Zeolite-loaded bacterial spore microcapsule, preparation thereof and application of zeolite-loaded bacterial spore microcapsule in wastewater remediation

Abstract

The invention provides a zeolite loaded bacterial spore microcapsule and a preparation method thereof, the microcapsule takes zeolite as a skeleton structure, bacterial spores are embedded in the skeleton formed by the zeolite, and nutrient substances are loaded on the outer surface of the skeleton; wherein the bacterial spores are bacillus pasteurii spores, and the nutrient substances comprise sodium succinate, inosine, Ni < 2 + > and Mn < 2 + >. The invention also provides application of the microcapsule in wastewater remediation. Through coordination of zeolite adsorption and bacterial spore activation, efficient and synchronous restoration of the lead-containing ammonia nitrogen composite wastewater is realized: on one hand, zeolite absorbs part of lead ions in the early stage to reduce the toxicity of the zeolite to spores and also absorbs ammonia nitrogen byproducts generated by urea hydrolysis by virtue of porous high adsorbability; on the other hand, the spores are resistant to a severe environment, germinate into nutritional cells after the lead concentration is reduced, catalyze urea hydrolysis to produce carbonate radicals and solidify residual lead into stable carbonate, and finally, the dual effects of lead deep solidification and ammonia nitrogen removal are achieved.

Description

Technical Field

[0001] This invention relates to the field of environmental engineering technology, and in particular to a zeolite-loaded bacterial spore microcapsule, its preparation method, and its application in wastewater remediation. Background Technology

[0002] In modern industrial and agricultural production, wastewater discharged from industries such as electroplating, battery manufacturing, and chemicals often carries large amounts of lead ions (Pb). 2+ Meanwhile, some production processes also involve ammonia nitrogen (NH4). + Lead and ammonia nitrogen pollution, a type of combined pollutant wastewater, has become a significant environmental problem threatening soil, aquatic ecosystems, and human health. Lead, a non-biodegradable heavy metal, accumulates through the food chain, inhibiting plant photosynthetic activity, damaging soil fertility, and harming the human nervous and digestive systems. Excessive ammonia nitrogen depletes dissolved oxygen in water, leading to oxygen deprivation and death of aquatic organisms. It may also be converted into nitrite and nitrate nitrogen, or react with chlorine to produce toxic substances such as chloramines and trihalomethanes, rendering water bodies unusable for drinking and exacerbating eutrophication disasters such as algal blooms.

[0003] Currently, the main remediation technologies for lead-containing wastewater include chemical precipitation, electrokinetic remediation, and bioremediation. Among these, microbial induced carbonate precipitation (MICP) technology in bioremediation has received widespread attention due to its advantages of being environmentally friendly, sustainable, and relatively low-cost. Its core mechanism involves urea-lysing bacteria hydrolyzing urea to produce carbonate ions (CO3-). 2- ), causing lead ions (Pb) 2+ Lead ions combine with carbonate ions to form stable carbonate bioprecipitates, thereby achieving lead immobilization. However, this technology faces two major bottlenecks: first, lead ions are extremely toxic to urea-lysing bacteria, significantly reducing urease activity and leading to decreased urea hydrolysis efficiency and poor lead immobilization; second, the urea hydrolysis process generates a large amount of ammonia nitrogen byproducts, which not only fails to solve the original ammonia nitrogen pollution but also exacerbates the risk of eutrophication, creating a contradiction of "remediating one type of pollution while triggering another." Furthermore, conventional urea-lysing bacteria have poor tolerance to harsh environments such as dryness, radiation, and acidity / alkalinity, making long-term storage difficult, and they struggle to grow and reproduce in environments with high concentrations of lead pollution, further limiting the practical application of MIP technology.

[0004] In the field of ammonia nitrogen removal, zeolite is widely used for the adsorption and treatment of ammonia nitrogen in water bodies due to its porous structure, large specific surface area, and excellent adsorption performance. However, the application of zeolite for wastewater containing both lead and ammonia nitrogen is quite limited: on the one hand, when used alone, it can only remove some lead ions through physical adsorption and cannot convert lead into stable solid precipitates for deep solidification, making it difficult to completely eliminate the toxicity risk of lead; on the other hand, if zeolite is simply mixed with conventional live bacteria (using the catalytic reaction of live bacteria to solidify lead) in an attempt to address lead pollution remediation, the strong toxicity of lead ions to the live bacteria will cause the bacteria to quickly become inactive and unable to perform their lead-fixing function, ultimately failing to simultaneously meet the dual needs of lead solidification and ammonia nitrogen removal.

