not constitute a limitation to the present invention.

[0070] Example 1: Domestication method of denitrification functional bacteria group S60.

[0071] Inoculate 5 mL of landfill leachate into a 100 mL conical flask containing sterile LB medium. Seal the flask with a sterile breathable membrane and incubate at 30°C with constant shaking at 150 rpm for 24 hours. Add another 5 mL of the bacterial suspension to the LB medium for activation. Once the medium becomes noticeably turbid, transfer the suspension to fresh LB medium for enrichment. Repeat this process twice.

[0072] Under sterile conditions, the enriched bacterial solution was transferred to an SBR containing 1 L of stage I culture medium, and the DO concentration was controlled at 4.0 mg / L by a gas flow meter. The culture was acclimated at 25 °C (as Figure 1 As shown). According to the NH4 + -N changes, the enrichment and acclimation process of the functional flora was divided into three stages, and different concentrations of NH4 were injected into each stage. + -N and COD. Stage I (0-5 days, NH4 + -N and COD were 50mg / L±2.0mg / L and 500mg / L±20mg / L, respectively. In stage II (6th-20th day, NH4 + -N and COD were 100mg / L±4.0mg / L and 1000mg / L±40mg / L respectively) and stage III (21st-60th day, NH4 + -N and COD were 200 mg / L±8.0 mg / L and 2000 mg / L±80 mg / L, respectively).

[0073] After 60 days of acclimation, a mixed bacterial community with HN-AD function under aerobic conditions was obtained, named S60 bacterial community. During the acclimation period, 400 mL of bacterial suspension was taken out every 24 hours and replaced with an equal amount of fresh HN medium. The bacterial suspension samples collected regularly were used to determine TN and NH4 + -N, NO3 - -N, NO2 --N and COD concentrations. In phase I, bacterial cultures were collected on days 0, 3, and 5 of acclimatization; in phase II, bacterial cultures were collected on days 10, 15, and 20 of acclimatization; and in phase III, bacterial cultures were collected on days 30, 45, and 60 of acclimatization. The nine sample points were named S0, S3, S5, S10, S15, S20, S30, S45, and S60, respectively. The collected bacterial cultures were centrifuged at 8000 rpm for 10 minutes, and the bacterial pellet was retained. After washing three times with sterile water, it was stored at -80°C for molecular biology analysis.

[0074] Example 2: Acclimation process of denitrification functional bacteria group S60.

[0075] Under aerobic conditions, the acclimation process is divided into three stages, and different concentrations of NH4 are injected into each stage. + -N and COD. In stage I, such as Figure 2a As shown, NH4 in SBR + -N concentration was 51.36 mg / L, and on the 3rd day, TN and NH4 + -N concentrations dropped to 38.15 mg / L and 31.05 mg / L, respectively. On the 5th day, NH4 + The removal rates of -N and COD reached 57.97% and 57.09% respectively ( Figure 2c shown).

[0076] On the 9th day, if Figure 2b As shown, TN and NH4 + -N increased slightly. On the 18th day, NO3 - -N concentration rose to 15.45 mg / L, and on the 20th day, TN and NO3 - -N concentrations dropped to 29.56 mg / L and 5.65 mg / L respectively, and the COD removal rate reached 87.76%.

[0077] In stage III, the influent NH4 + -N and COD concentrations increased to 200 mg / L and 2000 mg / L respectively. From the 21st to the 37th day of acclimation, NH4 + -N removal rate increased from 40.07% to 79.96%, NO3 - -N concentration gradually decreased from 42.14 mg / L to 10.58 mg / L, NO2 - -N concentration fluctuated in the range of 0.5mg / L to 5.23mg / L. From 55th to 60th day, NH4 + -N was almost completely removed, and the bacterial community was + -N removal reaches a stable state. In the stable state, the bacterial community has a strong ability to remove TN and NH4 +-N removal rates were 91.33% and 100% respectively, at which time NO2 - -N and NO3 - The HN-AD function was successfully domesticated at the concentration of 0.5 mg / L and 2.1 mg / L, and the S60 bacterial colony was named S60 and deposited in the General Microbiology Center of China Microorganism Culture Collection Committee with the deposit number: CGMCC NO.31650. Figure 3 As shown in the figure, Halomonas and Aliidiomarina were confirmed as the two dominant genera in the S60 bacterial community, accounting for a total of 89.81%.

