阅读说明：本技术 一种土壤硝酸还原酶活性测定的新方法 (Novel method for measuring activity of soil nitrate reductase ) 是由 刘涛 褚贵新 雷雨 于 2019-10-11 设计创作，主要内容包括：本发明属于土壤氮循环测定技术领域,公开了一种土壤硝酸还原酶活性测定的新方法,以硝酸钾(KNO<Sub>3</Sub>)为底物,在30℃恒温和厌氧条件下对土壤进行24小时(h)的还原培养,通过测定还原前后土壤硝态氮(NO<Sub>3</Sub><Sup>-</Sup>-N)含量并计算其差值得出每克(g)干土中被还原的NO<Sub>3</Sub><Sup>-</Sup>-N的量,以被还原的NO<Sub>3</Sub><Sup>-</Sup>-N的量表征该土壤的硝酸还原酶活性。本发明中的培养实验中,用普通锥形瓶和真空容器代替减压锥形瓶,用连续流动分析仪代替酚二磺酸比色法进行NO<Sub>3</Sub><Sup>-</Sup>-N含量的测定,与以往方法相比,可显著降低实验误差,提高测定结果的精确度和测试效率。(The invention belongs to the technical field of soil nitrogen cycle determination, and discloses a new method for determining soil nitrate reductase activity, which uses potassium nitrate (KNO) 3 ) As a substrate, the soil was subjected to reduction culture at a constant temperature of 30 ℃ under anaerobic conditions for 24 hours (h) by measuring nitrate Nitrogen (NO) in the soil before and after the reduction 3 ‑ N) and calculating the difference to yield the reduced NO per gram (g) of dry soil 3 ‑ Amount of N to reduced NO 3 ‑ The amount of-N characterizes the nitrate reductase activity of the soil. In the culture experiment of the invention, a common conical flask and a vacuum container are used for replacing a decompression conical flask, and a continuous flow analyzer is used for replacing a phenoldisulfonic acid colorimetric method for carrying out NO 3 ‑ Compared with the conventional method, the method for measuring the content of the N-component can obviously reduce experimental errors and improve the accuracy and the testing efficiency of the measurement result.)

1. A novel method for measuring the activity of a soil nitrate reductase is characterized by comprising the following steps: potassium nitrate (KNO)₃) As a substrate, carrying out reduction culture on soil for 24 hours at a constant temperature of 30 ℃ under an anaerobic condition;

by measuring nitrate Nitrogen (NO) before and after reduction₃ ^-N) content and calculating the difference to yield reduced NO per gram (g) of dry soil₃ ^-Amount of N to reduced NO₃ ^-The amount of-N characterizes the nitrate reductase activity of the soil.

2. The novel method for measuring the nitrate reductase activity in soil according to claim 1, wherein the novel method for measuring the nitrate reductase activity in soil specifically comprises:

firstly, accurately weighing 1.00g of air-dried soil sample and dried soil sample, placing the air-dried soil sample and dried soil sample into a 50 or 100mL conical flask, and sequentially adding 20mg of calcium carbonate (CaCO)₃) And 1mL of 1% KNO₃Shaking the solution evenly, and then adding 1mL of 1% glucose solution as a hydrogen donor;

secondly, placing the conical flask into a vacuum container, and closing a valve when the interior of the container is vacuumized to 70KPa by using a vacuum pump;

thirdly, placing the vacuum container into a biochemical incubator, and culturing for 24 hours at a constant temperature of 30 ℃;

fourthly, after the culture is finished, adding 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution into the conical flask, fully shaking up, and filtering the mixture into a sample bottle by using slow quantitative filter paper;

fifthly, using a continuous flow analyzer to measure NO in the standard curve solution and the sample filtrate₃ ^-Continuous batch measurement of-N to directly obtain NO in filtrate₃ ^--N concentration.

3. The novel method for measuring nitrate reductase activity in soil according to claim 2, wherein the following steps are carried out before the one-step process is carried out:

preparing soil: dividing each fresh soil sample into 2 parts, naturally drying one part, putting the other part into an oven, drying for 3 hours at 180 ℃ to inactivate soil enzymes, taking out and cooling to room temperature, and sieving the soil samples with a 1mm sieve;

preparing a soil culture reagent: 1% KNO₃The method comprises the following steps of (1) preparing a solution, a 1% glucose solution and a potassium aluminum sulfate saturated solution;

preparing a continuous flow analyzer on a computer to measure a reagent: 0.1% copper sulfate (CuSO)₄) Stock solution, 1% Zinc sulfate (ZnSO)₄) Stock solution, hydrazine sulfate mixed solution, color developing agent, 4% sodium hydroxide (NaOH) solution and phosphate buffer solution.

