Method for repairing nitrogen-polluted underground water by coupling bioremediation and underground water circulation well technology

Abstract

The invention relates to a method for repairing nitrogen-polluted underground water through coupling of bioremediation and an underground water circulation well technology, and belongs to the technical field of underground water polluted site control and remediation. A circulating well communicated with the ground surface is arranged in underground water in a nitrogen pollution area, an upper screen section and a packer are sequentially arranged in the circulating well from top to bottom, a sealing cover is arranged on a lower screen section, and aeration holes and the like are formed in a well mouth. The heterotrophic nitrification-aerobic denitrification flora S60 is fixed on a vinylon yarn carrier and then fixed in an underground water circulation well, so that a coupling remediation system is established and remediation work is completed. Through continuous oxygen supply of the surface air pump, under the driving effect of the circulating well on the underground water, the action range of the heterotrophic nitrification-aerobic denitrification flora S60 can be expanded, the denitrification treatment efficiency is improved, the remediation period is shortened, meanwhile, further pollution to the environment is reduced or avoided, and remediation of nitrogen pollutants in the underground water is effectively achieved.

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 biological remediation 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, the 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 need to adjust their oxygen supply conditions at any time to achieve ideal denitrification performance.

[0003] In response to the above problems, heterotrophic nitrification-aerobic denitrification (HN-AD) technology, as an emerging biological denitrification technology, shows great potential in underground environments due to its sustainable oxygen supply and easy control. The technology demonstrates efficient denitrification performance, adaptability to extreme environments, ability to utilize organic substrates, and the ability to simultaneously nitrify and denitrify. In order to further improve the denitrification efficiency and application scope of HN-AD technology, the present invention proposes a new in-situ groundwater remediation method that combines HN-AD technology with GCW technology, aiming to provide the required oxygen through GCW technology to optimize the denitrification effect and scope of action of HN-AD strains. Summary of the invention

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

[0005] In view of the many limitations and shortcomings of the prior art, the present invention aims to provide a coupling process combining groundwater circulation wells with heterotrophic nitrification-aerobic denitrification. The process is mainly used to repair nitrogen-contaminated groundwater, solving the problems of low denitrification efficiency, long repair cycle and easy secondary pollution in traditional methods. Through the implementation of the present invention, the treatment efficiency of nitrogen-contaminated groundwater can be effectively improved, the repair cycle can be shortened, and further pollution to the environment can be reduced or avoided.

[0006] 2. Technical solution:

[0007] The 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 silk loaded with heterotrophic nitrification-aerobic denitrification bacteria: placing the vinylon silk 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 pollution remediation is required;

[0010] Step 3, fixing the vinylon fiber 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 nitrification-aerobic denitrification bacterial community is bacterial community S60, which was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on August 12, 2024, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.31650. The test result is survival. The proposed classification name of the composite bacterial community S60 is Halomonas sp.

[0013] Furthermore, the height of the circulation well relative to the groundwater level was adjusted to 40-50 cm, and the aeration flow rate was 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, an aeration pipe passes through the aeration hole and enters the circulation well, an 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 through 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 invention provides a remediation device that couples a groundwater circulation well with a heterotrophic nitrification-aerobic denitrification process. The device is a biological denitrification reactor that is connected to the surface and is set in a groundwater environment in a nitrogen-polluted area. The biological denitrification reactor includes a groundwater circulation well and a vinylon fiber loaded with heterotrophic nitrification-aerobic denitrification bacteria.

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

[0019] Furthermore, the heterotrophic nitrification-aerobic denitrification bacterial community is bacterial community S60, and bacterial community S60 needs to be activated and fixed. The specific method is: putting the vinylon fiber into a sequencing batch bioreactor containing bacterial community S60, aerobic aeration to maintain the dissolved oxygen at 3.0-5.0 mg / L, and culturing at 20-30°C for 1-2 days.

