A method for recognizing and quantifying nitrogen conversion pathways in a sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation wastewater treatment process

Abstract

A kind of utilization 15 Nitrogen isotope tracing technology reveals the nitrogen transfer pathways and quantitative analysis methods in the biological treatment of wastewater involving sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation, belonging to the field of biological wastewater treatment. To distinguish the anaerobic ammonium oxidation, denitrification, and dissimilatory reduction to ammonium reactions in the sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation process, this invention designs an isotope tracing experiment, including a: a blank group; b: 15 NH4Cl, Na 14 NO2; c: 14 NH4Cl, Na 15 NO3; d: Na 15 NO3; e: Na 15 NO3. Take Na 15 The experimental group using NO3 as a substrate needs to use chemical methods to remove the NO3 produced during the reaction process. 15 NH4 + Transform into 30 The rate of dissimilatory reduction to ammonium was calculated using N2, combined with... 14 NH4Cl, Na 15 In the experimental group using NO3 as a substrate, the reaction process... 29 N2 and 30 The changes in N2 over time were analyzed to determine the rates of anaerobic ammonia oxidation and denitrification. This invention is economical, simple, and reliable.

Description

Technical Field

[0001] This invention relates to a method for identifying and quantifying nitrogen transformation pathways in the biological treatment of wastewater by sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation. It belongs to the field of wastewater biological treatment technology and is used to reveal the reaction mechanism of denitrification, anaerobic ammonia oxidation and dissimilatory reduction to ammonium in a sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation system. Background Technology

[0002] In recent years, with the acceleration of industrialization, the increase in nitrate content in regional water environments has become one of the main causes of water quality deterioration, and reducing nitrate concentration in water bodies has attracted widespread attention from researchers. Biological denitrification technology, as an economical and environmentally friendly wastewater treatment method, has been widely applied in wastewater treatment plants. Traditional denitrification processes require the consumption of organic carbon sources, but due to the low C / N ratio in many industrial wastewaters, this process requires external carbon sources, resulting in high treatment costs. Simultaneously, industrial wastewater contains large amounts of ammonia nitrogen and sulfides, which provides conditions for sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation for nitrogen and sulfur removal. The synergistic denitrification and sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation is a novel method. It utilizes sulfur autotrophic denitrification to reduce nitrate to nitrite, while sulfides are oxidized to elemental sulfur or sulfate. Then, nitrite and ammonia nitrogen undergo autotrophic denitrification under the action of anaerobic ammonia-oxidizing bacteria, generating nitrogen gas. Compared to full-process denitrification, this reaction reduces the production of greenhouse gas N2O, reduces treatment process steps, and lowers wastewater treatment costs, making it of great practical significance and broad application prospects. However, the complex reaction pathways within the sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation system make it difficult to determine the nitrogen transfer pathway, hindering its widespread application and promotion. Besides sulfur autotrophic denitrification and anaerobic ammonium oxidation, there is also a reaction in which nitrate is dissimilarly reduced to ammonium using sulfides as electron donors. This reaction occurs because sulfides inhibit NO and N₂O reductases in the denitrification stage, thereby suppressing the denitrification process and promoting the dissimilar reduction to ammonium.

