CN1979134A - Analysis method for detecting activity of nitrous acid reductase in soil - Google Patents
Analysis method for detecting activity of nitrous acid reductase in soil Download PDFInfo
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- CN1979134A CN1979134A CN 200510047888 CN200510047888A CN1979134A CN 1979134 A CN1979134 A CN 1979134A CN 200510047888 CN200510047888 CN 200510047888 CN 200510047888 A CN200510047888 A CN 200510047888A CN 1979134 A CN1979134 A CN 1979134A
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Abstract
The invention relates to an analysis method for testing soil nitrite reductase that includes the following steps: weighing soil air dried sample 3 portions in three test tubes, adding 2-2.5ml 0.20%-0.25% sodium nitrate solution into two test tubes, adding distilled water into another test tube, adjusting pH value to 7-8.5; adding 1-2ml 0.5%-1.0% grape sugar solution into three test tubes, adding distilled water to 5-10ml, sealing, shaking to uniformity, culturing for 24h at 30 degree centigrade constant temperature; adding potash alum saturated solution, shaking to uniformity, filtering, adding developer into filtrate, taking colorimetric determination at 520nm, testing extinction A; calculating NO2-N content in sample. Comparing to the existing method, the invention simplified process and make the result more stable and reliable.
Description
Technical field
The present invention relates to the detection of activity of nitrous acid reductase in soil, specifically a kind of analytical approach that detects activity of nitrous acid reductase in soil.
Background technology
Nitrous acid reductase in soil is one of two kinds of key enzymes in the plain denitrification process of soil N, its active power affects the discharging of greenhouse gases oxides of nitrogen, also is subjected to simultaneously influencing strongly of farmland management measure, nature or artificial disturbance and edaphic condition such as moisture, temperature, the soil texture.Therefore, the detection to activity of nitrous acid reductase is significant.But up to the present, the mensuration of relevant activity of nitrous acid reductase all is to breathe out Prokofiev work now with reference to [Soviet Union] Φ .X. basically, method in " soil enzyme activities " book that Zheng Hongyuan, Zhou Likai, Zhang Desheng translate, and this kind method complex operation step, need special-purpose reagent bottle and suction filtration device to vacuumize cultivation, and test findings makes it be difficult to the qualitative comparison and the quantitative examination of adapted soil activity of nitrous acid reductase with the uncertainty of differing texture soil due otherness and anaerobic culture degree on incubation time and sample weighting amount.
Summary of the invention
The object of the present invention is to provide a kind of analytical approach that detects activity of nitrous acid reductase in soil; This analytical approach is on the principle basis of forefathers' assay method, determination step to activity of nitrous acid reductase has carried out partly improving (it is improved by vacuumize the process that realizes anaerobic culture under certain pressure), thereby the complicacy and the uncertainty of anaerobic culture degree have been reduced, make analysis result more reliable and more stable, favorable reproducibility.
For achieving the above object, the technical solution used in the present invention is:
A kind of analytical approach of activity of nitrous acid reductase in soil,
1) take by weighing 3 parts of soil air-dry samples that 1g sieves respectively in 3 test tubes, add the sodium nitrite solution of 2-2.5ml 0.20%-0.25% in 2 test tubes wherein, the distilled water that adds equivalent in addition in 1 test tube contrasts as sample, and accent PH is 7-8.5; The glucose solution that adds 1-2ml0.5%-1.0% respectively in 3 test tubes adds to 5-10ml with distilled water again as hydrogen donor then, seals, and shakes up 30 ℃ of constant temperature anaerobic cultures 24 hours; Do reagent blank contrast (do not add soil, all the other are handled with sample) simultaneously;
2) after cultivation finishes, invisible spectro native liquid mixture is transferred in the triangular flask fully, adds 1ml potassium alum saturated solution again with distilled water; Suspension is carefully swayed, and use fine and close filter paper filtering;
3) get filtrate 1ml and add developer in the 50ml volumetric flask, water is settled to scale then, and light absorption value A is measured in colorimetric estimation under 520nm;
4) the light absorption value A1 of the NaNO2 solution of a series of concentration known of colorimetric estimation under 520nm, the light absorption value A ' preparation standard curve with the concentration and the mensuration of NaNO2 solution obtains slope b;
5), calculate the NO in the liquid that measures according to light absorption value A and slope b
2The content S of-N, S=A*b;
6) NO in the calculation sample
2The content of-N;
Computing formula: [S
1-(S
2-S
3) * 50*V
1/ 1000*V
2]/W=mgNO
2 -In-N/g soil 24h the formula: S
1Be the NO in the reagent blank
2The content mg/L of-N, S
2Be the NO in the sample
2The content mg/L of-N, S
3Be the NO in the sample contrast
2The content mg/L of-N, 50 is the liquor capacity ml of constant volume, V
1Be leaching liquor cumulative volume ml, V
2For drawing the volume (ml) of filtrate, W is for claiming soil sample heavy g, and 1000 is unit conversion factor.
