Method for identifying and quantifying nitrogen conversion path of iron-enhanced anaerobic ammonia oxidation system by using 15N isotope labeling technology

Abstract

The invention relates to a method for identifying and quantifying a nitrogen conversion path of an iron-enhanced anaerobic ammonia oxidation system by utilizing a 15N isotope labeling technology. According to the method, different 15N isotope substrate labeling schemes are designed, and the gas chromatography-isotope ratio mass spectrometry technology is combined, so that the composition difference of N2 isotopes generated in each denitrification path is accurately determined. Based on the generation rule and abundance ratio of 29N2 / 30N2, the contribution of Anammox, Feammox and NDFO to NH4 < + >-N oxidation and NOx <->-N reduction is quantitatively analyzed. The method can effectively reveal the activity change, individual contribution and synergistic action mechanism of each denitrification pathway in an iron-mediated complex reaction network, and provides powerful support for optimizing an iron-enhanced anaerobic ammonia oxidation system.

Description

Technical Field

[0001] This invention relates to a method utilizing 15 The method of identifying and quantifying nitrogen conversion pathways in iron-enhanced anaerobic ammonia oxidation systems using nitrogen isotope labeling technology belongs to the field of wastewater biological treatment technology. Background Technology

[0002] Anaerobic ammonium oxidation (Anammox) technology is considered a cost-effective and efficient novel wastewater denitrification method due to its advantages such as no need for aeration, no need for external carbon sources, and low sludge production. However, anaerobic ammonium-oxidizing bacteria grow slowly and are susceptible to the effects of heavy metals, organic matter, and pH. Iron, as one of the most widely distributed metallic elements in nature, plays an irreplaceable role in the physiological metabolism of microorganisms. Among them, anaerobic ammonium-oxidizing bacteria (AnAOB) show a particularly significant dependence on iron. AnAOB stores iron in its unique anaerobic ammonium oxidizing body and drives energy metabolism through three key iron-containing cofactors (ferrous heme c, iron-sulfur clusters, and iron-nickel proteins). Numerous studies have indicated that the appropriate addition of iron ions and iron oxides can enhance the activity of AnAOB by regulating the redox potential of the reaction system, promoting the activity of key enzymes in nitrogen metabolism, and promoting sludge granulation, thereby enhancing the denitrification performance and stability of the system. In addition, iron can act as an electron acceptor and electron donor, directly participating in nitrogen metabolism through anaerobic ferroammox and ferroautotrophic denitrification (NDFO) pathways, oxidizing NH4, respectively. + -N and reduction of NO x - -N (including NO2) - -N and NO3 - -N).

[0003] Iron-enhanced anaerobic ammonium oxidation systems improve nitrogen removal efficiency by increasing microbial metabolic activity and expanding reaction pathway diversity; however, the complex iron-mediated reaction network—including the coupling of Feammox, NDFO, and Anammox—leads to substrate competition and electron transport interactions between nitrogen removal pathways. Relying solely on conventional water quality indicators (NH4+) is insufficient. + -N, NO2 - -N, NO3 - The concentration changes of -N) make it difficult to analyze the activity thresholds, contribution ratios, and synergistic mechanisms of each pathway. Therefore, this invention introduces... 15 Nitrogen isotope labeling technology, through targeted tracking of different nitrogen forms ( 15 NH4 + -N、 15 NO2 - -N、 15 NO3 -The transformation trajectory and metabolic flow of Anammox and Feammox can overcome the limitations of traditional concentration monitoring. Specifically: (1) Design a method to target the interaction mechanism between Anammox and Feammox. 15 NH4 + -N and 14 NO2 - -N co-labeled system. In Fe 3+ The isotopic composition differences of generated N2 were determined by gas chromatography-isotope ratio mass spectrometry under conditions where the agent acts as an electron acceptor. The Anammox pathway involves... 15 NH4 + -N and 14 NO2 - -N coupling generation 29 N2, while the Feammox pathway is through 15 NH4 + -N direct( 15 NH4 + -N→ 30 N2) and indirect ( 15 NH4 + -N→ 15 NO x - -N→ 30 N2) generated 30 N2. Based on 29 N2 / 30 The production patterns and abundance ratios of N2 can determine the effects of two pathways on NH4. + (2) The interaction mechanism and contribution rate of -N oxidation; (3) Constructing for Anammox and NDFO. 14 NH4 + -N and 15 NO x - -N co-labeled system. In Fe 2+ Anammox, under the condition of an auxiliary electron donor, through 14 NH4 + -N and 15 NO2 - -N (can be indirectly derived from) 15 NO3 - -N generation) generation 29 N2, and 15 NO x - -N then Fe 2+ Generate electron donors 30 N2. Based on 29 N2 / 30 The production patterns and abundance ratios of N2 can determine the effects of two pathways on NO. x -The interaction mechanism and contribution rate of -N reduction. Summary of the Invention

