Application of salt-tolerant immobilized denitrifying functional bacteria agent in low-temperature salt-containing river water denitrification

Abstract

This invention discloses the application of a salt-tolerant immobilized denitrifying microbial agent in denitrification of low-temperature saline river water. The salt-tolerant immobilized denitrifying microbial agent is placed in a net bag and evenly suspended in the river channel. The salt-tolerant immobilized denitrifying microbial agent uses zeolite as the immobilization carrier, and the immobilized microorganisms are salt-tolerant denitrifying microbial agents enriched and screened from rivers flowing into the sea. The dosage of the salt-tolerant immobilized denitrifying microbial agent is 2 g·L⁻¹. ‑1 The dosage is the mass-to-volume ratio of the immobilized denitrifying bacterial agent to the river water. The low-temperature saline river water is saline river water with nitrate nitrogen as the main pollutant. The temperature of the low-temperature saline river water is 10℃~25℃. During the denitrification process, a carbon source is added to the river water to maintain denitrification efficiency. This invention has demonstrated its feasibility in practical engineering applications through pilot-scale experiments. It can effectively remediate river pollution and solve the problem of severe and difficult-to-remediate water pollution in saline rivers.

Description

Technical Field

[0001] This invention belongs to the field of surface water treatment, specifically relating to the application of a salt-tolerant immobilized denitrifying bacterial agent in denitrification of low-temperature saline river water. Background Technology

[0002] Currently, some progress has been made both domestically and internationally in isolating and screening denitrifying bacteria for river water purification. Studies have reported genera of bacteria capable of highly efficient denitrification, such as *Bacillus*, *Acinetobacter*, *Pseudomonas*, *Paracoccus*, and *Comamonas* (Wang Junli, 2025). However, these are mainly used for freshwater purification, and research on biological nitrogen removal from saline river water is still very limited. One study screened a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium, *Vibrio sinaloensis*, from an constructed wetland system, which can achieve 99.49% NH4+ removal at a salinity of 3%. + The removal rates of nitrogen (N) and total nitrogen (TN) can reach 61.99%. However, these studies mainly target ammonia nitrogen as the primary pollutant, while research on denitrification of river water under high salinity conditions, with nitrate nitrogen as the primary pollutant, is still very limited.

[0003] Since microbial removal of pollutants primarily relies on enzymatic catalysis, and enzymes are highly sensitive to temperature, enzyme activity decreases and pollutant removal efficiency declines below 15°C. Furthermore, nitrification and denitrification cease at temperatures below 8°C. Therefore, maintaining the denitrification capacity of microorganisms is crucial when river water temperature varies seasonally.

[0004] Microbial immobilization technology can mitigate the impact of the external environment on microorganisms, thereby improving their tolerance and the stability of pollutant degradation. Compared with suspended microbial agents, immobilized microorganisms are less likely to be lost in waterways, significantly reducing the dosage of microbial agents. Among them, adsorption immobilization technology using zeolite, diatomaceous earth, activated carbon, and sawdust as carriers is more suitable for long-term use in waterways due to the higher physical strength of the immobilized microbial particles. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and address the limited effectiveness of traditional microbial techniques in remediating saline, low-temperature surface water. It provides a salt-tolerant immobilized denitrifying bacterial agent for denitrification in low-temperature saline river water. Pilot-scale experiments have demonstrated its feasibility in practical engineering applications. This invention can effectively remediate river pollution and solve the problem of severe and difficult-to-remediate water pollution in saline rivers.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The application of a salt-tolerant immobilized denitrifying microbial agent in denitrification of low-temperature saline river water involves placing the salt-tolerant immobilized denitrifying microbial agent in a net bag and suspending it evenly in the river channel; the salt-tolerant immobilized denitrifying microbial agent uses zeolite as the immobilization carrier, and the immobilized microorganisms are salt-tolerant denitrifying microbial agents enriched and screened from rivers flowing into the sea.

[0008] Furthermore, the dosage of the salt-tolerant immobilized denitrifying bacterial agent is 2 g·L⁻¹. -1 The dosage is the mass-volume ratio of the immobilized denitrifying bacterial agent to the river water.

