A highly safe ammonium nitrogen 15 N detection method

Abstract

This invention discloses a highly safe ammonium nitrogen 15N The detection method relates to the field of stable isotope analysis technology. This method enriches ammonium nitrogen in the sample using a cation exchange resin, followed by dialysis desalting, and then... Fe3O4‑CeO2 Core-shell structured nanozymes catalyze sodium hypobromite NH4+ Quantitative oxidation to NO2‑ Subsequently, in an intelligent anaerobic microbial reactor, immobilized Pseudomonas bacteria with embedded dual-sensitive probes were used to... NO2‑ Targeted conversion N2O The reactor monitors and automatically adjusts the pH and microbial electron transfer activity inside the microspheres in real time to produce gas; N2O After purification and low-temperature enrichment of MOF materials, the samples were analyzed using an isotope ratio mass spectrometer. 15N Abundance. This method avoids the use of highly toxic substances, is highly safe, selective, and sensitive, and is suitable for detecting ammonium nitrogen in environmental samples. 15N Accurate detection.

Description

Technical Field

[0001] This invention belongs to the field of ecological environment material recycling and pollution control technology, specifically relating to a highly safe ammonium nitrogen... 15 N detection method. Background Technology

[0002] Nitrogen is one of the most complex elements in the cycling processes of terrestrial and aquatic ecosystems, with ammonium nitrogen (NH4) being a key component. + As the main form of inorganic nitrogen, it plays a key role in processes such as plant nutrition, water eutrophication, and greenhouse gas emissions. 15 Nitrogen isotope tracing is an effective method for studying nitrogen fate and transformation mechanisms, and for accurately determining ammonium nitrogen in environmental samples. 15 Nitrogen isotope abundance is of great significance for nitrogen cycle research and pollution source tracing.

[0003] Traditional ammonium nitrogen 15 Nitrogen (N) detection methods mainly include diffusion, distillation, and chemical oxidation. Diffusion and distillation are cumbersome and time-consuming, and inefficient when processing large batches of samples. While chemical oxidation offers fast detection, traditional methods require highly toxic reducing agents (such as sodium azide or iodine bromooxyoxygenate), posing a health risk to operators and generating toxic waste, resulting in poor environmental safety.

[0004] In recent years, research has developed a detection technique that combines chemical methods with denitrifying microorganisms, by detecting NH4 in samples. + Oxidized to NO2 - Then, denitrifying microorganisms are used to convert NO2 - The method involves converting microorganisms to N₂O for isotope detection. This avoids the use of multiple highly toxic reducing agents, improving operational safety. However, existing techniques still have the following shortcomings: strict requirements for microbial culture conditions; unstable strain activity leading to poor detection reproducibility; easy introduction of isotope fractionation during sample pretreatment; limited applicability to complex environmental matrices; detection sensitivity insufficient for trace sample analysis; potential side reactions during oxidation affecting detection accuracy; lack of real-time monitoring of the microbial transformation process; and the need for further improvement in gas purification efficiency.

[0005] To address the above problems, this invention provides an improved high-safety ammonium nitrogen solution. 15 The nitrogen detection method, by optimizing the oxidation reaction system, microbial treatment conditions, and detection process, and introducing several innovative technologies, further improves the safety, accuracy, and sensitivity of the detection. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a highly safe ammonium nitrogen. 15This nitrogen (N) detection method addresses the safety hazards of traditional methods using highly toxic reagents, while simultaneously improving recovery rate, accuracy, reproducibility, and sensitivity, enabling the detection of ammonium nitrogen in samples from complex environments. 15 Reliable determination of nitrogen isotope abundance.

[0007] Technical Solution: To achieve the above objectives, the present invention provides the following technical solution: S1. Sample pretreatment: Collect environmental samples to be tested, centrifuge or filter them, adjust the pH to 2.0-4.0, add cation exchange resin to enrich ammonium nitrogen, and then elute with elution buffer to obtain enriched ammonium salt solution. S2. Desalting treatment: The ammonium salt solution obtained in step S1 is desalted to remove high concentrations of salt ions from the eluent, resulting in a desalted ammonium salt solution. S3, Chemical Oxidation: Sodium hypobromite solution as an oxidant and Fe3O4-CeO2 core-shell nanozyme as a catalyst are added to the desalted ammonium salt solution obtained in step S2. The oxidation reaction is carried out under sealed conditions for 20-40 minutes at a temperature of 25-35℃. NH4... + Quantitative oxidation to NO2 - A treatment solution containing nitrite was obtained; the amount of sodium hypobromite solution added was such that the effective bromine concentration reached 0.5-2.0 g / L. S4. Intelligent Response Microbial Transformation: An intelligent anaerobic microbial reactor is used. A dedicated liquid nutrient substrate for denitrifying microorganisms is added to the reaction system. Inert gas is introduced for 20-30 minutes to maintain an anaerobic state. The pH of the treated liquid obtained in step S3 is adjusted to 6.5-7.5. Sodium alginate-chitosan composite microbial agent containing active denitrifying microorganisms and dual-sensitive probes is added to the reactor. The reactor is incubated at 25-35℃ for 2-6 hours. NO2 is converted to nitrogen through microbial denitrification. - The reaction is directionally converted into N2O gas. During the reaction, the pH value inside the microspheres and the electron transfer activity of microbial oxidation-reduction are monitored in real time through fiber optic sensors and micro electrochemical electrodes. The reactor is equipped with an intelligent control module, which automatically performs pH adjustment or nutrient factor supplementation when the monitoring signal deviates from the set threshold. The sodium alginate-chitosan composite microspheres contain 10 live bacteria. 9 -10 11 CFU / g of Pseudomonas, pH-sensitive fluorescent probes, and redox-sensitive electrochemical probes are covalently fixed to the microsphere carrier backbone to avoid probe signal inactivation due to high-temperature sterilization. S5. Gas purification and enrichment: Collect the N2O gas generated in step S4, remove impurity gases through a multi-stage purification column, and enrich it under low temperature conditions; the gas purification and enrichment step uses metal-organic framework materials for selective adsorption purification and enriches N2O under low temperature conditions. S6. Isotope Detection: The enriched N2O gas is introduced into an isotope ratio mass spectrometer to determine the nitrogen isotope composition of N2O. The ammonium nitrogen content in the sample is then calculated. 15 N isotope abundance; the isotope detection step uses an isotope ratio mass spectrometer to determine the nitrogen isotope composition of N2O.