[0004] A kind of utilization 15 A method for identifying and quantifying nitrogen conversion pathways in iron-enhanced anaerobic ammonium oxidation systems using nitrogen isotope labeling technology, characterized by employing a stable isotope pairing method, through the addition of... 15 N-labeled NH4 + -N, NO2 - -N, NO3 - -N and Fe, based on the differences in the reaction mechanisms and substrate utilization characteristics of Anammox, Feammox, and NDFO, were determined by measuring the products. 29 N2 and 30 The proportion of N2 was used to quantitatively analyze the denitrification contribution of each reaction pathway. The specific steps are as follows:

[0005] (1) Sample collection: The homogenized mud-water mixture of the iron-enhanced anaerobic ammonium oxidation system was collected in sterile plastic sampling bags and transported to the laboratory under refrigeration;

[0006] (2) Pre-culture substrate consumption: Sludge from the iron-enhanced anaerobic ammonium oxidation system was washed with simulated wastewater and then dispensed into serum bottles. Helium was used to purge the liquid and gas phases to remove oxygen and nitrogen, and the bottles were sealed with three-hole caps. The simulated wastewater consisted of 0.05 g / L MgSO4·7H2O, 0.05 g / L CaCl2·2H2O, 1.0 g / L NaHCO3, and 0.027 g / L KH2PO4. In addition, 1.5 mL of trace element solution was added per liter of simulated wastewater, consisting of 5 g / L... EDTA, 0.25 g / L CuSO4·5H2O, 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 were added. The serum bottles containing the sediment were placed in a constant temperature shaker and incubated for 24 h to consume the residual substrate from the original system. The incubation temperature was kept consistent with that of the original system.

[0007] (3) Isotope labeling scheme design: Anammox reaction substrates include NH4 + -N and NO2 - The introduction of -N and iron allows for the possible existence of Feammox and NDFO reaction pathways in the system, which oxidize NH4, respectively. + -N and reduction of NO2 - -N; Set up a T0 control group, adding only simulated wastewater; Fe was present in the bottom sediment of the experimental group. 3+ Under the condition of adding different substrates: T1 uses 15 NH4 + -N and14 NO2 - -N is used as the substrate; T2 uses 14 NH4 + -N、 15 NO2 - -N and Fe 2+ As a substrate; T3 substrate is 14 NH4 + -N、 15 NO3 - -N and Fe 2+ ;

[0008] (4) Experimental Procedure: Simulated wastewater purged with helium was added to the pre-cultured sediment. Helium was used to purge the liquid phase and headspace to remove oxygen and nitrogen, followed by sealing with a three-hole feed cap. One hole was used to add substrate and collect the liquid phase, another hole was used to collect headspace gas, and the third hole was used to connect a helium bag to balance the pressure and maintain the anaerobic environment. NH4 was injected using a syringe. + -N, NO2 - -N, NO3 - -N, Fe 2+ Sodium bicarbonate concentrate was added to the serum vial through the sample well; 5 mL of headspace gas was added to a syringe at intervals, sealed with a rubber stopper, and 4 mL of mud-water mixture was added to a 12 mL headspace vial. 0.1 mL of 7 mol / L ZnCl2 was added to the vial beforehand to terminate the reaction, and the vial was purged with helium to remove oxygen and nitrogen; the reaction was then analyzed using an isotope mass spectrometer. 29 N2 and 30 For N2, the liquid sample needs to be fully shaken until gas-liquid equilibrium is reached, and the upper gas is extracted for measurement; then the N2 content is calculated based on the sample volume and Henry's law.

