Anaerobic denitrifying bacterial agent as well as preparation method and application thereof

Abstract

The invention discloses an anaerobic denitrifying bacterial agent as well as a preparation method and application thereof, and belongs to the technical field of water environment treatment. The anaerobic denitrifying bacterium agent is prepared from the following components in parts by weight: 15 to 30 parts of sterile water, 15 to 60 parts of normal saline, 5 to 10 parts of micro-capsule embedded anaerobic denitrifying bacteria, 5 to 20 parts of a porous composite modified carrier, 10 to 30 parts of a carbon source, 4 to 7 parts of nutrient elements, 2.5 to 4 parts of a buffering agent and 1 to 5 parts of a stabilizer, the micro-capsule embedded anaerobic denitrifying bacteria are obtained by embedding anaerobic denitrifying bacteria with an alginic acid gel solution and a calcium chloride solution; the porous composite modified carrier is obtained by crosslinking modified nano iron oxide, modified nano titanium dioxide, a functional polymer solution and glutaraldehyde. The anaerobic denitrifying bacterial agent provided by the invention can effectively improve the efficiency of treating nitrogen pollution of a water body.

Description

Anaerobic denitrifying bacteria agents, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of water environment treatment technology, specifically relating to anaerobic denitrifying bacteria agents, their preparation methods, and applications. Background Technology

[0002] Nitrogen, one of the four essential elements in nature, undergoes a series of biochemical processes within the biosphere, migrating and transforming to maintain its internal balance. However, with the rapid development of industry and agriculture and the significant improvement in people's living standards, large amounts of nitrogen are being discharged into the biosphere, far exceeding its carrying capacity and natural purification capabilities, thus causing an imbalance in the biosphere's nitrogen cycle. This imbalance leads to the accumulation of a large number of intermediate products, ultimately resulting in nitrogen pollution problems, with water nitrogen pollution being particularly prominent.

[0003] Nitrogen pollution in water bodies primarily originates from human activities, with massive discharge volumes. Major sources include urban sewage, industrial wastewater, and agricultural wastewater. Nitrogen pollutants are mainly classified into two categories: organic nitrogen and inorganic nitrogen. Organic nitrogen specifically includes substances such as proteins, polypeptides, amino acids, and urea; while inorganic nitrogen mainly includes ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen.

[0004] Anaerobic denitrifying bacteria are microbial preparations that utilize anaerobic denitrifying bacteria for wastewater treatment and water remediation. These bacteria, operating in an anaerobic environment, use organic carbon sources as electron donors to reduce nitrates or nitrites to nitrogen gas, thus completing the denitrification process. Anaerobic denitrifying bacteria have advantages such as high efficiency, environmental friendliness, and economy, making them an important tool for treating nitrogen-containing wastewater. Therefore, this invention develops an anaerobic denitrifying bacteria agent, its preparation method, and its application to address the technical problems of existing anaerobic denitrifying bacteria agents lacking means to maintain long-term activity, stability, and adsorption performance, resulting in low efficiency and poor effectiveness in treating nitrogen pollution in water bodies. Summary of the Invention

[0005] The purpose of this invention is to provide anaerobic denitrifying bacteria agents, their preparation methods, and applications, in order to solve the technical problems of low efficiency and poor effectiveness in treating nitrogen pollution in water bodies in the prior art.

[0006] To achieve the above objectives, the present invention employs the following technical solution: an anaerobic denitrifying bacteria agent, comprising the following components by weight: 15-30 parts sterile water, 15-60 parts physiological saline, 5-10 parts microencapsulated anaerobic denitrifying bacteria, 5-20 parts porous composite modified carrier, 10-30 parts carbon source, 4-7 parts nutrients, 2.5-4 parts buffer, and 1-5 parts stabilizer; the microencapsulated anaerobic denitrifying bacteria are obtained by encapsulating anaerobic denitrifying bacteria in alginate solution and calcium chloride solution; the porous composite modified carrier is obtained by crosslinking modified nano iron oxide, modified nano titanium dioxide, functionalized polymer solution, and glutaraldehyde.

[0007] The carbon source is one or more of glucose, sucrose, and methanol; the nutrient elements are composed of the following components in parts by weight: 2-3 parts ammonium nitrate, 1-2 parts urea, 1-2 parts amino acids, 2-3 parts potassium dihydrogen phosphate, 2-3 parts dipotassium hydrogen phosphate, 0.1-0.3 parts ferrous sulfate, 0.1-0.3 parts zinc sulfate, and 0.1-0.3 parts manganese chloride; the buffer is composed of the following components in parts by weight: 2-4 parts sodium bicarbonate, 1-2 parts citric acid, 1-2 parts sodium citrate, 0.5-1 parts acetic acid, and 0.5-1 parts sodium acetate; the stabilizer is one or more of glycerol, trehalose, and hydroxymethyl cellulose.

