CN114910547A - A kind of method and application of detecting ammonium nitrogen 15N - Google Patents

Abstract

The invention belongs to the technical field of ecological environment substance circulation and pollution control, and particularly relates to a method for detecting ammonium nitrogen 15N and application thereof. The method comprises the following steps: NH in a sample to be tested ₄ ⁺ Oxidation to NO ₂ ^‑ Obtaining a treatment liquid, mixing the treatment liquid with denitrifying microorganisms to remove NO ₂ ^‑ Conversion to N ₂ O, detecting N ₂ Nitrogen isotope composition of O. The invention provides a method for detecting ammonium nitrogen by combining the characteristics of denitrifying microorganisms through a chemical method ¹⁵ Compared with the traditional method, the method saves the detection time, avoids using various highly toxic reducing agents, and is safer. The invention provides a method for detecting ammonium nitrogen ¹⁵ The N method has higher detection recovery rate, accuracy and precision, and is used for tracing ammonium nitrogen pollutionAnd nitrogen cycling processes provide important research tools.

Description

A kind of method and application of detecting ammonium nitrogen 15N

technical field

The invention belongs to the technical field of ecological environment material circulation and pollution control, and in particular relates to a method for detecting ammonium nitrogen 15N and its application.

Background technique

Nitrogen is one of the elements that constitute life on earth and is an important limiting factor for plant growth and crop yield. At present, the increase of nitrogen content in the ecological environment has caused a series of environmental problems such as eutrophication of surface water, toxicity of aquatic organisms, and haze weather. Ammonium nitrogen is one of the important forms of nitrogen in water, and nitrogen isotopic composition is not only an important means to identify nitrogen sources, but also an effective way to study nitrogen migration and transformation in terrestrial and aquatic ecosystems.

There are many methods for the determination of ammonium nitrogen and nitrogen isotopes in water and soil. The common method is to convert NH ₄ ⁺ into N ₂ or nitrogen-containing ammonium salts by pretreatment such as distillation, diffusion and ion exchange, and then combine with mass spectrometer to measure. The distillation method is to heat distillation by increasing the pH value of the solution, convert NH ₄ ⁺ into NH ₃ , and absorb the distillate with an acidic absorbent or acidic filter paper. Diffusion method, including micro-diffusion method, is to directly put water or soil extract in a container, add alkaline reagent to seal, and conduct shaking culture under normal temperature or heating conditions for several hours to ten days. The NH ₄ ⁺ in the sample is converted into It is NH ₃ , which is absorbed by filter paper or filter membrane with acid absorbent (such as H ₂ SO ₄ , KHSO ₄ , etc.) Determination. Ion-exchange chromatography is generally to enrich NH ₄ ⁺ in water or soil leaching solution through ion-exchange resin, and then combine with diffusion method to measure its N isotopic abundance. For samples with lower concentrations of NH ₄ ⁺ , in recent years, researchers have proposed a new analytical technique-chemical conversion method, which uses hypobromite to first oxidize NH ₄ ⁺ to nitrite (NO ₂ ^- ), Then use sodium azide-acetic acid buffer solution or reducing agent such as hydroxylamine hydrochloride to reduce NO ₂ ^- to form N ₂ O, and then combine with Precon or TraceGas to connect isotope mass spectrometer to measure δ ¹⁵ NN ₂ O, which is calculated by the calibration curve of standard solution The δ ¹⁵ N-NH ₄ ⁺ value in the original solution was obtained.

