CN112881506A - Method for measuring speed in process of dissimilatory reduction of nitrate radical in rice field soil into ammonium - Google Patents

Abstract

A method for measuring the process rate of dissimilatory reduction of nitrate to ammonium in rice field soil includes preparing slurry, isotopic marking and measuring the process rate of DNRA. The method utilizes K¹⁵NO₃For the tracer, strong oxidant is used to oxidize the ammonium nitrogen formed during DNRA process in sealed anaerobic culture system into ammonium nitrogen²⁹N₂And³⁰N₂then in a quantitative system by using a membrane sample introduction mass spectrometer¹⁵N signal strength (2)³⁰N₂+²⁹N₂) And determining the concentration of ammonium in the sample to be detected through a standard curve. And obtaining the speed of the DNRA process of the paddy field soil according to the linear fitting slope of the ammonium concentration change at three culture moments in the system. The method overcomes the defects of complex operation and easy error generation of the conventional method, and can quickly and accurately realize the direct determination of the DNRA process speed of the paddy field soil.

Description

Method for measuring speed in process of dissimilatory reduction of nitrate radical in rice field soil into ammonium

Technical Field

The invention belongs to the field of research on anaerobic nitrogen conversion processes of rice fields, and particularly relates to a method for measuring the rate of a process of dissimilatory reduction of nitrate radicals in rice fields into ammonium.

Background

The nitrate dissimilatory reduction to ammonium (DNRA) process refers to NO in media such as soil, water and sludge₃ ^-Is dissimilatorily reduced into NH by microorganisms under anaerobic conditions₄ ⁺The process of (1). In the thirties of the last century, DNRA was first discovered in pure culture systems of Clostridium wegener, and until 1975, studies have shown that the DNRA process can occur in systems of soil, sediments, and sludge. At present, most of the research on DNRA focuses on ocean and estuary sediments, and the research on DNRA in a paddy field soil system is relatively lacked, so that the DNRA is desirable in agriculture considering that the process is favorable for the retention of nitrogen in soil, and is worthy of intensive research. At present, the method for measuring the DNRA process rate is mainly based on¹⁵N isotope labeling and Gas Chromatography (GC) -Isotope Ratio Mass Spectrometry (IRMS), the method needs a complex degassing step, the operation is complicated, the error is large, the sample amount required in the determination process is large, the determination time is long, the efficiency is low, and a new method is urgently needed to be developed.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the defects of complex operation, easy error generation and low efficiency of the traditional method, the invention provides a method for measuring the speed of the process of dissimilatory reduction of nitrate radical in rice field soil into ammonium.