[0008] Preferably, the desalination treatment is performed by dialysis, with the dialysis bag having a molecular weight cutoff of 100-500 Da, and dialysis in deionized water at 4°C for 2-6 hours, during which the dialysis solution is replaced 1-3 times.

[0009] Preferably, the preparation method of the Fe3O4-CeO2 core-shell structured nanozyme is as follows: FeCl3·6H2O and FeCl2·4H2O are mixed at a molar ratio of 2:1, sodium citrate (0.4 times the weight of FeCl3·6H2O) is added, dissolved in ethylene glycol (40 times the weight of FeCl3·6H2O), and sodium acetate (2.4 times the weight of FeCl3·6H2O) is added. The mixture is stirred for 30 minutes. The mixture is then transferred to a reaction vessel and reacted at 200°C for 12 hours. After cooling, the Fe3O4 nanoparticles are magnetically separated and analyzed with ethanol. Wash with water and dry under vacuum at 60℃ to constant weight to obtain Fe3O4 nanoparticles; disperse the Fe3O4 nanoparticles in water at a weight of 1000 times the weight of the Fe3O4 nanoparticles, add Ce(NO3)3·6H2O at a weight of 4 times the weight of the Fe3O4 nanoparticles and hexamethylenetetramine at a weight of 10 times the weight of the Fe3O4 nanoparticles, and sonicate for 30 minutes; stir the mixture at 90℃ for 2 hours, magnetically separate the product, wash with ethanol and water, and dry under vacuum at 60℃ to obtain Fe3O4-CeO2 core-shell structured nanozyme.

[0010] Preferably, the preparation method of the sodium alginate-chitosan composite microspheres is as follows: (1) Activation of bacterial strain and preparation of bacterial suspension: Pseudomonas was inoculated into nutrient broth medium and cultured at 30°C with shaking for 24 hours. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, resuspended in sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 10. 1 CFU / mL, refrigerate at 4℃ for later use; (2) Preparation of carrier solution and immobilization of probe: Prepare a 3% (w / v) sodium alginate solution, filter it through a 0.22 μm filter membrane for sterilization, add covalently immobilized fluorescein isothiocyanate-labeled dextran and anthraquinone-2-sulfonate to the sterilized sodium alginate solution, stir well to obtain a mixed carrier solution; wherein the final concentration of fluorescein isothiocyanate-labeled dextran is 0.1 mg / mL, and the final concentration of anthraquinone-2-sulfonate is 0.5 mmol / L; (3) Initial preparation of microspheres: The mixed carrier solution was aseptically mixed with an equal volume of Pseudomonas bacterial suspension, and dripped into a 2% (w / v) sterile calcium chloride solution using a sterile syringe. The mixture was allowed to stand and solidify for 30 minutes to form calcium alginate gel microspheres. (4) Cross-linking and coating: The microspheres were transferred into a 0.5% (w / v) chitosan solution and cross-linked and coated at room temperature for 20 minutes to form sodium alginate-chitosan composite microspheres; the chitosan solution was dissolved in a 1% acetic acid solution and 0.02% (v / v) glutaraldehyde was added as a cross-linking agent. (5) Post-processing and storage: After cross-linking, the microspheres were washed three times with sterile physiological saline to obtain a viable count of 10. 1 CFU / g of compound micrococcal agent, stored at 4℃ for later use; The liquid nutrient substrate for denitrifying microorganisms contains glucose, potassium dihydrogen phosphate, magnesium sulfate, and yeast extract, with mass concentrations of 5-10 g / L, 1-2 g / L, 0.2-0.5 g / L, and 0.5-1 g / L, respectively.

[0011] Preferably, the anaerobic conditions are achieved by introducing an inert gas into the reaction system to replace the headspace and dissolved oxygen. The inert gas is helium, and the gas introduction time is 20-30 minutes, so that the oxygen concentration in the reaction system drops to below 0.2%.

[0012] Preferably, the metal-organic framework material is ZIF-8 with a pore size of 0.3-1.0 nm. The preparation method of the metal-organic framework material ZIF-8 is as follows: Zn(NO3)2·6H2O is dissolved in methanol with a volume equal to 20 times the weight of Zn(NO3)2·6H2O, denoted as solution A; 2-methylimidazolium with a weight equal to 2 times the weight of Zn(NO3)2·6H2O is dissolved in methanol with a volume equal to 20 times the weight of Zn(NO3)2·6H2O, denoted as solution B; solution B is quickly added to solution A, and the mixture is stirred at room temperature for 1 hour; the white precipitate is collected by centrifugation, washed three times with methanol, and dried under vacuum at 60°C for 12 hours to obtain ZIF-8 crystals.