Although the distillation method has a high nitrogen recovery rate, the decomposition of organic nitrogen is easy to generate cross-contamination of ¹⁵ N, and professional equipment such as Kjeldahl nitrogen analyzer and freeze dryer is required, and the process is complicated and difficult to operate outdoors; the diffusion method has a low recovery rate. , the cultivation time is long (usually 5-10 days), and condensed water is generated on the wall of the container during the cultivation process, which will produce isotopic fractionation; the ion exchange method is easily affected by strong ions, and the diffusion method is used in combination with the diffusion method. disadvantages. In addition, these methods require a large amount of samples. Generally, 500-2500 mL of samples such as rainwater, lake water, river water and well water need to be collected on site, which increases the difficulty of field sampling. Although the chemical conversion of hypobromite requires a small amount of sample, the first step to oxidize NH ₄ ⁺ to NO ₂ ^- requires the use of sodium arsenite (NaAsO ₂ ) to terminate the oxidation reaction and absorb excess hypobromite, NaAsO ₂ is highly toxic and easily soluble in water, causing water pollution. Human intake of arsenic may increase the risk of lung cancer, bladder cancer and skin cancer. Sodium azide-acetic acid buffer needs to be used in the second reduction process Reducing agents such as solution (azide and acetic acid buffer) or hydroxylamine hydrochloride (NH ₂ OH·HCl), among which azide acid is highly toxic and easy to explode, and hydroxylamine hydrochloride is also highly toxic, irritating to the skin, and easily harmful to the human body. certain risk. On the other hand, both azide acid and hydroxylamine hydrochloride contain N, which is easy to exchange nitrogen with the generated N ₂ O, thereby affecting the accuracy of the test results.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the present invention provides a method for detecting ammonium nitrogen 15N and its application. The detection of ammonium nitrogen ¹⁵ N by chemical-microbiological combined determination method has high accuracy and safety.

In a first aspect, the present invention provides a method for detecting ammonium nitrogen ¹⁵ N, comprising:

The NH ₄ ⁺ in the sample to be tested is oxidized to NO ₂ ^- to obtain a treatment solution, the treatment solution is mixed with denitrifying microorganisms to convert NO ₂ ^- into N ₂ O, and the nitrogen isotopic composition of N ₂ O is detected.

The invention adopts the chemical method combined with the microbial method to determine the δ ¹⁵ N value of ammonium nitrogen, the culture of bacteria can be carried out simultaneously with the ammonium nitrogen oxidation process, the completion time is easy to grasp, and the test is greatly shortened compared with the diffusion method and the ion exchange chromatography method. time. And according to this method, the concentration and isotope tests can be completed with a small sample volume (up to 15mL), which significantly reduces the workload of collecting samples in the field. At the same time, the present invention adopts the microbial method to replace the highly toxic reducing agent such as azide acid or hydroxylamine hydrochloride in the reduction process, the reaction waste liquid is easy to handle, and the harm to the human body and the environment is reduced.

The denitrifying microorganisms used in the present invention can be commercially available denitrifying microorganisms, which have the ability to convert NO ₂ ^- into N ₂ O, preferably Stenotrophomonas nitritireducens .

Further, the mixing of the treatment solution and the denitrifying microorganism to convert NO ₂ ⁻ into N ₂ O includes:

Cultivate the denitrifying microorganism with a concentration between 8×10 ⁸ cfu/ml and 1.6×10 ⁹ cfu/ml to obtain a denitrifying microorganism bacterial liquid; after adding an antifoaming agent, remove the denitrifying microorganism bacterial liquid by nitrogen of N ₂ O.

Further, the mixing reaction time of the treatment solution and the denitrifying microorganism lasts at least 1 hour.

Further, after the nitrogen isotopic composition of N ₂ O is detected, the isotope result of ammonium nitrogen ¹⁵ N can be obtained by calibrating with international standard samples.

Further, the medium used in the culturing process is TSB medium and/or NB medium; and/or, the culture conditions in the culturing process are a temperature of 22°C-30°C, and a rotational speed of 160rpm/min ~180rpm/min.

Further, in parts by weight, the TSB medium comprises: 100-200 parts of TSB, 1-5 parts of (NH ₄ ) ₂ SO ₄ and 20-30 parts of KH ₂ PO ₄ .

