An autotrophic denitrification bacterial agent and a preparation method thereof

Abstract

This invention relates to the field of environmental microbiology, specifically disclosing an autotrophic denitrifying bacterial agent and its preparation method. This autotrophic denitrifying bacterial agent employs a three-layer biomimetic design: a sulfur-calcium composite core, an inner shell gel layer, and an outer functional layer. Denitrifying Thiobacillus is loaded into the core, and Pseudomonas schlegelii is fixed in the outer layer, with a sodium alginate-xanthan gum gel layer providing physical isolation, achieving spatial partitioning and symbiosis of autotrophic and heterotrophic denitrifying bacterial communities. The sulfur-calcium composite core uses sulfur powder as an electron donor, and hydrophobically modified eggshell powder supplements alkalinity in situ. The inner shell layer incorporates ultrafine calcium carbonate, providing both mechanical reinforcement and pH buffering functions. The outer layer encapsulates sodium acetate-stearate microspheres for slow carbon source release. This autotrophic denitrifying bacterial agent exhibits high denitrification efficiency, pH stability, and strong resistance to hydraulic shearing under low carbon-to-nitrogen ratio conditions. The preparation process is mild, and the raw materials are inexpensive and readily available, making it suitable for deep denitrification in wastewater treatment and other scenarios.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to an autotrophic denitrifying bacteria agent and its preparation method. Background Technology

[0002] With the increasing severity of eutrophication in water bodies, advanced nitrogen removal from wastewater has become a research hotspot in the environmental field. Traditional heterotrophic denitrification processes rely on the addition of organic carbon sources, resulting in high operating costs, large sludge production, and low nitrogen removal efficiency when carbon sources are insufficient. Sulfate autotrophic denitrification technology, which uses elemental sulfur as an electron donor, does not require external organic carbon sources and has a low sludge yield, has attracted widespread attention in recent years.

[0003] Sulfur-autotrophic denitrifying bacteria use CO2 as a carbon source and obtain energy by oxidizing elemental sulfur to reduce nitrates to nitrogen gas. The reaction produces acid, causing a decrease in system pH and inhibiting bacterial activity. Current technologies typically use carbonate minerals such as limestone and dolomite as alkalinity supplements, but these suffer from slow dissolution rates, a mismatch between alkalinity release and acid production rates, and surface passivation after long-term operation. Furthermore, sulfur-autotrophic denitrifying bacteria have long generation cycles and slow reaction initiation, making initial nitrogen removal efficiency insufficient for engineering requirements.

[0004] To accelerate the start-up process, researchers attempted to couple heterotrophic denitrification with sulfur autotrophic denitrification, utilizing the rapid response characteristics of heterotrophic bacteria to compensate for the slow start-up of autotrophic bacteria. However, the two bacterial communities have significantly different requirements for environmental conditions such as carbon sources and dissolved oxygen. Simple mixing can easily lead to excessive proliferation of heterotrophic bacteria, inhibiting the colonization of autotrophic bacteria and resulting in poor long-term stability of the system.

[0005] Regarding carrier materials, chitosan-magnetic microspheres and PVA-sodium alginate gels are widely used, but their costs are high. Bentonite and limestone carriers are inexpensive, but their mechanical strength is poor and their functions are limited, making it impossible to simultaneously achieve electron donor slow release, alkalinity replenishment, and bacterial immobilization. Based on the above, this invention provides an autotrophic denitrifying bacteria agent and its preparation method. Summary of the Invention

[0006] To address the shortcomings of existing sulfur autotrophic denitrification technologies, such as slow start-up, insufficient alkalinity supply, high carrier costs, and complex preparation processes, this invention provides an autotrophic denitrifying bacterial agent and its preparation method.

[0007] In a first aspect, the present invention provides an autotrophic denitrifying bacteria agent, which adopts the following technical solution: An autotrophic denitrifying bacteria agent comprises, from the inside out: a sulfur-calcium complex core, an inner shell gel layer covering the surface of the sulfur-calcium complex core, and an outer functional layer covering the surface of the inner shell gel layer. The sulfur-calcium composite core comprises the following raw materials in parts by weight: 12-18 parts of sulfur autotrophic denitrifying bacteria suspension and 25-40 parts of sulfur-calcium composite carrier; The inner shell gel layer comprises the following raw materials in parts by weight: 1-2 parts sodium alginate, 0.2-0.5 parts xanthan gum, 1-2 parts ultrafine calcium carbonate, and 97-100 parts deionized water. The outer functional layer comprises the following raw materials in parts by weight: 10-15 parts of heterotrophic denitrifying bacteria suspension and 20-30 parts of outer gel solution.

[0008] Preferably, the sulfur-autotrophic denitrifying bacteria are denitrifying thiobacilli (Bacillus denitrificationus). Thiobacillus denitrificans The concentration of the sulfur-autotrophic denitrifying bacteria suspension is 1.5 × 10⁻⁶. 9 CFU / mL - 2.5 × 10 9 CFU / mL.

[0009] Preferably, the method for preparing the sulfur-calcium composite carrier is as follows: Eggshell powder is mixed with stearic acid and stirred evenly to obtain hydrophobic modified eggshell powder; sulfur powder is heated to 120-130℃ to melt, Tween-80 is added and stirred to dissolve, then hydrophobic modified eggshell powder is added and stirred evenly; after cooling to room temperature, sodium thiosulfate is added, stirred evenly, granulated, dried, and sulfur-calcium composite carrier is obtained.

[0010] Preferably, the preparation method of the sulfur-calcium composite carrier is as follows: Eggshell powder and stearic acid were mixed at a mass ratio of 1:0.03-0.08 and stirred at 50-70℃ and 200-400 rpm for 20-40 min to obtain hydrophobically modified eggshell powder. Sulfur powder was heated to 120-130℃ to melt, and 0.2-0.5% of Tween-80 (by mass of sulfur powder) was added. After dissolving by stirring at 200-400 rpm, 20-30% of hydrophobically modified eggshell powder (by mass of sulfur powder) was added, and stirred at 300-500 rpm for 10-20 min. After cooling to room temperature, 3-8% of sodium thiosulfate (by mass of sulfur powder) was added, and stirred at 200-300 rpm for 5-10 min. The mixture was then pressed into shape using a granulator, with the particle size controlled at 1.5-2.5 mm. Finally, it was dried at 55-65℃ until the moisture content was <5% to obtain a sulfur-calcium composite carrier.