[0009] (5) Identification of denitrification pathways: If nitrogen removal pathways are detected in experimental groups T1 and T2... 29 N2 indicates the presence of Anammox; if T1 detects it... 30 N2 indicates the presence of Feammox; if T2 is detected... 30 N2 indicates the presence of NDFO; if T3 is detected... 29 N2 indicates the presence of iron-autotrophic short-cut denitrification, which further provides NO2 as a substrate for anaerobic ammonium oxidation. - -N;

[0010] (6) Quantitative analysis of the contribution of the denitrification pathway:

[0011] Contribution of Feammox to ammonia nitrogen removal: Feammox (NH4) + -N)=

[0012] (1)V( 30 N2) / [V(29 N2)+V( 30 N2)]×100%

[0013] Anammox's contribution to ammonia nitrogen removal: Anammox (NH4) + -N)=100-

[0014] (2) Contribution Feammox (NH4 + -N)

[0015] Contribution of NDFO to nitrite removal: NDFO(NO2) - -N)=

[0016] (3)V( 30 N2) / [V( 29 N2)+V( 30 N2)]×100%

[0017] Anammox's contribution to nitrite removal: Anammox (NO2 - -N)=100-

[0018] (4) Contribution NDFO (NO2 - -N)

[0019] In the formula: V( 29 N2) refers to 29 N2 volume, ( 30 N2) refers to 30 N2 volume.

[0020] Innovation of this invention

[0021] (1) This invention addresses the differences in substrate utilization across different nitrogen conversion pathways by designing different... 15 The nitrogen isotope labeling scheme can accurately distinguish each denitrification pathway and accurately quantify the contribution of each pathway to nitrogen conversion by detecting the generation and distribution of labeled products.

[0022] (2) This invention can clearly reveal the nitrogen conversion mechanism of each reaction pathway in the denitrification process of the iron-enhanced anaerobic ammonium oxidation system, thus providing a scientific basis for the optimization of the denitrification process;

[0023] (3) It provides a new perspective for iron-enhanced anaerobic ammonium oxidation systems, which helps to understand the role mechanism of iron in the system and thus provides new ideas and methods for system optimization, such as adjusting the amount or form of iron added to further improve the denitrification efficiency of the system.

[0024] This invention introduces 15Nitrogen isotope labeling technology provides a precise tool for studying the denitrification mechanism of iron-enhanced anammox systems, effectively overcoming the limitations of traditional monitoring methods. Specifically, this invention can accurately distinguish and quantitatively analyze the contributions of different denitrification pathways such as Anammox, Feammox, and NDFO, thereby clarifying the activity thresholds and synergistic mechanisms of each pathway in the system. This not only helps to deepen the understanding of the multiple roles of iron in anammox systems but also provides a scientific basis for optimizing system operating conditions and improving denitrification efficiency. Furthermore, by accurately tracing the nitrogen transformation trajectory, this invention can reveal the details of substrate competition and electron transfer interactions in the complex iron-mediated reaction network, providing new ideas and methods for solving the problem of limited denitrification efficiency in practical applications. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the reaction apparatus used in the isotope labeling experiment according to an embodiment of the present invention;

[0026] In the diagram: 1--serum bottle; 2--three-hole filling cap; 3--sample injection and liquid phase collection port; 4--gas phase collection port; 5--helium bag connection port; 6--helium bag, connected to port 5;

[0027] Figure 2 This is experimental group T1 of the present invention. 29 N2 and 30 The change of N2 with reaction time (a) and the effect of Anammox and Feammox on NH4 + -N removal contribution (b);