[0008] Further, the preparation method of microencapsulated anaerobic denitrifying bacteria includes the following steps: (1) Activation and culture of bacteria: Prepare a pre-reduction culture medium, heat and boil or pass nitrogen gas through the pre-reduction culture medium before use, inoculate the anaerobic denitrifying bacteria into the pre-reduction culture medium, activate and culture under anaerobic conditions, then expand the culture, mix, and obtain a mixed suspension of anaerobic denitrifying bacteria; (2) Preparation of microencapsulation solution: Dissolve alginate in phosphate buffer to obtain alginate solution, mix the mixed suspension of anaerobic denitrifying bacteria with alginate solution evenly to obtain a mixed bacterial solution; (3) Preparation of microcapsules: Use a micro-dropper or syringe to drop the mixed bacterial solution into calcium chloride solution to obtain microcapsules, wash them first with phosphate buffer solution, then wash them with physiological saline, suspend the washed microcapsules in sterile physiological saline to obtain microencapsulated anaerobic denitrifying bacteria.

[0009] Further, the pre-reduction culture medium in step (1) consists of the following components by weight: 950-1000 parts deionized water, 1-1.2 parts KH2PO4, 1.5-1.8 parts K2HPO4, 0.5-0.6 parts NaCl, 0.2-0.3 parts MgSO4·7H2O, 0.1-0.12 parts CaCl2·2H2O, 0.5-0.6 parts yeast extract, 0.5-0.6 parts glucose, 1.8-2 parts sodium acetate, 1.8-2 parts sodium nitrate, and 10 parts trace element solution; the trace element solution consists of the following components by weight: 97-99 parts deionized water, 1 part KH2PO4, 1 part ... The anaerobic denitrifying bacteria consist of ionized water, 1-1.2 parts FeSO4·7H2O, 0.08-0.1 parts MnSO4·H2O, 0.08-0.1 parts CoCl2·6H2O, 0.05-0.06 parts CuSO4·5H2O, 0.05-0.06 parts ZnSO4·7H2O, 0.8-1 parts H3BO3, and 0.02 parts Na2MoO4·2H2O. The anaerobic denitrifying bacteria are one or more of *Pseudomonas schlegelii*, *Pseudomonas aeruginosa*, and *Paracoccus eluti*. All anaerobic denitrifying bacteria are derived from the China Center for Type Culture Collection (CCTCC), with *Pseudomonas schlegelii* having the designation CCTCC. HB20082814, *Pseudomonas aeruginosa* ID: CCTCAB 93066, *Paragonimococcus eluti* ID: CCTCC AB206665; activation culture temperature: 30-37℃, culture time: 24-48h; scale-up culture temperature: 30-37℃, culture time: 12-24h; the volume ratio of *Pseudomonas stearothermiae*, *Pseudomonas aeruginosa*, and *Paragonimococcus eluti* in the anaerobic denitrifying bacteria suspension is 1:1-2:2-3:1-2, and the concentration of anaerobic denitrifying bacteria is 10. 8 -10 9 CFU / mL; in step (2), the pH of the phosphate buffer is 7.0-7.4, the mass-volume concentration of the alginate solution is 1-3% (w / v), and the volume ratio of the anaerobic denitrifying bacteria mixed suspension to the alginate solution is 1:1-2; in step (3), the mass-volume concentration of calcium chloride is 2-10% (w / v), and the dripping speed is 1-2 drops / second.

[0010] Further, the preparation method of the porous composite modified carrier includes the following steps: S1, Preparation of modified nano-iron oxide: Fe(NO3)3·9H2O is dissolved in deionized water to obtain precursor solution one. Ammonia water is added to adjust the pH, and the reaction is stirred to form a sol. The sol is heated to form a gel. After drying, it is ground into nano-iron oxide powder, which is dispersed in nitric acid solution one, ultrasonically treated, and stirred to obtain modified nano-iron oxide solution. It is filtered to obtain modified nano-iron oxide powder, washed with deionized water, and then placed in an oven to dry to obtain modified nano-iron oxide powder; S2, Preparation of modified nano-titanium dioxide: Tetrabutyl titanate is dissolved in ethanol, and deionized water and nitric acid solution two are added dropwise, and stirred. The reaction is stirred to form a sol. The sol is heated to form a gel. After drying, it is ground into nano-titanium dioxide powder. The powder is dispersed in nitric acid solution 1, ultrasonically treated, and stirred to obtain a modified nano-titanium dioxide solution. The solution is filtered to obtain modified nano-titanium dioxide powder, washed with deionized water, and then dried in an oven to obtain modified nano-titanium dioxide powder. S3. Preparation of porous composite modified carrier: Polyvinyl alcohol is dissolved in phosphate buffer to obtain a polyvinyl alcohol solution. Glutaraldehyde is added to carry out a cross-linking reaction to obtain a functionalized polymer solution. Modified nano-iron oxide powder and modified nano-titanium dioxide powder are added to the functionalized polymer solution, mixed evenly, and spray-dried to obtain a porous composite modified carrier.