[0011] Preferably, the heterotrophic denitrifying bacteria is *Pseudomonas stearothermia* (…). Pseudomonas stutzeri The concentration of the heterotrophic denitrifying bacteria suspension is 4 × 10⁻⁶. 9 CFU / mL - 6 × 10 9 CFU / mL.

[0012] Preferably, the outer gel liquid comprises the following raw materials in parts by weight: 4-6 parts soluble starch, 1-2 parts sodium alginate, 0.5-1 part xanthan gum, 2-3 parts sodium acetate-stearic acid microspheres, and 80-90 parts deionized water.

[0013] Preferably, the method for preparing the sodium acetate-stearic acid microspheres is as follows: Sodium acetate was dissolved in deionized water to prepare a sodium acetate solution. Stearic acid was heated to melt and obtain molten stearic acid. Maltodextrin and emulsifier were added to the sodium acetate solution to obtain an aqueous phase. The molten stearic acid was mixed with the aqueous phase and sheared and emulsified to form an emulsion. The emulsion was spray-dried to obtain sodium acetate-stearic acid microspheres.

[0014] Preferably, the preparation method of the sodium acetate-stearic acid microspheres is as follows: Sodium acetate, stearic acid, maltodextrin, and Tween-80 were prepared in a mass ratio of 6-10:1.5-2.5:1:0.1-0.3. Sodium acetate was dissolved in deionized water to prepare a 40-50 wt% sodium acetate solution. Stearic acid was heated to 75-85℃ to melt, obtaining molten stearic acid. Maltodextrin and Tween-80 were added to the 40-50 wt% sodium acetate solution and stirred at 70-80℃ and 300-500 rpm to dissolve, obtaining an aqueous phase. Under a holding temperature of 80℃, molten stearic acid is mixed with an aqueous phase and sheared and emulsified at 8000-12000 rpm for 15-20 min to form an emulsion. The emulsion is then pumped into a spray dryer at a rate of 5-15 mL / min under a holding temperature of 70-80℃. The inlet air temperature of the spray dryer is 160-180℃ and the outlet air temperature is 70-80℃. After the feeding is completed, drying continues for 10-20 min to obtain sodium acetate-stearic acid microspheres.

[0015] Secondly, the present invention provides a method for preparing an autotrophic denitrifying bacteria agent, which adopts the following technical solution: A method for preparing an autotrophic denitrifying bacteria agent includes the following steps: S1. Mix the sulfur-calcium composite carrier with a sulfur-autotrophic denitrifying bacteria suspension, allow it to stand for adsorption, and dry it to obtain the sulfur-calcium composite core. S2. Add sodium alginate, xanthan gum and ultrafine calcium carbonate to deionized water, heat and stir, cool to room temperature to obtain inner shell gel liquid; S3. Spray the inner shell gel liquid onto the surface of the sulfur-calcium composite core to obtain intermediate A. Immerse intermediate A in calcium chloride solution for cross-linking and curing, filter, wash, and drain to form an inner shell gel layer and obtain inner shell coated particles. S4. Mix the heterotrophic denitrifying bacteria suspension with the outer layer gel solution to obtain the outer layer bacterial gel solution; S5. Spray the outer layer bacterial gel liquid onto the surface of the inner shell coated particles to obtain intermediate B. Immerse intermediate B in calcium chloride solution for cross-linking and solidification, filter, wash, and drain to form the outer functional layer, thus obtaining the autotrophic denitrifying bacteria agent.

[0016] Preferably, in step S1, the static adsorption temperature is 25-35℃, the static adsorption time is 12-24h, the drying temperature is 30-40℃, and the drying is carried out until the moisture content is <10%.

[0017] Preferably, in step S2, the heating temperature is 60-80℃, the stirring speed is 200-400rpm, and the stirring time is 20-40min.

[0018] Preferably, in step S3, the spraying rate is 1-3 mL / min, the spraying pressure is 0.05-0.15 MPa, and the spraying process is carried out with rolling stirring at 50-100 rpm; the mass fraction of calcium chloride solution is 1.5-2.5%; the cross-linking curing temperature is 20-30℃, and the cross-linking curing time is 10-20 min.

[0019] Preferably, in step S5, the spraying rate is 1-3 mL / min and the spraying pressure is 0.05-0.15 MPa.

[0020] Preferably, in step S5, the mass fraction of the calcium chloride solution is 1.5-2.5%; the crosslinking curing temperature is 20-30℃; and the crosslinking curing time is 15-30 min.

[0021] Thirdly, the present invention provides an application of the above-mentioned autotrophic denitrifying bacteria agent in wastewater treatment.

[0022] In summary, the present invention has the following beneficial effects: (1) The core-shell-outer layer three-level structure prepared in this invention loads sulfur-autotrophic denitrifying bacteria into the core and heterotrophic denitrifying bacteria into the outer functional layer, and achieves physical isolation and functional partitioning through the inner shell gel layer. The core efficiently performs autotrophic denitrification using sulfur as an electron donor, while the outer layer completes heterotrophic deep denitrification using a slow-release carbon source. The two pathways do not interfere with each other and work synergistically. The outer functional layer is formed by a high-speed shear-crosslinking solidification process, which has a rough surface and good bacterial loading capacity. The mud-water contact area and mass transfer efficiency are improved compared with traditional spherical particles. Ultrafine calcium carbonate is incorporated into the inner shell gel layer, which can be uniformly embedded in the sodium alginate-xanthan gum crosslinking network. On the one hand, it serves as a rigid skeleton to improve the particle's resistance to hydraulic shearing, thereby reducing the wear rate of the bacterial agent in the fluidized bed. On the other hand, it serves as a secondary pH buffer source, forming a dual-gradient alkalinity slow-release system with the core-modified eggshell powder, further stabilizing the reaction microenvironment and extending the shelf life of the bacterial agent.