[0078] Example 3: Activation and enrichment method of S60 bacterial population.

[0079] 10 mL of S60 bacterial culture solution stored at -80°C was added to a SBR containing 1000 mL of sterile activation medium. The DO concentration was controlled at 4.0 mg / L by bottom aeration and cultured at 25°C for 24 hours. After activation, the bacterial culture was centrifuged at 8000 rpm for 10 minutes, washed with pH 6.8 phosphate buffer, and resuspended in sterile water. The optical density of the cells was adjusted at 600 nm (OD 600 ) to 1.0±0.2 to prepare standardized bacterial suspension for subsequent experiments.

[0080] Related culture media:

[0081] (1) Sterile activation medium: (NH4)2SO4 0.472 g, CH3COONa 1.28 g, Weiss salt solution 50 mL, ultrapure water 950 mL.

[0082] (2) Weiss salt solution: K2HPO4 5.0 g, MgSO4·7H2O 2.5 g, NaCl 2.5 g, FeSO4·7H2O 0.05 g, MnSO4·4H2O 0.05 g, ultrapure water 1000 mL, pH 5.0.

[0083] Example 4: Denitrification effect of S60 bacterial colony after three passages.

[0084] Subculture once: Take out the S60 bacterial culture solution stored at -80℃ and add 10mL to a SBR containing 1000mL of sterile activation medium. Control the DO concentration to 4.0mg / L by bottom aeration and incubate at 25℃ for 24h. Centrifuge the cultured bacterial solution at 8000r / min for 10min, wash the cells with pH 6.8 phosphate buffer, and resuspend them in sterile water. Adjust the cell optical density at 600nm (OD 600) to 1.0±0.2, which is the bacterial solution 1 after one passage and activation.

[0085] Second passage: Take 10 mL of the bacterial solution 1 and add it to a SBR containing 1000 mL of sterile activation medium. Control the DO concentration to 4.0 mg / L by bottom aeration and incubate at 25°C for 24 h. Centrifuge the cultured solution at 8000 rpm for 10 min, wash the cells with pH 6.8 phosphate buffer, and resuspend them in sterile water. Adjust the optical density of the cells at 600 nm (OD 600 ) to 1.0±0.2, which is the bacterial solution 2 after secondary activation.

[0086] Subculture three times: Take 10 mL of culture 2 and add it to 1000 mL of sterile activation medium in an SBR. Control the DO concentration to 4.0 mg / L by bottom aeration and incubate at 25°C for 24 h. Centrifuge the cultured culture at 8000 rpm for 10 min, wash the cells with pH 6.8 phosphate buffer, resuspend them in sterile water, and adjust the optical density of the cells at 600 nm (OD 600 ) to 1.0±0.2, which is the bacterial solution 3 after three activations.

[0087] In the denitrification effect experiment, bacterial solutions 1, 2, and 3 were added to NH4 + -N and NO3 - -N and NO2 - -N as a mixed nitrogen source. In the SBR, the DO concentration was controlled to maintain at 4.0 mg / L, sodium acetate was used as the only carbon source, the C / N ratio was 10:1, the initial pH was 7.0, and the culture was carried out at 25°C for 72 h.

[0088] Related culture media mentioned above:

[0089] (1) Sterile activation medium: (NH4)2SO4 0.472 g, CH3COONa 1.28 g, Weiss salt solution 50 mL, ultrapure water 950 mL.