[0028] Figure 3 This is the T2 experimental group of the present invention. 29 N2 and 30 The change of N2 with reaction time (a) and the effect of Anammox and NDFO on NO2 - -N removal contribution (b);

[0029] Figure 4 In the T3 experimental group (a) and T30 control group (b) of the present invention embodiment, 29 N2 and 30 Changes in N2 over reaction time. Detailed Implementation

[0030] (1) Stable operation of the iron-enhanced anaerobic ammonium oxidation system: The laboratory-scale iron-enhanced anaerobic ammonium oxidation system has been operating stably for 150 days, with a total nitrogen removal efficiency of 87.2±5.4% and a sludge concentration of 4125±14 mg / L; the operation process uses simulated wastewater, NH4 + -N and NO2 --N was provided by NH4Cl and NaNO2, with concentrations of 200±0.64 mg / L and 150±0.77 mg / L, respectively. A total of 575 mg / L Fe2O3 was added to the system initially, and the Fe was distributed in the sludge and not lost with the wastewater. Other macroelements included 0.05 g / L MgSO4·7H2O, 0.05 g / L CaCl2·2H2O, 1.0 g / L NaHCO3, and 0.027 g / L KH2PO4. In addition, 1.5 mL of trace elements was added per liter of simulated wastewater; the composition of the trace elements was: 5 g / L EDTA, 0.25 g / L CuSO4·5H2O, 0... The reactor contained 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. The reactor temperature was maintained at 30 ± 2℃, and the cycle time was 24 h, including: 10 min of water inlet, 23 h of stirring, 40 min of sedimentation, and 10 min of water discharge, with a water discharge ratio of 2 / 3.

[0031] (2) Pre-culture substrate consumption: Sludge from the iron-enhanced anaerobic ammonium oxidation system in the stable operation phase was washed three times with nitrogen-free simulated wastewater and then dispensed into serum bottles. Figure 1 The liquid and gas phases of oxygen and nitrogen were purged with helium for 30 minutes and sealed with a three-hole feeding bottle cap. The serum bottle containing the sediment was placed in a constant temperature shaker for 24 hours to consume the substrate remaining in the original system. The culture temperature was kept the same as the original system at 30℃.

[0032] (3) Isotope labeling scheme design: The substrate for anaerobic ammonium oxidation reaction includes NH4 + -N and NO2 - The introduction of -N and iron allows for the possible existence of Feammox and NDFO reaction pathways in the system, which oxidize NH4, respectively. + -N and reduction of NO2 - -N; Set up a T0 control group, adding only simulated wastewater; Fe is present in the sediment. 3+ Under these conditions, experimental group T1 was set up, with 133 mg / L added. 15 NH4 + -N and 100 mg / L 14 NO2 - -N is used as a substrate, through detection 29 N2 and 30 N2 variation and distribution, determining the effects of Anammox and Feammox on NH4 + The contribution of -N removal; a T2 experimental group was set up, using 133 mg / L 14 NH4 + -N and 100 mg / L15 NO2 - -N is used as a substrate, through detection 29 N2 and 30 N2 variation and distribution, determining the effects of Anammox and NDFO on NO2. - The contribution of -N removal; based on the results of experiments T1 and T2, nitrogen conservation calculations were performed to determine the NH4+ removed by the anaerobic ammonium oxidation reaction. + -N and NO2 - -N concentration, to determine the NO2 removed. - -N / NH4 + Whether -N conforms to the Anammox stoichiometry of 1.32, and thus determines the NO3 generated by Anammox and Feammox. - -Does N supplement Anammox with NO2 through iron-autotrophic short-pathway denitrification? - -N; Set up the T3 experimental group with a substrate concentration of 30 mg / L. 14 NH4 + -N, 40mg / L 15 NO3 - -N and 200 mg / L Fe 2+ To further verify the existence of an iron-autotrophic short-cut denitrification pathway, a T30 control group was set up, which only added... 14 NH4 + -N and 15 NO3 - -N was used for comparison with the T3 experimental group to clarify Fe 2+ Its specific role in the denitrification process; each experiment was conducted in triplicate;