[0003] Currently, the main methods used to distinguish the role of microorganisms in the biological denitrification process of wastewater include the acetylene inhibition method, the direct measurement method, and... 15 Nitrogen isotope labeling tracer method. Among them, the first two methods can distinguish between denitrification and anaerobic ammonium oxidation reactions, but when there is a dissimilatory reduction to ammonium process in the system, the three reactions cannot be accurately distinguished. 15 The basic principle of nitrogen isotope labeling tracer method is to add a high abundance of nitrogen to the reaction system. 15 N-labeled compound solutions (e.g.) 15 NH4 + or 15 NO3 - ),according to 15 N-pairing technique, using isotope mass spectrometry to determine 29 N2 and30 Changes in N2 production can identify different N2 sources. This method first uses... 14 NH4 + and 15 NO3 - Distinguishing between anaerobic ammonium oxidation and denitrification processes, among which 30 All N2 is produced by the denitrification process, allowing for accurate calculation of the contributions of denitrification and anaerobic ammonium oxidation to nitrogen removal. Based on this, further calculations can be made... 15 NO3 - The treatment group, which measures the reaction process 15 NO3 - produce 15 NH4 + The rate of dissimilatory reduction to ammonium is determined by the following conditions. 15 NH4 + It needs to be converted through chemical oxidation. 30 N2 was measured; at the same time, the measured rate of dissimilatory reduction to ammonium was used to correct the denitrification and anaerobic ammonium oxidation, thereby calculating the reaction rates of denitrification, anaerobic ammonium oxidation and dissimilatory reduction to ammonium in the sulfur autotrophic denitrification coupled anaerobic ammonium oxidation system. Summary of the Invention

[0004] This invention is a method of utilizing 15 A method for determining the rates of anaerobic ammonium oxidation, denitrification, and dissimilatory reduction to ammonium in sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation in wastewater biological treatment. This method involves five isotope-labeled experimental groups, including two control groups, two experimental groups, and one test group. The rates of anaerobic ammonium oxidation, denitrification, and dissimilatory reduction to ammonium in the reaction process are determined using nitrogen isotope tracing technology. 29 N2 and 30 By detecting and analyzing N2, the reaction results of anaerobic ammonia oxidation, denitrification, and dissimilatory reduction to ammonium can be determined.

[0005] The technical solution of the present invention is as follows:

[0006] (1) Simulated wastewater from sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation

[0007] Simulated wastewater was prepared with the following composition: 0.46 g / L Na₂S·9H₂O, 0.05 g / L MgSO₄·7H₂O, 0.05 g / L CaCl₂·2H₂O, 1.0 g / L NaHCO₃, and 0.05 g / L KH₂PO₄. 1.0 ml of a trace element solution was added to each liter of simulated wastewater. The trace element solution consisted of: 1.5 g / L FeCl₃·6H₂O, 0.25 g / L CuSO₄·5H₂O, 10 g / L EDTA, 0.43 g / L ZnSO₄·7H₂O, 0.014 g / L H₃BO₄, 0.99 g / L MnCl₂·4H₂O, 0.22 g / L Na₂MoO₄·2H₂O, and 0.24 g / L CoCl₂·6H₂O.

[0008] (2) Experiment on pre-culture of sediment with sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation

[0009] The sulfur-autotrophic denitrification coupled with anaerobic ammonium oxidation sludge from the biological nitrogen removal system of the wastewater to be tested was washed three times with PBS. 2 ml of sludge was added to a 12 ml Labco glass culture flask, followed by three 3 mm diameter glass rollers. The flask was purged with helium for 10 minutes to remove dissolved oxygen from the liquid and headspace, and the original rubber stopper was replaced. The simulated wastewater was then purged with helium for 30 minutes to remove dissolved oxygen. 10 ml of simulated wastewater was then injected into the culture flask using a syringe, with an venting needle used to maintain pressure balance within the flask. After injection, it was ensured that no air bubbles remained in the flask. The culture flask was then incubated in a rotary culture apparatus for 24 hours at the same temperature as the original reaction system. This process consumed the NO originally present in the bottom sludge. x - ;

[0010] (3) Isotope labeling experiment

[0011] The isotope labeling experiment was set up in 5 groups, with the number of culture flasks in each group being 3 times the number of sampling points, that is, 3 replicates for each sample. The reaction substrate and sampling time for each group were set according to Table 1. 100 μL of each substrate was injected into the culture flask. During the injection, a guide needle was inserted. The length of the guide needle should be shorter than the syringe needle. For groups 1-5, 200 μL of 7mol / L ZnCl2 solution was added sequentially according to the sampling time to terminate the reaction.