[0023] (2) In this invention, the sulfur-calcium composite core is designed with a synergistic approach of sulfur powder, hydrophobically modified eggshell powder, and sodium thiosulfate: sulfur powder acts as the main electron donor, continuously providing electrons for autotrophic denitrification; the hydrophobically modified eggshell powder is uniformly dispersed in the sulfur matrix, slowly dissolving under denitrification acid production conditions, releasing alkalinity in situ and stabilizing the pH of the system; sodium thiosulfate acts as a water-soluble pore-forming agent and a fast-acting electron source, rapidly dissolving in the initial stage of use to form a continuous microporous network, significantly increasing the attachment sites for microorganisms and significantly shortening the start-up cycle of the inoculum. The synergistic effect of the three components enables the sulfur-calcium composite core to have the triple functions of electron donor, pH buffer, and biological carrier, effectively solving the problems of slow start-up, pH drop, and efficiency decay in traditional sulfur autotrophic denitrification.

[0024] (3) In this invention, the sodium acetate-stearic acid microspheres loaded on the outer functional layer use stearic acid as the hydrophobic wall material, sodium acetate as the carbon source core material, and maltodextrin as the stabilizer to form an enzyme-triggered response slow-release system. In the initial stage of use, the stearic acid shell resists water flow erosion and avoids sudden release of carbon source; as heterotrophic denitrifying bacteria colonize and proliferate and secrete lipase, the wall material is gradually enzymatically degraded, and sodium acetate is released steadily and slowly. The carbon source release rate is highly matched with the metabolic needs of the bacterial community, and the carbon source is effectively utilized. The residual COD in the effluent is effectively reduced compared with the direct addition of carbon source, which can effectively solve the problems of easy loss, easy exceeding of standards, and easy generation of sludge in traditional external carbon sources.

[0025] (4) This invention adopts a coupled mode of core autotrophic denitrification and outer heterotrophic denitrification, which can still achieve deep denitrification under low carbon-to-nitrogen ratio conditions. Core sulfur autotrophic denitrification uses reduced sulfur as an electron donor and does not require an external organic carbon source; outer heterotrophic denitrification relies on a slow-release carbon source to complete deep denitrification and nitrite removal. The dual-path synergy reduces the carbon-to-nitrogen ratio requirement of the system for denitrification, and is suitable for municipal effluent, low carbon-to-nitrogen ratio industrial wastewater, and rural decentralized sewage treatment scenarios.

[0026] (5) The preparation conditions used in this invention are mild and the process is simple. The sulfur-calcium composite core is made by melt granulation, which is safe and controllable. The inner shell gel layer is made by sodium alginate-calcium chloride crosslinking at room temperature, which does not damage microorganisms and has a high cell activity retention rate. The outer functional layer is formed in one step by high-speed shear-crosslinking and solidification, and the thickness and bacterial load can be flexibly adjusted.

[0027] (6) The bacterial agent prepared by this invention is in the form of microencapsulated particles, which can be directly added to fluidized bed bioreactors, anaerobic filters, etc., without the need for additional packing materials. The core-shell-outer layer structure can enhance the adhesion ability of the bacteria, reduce the loss of microorganisms, and improve the system's resistance to shock loads; at the same time, it has integrated functions such as slow release of electrons, in-situ pH stabilization, controlled release of carbon, and simultaneous denitrification, and there is no need to add alkalinity agents, carbon sources, pH adjusters, etc. after its addition. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0031] Among them, the ultrafine calcium carbonate was purchased from Jiangxi Kete Fine Powder Co., Ltd., specification: KT-0068; Soluble starch was purchased from Tianjin Jinhui Taiya Chemical Reagent Co., Ltd., specification: analytical grade; Soybean oil purchased from Guangzhou Weber Technology Co., Ltd., specifications: industrial refined grade, purity: 99%; Eggshell powder was purchased from Jiangsu Yihaotian Biotechnology Co., Ltd. Specifications: food grade, 200 mesh, purity: 99%. Sulfur powder was purchased from Xintai Taiwang Trade Co., Ltd. Specifications: Industrial grade, 325 mesh, purity: 99%. Nano-silica was purchased from Nanjing Baoket New Materials Co., Ltd., specification: PST-P10, hydrophilic, particle size: 20nm; The limestone powder was purchased from Shuanglong Powder Industry in Jingxian County, Anhui Province. Specification: ultrafine grade, 1250 mesh. Denitrifying Thiobacillus ( Thiobacillus denitrificans Purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number: HZB357313; Pseudomonas schrenckii ( Pseudomonas stutzeri Purchased from Wuhan Gray Algae Biotechnology Co., Ltd., product number: HZB146066.

[0032] Example 1 An autotrophic denitrifying bacteria agent comprises, from the inside out: a sulfur-calcium complex core, an inner shell gel layer covering the surface of the sulfur-calcium complex core, and an outer functional layer covering the surface of the inner shell gel layer. The sulfur-calcium composite core comprises the following raw materials in parts by weight: 12 parts of sulfur autotrophic denitrifying bacteria suspension and 25 parts of sulfur-calcium composite carrier; The inner shell gel layer comprises the following raw materials in parts by weight: 1 part sodium alginate, 0.2 parts xanthan gum, 1 part ultrafine calcium carbonate, and 97 parts deionized water; The outer functional layer comprises the following raw materials in parts by weight: 10 parts heterotrophic denitrifying bacteria suspension and 20 parts outer gel solution; The outer gel solution comprises the following raw materials in parts by weight: 4 parts soluble starch, 1 part sodium alginate, 0.5 parts xanthan gum, 2 parts sodium acetate-stearic acid microspheres, and 80 parts deionized water.

