CN113063922A - Culture device for determining nitrate conversion of flooded system based on MIMS method and application - Google Patents
Culture device for determining nitrate conversion of flooded system based on MIMS method and application Download PDFInfo
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- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 title description 6
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Abstract
A culture apparatus for measuring nitrate radical conversion of a flooded system based on MIMS method and application thereof, the apparatus comprises a cavity for simulating in-situ culture and a water inlet and outlet device; the cavity for simulating in-situ culture consists of an inner cavity and an outer cavity, the top of the inner cavity is provided with a speed-adjustable motor, and a triple magnetic rod connected with a speed-adjustable electrode is arranged in the inner cavity; the outer cavity is used for containing the overlying water and is internally provided with a sediment sampler; the water inlet and outlet device is a water inlet pipe and a water outlet pipe which are arranged at the top end of the sediment sampler, wherein the water inlet pipe is connected with the supply bottle; the water outlet pipe is used for sampling; the water inlet and outlet device is provided with a valve for controlling the flow speed. The device establishes a closed culture system which can be used for simulating in-situ culture conditions and realizing the culture without adding15The N marker is used for directly measuring the net denitrification rate in the system, and under the condition of not adding an external isotope nitrogen source, the N in the underestimation or overestimation flooding system is avoided to a great extent2The rate of generation of (c).
Description
Technical Field
The invention belongs to the field of a nitrogen conversion process in a flooding environment, and particularly relates to a culture device for determining nitrate conversion of a flooding system based on an MIMS method and application thereof.
Background
The denitrification process being activated Nitrogen (NO)3 -) Finally using inert nitrogen (N)2) The most main way for the form to leave the soil, water body and other internal organisms to circularly return to the atmosphere. Denitrification has been considered the only route to denitrification processes in the past, and until the 90's of the last century, the discovery of the Anammox process broke this traditional belief, Anammox (Anammox) refers to the anaerobic condition of the Anammox bacteria to produce NO3 -/NO2 -As an electron acceptor, reacting NH4 +Oxidation to N2The final product of (1) is also N2. The previous research methods related to the denitrification process of a flooding system mainly comprise an acetylene inhibition method, a difference method and15and (4) N-labeled tracing. The methods have certain problems when being practically applied to a flooding system: for example, the acetylene inhibition method is simple and quick, has low cost, but can seriously underestimate the soil denitrification rate, and is particularly not suitable for a flooded environment because acetylene is slowly diffused in the flooded environment; in the calculation process of the difference method, all unknown nitrogen fate is accumulated in the denitrification process, so that the result variation and uncertainty are large.15The N-tracer method is characterized by isotopic fractionation and labeling15N substrate and substrate sludge or soil itself15N is difficult to mix uniformly, and the obtained result can only qualitatively explain the destination of nitrogen but cannot accurately quantify the occurrence rate of various nitrogen conversion processes.
Currently used for flooding system N in international range2The method for measuring the content is N2the/Ar method, the basic assumption of which is: ar is inert gas, does not participate in biological reaction such as denitrification process, has the solubility in a flooding system only influenced by temperature and salinity, has very stable concentration, and therefore N2The change of the ratio of Ar to N can be directly reflected2Can be determined by measuring N in the water sample2The value of/Ar is calculated, and the dissolved state N in the water sample is calculated2The concentration of (c). Kana et al earlier applied MIMS method to flooding system N2Determination of/Ar, which is based onThe application environment is a marine water body, but the specific denitrification process cannot be distinguished. Furthermore, N is performed using MIMS2The key of the determination is to establish a relatively complex and precise closed culture system and prepare a standard water sample which is basically consistent with the environmental condition to be detected. Based on the above, the invention provides a Flow-through-based closed culture system, which can simulate in-situ culture conditions and can be used for being combined with15N-IPT technology is combined to distinguish and measure the contribution and occurrence potential of a specific denitrification process.
Disclosure of Invention
The technical problem to be solved is as follows: measuring N for MIMS2The difficult problems of establishing a complex and precise closed culture system and preparing a standard water sample consistent with the environmental condition to be detected are solved. The invention provides a culture device for determining nitrate radical conversion of a flooded system based on an MIMS method and application thereof, which can simulate in-situ culture conditions to the maximum extent and realize the effect of no addition of nitrate radical15N marker and directly determining the net denitrification rate in the system. Simultaneously through and15the respective contribution and occurrence potentials of denitrification and Anammox can also be distinguished and measured by using the N-IPT.