[0020] Furthermore, by adjusting the height of the circulation well relative to the groundwater level to 40-50 cm and controlling the aeration flow rate in the circulation well to 0.3-0.8 m 3 / h, forming a three-dimensional water circulation system, wherein the water circulation system allows the groundwater to be repaired to flow through the vinylon fibers loaded with heterotrophic nitrification-aerobic denitrification bacteria.

[0021] A third aspect of the present invention provides an application of a groundwater circulation well coupled heterotrophic nitrification-aerobic denitrification device in remediating a nitrogen-contaminated groundwater environment.

[0022] A fourth aspect of the present invention provides a method for acclimating a bacterial colony S60, comprising the following steps:

[0023] Step A, taking garbage leachate and inoculating it into a culture medium, performing activation treatment, and obtaining a bacterial solution;

[0024] Step B: Transfer the above bacterial solution to a sequencing batch bioreactor filled with culture medium, control the DO concentration to 3.0-5.0 mg / L by a gas flow meter, and cultivate at 20-30°C until the bacterial colony is resistant to NH4 + When the removal of -N reaches a stable state, the bacterial liquid is collected to obtain a composite bacterial community with heterotrophic nitrification-aerobic denitrification functions.

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

[0026] a. According to the NH4 + -N changes, the acclimation process is divided into three stages, namely stage I, stage II, and stage III; different concentrations of NH4 + -N and COD;

[0027] b. Transfer the bacterial solution obtained in step 1 to a sequencing batch bioreactor filled with the culture medium of stage I, control the DO concentration to 3.0-5.0 mg / L by a gas flow meter, and cultivate at 25°C;

[0028] c. During the acclimation process, remove 30% to 50% of the bacterial suspension every 24 hours and replace it with an equal amount of fresh culture medium;

[0029] d. The bacterial liquid samples collected regularly are used to determine TN and NH4 + -N and COD concentrations;

[0030] e. When TN, NH4 + When the concentration of -N and COD changes little, it can enter the next stage, that is, stage I enters stage II, and a higher concentration of NH4 is injected in stage II than in stage I. + -N and COD;

[0031] f. Repeat steps c and d until TN and NH4 + When the concentration of -N and COD changes little, it can enter the next stage, that is, stage II enters stage III, and a higher concentration of NH4 is injected in stage III than in stage II. + -N and COD;

[0032] g. Until the bacterial flora is resistant to NH4 + When the removal of -N reaches a stable state, the bacterial solution is collected to obtain a bacterial colony with HN-AD function;

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

[0034] Furthermore, in the present invention, a composite bacterial community with heterotrophic nitrification-aerobic denitrification function is obtained by acclimating for 60 days in the above step B, and is named bacterial community S60.

[0035] A fifth aspect of the present invention provides a method for applying bacterial colony S60 to remediate nitrogen pollutants in groundwater.

[0036] 3. The present invention achieves 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 dropped from 4.57mg / L to 2.40mg / L (at 120 days, NO3 - -N concentration dropped to 0.40 mg / L).

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

[0039] (3) The present invention ingeniously 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 the vinylon yarn loaded with S60 bacteria to maintain a stable repair effect during the groundwater repair process of up to 120 days, without the need to replace the vinylon yarn or add S60 bacteria. This can significantly reduce maintenance costs and improve 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, and 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 shows the changes in various nitrogen concentrations and carbon source utilization during the domestication of denitrification functional bacteria. Figure 2a NH4 + - Changes in N concentration; Figure 2b TN and NO2 in the process of domestication of denitrification functional bacteria - -N and NO3 - - Changes in N concentration; Figure 2c This is the change of chemical oxygen demand (COD) during the domestication of denitrification functional bacteria.

[0045] Figure 3This 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 It is a schematic diagram of the device for coupling the heterotrophic nitrification-aerobic denitrification process of the groundwater circulation well of the present invention.

[0049] Figure 7 This is the variation pattern of TN on the 0th, 1st, 2nd and 4th days during the operation of the simulated tank after the implementation of the present invention.