[0033] A method for preparing an autotrophic denitrifying bacteria agent includes the following steps: S1. Mix the sulfur-calcium composite carrier with a sulfur-autotrophic denitrifying bacteria suspension (the sulfur-autotrophic denitrifying bacteria suspension has a concentration of 1.5 × 10⁻⁶). 9 The mixture was prepared by mixing a CFU / mL suspension of denitrifying thiobacillus, allowing it to stand at 25°C for 12 hours to adsorb, and then drying it at 30°C until the water content of the system was 8% to obtain the sulfur-calcium composite core. S2. Add sodium alginate, xanthan gum and ultrafine calcium carbonate to deionized water, heat and stir at 60°C and 200 rpm for 20 min, and cool to room temperature to obtain inner shell gel liquid. S3. Spray the inner shell gel liquid onto the surface of the sulfur-calcium composite core at a rate of 1 mL / min and a pressure of 0.05 MPa. During the spraying process, the mixture is stirred at 50 rpm to obtain intermediate A. At a mass-volume ratio of 1 g: 10 mL, intermediate A is immersed in a 1.5 wt% calcium chloride solution and crosslinked and cured at 20°C for 10 min. After filtration through a 100-mesh filter cloth at normal pressure, it is washed twice with sterile deionized water, placed at room temperature, spread out to a thickness of 0.5 cm, and drained for 2 h to form an inner shell gel layer, thus obtaining inner shell coated particles. S4. Prepare a heterotrophic denitrifying bacteria suspension (the heterotrophic denitrifying bacteria suspension has a concentration of 4×10⁻⁶). 9 A suspension of *Pseudomonas stearothermia* (CFU / mL) was mixed with the outer gel solution to obtain the outer bacterial gel solution. S5. Spray the outer layer bacterial gel solution onto the surface of the inner shell coated particles at a rate of 1 mL / min and a pressure of 0.05 MPa. During the spraying process, the particles are stirred at 50 rpm to obtain intermediate B. At a mass-volume ratio of 1 g: 10 mL, intermediate B is immersed in a 1.5 wt% calcium chloride solution and cross-linked and cured at 20°C for 15 min. After filtration with a 100-mesh filter cloth under normal pressure, it is washed twice with sterile deionized water, placed at room temperature, spread out to a thickness of 0.5 cm, and drained for 4 h to form an outer functional layer, thus obtaining an autotrophic denitrifying bacterial agent.

[0034] The specific preparation method of the sulfur-calcium composite carrier is as follows: Eggshell powder and stearic acid were mixed at a mass ratio of 1:0.03 and stirred at 50℃ and 200 rpm for 20 min to obtain hydrophobically modified eggshell powder. Sulfur powder was heated to 120℃ to melt, and 0.2% of Tween-80 (by mass of sulfur powder) was added. After dissolving by stirring at 200 rpm, 20% of hydrophobically modified eggshell powder (by mass of sulfur powder) was added and stirred at 300 rpm for 10 min. After cooling to room temperature, 3% of sodium thiosulfate (by mass of sulfur powder) was added and stirred at 200 rpm for 5 min. The mixture was then pressed into shape using a granulator, with the particle size controlled at 1.5 mm. After drying at 55℃ to a moisture content of 4%, a sulfur-calcium composite carrier was obtained.

[0035] The specific preparation method of sodium acetate-stearic acid microspheres is as follows: Sodium acetate, stearic acid, maltodextrin, and Tween-80 were prepared in a mass ratio of 6:1.5:1:0.1. Sodium acetate was dissolved in deionized water to prepare a 40wt% sodium acetate solution. Stearic acid was heated to 75℃ to melt it, obtaining molten stearic acid. Maltodextrin and Tween-80 were added to the 40wt% sodium acetate solution and stirred at 70℃ and 300rpm to dissolve, obtaining an aqueous phase. Under the condition of holding at 70℃, the molten stearic acid was mixed with the aqueous phase and sheared and emulsified at 8000rpm for 15min to form an emulsion. The emulsion was pumped into a spray dryer at a rate of 5mL / min under the condition of holding at 70℃. The inlet air temperature of the spray dryer was 160℃ and the outlet air temperature was 70℃. After the feeding was completed, drying continued for 10min to obtain sodium acetate-stearic acid microspheres.

[0036] Example 2 An autotrophic denitrifying bacteria agent comprises, from the inside out: a sulfur-calcium complex core, an inner shell gel layer covering the surface of the sulfur-calcium complex core, and an outer functional layer covering the surface of the inner shell gel layer. The sulfur-calcium composite core comprises the following raw materials in parts by weight: 15 parts of sulfur autotrophic denitrifying bacteria suspension and 30 parts of sulfur-calcium composite carrier; The inner shell gel layer comprises the following raw materials in parts by weight: 1.5 parts sodium alginate, 0.4 parts xanthan gum, 1.5 parts ultrafine calcium carbonate, and 98 parts deionized water; The outer functional layer comprises the following raw materials in parts by weight: 12 parts heterotrophic denitrifying bacteria suspension and 25 parts outer gel solution; The outer gel liquid comprises the following raw materials in parts by weight: 5 parts soluble starch, 1.5 parts sodium alginate, 0.75 parts xanthan gum, 2.5 parts sodium acetate-stearic acid microspheres, and 85 parts deionized water.