For acid ground, when transferring PH to be 7-8.5, adopt to add CaCO
3Mode.(for calacareous soil, CaCO
3Need not to add, because the CaCO of soil itself
3Amount both can satisfy the optimal pH of microorganism)
Described developer both can adopt the equal-volume mixed liquor (Γ p и cc reagent) of sulfanilic acid and alpha-Naphthol, also can adopt the mixed liquor of sulfanilamide (SN) and N-(1-naphthyl)-ethylenediamine hydrochloric acid.
Use diameter 10-15mm during anaerobic culture, the simple glass test tube of long 150-180mm; In pedotheque, add the liquid of 5-10ml, promptly guarantee the liquid level that the above 5cm of native face is above; Used substrate NaNO
2The concentration of solution is 0.20%-0.25%, and its concentration and consumption are suitably adjusted according to the different of nitrate reductase activity; Described amount of liquid is the addition sum of substrate solution, glucose solution and distilled water.
The inventive method is improved according to being:
Nitrous acid reductase in soil is the same with the soil nitrate reductase, all is the reductase that participates in the soil denitrification process.All need the anaerobic culture condition in the two mensuration process.The mensuration of nitrate reductase activity is with KNO
3Be substrate, by adding 2, the 4-dinitrophenol dinitrophenolate suppresses the activity of nitrite reductase, detects NO in the unit interval
2 -The growing amount of-N.The mensuration of activity of nitrous acid reductase then is with NaNO in this method
2Be substrate, after anaerobic culture in 24 hours, by NO in the unit interval
2 -The reduction of-N characterizes.Existing result of study shows, adds 5-10ml water (height of topsoil liquid level is kept more than the 5cm) in the 1-5g pedotheque and cultivates the dissolved oxygen DO in the native aqueous mixtures that forms and charge into N
2The anaerobic condition that formed in three minutes is similar, and waterflooding 6-10 hour can be so that the O in the native aqueous mixtures
2Content reduces to 0.
Compare with the analytical approach in past, the present invention has following advantage:
1. equipment needed thereby is simple, and cost is low.Place the simple glass test tube of 10-15mm * 100-150mm (diameter * highly) to get final product load weighted pedotheque, and do not need to realize the special-purpose Dewar bottle of the band grinding port plug that vacuumizes with being used for of mentioning in the original method.
2. need not special-purpose vacuum extractor, keep certain liquid to realize anaerobic culture condition in the mensuration process more than the soil layer by making.Need carry out under certain pressure (10-12 millimeter of mercury) when originally vacuumizing in the method, activity of nitrous acid reductase has bigger otherness with the variation of vacuum tightness, so causes bigger systematic error easily in the operating process.And the present invention realizes anaerobic condition by making the liquid level of keeping certain height more than the pedotheque, has overcome in this respect significantly deficiency, has both simplified operation steps, has reduced systematic error simultaneously again.
3. easy and simple to handle.Sterile soil (180 ℃, 3 hours) in contrast need be set in original method, and this method only needs not add substrate solution with one, contrast gets final product all the other steps as sample with the same processing of pedotheque.
Embodiment
The reagent preparation:
1.0.20%-0.25% NaNO
2Solution: take by weighing 0.20-0.25g NaNO
2Be settled to 100ml with distilled water.
2.0.5%-1.0% glucose solution: take by weighing 0.5g-1.0g glucose, be settled to 100ml with distilled water.
3.CaCO
3, analytical reagent.
4. potassium alum saturated solution: aluminium potassium sulfate is dissolved in the distilled water, until supersaturation.
5. developer 1 (Γ p и cc reagent): with proportion is that 1.04 acetic acid is prepared 0.1% alpha-naphthylamine solution and 0.5% sulfanilic acid solution (b) respectively, with preceding equal-volume a, b is mixed.