[0012] For experiments 1-4, after the experiment, prepare the same number of 12ml Labco glass culture flasks. Purge the new flasks with helium for 30 minutes and then cap them to ensure an anaerobic environment. Draw 4ml of liquid from each of the flasks from the reactions that have terminated and inject it into a brand new flask. Simultaneously, use a venting needle to expel the same volume of gas to maintain pressure balance within the flask. Shake thoroughly until water-gas equilibrium is reached. Draw 50μL of gas and inject it into an isotope mass spectrometer to determine N2 production. Then, calculate the amount of N2 produced based on Henry's Law and the sample volume. 28 N2, 29 N2 and 30 N2;

[0013] Table 1 Isotope labeling experiments

[0014]

[0015] (4)NH4 + Convert to N2 experiment

[0016] After the fifth reaction group was completed, prepare the same number of 12ml Labco glass culture flasks. Take 4ml of liquid from the fifth culture flask (from which the reaction was terminated) and inject it into a brand new culture flask. Purge the liquid and headspace with helium for 15 minutes to remove dissolved oxygen, then seal the flask. Add 200μL of iodine hypobromide solution using a 1ml syringe, and react in a rotary incubator for 12 hours. Extract 50μL of gas and inject it into an isotope mass spectrometer to determine N2 production. Then, calculate the nitrogen generated during the reaction process based on Henry's Law and the sample volume. 15 NH4 + ;

[0017] Preparation of hypobromide iodine solution: Add 100 ml of liquid bromine to 600 ml of 16 mol / L NaOH while stirring. This process is carried out on ice. After the solution has been standing in a refrigerator at 4°C for one week, it is mixed with 0.2% KI at a ratio of 1:1 to prepare hypobromide iodine solution.

[0018] (5) Calculation of results

[0019] Group 1 and Group 2 were control experiments. In Group 1, if no detection was found... 29 N2 or 30 The production of N2 indicates that NO in the sediment was consumed during the pre-cultivation period. x - In group 2, if it can be detected 29 The generation of N2 and no 30 The generation of N2 proves that an anaerobic ammonia oxidation reaction occurred in the system; Group 4 provides supplementary data to verify the accuracy of the experimental process and results of Group 3.

[0020] Group 5 determined the product of dissimilatory reduction. 15 NH4 + occupy 15 NO3 - Maximum mole fraction F A :

[0021] F A = 15 NH 4+max / 15 NO3 -

[0022] Group 3 is the experimental group. Calculate the reaction rates of denitrification (DE), anaerobic ammonium oxidation (AN), and dissimilatory reduction to ammonium (DN) according to the following formulas:

[0023] p A 30 N2 = F A ×[p 29 N2+2×(1-F N -1 )×p 30 N2] / [F N -F A ]

[0024] Where, p A 30 N2 represents the product generated by anaerobic ammonium oxidation in the presence of the dissimilatory reduction to ammonium reaction. 30 The rate of N2, p 29 N2 represents 29 The rate of N2 production, p 30 N2 represents 30 The rate of N2 production, F N represent 15 NO3 - In total NO3 - The mole fraction in;

[0025] The reaction rates for the AN, DE, and DN pathways are calculated as follows:

[0026] AN = F N -1 ×(F N -F A ) -1 ×[p 29 N2-2×(1-F N )×p 30 N2]

[0027] DE = (p 30 N2-p A 30 N2)×F N -2

[0028] DN=(p 30 N2+p A 30 N2)×F N -1

[0029] The innovative aspects of this invention:

[0030] (1) The traditional method for determining the reaction pathway in biological denitrification is the direct measurement method, which involves estimating the reaction process by combining changes in substrate concentration with stoichiometry. This method can lead to significant errors in systems involving multiple reactions such as dissimilatory reduction to ammonium, denitrification, and anaerobic ammonium oxidation. 15 Nitrogen isotope tracing technology is more accurate than direct measurement methods because it distinguishes and labels the substrate.