[0037] A method for preparing an autotrophic denitrifying bacteria agent includes the following steps: S1. Mix the sulfur-calcium composite carrier with a sulfur-autotrophic denitrifying bacteria suspension (the sulfur-autotrophic denitrifying bacteria suspension has a concentration of 2.0 × 10⁻⁶). 9The mixture was prepared by mixing the sulfur-calcium composite core with a CFU / mL suspension of denitrifying thiobacillus and allowing it to stand at 30°C for 18 hours to adsorb. The mixture was then dried at 35°C until the water content of the system was 8%. S2. Add sodium alginate, xanthan gum and ultrafine calcium carbonate to deionized water, heat and stir at 70°C and 300 rpm for 30 min, and cool to room temperature to obtain inner shell gel liquid. S3. Spray the inner shell gel liquid onto the surface of the sulfur-calcium composite core at a rate of 2 mL / min and a pressure of 0.1 MPa. During the spraying process, the mixture is stirred at 80 rpm to obtain intermediate A. At a mass-volume ratio of 1 g: 15 mL, intermediate A is immersed in a 2.0 wt% calcium chloride solution and crosslinked and cured at 25°C for 15 min. After filtration through a 150-mesh filter cloth under normal pressure, it is washed twice with sterile deionized water, placed at room temperature, spread out to a thickness of 1 cm, and drained for 2.5 h to form an inner shell gel layer, thus obtaining inner shell coated particles. S4. Prepare a heterotrophic denitrifying bacteria suspension (the heterotrophic denitrifying bacteria suspension has a concentration of 5×10⁻⁶). 9 A suspension of *Pseudomonas stearothermia* (CFU / mL) was mixed with the outer gel solution to obtain the outer bacterial gel solution. S5. Spray the outer layer bacterial gel solution onto the surface of the inner shell coated particles at a rate of 2 mL / min and a pressure of 0.1 MPa. During the spraying process, the particles are stirred at 80 rpm to obtain intermediate B. At a mass-volume ratio of 1 g: 15 mL, intermediate B is immersed in a 2 wt% calcium chloride solution and cross-linked and cured at 25°C for 25 min. After filtration with a 150-mesh filter cloth under normal pressure, it is washed twice with sterile deionized water, placed at room temperature, spread out to a thickness of 1 cm, and drained for 4.5 h to form an outer functional layer, thus obtaining an autotrophic denitrifying bacterial agent.

[0038] The specific preparation method of the sulfur-calcium composite carrier is as follows: Eggshell powder and stearic acid were mixed at a mass ratio of 1:0.05 and stirred at 60℃ and 300 rpm for 30 min to obtain hydrophobically modified eggshell powder. Sulfur powder was heated to 125℃ to melt, and 0.3% of Tween-80 (by mass of sulfur powder) was added. After dissolving by stirring at 300 rpm, 25% of hydrophobically modified eggshell powder (by mass of sulfur powder) was added and stirred at 400 rpm for 15 min. After cooling to room temperature, 5% of sodium thiosulfate (by mass of sulfur powder) was added and stirred at 250 rpm for 8 min. The mixture was then granulated and pressed into shape with a particle size controlled at 2 mm. After drying at 60℃ to a moisture content of 4%, a sulfur-calcium composite carrier was obtained.

[0039] The specific preparation method of sodium acetate-stearic acid microspheres is as follows: Sodium acetate, stearic acid, maltodextrin, and Tween-80 were prepared in a mass ratio of 8:2:1:0.2. Sodium acetate was dissolved in deionized water to prepare a 45wt% sodium acetate solution. Stearic acid was heated to 80℃ to melt it, obtaining molten stearic acid. Maltodextrin and Tween-80 were added to the 45wt% sodium acetate solution and stirred at 75℃ and 400rpm to dissolve, obtaining an aqueous phase. Under the condition of holding at 75℃, the molten stearic acid was mixed with the aqueous phase and sheared and emulsified at 10000rpm for 18min to form an emulsion. The emulsion was pumped into a spray dryer at a rate of 10mL / min under the condition of holding at 75℃ for drying. The inlet air temperature of the spray dryer was 170℃ and the outlet air temperature was 75℃. After the feeding was completed, drying continued for 15min to obtain sodium acetate-stearic acid microspheres.

[0040] Example 3 An autotrophic denitrifying bacteria agent comprises, from the inside out: a sulfur-calcium complex core, an inner shell gel layer covering the surface of the sulfur-calcium complex core, and an outer functional layer covering the surface of the inner shell gel layer. The sulfur-calcium composite core comprises the following raw materials in parts by weight: 18 parts of sulfur autotrophic denitrifying bacteria suspension and 40 parts of sulfur-calcium composite carrier; The inner shell gel layer comprises the following raw materials in parts by weight: 2 parts sodium alginate, 0.5 parts xanthan gum, 2 parts ultrafine calcium carbonate, and 100 parts deionized water; The outer functional layer comprises the following raw materials in parts by weight: 15 parts heterotrophic denitrifying bacteria suspension and 30 parts outer gel solution; The outer gel solution comprises the following raw materials in parts by weight: 6 parts soluble starch, 2 parts sodium alginate, 1 part xanthan gum, 3 parts sodium acetate-stearic acid microspheres, and 90 parts deionized water.

[0041] A method for preparing an autotrophic denitrifying bacteria agent includes the following steps: S1. The sulfur-calcium composite carrier is mixed with a sulfur-autotrophic denitrifying bacteria suspension (the sulfur-autotrophic denitrifying bacteria suspension has a concentration of 2.5 × 10⁻⁶). 9 The mixture was prepared by mixing a CFU / mL suspension of denitrifying thiobacillus, allowing it to stand at 35°C for 24 hours to adsorb, and then drying it at 40°C until the water content of the system was 8% to obtain the sulfur-calcium composite core. S2. Add sodium alginate, xanthan gum and ultrafine calcium carbonate to deionized water, heat and stir at 80°C and 400 rpm for 40 min, and cool to room temperature to obtain inner shell gel liquid. S3. Spray the inner shell gel liquid onto the surface of the sulfur-calcium composite core at a rate of 3 mL / min and a pressure of 0.15 MPa. During the spraying process, the mixture is stirred at 100 rpm to obtain intermediate A. At a mass-volume ratio of 1 g: 20 mL, intermediate A is immersed in a 2.5 wt% calcium chloride solution and crosslinked and cured at 30℃ for 120 min. After filtration through a 200-mesh filter cloth at normal pressure, it is washed three times with sterile deionized water, placed at room temperature, spread out to a thickness of 1.5 cm, and drained for 3 h to form an inner shell gel layer, thus obtaining inner shell coated particles. S4. Prepare a heterotrophic denitrifying bacteria suspension (the heterotrophic denitrifying bacteria suspension has a concentration of 6×10⁻⁶). 9 A suspension of *Pseudomonas stearothermia* (CFU / mL) was mixed with the outer gel solution to obtain the outer bacterial gel solution. S5. Spray the outer layer bacterial gel solution onto the surface of the inner shell coated particles at a rate of 3 mL / min and a pressure of 0.15 MPa. During the spraying process, the particles are stirred at 100 rpm to obtain intermediate B. At a mass-volume ratio of 1 g: 20 mL, intermediate B is immersed in a 2.5 wt% calcium chloride solution and cross-linked and cured at 30°C for 30 min. After filtration through a 200-mesh filter cloth at normal pressure, it is washed three times with sterile deionized water, placed at room temperature, spread out to a thickness of 1.5 cm, and drained for 5 h to form an outer functional layer, thus obtaining an autotrophic denitrifying bacterial agent.