Preparation of standard solution: by KNO₃Preparing NO with concentration of 500mg/L₃ ^--N standard solutions;

preparation of a standard curve: respectively absorb NO₃ ^-Putting 0, 0.5, 1, 1.5, 2, 2.5 and 3mL of-N standard solution into a 50mL volumetric flask, and fixing the volume by using pure water to obtain NO with the concentration of 0, 5, 10, 15, 20, 25 and 30mg/L in sequence₃ ^--N standard curve solution.

4. The method of claim 1A novel method for measuring the activity of nitrate reductase in soil is characterized in that CaCO₃For regulating the pH value of soil>7 meets the condition of optimum pH value of the microorganism, and CaCO is not added when measuring nitrate reductase in alkaline soil₃。

5. The method for measuring the nitrate reductase activity in soil according to claim 1, wherein the pure water used for the reduction culture is sterilized and sonicated in advance to remove microorganisms, oxygen and carbon dioxide and stored in a sealed state;

the vacuum container is a container which can be vacuumized.

6. The method for measuring nitrate reductase activity in soil according to claim 2, wherein in the first step, the measured value of the filtrate extracted after the culture of the air-dried soil sample is used as NO in the reduced soil₃ ^-N concentration, measured value of filtrate extracted after the culture of the dried soil sample as NO before reduction₃ ^--N concentration.

7. The method of claim 1, wherein the filtered solution from the fourth step is first stored frozen at-18 ℃ if not immediately assayed.

8. The novel method for measuring nitrate reductase activity in soil according to claim 2, wherein in the fifth step, the nitrate reductase activity is calculated by the formula:

wherein C1 is NO in filtrate obtained by drying soil sample at 180 deg.C₃ ^--N content, C2 is NO in filtrate after air-dried soil-like culture₃ ^-N content, 53 is the final volume of the solution after culture, 1000 is the volume conversion coefficient, and W is the soil mass accurately weighed.

9. The novel method for determining nitrate reductase activity in soil as set forth in claim 2, wherein NO is measured in the standard curve of the fifth continuous flow analyzer₃ ^-The concentration gradient of-N is 0-50 mg/L.

10. A nitrate reductase activity measuring apparatus for carrying out the novel method for measuring nitrate reductase activity in soil according to claim 1.

Technical Field

The invention belongs to the technical field of soil nitrogen cycle determination, and particularly relates to a novel method for determining soil nitrate reductase activity.

Background

Soil denitrification is an important process of soil nitrogen cycle, and nitrate reductase is the final product nitrate Nitrogen (NO) for soil nitrification₃ ^--N) reduction to nitrite Nitrogen (NO)₂ ^--N) of a class of reductases,the activity of the compound can represent the strength of denitrification, indirectly influence the nitrogen loss of soil (nitrate leaching loss, gaseous nitrogen loss and the like), and also influence the utilization rate of nitrogen fertilizers in an agricultural ecological system. The basic principle of the method for measuring the activity of the soil nitrate reductase is that NO₃ ^--N conversion of NO catalyzed by nitrate reductase₂ ^--N, NO before and after reaction₃ ^-The difference of the-N content can represent the activity of the soil nitrate reductase.

Currently, the closest prior art:

in the conventional methods for measuring the nitrate reductase activity of soil, reference is made to "soil enzyme and its research method" (1986) written by guan-tuo et al and "soil and environmental microorganism research method" (2008) written by li sha et al, and the above methods are basically the same in principle and procedure, and both represent the nitrate reductase activity by measuring the blue reaction difference (i.e., absorbance difference) between nitrate nitrogen and phenoldisulfonic acid before and after reduction in the presence of a hydrogen donor and under anaerobic conditions, and the larger the absorbance difference, the higher the nitrate reductase activity.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel method for measuring the activity of soil nitrate reductase.

The invention is realized in such a way that a new method for measuring the activity of the soil nitrate reductase comprises the following steps:

with KNO₃As substrate, the soil was subjected to reduction culture at 30 ℃ under anaerobic conditions for 24 hours, by measuring NO before and after reduction₃ ^-The difference between the N contents and the reduced NO per g of dry soil₃ ^-The amount of N, in terms of reduced NO per g of dry soil₃ ^-The amount of-N is taken as the nitrate reductase activity of the soil.

Further, the new method for measuring the activity of the soil nitrate reductase specifically comprises the following steps:

firstly, accurately weighing 1.00g of air-dried soil sample and 180 ℃ dried soil sample which are sieved by a 1mm sieve, putting the air-dried soil sample and the 180 ℃ dried soil sample into a 50 or 100mL conical flask, and sequentially adding 20mg of CaCO₃And 1mL of 1% KNO₃Shaking the solution evenly, and then adding 1mL of 1% glucose solution;

secondly, placing the conical flask into a vacuum container, and closing a valve when the interior of the container is vacuumized to 70KPa by using a vacuum pump;

thirdly, placing the vacuum container into a biochemical incubator, and culturing for 24 hours at a constant temperature of 30 ℃;

fourthly, after the culture is finished, adding 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution into the conical flask, fully shaking up, and filtering the mixture into a sample bottle by using slow quantitative filter paper;

fifthly, using a continuous flow analyzer to continuously measure the nitrate nitrogen in the standard curve solution and the sample filtrate in batches, and directly obtaining NO in the filtrate₃ ^-N concentration (mg/L).