[0031] (2) The acetylene inhibition method uses high concentrations of acetylene to inhibit the activity of nitrous oxide reductase in sludge microorganisms, thereby preventing the denitrification process. By comparing the reaction without the addition of acetylene, the nitrogen loss caused by denitrification and anaerobic ammonia oxidation can be distinguished. However, the acetylene inhibition method has an insurmountable disadvantage: a small number of denitrifying microorganisms are not sensitive to acetylene. These microorganisms can promote the reduction of nitrous oxide to nitrogen gas in an environment with high carbon content and low nitrate concentration. Furthermore, acetylene is dangerous during storage. 15 The nitrogen isotope tracing technique does not interfere with the reaction process and the reaction operation is not dangerous.

[0032] (3) This method increases 15 NO3 - The experimental group calculated the rate of dissimilatory reduction to ammonium, and then combined it with... 14 NH4 + and 15 NO3 - Distinguishing between anaerobic ammonium oxidation and denitrification processes provides a more accurate understanding of the nitrogen transformation pathway in the sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation reaction system. Attached Figure Description

[0033] Figure 1 In group 3 of the present invention 29 N2, 30 How N2 changes over time;

[0034] Figure 2 This is group 4 of the embodiments of the present invention. 30 How N2 changes over time;

[0035] Figure 3 This is group 5 of the present invention. 15 NH4 +How it changes over time;

[0036] Figure 4 These are different concentrations in embodiments of the present invention. 15 NH4 + Transform into 30 The conversion rate of N2. Detailed Implementation

[0037] The patent application will be further explained with reference to the accompanying drawings and examples:

[0038] (1) Simulated wastewater from sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation

[0039] Simulated wastewater was prepared with the following composition: 0.46 g / L Na₂S·9H₂O, 0.05 g / L MgSO₄·7H₂O, 0.05 g / L CaCl₂·2H₂O, 1.0 g / L NaHCO₃, and 0.05 g / L KH₂PO₄. 1.0 ml of a trace element solution was added to each liter of simulated wastewater. The trace element solution consisted of: 1.5 g / L FeCl₃·6H₂O, 0.25 g / L CuSO₄·5H₂O, 10 g / L EDTA, 0.43 g / L ZnSO₄·7H₂O, 0.014 g / L H₃BO₄, 0.99 g / L MnCl₂·4H₂O, 0.22 g / L Na₂MoO₄·2H₂O, and 0.24 g / L CoCl₂·6H₂O.

[0040] (2) Experiment on pre-culture of sediment with sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation

[0041] Sulfate-autotrophic denitrification coupled with anaerobic ammonium oxidation sludge from a wastewater biological denitrification system was washed three times with PBS. Fifty-seven 12ml LabCO glass culture flasks were prepared, with 2ml of sludge and three 3mm diameter glass rollers added to each flask. The flasks were purged with helium for 10 minutes to remove dissolved oxygen from the liquid and headspace, and the original rubber stoppers were replaced. Simulated wastewater was then purged with helium for 30 minutes to remove dissolved oxygen. 10ml of simulated wastewater was then injected into each culture flask using a syringe, ensuring pressure balance within the flask during injection and guaranteeing no air bubbles remained after injection. The flasks were then incubated in a rotary culture system for 24 hours at the same temperature as the original reaction system. This process was intended to consume the NO present in the bottom sludge. x - ;

[0042] (3) Isotope labeling experiment

[0043] Five groups were set up for the isotope labeling experiment. The reaction substrate and sampling time for each group were set according to Table 1. 100 μL of each substrate was injected. During the injection, a guide needle was inserted. The length of the guide needle should be shorter than the syringe needle. For groups 1-5, 200 μL of 7 mol / L ZnCl2 solution was added sequentially according to the sampling time to terminate the reaction.