[0042] The specific preparation method of the sulfur-calcium composite carrier is as follows: Eggshell powder and stearic acid were mixed at a mass ratio of 1:0.08 and stirred at 70℃ and 400 rpm for 40 min to obtain hydrophobically modified eggshell powder. Sulfur powder was heated to 130℃ to melt, and 0.5% of Tween-80 (by mass of sulfur powder) was added. After dissolving by stirring at 400 rpm, 30% of hydrophobically modified eggshell powder (by mass of sulfur powder) was added and stirred at 500 rpm for 20 min. After cooling to room temperature, 8% of sodium thiosulfate (by mass of sulfur powder) was added and stirred at 300 rpm for 10 min. The mixture was then pressed into shape using a granulator, with the particle size controlled at 2.5 mm. After drying at 65℃ to a moisture content of 4%, a sulfur-calcium composite carrier was obtained.

[0043] The specific preparation method of sodium acetate-stearic acid microspheres is as follows: Sodium acetate, stearic acid, maltodextrin, and Tween-80 were prepared in a mass ratio of 10:2.5:1:0.3. Sodium acetate was dissolved in deionized water to prepare a 50wt% sodium acetate solution. Stearic acid was heated to 85℃ to melt it, obtaining molten stearic acid. Maltodextrin and Tween-80 were added to the 50wt% sodium acetate solution and stirred at 80℃ and 500rpm to dissolve, obtaining an aqueous phase. Under the condition of holding at 80℃, the molten stearic acid was mixed with the aqueous phase and sheared and emulsified at 12000rpm for 20min to form an emulsion. The emulsion was pumped into a spray dryer at a rate of 15mL / min under the condition of holding at 80℃. The inlet air temperature of the spray dryer was 180℃ and the outlet air temperature was 80℃. After the feeding was completed, drying continued for 20min to obtain sodium acetate-stearic acid microspheres.

[0044] Comparative Example 1 This comparative example provides a method for preparing an autotrophic denitrifying bacteria agent. The only difference from Example 2 is that steps S2 and S3 (i.e., no inner shell gel layer is prepared and no inner shell coating is performed) are omitted. In steps S4 and S5, the outer layer bacterial gel liquid is directly attached to the surface of the sulfur-calcium composite core. The other raw material types, amounts, and preparation process parameters are completely consistent with those of Example 2.

[0045] Specifically: A method for preparing an autotrophic denitrifying bacteria agent includes the following steps: S1. Mix the sulfur-calcium composite carrier with a sulfur-autotrophic denitrifying bacteria suspension (the sulfur-autotrophic denitrifying bacteria suspension has a concentration of 2.0 × 10⁻⁶). 9 The mixture was prepared by mixing the sulfur-calcium composite core with a CFU / mL suspension of denitrifying thiobacillus and allowing it to stand at 30°C for 18 hours to adsorb. The mixture was then dried at 35°C until the water content of the system was 8%. S2. Prepare a heterotrophic denitrifying bacteria suspension (concentration 5×10⁻⁶). 9 A suspension of *Pseudomonas stearothermia* (CFU / mL) was mixed with the outer gel solution to obtain the outer bacterial gel solution. S3. Spray the outer layer bacterial gel solution onto the surface of the sulfur-calcium composite core at a rate of 2 mL / min and a pressure of 0.1 MPa. During the spraying process, the mixture is stirred at 80 rpm to obtain intermediate B. At a mass-volume ratio of 1 g: 15 mL, intermediate B is immersed in a 2 wt% calcium chloride solution and cross-linked and cured at 25°C for 25 min. After filtration through a 150-mesh filter cloth at normal pressure, it is washed twice with sterile deionized water, placed at room temperature, spread out to a thickness of 1 cm, and drained for 4.5 h to form the outer functional layer, thus obtaining the autotrophic denitrifying bacterial agent.

[0046] Comparative Example 2 This comparative example provides a method for preparing an autotrophic denitrifying bacteria agent. The only difference from Example 2 is that ultrafine calcium carbonate is not added in step S2 (i.e., in the inner shell gel layer). The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0047] Comparative Example 3 This comparative example provides a method for preparing an autotrophic denitrifying bacteria agent. The only difference from Example 2 is that in step S2 (i.e., in the inner shell gel layer), ultrafine calcium carbonate is replaced with an equal mass of nano-silica. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0048] Comparative Example 4 This comparative example provides a method for preparing an autotrophic denitrifying bacteria agent. The only difference from Example 2 is that eggshell powder is replaced with an equal mass of limestone powder in the preparation process of the sulfur-calcium composite carrier. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0049] Comparative Example 5 This comparative example provides a method for preparing an autotrophic denitrifying bacteria agent. The only difference from Example 2 is that in step S4 (i.e., in the outer bacterial gel solution), sodium acetate-stearic acid microspheres are replaced with an equal mass of sodium acetate. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0050] Performance testing To verify the denitrification effect, structural stability, and cell activity of the autotrophic denitrifying bacteria prepared in this invention, the autotrophic denitrifying bacteria prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to uniform performance testing under the same testing conditions. The results were taken as the average of three parallel experiments. The specific testing contents are as follows: I. Preparation of Simulated Wastewater (1) Preparation of 1000 times concentrated solution of simulated wastewater Accurately weigh 303.5g of sodium nitrate, 5.0g of potassium dihydrogen phosphate, 10.0g of magnesium sulfate (MgSO4·7H2O), 3.0g of calcium chloride, and 100g of sodium bicarbonate. Dissolve them in deionized water, then add 1.0mL of trace element solution and bring the volume up to 1L to obtain a 1000-fold concentrated solution of simulated wastewater. The preparation method of the trace element solution is as follows: accurately weigh 10g of EDTA, 0.43g of zinc sulfate (ZnSO4·7H2O), 0.25g of copper sulfate (CuSO4·5H2O), 0.99g of manganese chloride (MnCl2·4H2O), 0.19g of nickel chloride (NiCl2·6H2O), 0.24g of cobalt chloride (CoCl2·6H2O), 0.014g of boric acid (H3BO3), and 0.22g of sodium molybdate (Na2MoO4·2H2O), add them to deionized water to dissolve, and make up to 1L to obtain the trace element solution.