[0050] Figure 8 This is the variation 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] Fig. 9 This is the variation 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] Fig.10 This is the variation pattern of TN on the 90th and 120th days during the operation of the simulated tank after the implementation of the present invention.

[0053] Fig.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] Fig.12 The NH4 on the 2nd, 4th, 8th and 12th days during the operation of the simulated tank after the implementation of the present invention + -The changing pattern of N.

[0055] Fig.13 The NH4 on the 16th, 20th, 28th and 36th days during the operation of the simulated tank after the implementation of the present invention + -The changing pattern of N.

[0056] Fig.14 The NH4 at 48, 60, 90 and 120 days during the operation of the simulated tank after the implementation of the present invention + -The changing pattern of N.

[0057] Fig.15 The NO3 on the 0th, 2nd, 8th and 16th days during the operation of the simulated tank after the implementation of the present invention --The changing pattern of N.

[0058] Fig.16 The NO3 on the 28th, 48th, 90th and 120th days during the operation of the simulated tank after the implementation of the present invention - -The changing pattern of N.

[0059] Fig.17 The NO2 on the 0th, 2nd, 8th and 16th days during the operation of the simulated tank after the implementation of the present invention - -The changing pattern of N.

[0060] Fig.18 The NO2 on the 28th, 48th, 90th and 120th days during the operation of the simulated tank after the implementation of the present invention - -The changing pattern of N. DETAILED DESCRIPTION

[0061] The present invention provides a coupling process combining groundwater circulation well and heterotrophic nitrification-aerobic denitrification, represented by composite bacterial community S60, which is deposited in China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC NO.31650.

[0062] Considering that in actual applications, the S60 bacterial community needs to be fixed on a carrier material and placed in a circulation well, its growth and metabolic activities in a complex groundwater environment will directly affect the remediation effect of the contaminated groundwater layer. Before actual site remediation application, indoor simulation experiments based on batch experimental data are very necessary for groundwater remediation. In other words, simulation experiments conducted in the laboratory are an important step in understanding and optimizing field application technologies.

[0063] During the preliminary investigation of the contaminated site, it was found that a groundwater monitoring well at the Guozhai domestic waste simple landfill in Leizhou City, Zhanjiang City, Guangdong Province was contaminated by NH4 + -N and NO3 --N pollution, with concentrations as high as 154mg / L and 4.57mg / L respectively, which belongs to Class V water standards. After comprehensive consideration, in order to effectively repair this pollution, this study adopted the coupling method of HN-AD process and groundwater circulation well technology. By injecting air into the circulation well, the groundwater circulation is driven to provide the required oxygen for the S60 bacterial community, promoting its growth and reproduction. At the same time, the circulation well is filled with vinylon silk. This material has the characteristics of large specific surface area, non-biological toxicity, flexibility and loose structure, which is conducive to the construction of biofilm reactors. In addition, the present invention provides the operating process parameters of the groundwater circulation well, and provides the influence of groundwater level and aeration flow on the circulation effect. Next, the TN and 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 device constructed by the present invention:

[0065] 1. Construction of simulation tank: The size of the simulation tank is 1.57m×0.1m×0.60m, with water inlet and outlet areas. 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. Control the groundwater level at 45cm and the flow rate at 0.6m / d. Connect the air pump to the gas flow meter at 0.7m 3 / h aeration flow rate to run the circulation well to ensure the continuous circulation of the water body. 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.

[0066] 2. Circulation well setting: Two circulation wells wrapped in gauze connected to the surface are set at one-third and two-thirds of the aquifer. Each well is composed of two inner and outer well pipes in a combined nested manner, equipped with an upper screen section, a packer and a lower screen section. A perforated flower pipe is set in the well to promote water flow in and out. The upper and lower screen sections are separated into upper and lower areas by a packer. The wellhead is provided with a cover and an aeration hole. The aeration pipe enters the circulation well from here. The air pump and the aeration head are connected through the aeration pipe to introduce air to promote water circulation, thereby 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 of the circulation well, it is necessary to ensure that the inner well of the circulation well is higher than the flower holes around the outer pipe.