[0044] For reactions 1-4, after the experiment, 54 new 12ml LabCO glass culture flasks were prepared. The new flasks were purged with helium for 30 minutes and then capped to ensure an anaerobic environment. 4ml of liquid was injected into each new culture flask, while the same volume of gas was expelled using a venting needle to maintain pressure balance. The flasks were shaken thoroughly until water-gas equilibrium was reached. 50μL of gas was injected into an isotope mass spectrometer to determine N2 production. The amount of N2 produced was then calculated based on Henry's Law and the sample volume. 28 N2, 29 N2 and 30 N2;

[0045]

[0046] Where K represents the Henry's law constant (mol L) -1 atm -1 ), p represents atmospheric pressure (1.0 atm), C dis V represents the N2 concentration measured by isotope mass spectrometry. H2O and V gas M represents the volume (mL) of the water sample and the upper air, respectively. N2 The value represents the molecular weight of N2, and T represents the reaction temperature (25℃).

[0047] The results of group 3 are as follows Figure 1 As shown, 29 N2 and 30 N2 gradually accumulates over time, among which 29 N2 is mainly produced by anaerobic ammonia oxidation. 30 N2 is mainly produced by denitrification; the results of group 4 are as follows: Figure 2 As shown, where 30 N2 is mainly produced by denitrification. 30 The trend of N2 change is consistent with that of group 2, proving the accuracy and reliability of the data from experimental group 3;

[0048] Table 1 Isotope Tracing Experiments

[0049]

[0050] (4)NH4 + Convert to N2 experiment

[0051] To calculate the rate of dissimilatory reduction to ammonium during the reaction, it is necessary to first analyze the samples in group 5. 15 NH4 + Transform into 30 N2; After the five sets of reactions were completed, 21 new 12ml LabCO glass culture flasks were prepared. 4ml of liquid was drawn and injected into each flask. The flasks were purged with helium gas to the liquid and headspace for 15 minutes, then the stoppers were closed. 200μL of iodine hypobromide solution was added using a 1ml syringe. The mixture was reacted in a rotary incubator for 12 hours. 50μL of gas was extracted and injected into an isotope mass spectrometer to determine N2 production. Subsequently, the amount of N2 produced was calculated using Henry's Law. 30 N2 production, 30 Twice the N2 production, which is generated during the reaction process. 15 NH4 + ;

[0052]

[0053] Where K represents the Henry's law constant (mol L) -1 atm -1 ), p represents atmospheric pressure (1.0 atm), C dis V represents the N2 concentration measured by isotope mass spectrometry. H2O and V gas M represents the volume (mL) of the water sample and the upper air, respectively. N2 The value represents the molecular weight of N2, and T represents the reaction temperature (25℃).

[0054] The test results of group 5 are as follows: Figure 3 As shown, 15 NH4 + After reaching its maximum value within 2 hours, the yield tends to stabilize; different concentrations 15 NH4 + Transform into 30 N2 conversion efficiency such as Figure 4 As shown, the maximum conversion rate in this experiment reached 95.2%. 15 NH4 + The lower the concentration, the higher the conversion rate;

[0055] Preparation of hypobromide iodine solution: Add 100 ml of liquid bromine to 600 ml of 16 mol / L NaOH while stirring (this process is carried out on ice). After standing in a refrigerator at 4°C for one week, mix it with 0.2% KI in a 1:1 ratio to prepare hypobromide iodine solution.

