CN102478525A - Method for determining soil nitrification potential - Google Patents
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Abstract
The invention relates to a method for determining the soil ammoxidation potential. According to the method, a soil suspension culture process is adopted, quantitative KClO3 and NaClO3 are added to inhibit the oxidation of NO2<-> into NO3<->, NO2<-> in soil is extracted by KCl after culturing for 24h; and the content of NO2<-> in the obtained filtrate after filtering is determined by adopting spectrophotometry, and the difference between the content of NO2<-> before the culture and the content of NO2<-> after the culture represents the soil nitrification potential. The method of the invention has the advantages of good repeatability, high reappearance, and simplicity and easy implementation.
Description
Technical field
The present invention relates to the mensuration of soil nitrification potentiality, a kind of specifically new soil nitrification (ammoxidation) acts on the assay method of potentiality.
Background technology
Nitrification (Nitrification) is meant that the ammonia (or ammonium) that under the effect of nitrobacteria, makes in the soil changes into the process of nitrate.Nitrification (NH
3→ NO
2 -→ NO
3 -) microbial metabolism comprise nitrification (being ammoxidation) and two processes of nitrosification; Carry out (document 1: He Jizheng by ammonia oxidizing bacteria and NOB two quasi-microorganism catalysis respectively; Zhang Limei, the ecological and nitrogen cycle progress of ammonia oxidation microbiological, Acta Ecologica Sinica; 2009,29 (1): 406-415).Ammoxidation is first reactions step of nitrification; Also being rate-limiting step, is the key link of global nitrogen cycle, with very relevant (the document 2:Prosser J I.Autotrophicnitrification in bacteria.Advances in Microbial Physiology of effective utilization of nitrogen; 1989; 30:125-181. document 3:Deboer W, Gunnewiek P J A K, Vecnhuis M; Etal.Nitrification at low pH by aggregated chemolithotropkicbacteria.Applied and Environmental Microbiology.1991,57:3600-3604.); About the problem of environmental pollution that causes because of nitrification, also receive researchers' attention day by day, it is significant therefore to measure nitrification.
The method that soil nitrification is measured on existing rank both at home and abroad adopts soil cultivation (document 4: Lu Rukun more; The soil agrochemistry analytical approach; Beijing: Chinese agriculture science and technology publishing house; 2000 :), key step comprises: 1) cultivate: get be equivalent to the 20g dry ground fresh soil in the container that size is fit to (wide-necked bottle of 100-250mL or triangular flask), in every bottle by the ratio adding (NH of 100g dry ground 25mg N
4)
2SO
4According to test objective, regulate soil moisture (general water holding capacity 60%).Be provided with simultaneously and do not add (NH
4)
2SO
4Control treatment, build bottleneck with vinyl film immediately then, 30 ℃ of constant temperature culture 5-20d.2) measure:, filter behind the concussion 1h with 100ml KCl solution (2mol/L) lixiviate soil sample; Get the some ml of filtrating, can adopt MgO-to decide nitrogen alloy (Vad alloy) way of distillation and measure or its NO of cadmium reduction-diazonium coupling colorimetric method for determining
3 -Content is to cultivate NO in the soil
3 -Content deducts NO in the original soil
3 -Content calculate soil nitrification intensity.
Use this method that soil nitrification has been measured following shortcoming: 1) to need to measure NO in the classic method
3 -Content, and current NO
3 -The Determination on content method is comparatively complicated and consuming time; 2) this method is with NO before and after cultivating
3 -The content difference is represented nitrification, and main representative is nitrosification in fact; 3) before cultivation, need to regulate soil moisture content, and between culture period, whenever added water, this process time and effort consuming at a distance from 2-3 days.