The technical scheme is as follows: a culture apparatus for measuring nitrate radical conversion of a flooded system based on MIMS method comprises a cavity for simulating in-situ culture and a water inlet and outlet device; the cavity for simulating in-situ culture consists of an inner cavity and an outer cavity, the top of the inner cavity is provided with a speed-adjustable motor, and a triple magnetic rod connected with a speed-adjustable electrode is arranged in the inner cavity; the outer cavity is used for containing the overlying water and is internally provided with a sediment sampler; the water inlet and outlet device is a water inlet pipe and a water outlet pipe which are arranged at the top end of the sediment sampler, wherein the water inlet pipe is connected with the supply bottle; the water outlet pipe is used for sampling; the water inlet and outlet device is provided with a valve for controlling the flow speed.
The device is applied to determination of net denitrification, denitrification and anaerobic ammonia oxidation of a flooded system.
The application steps are as follows: a. sampling: collecting undisturbed sediment/soil with the depth of 5-15 cm by using a sediment sampler, sealing the bottom of the sampler by using a rubber plug, collecting 20L of in-situ cover water or field surface water, and storing the water at low temperature by using an ice bag for subsequent culture;b. culturing: pouring in-situ overlying water or field surface water into an outer cavity of the device, vertically placing a sampler filled with an undisturbed sediment/soil sample into the device, immersing and standing for 8-12 h, wherein the water surface is 4-8 cm higher than the sampler, ventilating overlying water in the culture device by using a ventilating pump to ensure that the dissolved oxygen content of the overlying water in the culture device is consistent with the in-situ environment, and adjusting the temperature of the overlying water to be consistent with the actual water temperature during field sampling; before the sample to be detected is collected, one end of the water inlet pipe is connected to a supply bottle filled with the overlying water, and the supply bottle is placed at a higher position so that the overlying water in the bottle flows out under the action of siphon; in the whole operation process, no bubbles are generated in the closed culture column, and if bubbles exist in the culture device, the operation is required to be carried out again according to the flow; after the requirements are met, the speed-adjustable motor is started, the rotating speed of the motor is adjusted to control the rotating speed of the triple magnetic rod inside, and the rotation of the triple magnetic rod can drive the small magnetic rotor connected below each sampler cover in the outer cavity to rotate; collecting operation of a sample to be detected: after the culture system is debugged and the sediment/soil is kept stand, immediately collecting a water sample as a sample at 0h, and then collecting the overlying water at intervals, wherein each sampling point is provided with three repetitions; when sampling, firstly opening the water inlet valve to allow the water sample to flow out, discharging air at the front part of the water inlet pipe, then extending the water outlet pipe into the bottom of the sampling bottle to allow the overlying water to be injected into the sampling bottle until the overlying water overflows, then drawing out the water outlet pipe, and immediately sealing the sampling bottle; after sampling, 200 microliter of saturated ZnCl is added into a sampling bottle filled with a water sample by using a half micro-sampling needle2The microbial reaction was terminated and stored in a refrigerator at 4 ℃.
The determination method of the net denitrification rate comprises the following steps: determination of dissolved N in samples by MIMS2The concentration is linearly regressed with the culture time, and the slope of the regression line is the N in the sediment/soil overlying water2Rate of change of concentration in units of μmol N2 L-1h-1Further combining the dry weight of sediment/soil, unit g, with the volume of overlying water, unit L, the net denitrification rate (mu mol N g) can be obtained by conversion-1h-1)
In the formula 1, R is the net denitrification rate, mu mol N g-1h-1；SN2Is N2Rate of change in concentration of,. mu. mol N2 L-1h-1(ii) a V is the overlying water volume, L; w is the dry soil weight, g.