[0005] Therefore, developing a technical solution that can resist the toxic stress of lead ions on functional microorganisms, ensure the deep solidification effect of lead, and simultaneously adsorb the initial ammonia nitrogen or ammonia nitrogen byproducts generated during the remediation process, thereby achieving the synergistic treatment of lead- and ammonia nitrogen combined pollution wastewater, has become an urgent need in the field of environmental engineering for dealing with such complex pollution problems. It is also a key direction for promoting the practical application of combined pollution remediation technology and breaking through the existing technical bottlenecks. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a zeolite-loaded bacterial spore microcapsule and its preparation and application in wastewater remediation. It achieves efficient and simultaneous remediation of lead- and ammonia-nitrogen-contaminated wastewater through the "synergistic effect of zeolite adsorption and bacterial spore activation": on the one hand, the porous structure and high adsorption capacity of zeolite are used to adsorb some lead ions in the wastewater in the early stage to reduce its toxic stress on spores, while adsorbing ammonia nitrogen byproducts produced by urea hydrolysis; on the other hand, relying on the strong tolerance of bacterial spores to harsh environments, they germinate into vegetative cells after the lead concentration decreases, catalyze the hydrolysis of urea to produce carbonate ions, and solidify the remaining lead ions into stable carbonate precipitates, ultimately achieving the dual effect of deep lead solidification and ammonia nitrogen removal, solving the problem that existing technologies cannot simultaneously achieve anti-toxicity, lead solidification and ammonia nitrogen removal.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A zeolite-loaded bacterial spore microcapsule is disclosed. The microcapsule has a zeolite framework structure, with bacterial spores embedded within the framework. The outer surface of the framework is loaded with nutrients that promote spore germination. The bacterial spores are *Bacillus pasteurellii* spores, and the nutrients include sodium succinate, inosine, and Ni. 2+ and Mn 2+ .

[0008] As one of the preferred embodiments of the present invention, the bacterial spores are obtained in the following manner: (1) Take the frozen Bacillus pasteurellus strain, inoculate it into the activation medium, and incubate it at 28~32℃ and 170~190rpm for 22~26h to obtain the activated bacterial solution; (2) Inoculate the activated bacterial solution from step (1) into the spore-producing medium and continue to culture at 28-32℃ and 170-190rpm for 3-5 days to induce Bacillus pasteurellii to produce spores; (3) Sterilize the solution obtained in step (2) in a water bath at 75~85℃ for 15~25min, and then centrifuge the treated solution at 7000~9000rpm for 8~12min; collect the centrifuged precipitate, resuspend it with physiological saline to obtain a suspension of Bacillus pasteurellii spores.

[0009] As one of the preferred embodiments of the present invention, in step (1), the composition of the activation culture medium includes: 4.5~5.5 g / L beef extract, 4.5~5.5 g / L peptone, 9~11 mg / L MnSO4·H2O, 20~28 mg / L NiCl2·6H2O, and pH 8.5~9.0.

[0010] As one of the preferred embodiments of the present invention, in step (2), the sporulation culture medium comprises: 5~9 g / L sodium succinate, 3~7 g / L urea, and 3~4 mg / L Mn. 2+ pH 6.8~7.2.

[0011] As one of the preferred embodiments of the present invention, the particle size of the microcapsules is 2~5mm.

[0012] A method for preparing the above-mentioned microcapsules includes the following steps: (1) Raw material preparation Take a suspension of Bacillus pasteurellii spores, zeolite, sodium succinate, inosine, and Ni 2+ Source and Mn 2+ Source, backup; (2) Spore embedding Mix the Pasteurella multocida spore suspension with zeolite at a ratio of 0.8~1.2 mL / g of spore suspension volume to zeolite mass, and stir evenly to embed the bacterial spores inside the zeolite framework. (3) Granulation and molding The zeolite-spore mixture obtained in step (2) is added to a granulation device to obtain microspheres; (4) Initial drying The zeolite-spore microspheres obtained in step (3) were dried in an environment of 35~45℃ for 20~28h to remove free water from the system; (5) Nutrient load Sodium succinate, inosine, Ni2+ Source and Mn 2+ The source is dissolved in deionized water to prepare a nutrient solution, and then sprayed evenly onto the surface of the microspheres after drying in step (4). (6) Secondary drying After loading the microspheres with nutrients, they were dried again at 35-45℃ for 20-28 hours to obtain the desired zeolite-loaded bacterial spore microcapsules.

[0013] As one of the preferred embodiments of the present invention, in step (1), the spore concentration of the spore suspension is 2.0 × 10⁻⁶. 10 ~2.9×10 10 CFU / mL, Ni 2+ Source selection: NiCl2·6H2O, Mn 2+ The source is MnSO4·H2O.

[0014] As one of the preferred embodiments of the present invention, in step (5), the specific composition of the nutrient solution is: sodium succinate 5~8g / L, inosine 10~15mmol / L, NiCl2·6H2O 20~28mg / L, MnSO4·H2O 9~11mg / L.

[0015] An application of the above-mentioned microcapsules in wastewater remediation, wherein the wastewater is lead- and ammonia-nitrogen-contaminated wastewater; the ammonia nitrogen originates from the initial ammonia nitrogen in the wastewater and / or ammonia nitrogen byproducts generated during the remediation process.

[0016] As one of the preferred embodiments of the present invention, the microcapsules are added to wastewater at a solid-liquid ratio of 5%, and 30 mmol / L urea is added. The mixture is reacted at 28~32℃ and 170~190 rpm for 120 h to achieve simultaneous removal of lead ions and ammonia nitrogen.

[0017] As one of the preferred embodiments of the present invention, the initial lead ion concentration in the wastewater is 1~10 mmol / L.