[0009] Furthermore, the low-temperature saline river water is a saline river water body with nitrate nitrogen as the main pollutant.

[0010] Furthermore, the temperature of the low-temperature saline river water is 10℃~25℃.

[0011] Furthermore, during the denitrification process, carbon sources are added to the river water to maintain denitrification efficiency.

[0012] This invention also provides a method for preparing a salt-tolerant immobilized denitrifying functional bacterial agent, which uses a zeolite carrier adsorption combined with chitosan encapsulation method to immobilize the denitrifying functional bacterial community, including:

[0013] (1) Adsorption and biofilm formation: The zeolite carrier is immersed in the culture medium, inoculated with denitrifying functional bacteria, and cultured to allow the microorganisms to fully biofilm on the zeolite carrier. After washing, an immobilized carrier loaded with functional microorganisms is obtained.

[0014] (2) Coating and embedding: Immerse the immobilized carrier obtained in step (1) in chitosan solution, let it stand for coating, and then drain the remaining chitosan solution.

[0015] (3) Crosslinking and fixation: Sodium sulfate solution is added to the immobilized carrier after step (2) for immersion. After static fixation, the remaining sodium sulfate solution is discharged to obtain salt-tolerant immobilized denitrifying bacterial agent with immobilized denitrifying bacterial community.

[0016] Furthermore, the chitosan solution is prepared by dissolving chitosan with a degree of deacetylation of 90% in a 1% acetic acid solution, thereby achieving a chitosan concentration of 1.5%.

[0017] The concentration of the sodium sulfate solution is 35 g·L⁻¹. -1 .

[0018] Furthermore, in step (1):

[0019] The particle size of the zeolite carrier is 3 mm;

[0020] The culture medium is sterile TXD medium; during immersion, the liquid level of the culture medium is 5 cm above the zeolite carrier, and the medium is stirred 1-2 times a day during the culture period. The medium is cultured at 30°C for 5-6 days under sterile conditions.

[0021] The inoculation amount of the denitrifying functional bacterial culture into TXD medium is 5% of its volume.

[0022] Furthermore,

[0023] In steps (2) and (3):

[0024] In step (2), the static film covering time is 20 minutes, and the carrier is stirred every 5 minutes during the static period;

[0025] In step (3), the sodium sulfate solution is added and allowed to stand for 20 minutes, during which the carrier is stirred every 5 minutes.

[0026] The present invention also provides a salt-tolerant immobilized denitrifying bacterial agent, which is prepared by the aforementioned preparation method.

[0027] Compared with the prior art, the beneficial effects of the technical solution of the present invention are:

[0028] 1. This invention employs zeolite-based immobilization technology to improve low-temperature tolerance and structural stability. Zeolite particles provide a favorable microenvironment for microorganisms, mitigating the direct impact of low external temperatures on them. At 10℃, the nitrate nitrogen removal rate of the immobilized group (43.46%±3.66%) was significantly higher than that of the suspended bacterial agent control group (17.74%±5.68%), and it also reduced the accumulation of nitrite nitrogen, resulting in more stable effluent quality.

[0029] 2. This invention employs a net-suspension method for application, thereby reducing costs and preventing secondary loss. Compared to suspended microbial agents, net suspension makes it less likely for immobilized microorganisms to be lost in river flow, significantly reducing the amount of microbial agent required and maintaining a high biological density within a fixed space, without causing secondary pollution.

[0030] 3. By employing salt-tolerant denitrifying bacteria screened at the estuary, the bottleneck of biochemical treatment in saline water is overcome. This solves the problem of inhibited activity of traditional freshwater denitrifying bacteria in saline water, enabling effective removal of pollutants even at a salinity of 1.36.