The acetic acid of proportion 1.04 (acetic acid that is equivalent to percent by weight 35%) configuration: get the acetic acid of 175ml100%, be settled to 500ml, be 35%, the acetic acid of proportion 1.04.
Take by weighing 0.2g alpha-naphthylamine percent by weight 35%, the acetate dissolution of proportion 1.04 also is settled to 200ml, and this is (a) liquid.
Take by weighing 1.0g sulfanilic acid percent by weight 35%, the acetate dissolution of proportion 1.04 also is settled to 200ml, and this is (b) liquid.
Developer 2: take by weighing 2g sulfanilamide (SN) and 0.1gN-(1-naphthyl)-ethylenediamine hydrochloric acid and be dissolved in the 150ml distilled water, add the 20ml strong phosphoric acid, be cooled to room temperature, be settled to 200ml with distilled water.This liquid should be colourless solution, and needs preparation on the same day.
6. standard stock solution (250mgNO
2 --N/L): dissolving 1.2314gNaNO
2And be settled to 1000ml with distilled water, and being put in 4 ℃ of refrigerators and preserving, the holding time should not be of a specified duration excessively, is advisable to be no more than several weeks.
7. the preparation of typical curve: draw above-mentioned standard stock solution 1ml, be settled to 100ml, this is 2.5mgNO
2 -The solution of-N/L.Draw this liquid 0,1,2,3,4,5ml more respectively in the 50ml volumetric flask, add a small amount of distilled water and 4ml developer, and use the distilled water constant volume.The amount that this standard series contains nitrite nitrogen is respectively 0,0.05,0.1,0.15,0.2,0.25mgNO
2 --N/L.The NaNO of a series of concentration known of colorimetric estimation under 520nm
2The light absorption value A1 of solution is with NaNO
2The concentration of solution and the light absorption value of mensuration A ' preparation standard curve obtain slope b;
Operation steps:
1. take by weighing 3 parts of soil air-dry samples that 1g crosses 1mm sieve in 3 test tubes, add 20mgCaCO respectively
3(for calacareous soil, CaCO
3Need not to add, because the CaCO of soil itself
3Amount both can satisfy the optimal pH of microorganism), carefully mix the sodium nitrite solution that the back adds 2-2.5ml0.20%-0.25% in 2 test tubes wherein, the distilled water of adding equivalent contrasts as sample in 1 test tube in addition.The glucose solution that adds 1-2ml 0.5%-1.0% respectively in 3 test tubes adds to 5-10ml with distilled water again as hydrogen donor then, and air-locked beyond the Great Wall rubber plug shakes up, and places 30 ℃ of constant temperature ovens to cultivate 24 hours.Do reagent blank contrast (do not add soil, all the other are handled with sample) simultaneously.
2. after cultivating end, invisible spectro native liquid mixture is transferred in the 100ml triangular flask fully, adds 1ml potassium alum saturated solution again with 50ml distilled water.Suspension is carefully swayed, and use fine and close filter paper filtering.
3. get filtrate 1ml in the 50ml volumetric flask, add a small amount of distilled water and 4ml developer, water is settled to scale then.After 15 minutes, colorimetric under 520nm is measured light absorption value A.
4. according to light absorption value A and slope b, calculate the NO in the liquid that measures
2The content S of-N, S=A*b;
5. NO in the calculation sample
2The content of-N;
Computing formula: [S
1-(S
2-S
3) * 50*V
1/ 1000*V
2]/W=mgNO
2 --N/g soil 24h
In the formula: S
1Be the NO in the reagent blank
2The content of-N (mg/L) S
2Be the NO in the sample
2The content of-N (mg/L), S
3Be the NO in the sample contrast
2The content of-N (mg/L), 50 is the liquor capacity (ml) of constant volume, V
1Be leaching liquor cumulative volume (ml), V
2For drawing the volume (ml) of filtrate, W is for claiming soil sample heavy (g), and 1000 is unit conversion factor;
Embodiment 1
Select four kinds of pedotheques for use through different disposal, wherein 1 is contrast, 2,3, No. 4 samples amount of adding certain compound strengthens successively, 25 ℃ of temperature, after cultivating for 1 week under the condition of soil moisture content maintenance 20% (to dry base), detect of the influence of the addition of this compound to activity of nitrous acid reductase in soil.The concrete analysis step is as follows:
1. take by weighing 3 parts of soil air-dry samples that 1g crosses 1mm sieve in 3 test tubes, add 20mgCaCO respectively
3, careful mixing the sodium nitrite solution that the back adds 2ml 0.25% in 2 test tubes wherein, the distilled water of adding equivalent contrasts as sample in 1 test tube in addition.The glucose solution that adds 2ml 0.5% respectively in 3 test tubes adds distilled water 2ml again as hydrogen donor then, and air-locked beyond the Great Wall rubber plug shakes up, and places 30 ℃ of constant temperature ovens to cultivate 24 hours.Do reagent blank contrast (do not add soil, all the other are handled with sample) simultaneously
2. after cultivating end, invisible spectro native liquid mixture is transferred in the 100ml triangular flask fully, adds 1ml potassium alum saturated solution again with 50ml distilled water.Suspension is carefully swayed, and use fine and close filter paper filtering.