[0013] Preferably, in the gas purification and enrichment step, a multi-stage purification system is used, in which the gas passes through an alkali asbestos column, an anhydrous magnesium perchlorate column and an MOF packed column in sequence, and then enters a liquid nitrogen cold trap filled with MOF material for enrichment. The intelligent anaerobic microbial reactor integrates a signal acquisition unit, a central processing unit, and an execution unit. The signal acquisition unit transmits fluorescence and electrochemical monitoring data in real time via fiber optic sensors and microelectrodes. The central processing unit compares the data with preset thresholds. When the pH deviates from the 6.5-7.5 range or the microbial redox electron transport activity decreases by more than 10%, the execution unit is triggered to automatically pump dilute hydrochloric acid or dilute sodium hydroxide solution into the system to adjust the pH. By monitoring the microbial redox electron transport activity, the reactor reflects the denitrifying microbial metabolic activity in real time, ensuring NO2... - Conversion efficiency.

[0014] Preferably, the cation exchange resin is a strong acid styrene-based cation exchange resin, the eluent is a 1-3 mol / L NaCl or KCl solution, and the elution flow rate is 0.5-2.0 mL / min; in the chemical oxidation step, the nanozyme catalyst is recycled and reused through magnetic recovery.

[0015] Preferably, the isotope ratio mass spectrometer simultaneously detects the ion current intensities of the N2O molecular ion peak at m / z 44, 45, and 46, and calculates the values ​​based on international standard reference materials. 15 Percentage of N atoms.

[0016] The beneficial effects of this invention are as follows: 1. This invention is the first to propose an integrated pretreatment technology based on ion exchange pre-enrichment and matrix removal. This invention uses cation exchange resin to selectively enrich ammonium nitrogen in environmental samples while simultaneously removing soluble organic matter and metal ions that interfere with detection. Compared with traditional pretreatment methods, this technology not only increases the concentration of ammonium nitrogen to meet the detection requirements of trace samples, but more importantly, it eliminates the interference of complex environmental matrices on subsequent oxidation reactions and microbial activity, significantly improving the accuracy and reproducibility of detection. By optimizing elution conditions and flow rates, the ammonium nitrogen recovery rate can reach over 98%, without producing isotope fractionation effects.

[0017] 2. This invention constructs a two-stage reaction system that synergistically integrates catalytic oxidation and microbial transformation. In the oxidation stage, by introducing Fe3O4-CeO2 core-shell structured nanozymes as biomimetic catalysts, the activation energy required for the oxidation reaction is reduced, allowing sodium hypobromite to convert NH4+ under mild conditions. + Quantitative oxidation to NO2 -This method avoids high-temperature and high-pressure operations and reduces the generation of byproducts. During the microbial transformation stage, a stabilized bacterial agent prepared using immobilization technology encapsulates denitrifying microorganisms within sodium alginate-chitosan composite microspheres with excellent mass transfer properties. This significantly enhances the strain's tolerance to environmental pH and temperature fluctuations and extends the agent's lifespan. The immobilized agent can be reused 5-8 times with an activity retention rate exceeding 85%, substantially reducing detection costs.

[0018] 3. This invention designs an online detection system combining gas purification and low-temperature enrichment. Before N2O gas enters the mass spectrometer, it is passed sequentially through an alkali asbestos column (to remove CO2) and an anhydrous magnesium perchlorate column (to remove water vapor), effectively eliminating the influence of interfering gases on isotope determination. Subsequently, a liquid nitrogen cold trap is used to enrich the purified N2O at low temperature, increasing the gas concentration entering the mass spectrometer and significantly enhancing the intensity of the detection signal. This design improves the detectable lower limit of ammonium nitrogen concentration by one order of magnitude compared to traditional methods, providing a technical means for the study of samples from oligotrophic environments.

[0019] 4. This invention introduces nanozyme enhancement technology into the chemical oxidation step, using Fe3O4-CeO2 core-shell structured nanozymes as biomimetic catalysts to replace traditional chemical catalysts. The nanozymes exhibit oxidase-like activity and can efficiently catalyze NH4+ at room temperature and pressure. + To NO2 - The conversion can reduce the reaction temperature to 25-35℃ and shorten the reaction time to 20-40 minutes. Compared with the existing technology, this innovation has the following advantages: (1) no metal salt catalyst is needed, avoiding the inhibitory effect of heavy metal ions on the subsequent microbial activity; (2) nanozymes can be magnetically recovered and reused, reducing the cost of use; (3) the reaction conditions are milder, and energy consumption is reduced; (4) the enzyme-like activity of nanozymes has substrate specificity, the incidence of side reactions is low, and the purity of oxidation products is improved. By controlling the particle size, morphology and surface modification of nanozymes, the catalytic efficiency and selectivity can be further optimized.

[0020] 5. Based on microbial immobilization technology, this invention develops intelligent responsive composite immobilized microspheres. These microspheres use sodium alginate-chitosan as a matrix and internally embed: (1) a pH-sensitive fluorescent probe (such as fluorescein isothiocyanate-labeled dextran) for real-time monitoring of pH changes in the microenvironment within the microspheres; and (2) a redox-sensitive electrochemical probe for monitoring NO2. -(3) Slow-release nutrients provide a continuous energy supply for microorganisms. During the microbial transformation process, fluorescence signals are collected in real time by fiber optic sensors, electrochemical signals are monitored by microelectrodes, and the signals are wirelessly transmitted to the data processing terminal to realize online monitoring of microbial metabolic activity. When the pH inside the microsphere deviates from the optimal range or the electron transfer activity decreases, the system automatically adjusts the pH of the reaction system or adds nutrients to maintain the optimal activity state of the microorganisms. This technology improves the stability of the microbial transformation process, reduces the batch-to-batch variation coefficient, and significantly improves the reliability of the detection results.