[0068] 3. Construction of biological denitrification reactor: Vinylon was placed in a sequencing batch bioreactor containing 1L of simulated tank experimental activation medium, inoculated with S60 bacterial solution at a 4% inoculation rate, 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 ratio reaches about 1.0, the bacterial flora is immobilized on the carrier. After that, the vinylon yarn is wound around the aeration pipe and fixed in the circulation well to construct a biological denitrification reactor suitable for underground environment.

[0069] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and process are given, which are 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] Take 5 mL of landfill leachate and inoculate it into a conical flask containing 100 mL of sterile LB medium, seal it with a sterile breathable membrane, and culture it at 30°C with a constant temperature of 150 r / min for 24 hours. Take another 5 mL of bacterial suspension and add it to the LB medium for activation. When the medium becomes obviously turbid, transfer it to fresh LB medium for enrichment, and repeat the operation 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 (eg Figure 1 According to the NH4 + -N changes, the enrichment and domestication 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 are 200mg / L±8.0mg / L and 2000mg / L±80mg / 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 stage I, bacterial liquid was collected on the 0th, 3rd, and 5th day of acclimatization; in stage II, bacterial liquid was collected on the 10th, 15th, and 20th day of acclimatization; in stage III, bacterial liquid was collected on the 30th, 45th, and 60th day of acclimatization. The 9 sample points were named S0, S3, S5, S10, S15, S20, S30, S45, and S60, respectively. The collected bacterial liquid was centrifuged at 8000r / min for 10min, the bacterial precipitate was retained, and after washing 3 times with sterile water, it was stored in a -80℃ refrigerator for molecular biology analysis.

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

[0075] Under aerobic conditions, the acclimation process was divided into three stages, with different concentrations of NH4 injected in 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 third day, TN and NH4 + -N concentrations dropped to 38.15mg / L and 31.05mg / L, respectively. On the 5th day, NH4 + The removal rates of -N and COD reached 57.97% and 57.09% respectively (e.g. 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 acclimatization, 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 the 55th to the 60th day, NH4 + -N was almost completely removed, and the bacterial flora was + -N removal reaches a stable state. In the stable state, the bacterial community has a high efficiency in removing TN and NH4 + -N removal rates were 91.33% and 100% respectively, at which time NO2 - -N and NO3 - -N concentration was 0.5mg / L and 2.1mg / L, and the bacterial flora with HN-AD function was successfully domesticated and named S60 bacterial flora. The S60 bacterial flora was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number: CGMCC NO.31650. Figure 3 As shown, 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 flora.

[0079] Take 10mL of the S60 bacterial culture solution stored at -80℃ and add it to the SBR containing 1000mL of sterile activation medium. The DO concentration is controlled to 4.0mg / L by bottom aeration and cultured at 25℃ for 24h. After activation, centrifuge the bacterial solution at 8000r / min for 10min, wash the bacteria with pH 6.8 phosphate buffer, resuspend them in sterile water, and adjust the cell optical density at 600nm (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 flora after three passages.

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

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

[0086] Subculture three times: Take 10mL of bacterial solution 2 and add it to SBR containing 1000mL of sterile activation medium. Control the DO concentration to 4.0mg / L by bottom aeration and culture at 25℃ for 24h. Centrifuge the cultured bacterial solution at 8000r / min for 10min, wash the bacteria with pH 6.8 phosphate buffer, and resuspend them in sterile water. Adjust the cell optical density at 600nm wavelength (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 was used as a mixed nitrogen source in the culture medium. In the SBR, the DO concentration was controlled to be maintained at 4.0 mg / L, sodium acetate was used as the only carbon source, the C / N ratio was 10:1, the initial pH value was 7.0, and the culture was carried out at 25°C for 72 h.

[0088] Related culture media:

[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 subcultures

[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: Methods for constructing simulation tanks, circulation wells, bioreactors and experimental devices.

