CN101294191A - Analysis method for testing ammonium oxidizing enzyme activity in soil - Google Patents
Analysis method for testing ammonium oxidizing enzyme activity in soil Download PDFInfo
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- CN101294191A CN101294191A CNA2007100111199A CN200710011119A CN101294191A CN 101294191 A CN101294191 A CN 101294191A CN A2007100111199 A CNA2007100111199 A CN A2007100111199A CN 200710011119 A CN200710011119 A CN 200710011119A CN 101294191 A CN101294191 A CN 101294191A
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Abstract
The invention relates to an analytical method for detecting the activity of ammonium oxidase in soil. The analytical method comprises the following steps: first, n parts of fresh soil samples are weighed and placed in triangular flasks; a substrate ammonium sulfate solution is added in the triangular flasks, the number of which is n minus 1; equivalent distilled water is added in the other test tube to replace the substrate as sample contrast; a sodium chlorate solution is added in all the triangular flasks, shaken uniformly, and cultivated at a constant temperature after capping the tasks with cocks; second, after the cultivation, a potassium chloride solution is added, vibrated and filtered; third, a given amount of filter liquor is sucked into a color comparison test tube; an ammonium chloride buffer solution and a color-developing agent are added, mixed uniformly and developed; fourth, a spectrophotometric method is used for detecting an absorbance value A by color comparison at 520nm; fifth, the content of nitrite nitrogen is calculated according to a standard curve, and then the activity of the ammonia oxidation enzyme is calculated. The analytical method has the following advantages: 1) the operational procedure is simplified, and the uncertainty is reduced during the experiment process; 2) the dependency on requirements for equipment is reduced; 3) the method has high accuracy, and is easy to operate; 4) the result is stable and reliable, and the reproducibility is good.
Description
Technical field
The present invention relates to the mensuration of ammonium oxidizing enzyme activity in the soil, specifically a kind of analytical procedure that detects ammonium oxidizing enzyme activity in the soil.
Background technology
The ammonium nitrogen that ammonium oxidizing enzyme in the soil can will form in the Soil Nitrogen metabolic process is oxidized to nitrite nitrogen, further is oxidized to nitric nitrogen under the effect of other enzyme, causes the loss of nitrogen, reduces the utilising efficiency of nitrogen.
Therefore, the power of ammonium oxidizing enzyme activity has influence on the size of nitrogen loss in the Soil Nitrogen metabolic process in the soil, the utilising efficiency of remote effect nitrogenous fertilizer.The activity of ammonium oxidizing enzyme is subjected to influencing strongly of edaphic condition such as moisture, temperature, the soil texture in the soil, also is subjected to the influence of farming operation system, control measures and Different Crop simultaneously.Therefore, the check to ammonium oxidizing enzyme activity in the soil has very important significance.And up to the present, the mensuration of relevant ammonium oxidizing enzyme activity does not form a reliable method, just the measuring method with other enzyme is that similar mensuration is carried out on the basis, the otherness and the uncertainty of different cultivations batch have so just been caused, make it be difficult to the qualitative comparison and the quantitative examination of ammonium oxidizing enzyme activity in the adapted soil, reduced conventional efficient.
Summary of the invention
The object of the present invention is to provide the detection method of ammonium oxidizing enzyme activity in a kind of soil; This method is on the principle basis of using for reference other enzyme testing method, characteristic according to ammonium oxidizing enzyme, determination step and coloration method to other enzymic activity improve, formed the method for suitable mensuration ammonium oxidizing enzyme activity, this method has reduced the complicacy of testing sequence, make analytical results more accurately reliable, it is better to analyze circulation ratio.