The technical scheme is as follows: a method for measuring the speed of the process of dissimilatory reduction of nitrate radical into ammonium in rice field soil comprises the following steps: (1) preparing slurry: mixing a fresh soil sample screened by a 2mm sieve with water according to a mass ratio of 1:7 to prepare slurry, filling helium for 30min, then respectively filling the slurry subjected to helium exposure treatment into 12mL headspace bottles in an anaerobic helium environment, screwing bottle caps, ensuring that no bubbles exist in the bottles, and then placing the slurry in a rotary incubator to pre-culture in a temperature and dark manner until nitrate radicals in the sample are exhausted; (2) isotope labeling: under anaerobic environment, a micro-sampling needle is used to puncture a bottle cap spacer, and 0.2mL of 6 mmol.L is added to the slurry sample after the pre-culture is finished^{-1 15}N-KNO₃Wherein¹⁵The N abundance is 99 percent, and the equal volume of slurry is discharged by using a needle head and is added¹⁵N-KNO₃The slurry sample is placed at the speed of 6-9 r.min^-1Rotary incubatorCulturing at room temperature in dark place; (3) determination of the process rate of dissimilatory reduction of nitrate to ammonium (DNRA) in rice field soil: after culturing for 0, 3 and 6 hours, 200. mu.L of 7 mol. multidot.L was injected into the culture system using a micro-needle^-1Terminating the culture by using zinc chloride, transferring the slurry into a 100mL wide-mouth bottle, filling helium for 30-60 min again, and discharging the sludge generated by denitrification and anaerobic ammonia oxidation²⁹N₂、³⁰N₂Then subpackaging the slurry into 12mL headspace bottles again, adding 0.2mL oxidant into the subpackaged slurry sample, shaking uniformly, and oxidizing ammonium nitrogen formed in the DNRA process into ammonium nitrogen²⁹N₂And³⁰N₂then, all samples are put at 1500-2000 r.min^-1Centrifuging at a speed of 5-8 min, and finally determining the dissolved N in the supernatant by using a membrane sample introduction mass spectrometer₂Isotope signal intensity; (4) standard curve plotting and DNRA process rate calculation: respectively preparing 0, 2, 4, 6, 8, 10, 20 and 50 mu mol.L^-1Is/are as follows¹⁵NH₄Cl solution of wherein¹⁵The N abundance is 99.5 percent, the N abundance is distributed into 12mL headspace bottles, and 0.2mL oxidant is added into each headspace bottle by a micro-sampling needle¹⁵NH₄ ⁺Oxidation to N₂Said N is₂Is composed of²⁹N₂And³⁰N₂determination of water by MIMS¹⁵N signal strength of said¹⁵N signal strength equals to two times³⁰N₂Signal strength plus²⁹N₂Signal strength, in 2³⁰N₂+²⁹N₂Expression, use of the obtained¹⁵N signal intensity and¹⁵NH₄standard curves were plotted for Cl concentration, with three replicates per concentration set; in the actual slurry sample¹⁵NH₄ ⁺The concentration was determined, in part, by plotting a standard curve, and the rate of DNRA progress in the sample was calculated as follows:

wherein R is_DNRAFor the rate of the DNRA process, the unit nmol g^-1·h^-1；

Is composed of¹⁵NH₄ ⁺The concentration and the culture time are linearly regressed to obtain the slope, and the slope is¹⁵NH₄ ⁺Production rate of (D) in nmol. mL^-1·h^-1(ii) a V is the volume of the headspace bottle, in mL; w is the dry soil weight in g.

The oxidant is sodium hypobromite iodine solution.

Has the advantages that: the method overcomes the defects of complex operation, easy error generation and low efficiency of the conventional method, and can realize the rapid and accurate determination of the DNRA process speed of the paddy field soil by adopting an isotope tracing means based on MIMS. The method can quickly and accurately measure the DNRA process speed under the treatment of different carbon-nitrogen ratios of the paddy soil, has simple and convenient operation flow and lower standard error, and can realize batch treatment of samples.

Drawings

FIG. 1 is a flowchart of the operation of a DNRA process rate determination based on MIMS;

FIG. 2 is a schematic isotopic representation; 1: micro-sampling needle, 2: needle head, 3: a headspace bottle with a silicon rubber spacer;

FIG. 3 Standard Curve (from)¹⁵N signal intensity and¹⁵NH₄ ⁺linear regression of concentration);

FIG. 4 is a graph of the DNRA process rates for a variety of carbon to nitrogen ratios in a case of mature rice; error bars in the figure are standard errors of the mean of the experimental results.

Detailed Description

Example 1

1) Slurry preparation

Fresh soil samples screened through a 2mm sieve and water were mixed at a ratio of 1:7, charging helium for 30-60 min to reduce background O in the slurry₂. Then, filling the slurry subjected to aeration (He) treatment into 12mL headspace bottles in an anaerobic helium environment, screwing the bottle caps tightly, ensuring that the bottles are free of bubbles, and then placing the slurry into an inner chamber of a rotary incubator for pre-culturing for one week in a warm and dark place (according to the concentration of nitrate nitrogen in the background in soil)Degree sets the corresponding duration).