Further, before the previous step, the following steps are carried out:

preparing soil: dividing each fresh soil sample into 2 parts, naturally drying one part, drying the other part at 180 ℃ for 3 hours to inactivate soil enzymes, and sieving the soil samples by a 1mm sieve;

preparing a reagent required by soil culture: 1% KNO₃The method comprises the following steps of (1) preparing a solution, a 1% glucose solution and a potassium aluminum sulfate saturated solution;

preparing reagents required by the determination on a continuous flow analyzer: 0.1% CuSO₄Stock solution, 1% ZnSO₄Stock solution, hydrazine sulfate mixed solution, color developing agent, 4% NaOH solution and phosphate buffer solution.

Preparation of standard solution: by KNO₃Preparing NO with concentration of 500mg/L₃ ^--N standard solutions;

preparation of a standard curve: respectively absorb NO₃ ^-Putting 0, 0.5, 1, 1.5, 2, 2.5 and 3mL of-N standard solution into a 50mL volumetric flask, and fixing the volume by using pure water to obtain NO with the concentration of 0, 5, 10, 15, 20, 25 and 30mg/L in sequence₃ ^--N standard curve solution.

Further, due to CaCO₃The effect of the method is to adjust the pH value of the soil>7 to satisfy the optimum pH condition of the microorganism, therefore, CaCO may not be added in the measurement of nitrate reductase in alkaline soil₃(performed in the first step).

Further, pure water for reduction culture is sterilized and sonicated in advance to remove microorganisms, oxygen and carbon dioxide and stored in a sealed state.

Further, the vacuum vessel is any vessel that can be evacuated, such as a vacuum dryer.

Further, in the first step, the measured value of the filtrate extracted after the air-dried soil sample is the reduced NO₃ ^-N concentration, measured value of filtrate extracted after the culture of the dried soil sample is NO before reduction₃ ^--N concentration.

Further, the solution after the fourth filtration step may be stored frozen at-18 ℃ if it is not measured immediately.

Further, in the fifth step, the nitrate reductase activity is calculated by the formula:

wherein C1 is NO in filtrate obtained by drying soil sample at 180 deg.C₃ ^-N content (mg/L), C2 is NO in filtrate after air-dried soil-like culture₃ ^-N content (mg/L), 53 is the final volume of the solution after culture, 1000 is the volume conversion coefficient, and W is the accurately weighed soil mass (g).

Further, NO in the standard curve measured by the fifth continuous flow analyzer₃ ^-The concentration gradient of-N is 0-50 mg/L.

Another object of the present invention is to provide a novel method for measuring nitrate reductase activity in soil and to provide a nitrate reductase activity measuring apparatus.

In summary, the advantages and positive effects of the invention are:

the invention improves the culture and determination parts of the soil nitrate reductase activity, and the concentrated vacuum pumping of all culture bottles not only shortens the operation time, but also enables the samples to be in the same reduction condition, and the NO before and after reduction can be rapidly determined by using a professional instrument₃ ^-The improvement can obviously reduce the operation error, so that the determination result is more accurate and reliable, and meanwhile, the determination efficiency is obviously improved, and the method is particularly suitable for the mass determination of the nitrate reductase activity of the soil.

The culture experiment in the invention does not need a special decompression triangular flask used in the prior method, only needs a common conical flask and a vacuum-pumping typeContainer replacement; with the continuous flow analyzer in colleges and universities and scientific research units, NO can be carried out by using the continuous flow analyzer₃ ^-And the mass measurement of the-N can obviously reduce errors and improve the precision and the measurement efficiency.

Drawings

FIG. 1 is a flow chart of a novel method for measuring nitrate reductase activity of soil according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the prior art, each sample needs a special decompression conical flask, the time for vacuumizing each decompression conical flask is 3min, if the sample amount is large, the time for the step is too long, and the efficiency is obviously reduced. The prior art does not address the vacuum pressure range, which causes different anaerobic levels in each reduced pressure flask, which causes different reduction levels during the incubation process, increasing the uncertainty of the assay results. In the prior art, a phenoldisulfonic acid colorimetric method is used for determination, more than 6 hours are needed for phenoldisulfonic acid configuration, the method is long in time consumption in a water bath evaporation link, a developed sample is generally required to be subjected to absorbance value determination in a short time, when a large number of samples are developed, the absorbance value error is increased, and the accuracy and the determination efficiency of an experimental result are reduced.