[0021] 6. In the gas purification step, the present invention introduces metal-organic framework materials as a novel gas purification medium. ZIF-8, a metal-organic framework material with high specific surface area and adjustable pore size, is used to replace or supplement the traditional purification column packing. Metal-organic framework materials have high selective adsorption capacity for N2O, which can effectively remove trace impurity gases (such as residual CO2, water vapor, volatile organic compounds, etc.) while avoiding N2O adsorption loss. The specific application method is: after the traditional alkali asbestos and magnesium perchlorate purification column, a micro purification column filled with metal-organic framework is connected in series, or metal-organic framework materials are filled in a low-temperature enrichment cold trap to achieve the integration of purification and enrichment. Metal-organic framework materials have the following advantages: (1) Specific surface area as high as 1000-4000 m 2 / g, with a large adsorption capacity and improved purification efficiency; (2) the pore size can be precisely controlled to achieve selective sieving of N2O molecules (kinetic diameter 0.33nm); (3) it can be repeatedly regenerated and used; (4) the structure is stable under low temperature conditions (-196℃ to room temperature) and is suitable for cold trap environments. After adopting metal-organic framework technology, the gas purification time is shortened, the N2O recovery rate is improved, and the removal rate of impurity gases is higher, which significantly improves the signal-to-noise ratio and accuracy of isotope detection. Attached Figure Description

[0022] Figure 1 This is a schematic flowchart of the detection method of the present invention.

[0023] Figure 2 This is a schematic diagram of the Fe3O4-CeO2 core-shell nanozyme of the present invention.

[0024] Figure 3 This is a schematic diagram of the structure of the intelligent responsive immobilized bacterial agent microspheres of the present invention.

[0025] Figure 4 This is a schematic diagram of the structure of the MOF-enhanced gas purification and enrichment module of the present invention. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Sample collection: 1 L of surface water from a lake was collected, filtered through a 0.45 μm filter membrane to remove suspended particulate matter, and the pH was adjusted to 3.0 with dilute hydrochloric acid.

[0028] Ion exchange enrichment: The pretreated water sample was passed through an exchange column packed with 5 mL of strong acid styrene-based cation exchange resin (type 001×7) at a flow rate of 1.5 mL / min. The resin was then washed with 30 mL of deionized water to remove impurities. Finally, ammonium nitrogen was eluted with 40 mL of 2.0 mol / L NaCl solution at a flow rate of 1.0 mL / min, and the eluent was collected.

[0029] Desalting treatment: Transfer the above eluent to a dialysis bag with a molecular weight cutoff of 100 Da, and dialyze at 4°C in 20 times its volume of deionized water for 4 hours, changing the dialysate twice during the process to remove high concentrations of Cl. - and Na + After dialysis, the solution in the dialysis bag was concentrated to 80% of its original volume under reduced pressure at 60°C, and the ammonium nitrogen concentration was measured. After desalting, the solution conductivity decreased from the initial 180 mS / cm to below 2.5 mS / cm, the ammonium nitrogen recovery rate was 96.2%, and there was no significant isotope fractionation effect.

[0030] Nanozyme-catalyzed oxidation: 20 mL of the desalted solution was placed in a sealed reaction flask. 5 mg of Fe3O4-CeO2 core-shell nanozyme (approximately 50 nm in diameter) was added, along with 0.3 mL of freshly prepared sodium hypobromite solution (effective bromine content 100 g / L), bringing the effective bromine concentration in the reaction system to approximately 1.5 g / L. The reaction flask was sealed and the mixture was shaken and reacted in a 30°C constant temperature water bath for 30 minutes. NH4+ was then removed. + Oxidized to NO2 - After the reaction is complete, an external magnetic field is applied to recover the nanozyme for later use. Measurements show that NH4... + Conversion rate 99.3%, NO2 - The yield was 98.7%.

[0031] Intelligent Response Microbial Transformation: The oxidized solution was transferred to an intelligent anaerobic microbial reactor. Simultaneously, a liquid nutrient substrate specifically for denitrifying microorganisms was added to the reaction system. High-purity helium gas (99.999%) was introduced to displace dissolved oxygen in the headspace and solution for 20 minutes, reducing the oxygen concentration in the reaction system to below 0.1%. The pH was adjusted to 7.0 using sterile phosphate buffer, and 3 g of immobilized Pseudomonas aspertutzeri in the form of sodium alginate-chitosan composite microspheres was added. The microspheres contained a fluorescein isothiocyanate-labeled dextran pH-sensitive fluorescent probe and an anthraquinone-2-sulfonate redox-sensitive electrochemical probe, with the probes covalently fixed to the microsphere carrier framework. The reactor incorporated a fiber optic sensor and a micro-electrochemical electrode to monitor the internal pH and microbial redox electron transport activity in real time. After anaerobic incubation at 30°C for 3 hours, the microbial agent converted NO2... - The gas is directionally converted into N2O gas. When the monitoring system detects a pH deviation from the 6.5-7.5 range or a decrease in microbial redox electron transport activity of more than 10%, the intelligent control module automatically adjusts the pH. After cultivation, the headspace gas in the reactor is collected using an airtight syringe.