[0051] (2) Preparation of working solution for low carbon-nitrogen ratio simulated anoxic wastewater Take 1 mL of the 1000-fold concentrated solution of the above simulated wastewater and dilute it to 1 L with deionized water to obtain the low C / N ratio simulated anoxic wastewater working solution. The initial total nitrogen (TN) concentration of this low C / N ratio simulated anoxic wastewater working solution is 50 mg / L, the C / N ratio is ≤1, and the initial pH value is 7.0.

[0052] II. Testing Conditions Measure 500 mL of the low C / N ratio simulated anoxic wastewater solution and place it in a 500 mL anaerobic bottle. Add 2.5 g of autotrophic denitrifying bacteria agent at an inoculation rate of 5 g / L. After sealing, place the bottle in a 25℃ constant temperature incubator for anaerobic batch static culture for 72 h.

[0053] III. Testing Items and Methods (1) Total nitrogen (TN) removal rate detection Referring to HJ 636-2012 "Determination of Total Nitrogen in Water by Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometry", the total nitrogen concentration in water samples at the initial culture time (0h), 24h, 48h, and 72h was measured respectively. The total nitrogen removal rate was calculated according to the formula: Total nitrogen removal rate (%) = (initial total nitrogen concentration - total nitrogen concentration at the time of detection) / initial total nitrogen concentration × 100%.

[0054] (2) Nitrite nitrogen (NO2) - -N) Accumulation rate detection The concentration of nitrite nitrogen in the water sample after 72 hours of cultivation was detected according to GB / T 7493-1987 "Determination of Nitrite Nitrogen in Water Quality - Spectrophotometric Method". The nitrite accumulation rate was calculated to determine the degree of denitrification. The calculation formula is as follows: Nitrite accumulation rate (%) = Nitrite nitrogen concentration / (Initial total nitrogen concentration - 72h total nitrogen concentration) × 100%. When the total nitrogen removal rate is less than 10%, this indicator is not used as an evaluation basis.

[0055] (3) Detection of pH stability of effluent A precision pH meter was used to measure the pH value of the effluent after 72 hours of cultivation to determine the acid-base buffering capacity of the autotrophic denitrifying bacteria agent on the reaction system.

[0056] (4) Detection of bacterial cell storage stability (viable cell retention rate) Referring to the plate count method in GB 4789.2-2022 "National Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count", the initial total viable count of the autotrophic denitrifying bacteria agent was tested. After storing the agent in a sealed container at 25℃ in the dark for 90 days, the total viable count was tested again, and the viable count retention rate was calculated using the following formula: Viable count retention rate (%) = (Number of viable bacteria after 90 days of storage / Initial number of viable bacteria) × 100%.

[0057] (5) Test of water shear failure rate The autotrophic denitrifying bacteria agent to be tested was first pre-sieved using a 1.2mm standard sieve to remove particles smaller than 1.2mm. 5g of the sieve residue was accurately weighed and placed in a 250mL Erlenmeyer flask containing 100mL of deionized water. The flask was shaken at 25℃ and 200rpm for 72 hours to simulate hydraulic shear. After shaking, all the material in the flask was transferred to a 1.2mm standard sieve. The sieve residue was slowly rinsed with deionized water until the filtrate was clear. The undersize material was collected and dried at 105℃ to constant weight. The mass of the undersize material was then weighed. The hydraulic shear breakage rate was calculated using the following formula: Hydraulic shear breakage rate (%) = (mass of undersize material / 5g) × 100%.

[0058] IV. Test Results The specific test results are shown in Table 1.

[0059] Table 1 Performance test results of autotrophic denitrifying bacteria agent As shown in Table 1, the autotrophic denitrifying bacteria agents prepared in Examples 1-3 of this invention exhibit excellent overall performance, with Example 2 showing the best effect, outperforming Comparative Examples 1-5 in all indicators. A detailed comparison and analysis of each comparative example with Example 2 is as follows: A comparison between Comparative Example 1 and Example 2 shows that Comparative Example 1, which did not have an inner shell gel layer, experienced a significant decrease in the denitrification effect, pH buffering capacity, cell survival stability, and structural shear resistance of the autotrophic denitrifying bacteria. This indicates that the inner shell gel layer is a key structure for achieving functional zoning, acid-base buffering, and cell protection.

[0060] Comparing Comparative Example 2 with Example 2, it can be seen that without the addition of ultrafine calcium carbonate to the inner shell gel layer of Comparative Example 2, the denitrification efficiency of the autotrophic denitrifying bacteria was significantly reduced, the system was prone to acidification, and the stability and mechanical strength of the bacteria cells also deteriorated significantly. This indicates that ultrafine calcium carbonate plays a key role in alkalinity replenishment and structural enhancement.

[0061] As can be seen from the comparison between Comparative Example 3 and Example 2, after replacing ultrafine calcium carbonate with nano-silica, the overall performance of the autotrophic denitrifying bacteria agent in Comparative Example 3 is still significantly inferior to that in Example 2, indicating that the function of calcium carbonate cannot be simply replaced by ordinary inert fillers.