[0031] 4. With nitrate nitrogen as the main target pollutant, it can simultaneously remove nitrogen through aerobic denitrification under immobilized and low-temperature saline conditions. This solves the problem that most studies have focused on exploring the nitrification and denitrification reactions of denitrifying microorganisms under anaerobic conditions, and it is difficult to simultaneously tolerate salt and cold. Attached Figure Description

[0032] Figure 1 A graph showing the changes in nitrate nitrogen concentration in river water;

[0033] Figure 2 A graph showing the changes in nitrite nitrogen concentration in river water;

[0034] Figure 3 COD of river water Mn Concentration change graph;

[0035] Figure 4 This is a graph showing the average total nitrogen removal rate at different temperatures in river water. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0037] This embodiment provides an application of salt-tolerant immobilized denitrifying bacterial agent in low-temperature saline river water denitrification. The salt-tolerant immobilized denitrifying bacterial agent is placed in a pore size of approximately 1 mm. 2 Inside the net, the bacteria are evenly suspended in the river channel; the salt-tolerant immobilized denitrifying bacterial agent uses zeolite as the immobilization carrier, and the immobilized microorganisms are salt-tolerant denitrifying bacterial agents enriched and screened from rivers at the estuary.

[0038] Preferably, the preparation method of the immobilized denitrifying functional bacterial agent is as follows: the denitrifying functional bacterial agent is adsorbed and immobilized using a zeolite carrier adsorption + chitosan encapsulation method. The chitosan solution is prepared by dissolving 1.5% (w / w) chitosan with a 90% degree of deacetylation in a 1% (w / w) acetic acid solution, and the sodium sulfate solution concentration is 35 g·L⁻¹. -1The specific procedure is as follows: Take an appropriate amount of zeolite with a particle size of approximately 3 mm into a beaker, add sterile TXD culture medium until it covers the carrier by about 5 cm, and transfer the denitrifying functional bacterial culture to the culture medium at a 5% inoculation rate under sterile conditions. Seal the beaker opening with sealing film and rubber bands to prevent foreign matter from entering. Stir 1-2 times a day to ensure sufficient microbial biofilm formation, and incubate in a biochemical incubator at 30℃ for 5-6 days. Remove the fully biofilmed immobilized carrier and gently rinse it with sterile water to obtain the immobilized carrier loaded with functional microorganisms. Then, embed the zeolite loaded with functional microorganisms using the chitosan solution described above: Pour chitosan solution into the beaker containing the zeolite loaded with functional microorganisms until it covers the carrier, and let it stand for 20 minutes. This is the coating process. During this time, gently stir the carrier every 5 minutes to ensure more uniform mixing. After 20 minutes, discard the chitosan solution. Next, pour sodium sulfate solution into the beaker until it covers the carrier, and let it stand for 20 minutes. This process is the fixation process. During this process, gently stir the carrier every 5 minutes to make the mixture more uniform. After 20 minutes, pour off the sodium sulfate solution. The carrier in the beaker is the immobilized denitrifying functional bacterial agent with immobilized functional bacterial community.

[0039] Preferably, this embodiment compares the effects of adding immobilized denitrifying bacterial agents and suspended bacterial agents at 2 cm·s. -1 The study investigated the pollutant removal efficiency of immobilized denitrifying bacteria in saline river water with high nitrate nitrogen content under circulating flow conditions. This demonstrated the effective removal of pollutants from the immobilized denitrifying bacteria, and specifically included the following steps:

[0040] In two effective volumes of 0.09m³ 3 In the simulated river pilot-scale device, saline river water with an effective depth of 0.3m and a salinity of 1.36 was added. The specific experimental conditions were: (1) 2g·L⁻¹ was added to the immobilized group G. -1 (2) The control group was given the same amount of suspended bacterial agent as the immobilized group. The immobilization carrier of the immobilized denitrifying bacterial agent was zeolite, and the microorganisms immobilized by the immobilized denitrifying bacterial agent were highly efficient salt-tolerant denitrifying bacterial agents obtained by enrichment and screening from rivers at the estuary.

[0041] The experiment was divided into two phases: the first phase was the cooling phase, in which 25℃, 20℃, 16℃, 12℃ and 10℃ were selected as set temperatures to simulate the temperature change from summer to winter under actual conditions by cooling.

[0042] The second stage is the warming stage, with set warming temperatures of 16℃, 20℃, and 25℃ to simulate the warming trend in spring after a winter low temperature (minimum temperature 10℃, minimum salinity limit 1.36%). During the cooling stage, 25℃ is the optimal survival temperature for microorganisms among the set temperatures, and 5 cycles are set. Temperatures of 20℃ and 16℃ are lower than 25℃, requiring an investigation of the long-term adaptability of microorganisms during cooling, and 10 cycles are set for each. The last cycle at 16℃ is a multi-carbon source cycle. Temperatures of 12℃ and 10℃ are similar, and in preliminary experiments, the pollutant removal effects were similar; therefore, 5 normal cycles and 1 multi-carbon source cycle are set for 12℃, and 10 cycles are set for 10℃.