[0018] Design concept and principles: Addressing the three major challenges in the remediation of lead- and ammonia-nitrogen combined pollutant wastewater—lead ion poisoning of microorganisms, ammonia-nitrogen byproducts generated by MIP technology, and the difficulty of using a single material to simultaneously treat both types of pollution—this invention focuses on the synergistic effect of "anti-toxicity, lead fixation, and ammonia-nitrogen removal," constructing a "zeolite-loaded bacterial spore microcapsule" system. First, bacterial spores (rather than conventional live bacteria) are selected, utilizing their strong tolerance to heavy metals and harsh environments during dormancy to solve the problems of microbial inactivation due to lead poisoning and difficulty in long-term storage. Then, zeolite is used as both a carrier and adsorbent. On one hand, its porous structure and large specific surface area adsorb some lead ions from the wastewater in the early stages to reduce toxicity and create a safe environment for spore germination. Simultaneously, it adsorbs ammonia-nitrogen byproducts generated by urea hydrolysis, avoiding secondary pollution. On the other hand, its porous structure provides attachment space for spore germination and vegetative cell reproduction. Finally, through a synergistic model of "adsorption first + biomineralization follow-up," zeolite first physically adsorbs and alleviates lead toxicity, then activates spores to transform into vegetative cells that catalyze urea hydrolysis, achieving deep lead fixation and simultaneously resolving lead and ammonia-nitrogen pollution.

[0019] In terms of specific working principle, the core function of zeolite is reflected in three aspects: first, it preferentially adsorbs some Pb through ion exchange and physical adsorption. 2+ Firstly, it reduces the initial concentration of lead ions in the water, alleviating the toxic stress on spores; secondly, it relies on the effect of NH4+. + The high selective adsorption capacity simultaneously removes existing ammonia nitrogen and subsequent ammonia nitrogen byproducts from urea hydrolysis in wastewater; thirdly, the porous structure constructs "micro-reaction chambers" to provide a place for nutrient cells to attach and reproduce, ensuring the continuous progress of the reaction. Bacterial spores rely on their dormant state characteristics to thrive in high Pb environments. 2+ Once the lead concentration is reduced by zeolite adsorption in the environment, the cells germinate into vegetative cells under the induction of nutrient solution (sodium succinate, inosine, etc.) on the surface of the microcapsules. These cells secrete urease to catalyze the hydrolysis of urea: first, urea is converted into NH3 and CO2, and then NH3 dissolves in water to convert into NH4. + With OH - CO2 then reacts with OH - Combine to generate CO3 2- Ultimately, CO3 2- With the remaining Pb 2+ The combination forms stable carbonate bioprecipitates (such as PbCO3), thus completing the deep solidification of lead.

[0020] The entire microcapsule system forms a closed-loop synergistic effect: in the early stage, zeolite adsorption "paves the way" for spore germination, and in the later stage, spore activation and solidification of residual lead and zeolite simultaneous treatment of ammonia nitrogen byproducts not only solve the toxicity and byproduct problems of MIP technology, but also make up for the deficiency that single zeolite cannot deeply solidify lead, and finally achieve efficient and simultaneous remediation of lead-containing ammonia nitrogen composite polluted wastewater.

[0021] The advantages of this invention compared to the prior art are: (1) Strong resistance to toxins, breaking through the bottleneck of microbial inactivation. This invention utilizes *Bacillus pasteurellii* spores as its core functional component. Spores, as a stress response mode for bacteria to resist harsh external environments, exhibit extremely strong resistance in their dormant state to bactericidal agents such as heat, dryness, radiation, acids, alkalis, organic solvents, and heavy metal ions, making them the most resilient cells discovered in nature. Combined with the pre-adsorption effect of zeolite on lead ions, this further reduces Pb levels in water. 2+ The concentration reduces the toxic stress on spores and effectively solves the key problem in traditional MIP technology that "lead ions inhibit urease activity and cause rapid inactivation of live bacteria". Even in environments with high lead pollution concentrations of 1~10 mmol / L, the survival and subsequent activity of functional microorganisms can still be guaranteed.

[0022] (2) The carrier has multiple functions, providing efficient and stable support for repair. Zeolite was selected as the microcapsule framework, and its porous structure and large specific surface area have three core functions: First, it provides a stable attachment site for spore germination and the growth and reproduction of vegetative cells, preventing the loss of microorganisms and constructing a "micro-reaction chamber" to ensure the continuous catalytic reaction; second, it can serve as a slow-release carrier for nutrients, and together with the sodium succinate, inosine and other nutrients loaded on the surface of the microcapsules, it provides sufficient nutrition for spore germination; third, with its excellent adsorption performance, it can remove heavy metals and ammonia nitrogen, forming a synergistic closed loop of "adsorption-germination-solidification".

[0023] (3) Simultaneous treatment of two types of pollution to prevent secondary pollution Zeolite, as a material that combines adsorption and carrier functions, can simultaneously adsorb Pb from wastewater. 2+ With NH4 + In the initial stage of remediation, Pb in the water is reduced through physical adsorption and ion exchange. 2+ The initial concentration creates a low-toxicity environment for spore germination; during the biomineralization stage, when spores germinate into vegetative cells, they catalyze the hydrolysis of urea to produce CO3. 2- To solidify the remaining Pb 2+ (When a stable carbonate precipitate is formed, the lead removal efficiency reaches over 99%), zeolite can simultaneously adsorb NH4 produced by urea hydrolysis. + The byproducts also remove the original ammonia nitrogen in the wastewater, with an ammonia nitrogen removal efficiency of 72% to 93%, completely avoiding the contradiction of traditional MIP technology "repairing lead pollution but causing secondary ammonia nitrogen pollution".