[0090] (2) Weiss salt solution: K2HPO4 5.0 g, MgSO4·7H2O 2.5 g, NaCl 2.5 g, FeSO4·7H2O 0.05 g, MnSO4·4H2O 0.05 g, ultrapure water 1000 mL, pH 5.0.

[0091] (3) Ammonia nitrogen, nitrate and nitrite mixed nitrogen source culture medium: (NH4)2SO4 0.943 g, KNO3 0.144 g, NaNO2 0.099 g, CH3COONa 3.072 g, Vickers salt solution 50 mL, ultrapure water 950 mL.

[0092] The results are shown in Table 1.

[0093] Table 1 Denitrification effect of S60 bacterial colony after three passages

[0094] Number of passages <![CDATA[NH4 + -N removal rate]]> TN removal rate 1 100％ 87.56％ 2 100％ 86.98％ 3 99.26％ 86.25％

[0095] The above results show that the denitrification effect of the S60 bacterial community is still very stable after three generations, that is, the S60 bacterial community can be stably inherited.

[0096] Example 5: Construction methods of simulation tanks, circulation wells, bioreactors and experimental devices.

[0097] 1. Simulation Tank Construction: The simulation tank measures 1.57m x 0.1m x 0.60m and is equipped with an inlet and outlet area. Medium sand with a mesh size of 26-40 is added to the simulation tank to a height of 55cm and compacted during filling to form a simulated aquifer. The groundwater flow rate is controlled at 0.6m / d, and a circulation well is operated by controlling the groundwater level and the aeration flow rate of the air pump.

[0098] Among them, the peristaltic pump is used to transport wastewater to the simulation tank and control the flow rate of groundwater; the air pump is used to introduce air into the circulation well, continuously provide oxygen to S60, and provide power for the three-dimensional circulation of groundwater in the aquifer.

[0099] 2. Construction of Circulation Wells: Two gauze-wrapped circulation wells connected to the surface were installed at one-third and two-thirds of the aquifer. Each well consisted of two nested well pipes, one inside and one outside. Each well had an upper screen section, a packer, and a lower screen section. Perforated floral pipes were installed within the well to facilitate water flow. The upper and lower screen sections were separated by a packer into upper and lower zones. The wellhead was capped and equipped with aeration holes. Aeration pipes entered the circulation well from here and were connected to an air pump and aeration head to introduce air to promote water circulation, thus forming a three-dimensional water circulation system underground.

[0100] Figure 4 b provides the size and proportion of the circulation well. Those skilled in the art should know that the size of the circulation well can be adjusted according to the actual situation on site. During the adjustment process, it is necessary to ensure that the inner well of the circulation well is higher than the holes around the outer pipe.

[0101] 3. Construction of bioreactor:

[0102] The vinylon yarn (such as Figure 4 c) Place the SBR containing 1 L of simulated tank experimental activation medium, inoculate S60 bacterial suspension at a 4% inoculum volume, control the DO concentration to 4.0 mg / L through bottom aeration, and culture at 25°C for 24 h. The OD 600about 1.0. At this time, the vinylon filaments are taken out and wound on the aeration pipe, and fixed in the circulating well (e.g. Figure 4 b) in the application.

[0103] Vinylon filaments: a common name for those skilled in the art, referring to a three-dimensional elastic material, wherein the three-dimensional elastic material used in the application has the following parameters: diameter 150 mm, and the amount used is 2 meters per cubic meter.

[0104] The simulated tank experiment activation medium is: (NH4)2SO40.727 g, KNO30.033 g, CH3COONa 2.03 g, Vilsmeier salt solution 50 mL, ultrapure water 950 mL, pH value 5.73, 121 ℃ high-pressure sterilization for 30 min. Among them, the Vilsmeier salt solution is: K2HPO45.0 g, MgSO4·7H2O 2.5 g, NaCl 2.5 g, FeSO4·7H2O 0.05 g, MnSO4·4H2O 0.05 g, ultrapure water 1000 mL, pH value 5.0.