[0043] Adjusting COD in river water using C6H5Na3O7 and KNO3 Mn NO3 - The initial concentration of -N is adjusted to a set concentration at each stage, i.e., the normal cycle COD Mn 50 mg·L -1 NO3 - -N 10.0 mg·L -1 High carbon source cycle COD Mn 70 mg·L -1 NO3 - -N 10.0 mg·L -1 The pollutant concentration was based on water quality monitoring data from the estuaries of several rivers in North China, where nitrate nitrogen was the main pollutant, monitored from July 2023 to August 2024. During the experiment, due to water circulation, the river water in the reactor had ample contact with air, resulting in sufficient dissolved oxygen (5.0-9.0 mg / L). -1 Therefore, no aeration device was installed. The test cycle was set to run for 3 days, and COD was measured every 24 hours. Mn NO3 - -N, NO2 - -N concentration, and add C6H5Na3O7 at the end of each cycle to reduce COD. Mn Restored to 50 mg·L -1 The level of KNO3 is increased by adding KNO3 to make NO3 - -N was restored to 10 mg·L⁻¹ -1 The level. The fixed C / N ratio involved in this embodiment is 5~7.

[0044] NO3 in reactor influent and effluent under different temperature conditions - -N concentration such as Figure 1 As shown. The immobilized group, treated with immobilized denitrifying bacteria, showed NO3 levels at 25°C. -The removal rate of NO3- was 93.69% ± 5.21%, higher than that of the control group (80.18% ± 7.34%), and the effluent quality of the immobilized group was more stable. As the water temperature decreased, NO3-... - -N removal rate gradually decreased until NO3 removal rate in the immobilized group reached 10℃. - -N removal rate decreased to 37.61%±8.44%, while NO3 removal rate in the control group was lower. - -N removal rate was only 17.74% ± 5.68%. Under various temperature conditions, the immobilized NO3... - -N removal was significantly better in the immobilized group than in the control group. Notably, at 10℃, after a 5-cycle adaptation phase, NO3- removal was significantly lower in the subsequent 5 cycles. - -N increased from 31.77%±7.85% to 43.46%±3.66%, indicating that the immobilized denitrifying bacterial agent had better adaptability to low-temperature conditions compared to the suspended bacterial agent (control group). When the temperature rose back to 25℃, the NO3- in the immobilized group... - -N removal rate was 91.21% ± 2.01%, compared to the control group's NO3 removal rate. - -N removal rate was 85.42% ± 6.60%, compared to NO3 removal rate at 25℃ during the cooling phase. - The similar nitrogen removal rates indicate that the immobilized denitrifying bacterial agent has good temperature adaptability.

[0045] NO2 concentration in each reaction cycle in the reactor under different temperature conditions - -N cumulative amount and COD Mn The changes in influent and effluent concentrations are as follows: Figure 2 and 3 As shown, the optimal temperatures for microbial survival are 25°C and 20°C during the temperature decrease phase. At these temperatures, the immobilized group accumulates a small amount of NO2 in each cycle. - After being treated with -N, the nitrogen was gradually denitrified, resulting in better effluent quality. In contrast, the control group had weaker denitrification capacity and accumulated a large amount of NO2 after each experimental cycle. - -N. The reason for this may be that the zeolite particles in the immobilized group provided a better microenvironment for the microorganisms, resulting in stronger microbial cell activity in the immobilized denitrifying bacterial agent. Compared to the control group, the immobilized denitrifying bacterial agent is more likely to enrich and maintain the structural stability of highly active denitrifying bacterial communities, thus reducing NO2. - -N reduction is more complete. When the temperature drops below 16℃, NO2... - The accumulation of -N decreased significantly, mainly due to NO3. - -N reduction is inhibited by low temperature, NO3 - The result is a decrease in NO2 removal rate. NO2 levels in the immobilized group and control group during the heating phase. --N cumulative amounts were all below 2.00 mg·L⁻¹. -1 The temperature was significantly lower than during the initial cooling phase of the experiment, indicating that the denitrification capacity of the microorganisms in the reaction system had improved after long-term domestication.