3. get filtrate 1ml in the 50ml volumetric flask, add a small amount of distilled water and 4ml developer 1 (Γ p и cc reagent), water is settled to scale then.After 15 minutes, colorimetric under 520nm is measured light absorption value A.
4. according to light absorption value A and slope b, calculate the NO in the liquid that measures
2The content S of-N, S=A*b.
5. NO in the calculation sample
2The content of-N.
Test findings is as follows:
| Handle number | Repeat | Activity of nitrous acid reductase (mgNO 2 --N/g·24h) | Standard deviation | The significance of difference (P＜0.01) | |
| Measured value | Average | ||||
| 1 | Repeat 1 | 0.40 | 0.43 | 0.02 | C |
| Repeat 2 | 0.43 | ||||
| Repeat 3 | 0.45 | ||||
| 2 | Repeat 1 | 0.55 | 0.56 | 0.01 | B |
| Repeat 2 | 0.56 | ||||
| Repeat 3 | 0.56 | ||||
| 3 | Repeat 1 | 0.61 | 0.62 | 0.01 | A |
| Repeat 2 | 0.63 | ||||
| Repeat 3 | 0.62 | ||||
| 4 | Repeat 1 | 0.64 | 0.64 | 0.01 | A |
| Repeat 2 | 0.63 | ||||
| Repeat 3 | 0.64 | ||||
By data in the table as can be seen, result difference was remarkable between each was handled, and lower standard difference shows that the deviation between this analytical approach result is little, test precision height, favorable reproducibility.
Embodiment 2
The pedotheque of four kinds of different disposal is carried out the mensuration of activity of nitrous acid reductase, and the concrete analysis step is as follows: substrate NaNO
2Concentration be 0.20%, add 2ml; The concentration of glucose is 1% adding 1ml, adds 5ml distilled water then, adds developer 2 during colour developing, and all the other operation stepss are with embodiment 1.Test findings is as follows:
| Handle number | Repeat | Activity of nitrous acid reductase (mgNO 2 --N/g·24h) | Standard deviation | The significance of difference (P＜0.01) | |
| Measured value | Average | ||||
| Sample 1 | Repeat 1 | 0.20 | 0.20 | 0.01 | D |
| Repeat 2 | 0.21 | ||||
| Repeat 3 | 0.20 | ||||
| Sample 2 | Repeat 1 | 0.25 | 0.27 | 0.02 | C |
| Repeat 2 | 0.27 | ||||
| Repeat 3 | 0.28 | ||||
| Sample 3 | Repeat 1 | 0.38 | 0.38 | 0.01 | B |
| Repeat 2 | 0.39 | ||||
| Repeat 3 | 0.38 | ||||
| Sample 4 | Repeat 1 | 0.52 | 0.55 | 0.03 | A |
| Repeat 2 | 0.55 | ||||
| Repeat 3 | 0.58 | ||||
Data have shown the stability of analysis result equally in the table, and the difference between each processing reaches the utmost point level of signifiance.