In view of the problems of the prior art, the present invention provides a new method for measuring the activity of soil nitrate reductase, and the present invention is described in detail with reference to the accompanying drawings.

As shown in fig. 1, before the novel method for measuring the nitrate reductase activity of soil provided by the embodiment of the present invention is performed, the following steps are performed:

preparing soil: dividing each fresh soil sample into 2 parts, naturally drying one part, drying the other part at 180 ℃ for 3 hours to inactivate soil enzymes, and sieving the soil samples with a 1mm sieve.

Preparing a reagent required by soil culture: 1% KNO₃Solution, 1% glucose solution and saturated solution of potassium aluminum sulfate.

Preparing reagents required by the determination on a continuous flow analyzer: 0.1% CuSO₄Stock solution, 1% ZnSO₄Stock solution, hydrazine sulfate mixed solution, color developing agent, 4% NaOH solution and phosphate buffer solution.

Preparation of standard solution: by KNO₃Preparing NO with concentration of 500mg/L₃ ^--N standard solution.

Preparation of a standard curve: respectively absorb NO₃ ^-Putting 0, 0.5, 1, 1.5, 2, 2.5 and 3mL of-N standard solution into a 50mL volumetric flask, and fixing the volume by using pure water to obtain NO with the concentration of 0, 5, 10, 15, 20, 25 and 30mg/L in sequence₃ ^--N standard curve solution.

The method comprises the following specific steps:

s101, weighing 1.00g of air-dried soil sample and 180-DEG C dried soil sample which are sieved by a 1mm sieve, putting the air-dried soil sample and the 180-DEG C dried soil sample into a 50 or 100mL conical flask, and sequentially adding 20mg of CaCO₃And 1mL of 1% KNO₃The solution was shaken up and 1mL of 1% glucose solution was added.

S102, placing the conical flask into a vacuum container, and closing a valve when the interior of the container is vacuumized to 70KPa by using a vacuum pump.

S103, placing the vacuum container into a biochemical incubator, and culturing for 24 hours at a constant temperature of 30 ℃.

S104, after the culture is finished, adding 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution into the conical flask, fully shaking up, and filtering by using slow quantitative filter paper to a sample bottle.

S105, continuously measuring the nitrate nitrogen in the standard curve solution and the sample filtrate in batches by using a continuous flow analyzer to directly obtain NO in the filtrate₃ ^-N concentration (mg/L).

In the present example, the calculation formula of nitrate reductase activity is:

wherein C1 is NO in filtrate obtained by drying soil sample at 180 deg.C₃ ^-N content (mg/L), C2 is NO in filtrate after air-dried soil-like culture₃ ^-N content (mg/L), 53 is the final volume (mL) of the solution after culture, 1000 is the volume conversion factor, and W is the accurately weighed soil mass (g).

In the examples of the present invention, since CaCO₃The effect of the method is to adjust the pH value of the soil>7 to satisfy the optimum pH condition of the microorganism, therefore, CaCO may not be added in the measurement of nitrate reductase in alkaline soil₃(performed in the first step).

In the present example, the pure water used for the reduction culture was previously sterilized and sonicated to remove microorganisms, oxygen and carbon dioxide and stored in a sealed state.

In the embodiment of the present invention, the vacuum container is any container that can be evacuated, such as a vacuum dryer.

In the examples of the present invention, the measured value of the filtrate extracted after the culture of the air-dried soil sample was reduced NO₃ ^-N concentration, measured value of filtrate extracted after the culture of the dried soil sample is NO before reduction₃ ^--N concentration.

In the embodiment of the invention, in a standard working curve measured by a continuous flow analyzer, the concentration gradient range of nitrate nitrogen is 0-50 mg/L.

In the present example, the filtered solution was stored frozen at-18 ℃ if it could not be measured immediately.

In the embodiment of the invention, the method is improved according to the following steps: the reduction culture under anaerobic condition is the necessary condition for measuring nitrate reductase with KNO₃For substrate, after 24h incubation, by measuring NO before and after reduction₃ ^-The difference in the-N content gives the reduced NO per gram of dry soil₃ ^-Amount of N to reduced NO₃ ^-The amount of N characterizing the soil nitrate reductase activity. The natural air-dried soil used by the invention is KNO₃As substrate, carrying out 24h anaerobic culture at 30 ℃, and catalytically reducing nitric acid under the action of nitrate reductasePart of NO in Potassium₃ ^--N. The nitrate reductase dried in the soil at 180 ℃ is inactivated at high temperature so that the nitrate reductase cannot catalyze and reduce the substrate, and therefore, the difference between the two can be used for representing the activity of the nitrate reductase in the soil.