[0046] like Figure 3 As shown, under the conditions of 12℃, 16℃, 20℃, and 25℃ during the cooling and heating stages, when COD Mn The dosage is 50 mg·L -1 At that time, COD Mn The removal rates of both groups reached over 90%, and there was little difference between the immobilized group and the control group. During the final cycle of the cooling phase at 16℃ and 12℃, the carbon source addition was increased to 70 mg·L⁻¹. -1 COD at 16℃ Mn The removal rate can still reach 95%, and the immobilized NO3 group - The concentration of -N in the effluent decreased to 1.61 mg·L⁻¹. -1 NO3 - -N removal rate reached 85.02%, significantly higher than COD. Mn 50 mg·L -1 At 73.71%, this indicates that the main limiting factor for pollutant removal at this point is the content of the carbon source, i.e., the electron donor. Previous studies have shown that, assuming all other conditions remain constant, carbon source scarcity is a key factor restricting the denitrification process. However, at 12℃, increasing the initial carbon source concentration during the periodic COD... Mn The removal rate was only 70%, indicating a decrease in microbial cell activity in the reactor and a reduction in COD. Mn The utilization rate decreased. Over ten cycles at 10℃, the COD of the immobilized group... Mn The removal rate (73.24%±4.22%) was similar to that of the control group (76.64%±3.59%), indicating that pollutant removal was mainly limited by temperature.

[0047] The average TN removal under different temperature conditions and within the same temperature cycle is as follows: Figure 4 As shown, under all temperature conditions, the TN removal rate of the immobilized group was higher than that of the control group. However, the TN removal efficiency gradually decreased with decreasing temperature. This is because low temperature significantly inhibits biological activity, and the inhibitory effect of biological enzymes at low temperatures impairs the function of the microbial electron transport system that relies on biocatalysis for redox reactions, ultimately leading to a decrease in electron transport efficiency. Electron transport efficiency affects the synthesis of intracellular energy molecules (such as ATP), and its reduction leads to insufficient energy supply within microbial cells, thereby weakening the activity of microbial processes such as nitrogen conversion and NO3-. - -N and NO2 -The denitrification rate of -N is limited. At the same time, under low temperature conditions, when the flow rate is relatively constant, the decrease in cell activity will reduce the consumption of dissolved oxygen in the system, resulting in an increase in dissolved oxygen concentration. The increase in dissolved oxygen concentration will inhibit the denitrification efficiency of denitrifying bacteria.

[0048] Preferably, this embodiment provides a method for screening denitrifying functional bacterial communities, including:

[0049] S1. Enrichment of denitrifying bacteria: Water samples were taken from the river channel at the mouth of Bohai Bay in Binhai New Area, Tianjin. The samples were filtered through a 0.45μm filter membrane. The filter membrane containing the microorganisms was placed in marine bacterial culture medium 2216E and placed in a constant temperature shaking incubator for expansion culture at 30℃ for 3 days. After that, the samples were transferred to enrichment and screening culture medium for screening and domestication culture.

[0050] The above-mentioned marine bacterial culture medium 2216E was obtained from Qingdao Haibo Biotechnology Co., Ltd., and its composition was as follows: 5.00 g peptone, 1.00 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, 0.008 g disodium hydrogen phosphate, and distilled water was added to 1 L, with pH=7.6±0.2.

[0051] S2. Screening of denitrifying functional bacteria: The enriched bacterial solution obtained in step S1 was transferred to enrichment screening medium TXD for culture. After three days of expansion culture at room temperature, 1 mL (1% transfer ratio) of the suspension was transferred to 100 mL of sterile enrichment screening medium TXD and placed in a constant temperature shaking incubator at 30°C for 3 days. The transfer culture was repeated 10 times.