[0011] Further, in S1, the concentration of precursor solution one is 0.1-1M, the pH is adjusted to 10 with ammonia, the reaction is stirred for 24-26 hours, and the sol is heated to 80-82℃; the concentration of nitric acid solution one is 0.2-0.7M; the mixture is washed 3-5 times with deionized water until the pH of the washing solution is neutral, and the drying temperature in the oven is 60-100°C for 24-30 hours; in S2, the volume ratio of tetrabutyl titanate, ethanol, and deionized water is 1:10-12:3-5, the concentration of nitric acid solution two is 0.1-0.5M, and the volume ratio of deionized water to nitric acid solution two is 1:0.1-0.12. The concentration of liquid 1 is 0.2-0.7M; wash with deionized water 3-5 times until the pH of the washing solution is neutral; the drying temperature of the oven is 60-100°C and the drying time is 24-30h; the pH of the phosphate buffer in S3 is 7-7.4, the concentration of the polyvinyl alcohol solution is 10-30% (w / v), the mass concentration of glutaraldehyde is 1-5%, the amount of glutaraldehyde is 10-20% (v / v) of the polymer solution, the crosslinking reaction time is 30-60min, and the mass ratio of modified nano iron oxide powder, modified nano titanium dioxide powder and polyvinyl alcohol is 0.1-0.3:0.1-0.3:1.

[0012] The preparation method of anaerobic denitrifying bacteria agent includes the following steps: M1, fixation: grinding the porous composite modified carrier into powder, adding physiological saline to prepare a porous composite modified carrier suspension, adding microencapsulated anaerobic denitrifying bacteria to the porous composite modified carrier suspension, and allowing it to stand to obtain an anaerobic denitrifying bacteria carrier mixture; M2, mixing: dissolving nutrients and buffers in sterile water, stirring until completely dissolved, then adding carbon source and stabilizer, continuing to stir and mix evenly, then adding the anaerobic denitrifying bacteria carrier mixture, stirring and mixing evenly to obtain the anaerobic denitrifying bacteria agent.

[0013] Furthermore, the porous composite modified carrier in M1 has a particle size of 10-100 μm and is left to stand for 2-4 hours.

[0014] This invention also provides the application of anaerobic denitrifying bacteria agents in water treatment.

[0015] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows: 1. The present invention, by immobilizing anaerobic denitrifying bacteria through microencapsulation and porous composite modified carrier, enables them to maintain high activity and improve denitrification efficiency. The porous composite modified carrier has good stability and adsorption performance, which can firmly fix the anaerobic denitrifying bacteria and prevent their loss. At the same time, the nutrients and buffers in the bacterial agent can provide the nutrients and suitable pH environment required for the growth of the bacteria, which is conducive to the growth and metabolism of the bacteria, thereby improving the efficiency of wastewater treatment.

[0016] 2. This invention provides a protective environment for anaerobic denitrifying bacteria through microencapsulation technology. Alginate, as a natural polymer material, has good biocompatibility and film-forming properties. Calcium ions in calcium chloride solution can combine with carboxyl groups in alginate to form a stable gel structure, thereby encapsulating anaerobic denitrifying bacteria in microcapsules, which helps improve the survival rate of anaerobic denitrifying bacteria in complex environments. In addition, microencapsulation technology can enhance the stability of anaerobic denitrifying bacteria, enabling them to maintain their activity during storage and transportation, which is beneficial for the long-term preservation of anaerobic denitrifying bacteria.