Summary of the invention
The purpose of this invention is to provide a kind of good reproducibility, reappearance is high, and the method for simple mensuration soil nitrification potentiality.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is following:
Adopt the soil supension cultivation to replace traditional soil cultivation, and add quantitative KClO
3Or NaClO
3Suppress NO
2 -Be oxidized to NO
3 -, cultivate behind the 24h with the NO in the KCl lixiviate soil
2 -After the filtration, adopt the NO in the spectrophotometry filtrating
2 -Content is at last with NO before and after cultivating
2 -The content difference is represented the soil nitrification potentiality.Concrete steps are following:
1) takes by weighing sieve (2mm) fresh native 5g, place the triangular flask of 100ml, add 20ml and contain 1mM (NH
4)
2SO
4Phosphate buffer solution (PBS) solution and the KClO of 1ml
3(0.2-0.4mol/L), mixing is sealed vinyl film, pricks 4-5 aperture, is placed on then and cultivates 24h in the isothermal vibration incubator.Other has the KCl extracting solution of original soil to be kept under-20 ℃, waits and measures NO together after cultivate finishing
2 -Content;
2) take by weighing the pedotheque that sieves in right amount simultaneously, carry out the mensuration of soil moisture according to the requirement of GB7172;
3) after cultivation finishes,, filter behind the concussion 1h with 10ml KCl (2mol/L) lixiviate soil;
4) typical curve preparation: with one group of 7 25ml volumetric flask, add the nitrite titer of different amounts respectively, and the corresponding KCl solution (2mol/L) that adds 2ml, in each volumetric flask, add the 5ml developer, fully mix, leave standstill 15min at ambient temperature.Use spectrophotometer to be 540nm place colorimetric, NO at wavelength
2 -Content (μ g) is drawn calibration curve for horizontal ordinate;
5) get filtrating 2ml and place the volumetric flask of 25ml, add the 5ml developer and fully mix, leave standstill 15min at ambient temperature after, with spectrophotometer at its NO of 540nm place colorimetric estimation
2 -Concentration.
Test findings is calculated:
N=(C
Original soil-C
Compost)/(5 * (1-W))
In the formula: N---soil nitrification potentiality (ug/g24h)
C
Original soil---NO in the original soil
2 -Amount (ug)
C
Compost---NO in the corresponding compost
2 -Amount (ug)
W---the water cut (g) of bright soil
The principle that this method relates to:
1.ClO
3 -Radical ion suppresses NO
2 -Be oxidized to NO
3 -:
2.NO
2 -Root and sulfanilamide (SN) reaction generate diazo salt, generate the aubergine dyestuff with hydrochloric acid N-(1-naphthyl)-ethylenediamine coupling again, and in the finite concentration scope, nitrite nitrogen concentration and absorbance meet Lambert-Beer's law.
Innovative point of the present invention is following:
1) adopt the soil supension cultivation: (about 12h) adjusted soil moisture content again after traditional cultural method need at first be measured soil moisture content, and in incubation, added water, and above step is gone in this method saving, and incubation time is obviously short than classic method.
Add KClO when 2) cultivating
3: at traditional cultural method with NO before and after cultivating
2 -And NO
3 -Mainly represent in fact the content difference be nitrosification by the border; And this method is through adding KClO
3Suppress NO to reach
2 -Be oxidized to NO
3 -, again through measuring NO
2 -Content represents soil nitrification more accurate.
3) adopt spectrophotometric colorimetric method for determining NO
2 -Content: this method is measured NO
2 -Content saves time easy, can more large batch of mensuration pedotheque (100-200), and the way of distillation can only reach 40 sample numbers every day.
Use this method that the soil nitrification potentiality is measured, following advantage arranged:
1) time saving and energy saving, easy and simple to handle:; This method saves some steps than classic method and incubation time is short, uses spectrophotometric colorimetric method for determining NO
2 -The content operation is more easy, can measure pedotheque in enormous quantities;
2) degree of accuracy is high, accuracy good: the solid-state (NH that every part of pedotheque of classic method adds
4)
2SO
4Measure lowlyer, about about 5mg, need ten thousand/balance weighing, be difficult for weighing and can cause, and this method adopts the solution mode to add (NH than mistake
4)
2SO
4, make addition more accurate; Use AAS to compare with the distillation titrimetry in this method, precision is higher as a result for mensuration.