[0024] (4) The process is simple and easy to control, making it convenient for industrialization. The microcapsule preparation (spore encapsulation-granulation-drying-nutrient loading) and wastewater remediation method of this invention is reasonably conceived, requiring no complex equipment or harsh operating conditions: key parameters are easy to control during the preparation process; when applied, only microcapsules need to be added at a solid-liquid ratio of 5% and supplemented with 30 mmol / L urea, and the remediation of lead-ammonia-nitrogen composite polluted wastewater can be completed within 120 hours. It is suitable for actual wastewater treatment in industries such as electroplating and battery manufacturing, and is simple to operate, cost-controllable, and easy to scale up and apply. Attached Figure Description

[0025] Figure 1 These are scanning electron microscope (SEM) images of zeolite (image a: ×2000x; image b: ×1000x; image c: ×4000x; image d: ×5000x). Figure 2 This is a scanning electron microscope (SEM) section of zeolite-loaded bacterial spore microcapsules; Figure 3 This is a graph showing the wastewater remediation effect when the lead ion concentration is 1 mmol / L (showing the trends of lead ion removal efficiency, ammonia nitrogen removal efficiency, and wastewater pH value within 0-120 h of reaction). Figure 4 This is a graph showing the wastewater remediation effect when the lead ion concentration is 5 mmol / L (showing the trends of lead ion removal efficiency, ammonia nitrogen removal efficiency, and wastewater pH value within 0~120h of reaction). Figure 5 This is a graph showing the wastewater remediation effect when the lead ion concentration is 10 mmol / L (showing the trends of lead ion removal efficiency, ammonia nitrogen removal efficiency, and wastewater pH value within 0-120 h of reaction). Figure 6 This is a graph showing the changes in urease activity and ammonium ion concentration in Bacillus pasteurellis spores (showing spore urease activity, bacterial concentration, and NH4+ concentration during 0-120 hours of culture). + (Dynamic changes in concentration) Figure 7 This is a graph showing the germination rate of Bacillus pasteurellis spores under different lead ion concentrations (showing Pb). 2+ (Changes in spore germination rate at concentrations of 0–10 mmol / L) Figure 8 This is an adsorption kinetics curve of zeolite for lead ions of different concentrations (showing the initial Pb). 2+ Pb concentrations of zeolite at concentrations of 1 mmol / L, 5 mmol / L, and 10 mmol / L within the range of 0–400 min 2+ (Changes in adsorption capacity) Figure 9 This is an adsorption kinetic curve of zeolite for different concentrations of ammonium ions (showing the initial NH4+). +At concentrations of 10 mmol / L, 30 mmol / L, and 60 mmol / L, the NH4+ ions generated by zeolite during the adsorption process... + (Changes in adsorption amount). Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and instruments used below are conventional reagents and instruments in the art; the experimental methods used, unless otherwise specified, are conventional methods in the art and will not be described in detail further.

[0027] Example 1 The method for obtaining a type of Bacillus pasteurellii spore in this embodiment is as follows: (1) Take the frozen Bacillus pasteurellii strain, inoculate it into the activation medium, and incubate it at 30℃ and 180 rpm for 24 h to obtain the activated bacterial solution. The activation medium consists of: 5.0 g / L beef extract, 5.0 g / L peptone, 10 mg / L MnSO4·H2O, 24 mg / L NiCl2·6H2O, pH 8.8.

[0028] (2) The activated bacterial solution from step (1) was inoculated into the sporulation medium at a 1% inoculation ratio, and the culture was continued at 30℃ and 180 rpm for 4 days to induce *Bacillus pasteurellii* to produce spores. The sporulation medium consisted of: 7 g / L sodium succinate, 5 g / L urea, and 3.6 mg / L Mn. 2+ pH 7.0.

[0029] (3) The solution obtained from step (2) was sterilized in an 80°C water bath for 20 min, and then centrifuged at 8000 rpm for 10 min; the centrifuged precipitate was collected, resuspended and diluted with physiological saline to obtain a spore concentration of 2.3 × 10⁻⁶. 10 CFU / mL Bacillus pasteurellium spore suspension.

[0030] Example 2 The method for obtaining a type of Bacillus pasteurellii spore in this embodiment is as follows: (1) Take the frozen Bacillus pasteurellii strain, inoculate it into the activation medium, and incubate it at 28℃ and 170 rpm for 22 h to obtain the activated bacterial solution. The activation medium consists of: 4.5 g / L beef extract, 4.5 g / L peptone, 9 mg / L MnSO4·H2O, 20 mg / L NiCl2·6H2O, pH 8.5.

[0031] (2) The activated bacterial solution from step (1) was inoculated into the sporulation medium at a 1% inoculation ratio, and the culture was continued at 28℃ and 170 rpm for 3 days to induce *Bacillus pasteurellii* to produce spores. The sporulation medium consisted of: 5 g / L sodium succinate, 3 g / L urea, and 3 mg / L Mn. 2+ pH 6.8.