The determination method of denitrification and anaerobic ammonia oxidation comprises the following steps: firstly, preparing a sediment/soil slurry culture system or an undisturbed sediment/soil and field surface water closed culture system, and carrying out pre-culture for 1-7 d to exhaust background NO in the system3 -And O2(ii) a Then, the pre-cultured soil columns are divided into 3 groups, and the following operations are respectively carried out: adding 100 mu M of the product with the abundance of 99.5% into the system15NH4 +(ii) a ② adding 100 mu M abundance 99.5 percent into the system15NH4 +And 100. mu.M14NO3 -(ii) a ③ adding 99.3 percent of abundance into the system15NO3 -(ii) a Among them, the treatment (i) is a negative control group for the purpose of verifying background NO in the system after preculture3 -Whether the consumption is complete; if the anaerobic conditions required for the experiment are reached, no observation is made30N2A significant accumulation of (c); treatment II is a positive control group, the purpose is to verify whether the anaerobic ammonia oxidation process occurs, if the anaerobic ammonia oxidation process exists, the anaerobic ammonia oxidation process can be observed29N2A significant accumulation of (c); the third treatment is a normal treatment group which is used for measuring the N generated by denitrification and anaerobic ammonia oxidation in the system2An amount; according to the requirement of processing the third step, after adding isotope labels, immediately collecting the water sample as a sample at 0h, then sampling at intervals, repeating 3 times, and determining different components N in the water sample during the culture period based on the MIMS method2Variation of dissolved amount and use of N2The random pairing principle of the generation process calculates the rates of denitrification and Anammox, and the formula is as follows:
d in formula 2 and formula 3totalAnd AtotalN produced by denitrification and anammox in the sample2Amount, unit μmol N2 L-1；P29And P30In systems separately measured for MIMS29N2And30N2amount of (d) in units of. mu. mol N2 L-1；FnFor adding isotope into the system15NO3 -Is NO3 -The ratio of the total concentration; each sample N thus measured2Performing linear regression on the yield and the time point and combining a random pairing principle to obtain the denitrification and anaerobic ammonia oxidation generation potentials of the sample; assuming that no other denitrification processes exist in the system, the respective denitrification contributions of denitrification and anammox can be calculated according to the denitrification and anammox rates.
Has the advantages that: the device establishes a closed culture system which can be used for simulating in-situ culture conditions and realizing the culture without adding15The N marker is used for directly measuring the net denitrification rate in the system, and under the condition of not adding an external isotope nitrogen source, the N in the underestimation or overestimation flooding system is avoided to a great extent2The rate of generation of (c). And by reaction with15N-IPT combination, the culture system is pre-cultured, and N generated in the system is measured under three groups of experimental treatments2Can distinguish and determine the respective contribution and occurrence potentials of denitrification and Anammox.
Drawings
FIG. 1 is a schematic view of a culture apparatus;
FIG. 2 clean denitrification process producing soluble N2A schematic diagram;
FIG. 3 net denitrification rates for three paddy soils;
FIG. 415The culture process of the N-IPT method is shown schematically;
FIG. 5 the denitrification and Anammox potentials of the six rice soils.
Detailed Description
Example 1
This example will provide a MIMS based culture device for net denitrification, denitrification and Anammox in flooded systems.
Referring to fig. 1 and fig. 2, the culture device for measuring the net denitrification, denitrification and Anammox of the wetland and paddy soil in the embodiment is shown, which comprises a cavity for simulating in-situ culture and a water inlet and outlet system (fig. 1); in the embodiment, the culture cavity is divided into an inner cavity and an outer cavity, the inner cavity consists of a speed-adjustable motor and a triple magnetic rod, the motor in the culture device is started, the inner triple magnetic rod is rotated at a proper speed, and the rotation of the magnetic rod can drive a small and medium-sized magnetic rotor of a non-disturbance sediment sampler to uniformly mix dissolved gas (shown in figure 2) generated in the culture process and the flow of a simulated water body; the outer cavity is mainly used for accommodating a plurality of undisturbed sediment samplers and covering water, the undisturbed sediment samplers are made of PVC hollow cylinders, and the inner diameter, the outer diameter and the height of each undisturbed sediment sampler are 8cm, 9 cm and 27cm respectively. After the sampler collects sediment/soil, the bottom of the sampler is sealed by a rubber plug and is vertically placed in an outer cavity filled with in-situ upper cover water. And (4) moderate ventilation is carried out, so that the water dissolved oxygen on the outer cavity is kept consistent with the in-situ water flooding environment as far as possible, and the temperature of the overlying water is adjusted to the actual water temperature during field sampling. The water inlet and outlet system in this embodiment is a rubber hose installed on the top of the undisturbed sediment sampler for water inlet and outlet. One end of the water inlet pipe is connected with a supply bottle (which supplies the upper water lost in the later sampling of the culture column through the siphon action) arranged at a higher position, and the water inlet can be regulated by a water stop clamp; the outlet pipe is used for the later stage sample, and the stagnant water presss from both sides and is used for controlling the velocity of flow of water.
The embodiment can not only realize no addition based on the MIMS method15Direct determination of net denitrification rate in the system by N-labeling, and the like15The N-IPT technology is used for jointly measuring the occurrence potentials of denitrification and Anammox and distinguishing the respective contributions of denitrification and Anammox to the denitrification process.