[0033] (4) Experimental procedure: The simulated wastewater after helium purging was added to the pre-cultured sediment, and helium purging was continued for 30 minutes to remove oxygen and nitrogen from the liquid phase and headspace. Then, the mixture was sealed with a three-hole feed cap. Figure 1 One orifice is used to add substrate and collect liquid phase, another orifice is used to collect headspace gas, and a third orifice is used to connect a helium bag to equalize the gas pressure and maintain the anaerobic environment; NH4 is injected using a syringe. + -N, NO2 - -N, NO3 - -N, Fe 2+ The sodium bicarbonate concentrate was added to the serum vial through the sample well. At hours 0, 4, 4, 6, 10, and 24, 5 mL of headspace gas was added to a syringe and sealed with a rubber stopper. Simultaneously, 4 mL of the mud-water mixture was added to a 12 mL headspace vial. 0.1 mL of 7 mol / L ZnCl2 was added to the vial beforehand to terminate the reaction, and the vial was purged with helium for 3 min to remove oxygen and nitrogen. Isotope mass spectrometry was used to determine the concentration of the sample. 29 N2 and30 For N2, the liquid sample needs to be fully shaken until gas-liquid equilibrium is reached, and the upper gas is extracted for measurement; then the N2 content is calculated based on the sample volume and Henry's law.

[0034] (4) Calculation of results

[0035] No detection was found in the T0 control group. 29 N2 and 30 N2; T1 experimental group along the route 29 N2 and 30 N2 such as Figure 2 As shown in Figure a, the effects of Anammox and Feammox on NH4 at each reaction point are calculated according to formulas (1) and (2). + -N removal contribution ( Figure 2 b) T2 experimental group along the route 29 N2 and 30 N2 such as Figure 3 As shown in Figure a, the effects of Anammox and Feammox on NH4 at each reaction point are calculated according to formulas (3) and (4). + -N removal contribution ( Figure 3 b) Based on the results of experiments T1 and T2, perform nitrogen conservation calculations: NH4 removed by Anammox + -N and NO2 - -N were 101.5 and 88.6 mg / L, respectively, and the removed NO2 - -N and NH4 + The -N ratio was 0.81, lower than the theoretical value of 1.32, indicating that the NO3 generated by Anammox and Feammox... - -N may supplement NO2 through the iron autotrophic short-pathway denitrification pathway. - -N, thus becoming NO2 - Potential sources of -N; further generated based on the T3 experimental group. 29 N2 confirms that iron-autotrophic short-cut denitrification provides Anammox with NO2 as a substrate. - -N; while the T30 control group was almost undetectable in the early stages. 29 N2 is generated, but the Feammox action will reduce the inherent Fe in the system. 3+ Reduced to Fe 2+ Subsequently, during the 10-24 hour period, Anammox was also generated through iron autotrophic short-range denitrification coupled with nitrification. 29 N2;

[0036] Contribution Feammox (NH4 + -N)=V( 30 N2) / [V( 29 N2)+V( 30[N2)]×100% (1)

[0037] Contribution Anammox (NH4 + -N)=100-Contribution Feammox (NH4 + -N) (2)

[0038] Contribution NDFO (NO2 - -N)=V( 30 N2) / [V( 29 N2)+V( 30 [N2)]×100% (3)

[0039] Contribution Anammox (NO2 - -N)=100-Contribution Anammox (NO2 - -N) (4)

[0040] In the formula: V( 29 N2) refers to 29 N2 volume, ( 30 N2) refers to 30 N2 volume.