[0032] (3) The solution obtained from step (2) was sterilized in a 75°C water bath for 15 min, and then centrifuged at 7000 rpm for 8 min; the centrifuged precipitate was collected, resuspended and diluted with physiological saline to obtain a spore concentration of 2.0 × 10⁻⁶. 10 CFU / mL Bacillus pasteurellium spore suspension.

[0033] Example 3 The method for obtaining a type of Bacillus pasteurellii spore in this embodiment is as follows: (1) Take the frozen Bacillus pasteurellii strain, inoculate it into the activation medium, and incubate it at 32℃ and 190 rpm for 26 h to obtain the activated bacterial solution. The activation medium consists of: 5.5 g / L beef extract, 5.5 g / L peptone, 11 mg / L MnSO4·H2O, 28 mg / L NiCl2·6H2O, pH 9.0.

[0034] (2) The activated bacterial solution from step (1) was inoculated into the sporulation medium at a 1% inoculation ratio, and the culture was continued at 32℃ and 190 rpm for 5 days to induce *Bacillus pasteurellii* to produce spores. The sporulation medium consisted of: 9 g / L sodium succinate, 7 g / L urea, and 4 mg / L Mn. 2+ pH 7.2.

[0035] (3) The solution obtained from step (2) was sterilized in an 85°C water bath for 25 min, and then centrifuged at 9000 rpm for 12 min; the centrifuged precipitate was collected, resuspended and diluted with physiological saline to obtain a spore concentration of 2.9 × 10⁻⁶. 10 CFU / mL Bacillus pasteurellium spore suspension.

[0036] Example 4 This embodiment provides a zeolite-loaded bacterial spore microcapsule with zeolite as the framework structure, wherein bacterial spores are embedded inside the zeolite framework, and the outer surface of the framework is loaded with nutrients that can promote spore germination.

[0037] Preparation method: (1) Raw material preparation Take the spore suspension of Bacillus pasteurellii from Example 1 (spore concentration 2.3 × 10⁻⁶). 10(CFU / mL) and zeolite, sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O, for later use.

[0038] (2) Spore embedding Mix 50 mL of Pasteurella multocida spore suspension with 50 g of zeolite and stir well to embed the bacterial spores inside the zeolite framework.

[0039] (3) Granulation and molding The zeolite-spore mixture obtained in step (2) was added to a granulator to obtain microspheres with a particle size of 3.5 mm.

[0040] (4) Initial drying The zeolite-spore microspheres obtained in step (3) were dried at 40°C for 24 hours to remove free water from the system.

[0041] (5) Nutrient load Sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O were dissolved in deionized water to prepare a nutrient solution. The nutrient solution contained 7 g / L sodium succinate, 13 mmol / L inosine, 24 mg / L NiCl2·6H2O and 10 mg / L MnSO4·H2O.

[0042] According to the spraying ratio of "1mL nutrient solution / 2g microspheres", the prepared nutrient solution is evenly sprayed onto the surface of the microspheres after drying in step (4).

[0043] (6) Secondary drying The nutrient-loaded microspheres were dried again at 40°C for 24 hours to obtain the desired zeolite-loaded bacterial spore microcapsules (average particle size 3.5 mm).

[0044] Example 5 This embodiment provides a zeolite-loaded bacterial spore microcapsule with zeolite as the framework structure, wherein bacterial spores are embedded inside the zeolite framework, and the outer surface of the framework is loaded with nutrients that can promote spore germination.

[0045] Preparation method: (1) Raw material preparation Take the Bacillus pasteurellium spore suspension from Example 2 (spore concentration 2.0 × 10⁻⁶). 10 (CFU / mL) and zeolite, sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O, for later use.

[0046] (2) Spore embedding Mix 40 mL of Pasteurella multocida spore suspension with 50 g of zeolite and stir well to embed the bacterial spores inside the zeolite framework.

[0047] (3) Granulation and molding The zeolite-spore mixture obtained in step (2) was added to a granulator to obtain microspheres with a particle size of 2 mm.

[0048] (4) Initial drying The zeolite-spore microspheres obtained in step (3) were dried at 35°C for 20 hours to remove free water from the system.

[0049] (5) Nutrient load Sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O were dissolved in deionized water to prepare a nutrient solution. The nutrient solution contained 5 g / L sodium succinate, 10 mmol / L inosine, 20 mg / L NiCl2·6H2O and 9 mg / L MnSO4·H2O.

[0050] According to the spraying ratio of "1mL nutrient solution / 2g microspheres", the prepared nutrient solution is evenly sprayed onto the surface of the microspheres after drying in step (4).

[0051] (6) Secondary drying The nutrient-loaded microspheres were dried again at 35°C for 20 hours to obtain the desired zeolite-loaded bacterial spore microcapsules (average particle size 2 mm).

[0052] Example 6 This embodiment provides a zeolite-loaded bacterial spore microcapsule with zeolite as the framework structure, wherein bacterial spores are embedded inside the zeolite framework, and the outer surface of the framework is loaded with nutrients that can promote spore germination.

[0053] Preparation method: (1) Raw material preparation Take the spore suspension of Bacillus pasteurellii from Example 3 (spore concentration 2.9 × 10⁻⁶). 10 (CFU / mL) and zeolite, sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O, for later use.

