CN110643676A - Novel method for measuring activity of soil nitrate reductase - Google Patents
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Abstract
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 reduction3 ‑N) and calculating the difference to yield the 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. 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 NO3 ‑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.
Description
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.
The specific steps in the prior art are as follows:
1g of soil sample is put into a 100mL pressure-reducing triangular flask, and 20mg of CaCO is added3And 1mL KNO3Carefully mixing the solutions, adding 1ml of glucose solution as hydrogen donor, pumping for 3min, shaking, and culturing in a 30 deg.C constant temperature incubator for 24 h; after the culture is finished, adding 50mL of deionized water and 1mL of potassium aluminum solution; and (3) putting 20mL of filtrate into a porcelain evaporation vessel or a conical flask, evaporating to dryness in a water bath, adding 2mL of phenol disulfonic acid solution for dissolving for 10min, adding 15mL of deionized water, adjusting to yellowish with 10% of NaOH solution, transferring to a 50mL volumetric flask, fixing the volume with distilled water, and carrying out color comparison at 400-500 nm by using a spectrophotometer. While sterile soil (oven dried at 180 ℃ for 3h) was used as a control.
Suck 50mL KNO3Evaporating the standard solution (100mg/L) in porcelain evaporating dish with water bath, adding 2mL phenoldisulfonic acid solution into the residue, treating for 10min, adding 15mL distilled water, and diluting to 500mL to obtain 10mg/L NO3 --N solution, sucking 5-40 mL of the solution into a 50mL volumetric flask, adjusting to yellowish with 10% NaOH solution, and distillingCarrying out color comparison at 400-500 nm after water constant volume, drawing a standard curve by taking absorbance as ordinate and concentration as abscissa for calculating NO in soil sample3 --N content. Nitrate reductase Activity with reduced NO per g soil after 24h3 --quantity of N.
In summary, the problems of the prior art are as follows:
(1) 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;
(2) the prior art does not describe the vacuum pressure range, which causes different anaerobic degrees in each decompression bottle, causes different reduction degrees in the culture process and increases the uncertainty of the measurement result;
(3) 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 conclusion, the existing measuring method is complicated in the actual operation process, low in measuring efficiency, especially incapable of meeting the test requirement on a large number of samples in a short time, poor in reproducibility due to large measuring errors and difficult to obtain accurate data.
The difficulty of solving the technical problems is as follows:
the decompression conical flask is replaced by a conventional conical flask, samples in the same batch can be placed in the same vacuum container, and the samples are collectively vacuumized by using a vacuum pump with a manometer, so that the vacuumizing time can be remarkably saved, and the vacuum container and the vacuum pump with the manometer are common laboratory equipment, so that the technical problem is easily solved; although the phenoldisulfonic acid colorimetric method is a classic method for measuring nitrate nitrogen, the method is relatively backward, the time consumption for phenoldisulfonic acid configuration and water bath evaporation is relatively long, the measuring efficiency is low, a large number of samples can be measured in a short time by using a continuous flow analyzer, the measuring efficiency can be obviously improved, the operation error is reduced, the testing precision is improved, the problem that the continuous flow analyzer needs to be arranged is solved, and the instrument is more and more popularized in universities and scientific research institutions in recent years.
The significance of solving the technical problems is as follows:
the problems of unstable data, poor reproducibility and large error caused by factors such as poor control of reduction conditions, long time consumption in the operation process and the like are always problems in enzyme activity determination. The invention has the significance that the test precision and efficiency of the nitrate reductase can be obviously improved through the improvement on the technology, and the invention is particularly suitable for the mass measurement of the soil nitrate reductase.
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.
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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.
In the prior art, each sample needs to be vacuumized, the consumed time is long, and the determination efficiency is greatly reduced. Because the prior art does not determine the vacuum pressure range, the anaerobic degree of each sample is different, so that the reduction difference in the culture process is caused, and finally the result is uncertain, and the operation time can be obviously saved by putting the samples into the vacuum container for collective vacuum pumping. The phenoldisulfonic acid colorimetric method applied in the prior art is long in time consumption and low in determination efficiency, the test requirements for large-batch samples in a short time cannot be met, the reproducibility is poor due to large determination errors, accurate data are difficult to obtain, the determination efficiency can be remarkably improved by applying a continuous flow analyzer, the errors are reduced, and the test precision is improved.
The invention is further described with reference to specific examples.
Example 1
Compared with the prior art, the new method for measuring the activity of the soil nitrate reductase provided by the embodiment 1 of the invention comprises the following steps:
preparing soil: in the embodiment, the soil sample is collected from a 0-20 cm soil layer of a test station of the college of agriculture of the river university, and impurities are removed and the soil sample is sieved by a 1mm sieve. The soil sample is divided into 2 parts, one part is naturally dried (7-10 days), and the other part is dried for 3 hours at 180 ℃ to inactivate soil enzymes.