[0052] The above-mentioned enrichment and screening medium TXD was prepared with the following ratios: 6.00 g trisodium citrate, 3.00 g KNO3, 0.05 g K2HPO4, 0.20 g KH2PO4, 0.20 g MgSO4·7H2O, 30.00 g sodium chloride, 0.5 mL trace element concentrate, and distilled water added to 1 L, pH = 7.0.

[0053] The concentration of the above trace elements is as follows: FeSO4·7H2O 5 g, EDTA 50 g, ZnSO4·7H2O 3.93 g, H3BO3 11 g, MnSO4·H2O 4.32 g, CoCl2·6H2O 1.61 g, CuSO4·5H2O 1.57 g, (NH4)6Mo7O24 Add 1.1 g of 4H2O and distilled water to a final volume of 1 L, pH=6.0.

[0054] This invention is not limited to the embodiments described above. The above description of specific embodiments is intended to illustrate and explain the technical solutions of this invention. The specific embodiments described above are merely illustrative and not restrictive. Without departing from the spirit and scope of the claims, those skilled in the art can make many specific modifications based on the teachings of this invention, and these modifications all fall within the scope of protection of this invention.

Claims

1. The application of a salt-tolerant immobilized denitrifying bacterial agent in low-temperature saline river water denitrification, characterized in that, Salt-tolerant immobilized denitrifying microbial agents are placed in a net bag and evenly suspended in the river channel; the salt-tolerant immobilized denitrifying microbial agents use zeolite as the immobilization carrier, and the immobilized microorganisms are salt-tolerant denitrifying microbial agents enriched and screened from rivers at the estuary.

2. The application according to claim 1, characterized in that, The dosage of the salt-tolerant immobilized denitrifying functional bacteria agent is 2 g·L -1 , and the dosage is the mass-volume ratio of the immobilized denitrifying functional bacteria agent to river water.

3. The application according to claim 1, characterized in that, The low-temperature saline river water refers to saline river water bodies with nitrate nitrogen as the main pollutant.

4. The application according to claim 1 or 3, characterized in that, The temperature of the low-temperature saline river water is 10℃~25℃.

5. The application according to claim 1, characterized in that, During the denitrification process, carbon sources are added to the river water to maintain denitrification efficiency.

6. A method for preparing a salt-tolerant immobilized denitrifying bacterial agent, characterized in that, The denitrifying bacterial community was immobilized using a method combining zeolite carrier adsorption and chitosan encapsulation, including: (1) Adsorption and biofilm formation: The zeolite carrier is immersed in the culture medium, inoculated with denitrifying functional bacteria, and cultured to allow the microorganisms to fully biofilm on the zeolite carrier. After washing, an immobilized carrier loaded with functional microorganisms is obtained. (2) Coating and embedding: Immerse the immobilized carrier obtained in step (1) in chitosan solution, let it stand for coating, and then drain the remaining chitosan solution. (3) Crosslinking and immobilization: Sodium sulfate solution is added to the immobilized carrier after step (2) for immersion. After static fixation, the remaining sodium sulfate solution is discharged to obtain salt-tolerant immobilized denitrifying bacterial agent with immobilized denitrifying bacterial community.

7. The preparation method according to claim 6, characterized in that, The chitosan solution is prepared by dissolving chitosan with a degree of deacetylation of 90% in a 1% acetic acid solution, so that the chitosan concentration is 1.5%. The concentration of the sodium sulfate solution is 35 g L -1 .

8. The preparation method according to claim 6, characterized in that, In step (1): The particle size of the zeolite carrier is 3 mm; The culture medium is sterile TXD medium; during immersion, the liquid level of the culture medium is 5 cm above the zeolite carrier, and the medium is stirred 1-2 times a day during the culture period. The medium is cultured at 30°C for 5-6 days under sterile conditions. The inoculation amount of the denitrifying functional bacterial culture into TXD medium is 5% of its volume.

9. The preparation method according to claim 6, characterized in that, In steps (2) and (3): In step (2), the static film covering time is 20 minutes, and the carrier is stirred every 5 minutes during the static period; In step (3), the sodium sulfate solution is added and allowed to stand for 20 minutes, during which the carrier is stirred every 5 minutes.

10. A salt-tolerant immobilized denitrifying bacterial agent, characterized in that, It is prepared by the preparation method according to any one of claims 6 to 9.

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