[0054] (2) Spore embedding Mix 60 mL of Pasteurella multocida spore suspension with 50 g of zeolite and stir well to embed the bacterial spores inside the zeolite framework.

[0055] (3) Granulation and molding The zeolite-spore mixture obtained in step (2) was added to a granulator to obtain microspheres with a particle size of 5 mm.

[0056] (4) Initial drying The zeolite-spore microspheres obtained in step (3) were dried at 45°C for 28 hours to remove free water from the system.

[0057] (5) Nutrient load Sodium succinate, inosine, NiCl2·6H2O and MnSO4·H2O were dissolved in deionized water to prepare a nutrient solution. The nutrient solution contained 8 g / L sodium succinate, 15 mmol / L inosine, 28 mg / L NiCl2·6H2O and 11 mg / L MnSO4·H2O.

[0058] According to the spraying ratio of "1mL nutrient solution / 2g microspheres", the prepared nutrient solution is evenly sprayed onto the surface of the microspheres after drying in step (4).

[0059] (6) Secondary drying The nutrient-loaded microspheres were dried again at 45°C for 28 hours to obtain the desired zeolite-loaded bacterial spore microcapsules (average particle size 5 mm).

[0060] Comparative Example 1 The microcapsule in this comparative example is basically the same as that in Example 4, except that "Pasteurella spores" are replaced with "logarithmic-phase Bacillus pasteurellii live bacteria" (i.e., dormant spores are not used, and conventional live bacteria are used directly). The remaining preparation steps (zeolite dosage, mixing ratio, nutrient solution loading, drying conditions, etc.) are the same as those in Example 4.

[0061] Comparative Example 2 This comparative example of a microcapsule is basically the same as that in Example 4, the main difference being that the surface of the microcapsule is not loaded with sodium succinate, inosine, and Ni. 2+ and Mn 2+ The nutrient solution (i.e., only spores and zeolite are embedded, without the nutrients required for germination) is used, and the remaining preparation steps (spore amount, zeolite amount, mixing ratio, drying conditions, etc.) are the same as in Example 4.

[0062] Comparative Example 3 This comparative example uses a single zeolite adsorbent (the same zeolite raw material as in Example 4), without encapsulating Bacillus pasteurellii spores, and without loading sodium succinate, inosine, or Ni. 2+ Mn 2+ Nutrient solution.

[0063] Experimental Example 1 This experimental example is used to verify the compatibility of zeolite structures and the spore encapsulation effect in the microcapsules of the present invention.

[0064] I. Microscopic morphology of zeolite The zeolite was observed using a scanning electron microscope (SEM), and the results are as follows: Figure 1 As shown, the zeolite surface exhibits a rich porous structure with a rough surface and obvious grooves. This structure can provide sufficient spore attachment sites and a microenvironment for spore germination.

[0065] II. Verification of Spore Embedding A slice of the microcapsule of the present invention (Example 4) was observed by scanning electron microscopy (SEM), and the results are as follows. Figure 2 As shown: spores are evenly distributed inside the porous framework of zeolite, confirming that zeolite can achieve stable spore encapsulation.

[0066] Experiment Example 2 This experimental example is used to verify the remediation effect of the zeolite-loaded bacterial spore microcapsules of the present invention on wastewater with different concentrations of lead and ammonia nitrogen combined pollution, and at the same time supplement the quantitative data of microbial function to explain the remediation mechanism.

[0067] I. Verification of Wastewater Remediation Effect 1. Prepare wastewater solutions with lead ion concentrations of 1 mmol / L, 5 mmol / L, and 10 mmol / L respectively, adjust the initial pH to 3, and add 30 mmol / L urea to each wastewater solution. Stir until completely dissolved and set aside.

[0068] 2. According to the solid-liquid ratio of 5% (microcapsule mass: wastewater volume), the microcapsules prepared in this invention (taking the microcapsules prepared in Example 4 as an example) were added to the lead-containing wastewater of different concentrations above, and placed in a constant temperature shaking reaction at 28~32℃ and 180rpm for 120h.

[0069] 3. Samples were taken at 0h, 24h, 48h, 72h, and 120h of the reaction to determine the residual lead ions (Pb) in the wastewater. 2+ The concentration and pH value of urea were measured; and after 120 hours of reaction, the concentration of residual urea and residual ammonia nitrogen (NH4) were additionally measured. + )concentration.

[0070] 4. Calculate the efficiency using the following formula: Lead ion removal efficiency = [(initial lead ion concentration - residual lead ion concentration after 120h) / initial lead ion concentration] × 100%; Theoretical ammonia nitrogen concentration = 2 × (initial urea concentration - 120h remaining urea concentration); Ammonia nitrogen removal efficiency = [(theoretical ammonia nitrogen concentration - measured ammonia nitrogen concentration over 120 hours) / theoretical ammonia nitrogen concentration] × 100%.

[0071] It should be noted that 1 mol of urea hydrolyzes to produce 2 mol of NH4. + and 1 mol CO3 2- NH4 produced by urea hydrolysis +Adsorbed by zeolite, therefore theoretically NH4 + and measured NH4 + There are differences in concentration.

[0072] The results are as follows Figure 3 , 4 As shown in Figures 5 and 1.