For achieving the above object, the technical solution used in the present invention is:
A kind of analytical procedure that detects ammonium oxidizing enzyme activity in the soil,
1) taking by weighing the bright earth sample n of 5-10g part inserts respectively in n the triangular flask, n 〉=2, the substrate ammonium sulfate working fluid that in n-1 triangular flask wherein, adds 5-15mL 1-10mM, the distilled water that adds equivalent in other 1 test tube replaces substrate to contrast as sample, the sodium chlorate solution's (sodium chlorate solution who adds in the test is used for suppressing the oxidation of nitrite nitrogen to nitric nitrogen) who in all triangular flasks, adds 0.2-0.5mL1.0-2.0M then, mixing, build bottle stopper, be placed on constant temperature culture 2 ± 0.2h in 37 ± 1 ℃ of constant temperature culture; Do the reagent blank contrast simultaneously;
2) after cultivation finishes, add 5-20mL 1.8-2moLKCL solution respectively in triangular flask, mixing behind vibration 1 ± 0.5h, filters;
3) draw 5-10ml filtrate respectively in the colorimetric test tube, add 3-5ml 0.19M-0.2M chloride buffer solution and 2-5ml developer, mixing, colour developing 15 ± 5min;
4) measure absorbance A with spectrophotometry 520nm place;
5) make the working standard curve: prepare a series of nitrite nitrogen standardized solution, draw a series of standardized solution 5-10ml respectively in different colorimetric test tubes, add 3-5ml 0.19-0.2M chloride buffer solution and 2-5ml developer respectively, mixing, colour developing 15 ± 5min measures absorbance A with sample at 520nm '; Make the working standard curve with the series concentration of nitrite nitrogen solution and the light absorption value A ' of mensuration, obtain slope b;
6), calculate the concentration S of the nitrite nitrogen in the filtrate of measuring, S=A*b according to light absorption value A and slope b;
7) calculation sample Central Asia nitrate nitrogen content;
8) calculate ammonium oxidizing enzyme activity:
Calculation formula is: (S-C) * V
1* V
2/ (V
3.W)=μ g NO
2-N/g dry ground .2h
In the formula: S is mean value (the mg NO of sample determination
2-N/L); C is control value (mgNO
2-N/L); V
1Be the liquid volume in the colorimetric test tube (mL); V
2Volume (mL) for extracting solution; V
3Be the filtrate volume of drawing (mL); W is dry ground quality (g).
Described making working standard curve process is to draw 1000mg NO
2The nitrite nitrogen standard stock solution 5mL of-N/L, constant volume is to 500mL, and this is 10mg NO
2The solution of-N/L; Draw this liquid 0,2,4,8 more respectively, 10mL behind the adding 5-15mL 1.8-2M KCI solution, uses the distilled water constant volume, if any sedimentation and filtration in the 100mL volumetric flask; The amount that this standard series contains nitrite nitrogen is respectively 0,0.2, and 0.4,0.8 and 1.0mg NO
2-N/L; Draw a series of standardized solution 5-10ml respectively in different colorimetric test tubes, add 3-5ml 0.19-0.2M chloride buffer solution and 2-5ml developer, mixing, colour developing 15 ±
5Min measures absorbance A with sample at 520nm '; Make the working standard curve with the series concentration of nitrite nitrogen solution and the light absorption value A ' of mensuration, obtain slope b.
The employed distilled water of described experiment before the use, need be heated to the O in the boiling expeling water
2, sealing is cooled to room temperature then; Developer sulfanilamide (SN) and N-(how basic 1-is)-ethylenediamine salt acid solution is necessary for colourless and is preparation on the same day, and their adding is excessive with respect to nitrite anions;
The improved foundation of the inventive method
Ammonium oxidizing enzyme in the soil is to participate in nitrogen in the soil to be oxidized to the general name of all enzymes of nitrite nitrogen process from ammonium nitrogen, needs the anaerobism culture condition in the mensuration process.Measuring nitric acid reductase activity is with saltpetre (KNO
3) be substrate, by adding 2, the 4-dinitrophenol suppresses the activity of nitrite reductase, detects NO in the unit time
2 -The growing amount of-N; The measuring method of activity of nitrous acid reductase then is with Sodium Nitrite (NaNO
2) be substrate, cultivate through 24 hours anaerobism, by NO in the unit time
- 2 -The reduction of N characterizes; Identical therewith, the mensuration of present method soil ammonium oxidizing enzyme activity is with ammonium sulfate ((NH
4)
2SO
4) be substrate, through the constant temperature culture of 2h, by the NO of Units of Account unit soil in the time
2 -The growing amount of-N characterizes the activity of ammonium oxidizing enzyme.This is tested native face and is submerged in below the liquid level, and purpose is exactly to create the good foster environment of detesting for culture experiment.
Measuring principle used in the present invention is:
Ammonium oxidizing enzyme reaction needed in the soil is carried out in detesting foster environment, and the soil of adding is submerged in below the liquid level, and ammonium nitrogen wherein can only oxidized generation nitrite nitrogen.Therefore, this test intended is by reaction back NO
2 -The growing amount of-N characterizes the soil ammonium oxidizing enzyme activity.