Further, the treatment solution obtained by oxidizing NH ₄ ⁺ in the sample to be tested to NO ₂ ^- includes:

BrO ^- was used to oxidize NH ₄ ⁺ in the sample to be tested to NO ₂ ^- , and Na ₂ S ₂ O ₃ was used to terminate the oxidation reaction after the reaction.

Further, the use of BrO ^- to oxidize NH ₄ ⁺ in the test sample to NO ₂ ^- is carried out at room temperature, and the reaction time is 30-45 min.

Further, the concentration of Na ₂ S ₂ O ₃ is 0.1-0.3 mol/L, and the dosage is 20-50 μL, preferably 0.2 mol/L, and the dosage is 20 μL.

The invention uses sodium thiosulfate instead of sodium arsenite to terminate the oxidation reaction, and further reduces the harm to the human body and the environment.

Further ^, the preparation method of described BrO- is as follows:

Using sodium bromate and sodium bromide as raw materials, the acid is mixed to produce Br ₂ , and the base is mixed to obtain BrO ^- after 5 to 10 minutes, preferably 5 minutes.

Further ^, the consumption of the BrO- is 6-15% of the sample to be tested, preferably 10%.

Further, the mass ratio of the sodium bromate and sodium bromide is (0.4~0.8): (4~6).

After the treatment solution is obtained by oxidizing NH ₄ ⁺ in the sample to be tested to NO ₂ ^- , the method further includes: adjusting the pH of the treatment solution to 5-12.

The pH of the treatment liquid is preferably 8 to 11.

Further, the nitrogen isotopic composition of N ₂ O is measured by a trace gas pre-concentration device-isotope ratio mass spectrometer to obtain the amount of ammonium nitrogen ¹⁵ N.

Further, for the sample to be tested, if the total NH ₄ ⁺ concentration in the sample to be tested is relatively high, it is diluted to 1-20 μmol/L, preferably 10-15 μmol/L.

The present invention further provides the application of the method in the detection of ^ammonium nitrogen15N in soil, rainwater, surface water, groundwater, sediment, atmospheric aerosol or isotope tracer experiments.

The present invention has the following beneficial effects:

The invention provides a method for determining the δ ¹⁵ N value of ammonium nitrogen by a chemical method combined with a microbial method. The process of converting NO ₂ ^- into N ₂ O is completed by denitrifying microorganisms. Compared with other existing methods, the use of The highly toxic reducing agent has higher safety; the instruments and reagents used are also more conventional, and the detection cost is reduced; at the same time, it still has a high recovery rate and accuracy, and has the advantages of scientificity, simplicity and operability. The promotion has broad prospects.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, bacteria, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Four reference materials USGS25, USGS26, USGS-N1 and RSIL-N7373 were purchased from USGS/Reston Stable Isotope Laboratory.

Example 1

The present embodiment provides a chemical-microbiological joint determination method for ammonium nitrogen δ ¹⁵ N, comprising the following steps:

(1) Microbial colony enrichment

Weigh 3g Tryptone Soy Broth Agar (TSB), 0.05g (NH ₄ ) ₂ SO ₄ , 0.49g KH ₂ PO ₄ , 200mL deionized water, dissolve and mix well, and dispense 120mL into 250mL glass bottles with breathable stoppers medium, autoclave at 121 °C for 30 min to form microbial colonies (using Stenotrophomonas nitritireducens , numbered ATCC No. BAA-12, purchased from ATCC) to enrich the TSB culture medium; inoculate a single colony of Stenotrophomonas nitritireducens into a bottle of TSB culture cultured in a shaker (30°C, 180rpm/min) for 1-2 days;

(2) Determine the NH ₄ ⁺ concentration in the sample and the sample volume

The concentration of NH ₄ ⁺ in the rainwater sample was determined by flow injection analyzer to be 165.71 μmol/L, diluted 15 times, and 2 mL was taken for subsequent reaction;

(3) Oxidation reaction - NH ₄ ⁺ in the sample is oxidized to NO ₂ ^-

First prepare BrO ^- stock solution: BrO ^- stock solution: 0.6g sodium bromate (sodium bromate) and 5g sodium bromide (sodium bromide) dissolved in 250mL deionized water; add 0.2mL stock solution to 10mL deionized water, then 0.6 mL of 6M HCl was added to generate Br ₂ , and after 5 minutes of dark reaction, 10 mL of 10 M NaOH was rapidly added to generate BrO ⁻ , forming a BrO ⁻ working solution.