[0105] 4. Construction of experimental device: According to the classification and naming standards of soil in the appendix of the “Water Supply Hydrogeological Exploration Specification”, the medium of the aquifer in this experiment is selected to be medium sand (26-40 mesh) (e.g. Figure 4 a). As shown in Figure 5 and Figure 6 , the front of the simulation tank is provided with 32 branch holes as sampling holes, which are distributed in four rows (A-D rows), and each row contains 8 sampling holes (numbered from A1 to A8, and so on). Each sampling hole is connected by a pressure measuring tube for measuring water level changes. In order to monitor the water level changes of each sampling point, a three-way water valve is installed in each sampling hole: one end is connected to the sampling hole of the simulation tank, one end is fixed to the pressure plate through a silica gel hose, and the other end is used as a sampling port and connected to a double-head pagoda valve to control water outlet. Two groundwater circulation wells are placed at monitoring points 3 and 6 of the simulation tank. Before the experiment, medium sand is added to the simulation tank to a height of 55 cm. After filling, saturated calcium carbonate solution is injected from the bottom of the simulation device. Every time the water level rises 2-5 cm, the water injection is stopped and stabilized for a period of time, and then the water injection is continued until the water level covers the surface of the sand layer, and it is allowed to stabilize overnight. After the sand layer is completely saturated, stop water injection, adjust the groundwater level using a peristaltic pump and a drain pipe, and after 24 h of stabilization, the simulation experiment is started.

[0106] Example 6: Exploration of the influence of groundwater level and aeration flow on the groundwater circulation effect.

[0107] The present application Figure 4b provides the size and proportion of the circulation well. Those skilled in the art should know that the size of the circulation well can be adjusted according to the actual situation on site. When the size of the circulation well is adjusted, the relative height of the circulation well provided by the present invention relative to the groundwater level and the aeration flow in the circulation well should also be adjusted accordingly, so as to eventually form a three-dimensional water circulation system and allow the groundwater to be repaired to flow through the vinylon yarn loaded with heterotrophic nitrification-aerobic denitrification bacteria.

[0108] The present invention is based on Figure 4 The experiment was conducted by adjusting the size of the circulation well in b to explore the influence of groundwater level and aeration flow on the groundwater circulation effect.

[0109] In the actual field experiment, since the natural groundwater level cannot be adjusted, the depth of the aeration well underground needs to be adjusted to simulate the change of the groundwater level relative to the aeration well position. Tap water is used to simulate groundwater, and the groundwater flow rate is controlled at 0.6m / d. The aeration position remains unchanged at 5cm above the bottom edge of the inner well. The initial groundwater level is adjusted to 35cm, 40cm, 45cm and 50cm respectively. At different aeration flow rates of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8m 3 / h, and after stable operation for 5 minutes, observe and record the changes in the water level height in each pressure measuring tube before and after aeration.

[0110] When the groundwater level is 35cm, the aeration flow rate is 0.1m 3 / h~0.8m 3 / h range, the water level heights at each monitoring point remained almost unchanged, and no groundwater circulation was formed.

[0111] When the groundwater level is 40cm, the groundwater level is flush with the lower edge of the sieve hole on the outer well of the aeration well, and the aeration flow rate is ≤0.6m 3 / h, groundwater circulation cannot be formed. However, the aeration flow rate is adjusted to 0.7m 3 / h and 0.8m 3 / h. After 5 minutes of stable operation, the circulation flow state of groundwater is basically stable and a stable cycle can be formed.

[0112] When the groundwater level is 45cm, the water level is aligned with the center of the sieve hole on the upper part of the aeration well. 3 / h aeration flow rate can form a cycle. On this basis, the greater the aeration flow rate, the larger the range of the cycle. 3 / h has the best circulation effect.

[0113] When the groundwater level is 50cm, the groundwater level is flush with the upper edge of the sieve hole on the outer well of the aeration well. 3 / h~0.8m 3 / h range of experiments found that only when the aeration flow rate was 0.3m 3 / h and 0.4m 3 / h, groundwater can form a cycle in the simulation tank. At the same time, when the aeration flow rate is ≥0.5m 3 / h, the maximum water level of the water flow exceeds the surface of the air-filling zone, thus affecting the circulation effect.