Compare with the analytical procedure of other enzyme, advantage of the present invention mainly contains:
1) required equipment simple, reduced experimentation cost.Culture experiment among the present invention is to carry out in the triangular flask of 50mL; Minimizing is to the dependency of equipment requirements.
2) method accuracy height is easy to operate.Employed method colour developing is simple, the accuracy that has improved sample determination.
3) simple to operate, the complicacy of minimizing operating process; Analytical results is reliable and stable, favorable reproducibility.The present invention has simplified operation steps, has reduced instability of experiment process; The mensuration of other enzymes need be provided with sterile soil in contrast, and the present invention only needs a sample contrast that does not add substrate.Simultaneously and since involved in the present invention to cultural method reduced the complicacy and the uncertainty of anaerobism culturing process, so reproducibility of analysis results is very good.
Embodiment
The reagent preparation:
1. substrate storing solution (10mM): after taking by weighing the dissolving of 1.3214g ammonium sulfate, fixed molten to 1000ml with distilled water.
2. substrate working fluid (1mM): draw above-mentioned substrate storing solution 100ml, dilute with water is fixed molten to 1000ml.Amount to that nitrogen content is 0.0280mg/ml in the substrate.
3.1.5M sodium chlorate solution: take by weighing 15.97g NaCIO
3, behind dissolved in distilled water, fixed molten to 100ml.
4.2M KCI solution: take by weighing 149.12g KCI in distilled water, fixed molten to 1000ml.
5. ammonium chloride buffer (0.19M, pH 8.5): dissolving 10g ammonium chloride is used NH in distilled water
4OH regulates pH to 8.5, and is fixed molten to 1000ml.
6. developer: take by weighing 2g sulfanilamide (SN) and 0.1g N-(1-how base)-quadrol hydrochloric acid and be dissolved in the 150ml distilled water, add the 20ml strong phosphoric acid again, be cooled to after the room temperature fixed molten to 200ml with distilled water.This solution should be colourless, and must preparation on the same day.
7. standard reserving solution (1000mg NO
2-N/L): dissolving 4.9257g NaNO
2And fixed molten with distilled water to 1000ml, be put in 4 ℃ of refrigerators and preserve, the shelf time is unsuitable long, is advisable to be no more than several weeks.
8. the preparation of typical curve: draw above-mentioned standard stock solution 5mL, fixed molten to 500mL, this is 10mg NO
2The solution of-N/L.Draw this liquid 0,2,4,8 more respectively, 10mL is in the 100mL volumetric flask, behind the KCI solution of adding 5-15mL 2M, with distilled water fixed molten (if any sedimentation and filtration).The amount that this standard series contains nitrite nitrogen is respectively 0,0.2, and 0.4,0.8 and 1.0mg NO
2-N/L.
Operation steps:
1) takes by weighing the bright soil of 5g and insert (n 〉=2) in 4 50mL triangular flasks respectively for 4 parts, the substrate ammonium sulfate working fluid that in 3 triangular flasks wherein, adds 10mL 1mM, the distilled water that adds equivalent in other 1 test tube replaces substrate to contrast as sample, in all triangular flasks, add 0.2mL 1.5M sodium chlorate solution then, mixing, build bottle stopper, be placed on constant temperature culture 2h in 37 ℃ of constant temperature culture; Do the sample contrast simultaneously;
2) after cultivation finishes, add 20ml 2moLKCL solution, mixing behind the vibration 1h, filters;
3) draw 5ml filtrate in the colorimetric test tube, add 3ml 0.19M chloride buffer solution and 2ml developer, mixing, colour developing 15min;
4) measure absorbance A with spectrophotometry 520nm place;
5) make the working standard curve: the nitrite nitrogen standardized solution 5ml that draws a series of concentration respectively adds 3ml 0.19M chloride buffer solution and 2ml developer in the colorimetric test tube, mixing, colour developing 15min measures absorbance A with sample at 520nm '.Series concentration 0,0.2,0.4,0.8 and 1.0mg NO with nitrite nitrogen solution
2The light absorption value A ' of-N/L and mensuration makes the working standard curve, obtains slope b;
6), calculate the concentration S of the nitrite nitrogen in the solution of measuring, S=A*b according to light absorption value A and slope b
7) calculation sample Central Asia nitrate nitrogen content;
8) calculate ammonium oxidizing enzyme activity:
Calculation formula is: (S-C) * V
1* V
2/ (V
3.W)=μ g NO
2-N/g dry ground .2h
In the formula: S is mean value (the mg NO of sample determination
2-N/L); C is control value (mgNO
2-N/L); V
1Be the liquid volume in the colorimetric test tube (mL); V
2Volume (mL) for extracting solution; V
3Be the filtrate volume of drawing (mL); W is dry ground quality (g).