Next, 2 mL of the rainwater sample tested in step (2) and 2 mL of 10 μmol/L USGS25, USGS26, and IAEA-N1 standard samples were added to a 20 mL headspace vial that had been baked at 450 °C for 4 h, and then 0.2 mL of hypobromous acid ( BrO ^- ) working solution, shake at 200 rpm/min on a shaker for 30 min, and add 20 μL of 0.2 mol/L Na ₂ S ₂ O ₃ after the reaction to stop the oxidation reaction;

(4) pH adjustment after oxidation of NH ₄ ⁺ to NO ₂ ^- in the sample

Add a few drops of 6M HCl to adjust the pH of the samples and standards after the reaction was terminated in step (3) to about 11, and seal them with sealing film for use;

(5) Add the cell culture to the headspace bottle, purify the upper space and remove the N ₂ O in the microbial liquid

Steps after enrichment culture is completed (1) Add a few drops of antifoaming agent to the microbial culture bottle, dispense 4mL into a 20mL headspace bottle and seal, use a nitrogen purger to purge, and insert the headspace bottle upside down on a nitrogen needle In addition, insert a 6cm long 25gauge needle on the silica gel pad of the bottle mouth, the flow rate is 20-40 mL/min, and replace the air in the headspace bottle and the N ₂ O in the bacterial liquid with high-purity nitrogen, and after purging for 30 minutes Remove the headspace bottle for later use;

(6) Microorganisms convert NO ₂ ^- into N ₂ O

Use an air-tight syringe to take 2 mL of the sample and standard product in step (4) and add them to the headspace bottle of bacterial liquid that has been purged in step (5). Fully mix the sample and bacterial liquid, invert for 1 hour, and add 0.20 mL of NaOH with a concentration of 10 mol/L after the reaction to terminate the reaction and absorb the generated CO ₂ ;

At the same time, use an airtight syringe to take 0.10 mL of a 2 mg/L RSIL-N7373 standard sample (total nitrogen content of 0.2 μg) and react with the bacterial solution;

(7) N ₂ O nitrogen isotope determination

The N ₂ O gas generated by the headspace bottle obtained in step (6) is measured by TraceGas trace gas analyzer-isotope ratio mass spectrometer (IRMS) to obtain the measured value of N ₂ O nitrogen isotopic composition;

(8) NH ₄ ⁺ -δ ¹⁵ N test result correction

Through instrument measurement, the results obtained in step (7) are shown in Table 1:

Table 1 Actual, measured and corrected values of δ ¹⁵ N for standard and rainwater NH ₄ ⁺

Note: ^a is NaNO ₂ , nitrite standard sample, the value represents δ ¹⁵ N -NO ₂ ^-

It can be seen from the results in Table 1 that the measured value of RSIL-N7373 is δ ¹⁵ N=-80.26±0.23‰, which is very close to its true value of -79.6‰, indicating that the microbial cells are reliable for the reduction of nitrite to N ₂ O. . Linear regression was performed between the measured value and the true value of the ammonium nitrogen standard sample, and the correction equation was obtained as T=1.0948*M+2.7744, where T was the true value, M was the measured value, and R=0.9999, indicating that the linear relationship was good. According to the correction equation and the measured value of rainwater NH ₄ ⁺ -δ ¹⁵ N, the actual value of rainwater NH ₄ ⁺ -δ ¹⁵ N is -7.12‰. According to the literature, this value traces back to the source indicating that the NH ₄ ⁺ in rainwater mainly comes from animal excretion Ammonia released from the substance.