[0114] By adjusting the initial groundwater level to 35cm, 40cm, 45cm and 50cm, the experimental results show that the groundwater level is controlled at 45cm and the aeration flow rate is controlled at 0.7m 3 / h, the best groundwater circulation effect can be achieved (as shown in Table 2).

[0115] Table 2 Influence of groundwater level and aeration flow on groundwater circulation effect

[0116]

[0117]

[0118] Note: < indicates that circulation cannot be formed; = indicates that circulation can be formed and the maximum water level does not exceed the surface of the vadose zone; > indicates that circulation can be formed and the maximum water level exceeds the surface of the vadose zone.

[0119] Example 7: Simulation tank operation method and parameters.

[0120] The experimental water was prepared with reference to the water chemical parameters of the groundwater at the Guozhai domestic waste simple landfill in Leizhou City, Zhanjiang City, Guangdong Province. The tap water was used to prepare saturated calcium carbonate water to simulate the groundwater. The pH value was 5.73, and the NH4 + -N is 154mg / L, NO3 - The nitrogen-N content was 4.57 mg / L, and 5% Vickers salt solution was added. Sodium acetate solution was injected into the circulation well every two days. Water was introduced from the bottom right side of the sand box to saturate the sand box, simulating a contaminated aquifer. Once the simulated contaminated groundwater was roughly evenly distributed throughout the sand box, water was stopped and the water level was maintained stable to prevent the formation of a hydraulic gradient.

[0121] The vinylon silk carrier loaded with S60 bacteria was fixed in the circulation well, and the groundwater level was controlled at 45 cm, the groundwater flow rate was 0.6 m / d, and the flow rate was 0.7 m / d. 3 After starting the device, water samples were taken from the sampling holes on the side of the sand box at 1d, 2d, 4d, 8d, 12d, 16d, 20d, 28d, 36d, 48d, 60d, 90d and 120d, and the TN and NH4 contents in the simulated aquifer were recorded and analyzed. + -N, NO3- -N and NO2 - -N's repair effects, processes, and rules.

[0122] Example 8: Simulating the changing pattern of TN during tank operation.

[0123] like Figures 7-10 As shown in the study of remediating contaminated aquifers using a coupled HN-AD process and groundwater circulation well technology, the TN concentration patterns of an indoor simulated test tank were monitored over 120 days. Over time, the remediation effect was most pronounced on the left side of the test tank. This is because the water flow is from right to left, with groundwater contaminated with mixed nitrogen sources first passing through the right circulation well for initial treatment before flowing to the left circulation well for further treatment. This design not only achieves gradual improvement in water quality but also optimizes the overall system's efficiency and effectiveness through phased treatment. At the start of the experiment (day 0), the entire test tank was completely contaminated with mixed nitrogen sources, with a TN concentration of 159.5 mg / L. After one day of operation, the TN concentration in the test tank generally showed a downward trend, demonstrating the effectiveness of this coupled technology in removing groundwater contaminated with mixed nitrogen sources. By the second day of operation, the TN concentration at the outlet had decreased to 82.53 mg / L, with a removal efficiency of 48.26%. However, the TN concentration remained high at 129.49 mg / L at the lower right portion of the inlet, due to the mixed nitrogen source contaminated groundwater entering the simulation tank from this location. By the fourth day of operation, experimental data showed that the TN concentration at the outlet had further decreased to 40.28 mg / L, and the removal efficiency had increased to 74.75%. However, the TN concentration on the right side remained significantly higher than on the left side, revealing the spatial unevenness of nitrogen pollution removal efficiency in the early stages. By the 12th day of operation, TN concentrations decreased significantly, despite the circulation radius not directly covering both ends of the sand box. This is likely due to the S60 bacterial colony being stripped by air during aeration and entering the simulation tank medium, where it slowly undergoes nitrification or denitrification. By the 28th day of operation, the difference in TN concentration between the left and right sides of the tank had significantly decreased, indicating that the mixed nitrogen source contaminated groundwater had begun to distribute evenly within the tank, and the removal efficiency at the outlet reached 72.90%. From the 28th day to the 120th day at the end of the experiment, the TN concentration in the simulation tank changed little. On the 120th day, the TN concentration at position A1 was 43.14 mg / L, with a removal efficiency of 73%, indicating that nitrogen removal in the simulation tank and the TN concentration in the incoming water were roughly balanced. As the experimental period extended, the TN concentration in the tank became less noticeable.