Example 2
The embodiment will utilize the device of embodiment 1 to show a specific implementation effect of a clean denitrification culture device based on MIMS determination flooding system, which is as follows:
collecting 0-20cm of surface layer paddy soil of Changchun Jilin and Jiangxi Yingtan, culturing by a Flow-through culture device, and determining N in a sample2The concentration of the water is obtained to obtain the net denitrification rate of the rice soil.
1) Earth pillar Assembly and Pre-culture
Weighing 250g of fresh rice soil passing through a 2mm sieve, adding the fresh rice soil into a non-disturbance sediment sampler, adding deionized water to fill the sampler, sealing the bottom of the sampler by using a rubber plug, checking whether a closed culture column generates bubbles, vertically placing the sampler into a Flow-through filled with deionized water after confirming that no bubbles exist, and immersing for pre-culture for 1-7 d. And (3) properly ventilating the upper water in the culture device by using a ventilation pump, and adjusting the temperature to be consistent with the actual water temperature sampled in the field.
2) Cultivation process
When the beginning sample, at first close the breather pump, stretch to the lid of cultivateing the post and screw up under water, the lid below is provided with rubber seal, can guarantee its leakproofness, cultivates the lid of post and screws up the back, begins to connect inlet tube and outlet pipe, places the supply bottle (for the upper covering water of the loss of sample in the supply cultivateing the post) that links to each other with the inlet tube in higher position. The operation process needs to ensure that no air bubbles are generated in the closed culture column, and if the air bubbles are generated, the operation needs to be carried out again according to the flow until the requirements are met. Then, the motor in the culture device is started, the proper speed is adjusted to rotate the triple magnetic rod inside, and the rotation of the magnetic rod can correspondingly drive the culture column cover to be connected with the rotation of the small-sized magnetic rotor, so that the dissolved gas generated in the culture process can be uniformly mixed with the flow of simulated river water, and the determination of the net denitrification rate more conforming to the in-situ condition is facilitated.
3) Sample collection
After the culture system is debugged and the sediment/soil is stood, immediately collecting a water sample as a sample at the time of 0h, and then sampling at the time of 6 h, 18 h, 24 h and 30h respectively, wherein each sampling point is repeated in three. During sampling, no air bubbles are generated in the sampling bottle, and if the air bubbles exist, the sampling needs to be carried out again. While taking a sampleThe water stopping clamp of the water inlet pipe is firstly opened, a part of water sample flows out (air in the front of the water inlet pipe is discharged), then the water outlet pipe stretches into the bottom of the sampling bottle, the flow rate of the water outlet pipe can be adjusted through the water stopping clamp, so that the overlying water is slowly injected into the sampling bottle until overflowing, the water outlet pipe is slowly pumped out, the cover of the sampling bottle is immediately screwed down, and the air pollution to the water sample is avoided. After sampling, 200 mu L of saturated ZnCl needs to be added into a sampling bottle filled with a water sample by using a semi-micro sample injection needle2The microbial reaction was terminated and stored in a refrigerator at 4 ℃.
4) Sample determination and calculation
Determination of end product N in net denitrification process based on MIMS method after sample collection2The dissolution concentration in water is linearly regressed with the culture time, and the slope of the regression line is the N in the water covering the sediment or the soil column2Rate of change of concentration (. mu. mol N)2 L-1h-1) Further combining the dry weight (g) of soil in the soil column with the volume (L) of overlying water, the net denitrification rate (mu mol N g) can be converted-1h-1)
In the formula 1, R is the net denitrification rate, mu mol N g-1h-1；SN2Is N2Rate of change of concentration,. mu.mol N2 L-1h-1(ii) a V is the overlying water volume, L; w is the dry soil weight, g.
From FIG. 3, it can be seen that N is present in the culture system2The concentration has good linear relation with the time, and the net denitrification rates of the black soil, the red sandy soil and the red clay are respectively 1.02 +/-0.28, 1.44 +/-0.07 and 2.15 +/-0.36 nmol N g-1h-1. Former collected undisturbed soil samples from rice field by PVC column and carried out urea mixed application and surface application treatment indoors to obtain measured N2The generation rate is 0.92 to 1.66nmol N g-1h-1This is consistent with the experimentally measured net denitrification rate over a range of values, indicating that the Flow-through system can be simulated in situ by indoor simulationThe situation reflects a net denitrification rate that approximates the in situ case.