2) Isotopic labeling

Under anaerobic environment, a micro-sampling needle is used to puncture a bottle cap spacer, and 0.2mL of 6 mmol.L is added to the slurry sample after the pre-culture is finished^{-1 15}N-KNO₃(¹⁵99.5% of N abundance), and simultaneously discharging the equal volume of slurry by using a short needle head and adding the slurry¹⁵N-KNO₃The slurry sample is placed at a speed of 8 r.min^-1The rotary incubator is used for culturing at room temperature in a dark place.

3) DNRA Process Rate determination

After culturing for 0, 3 and 6 hours, 200. mu.L of 7 mol. multidot.L was injected into the culture system using a micro-needle^-1The culture was terminated with zinc chloride. Then transferring the slurry to a 100mL wide-mouth bottle, filling helium for 30-60 min again, and discharging the slurry generated by denitrification and anaerobic ammonia oxidation²⁹N₂、³⁰N₂The slurry was then re-dispensed into clean 12mL headspace bottles. Then 0.2mL of oxidant (sodium hypobromite iodine solution) is added into the subpackaged slurry sample and uniformly shaken to oxidize ammonium nitrogen formed in the DNRA process into ammonium nitrogen²⁹N₂And³⁰N₂. Then all samples are put at 1500-2000 r.min^-1Centrifuging at a speed of 5-8 min, and finally measuring the dissolved N in the supernatant by using MIMS₂Isotope signal intensity. The preparation method of the sodium hypobromite iodine solution comprises the following steps: 120mL of bromine water was added dropwise to 600mL of 16 mol. L^-1And (3) putting the container filled with the NaOH solution into an ice-water mixture at the temperature of below 5 ℃, continuously stirring the mixture in the whole process, keeping the temperature of the ice-water mixture below 5 ℃, putting the mixed solution into a refrigerator at 4 ℃, standing the mixed solution for one week, taking out the mixed solution, filtering the mixed solution by using a separating funnel, taking supernatant, mixing the supernatant with a 0.2 percent KI solution in the same volume, and storing the mixed solution in the refrigerator at 4 ℃ to obtain the sodium hypobromite iodine solution.

4) Standard Curve plotting and DNRA Process Rate calculation

Respectively preparing 0, 2, 4, 6, 8, 10, 20 and 50 mu mol.L^-1Is/are as follows¹⁵NH₄Cl(¹⁵99.5 percent of N abundance) and subpackaged in 12mL headspace bottles, and a micro-sampling needle is used for injecting the solution into each headspace bottleAdding 0.2mL of oxidant¹⁵NH₄ ⁺Oxidation to N₂(²⁹N₂And³⁰N₂) Determination of water by MIMS¹⁵N signal strength (2)³⁰N₂+²⁹N₂) Using the obtained¹⁵N signal intensity and¹⁵NH₄cl concentration standard curves were plotted, with three replicates per concentration setting. In the actual slurry sample¹⁵NH₄ ⁺The concentration was determined, in part, by plotting a standard curve, and the rate of the DNRA process in the sample was calculated as follows:

wherein R is_DNRAFor the rate of the DNRA process, nmol.g^-1·h^-1；

Is composed of¹⁵NH₄ ⁺The slope is obtained by linear regression of the concentration versus incubation time (i.e.¹⁵NH₄ ⁺Production rate of (b), nmol. mL^-1·h^-1(ii) a V is the volume of the headspace bottle, mL; w is the dry soil weight, g.

Measurement of MIMS by Standard Curve¹⁵Conversion of N Signal to¹⁵NH₄ ⁺The concentration is calculated by using a formula to obtain the DNRA process speed of the normally mature rice soil under different carbon-nitrogen ratio treatments, and the DNRA process speed under different treatments is found to be between 2.29 +/-0.01 and 3.78 +/-0.01 nmol NH₄ ⁺·g^-1·h^-1. Wherein the DNRA rate under control treatment was 2.29. + -. 0.01nmol NH₄ ⁺·g^-1·h^-1(ii) a Compared with the control soil, the DNRA rate is increased along with the increase of C/N, when the C/N of the culture system is higher than 12, the DNRA process rate is obviously higher than that of the control, and the research result is consistent with the experimental result of the prior people under the pure bacteria culture system.