It can be seen that it is completely feasible to use the method of the present invention to test the ammonium nitrogen isotopic composition of rainwater.

Example 2

The present embodiment provides a chemical-microbiological joint determination method for ammonium nitrogen δ ¹⁵ N, and the specific operation steps are as follows:

(1) Weigh 3g Tryptone Soy Broth Agar (TSB), 0.05g (NH ₄ ) ₂ SO ₄ , 0.49g KH ₂ PO ₄ , 200 mL deionized water, dissolve and mix well, and dispense 35 mL into 50 mL centrifuge tubes medium, autoclave at 121 °C for 30 min to form a microbial colony enriched TSB culture solution; inoculate a single colony of Stenotrophomonas nitritireducens into the TSB culture solution, and culture on a shaker (26 °C, 160 rpm/min) for 2-3 days;

(2) Determine the NH ₄ ⁺ concentration in the sample and the sample volume

The NH ₄ ⁺ concentration in the 2M KCl soil extract was determined by flow injection analyzer to be 22.5 μmol/L, diluted 2 times, and 2 mL was taken for subsequent reaction;

Steps (3)-(4) are the same as in Example 1.

(5) Add the cell culture to the headspace bottle, purify the upper space and remove the N ₂ O in the microbial liquid

Pour multiple enrichment and culture steps (1) Stenotrophomonas nitritireducens culture tube into a sterilized Erlenmeyer flask and mix well, add a few drops of antifoaming agent, dispense 4mL into a 20mL headspace bottle and seal it with nitrogen. Purge with the purging instrument, insert the headspace bottle upside down on the nitrogen needle, and insert a 6cm long 25gauge needle on the silicone pad of the bottle mouth, the flow rate is 20-40 mL/min, put the air and bacterial liquid in the headspace bottle into the air and bacterial liquid in the headspace bottle. The N ₂ O was replaced with high-purity nitrogen, and after purging for 30 min, the headspace vial was removed for use;

Steps (6)-(7) are the same as in Example 1.

(8) NH ₄ ⁺ -δ ¹⁵ N test result correction

After measuring by the instrument, the results obtained in step (7) are shown in Table 2:

Table 2 The true value, measured value and corrected value of δ ¹⁵ N of standard and soil extract NH ₄ ⁺

Note: ^a is NaNO ₂ , nitrite standard sample, the value represents δ ¹⁵ N -NO ₂ ^-

From the results in Table 2, it can be seen that the measured value of RSIL-N7373 is δ ¹⁵ N=-79.2±0.11‰, which is very close to the real value of -79.6‰, indicating that the microbial cells are reliable for the reduction of nitrite to N ₂ O. . Linear regression was performed between the measured value and the true value of the ammonium nitrogen standard sample, and the correction equation was obtained as T=1.1116*M+2.153, where T was the true value, M was the measured value, and R=0.9998, indicating a good linear relationship. According to the calibration equation and the measured value of NH ₄ ⁺ -δ ¹⁵ N in the soil extract, the actual value of NH ₄ ⁺ -δ ¹⁵ N in the soil extract is -0.55‰. According to the literature, the traceability of this value indicates that the NH ₄ in the soil ⁺ Mainly ammonia released from fertilizers.

It can be seen that it is completely feasible to use the method of the present invention to test the isotopic composition of ammonium nitrogen in the soil leaching solution, and has a high accuracy.

Example 3

A chemical-microorganism joint determination method for ammonium nitrogen δ ¹⁵ N, the specific operation steps are:

Step (1) is the same as in Example 2.

(2) Determine the NH ₄ ⁺ concentration in the sample and the sample volume

The concentration of NH ₄ ⁺ in the river water was determined to be 5.5 μmol/L by flow injection analyzer, and 5 mL was taken for subsequent reaction;

Steps (3)-(5) are the same as in Example 2.