[0017] 3. This invention prepares nano-iron oxide and titanium dioxide using a sol-gel method, improving their specific surface area and activity. Further modification of the nanoparticles is achieved through ultrasonic-assisted dispersion and nitric acid solution treatment, forming more carboxyl active sites and enhancing their surface activity and stability. Polyvinyl alcohol is cross-linked with glutaraldehyde to form a three-dimensional network structure, providing a basis for a porous carrier. The modified nano-iron oxide and titanium dioxide are then incorporated into the polyvinyl alcohol network, enhancing the carrier's adsorption performance. Furthermore, spray drying technology is used to form a uniform porous structure, improving the carrier's mechanical strength and stability. The porous composite modified carrier possesses a high specific surface area and abundant pore structure, providing numerous adsorption sites and growth space for anaerobic denitrifying bacteria, facilitating their immobilization and proliferation, enhancing denitrification, and effectively removing nitrates and nitrites from water. In addition, the modified nano-iron oxide and titanium dioxide exhibit excellent adsorption and reduction capabilities, effectively removing heavy metal ions from water. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Embodiments

[0019] This embodiment discloses an anaerobic denitrifying bacteria agent, which is composed of the following components by weight: 15g sterile water, 15g physiological saline, 5g microencapsulated anaerobic denitrifying bacteria, 5g porous composite modified carrier, 10g glucose, 1g ammonium nitrate, 0.5g urea, 0.5g amino acids, 1g potassium dihydrogen phosphate, 1g dipotassium hydrogen phosphate, 0.05g ferrous sulfate, 0.05g zinc sulfate, 0.05g manganese chloride, 1g sodium bicarbonate, 0.5g citric acid, 0.5g sodium citrate, 0.25g acetic acid, 0.25g sodium acetate, and 10g glycerol; the microencapsulated anaerobic denitrifying bacteria are obtained by encapsulating anaerobic denitrifying bacteria in alginate solution and calcium chloride solution; the porous composite modified carrier is obtained by crosslinking modified nano iron oxide, modified nano titanium dioxide, functionalized polymer solution, and glutaraldehyde.

[0020] The preparation method of microencapsulated anaerobic denitrifying bacteria includes the following steps: (1) Activation and culture of the strain: Prepare a pre-reduction culture medium composed of the following components by weight: 950g deionized water, 1g KH2PO4, 1.5g K2HPO4, 0.5g NaCl, 0.2g MgSO4·7H2O, 0.1g CaCl2·2H2O, 0.5g yeast extract, 0.5g glucose, 1.8g sodium acetate, 1.8g sodium nitrate and 10g trace element solution; wherein the trace element solution is composed of the following components by weight: 97g deionized water, 1g FeSO4·7H2O, 0.08g MnSO4·H2O, 0.08g CoCl2·6H2O, 0.05g CuSO4·5H2O, 0.05g ZnSO4·7H2O, 0.8g H3BO3 and 0.02g Na2MoO4·2H2O; The pre-reduced culture medium was heated and boiled or purged with nitrogen before use. Pseudomonas schrenckii, Pseudomonas aeruginosa and Paracoccus ilei were inoculated into the pre-reduced culture medium and activated under anaerobic conditions at 30°C for 24 h. Then, the culture was expanded at 30°C for 12 h. The bacterial suspensions of Pseudomonas schrenckii, Pseudomonas aeruginosa and Paracoccus ilei were mixed in a ratio of 1:1:2:1 to obtain 500 mL of anaerobic denitrifying bacteria mixed suspension; (2) Preparation of microencapsulation solution: 10 g of alginate was dissolved in 1 L In a phosphate buffer solution with a pH of 7.0, an alginate gel solution was obtained. The anaerobic denitrifying bacteria mixed suspension was mixed evenly with the alginate gel solution to obtain a mixed bacterial gel solution. (3) Preparation of microcapsules: Using a micro-dropper, the mixed bacterial gel solution was dropped into a 2% (w / v) calcium chloride solution at a dropping speed of 1 drop / second to obtain microcapsules. The microcapsules were first washed with phosphate buffer solution and then washed with physiological saline. The washed microcapsules were suspended in sterile physiological saline to obtain microcapsule-embedded anaerobic denitrifying bacteria.