Claims (2)

1. Rice productThe method for measuring the rate of dissimilatory reduction of nitrate to ammonium in field soil is characterized by comprising the following steps of: (1) preparing slurry: mixing a fresh soil sample which is sieved by a 2mm sieve with water according to a mass ratio of 1:7 to prepare slurry, filling helium for 30-60 min, then respectively filling the slurry subjected to helium exposure treatment into 12mL headspace bottles in an anaerobic helium environment, screwing bottle caps, ensuring that no bubbles exist in the bottles, and then placing the slurry in an inner chamber of a rotary incubator to pre-culture in a temperature and light-proof manner until nitrate radicals in the sample are exhausted; (2) isotope labeling: under anaerobic environment, a micro-sampling needle is used to puncture a bottle cap spacer, and 0.2mL of 6 mmol.L is added to the slurry sample after the pre-culture is finished^-115N-KNO₃Wherein¹⁵The N abundance is 99.5 percent, and the equal volume of slurry is discharged by using a needle head and is added completely¹⁵N-KNO₃The slurry sample is placed at the speed of 6-9 r.min^-1The rotary incubator is used for culturing at room temperature in a dark place; (3) determination of the process rate of dissimilatory reduction of nitrate to ammonium (DNRA) in rice field soil: after culturing for 0, 3 and 6 hours, 200. mu.L of 7 mol. multidot.L was injected into the culture system using a micro-needle^-1Terminating the culture by using zinc chloride, transferring the slurry into a 100mL wide-mouth bottle, filling helium for 30-60 min again, and discharging the sludge generated by denitrification and anaerobic ammonia oxidation²⁹N₂、³⁰N₂Then subpackaging the slurry into 12mL headspace bottles again, adding 0.2mL oxidant into the subpackaged slurry sample, shaking uniformly, and oxidizing ammonium nitrogen formed in the DNRA process into ammonium nitrogen²⁹N₂And³⁰N₂then, all samples are put at 1500-2000 r.min^-1Centrifuging at a speed of 5-8 min, and finally determining the dissolved N in the supernatant by using a membrane sample introduction mass spectrometer₂Isotope signal intensity; (4) standard curve plotting and DNRA process rate calculation: respectively preparing 0, 2, 4, 6, 8, 10, 20 and 50 mu mol.L^-1Is/are as follows¹⁵NH₄Cl solution of wherein¹⁵The N abundance is 99 percent, the mixture is respectively filled into 12mL headspace bottles, and 0.2mL oxidant is added into each headspace bottle by a micro-sampling needle¹⁵NH₄ ⁺Oxidation to N₂Said N is₂Is composed of²⁹N₂And³⁰N₂determination of water by MIMS¹⁵Strong N signalDegree of, the¹⁵N signal strength equals to two times³⁰N₂Signal strength plus²⁹N₂Signal strength, in 2³⁰N₂+²⁹N₂Expression, use of the obtained¹⁵N signal intensity and¹⁵NH₄standard curves were plotted for Cl concentration, with three replicates per concentration set; in the actual slurry sample¹⁵NH₄ ⁺The concentration was determined, in part, by plotting a standard curve, and the rate of DNRA progress in the sample was calculated as follows:

wherein R is_DNRAFor the rate of the DNRA process, the unit nmol g^-1·h^-1；

Is composed of¹⁵NH₄ ⁺The concentration and the culture time are linearly regressed to obtain the slope, and the slope is¹⁵NH₄ ⁺Production rate of (D) in nmol. mL^-1·h^-1(ii) a V is the volume of the headspace bottle, in mL; w is the dry soil weight in g.

2. The method for determining the reduction rate of nitrate in paddy soil to ammonium by dissimilatory reaction according to claim 1, wherein the oxidizing agent is a solution of sodium hypobromite and iodine.

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