Claims (4)
1. the analytical approach of an activity of nitrous acid reductase in soil is characterized in that:
1) take by weighing 3 parts of soil air-dry samples that 1g sieves respectively in 3 test tubes, add the sodium nitrite solution of 2-2.5ml 0.20%-0.25% in 2 test tubes wherein, the distilled water that adds equivalent in addition in 1 test tube contrasts as sample, and accent PH is 7-8.5; The glucose solution that adds 1-2ml0.5%-1.0% respectively in 3 test tubes adds to 5-10ml with distilled water again as hydrogen donor then, seals, and shakes up 30 ℃ of constant temperature anaerobic cultures 24 hours; Do the reagent blank contrast simultaneously;
2) after cultivation finishes, invisible spectro native liquid mixture is transferred in the triangular flask fully, adds 1ml potassium alum saturated solution again with distilled water; Sway, filter;
3) get filtrate 1ml and add developer in the 50ml volumetric flask, water is settled to scale then, and light absorption value A is measured in colorimetric estimation under 520nm;
4) the light absorption value A1 of the NaNO2 solution of a series of concentration known of colorimetric estimation under 520nm, the light absorption value A ' preparation standard curve with the concentration and the mensuration of NaNO2 solution obtains slope b;
5), calculate the NO in the liquid that measures according to light absorption value A and slope b
2The content S of-N, S=A*b;
6) NO in the calculation sample
2The content of-N;
Computing formula: [S
1-(S
2-S
3) * 50*V
1/ 1000*V
2]/W=mgNO
2 -In-N/g soil 24h the formula: S
1Be the NO in the reagent blank
2The content mg/L of-N, S
2Be the NO in the sample
2The content mg/L of-N, S
3Be the NO in the sample contrast
2The content mg/L of-N, 50 is the liquor capacity ml of constant volume, V
1Be leaching liquor cumulative volume ml, V
2For drawing the volume (ml) of filtrate, W is for claiming soil sample heavy g, and 1000 is unit conversion factor.
2. analytical approach according to claim 1 is characterized in that: for acid ground, when transferring PH to be 7-8.5, adopt to add CaCO
3Mode.
3. analytical approach according to claim 1 is characterized in that: described developer both can adopt the equal-volume mixed liquor of sulfanilic acid and alpha-Naphthol, also can adopt the mixed liquor of sulfanilamide (SN) and N-(1-naphthyl)-ethylenediamine hydrochloric acid.
4. analytical approach according to claim 1 is characterized in that: use diameter 10-15mm during anaerobic culture, the simple glass test tube of long 150-180mm; In pedotheque, add the liquid of 5-10ml, promptly guarantee the liquid level that the above 5cm of native face is above.
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|---|---|---|---|
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|---|---|---|---|
| CNB2005100478885A CN100494994C (en) | 2005-11-30 | 2005-11-30 | Analysis method for detecting activity of nitrous acid reductase in soil |
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|---|---|
| CN1979134A true CN1979134A (en) | 2007-06-13 |
| CN100494994C CN100494994C (en) | 2009-06-03 |
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596540A (en) * | 2016-12-20 | 2017-04-26 | 江苏食品药品职业技术学院 | Chromogenic agent and watered cow milk determination method |
| CN109682808A (en) * | 2019-03-07 | 2019-04-26 | 济南大学 | A kind of improved faintly acid water body Central Asia nitrate nitrogen content measuring method |
| CN111474127A (en) * | 2020-04-27 | 2020-07-31 | 济南大学 | Improved nitrite reductase assay |
| CN113624755A (en) * | 2021-08-13 | 2021-11-09 | 九江学院 | Method for rapidly determining recovery effect of degraded soil |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU657344A1 (en) * | 1975-11-21 | 1979-04-15 | Почвенный Инстииут Им.В.В.Докучаева | Method of preparing soil sample for determining activity of extracellular nitratereductases and nitritereductases |
| CN1680587A (en) * | 2005-01-27 | 2005-10-12 | 中国科学院等离子体物理研究所 | Rapid detection of lipase activity of crop seed |
-
2005
- 2005-11-30 CN CNB2005100478885A patent/CN100494994C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596540A (en) * | 2016-12-20 | 2017-04-26 | 江苏食品药品职业技术学院 | Chromogenic agent and watered cow milk determination method |
| CN109682808A (en) * | 2019-03-07 | 2019-04-26 | 济南大学 | A kind of improved faintly acid water body Central Asia nitrate nitrogen content measuring method |
| CN111474127A (en) * | 2020-04-27 | 2020-07-31 | 济南大学 | Improved nitrite reductase assay |
| CN113624755A (en) * | 2021-08-13 | 2021-11-09 | 九江学院 | Method for rapidly determining recovery effect of degraded soil |
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| Publication number | Publication date |
|---|---|
| CN100494994C (en) | 2009-06-03 |
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