[0021] The preparation method of the porous composite modified carrier includes the following steps: S1, Preparation of modified nano-iron oxide: Dissolve 4.04g Fe(NO3)3·9H2O in 100mL deionized water to obtain 0.1M precursor solution one. Add ammonia to adjust the pH to 10, stir and react for 24h to form a sol. Heat the sol to 80℃ to form a gel. After drying, grind it into nano-iron oxide powder, disperse it in 0.2M nitric acid solution one, sonicate and stir to obtain modified nano-iron oxide solution. Filter it to obtain modified nano-iron oxide powder, wash it three times with deionized water, and then put it in an oven to dry at 60°C for 24h to obtain modified nano-iron oxide powder; S2, Preparation of modified nano-titanium dioxide: Dissolve 10mL tetrabutyl titanate in 100mL ethanol, add 30mL deionized water and 0.1M nitric acid solution one. 1 mL of nitric acid solution II was stirred to form a sol. The sol was heated to 80°C to form a gel. After drying, it was ground into nano-titanium dioxide powder and dispersed in nitric acid solution I. The solution was ultrasonically treated and stirred to obtain a modified nano-titanium dioxide solution. The solution was filtered to obtain modified nano-titanium dioxide powder, which was washed three times with deionized water and then placed in an oven for drying at 60°C for 24 hours to obtain modified nano-titanium dioxide powder. S3. Preparation of porous composite modified carrier: 10 g of polyvinyl alcohol was dissolved in 100 mL of phosphate buffer to obtain a 10% (w / v) polyvinyl alcohol solution. 10 mL of 1% glutaraldehyde was added for crosslinking reaction for 30 min to obtain a functionalized polymer solution. 1 g of modified nano-iron oxide powder and 1 g of modified nano-titanium dioxide powder were added to the functionalized polymer solution and mixed evenly. The solution was then spray-dried to obtain a porous composite modified carrier.

[0022] The preparation method of anaerobic denitrifying bacteria agent includes the following steps: M1, fixation: Grind the porous composite modified carrier into powder, add physiological saline to prepare a porous composite modified carrier suspension, add microencapsulated anaerobic denitrifying bacteria to the porous composite modified carrier suspension, let stand for 2 hours to obtain an anaerobic denitrifying bacteria carrier mixture; M2, mixing: Dissolve nutrients and buffer in sterile water, stir until completely dissolved, then add carbon source and stabilizer, continue stirring and mixing evenly, then add the anaerobic denitrifying bacteria carrier mixture, stir and mix evenly to obtain the anaerobic denitrifying bacteria agent.

[0023] This invention also provides the application of anaerobic denitrifying bacteria in water treatment. Examples

[0024] This embodiment discloses an anaerobic denitrifying bacterial agent, composed of the following components by weight: 30g sterile water, 60g physiological saline, 10g microencapsulated anaerobic denitrifying bacteria, 20g porous composite modified carrier, 10g glucose, 10g sucrose, 10g methanol, 1.5g ammonium nitrate, 1g urea, 1g amino acids, 1.5g potassium dihydrogen phosphate, 1.5g dipotassium hydrogen phosphate, 0.15g ferrous sulfate, and 0.15g zinc sulfate. The microencapsulated anaerobic denitrifying bacteria are obtained by encapsulating anaerobic denitrifying bacteria in alginate solution and calcium chloride solution; the porous composite modified carrier is obtained by crosslinking modified nano iron oxide, modified nano titanium dioxide, functionalized polymer solution and glutaraldehyde.

[0025] The preparation method of microencapsulated anaerobic denitrifying bacteria includes the following steps: (1) Activation and culture of the strain: Prepare a pre-reduction culture medium composed of the following components by weight: 1000g deionized water, 1.2g KH2PO4, 1.8g K2HPO4, 0.6g NaCl, 0.3g MgSO4·7H2O, 0.12g CaCl2·2H2O, 0.6g yeast extract, 0.6g glucose, 2g sodium acetate, 2g sodium nitrate and 10g trace element solution; wherein the trace element solution is composed of the following components by weight: 99g deionized water, 1.2g FeSO4·7H2O, 0.1g MnSO4·H2O, 0.1g CoCl2·6H2O, 0.06g CuSO4·5H2O, 0.06g ZnSO4·7H2O, 1g H3BO3 and Na2MoO4·2H2O; the pre-reduced medium was heated and boiled or purged with nitrogen before use. Pseudomonas schrenckii, Pseudomonas aeruginosa and Paracoccus ilei were inoculated into the pre-reduced medium and activated under anaerobic conditions at 37°C for 48 h. Then the culture was expanded at 37°C for 24 h. The bacterial suspensions of Pseudomonas schrenckii, Pseudomonas aeruginosa and Paracoccus ilei were mixed in a ratio of 1:2:3:2 to obtain 800 mL of anaerobic denitrifying bacteria mixed suspension; (2) Preparation of microencapsulation solution: 30 g of alginate was dissolved in 1 L In a phosphate buffer solution with a pH of 7.4, an alginate gel solution was obtained. The anaerobic denitrifying bacteria mixed suspension was mixed evenly with the alginate gel solution to obtain a mixed bacterial gel solution. (3) Microcapsules were prepared: Using a syringe, the mixed bacterial gel solution was dripped into a 10% (w / v) calcium chloride solution at a dripping speed of 1-2 drops / second to obtain microcapsules. The microcapsules were first washed with phosphate buffer solution and then washed with physiological saline. The washed microcapsules were suspended in sterile physiological saline to obtain microcapsule-embedded anaerobic denitrifying bacteria.