[0097] 1. Construction of simulation tank: The size of the simulation tank is 1.57m×0.1m×0.60m, with water inlet and outlet areas. 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. Control the groundwater flow rate to 0.6m / d, and operate the circulation well by controlling the groundwater level and the aeration flow 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 for S60, and provide power for the three-dimensional circulation of groundwater in the aquifer.

[0099] 2. Construction of circulation wells: Two circulation wells wrapped in gauze connected to the surface are set at one-third and two-thirds of the aquifer. Each well is composed of two inner and outer well pipes in a nested combination, equipped with an upper screen section, a packer and a lower screen section. A perforated flower pipe is set in the well to promote water flow in and out. The upper and lower screen sections are separated into upper and lower areas by a packer. The wellhead is capped and equipped with aeration holes. The aeration pipe enters the circulation well from here. The air pump and the aeration head are connected through the aeration pipe to introduce air to promote water circulation, thereby forming a three-dimensional water circulation system underground.

[0100] 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. During the adjustment of the circulation well, it is necessary to ensure that the inner well of the circulation well is higher than the flower holes around the outer pipe.

[0101] 3. Construction of bioreactor:

[0102] The vinylon yarn (such as Figure 4 c) Place the mixture in a SBR containing 1 L of activation medium for the simulated tank experiment, inoculate with 4% of the S60 bacterial suspension, control the DO concentration to 4.0 mg / L through bottom aeration, and culture at 25°C for 24 h. 600 At this time, take out the vinylon yarn and wrap it around the aeration pipe and fix it in the circulation well (such as Figure 4 b) In.

[0103] Vilen silk: A common name used by technicians in this field, referring to three-dimensional elastic materials. The parameters of the three-dimensional elastic material used in the present invention are: diameter 150mm, usage amount is 2 meters per cubic meter.

[0104] Activation medium for simulated tank experiment: (NH4)2SO4 0.727g, KNO3 0.033g, CH3COONa 2.03g, Weiss salt solution 50mL, ultrapure water 950mL, pH value is 5.73, autoclave at 121℃ for 30min. Among them, Weiss salt solution: K2HPO45.0g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05g, MnSO4·4H2O0.05g, ultrapure water 1000mL, pH value is 5.0.

[0105] 4. Construction of experimental device: According to the classification and naming standards of soil in the appendix of the "Specifications for Water Supply Hydrogeological Exploration", this experiment chooses to use medium sand (26-40 mesh) as the aquifer medium (such as Figure 4 a). Figure 5 and Figure 6As shown in the figure, there are 32 branch holes on the front of the simulation tank, which are set as sampling holes. These sampling holes are distributed in four rows (AD 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 to measure the water level change. In order to monitor the water level change at each sampling point, each sampling hole is equipped with a three-way water diverter valve: one end is connected to the sampling hole of the simulation tank, one end is fixed to the pressure measuring plate through a silicone hose, and the other end is used as a sampling port, connected to a double-headed pagoda ball valve to control the water outflow. Two groundwater circulation wells are placed at monitoring points 3 and 6 of the simulation tank. Before the experiment, medium sand was added to the simulation tank to a height of 55 cm. After filling, saturated calcium carbonate solution was injected from the bottom of the simulation device. Whenever the water level rises by 2 to 5 cm, the water injection is suspended 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 is allowed to stabilize overnight. When the sand layer is fully saturated, water injection is stopped, and the groundwater level is adjusted using a peristaltic pump and a drainage pipe. After 24 hours of stabilization, the simulation experiment begins.