[0032] MOF-enhanced gas purification and enrichment: The collected gas was sequentially passed through a pre-purification column (5 mm inner diameter, 15 cm length) filled with alkali asbestos and anhydrous magnesium perchlorate to remove CO2 and water vapor. The pre-purified gas was then passed through a micro-purification column (3 mm inner diameter, 5 cm length) filled with ZIF-8 MOF material (200 mg) for further removal of trace impurities. The purified gas was then passed through a liquid nitrogen cold trap (-196℃) filled with MOF material for enrichment for 15 minutes, achieving selective adsorption and enrichment of N2O. After enrichment, the cold trap was rapidly heated to 50℃ to release N2O gas.

[0033] Isotope detection: The released N₂O gas was introduced into a ThermoScientific Delta V Advantage isotope ratio mass spectrometer using helium as a carrier, and the ion current intensities of the N₂O molecular ion peaks at m / z 44, 45, and 46 were simultaneously detected. The δ¹⁴⁶ of ammonium nitrogen in the sample was calculated based on calibration with international standard reference materials. 15 N value and 15 Percentage of N atoms.

[0034] Example 2: Sample collection: 1 L of surface water from a lake was collected, filtered through a 0.45 μm filter membrane to remove suspended particulate matter, and the pH was adjusted to 3.0 with dilute hydrochloric acid.

[0035] Ion exchange enrichment: The pretreated water sample was passed through an exchange column containing 5 mL of strong acid styrene-based cation exchange resin (type 001×7) at a flow rate of 1.5 mL / min. The resin was then washed with 30 mL of deionized water to remove impurities. Finally, ammonium nitrogen was eluted with 40 mL of 1.0 mol / L NaCl solution at a flow rate of 0.5 mL / min, and the eluent was collected.

[0036] Desalting treatment: Transfer the above eluent to a dialysis bag with a molecular weight cutoff of 500 Da, and dialyze at 4°C in 15 times its volume of deionized water for 2 hours, changing the dialysate once during the process to remove high concentrations of Cl. - and Na + After dialysis, the solution in the dialysis bag was concentrated to 80% of its original volume under reduced pressure at 60°C, and the ammonium nitrogen concentration was measured. After desalting, the solution conductivity decreased from the initial 180 mS / cm to below 3.2 mS / cm, the ammonium nitrogen recovery rate was 95.1%, and there was no significant isotope fractionation effect.

[0037] Nanozyme-catalyzed oxidation: 20 mL of the desalted solution was placed in a sealed reaction flask. 5 mg of Fe3O4-CeO2 core-shell nanozyme (approximately 50 nm in diameter) was added, along with 0.2 mL of freshly prepared sodium hypobromite solution (effective bromine content 100 g / L), bringing the effective bromine concentration in the reaction system to approximately 1.0 g / L. The reaction flask was sealed and the mixture was shaken and reacted in a 25°C water bath for 20 minutes. NH4+ was then removed. + Oxidized to NO2 - After the reaction is complete, an external magnetic field is applied to recover the nanozyme for later use. Measurements show that NH4... + Conversion rate 97.8%, NO2 - The yield was 96.5%.

[0038] Intelligent Response Microbial Transformation: The oxidized solution was transferred to an intelligent anaerobic microbial reactor. Simultaneously, a liquid nutrient substrate specifically for denitrifying microorganisms was added to the reaction system. High-purity helium gas (99.999%) was introduced to displace dissolved oxygen in the headspace and solution for 25 minutes, reducing the oxygen concentration in the reaction system to below 0.2%. The pH of the oxidized solution was adjusted to 7.0 using sterile phosphate buffer, and 3 g of immobilized Pseudomonas stutzeri in the form of sodium alginate-chitosan composite microspheres was added. The microspheres contained a fluorescein isothiocyanate-labeled dextran pH-sensitive fluorescent probe and an anthraquinone-2-sulfonate redox-sensitive electrochemical probe, with the probes covalently fixed to the microsphere carrier framework. The reactor incorporated a fiber optic sensor and a micro-electrochemical electrode to monitor the internal pH of the microspheres and the redox electron transport activity of the microorganisms in real time. After anaerobic incubation at 25°C for 2 hours, the microbial agent converted NO2... -The gas is directionally converted into N2O gas. When the monitoring system detects a pH deviation from the 6.5-7.5 range or a decrease in microbial redox electron transport activity of more than 10%, the intelligent control module automatically adjusts the pH. After cultivation, the headspace gas in the reactor is collected using an airtight syringe.

[0039] MOF-enhanced gas purification and enrichment: The collected gas was sequentially passed through a pre-purification column (5 mm inner diameter, 15 cm length) filled with alkali asbestos and anhydrous magnesium perchlorate to remove CO2 and water vapor. The pre-purified gas was then passed through a micro-purification column (3 mm inner diameter, 5 cm length) filled with ZIF-8 MOF material (200 mg) for further removal of trace impurities. The purified gas was then passed through a liquid nitrogen cold trap (-196℃) filled with MOF material for enrichment for 15 minutes, achieving selective adsorption and enrichment of N2O. After enrichment, the cold trap was rapidly heated to 50℃ to release N2O gas.

[0040] Isotope detection: The released N₂O gas was introduced into a ThermoScientific Delta V Advantage isotope ratio mass spectrometer using helium as a carrier, and the ion current intensities of the N₂O molecular ion peaks at m / z 44, 45, and 46 were simultaneously detected. The δ¹⁴⁶ of ammonium nitrogen in the sample was calculated based on calibration with international standard reference materials. 15 N value and 15 Percentage of N atoms.

[0041] Example 3: Sample collection: 1 L of surface water from a lake was collected, filtered through a 0.45 μm filter membrane to remove suspended particulate matter, and the pH was adjusted to 3.0 with dilute hydrochloric acid.