[0026] The preparation method of the porous composite modified carrier includes the following steps: S1, Preparation of modified nano-iron oxide: Dissolve 40.4g Fe(NO3)3·9H2O in 100mL deionized water to obtain 1M precursor solution one. Add ammonia water to adjust the pH to 10, stir and react for 26h to form a sol. Heat the sol to 82℃ to form a gel. After drying, grind it into nano-iron oxide powder, disperse it in 0.7M nitric acid solution one, sonicate and stir to obtain modified nano-iron oxide solution. Filter it to obtain modified nano-iron oxide powder, wash it 5 times with deionized water, and then put it in an oven to dry at 100°C for 30h to obtain modified nano-iron oxide powder; S2, Preparation of modified nano-titanium dioxide: Dissolve 10mL tetrabutyl titanate in 120mL ethanol, add 50mL deionized water and 0.5M nitric acid solution one. 1.2 mL of nitric acid solution II was stirred to form a sol. The sol was heated to 82°C to form a gel. After drying, it was ground into nano-titanium dioxide powder, which was dispersed in nitric acid solution I, sonicated, and stirred to obtain a modified nano-titanium dioxide solution. The solution was filtered to obtain modified nano-titanium dioxide powder, which was washed 5 times with deionized water and then placed in an oven to dry at 100°C for 30 hours to obtain modified nano-titanium dioxide powder. S3. Preparation of porous composite modified carrier: 30 g of polyvinyl alcohol was dissolved in 100 mL of phosphate buffer to obtain a 30% (w / v) polyvinyl alcohol solution. 20 mL of 5% glutaraldehyde was added to carry out a crosslinking reaction for 60 min to obtain a functionalized polymer solution. 3 g of modified nano-iron oxide powder and 3 g of modified nano-titanium dioxide powder were added to the functionalized polymer solution and mixed evenly. The solution was then spray-dried to obtain a porous composite modified carrier.

[0027] The preparation method of anaerobic denitrifying bacteria agent includes the following steps: M1, fixation: The powder of porous composite modified carrier is ground into physiological saline to prepare a porous composite modified carrier suspension. The microencapsulated anaerobic denitrifying bacteria are dripped into the porous composite modified carrier suspension and allowed to stand for 4 hours to obtain an anaerobic denitrifying bacteria carrier mixture; M2, mixing: Nutrient elements and buffer are dissolved in sterile water and stirred until completely dissolved. Then, carbon source and stabilizer are added and stirred until uniform. Then, the anaerobic denitrifying bacteria carrier mixture is added and stirred until uniform to obtain the anaerobic denitrifying bacteria agent.

[0028] This invention also provides the application of anaerobic denitrifying bacteria agents in water treatment.

[0029] Comparative Example 1: Compared with Example 2, in the preparation process of the anaerobic denitrifying bacteria agent, the microencapsulated anaerobic denitrifying bacteria in Comparative Example 1 were replaced with anaerobic denitrifying bacteria, while other conditions remained unchanged.

[0030] Comparative Example 2: Compared with Example 2, Comparative Example 2 did not add a porous composite modified carrier during the preparation of the anaerobic denitrifying bacteria agent, and all other conditions remained unchanged.

[0031] Comparative Example 3: Compared with Example 2, Comparative Example 3 did not add any nutrients during the preparation of the anaerobic denitrifying bacteria agent, and all other conditions remained unchanged.

[0032] The performance of the anaerobic denitrifying bacteria agents prepared in Examples 1-2 and Comparative Examples 1-3 was monitored in the experimental cases.

[0033] Low-carbon wastewater with a total nitrogen concentration of 500 mg / L was prepared using potassium nitrate as the nitrogen source. Anaerobic denitrifying bacteria agents prepared in Examples 1-2 and Comparative Examples 1-3 were added to the wastewater, respectively. The total nitrogen content in the wastewater was measured after 96 hours, and the denitrification efficiency of each denitrifying bacteria agent was calculated. The total nitrogen content was determined according to HJ 636-2012 "Determination of Total Nitrogen in Water Quality - Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometer". The denitrification experimental results are shown in Table 1: Group Total Nitrogen Content (mg / L) Denitrification Efficiency (%) Example 1 6.5 98.7 Example 2 4.5 99.1 Comparative Example 1 175 65 Comparative Example 2 160 68 Comparative Example 3 90 82 As shown in Table 1, the test results indicate that the anaerobic denitrifying bacteria agents prepared in Examples 1-2 of this invention can effectively improve the efficiency of treating nitrogen pollution in water bodies. A comparison between Comparative Examples 1-3 and Examples 1-2 shows that adding microencapsulated anaerobic denitrifying bacteria, porous composite modified carriers, and nutrients can effectively improve the efficiency of treating nitrogen pollution in water bodies.