Claims

1. A method of utilizing 15 A method for identifying and quantifying nitrogen conversion pathways in iron-enhanced anaerobic ammonia oxidation systems using nitrogen isotope labeling technology, characterized in that... The specific steps are as follows: (1) Sample collection: The homogenized mud-water mixture of the iron-enhanced anaerobic ammonium oxidation system was collected in sterile plastic sampling bags and transported to the laboratory under refrigeration; (2) Pre-culture substrate consumption: Sludge from the iron-enhanced anaerobic ammonium oxidation system was washed with simulated wastewater and then dispensed into serum bottles. Helium was used to purge the liquid and gas phases to remove oxygen and nitrogen, and the bottles were sealed with three-hole feeding caps. The simulated wastewater consisted of 0.05 g / L MgSO4·7H2O, 0.05 g / L CaCl2·2H2O, 1.0 g / L NaHCO3, and 0.027 g / L KH2PO4. In addition, 1.5 mL of trace element solution was added per liter of simulated wastewater, consisting of 5 g / L... EDTA, 0.25 g / L CuSO4·5H2O, 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; serum bottles containing bottom sediment were placed in a constant temperature shaker and incubated for 24 h. (3) Isotope labeling scheme design: Anammox reaction substrates include NH4 + -N and NO2 - The introduction of -N and iron allows for the possible existence of Feammox and NDFO reaction pathways in the system, which oxidize NH4, respectively. + -N and reduction of NO2 - -N; Set up a T0 control group, adding only simulated wastewater; Fe was present in the bottom sediment of the experimental group. 3+ Under the condition of adding different substrates: T1 uses 15 NH4 + -N and 14 NO2 - -N is used as the substrate; T2 uses 14 NH4 + -N、 15 NO2 - -N and Fe 2+ As a substrate; T3 substrate is 14 NH4 + -N、 15 NO3 - -N and Fe 2+ ; (4) Experimental Procedure: Simulated wastewater purged with helium was added to the pre-cultured sediment. Helium was used to purge the liquid phase and headspace to remove oxygen and nitrogen, followed by sealing with a three-hole feed cap. One hole was used to add substrate and collect the liquid phase, another hole was used to collect headspace gas, and the third hole was used to connect a helium bag to balance the pressure and maintain the anaerobic environment. NH4 was injected using a syringe. + -N, NO2 - -N, NO3 - -N, Fe 2+ Sodium bicarbonate concentrate was added to the serum vial through the sample well; 5 mL of headspace gas was added to a syringe at intervals, sealed with a rubber stopper, and 4 mL of mud-water mixture was added to a 12 mL headspace vial. 0.1 mL of 7 mol / L ZnCl2 was added to the vial beforehand to terminate the reaction, and the vial was purged with helium to remove oxygen and nitrogen; the reaction was then analyzed using an isotope mass spectrometer. 29 N2 and 30 For N2, the liquid sample needs to be fully shaken until gas-liquid equilibrium is reached, and the upper gas is extracted for measurement; then the N2 content is calculated based on the sample volume and Henry's law. (5) Identification of denitrification pathways: If nitrogen removal pathways are detected in experimental groups T1 and T2... 29 N2 indicates the presence of Anammox; if T1 detects it... 30 N2 indicates the presence of Feammox; if T2 is detected... 30 N2 indicates the presence of NDFO; if T3 is detected... 29 N2 indicates the presence of iron-autotrophic short-cut denitrification, which further provides NO2 as a substrate for anaerobic ammonium oxidation. - -N; (6) Quantitative analysis of the contribution of the denitrification pathway: Contribution of Feammox to ammonia nitrogen removal: Feammox (NH4) + -N)=V( 30 N2) / [V( 29 N2)+V( 30 [N2)]×100% (1) Anammox's contribution to ammonia nitrogen removal: Anammox (NH4) + -N)=100-Contribution Feammox (NH4 + -N) (2) Contribution of NDFO to nitrite removal: NDFO(NO2) - -N)=V( 30 N2) / [V( 29 N2)+V( 30 [N2)]×100% (3) Anammox's contribution to nitrite removal: Anammox (NO2 - -N)=100-Contribution NDFO (NO2 - -N)(4) In the formula: V( 29 N2) refers to 29 N2 volume, ( 30 N2) refers to 30 N2 volume.

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