[0042] Ion exchange enrichment: The pretreated water sample was passed through an exchange column packed with 5 mL of strong acid styrene-based cation exchange resin (type 001×7) at a flow rate of 1.5 mL / min. The resin was then washed with 30 mL of deionized water to remove impurities. Finally, ammonium nitrogen was eluted with 40 mL of 3.0 mol / L NaCl solution at a flow rate of 2.0 mL / min, and the eluent was collected.

[0043] Desalting treatment: Transfer the above eluent to a dialysis bag with a molecular weight cutoff of 100 Da, and dialyze at 4°C in 25 times its volume of deionized water for 6 hours, changing the dialysate 3 times during the process to remove high concentrations of Cl. - and Na + After dialysis, the solution in the dialysis bag was concentrated to 80% of its original volume, and the ammonium nitrogen concentration was measured. After desalting, the solution conductivity decreased from the initial 210 mS / cm to below 2.0 mS / cm, and the ammonium nitrogen recovery rate was 97.3%.

[0044] Nanozyme-catalyzed oxidation: 20 mL of the desalted solution was placed in a sealed reaction flask. 5 mg of Fe3O4-CeO2 core-shell nanozyme (approximately 50 nm in diameter) was added, along with 0.4 mL of freshly prepared sodium hypobromite solution (effective bromine content 100 g / L), bringing the effective bromine concentration in the reaction system to approximately 2.0 g / L. The reaction flask was sealed and the mixture was shaken and reacted in a 35°C water bath for 40 minutes. NH4+ was then removed. + Oxidized to NO2 - After the reaction is complete, an external magnetic field is applied to recover the nanozyme for later use. Measurements show that NH4... + Conversion rate 99.6%, NO2 - The yield was 99.1%.

[0045] Intelligent Response Microbial Transformation: The oxidized solution was transferred to an intelligent anaerobic microbial reactor. The pH was adjusted to 7.0 using sterile phosphate buffer. Simultaneously, a liquid nutrient substrate specifically for denitrifying microorganisms was added to the reaction system. High-purity helium gas (99.999%) was introduced to displace dissolved oxygen in the headspace and solution for 20 minutes, reducing the oxygen concentration in the reaction system to below 0.05%. Immobilized Pseudomonas asstutzeri in the form of sodium alginate-chitosan composite microspheres were added. The microspheres contained a fluorescein isothiocyanate-labeled dextran pH-sensitive fluorescent probe and an anthraquinone-2-sulfonate redox-sensitive electrochemical probe, with the probes covalently fixed to the microsphere carrier framework. The reactor incorporated a fiber optic sensor and a micro-electrochemical electrode to monitor the internal pH and microbial redox electron transport activity in real time. After anaerobic incubation at 35°C for 6 hours, the microbial agent converted NO2... - The gas is directionally converted into N2O gas. When the monitoring system detects a pH deviation from the 6.5-7.5 range or a decrease in microbial redox electron transport activity of more than 10%, the intelligent control module automatically adjusts the pH. After cultivation, the headspace gas in the reactor is collected using an airtight syringe.

[0046] MOF-enhanced gas purification and enrichment: The collected gas was sequentially passed through a pre-purification column (5 mm inner diameter, 15 cm length) filled with alkali asbestos and anhydrous magnesium perchlorate to remove CO2 and water vapor. The pre-purified gas was then passed through a micro-purification column (3 mm inner diameter, 5 cm length) filled with ZIF-8 MOF material (200 mg) for further removal of trace impurities. The purified gas was then passed through a liquid nitrogen cold trap (-196℃) filled with MOF material for enrichment for 15 minutes, achieving selective adsorption and enrichment of N2O. After enrichment, the cold trap was rapidly heated to 50℃ to release N2O gas.

[0047] Isotope detection: The released N₂O gas was introduced into a ThermoScientific Delta V Advantage isotope ratio mass spectrometer using helium as a carrier, and the ion current intensities of the N₂O molecular ion peaks at m / z 44, 45, and 46 were simultaneously detected. The δ¹⁴⁶ of ammonium nitrogen in the sample was calculated based on calibration with international standard reference materials. 15 N value and 15 Percentage of N atoms.

[0048] Comparative Example 1: Same as Example 1, except that no desalting treatment is performed after elution, and the high-salt eluent is directly used in subsequent steps.

[0049] Comparative Example 2: Same as Example 1, except that sodium hypobromite (1.0 g / L available chlorine) and MnSO4·H2O (5 mg / L) were added to the desalted solution as catalysts, and the reaction was carried out in a water bath at 60°C for 60 minutes.

[0050] Comparative Example 3: Same as Example 1, except that the oxidized solution was transferred to a regular culture flask, the pH was adjusted to 7.0 with sterile phosphate buffer, and an equal volume of free Pseudomonas stutzeri bacterial suspension (final concentration 10) was added. 9 CFU / mL), and cultured at 30℃ with constant shaking for 3 hours (anaerobic conditions were not controlled, and no probes or monitoring systems were added).

[0051] Comparative Example 4: Same as Example 1, except that purification is performed only through an alkali asbestos column and anhydrous magnesium perchlorate column, without passing through the ZIF-8MOF micro-purification column, and directly enters the liquid nitrogen cold trap (the cold trap is not filled with MOF material) for enrichment for 15 minutes.

[0052] Comparative Example 5: Referring to the "Stable Nitrogen Isotope Determination of Ammonium Nitrogen in Water by Distillation-Diffusion Method" (HJ 202X, Simulation Method), 1 L of lake water from the same batch was taken. After MgO distillation, boric acid absorption, and diffusion enrichment, the ammonium salt was converted into N2 and then introduced into an isotope mass spectrometer for δ-ray distillation. 15 N value.