[0034] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0035] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to specific implementations. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An anaerobic denitrifying bacteria agent, characterized in that, It is composed of the following components in parts by weight: 15-30 parts sterile water, 15-60 parts physiological saline, 5-10 parts microencapsulated anaerobic denitrifying bacteria, 5-20 parts porous composite modified carrier, 10-30 parts carbon source, 4-7 parts nutrients, 2.5-4 parts buffer and 1-5 parts stabilizer; the microencapsulated anaerobic denitrifying bacteria are obtained by encapsulating anaerobic denitrifying bacteria in alginate solution and calcium chloride solution; the porous composite modified carrier is obtained by crosslinking modified nano iron oxide, modified nano titanium dioxide, functionalized polymer solution and glutaraldehyde.

2. The anaerobic denitrifying bacteria agent according to claim 1, characterized in that, The carbon source is one or more of glucose, sucrose, and methanol; the nutrient elements are composed of the following components in parts by weight: 2-3 parts ammonium nitrate, 1-2 parts urea, 1-2 parts amino acids, 2-3 parts potassium dihydrogen phosphate, 2-3 parts dipotassium hydrogen phosphate, 0.1-0.3 parts ferrous sulfate, 0.1-0.3 parts zinc sulfate, and 0.1-0.3 parts manganese chloride; the buffer is composed of the following components in parts by weight: 2-4 parts sodium bicarbonate, 1-2 parts citric acid, 1-2 parts sodium citrate, 0.5-1 parts acetic acid, and 0.5-1 parts sodium acetate; the stabilizer is one or more of glycerol, trehalose, and hydroxymethyl cellulose.

3. The anaerobic denitrifying bacteria agent according to claim 1, characterized in that, The preparation method of microencapsulated anaerobic denitrifying bacteria includes the following steps: (1) Activation and culture of bacteria: Prepare a pre-reduction culture medium. Before use, the pre-reduction culture medium is heated and boiled or purged with nitrogen. The anaerobic denitrifying bacteria are inoculated into the pre-reduction culture medium and activated under anaerobic conditions. Then, the culture is expanded and mixed to obtain a mixed suspension of anaerobic denitrifying bacteria; (2) Preparation of microencapsulation solution: Alginate is dissolved in phosphate buffer to obtain an alginate solution. The mixed suspension of anaerobic denitrifying bacteria is mixed evenly with the alginate solution to obtain a mixed bacterial solution; (3) Preparation of microcapsules: Using a micro-dropper or syringe, the mixed bacterial solution is dripped into a calcium chloride solution to obtain microcapsules. The microcapsules are washed with phosphate buffer solution and then with physiological saline. The washed microcapsules are suspended in sterile physiological saline to obtain microencapsulated anaerobic denitrifying bacteria.

4. The anaerobic denitrifying bacteria agent according to claim 3, characterized in that, The pre-reduction culture medium in step (1) consists of the following components by weight: 950-1000 parts deionized water, 1-1.2 parts KH₂PO₄, 1.5-1.8 parts K₂HPO₄, 0.5-0.6 parts NaCl, 0.2-0.3 parts MgSO₄·7H₂O, 0.1-0.12 parts CaCl₂·2H₂O, 0.5-0.6 parts yeast extract, 0.5-0.6 parts glucose, 1.8-2 parts sodium acetate, 1.8-2 parts sodium nitrate, and 10 parts trace element solution; the trace element solution consists of the following components by weight: 97-99 parts deionized water, 1-1.2 parts FeSO₄·7H₂O, 0.08-0.1 parts MnSO₄·H₂O, 0.0 The anaerobic denitrifying bacteria consist of 0.1 parts CoCl2·6H2O, 0.05-0.06 parts CuSO4·5H2O, 0.05-0.06 parts ZnSO4·7H2O, 0.8-1 parts H3BO3, and 0.02 parts Na2MoO4·2H2O. The anaerobic denitrifying bacteria are one or more of *Pseudomonas schlegelii*, *Pseudomonas aeruginosa*, and *Paracoccus eluti*. The activation culture temperature is 30-37℃, and the culture time is 24-48 h. The scale-up culture temperature is 30-37℃, and the culture time is 12-24 h. The volume ratio of *Pseudomonas schlegelii*, *Pseudomonas aeruginosa*, and *Paracoccus eluti* in the anaerobic denitrifying bacteria suspension is 1:1-2:2-3:1-2, and the concentration of the anaerobic denitrifying bacteria is 10. 8 -10 9 CFU / mL.