[0062] Comparing Comparative Example 4 with Example 2, it can be seen that Comparative Example 4 uses limestone powder instead of modified eggshell powder, and the denitrification effect and pH stability of the autotrophic denitrifying bacteria are significantly reduced, proving that modified eggshell powder has obvious advantages in slow-release alkalinity and improved biocompatibility.

[0063] Comparing Comparative Example 5 with Example 2, it can be seen that when sodium acetate is used instead of sodium acetate-stearic acid microspheres in the outer gel of Comparative Example 5, the denitrification efficiency is significantly reduced and the hydraulic shear failure rate is significantly increased. This indicates that sodium acetate-stearic acid microspheres not only provide a slow-release carbon source, but their solid microsphere morphology can also enhance the mechanical strength of the outer layer and maintain structural integrity.

[0064] In summary, this invention, through the synergistic effect of the sulfur-calcium composite core, the inner shell gel layer, and the outer functional layer, and in conjunction with key designs such as modified eggshell powder, ultrafine calcium carbonate, and slow-release carbon source microspheres, enables the prepared autotrophic denitrifying bacteria agent to simultaneously possess advantages such as high nitrogen removal efficiency, low nitrite accumulation, stable system pH, high bacterial survival rate, and good resistance to hydraulic shear.

[0065] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An autotrophic denitrifying bacteria agent, characterized in that, From the inside out, it includes: a sulfur-calcium composite core, an inner shell gel layer covering the surface of the sulfur-calcium composite core, and an outer functional layer covering the surface of the inner shell gel layer; The sulfur-calcium composite core comprises the following raw materials in parts by weight: 12-18 parts of sulfur autotrophic denitrifying bacteria suspension and 25-40 parts of sulfur-calcium composite carrier; The inner shell gel layer comprises the following raw materials in parts by weight: 1-2 parts sodium alginate, 0.2-0.5 parts xanthan gum, 1-2 parts ultrafine calcium carbonate, and 97-100 parts deionized water. The outer functional layer comprises the following raw materials in parts by weight: 10-15 parts of heterotrophic denitrifying bacteria suspension and 20-30 parts of outer gel solution.

2. The autotrophic denitrifying bacteria agent according to claim 1, characterized in that, The sulfur-autotrophic denitrifying bacteria are denitrifying thiobacilli; the concentration of the sulfur-autotrophic denitrifying bacteria suspension is 1.5 × 10⁻⁶. 9 CFU / mL -2.5×10 9 CFU / mL.

3. The autotrophic denitrifying bacteria agent according to claim 1, characterized in that, The method for preparing the sulfur-calcium composite carrier is as follows: Eggshell powder and stearic acid were mixed and stirred evenly to obtain hydrophobically modified eggshell powder. Sulfur powder was heated to 120-130℃ to melt, Tween-80 was added and stirred to dissolve, then hydrophobically modified eggshell powder was added and stirred evenly. After cooling to room temperature, sodium thiosulfate was added and stirred evenly. The mixture was granulated, dried, and obtained a sulfur-calcium composite carrier.

4. The autotrophic denitrifying bacteria agent according to claim 1, characterized in that, The heterotrophic denitrifying bacteria are *Pseudomonas schlegelii*; the concentration of the heterotrophic denitrifying bacteria suspension is 4 × 10⁻⁶. 9 CFU / mL - 6 × 10 9 CFU / mL.

5. The autotrophic denitrifying bacteria agent according to claim 1, characterized in that, The outer gel liquid comprises the following raw materials in parts by weight: 4-6 parts soluble starch, 1-2 parts sodium alginate, 0.5-1 part xanthan gum, 2-3 parts sodium acetate-stearic acid microspheres, and 80-90 parts deionized water.

6. The autotrophic denitrifying bacteria agent according to claim 5, characterized in that, The preparation method of the sodium acetate-stearic acid microspheres is as follows: Sodium acetate was dissolved in deionized water to prepare a sodium acetate solution. Stearic acid was heated to melt and obtain molten stearic acid. Maltodextrin and emulsifier were added to the sodium acetate solution to obtain an aqueous phase. The molten stearic acid was mixed with the aqueous phase and sheared and emulsified to form an emulsion. The emulsion was spray-dried to obtain sodium acetate-stearic acid microspheres.

7. A method for preparing an autotrophic denitrifying bacteria agent as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Mix the sulfur-calcium composite carrier with a sulfur-autotrophic denitrifying bacteria suspension, allow it to stand for adsorption, and dry it to obtain the sulfur-calcium composite core. S2. Add sodium alginate, xanthan gum and ultrafine calcium carbonate to deionized water, heat and stir, and cool to room temperature to obtain inner shell gel liquid. S3. Spray the inner shell gel liquid onto the surface of the sulfur-calcium composite core to obtain intermediate A. Immerse intermediate A in calcium chloride solution for cross-linking and curing, filter, wash, and drain to form an inner shell gel layer and obtain inner shell coated particles. S4. Mix the heterotrophic denitrifying bacteria suspension with the outer layer gel solution to obtain the outer layer bacterial gel solution; S5. Spray the outer layer bacterial gel liquid onto the surface of the inner shell coated particles to obtain intermediate B. Immerse intermediate B in calcium chloride solution for cross-linking and solidification, filter, wash, and drain to form the outer functional layer, thus obtaining the autotrophic denitrifying bacteria agent.

8. The method for preparing the autotrophic denitrifying bacteria agent according to claim 7, characterized in that, In step S5, the spraying rate is 1-3 mL / min and the spraying pressure is 0.05-0.15 MPa.

9. The method for preparing the autotrophic denitrifying bacteria agent according to claim 7, characterized in that, In step S5, the mass fraction of calcium chloride solution is 1.5-2.5%; the cross-linking curing temperature is 20-30℃; and the cross-linking curing time is 15-30 min.

10. The application of an autotrophic denitrifying bacteria agent as described in any one of claims 1-6 in wastewater treatment.

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