[0073] Table 1 Results of wastewater remediation using microcapsules according to the present invention

[0074] II. Quantitative Detection of Microbial Function (Sporurase Activity and Germination Rate) To verify the supporting role of spore function in the repair effect, the following tests were conducted simultaneously: 1. Take the spore suspension of the present invention (Example 1) and inoculate it at a ratio of 1% into a nutrient medium containing 50 mmol / L urea (sodium succinate 7 g / L, inosine 13 mmol / L, NiCl2·6H2O 24 mg / L, MnSO4·H2O 10 mg / L). Incubate at 30℃ and 180 rpm for 120 h. Measure urease activity and NH4+ every 24 h. + concentration.

[0075] The results are as follows Figure 6 As shown: NH4 + The concentration of NH4+ continued to increase over time, entering a rapid rise phase from 30 to 60 hours (synchronizing with the peak of urease activity), and reaching a peak at 120 hours. + The concentration reached 98.14 mmol / L, which is close to the theoretical value for complete hydrolysis of urea (100 mmol / L), confirming that the spores can efficiently secrete urease to catalyze the hydrolysis of urea after germination; 2. Take the spore suspension of the present invention (Example 1) and add it to the nutrient medium (sodium succinate 7g / L, inosine 13mmol / L, NiCl2·6H2O 24mg / L, MnSO4·H2O 10mg / L) with lead ion concentrations of 0mmol / L, 2mmol / L, 5mmol / L, 7mmol / L, 9mmol / L, and 10mmol / L respectively. After incubation at 30℃ for 48h, plate counting is performed.

[0076] The results are as follows Figure 7 As shown, the spore germination rate decreased slightly with increasing lead concentration, but the germination rate still reached 75% at a lead concentration of 10 mmol / L, confirming the spores' resistance to high lead environments.

[0077] The results of this experiment show that the microcapsules of the present invention have excellent removal effects (over 99%) on lead ions in the concentration range of 1~10 mmol / L, and the ammonia nitrogen removal efficiency reaches 72%~93%, achieving simultaneous and efficient repair of lead and ammonia nitrogen; combined with microbial functional data, it can be seen that the high toxicity resistance and high urease activity of the spores are the core guarantee for the repair effect.

[0078] Experimental Example 3 This experimental example is used to compare the wastewater remediation effects of the present invention with those of Comparative Examples 1-3 to clarify the role of the core components, while also providing empirical evidence of the synergistic effect of the carrier.

[0079] I. Comparison of Wastewater Remediation Effects 1. Four groups of experiments were set up, namely the microcapsule group of the present invention (Example 4, zeolite + spores + nutrient solution), the microcapsule group of Comparative Example 1 (zeolite + logarithmic phase live bacteria + nutrient solution), the microcapsule group of Comparative Example 2 (zeolite + spores + no nutrient solution), and the zeolite group of Comparative Example 3 (single zeolite).

[0080] 2. Prepare wastewater with a lead ion concentration of 5 mmol / L, adjust the initial pH to 3, add 30 mmol / L urea, and stir until completely dissolved.

[0081] 3. Add each group of materials at a solid-liquid ratio of 5% (microcapsule / zeolite mass: wastewater volume), and place them in a constant temperature shaking reaction at 30℃ and 180rpm for 120h.

[0082] 4. Following the method in Experiment Example 1, determine the lead ion removal efficiency and ammonia nitrogen removal efficiency after 120 hours.

[0083] The results are shown in Table 2.

[0084] Table 2 Wastewater remediation results for each group of materials

[0085] II. Empirical Evidence of Carrier Synergistic Effect (Verification of Zeolite Adsorption Kinetics) To clarify the synergistic function of zeolite in "early-stage detoxification + late-stage ammonia nitrogen removal", zeolite adsorption experiments were conducted separately: Take 5g of the zeolite used in this invention and add Pb separately. 2+ Concentrations 1 mmol / L / 5 mmol / L / 10 mmol / L, NH4 + In solutions with concentrations of 10 mmol / L, 30 mmol / L, and 60 mmol / L, the reaction was carried out at 30 °C and 140 rpm for 6 h, and the adsorption amount was measured every 30 min.

[0086] The results are shown in Figures 8 and 9: Zeolite-Pb 2+The equilibrium adsorption capacity increased from 41.4 mg / g to 360.8 mg / g with increasing initial concentration. The adsorption capacity increased rapidly in the initial adsorption phase (0–60 min) and then tended to reach equilibrium after 120 min. Figure 8 ); for NH4 + The equilibrium adsorption capacity increased from 29.3 mg / g to 145.5 mg / g, and the adsorption trend was similar to that of Pb. 2+ Consistency Figure 9 This confirms that zeolite can rapidly adsorb initial Pb. 2+ (Reducing spore toxicity) and NH4 produced by urea hydrolysis + (To avoid secondary pollution).

[0087] The results of this experiment show that the remediation effect of this invention is significantly better than that of the comparative examples: compared with comparative example 1 (live bacteria replacing spores), it highlights the anti-toxic advantage of spores in high lead environments; compared with comparative example 2 (no nutrient solution), it confirms the promoting effect of nutrient solution on spore germination and urea hydrolysis; compared with comparative example 3 (single zeolite), it demonstrates the synergistic effect of "zeolite adsorption + spore biomineralization", which greatly improves the lead ion removal efficiency and the simultaneous treatment capacity of ammonia nitrogen; at the same time, combined with zeolite adsorption kinetic data, it further verifies the necessity of the synergistic effect of the carrier.