Embodiment 1
The employed black earth of present embodiment picks up from the ecological testing station of the Helen of the Chinese Academy of Sciences, establishes three different processing: No. 1 position contrast, both without any processing with add any soil additive at 25 ℃ of normal soils of cultivating more than the 24h; No. 2 for adding nitrification inhibitor DCD and the soil more than 25 ℃ of cultivation 24h; No. 3 for adding nitrification inhibitor DMPP and the soil more than 25 ℃ of cultivation 24h.Soil moisture content is 20% (wind desiceted soil is heavy).Add the soil of nitrification inhibitor No. 2 and No. 3, wherein the addition of two kinds of inhibitor all is 50ppm, detects the ammonium oxidizing enzyme activity of three kinds of processing with aforesaid method.
It is as follows that every kind of soil must be made a concrete analysis of step:
1) takes by weighing the bright soil of 5g and insert (n 〉=2) in 4 50mL triangular flasks respectively for 4 parts, the substrate ammonium sulfate working fluid that in 3 triangular flasks wherein, adds 10mL 1mM, the distilled water that adds equivalent in other 1 test tube replaces substrate to contrast as sample, in all triangular flasks, add 0.2mL 1.5M sodium chlorate solution then, mixing, build bottle stopper, be placed on constant temperature culture 2h in 37 ℃ of constant temperature culture; Do the sample contrast simultaneously;
2) after cultivation finishes, add 20ml 2moLKCL solution, mixing behind the vibration 1h, filters;
3) draw 5ml filtrate in the colorimetric test tube, add 3ml 0.19M chloride buffer solution and 2ml developer, mixing, colour developing 15min;
4) measure absorbance A with spectrophotometry 520nm place;
5) make the working standard curve: the nitrite nitrogen standardized solution 5ml that draws a series of concentration respectively adds 3ml 0.19M chloride buffer solution and 2ml developer in the colorimetric test tube, mixing, colour developing 15min measures absorbance A with sample at 520nm '.Series concentration 0,0.2,0.4,0.8 and 1.0mg NO with nitrite nitrogen solution
2The light absorption value A ' of-N/L and mensuration makes the working standard curve, obtains slope b;
6), calculate the concentration S of the nitrite nitrogen in the solution of measuring, S=A*b according to light absorption value A and slope b
7) calculation sample Central Asia nitrate nitrogen content;
8) calculate ammonium oxidizing enzyme activity
Test-results is as follows:
By data in the table as can be seen, result difference reached conspicuous level between each was handled, and less standard difference shows that the deviation between this analytical procedure result is less, and tolerance range is higher, favorable reproducibility.
Embodiment 2
The employed three kinds of different brown earth of planting plant of present embodiment are all adopted in ecological experiment station, Chinese Academy of Sciences Shenyang: wherein No. 4 soil preceding crops are paddy rice, and No. 5 soil preceding crops are corn, and No. 6 soil preceding crops are soybean.The soil of three kinds of different cropping systems is 20% (wind desiceted soil is heavy) in water content all, and temperature is measured its ammonium oxidizing enzyme activity cultivating more than the 24h under 25 ℃ of conditions, and concrete steps are as follows: the substrate ammonium sulfate concentrations is 2mM, adds 5mL, and all the other steps are with example 1.
Test-results is as follows:
Data have shown the stability and the higher precision of analytical results equally in the table.