Preparing a reagent required by soil culture: 1% KNO3Solution (weighing 1.00g KNO3To 100mL with pure water), 1% glucose solution (1.00 g glucose was weighed to 100mL with pure water), CaCO3(white powder) and a potassium aluminum sulfate saturated solution (potassium aluminum sulfate is dissolved in 100mL of pure water to be supersaturated, namely crystals are ensured to be separated out in the solution).
The preparation of reagents required by the continuous flow analyzer in the invention: 0.1% CuSO4Stock solution (0.10 g CuSO)4Pure water to 100mL), 1% ZnSO4Stock solution (1.00 g ZnSO)4Pure water to 100mL), hydrazine sulfate mixture (7 mL of 0.1% CuSO4Stock solution, 5mL of 1% ZnSO4Stock solution and 3.00g of hydrazine sulfate were dissolved in 500mL of pure water and mixed well), developer (5.00 g of sulfanilamide, 0.25g N- (1-naphthyl) ethylenediamine dihydrochloride, and 50mL of phosphoric acid were diluted to 500mL with pure water), 4% NaOH solution (20 g of NaOH was diluted to 500mL with pure water), phosphate buffer (3 mL of phosphoric acid and 4.00g of tetrasodium dodecaphosphate were dissolved in 1000mL of pure water and mixed well).
In the prior art, reagents required for the colorimetric determination of the phenoldisulfonic acid are prepared: 10% NaOH solution (10g NaOH is made to 100mL with pure water), phenol disulfonic acid (3 g redistilled phenol is mixed with 37g concentrated sulfuric acid and heated and refluxed for 6h on a boiling water bath).
Preparing a nitrate nitrogen standard solution: 3.6090g KNO is accurately weighed3Adding pure water to a constant volume of 1L to obtain NO with a concentration of 500mg/L3 --N standard solution, refrigerated storage.
The preparation of the nitrate nitrogen standard curve of the invention: 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 preparation of nitrate nitrogen standard curve in the prior art: adding NO3 -Diluting the standard solution by 5 times to obtain 100mg/L NO3 -And (4) sucking 50mL of the solution out of the N solution, placing the solution in a porcelain evaporation dish, evaporating the solution on a boiling water bath to dryness, treating the residue with 2mL of phenol disulfonic acid for 10min, and adding 15mL of distilled water to reach the constant volume of 500 mL. Respectively sucking 0, 5, 10, 15, 20, 30, and 40mL of the solution into a 50mL volumetric flask, adjusting to yellowish with 10% NaOH solution, and diluting to constant volume with pure water to obtain NO with concentration of 0, 1, 2, 3, 4, 6, and 8mg/L3 --N standard curve solution, colorimeted with a spectrophotometer at 450nm, in absorbance values and NO3 --N concentration value fitting equation.
The method comprises the following specific operation steps: accurately weighing 7 parts of 1.00g of air-dried soil sample and dried soil sample, putting the air-dried soil sample and the dried soil sample into a 50mL conical flask, and sequentially adding 20mg of CaCO3And 1mL of 1% KNO3The solution was shaken up and 1mL of 1% glucose solution was added. Putting the conical flask into a vacuum drier, vacuumizing to 70KPa by using a vacuum pump, closing a valve, putting the conical flask into a biochemical incubator, and performing constant-temperature culture for 24 hours at the temperature of 30 ℃. After the culture, 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution are added into the conical flask, and the mixture is fully shaken and filtered into a sample bottle by slow quantitative filter paper. NO in standard curve solution and sample filtrate using continuous flow analyzer3 -Continuous batch measurement of-N to directly obtain NO in filtrate3 -N concentration (mg/L). And then the activity of the soil nitrate reductase is calculated by a formula and is expressed by mg of reduced nitrate nitrogen in 24h per g of dry soil.
The prior art comprises the following specific operation steps: accurately weighing 1.00g of air-dried soil sample and 7 parts of dried soil sample into a 100mL pressure-reducing conical flask, and sequentially adding 20mg of CaCO3And 1mL of 1% KNO3Adding 1mL of 1% glucose solution, shaking, pumping each pressure-reducing conical flask for 3min by a vacuum pump, placing in the same biochemical incubator, and culturing at constant temperature of 30 deg.C for 24 h. After the completion of the culture, 50mL of pure water and 1mL of a saturated solution of aluminum potassium sulfate were added to the flask. Measuring 20mL of solution in each conical flask, placing the solution in an evaporating dish for water bath evaporation, treating residues with 2mL of phenol disulfonic acid for 10min, adding 15mL of distilled water, adjusting the residues to yellowish with 10% of NaOH solution, transferring the solution into a 50mL volumetric flask, measuring the volume with pure water, carrying out color comparison at 450nm by using a spectrophotometer, and calculating NO reduced in 24h per g of dry soil by using the nitrate reductase activity according to a standard curve fitting formula and a nitrate reductase calculation formula3 -Mg of-N denotes.