[0056] (5) Results Calculation and Analysis

[0057] Group 1 and Group 2 served as control experiments; no abnormalities were detected in Group 1. 29 N2 or30 The significant production of N2 indicates that NO in the sediment was consumed during the pre-culture period. x - In group 2, it was detected 29 The generation of N2 and no 30 The generation of N2 proves that an anaerobic ammonia oxidation reaction occurred within the system; the N2 generated by group 4 30 N2 and added 15 NO3 - The matching proves that the experimental process and calculations are accurate;

[0058] Group 5 determined the product of dissimilatory reduction. 15 NH4 + The occupation 15 NO3 - Maximum mole fraction (F) A ):

[0059] F A = 15 NH 4+max / 15 NO3 -

[0060] Group 3 is the experimental group. Calculate the reaction rates of denitrification (DE), anaerobic ammonium oxidation (AN), and dissimilatory reduction to ammonium (DN) using the following formulas:

[0061] p A 30 N2 = F A ×[p 29 N2+2×(1-F N -1 )×p 30 N2] / [F N -F A ]

[0062] Where, p A 30 N2 represents the product generated by AN in the presence of the DN reaction. 30 The rate of N2, p 29 N2 represents 29 N2 generation rate, p 30 N2 represents 30 N2 production rate, F N represent 15 NO3 - In total NO3 - The mole fraction in;

[0063] The reaction rates for the AN, DE, and DN pathways are calculated as follows:

[0064] AN = F N-1 ×(F N -F A ) -1 ×[p 29 N2-2×(1-F N )×p 30 N2]

[0065] DE = (p 30 N2-p A 30 N2)×F N -2

[0066] DN=(p 30 N2+p A 30 N2)×F N -1

[0067] Calculations showed that the reaction rates of anammox, denitrification, and dissimilatory reduction to ammonium in this organism were 0.36 μmol / h, 0.48 μmol / h, and 0.57 μmol / h, respectively. Among them, anammox and denitrification processes can lead to direct nitrogen loss, while dissimilatory reduction to ammonium provides substrates for anammox and complete denitrification.

[0068] This experiment demonstrates that isotope labeling technology can trace the source, migration, and transformation of nitrogenous substances in the sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation wastewater treatment process. It provides a theoretical basis for the application of isotope labeling tracing technology in the study of the deep denitrification mechanism of urban wastewater and provides a reliable basis for the optimization of reaction conditions and operating conditions.