[0053] The statistical results of the experiment are shown in the table below: Table 1. Test results of the examples and comparative examples

[0054] Based on the results above, in Comparative Example 1 (without desalination), the N2O yield was only 10.2%, indicating that the high-salt environment severely inhibited microbial transformation activity. Examples 1-3, through desalination treatment, significantly improved the subsequent microbial transformation efficiency, with N2O yields all >96%. Comparative Example 2 (using MnSO4 catalyst) required a reaction time of 60°C for 60 minutes, and NH4... + Conversion rate (94.2%) and NO2 - The yields (91.5%) were all lower than those of the examples; the examples achieved a conversion rate of ≥97.8% under conditions of 25-35℃ and 20-40 minutes, with a milder and more efficient reaction. Comparative Example 3 (free cells + aerobic) had an N2O yield of only 25.4% and an RSD as high as 8.5%, indicating that immobilization, anaerobic conditions, and real-time monitoring are crucial for stability and conversion efficiency; Examples 1-3 showed N2O yields >96% and RSD ≤1.6%, demonstrating excellent reproducibility. Comparative Example 4 (without MOF purification column and cold trap filling) had a recovery rate of 95.3%, slightly lower than the examples (≥98.9%), indicating that MOF materials further improved gas purification efficiency and recovery rate. Comparative Example 5 (distillation-diffusion method) showed a recovery rate of 91.2% and an RSD of 3.8%, with a detection time of approximately 12 hours. Examples 1-3 demonstrated superior recovery (≥98.9%), precision (RSD≤1.6%), and detection efficiency (≤6.5 hours), showcasing the comprehensive advantages of this technical solution. In summary, the technical solution proposed in this invention achieves efficient enrichment, high-precision conversion, and high-sensitivity isotope analysis of ammonium nitrogen in environmental samples through a complete process of ion exchange enrichment, desalination, nanozyme-catalyzed oxidation, intelligent immobilized microbial transformation, MOF-enhanced gas purification and enrichment, and isotope mass spectrometry detection. Examples 1-3 significantly outperformed the comparative examples in terms of conversion efficiency, recovery rate, reproducibility, and detection time, verifying the method's advancement, stability, and practicality.

[0055] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A highly safe ammonium nitrogen 15 The N detection method is characterized by, Includes the following steps: S1. Sample pretreatment: Collect environmental samples to be tested, centrifuge or filter them, adjust the pH to 2.0-4.0, add cation exchange resin to enrich ammonium nitrogen, and then elute with elution buffer to obtain enriched ammonium salt solution. S2. Desalting treatment: The ammonium salt solution obtained in step S1 is desalted to remove high concentrations of salt ions from the eluent, resulting in a desalted ammonium salt solution. S3, Chemical Oxidation: Sodium hypobromite solution as an oxidant and Fe3O4-CeO2 core-shell nanozyme as a catalyst are added to the desalted ammonium salt solution obtained in step S2. The oxidation reaction is carried out under sealed conditions for 20-40 minutes at a temperature of 25-35℃. NH4... + Quantitative oxidation to NO2 - A treatment solution containing nitrite was obtained; the amount of sodium hypobromite solution added was such that the effective bromine concentration reached 0.5-2.0 g / L. S4. Intelligent Response Microbial Transformation: An intelligent anaerobic microbial reactor is used. A dedicated liquid nutrient substrate for denitrifying microorganisms is added to the reaction system. Inert gas is introduced for 20-30 minutes to maintain an anaerobic state. The pH of the treated liquid obtained in step S3 is adjusted to 6.5-7.

5. Sodium alginate-chitosan composite microbial agent containing active denitrifying microorganisms and dual-sensitive probes is added to the reactor. The reactor is incubated at 25-35℃ for 2-6 hours. NO2 is converted to nitrogen through microbial denitrification. - The reaction is directionally converted into N2O gas. During the reaction, the pH value inside the microspheres and the electron transfer activity of microbial oxidation-reduction are monitored in real time through fiber optic sensors and micro electrochemical electrodes. The reactor is equipped with an intelligent control module, which automatically performs pH adjustment or nutrient factor supplementation when the monitoring signal deviates from the set threshold. The sodium alginate-chitosan composite microspheres contain 10 live bacteria. 9 -10 11 CFU / g of Pseudomonas, pH-sensitive fluorescent probes, and redox-sensitive electrochemical probes are covalently fixed to the microsphere carrier backbone to avoid probe signal inactivation due to high-temperature sterilization. S5. Gas purification and enrichment: Collect the N2O gas generated in step S4, remove impurity gases through a multi-stage purification column, and enrich it under low temperature conditions; the gas purification and enrichment step uses metal-organic framework materials for selective adsorption purification and enriches N2O under low temperature conditions. S6. Isotope Detection: The enriched N2O gas is introduced into an isotope ratio mass spectrometer to determine the nitrogen isotope composition of N2O. The ammonium nitrogen content in the sample is then calculated. 15 N isotope abundance; the isotope detection step uses an isotope ratio mass spectrometer to determine the nitrogen isotope composition of N2O.

2. The method according to claim 1, characterized in that, The desalination process is performed using dialysis. The dialysis bag has a molecular weight cutoff of 100-500 Da. Dialysis is carried out in deionized water at 4°C for 2-6 hours, during which the dialysis solution is replaced 1-3 times.