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

[0107] The present invention Figure 4 The size and proportion of the circulation well are provided. 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 to the groundwater level and the aeration flow rate in the circulation well provided by the present invention should also be adjusted accordingly, so that a three-dimensional water circulation system can be formed eventually, and the groundwater to be repaired can flow through the vinylon silk 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 to be 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, 0.8m 3 / h. After 5 minutes of stable operation, 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 at the top 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 circulate 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 Effects of groundwater level and aeration flow on groundwater circulation

[0116]

[0117]

[0118] Note: < means that circulation cannot be formed; = means that circulation can be formed and the maximum water level does not exceed the surface of the vadose zone; > means 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 according 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 - -N is 4.57 mg / L, and 5% Vickers salt solution is added. Sodium acetate solution is injected into the circulation well every 2 days. Water is introduced from the bottom of the right side of the sand box to saturate the sand box to simulate the contaminated aquifer. After the simulated contaminated groundwater is roughly evenly distributed in the sand box, water is stopped and the water level is kept stable without the formation of 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 in the simulated aquifer were recorded and analyzed. + -N、NO3 - -N and NO2 - -N's repair effects, processes and rules.

[0122] Example 8: Simulate the variation of TN during cell operation.

[0123] like Figures 7-10As shown in the figure, in the study of the coupling method of HN-AD process and groundwater circulation well technology to remediate contaminated aquifers, the change pattern of TN concentration was observed by monitoring the operation of the indoor simulation experimental tank within 120 days. It can be observed over time that the remediation effect on the left side of the simulation tank is the most significant. This is because the direction of water flow is from right to left, and the groundwater contaminated by mixed nitrogen sources first passes through the circulation well on the right for preliminary treatment, and then flows to the circulation well on the left for further treatment. This design not only achieves a gradual improvement in water quality, but also optimizes the treatment efficiency and effect of the entire system through staged treatment. At the beginning of the experiment (0d), the entire simulation tank was completely contaminated by the mixed nitrogen source, and the TN concentration was 159.5mg / L. After the simulation tank was running for 1d, the concentration of TN in the tank generally showed a downward trend, showing the effectiveness of this coupling technology in removing groundwater contaminated by mixed nitrogen sources. When it was running for 2d, the TN concentration at the outlet was reduced to 82.53mg / L, and the removal rate was 48.26%. However, the TN concentration in the lower right part of the water inlet is still high, reaching 129.49 mg / L, which is due to the mixed nitrogen source contaminated groundwater entering the simulation tank from this position. When running to the 4th day, the experimental data showed that the TN concentration at the outlet further dropped to 40.28 mg / L, and the removal rate increased to 74.75%, but the TN concentration on the right side was still significantly higher than that on the left side, revealing the uneven spatial distribution of nitrogen pollution removal efficiency in the early stage. When running to the 12th day, although the circulation radius did not directly cover both ends of the sand box, the TN concentration still dropped significantly, which may be due to the S60 bacteria being blown off by air during aeration, entering the simulation tank medium and slowly nitrifying or denitrifying. When running to the 28th day, the difference in TN concentration on the left and right sides of the tank decreased significantly, indicating that the mixed nitrogen source contaminated groundwater began to be evenly distributed in the tank, and the removal rate 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. At the 120th day, the TN concentration at position A1 was 43.14 mg / L, and the removal rate was 73%, indicating that the removal of nitrogen in the simulation tank and the TN concentration in the new water were roughly balanced. As the experimental period was extended, the change of TN concentration in the tank was no longer obvious.

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

[0125] like Figures 11 to 14 As shown, during the 120-day operation, NH4 + -N concentration changes, 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 one day of operation, the NH4 + -N concentration dropped 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 34.78 mg / 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 NH4 + -N concentration is low, and the NH4 + -N concentration is also lower. This observation further demonstrates the effectiveness of this coupling technique 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 + The -N concentration is also relatively uniform.

[0126] When running to 28 days, the NH4 + -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 when the experiment ended, the NH4 + -N concentration changes very little, similar to 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. 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, after the simulation tank continues to operate for 1 day, 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 easy to cause secondary pollution. Therefore, this method is suitable for low-concentration nitrogen pollution 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, which helps to optimize treatment parameters in practical applications, thereby improving the efficiency and effectiveness of the treatment system.