[0124] Example 9: NH4 during the simulation tank operation + -The changing pattern of N.

[0125] like Figures 11-14 As shown, during the 120-day operation, NH4+ -N concentration variation, experimental results show that NH4 + The distribution of -N concentration gradually decreases from the water inlet to the water outlet. In particular, the repair effect on the left side of the simulation tank is more significant. At the beginning of the experiment (0d), the NH4 + The initial concentration of -N was 154 mg / L. After only 1 day of operation, the NH4 + -N concentration decreased to 84.73mg / L, and the removal rate reached 44.98%. During this period, NH4 + The significant difference in -N concentration is mainly due to the secondary biological treatment of the mixed nitrogen source contaminated groundwater in the left circulation well. Similar to the change in TN concentration, the distribution of pollutants at this time shows spatial inhomogeneity. + -N concentration difference decreased, and by the 4th day, the NH4 + -N concentration further decreased to 34.78mg / L, and the removal rate increased to 77.42%. Although the initial test results showed that there was heterogeneity in the environment, the data on the 4th day showed that the NH4 + -N concentration is low, and the NH4 + -N concentration is also low. This observation further proves that the coupling technology is effective in promoting the NH4 + -N pollutants are gradually evenly distributed and effectively removed. From the 4th day to the 20th day, the NH4 + -N removal rate was maintained between 74.49% and 77.79%, and the NH4 + -N concentration is also relatively uniform.

[0126] When running to 28 days, the NH4 on the left and right sides of the tank + -N concentration difference is significantly reduced, and its distribution begins to be uniform. At this time, the NH4 + -N concentration dropped to 35.12 mg / L, and the removal rate increased to 77.19%. From the 28th day to the 120th day at the end of the experiment, the NH4 + -N concentration changes very little, and this change is similar to the TN concentration. + -N concentration was 33.85 mg / L, achieving a removal rate of 78.02%. This shows that the S60 bacterial community and GCW coupling technology are effective in reducing NH4 + -N load has obvious effect. However, this technology fails to reduce NH4 +-N concentration dropped to the Class I water standard specified in the Groundwater Quality Standard (GB / T14848-2017), which is below 0.02 mg / L. This finding emphasizes that under the existing technical framework, the NH4 + The challenge of deep removal of NH4-N also highlights the need for further research and improvement of this technology to achieve more stringent environmental protection standards. + -N concentration, continue to add sodium hypochlorite at a rate of 2g / L. Under the action of sodium hypochlorite, the simulation tank continues to run for 1 day, and the NH4 + -N concentration dropped from 33.85mg / L to 0mg / L, which meets the Class I water standard specified in the Groundwater Quality Standard (GB / T 14848-2017). Sodium hypochlorite can effectively reduce NH4 + -N concentration, but it is prone to secondary pollution. Therefore, this method is suitable for low-concentration nitrogen-contaminated environments, and a small amount can achieve the purpose of denitrification. In addition, this study provides an important reference for similar contaminated groundwater treatment technologies, helping to optimize treatment parameters in practical applications, thereby improving the efficiency and effectiveness of treatment systems.

[0127] Example 10: NO3 during the simulation tank operation - -The changing pattern of N.