Example 3
In this example, the device of example 1 is used to demonstrate the specific implementation effect of a device for measuring denitrification and Anammox culture of a flooding system based on MIMS, which is specifically as follows:
similar to the column assembly and preculture and culture procedure in example 2. Dividing the soil columns after the pre-culture into 3 groups, and respectively carrying out the following operations: adding 100 μ M to the system15NH4 +(abundance 99.5%); ② add 100 μ M to the system15NH4 +(abundance 99.5%) and 100. mu.M14NO3 -(ii) a Adding to the system15NO3 -(abundance 99.3%). Among them, the treatment (i) is a negative control group for the purpose of verifying background NO in the system after preculture3 -Whether it has been completely consumed. If the anaerobic conditions required for the experiment are reached, no observation is made30N2A significant accumulation of (c); treatment (positive control) for the purpose of verifying whether the Anramox process occurred, if it existed, it was observed29N2A significant accumulation of (c); the processing unit is a normal processing group which is used for measuring the N generated by denitrification and Anammox in the system2Amount of the compound (A). According to the requirement of processing the third step, after adding isotope labeling, immediately collecting a water sample as a sample at the time of 0h, then respectively sampling at the time of 6 h, 18 h, 24 h and 30h, repeating 3 times each time, and determining different components N in the water sample during the culture period based on the MIMS method2Variation of dissolved amount and use of N2The stochastic pairing principle of the generation process calculates the occurrence potential of denitrification and Anammox. The formula is as follows:
d in formula 2 and formula 3totalAnd AtotalN generated for denitrification and Anammox in samples respectively2Amount, μmol N2L-1；P29And P30In systems separately measured for MIMS29N2And30N2amount of (d), μmol N2 L-1；FnFor the addition of NO3 -In15NO3 -The ratio of (a) to (b).
The experimental soil is 0-20cm of surface soil under a rice planting system of a normal-maturing agricultural ecological experimental station, and six treatments of the experiment are astragalus sinicus returning, astragalus sinicus returning and nitrogen fertilizer returning, broad bean straw returning and nitrogen fertilizer returning, rice and wheat crop rotation and single cropping rice respectively.
In the six treated paddy soils (FIG. 5), the denitrification and Anammox rates ranged from (1.01. + -. 0.35) to (1.53. + -. 0.42) and (-0.09. + -. 0.10) to (0.05. + -. 0.04) nmol N g-1h-1. The contribution of Anammox to the denitrification process is low compared to the denitrification potential, and for straw-only return-to-field treatments, application of nitrogen fertilizer significantly increases the denitrification potential.
Experiments of examples 2 and 3 completely show that the Flow-through device of the invention can not only simulate in-situ culture conditions to the maximum extent and obtain denitrification rate approximate to the in-situ condition, but also can determine respective contributions of occurrence potential and specific denitrification processes.
Although the invention has been described in detail above with reference to specific embodiments and illustrative embodiments, it is apparent that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed. The invention provides important technical and method support for promoting the research of the soil nitrogen cycle process of the flooding ecosystem.
Claims (5)
1. A culture apparatus for measuring nitrate radical conversion of a flooded system based on MIMS method is characterized in that the apparatus comprises a cavity for simulating in-situ culture and a water inlet and outlet device; the cavity for simulating in-situ culture consists of an inner cavity and an outer cavity, the top of the inner cavity is provided with a speed-adjustable motor, and a triple magnetic rod connected with a speed-adjustable electrode is arranged in the inner cavity; the outer cavity is used for containing the overlying water and is internally provided with a sediment sampler; the water inlet and outlet device is a water inlet pipe and a water outlet pipe which are arranged at the top end of the sediment sampler, wherein the water inlet pipe is connected with the supply bottle; the water outlet pipe is used for sampling; the water inlet and outlet device is provided with a valve for controlling the flow speed.
2. Use of the apparatus of claim 1 for determining net nitrogen removal, denitrification and anammox in a flooded system.