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

[0128] like Fig.15 , 16 As shown, during the 120-day operation, NO3 - -N concentration changes. At the beginning of the experiment (0d), NO3 - -N concentration was 4.57mg / L. After 2 days of operation, the total NO3 - -N concentration decreased slightly. On the 8th day of operation, 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 of operation, NO3 - -N concentration further dropped 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 0.40mg / L. This process shows that the simulation tank has a strong effect on NO3 - -N processing capacity gradually increases, eventually achieving a significant reduction in NO3 --N concentration can reach the Class I water standard specified in the Groundwater Quality Standard (GB / T 14848-2017), that is, NO3 - -N concentration should not exceed 2.0mg / L.

[0129] Example 11: NO2 during the simulation tank operation - -The changing pattern of N.

[0130] like Fig.17 , 18 As shown, during the 120-day operation, NO2 - -N concentration changes. With the NH4 + -N is partially oxidized to NO3 - -N, and NO3 - -N is further reduced to NO2 - -N or N2, with different levels of NO2 appearing at different time points - -N accumulation. On the 28th day, NO2 - -N accumulation was the most significant, with a concentration as high as 1.6 mg / L. - -N accumulation phenomenon is reduced, and NO2 - -N concentration meets the requirements of Class III water standard in the Groundwater Quality Standard (GB / T 14848-2017), that is, NO2 - -N concentration does not exceed 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 vinylon silk loaded with heterotrophic nitrification-aerobic denitrification bacteria: placing the vinylon silk 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; Step 2: In the area where groundwater nitrogen pollution remediation is required, one or more groundwater circulation wells are set up; Step 3, fixing the vinylon fiber 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 and the aeration flow rate in the circulation well to form a water circulation system, so that the groundwater to be repaired flows through the vinylon fiber loaded with heterotrophic nitrification-aerobic denitrification bacteria to repair the nitrogen-contaminated groundwater.

2. The method according to claim 1, characterized in that The heterotrophic nitrification-aerobic denitrification bacterial community is bacterial community S60, and bacterial community S60 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCCNO.31650.

3. The method according to claim 1, characterized in that Adjust the height of the circulation well relative to the groundwater level to 40-50cm, and control the aeration flow rate to 0.3-0.8m 3 / h.

4. 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.

5. The method according to claim 4, characterized in that Sodium acetate solution was injected into the circulation well every 2 days.

6. The method according to claim 1, characterized in that The wellhead of the circulation well is provided with a cover and an aeration hole, an aeration pipe passes through the aeration hole and enters the circulation well, one end of the aeration pipe outside the circulation well is connected to an air pump, and one end inside the circulation well is connected to an aeration head; wherein, air is introduced into the circulation well through the air pump to keep the dissolved oxygen in the circulation well at 2.0-6.0 mg / L.

7. A groundwater circulation well coupled heterotrophic nitrification-aerobic denitrification device, characterized in that: The device is a biological denitrification reactor connected to the ground surface and arranged in a groundwater environment in a nitrogen-polluted area. The biological denitrification reactor comprises a groundwater circulation well and vinyl fibers loaded with heterotrophic nitrification-aerobic denitrification bacteria.

8. The device according to claim 7, characterized in that The heterotrophic nitrification-aerobic denitrification bacterial community is bacterial community S60, and bacterial community S60 needs to be activated and fixed. The specific method is: putting the vinylon silk into a sequencing batch bioreactor containing bacterial community S60, aerobic aeration to maintain the dissolved oxygen at 3.0-5.0 mg / L, and culturing at 20-30° C. for 1-2 days.

9. The device according to claim 7, characterized in that By adjusting the height of the circulation well relative to the groundwater level to 40-50 cm and controlling the aeration flow rate in the circulation well to 0.3-0.8 m 3 / h, forming a three-dimensional water circulation system, allowing the groundwater to be repaired to flow through the bacterial community S60.

10. Use of the groundwater circulation well coupled heterotrophic nitrification-aerobic denitrification device as claimed in any one of claims 7 to 9 in remediating nitrogen-contaminated groundwater environments.

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