5. The anaerobic denitrifying bacteria agent according to claim 3, characterized in that, In step (2), the pH of the phosphate buffer solution is 7.0-7.4, the mass-volume concentration of the alginate solution is 1-3% (w / v), and the volume ratio of the anaerobic denitrifying bacteria mixed suspension to the alginate solution is 1:1-2; in step (3), the mass-volume concentration of calcium chloride is 2-10% (w / v), and the dripping speed is 1-2 drops / second.

6. The anaerobic denitrifying bacteria agent according to claim 1, characterized in that, The preparation method of the porous composite modified carrier includes the following steps: S1, Preparation of modified nano iron oxide: Fe(NO3)3·9H2O is dissolved in deionized water to obtain precursor solution one. Ammonia water is added to adjust the pH, and the reaction is stirred to form a sol. The sol is heated to form a gel. After drying, it is ground into nano iron oxide powder, which is dispersed in nitric acid solution one, ultrasonically treated, and stirred to obtain modified nano iron oxide solution. The solution is filtered to obtain modified nano iron oxide powder, washed with deionized water, and then dried in an oven to obtain modified nano iron oxide powder; S2, Preparation of modified nano titanium dioxide: Tetrabutyl titanate is dissolved in ethanol, and deionized water and nitric acid solution two are added dropwise. The reaction is stirred. The process involves: forming a sol, heating the sol to form a gel, drying and grinding it into nano-titanium dioxide powder, dispersing it in nitric acid solution, ultrasonicating and stirring to obtain a modified nano-titanium dioxide solution, filtering it to obtain modified nano-titanium dioxide powder, washing it with deionized water, and then drying it in an oven to obtain modified nano-titanium dioxide powder; S3, preparing a porous composite modified carrier: dissolving polyvinyl alcohol in phosphate buffer to obtain a polyvinyl alcohol solution, adding glutaraldehyde to carry out a crosslinking reaction to obtain a functionalized polymer solution, adding modified nano-iron oxide powder and modified nano-titanium dioxide powder to the functionalized polymer solution, mixing them evenly, and spray drying them to obtain a porous composite modified carrier.

7. The anaerobic denitrifying bacteria agent according to claim 6, characterized in that, In S1, the concentration of precursor solution one is 0.1-1M, the pH is adjusted to 10 with ammonia, the reaction is stirred for 24-26 hours, and the sol is heated to 80-82℃; the concentration of nitric acid solution one is 0.2-0.7M; washing with deionized water 3-5 times until the pH of the washing solution is neutral, drying in an oven at 60-100°C for 24-30 hours; in S2, the volume ratio of tetrabutyl titanate, ethanol, and deionized water is 1:10-12:3-5, the concentration of nitric acid solution two is 0.1-0.5M, the volume ratio of deionized water to nitric acid solution two is 1:0.1-0.12, and the concentration of nitric acid solution one is... The concentration is 0.2-0.7M; wash with deionized water 3-5 times until the pH of the washing solution is neutral; the drying temperature of the oven is 60-100°C and the drying time is 24-30h; the pH of the phosphate buffer in S3 is 7-7.4, the concentration of the polyvinyl alcohol solution is 10-30% (w / v), the mass concentration of glutaraldehyde is 1-5%, the amount of glutaraldehyde is 10-20% (v / v) of the polymer solution, the crosslinking reaction time is 30-60min, and the mass ratio of modified nano iron oxide powder, modified nano titanium dioxide powder and polyvinyl alcohol is 0.1-0.3:0.1-0.3:

1.

8. A method for preparing anaerobic denitrifying bacteria, characterized in that, The process includes the following steps: M1, Fixation: The porous composite modified carrier is ground into powder, added to physiological saline to prepare a porous composite modified carrier suspension, and microencapsulated anaerobic denitrifying bacteria are dripped into the porous composite modified carrier suspension and allowed to stand to obtain an anaerobic denitrifying bacteria carrier mixture; M2, Mixing: Nutrients and buffers are dissolved in sterile water and stirred until completely dissolved. Then, carbon source and stabilizer are added and stirred until homogeneous. Then, the anaerobic denitrifying bacteria carrier mixture is added and stirred until homogeneous to obtain an anaerobic denitrifying bacteria agent.

9. The method for preparing the anaerobic denitrifying bacteria agent according to claim 8, characterized in that, The porous composite modified carrier in M1 has a particle size of 10-100 μm and is left to stand for 2-4 hours.

10. The application of the anaerobic denitrifying bacteria agent according to any one of claims 1 to 9 in water treatment.