3. Use according to claim 2, characterized by the steps of: a. sampling: collecting undisturbed sediment/soil with the depth of 5-15 cm by using a sediment sampler, sealing the bottom of the sampler by using a rubber plug, collecting 20L of in-situ cover water or field surface water, and storing the water at low temperature by using an ice bag for subsequent culture; b. culturing: pouring in-situ overlying water or field surface water into an outer cavity of the device, vertically placing a sampler filled with an undisturbed sediment/soil sample into the device, immersing and standing for 8-12 h, wherein the water surface is 4-8 cm higher than the sampler, ventilating overlying water in the culture device by using a ventilating pump to ensure that the dissolved oxygen content of the overlying water in the culture device is consistent with the in-situ environment, and adjusting the temperature of the overlying water to be consistent with the actual water temperature during field sampling; before the sample to be detected is collected, one end of the water inlet pipe is connected to a supply bottle filled with the overlying water, and the supply bottle is placed at a higher position so that the overlying water in the bottle flows out under the action of siphon; in the whole operation process, no bubbles are generated in the closed culture column, and if bubbles exist in the culture device, the operation is required to be carried out again according to the flow; after the requirements are met, the speed-adjustable motor is started, the rotating speed of the motor is adjusted to control the rotating speed of the triple magnetic rod inside, and the rotation of the triple magnetic rod can drive the small magnetic rotor connected below each sampler cover in the outer cavity to rotate; collecting operation of a sample to be detected: after the culture system is debugged and the sediment/soil is kept stand, immediately collecting a water sample as a sample at 0h, and then collecting the overlying water at intervals, wherein each sampling point is provided with three repetitions; getWhen in sampling, firstly opening the water inlet valve to allow the water sample to flow out, discharging air at the front part of the water inlet pipe, then extending the water outlet pipe into the bottom of the sampling bottle to allow the overlying water to be injected into the sampling bottle until the overlying water overflows, then drawing out the water outlet pipe, and immediately sealing the sampling bottle; after sampling, 200 microliter of saturated ZnCl is added into a sampling bottle filled with a water sample by using a half micro-sampling needle2The microbial reaction was terminated and stored in a refrigerator at 4 ℃.
4. Use according to claim 2, characterized in that the net denitrification rate is determined by: determination of dissolved N in samples by MIMS2The concentration is linearly regressed with the culture time, and the slope of the regression line is the N in the sediment/soil overlying water2Rate of change of concentration in units of μmol N2 L-1h-1Further combining the dry weight of sediment/soil, unit g, with the volume of overlying water, unit L, the net denitrification rate (mu mol N g) can be obtained by conversion-1h-1)
In the formula 1, R is the net denitrification rate, mu mol N g-1h-1；
Is N2Rate of change in concentration of,. mu. mol N2 L-1h-1(ii) a V is the overlying water volume, L; w is the dry soil weight, g.
5. The use according to claim 2, characterized in that the denitrification and anammox assay is performed by: firstly, preparing a sediment/soil slurry culture system or an undisturbed sediment/soil and field surface water closed culture system, and carrying out pre-culture for 1-7 d to exhaust background NO in the system3 -And O2(ii) a Then, the pre-cultured soil columns are divided into 3 groups, and the following operations are respectively carried out: adding 100 mu M of the product with the abundance of 99.5% into the system15NH4 +(ii) a ② adding 100 mu M abundance 99.5 percent into the system15NH4 +And 100. mu.M14NO3 -(ii) a ③ adding 99.3 percent of abundance into the system15NO3 -(ii) a Among them, the treatment (i) is a negative control group for the purpose of verifying background NO in the system after preculture3 -Whether the consumption is complete; if the anaerobic conditions required for the experiment are reached, no observation is made30N2A significant accumulation of (c); treatment II is a positive control group, the purpose is to verify whether the anaerobic ammonia oxidation process occurs, if the anaerobic ammonia oxidation process exists, the anaerobic ammonia oxidation process can be observed29N2A significant accumulation of (c); the third treatment is a normal treatment group which is used for measuring the N generated by denitrification and anaerobic ammonia oxidation in the system2An amount; according to the requirement of processing the third step, after adding isotope labels, immediately collecting the water sample as a sample at 0h, then sampling at intervals, repeating 3 times, and determining different components N in the water sample during the culture period based on the MIMS method2Variation of dissolved amount and use of N2The random pairing principle of the generation process calculates the rates of denitrification and Anammox, and the formula is as follows:
d in formula 2 and formula 3totalAnd AtotalN produced by denitrification and anammox in the sample2Amount, unit μmol N2 L-1；P29And P30In systems separately measured for MIMS29N2And30N2amount of (d) in units of. mu. mol N2 L-1；FnFor adding isotope into the system15NO3 -Is NO3 -The ratio of the total concentration; each sample N thus measured2Performing linear regression on the yield and the time point and combining a random pairing principle to obtain the denitrification and anaerobic ammonia oxidation generation potentials of the sample; assuming that no other denitrification processes exist in the system, the respective denitrification contributions of denitrification and anammox can be calculated according to the denitrification and anammox rates.
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