[0088] In summary, this invention constructs a highly efficient synergistic remediation system through a design of "zeolite-encapsulated Bacillus pasteurellii spores + surface-loaded nutrient solution": the porous structure of zeolite provides an encapsulation and germination environment for the spores, and its adsorption capacity can reduce lead toxicity in the early stage and remove ammonia nitrogen byproducts in the later stage; the high toxicity resistance and high urease activity of the spores ensure deep lead mineralization. Experiments have confirmed that this microcapsule has excellent remediation effect on lead-containing ammonia nitrogen wastewater with concentrations of 1-10 mmol / L (Pb). 2+ Removal rate ≥99.1%, NH4 + With a removal rate of ≥72%, and the synergistic effect of "zeolite-spore-nutrient solution" is indispensable, the remediation efficiency is significantly higher compared to solutions with single zeolite or those lacking key components, making it highly valuable for the treatment of complex polluted wastewater.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A zeolite-supported bacterial spore microcapsule, characterized by, The microcapsule has a zeolite as a skeleton structure, and the skeleton is internally embedded with bacterial spores and externally loaded with nutrients; wherein the bacterial spores are Bacillus pasteurii spores, and the nutrients include sodium succinate, inosine, Ni 2+ , and Mn 2+ .

2. The microcapsule according to claim 1, wherein The bacteria spores are obtained as follows: (1) Take the frozen Bacillus pasteurii strain, inoculate into the activation culture medium, and incubate at 28-32℃ and 170-190 rpm for 22-26 hours to obtain the activated bacteria solution; (2) The activated bacteria solution of step (1) is inoculated into the spore production culture medium, and incubated at 28-32℃ and 170-190 rpm for 3-5 days to induce the Bacillus pasteurii to produce spores; (3) The solution obtained in step (2) is sterilized in a 75-85℃ water bath for 15-25 minutes, and then the treated solution is centrifuged at 7000-9000 rpm for 8-12 minutes; The centrifugal precipitate is resuspended with normal saline to obtain the Bacillus pasteurii spore suspension.

3. The microcapsule according to claim 2, wherein In step (1), the composition of the activation culture medium includes 4.5-5.5 g / L beef extract, 4.5-5.5 g / L peptone, 9-11 mg / L MnSO4·H2O, 20-28 mg / L NiCl2·6H2O, and pH 8.5-9.

0.

4. The microcapsule of claim 2, wherein The composition of the sporulation medium in step (2) includes: 5~9g / L sodium succinate, 3~7g / L urea, 3~4mg / L Mn 2+ , pH 6.8~7.

2.

5. The microcapsule according to any one of claims 1 to 4, wherein The particle size of the microcapsule is 2-5 mm.

6. A process for the preparation of microcapsules according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) Raw material preparation Take bacillus spores suspension, zeolite, sodium succinate, inosine, Ni 2+ source and Mn 2+ source, backup; (2) Spore embedding The Bacillus pasteurii spore suspension and zeolite are mixed in a ratio of 0.8-1.2 mL / g of the volume of the spore suspension to the mass of the zeolite, and stirred uniformly to embed the bacterial spores in the zeolite framework; (3) Granulation and molding The zeolite-spore mixture obtained in step (2) is added to a granulation device to prepare microspheres; (4) Primary drying The zeolite-spore microspheres obtained in step (3) are dried in an environment of 35-45℃ for 20-28 hours to remove free water in the system; (5) Nutrient loading Na succinate, inosine, Ni 2+ source and Mn 2+ The source is dissolved in deionized water, a nutrient solution is prepared, and it is uniformly sprayed onto the surface of the microspheres after drying in step (4). (6) Secondary drying The microspheres loaded with nutrients are dried again in an environment of 35-45℃ for 20-28 hours to obtain the target zeolite-loaded bacterial spore microcapsules.

7. The preparation method according to claim 6, characterized in that, The spore concentration of the spore suspension in step (1) is 2.0 x 10 10 2.9 x 10 10 CFU / mL, Ni 2+ The source of Ni is selected from NiCl2-6H2O, Mn 2+ The source of Mn is selected from MnSO4-H2O.

8. The preparation method according to claim 6, characterized in that, In step (5), the specific composition of the nutrient solution is: sodium succinate 5-8 g / L, inosine 10-15 mmol / L, NiCl2·6H2O 20-28 mg / L, and MnSO4·H2O 9-11 mg / L.

9. Use of microcapsules according to any one of claims 1 to 5 for the remediation of waste water, characterized in that The wastewater is lead-containing ammonia-nitrogen composite contaminated wastewater; the ammonia-nitrogen is derived from the initial ammonia-nitrogen in the wastewater and / or the ammonia-nitrogen byproduct generated in the remediation process.

10. Use according to claim 9, characterized in that, The microcapsules are added to the wastewater at a solid-liquid ratio of 5%, and 30 mmol / L urea is added, and the reaction is carried out at 28-32℃ and 170-190 rpm for 120 hours to realize the synchronous removal of lead ions and ammonia-nitrogen.

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