3. The method according to claim 1, characterized in that, The preparation method of the Fe3O4-CeO2 core-shell structured nanozyme is as follows: FeCl3·6H2O and FeCl2·4H2O are mixed at a molar ratio of 2:

1. Sodium citrate (0.4 times the weight of FeCl3·6H2O) is added and dissolved in ethylene glycol (40 times the weight of FeCl3·6H2O). Sodium acetate (2.4 times the weight of FeCl3·6H2O) is added, and the mixture is stirred for 30 minutes. The mixture is then transferred to a reaction vessel and reacted at 200℃ for 12 hours. After cooling, the Fe3O4 nanoparticles are magnetically separated and analyzed using ethanol and water. Wash and vacuum dry at 60℃ to constant weight to obtain Fe3O4 nanoparticles; disperse the Fe3O4 nanoparticles in water at 1000 times their weight, add Ce(NO3)3·6H2O at 4 times their weight and hexamethylenetetramine at 10 times their weight, and ultrasonically disperse for 30 minutes; stir the mixture at 90℃ for 2 hours, magnetically separate the product, wash with ethanol and water, and vacuum dry at 60℃ to obtain Fe3O4-CeO2 core-shell structured nanozyme.

4. The method according to claim 1, characterized in that, The preparation method of the sodium alginate-chitosan composite microspheres is as follows: (1) Activation of bacterial strain and preparation of bacterial suspension: Pseudomonas was inoculated into nutrient broth medium and cultured at 30°C with shaking for 24 hours. The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline, resuspended in sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 10. 1 CFU / mL, refrigerate at 4℃ for later use; (2) Preparation of carrier solution and immobilization of probe: Prepare a 3% (w / v) sodium alginate solution, filter it through a 0.22 μm filter membrane for sterilization, add covalently immobilized fluorescein isothiocyanate-labeled dextran and anthraquinone-2-sulfonate to the sterilized sodium alginate solution, stir well to obtain a mixed carrier solution; wherein the final concentration of fluorescein isothiocyanate-labeled dextran is 0.1 mg / mL, and the final concentration of anthraquinone-2-sulfonate is 0.5 mmol / L; (3) Initial preparation of microspheres: The mixed carrier solution was aseptically mixed with an equal volume of Pseudomonas bacterial suspension, and dripped into a 2% (w / v) sterile calcium chloride solution using a sterile syringe. The mixture was allowed to stand and solidify for 30 minutes to form calcium alginate gel microspheres. (4) Cross-linking and coating: The microspheres were transferred into a 0.5% (w / v) chitosan solution and cross-linked and coated at room temperature for 20 minutes to form sodium alginate-chitosan composite microspheres; the chitosan solution was dissolved in a 1% acetic acid solution and 0.02% (v / v) glutaraldehyde was added as a cross-linking agent. (5) Post-processing and storage: After cross-linking, the microspheres were washed three times with sterile physiological saline to obtain a viable count of 10. 1 CFU / g of compound micrococcal agent, stored at 4℃ for later use; The liquid nutrient substrate for denitrifying microorganisms contains glucose, potassium dihydrogen phosphate, magnesium sulfate, and yeast extract, with mass concentrations of 5-10 g / L, 1-2 g / L, 0.2-0.5 g / L, and 0.5-1 g / L, respectively.

5. The method according to claim 1, characterized in that, The anaerobic conditions are achieved by introducing an inert gas, helium, into the reaction system to replace the headspace and dissolved oxygen. The gas is introduced for 20-30 minutes to reduce the oxygen concentration in the reaction system to below 0.2%.

6. The method according to claim 1, characterized in that, The metal-organic framework material is ZIF-8 with a pore size of 0.3-1.0 nm. The preparation method of ZIF-8 is as follows: Zn(NO3)2·6H2O is dissolved in methanol at a volume 20 times the weight of Zn(NO3)2·6H2O, denoted as solution A; 2-methylimidazolium at a weight 2 times the weight of Zn(NO3)2·6H2O is dissolved in methanol at a volume 20 times the weight of Zn(NO3)2·6H2O, denoted as solution B; solution B is quickly added to solution A, and the mixture is stirred at room temperature for 1 hour; the white precipitate is collected by centrifugation, washed three times with methanol, and dried under vacuum at 60°C for 12 hours to obtain ZIF-8 crystals.

7. The method according to claim 1, characterized in that, In the gas purification and enrichment step, a multi-stage purification system is used, which sequentially passes the gas through an alkali asbestos column, an anhydrous magnesium perchlorate column and a MOF-filled column, and then enters a liquid nitrogen cold trap filled with MOF material for enrichment. The intelligent anaerobic microbial reactor integrates a signal acquisition unit, a central processing unit, and an execution unit. The signal acquisition unit transmits fluorescence and electrochemical monitoring data in real time via fiber optic sensors and microelectrodes. The central processing unit compares the data with preset thresholds. When the pH deviates from the 6.5-7.5 range or the microbial redox electron transport activity decreases by more than 10%, the execution unit is triggered to automatically pump dilute hydrochloric acid or dilute sodium hydroxide solution into the system to adjust the pH. By monitoring the microbial redox electron transport activity, the reactor reflects the denitrifying microbial metabolic activity in real time, ensuring NO2... - Conversion efficiency.

8. The method according to claim 1, characterized in that, The cation exchange resin is a strong acid styrene-based cation exchange resin, the eluent is a 1-3 mol / L NaCl or KCl solution, and the elution flow rate is 0.5-2.0 mL / min; in the chemical oxidation step, the nanozyme catalyst is recovered and reused through magnetic recovery.

9. The method according to claim 1, characterized in that, The isotope ratio mass spectrometer simultaneously detects the ion current intensities of the N2O molecular ion peaks at m / z 44, 45, and 46, and calculates them based on international standard reference materials. 15 Percentage of N atoms.

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