In step (4), 5 mL of the river water sample tested in step (2) needs to be taken and then 0.5 mL of hypobromous acid (BrO ^- ) working solution is added for oxidation reaction;

(6) Microorganisms convert NO ₂ ^- into N ₂ O

Use an air-tight syringe to take 5 mL of the sample in step (4), and add 2 mL of the standard to the headspace bottle of bacterial liquid that has been purged in step (5) (total ammonium nitrogen is about 0.2 μg), and suck out the volume of gas, mix the sample and bacterial liquid thoroughly, invert and cultivate overnight, add 0.20 mL of NaOH with a concentration of 10 mol/L after the reaction to terminate the reaction and absorb the generated CO ₂ ; at the same time, use an air-tight syringe to take 2 mg/L The RSIL-N7373 standard sample 0.10mL (total nitrogen is 0.2μg);

Steps (7)-(8) are the same as in Example 2.

The result showed that the real value of NH ₄ ⁺ -δ ¹⁵ N in the river water was -9.59 ‰, indicating that the NH ₄ ⁺ in the river water mainly came from the ammonia released by the animal excrement of the surrounding farms.

It can be seen that it is completely feasible to use the method of the present invention to test the NH ₄ ⁺ -δ ¹⁵ N value in the water body, and it has a high accuracy.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (10)

1. a method for detecting ammonium nitrogen 15N, is characterized in that, comprises: The NH ₄ ⁺ in the sample to be tested is oxidized to NO ₂ ^- to obtain a treatment solution, the treatment solution is mixed with denitrifying microorganisms to convert NO ₂ ^- into N ₂ O, and the nitrogen isotopic composition of N ₂ O is detected. 2. The method according to claim 1, wherein the denitrifying microorganism is nitrite-reducing oligotrophomonas. 3. The method according to claim 1, wherein the mixing of the treatment solution and the denitrifying microorganism to convert NO ₂ ^- into N ₂ O comprises: Cultivate the denitrifying microorganism with a concentration between 8×10 ⁸ cfu/ml and 1.6×10 ⁹ cfu/ml to obtain a denitrifying microorganism bacterial liquid; after adding an antifoaming agent, remove the denitrifying microorganism bacterial liquid by nitrogen of N ₂ O. 4. method according to claim 3, is characterized in that, the substratum that adopts in the process of described cultivation is TSB substratum and/or NB substratum; And/or, the culture condition in the process of described cultivation is The temperature is 22℃-30℃, and the rotation speed is 160rpm/min~180rpm/min. 5 . The method according to claim 1 , wherein the oxidizing NH ₄ ⁺ in the sample to be tested to NO ₂ ^- to obtain the treatment liquid comprises: 5 . BrO ^- was used to oxidize NH ₄ ⁺ in the sample to be tested to NO ₂ ^- , and Na ₂ S ₂ O ₃ was used to terminate the oxidation reaction after the reaction. 6. method according to claim 5, is characterized in that ^, the preparation method of described BrO- is as follows: Using sodium bromate and sodium bromide as raw materials, the acid is mixed to produce Br ₂ , and the base is mixed after 5-10 minutes to obtain BrO ^- . 7. method according to claim 5 is characterized in that, the consumption of described BrO is 6 ^～ 15% of described sample to be tested. 8. The method according to claim 5, characterized in that, after the oxidation reaction is terminated with Na ₂ S ₂ O ₃ , the pH of the treatment solution is adjusted to 5-12; the Na ₂ S ₂ O The concentration of ₃ is 0.1 to 0.3 mol/L. 9 . The method according to claim 1 , wherein the amount of ammonium nitrogen ¹⁵ N is obtained by measuring the nitrogen isotopic composition of N ₂ O by a trace gas pre-concentration device-isotope ratio mass spectrometer. 10 . 10. Application of the method according to any one of claims ¹ to 9 in the detection of ammonium nitrogen15N in soil, rainwater, surface water, groundwater, sediment, atmospheric aerosol or isotope tracer experiments.