The test results are as follows:
as can be seen from the above table, the error of the nitrate reductase activity of soil measured by the prior art is large, the repeatability is not good, the standard deviation can be reduced by 79.26% by the method of the present invention, and the nitrate reductase activity measured by the present invention is slightly higher than that of the prior art, which is closely related to the determination of the vacuum pressure to make the soil in a stable reducing environment and the reduction of the operation error by the measurement of a flow analyzer in the present invention.
Example 2 the novel method for measuring the nitrate reductase activity of soil provided by the embodiment of the present invention comprises:
preparing soil: collecting soil samples of 0-20 cm soil layers, removing impurities and sieving the soil samples with a sieve of 1 mm. Each sample is divided into 2 parts, one part is naturally dried (7-10 days), and the other part is dried for 3 hours at 180 ℃ to inactivate soil enzymes.
Preparing a reagent required by soil culture: 1% KNO3Solution (weighing 1.00g KNO3To 100mL with pure water), 1% glucose solution (1.00 g glucose was weighed to 100mL with pure water), CaCO3(white powder) and a potassium aluminum sulfate saturated solution (potassium aluminum sulfate is dissolved in 100mL of pure water to be supersaturated, namely crystals are ensured to be separated out in the solution).
Preparing a reagent on a continuous flow analyzer: 0.1% CuSO4Stock solution (0.10 g CuSO)4Pure water to 100mL), 1% ZnSO4Stock solution (1.00 g ZnSO)4Pure water to 100mL), hydrazine sulfate mixture (7 mL of 0.1% CuSO4Stock solution, 5mL of 1% ZnSO4Stock solution and 3.00g of hydrazine sulfate were dissolved in 500mL of pure water and mixed well), developer (5.00 g of sulfanilamide, 0.25g N- (1-naphthyl) ethylenediamine dihydrochloride, and 50mL of phosphoric acid were diluted to 500mL with pure water), 4% NaOH solution (20 g of NaOH was diluted to 500mL with pure water), phosphate buffer (3 mL of phosphoric acid and 4.00g of tetrasodium dodecaphosphate were dissolved in 1000mL of pure water and mixed well).
Preparing a nitrate nitrogen standard solution: 3.6090g KNO is accurately weighed3Adding pure water to a constant volume of 1L to obtain NO with a concentration of 500mg/L3 --N standard solution, refrigerated storage.
Preparing a nitrate nitrogen 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 operation steps: accurate and accurateWeighing 1.00g of air-dried soil sample and dried soil sample, placing into a 50mL conical flask, sequentially adding 20mg of CaCO3And 1mL of 1% KNO3The solution was shaken up and 1mL of 1% glucose solution was added. The flask was placed in a vacuum desiccator and the valve was closed when the vacuum was pumped down to 70 KPa. And (3) putting the vacuum drier into a biochemical incubator, and culturing for 24h at a constant temperature of 30 ℃. After the culture, 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution are added into the conical flask, and the mixture is fully shaken and filtered into a sample bottle by slow quantitative filter paper. NO in standard curve solution and sample filtrate using continuous flow analyzer3 -Continuous batch measurement of-N to directly obtain NO in filtrate3 -N concentration (mg/L). And then the activity of the soil nitrate reductase is calculated by a formula and is expressed by mg of reduced nitrate nitrogen in 24h per g of dry soil.
The invention is further described below in connection with the experiments.
The soil samples used in example 2 were collected from northern Xinjiang farmland, calcareous soil with three textures of sandy soil, loam and clay of 0-20 cm soil layers was collected respectively, and the nitrate reductase activity in the soil samples with the three textures was detected by the method of the present invention.
Removing impurities from the soil samples, sieving with a 1mm sieve, dividing each soil sample into 2 parts, naturally air drying one part, and oven drying the other part at 180 deg.C for 3 hr. Weighing 1.00g of air-dried soil sample and four parts of dried soil sample in 50mL conical flask for each kind of geological soil, adding 1mL of 1% KNO3The solution was shaken up and 1mL of 1% glucose solution was added. Putting the conical flask into a vacuum dryer, coating a layer of vaseline on the frosted part of the opening of the dryer, covering the vacuum dryer with a cover, vacuumizing the interior of the dryer to 70KPa by using a vacuum pump, closing a valve, putting the dryer into a biochemical incubator, and performing constant-temperature culture at 30 ℃ for 24 hours.
After the culture, 50mL of pure water and 1mL of aluminum potassium sulfate saturated solution were added to the flask, and the mixture was thoroughly shaken and filtered through slow quantitative filter paper into a 50mL sample bottle.
Using continuous flow analyzer to measure NO in standard curve solution and the above filtrate3 --N is measured continuously to giveNO in filtrate3 --the concentration of N.
The NO reduced in 24h per g of dry soil is calculated by using a formula3 -Mg of-N denotes.
The test results are as follows:
in the table, the difference of the nitrate reductase activity among the soils with different textures reaches an obvious level, and the standard deviation and the standard error can also prove that the result obtained by the method has good repeatability and higher accuracy.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
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.
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