[0128] like Figure 15 、 16 As shown, during the 120-day operation, NO3 - -N concentration variation. At the beginning of the experiment (0d), NO3 - -N concentration is 4.57mg / L. When running for 2 days, the total NO3 - -N concentration decreased slightly. On the 8th day, NO3 - -N concentration increased significantly, and the concentration at the outlet was 7.40 mg / L. This is because the S60 bacteria can only convert part of the NH4 + -N is oxidized to NO3 - -N, part of NO3 - -N is further reduced to NO2 - -N or N2. Although NO3 - -N was continuously injected into the tank, but by the 16th day, NO3 - -N accumulation decreased. On the 28th day, NO3 - -N concentration further decreased to 2.40 mg / L. From the 28th day, NO3 - -N accumulation began to decrease gradually, and at the 120th day, NO3 -- N concentration dropped to 0.40 mg / L. This process showed that the simulated tank had gradually enhanced the treatment capacity of NO3 - - N, and finally achieved a significant reduction of NO3 - - N concentration. The effect of NO2 - - N concentration was observed, which could reach the Class I water standard of the Groundwater Quality Standard (GB / T 14848-2017), i.e., NO3 - - N concentration was not more than 2.0 mg / L.

[0129] Example 11: Change rule of NO2 - - N during the operation of the simulated tank.

[0130] As shown in Figs. Figure 17 , 18 , the change rule of NO2 - - N concentration was observed during the operation of 120 days. With the oxidation of NH4 + - N to NO3 - - N, NO3 - - N was further reduced to NO2 - - N or N2, and NO2 - - N accumulation occurred at different degrees at different time points. At the 28th day, the accumulation of NO2 - - N was most significant at the left side of the circulation well, with a concentration of 1.6 mg / L. At the 120th day, the accumulation of NO2 - - N was reduced, and the NO2 - - N concentration in the tank reached the Class III water standard of the Groundwater Quality Standard (GB / T 14848-2017), i.e., NO2 - - N concentration was not more than 1.0 mg / L.

Claims

1. A method for remediating nitrogen-contaminated groundwater by coupling bioremediation with groundwater circulation well technology, characterized in that: The following steps are involved: Step 1: preparing a vinylon yarn loaded with heterotrophic nitrifying-aerobic denitrifying bacteria: placing the vinylon yarn into a sequencing batch bioreactor containing heterotrophic nitrifying-aerobic denitrifying bacteria, aerobic aeration to maintain dissolved oxygen at 3.0-5.0 mg / L, and culturing at 20-30° C. for 1-2 days; wherein the heterotrophic nitrifying-aerobic denitrifying bacteria is bacteria group S60, which is deposited in the General Microorganism Center of the China National Center for Microbiological Culture Collection with a deposit number of CGMCC NO. 31650; Step 2: In an area where groundwater nitrogen contamination remediation is required, one or more groundwater circulation wells are set up; the wellhead of the circulation well is provided with a cap and an aeration hole, an aeration pipe passes through the aeration hole and enters the circulation well, and the aeration pipe is connected to an air pump at one end outside the circulation well and an aeration head at one end inside the circulation well; wherein air is introduced into the circulation well by the air pump to maintain the dissolved oxygen in the circulation well at 2.0-6.0 mg / L; Step 3, fixing the vinylon yarn loaded with heterotrophic nitrification-aerobic denitrification bacteria in step 1 inside the groundwater circulation well; Step 4: Adjust the height of the circulation well relative to the groundwater level to 40-50 cm and the aeration flow rate in the circulation well to 0.3-0.8 m 3 / h, forming a water circulation system, allowing the groundwater to be repaired to flow through the vinylon fibers loaded with heterotrophic nitrification-aerobic denitrification bacteria to repair the nitrogen-contaminated groundwater.

2. The method according to claim 1, characterized in that One or more of glycogen, alcohols, and acids are added to the circulation well as a carbon source every 1 to 10 days.

3. The method according to claim 2, characterized in that Sodium acetate solution was injected into the circulation well every 2 days.

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