Claims

1. A method for identifying and quantifying nitrogen transformation pathways in wastewater treatment processes involving sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation, characterized in that, Using stable isotope labeling and pairing techniques, by adding 15 N-marked 15 NH4 + or 15 NO x - Based on the differences in substrate utilization during denitrification, dissimilatory reduction to ammonium, and anaerobic ammonia oxidation, the rates of each reaction were determined as follows: (1) Simulated wastewater from sulfur autotrophic denitrification coupled with anaerobic ammonia oxidation Simulated wastewater was prepared with the following composition: 0.46 g / L Na₂S·9H₂O, 0.05 g / L MgSO₄·7H₂O, 0.05 g / L CaCl₂·2H₂O, 1.0 g / L NaHCO₃, and 0.05 g / L KH₂PO₄. 1.0 ml of a trace element solution was added to each liter of simulated wastewater. The trace element solution consisted of: 1.5 g / L FeCl₃·6H₂O, 0.25 g / L CuSO₄·5H₂O, 10 g / L EDTA, 0.43 g / L ZnSO₄·7H₂O, 0.014 g / L H₃BO₄, 0.99 g / L MnCl₂·4H₂O, 0.22 g / L Na₂MoO₄·2H₂O, and 0.24 g / L CoCl₂·6H₂O. (2) Experiment on pre-culture of sediment with sulfur autotrophic denitrification coupled with anaerobic ammonium oxidation The sulfur-autotrophic denitrification coupled with anaerobic ammonium oxidation sludge from the biological nitrogen removal system of the wastewater to be tested was washed three times with PBS. 2 ml of sludge was added to a 12 ml Labco glass culture flask, followed by three 3 mm diameter glass rollers. The flask was purged with helium for 10 minutes to remove dissolved oxygen from the liquid and headspace, and the original rubber stopper was replaced. The simulated wastewater was then purged with helium for 30 minutes to remove dissolved oxygen. 10 ml of simulated wastewater was then injected into the culture flask using a syringe, with an venting needle used to maintain pressure balance within the flask. After injection, it was ensured that no air bubbles remained in the flask. The culture flask was then incubated in a rotary culture apparatus for 24 hours at the same temperature as the original reaction system. This process consumed the NO originally present in the bottom sludge. x - ; (3) Isotope labeling experiment The isotope labeling experiment was set up in 5 groups, with the number of culture flasks in each group being 3 times the number of sampling points, that is, 3 replicates for each sample. The reaction substrate and sampling time for each group were set according to Table 1. 100 μL of each substrate was injected into the culture flask. During the injection, a guide needle was inserted. The length of the guide needle should be shorter than the syringe needle. For groups 1-5, 200 μL of 7mol / L ZnCl2 solution was added sequentially according to the sampling time to terminate the reaction. For experiments 1-4, after the experiment, prepare the same number of 12ml Labco glass culture flasks. Purge the new flasks with helium for 30 minutes and then cap them to ensure an anaerobic environment. Draw 4ml of liquid from each of the flasks from the reactions that have terminated and inject it into a brand new flask. Simultaneously, use a venting needle to expel the same volume of gas to maintain pressure balance within the flask. Shake thoroughly until water-gas equilibrium is reached. Draw 50μL of gas and inject it into an isotope mass spectrometer to determine N2 production. Then, calculate the amount of N2 produced based on Henry's Law and the sample volume. 28 N2, 29 N2 and 30 N2; (4)NH4 + Convert to N2 experiment After the fifth reaction group was completed, prepare the same number of 12ml Labco glass culture flasks. Take 4ml of liquid from the fifth culture flask (from which the reaction was terminated) and inject it into a brand new culture flask. Purge the liquid and headspace with helium for 15 minutes to remove dissolved oxygen, then seal the flask. Add 200μL of iodine hypobromide solution using a 1ml syringe, and react in a rotary incubator for 12 hours. Extract 50μL of gas and inject it into an isotope mass spectrometer to determine N2 production. Then, calculate the nitrogen generated during the reaction process based on Henry's Law and the sample volume. 15 NH4 + ; Preparation of hypobromide iodine solution: Add 100 ml of liquid bromine to 600 ml of 16 mol / L NaOH while stirring. This process is carried out on ice. After the solution has been standing in a refrigerator at 4°C for one week, it is mixed with 0.2% KI at a ratio of 1:1 to prepare hypobromide iodine solution. (5) Calculation of results Group 1 and Group 2 were control experiments. In Group 1, if no detection was found... 29 N2 or 30 The production of N2 indicates that NO in the sediment was consumed during the pre-cultivation period. x - In group 2, if it can be detected 29 The generation of N2 and no 30 The generation of N2 proves that an anaerobic ammonia oxidation reaction occurred in the system; Group 4 provides supplementary data to verify the accuracy of the experimental process and results of Group 3. Group 5 determined the production of dissimilatory reduction 15 NH4 + occupy 15 NO3 - Maximum mole fraction F A : F A = 15 NH 4+max / 15 NO3 - Group 3 is the experimental group. Calculate the reaction rates of denitrification (DE), anaerobic ammonium oxidation (AN), and dissimilatory reduction to ammonium (DN) according to the following formulas: p A 30 N2=F A ×[p 29 N2+2×(1-F N -1 )×p 30 N2] / [F N -F A ] Where, p A 30 N2 represents the product generated by anaerobic ammonium oxidation in the presence of the dissimilatory reduction to ammonium reaction. 30 The rate of N2, p 29 N2 represents 29 The rate of N2 production, p 30 N2 represents 30 The rate of N2 production, F N represent 15 NO3 - In total NO3 - The mole fraction in; The reaction rates for the AN, DE, and DN pathways are calculated as follows: AN=F N -1 ×(F N -F A ) -1 ×[p 29 N2-2×(1-F N )×p 30 N2] DE=(p 30 N2-p A 30 N2)×F N -2 DN=(p 30 N2+p A 30 N2)×F N -1 。

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