Claims (4)
1. analytical procedure that detects ammonium oxidizing enzyme activity in the soil is characterized in that:
1) taking by weighing 5-10g fresh soil sample product n part inserts respectively in n the triangular flask, n 〉=2, the substrate ammonium sulfate working fluid that in n-1 triangular flask wherein, adds 5-15mL 1-10mM, the distilled water that adds equivalent in other 1 test tube replaces substrate to contrast as sample, the sodium chlorate solution's (sodium chlorate solution who adds in the test is used for suppressing the oxidation of nitrite nitrogen to nitric nitrogen) who in all triangular flasks, adds 0.2-0.5mL 1.0-2.0M then, mixing, build bottle stopper, be placed on constant temperature culture 2 ± 0.2h in 37 ± 1 ℃ of constant temperature culture; Do the reagent blank contrast simultaneously;
2) after cultivation finishes, add 5-20mL 1.8-2moLKCL solution respectively in triangular flask, mixing behind vibration 1 ± 0.5h, filters;
3) draw 5-10ml filtrate respectively in the colorimetric test tube, add 3-5ml 0.19-0.2M chloride buffer solution and 2-5ml developer, mixing, colour developing 15 ± 5min;
4) measure absorbance A with spectrophotometry 520nm place;
5) make the working standard curve: prepare a series of nitrite nitrogen standardized solution, draw a series of standardized solution 5-10ml respectively in different colorimetric test tubes, add 3-5ml 0.19-0.2M chloride buffer solution and 2-5ml developer respectively, mixing, colour developing 15 ± 5min measures absorbance A with sample at 520nm '; Make the working standard curve with the series concentration of nitrite nitrogen solution and the light absorption value A ' of mensuration, obtain slope b;
6), calculate the concentration S of the nitrite nitrogen in the filtrate of measuring, S=A*b according to light absorption value A and slope b;
7) calculation sample Central Asia nitrate nitrogen content;
8) calculate ammonium oxidizing enzyme activity:
Calculation formula is: (S-C) * V
1* V
2/ (V
3.W)=μ g NO
2-N/g dry ground .2h
In the formula: S is mean value (the mg NO of sample determination
2-N/L); C is control value (mgNO
2-N/L); V
1Be the liquid volume in the colorimetric test tube (mL); V
2Volume (mL) for extracting solution; V
3Be the filtrate volume of drawing (mL); W is dry ground quality (g).
2. analytical procedure according to claim 1 is characterized in that: described making working standard curve process is to draw 1000mg NO
2The nitrite nitrogen standard stock solution 5mL of-N/L, constant volume is to 500mL, and this is 10mg NO
2The solution of-N/L; Draw this liquid 0,2,4,8 more respectively, 10mL behind the adding 5-15mL 1.8-2M KCI solution, uses the distilled water constant volume, if any sedimentation and filtration in the 100mL volumetric flask; The amount that this standard series contains nitrite nitrogen is respectively 0,0.2, and 0.4,0.8 and 1.0mgNO
2-N/L; Draw a series of standardized solution 5-10ml respectively in different colorimetric test tubes, add 3-5ml0.19-0.2M chloride buffer solution and 2-5ml developer, mixing, colour developing 15 ± 5min measures absorbance A with sample at 520nm '; Make the working standard curve with the series concentration of nitrite nitrogen solution and the light absorption value A ' of mensuration, obtain slope b.
3. analytical procedure according to claim 1 is characterized in that: the employed distilled water of described experiment before the use, need be heated to the O in the boiling expeling water
2, sealing is cooled to room temperature then.
4. analytical procedure according to claim 1 is characterized in that: developer sulfanilamide (SN) and N-(how basic 1-is)-ethylenediamine salt acid solution is necessary for colourless and is preparation on the same day, and their adding is excessive with respect to nitrite anions.
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|---|---|---|---|
| CNA2007100111199A CN101294191A (en) | 2007-04-27 | 2007-04-27 | Analysis method for testing ammonium oxidizing enzyme activity in soil |
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|---|---|---|---|
| CNA2007100111199A CN101294191A (en) | 2007-04-27 | 2007-04-27 | Analysis method for testing ammonium oxidizing enzyme activity in soil |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102353713A (en) * | 2011-07-06 | 2012-02-15 | 中国科学院长春应用化学研究所 | Detection methods for activity and substrate concentration of oxidase |
| CN102478525A (en) * | 2010-11-30 | 2012-05-30 | 中国科学院沈阳应用生态研究所 | Method for determining soil nitrification potential |
-
2007
- 2007-04-27 CN CNA2007100111199A patent/CN101294191A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102478525A (en) * | 2010-11-30 | 2012-05-30 | 中国科学院沈阳应用生态研究所 | Method for determining soil nitrification potential |
| CN102353713A (en) * | 2011-07-06 | 2012-02-15 | 中国科学院长春应用化学研究所 | Detection methods for activity and substrate concentration of oxidase |
| CN102353713B (en) * | 2011-07-06 | 2014-08-27 | 中国科学院长春应用化学研究所 | Detection methods for activity and substrate concentration of oxidase |
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Open date: 20081029 |