Points for attention:
1) used glassware needs thoroughly to clean in the test, and with the ionized water flushing, to guarantee not having nitrate ion and nitrite ion exists.
2) concentration of suppressant chlorate anions and consumption are applicable to that the content of organic matter is the soil of 1.0%-3.5%.If carry out the evaluation of the higher soil nitrification potentiality of the content of organic matter, then need increase the concentration of suppressant, because organic matter possibly have non-obligate suction-operated to suppressant with this method.
Embodiment
The present invention adopts traditional soil supension cultivation, and the combined chloride acid ion suppresses the principle of nitrite anions oxidation and directly measures the soil nitrification potentiality, good reproducibility of the present invention, and reappearance is high, and is simple.
The configuration of important reagent:
PBS solution preparation: take by weighing NaCl8.0g, KCl 2.0g, Na respectively
2HPO
40.2g, NaH
2PO
40.2g (NH
4)
2SO
40.132g to beaker, add the sterilization deionized water dissolving, be transferred to back constant volume in the 1000ml volumetric flask, contain (the NH of 1mmol/L in this solution
4)
2SO
4
The developer preparation:
Solution 1: take by weighing 0.4g sulfanilamide (SN) (C
6H
8N
2O
2S), put into the beaker that fills about 50ml water, add 40ml hydrochloric acid then, stirring and dissolving is transferred in the 200ml volumetric flask again and is also used the deionized water constant volume, keeps in Dark Place.
Solution 2: take by weighing how ethylenediamine-hydrochloride (C of 0.1g
12H
16N
2C
122HCl), put into fully stirring and dissolving of beaker, transfer to the brown volumetric flask of 100ml again and use the deionized water constant volume, keep in Dark Place.
Chromophoric solution: draw 80ml solution 1 respectively and place the brown volumetric flask of 100ml, keep in Dark Place after fully mixing, should join first usefulness at present with 20ml solution 2.
Nitrite mark liquid preparation: take by weighing sodium nitrite (GB 633, the standard reagent) 0.1500g of under 115 ± 5 ℃, drying, in the 500ml volumetric flask, add constant volume behind the deionized water dissolving to constant weight.This solution contains the standard reserving solution of nitrite ion 200mg/L.In the 200mL volumetric flask, use the deionized water constant volume with pipette, extract sodium nitrite standard inventory solution 5ml.This solution is standard operation solution, contains nitrite ion 5ug/mL, joins in the time of should using at present.
Embodiment 1: the recovery of measuring this method
Detailed process is following:
1) gathers ecological station, Shenyang applied nitrogen and handle pedotheque, at first remove foreign material, fully mix, cross the 2mm sieve;
2) get the pedotheque that sieves in right amount in the aluminium box, put into baking oven after weighing, in 105 ℃ of oven dry 12h down, the quality of the oven dry of weighing afterwards soil;
3) take by weighing 12 parts and sieve the fresh native 5g in back in the 100ml triangular flask, wherein 4 parts of addings contain 1mmol/L (NH
4)
2SO
4PBS solution 20ml, each 4 parts add respectively and contain 10ug, 20ug NaNO in addition
2The same solution 20ml, in eight triangular flasks, add the KClO of 0.2M respectively
3Solution, fully mixing.Seal vinyl film, and prick 4-5 aperture, place to shake under the dark condition in the isothermal vibration incubator and cultivate 24h;
4) after cultivation finishes, remove the vinyl film on the triangular flask, add 10ml KCl solution (2mol/L) lixiviate concussion 1h then, the employing quantitative filter paper is filled in the white plastic bottle;
5) typical curve: draw standard operation solution 0,0.2,0.4,0.6,0.8,1.0,1.2ml respectively in the 25ml volumetric flask; And the corresponding KCl solution (2mol/L) that adds 2ml; Add the 5ml developer then; Fully mix, room temperature is used the deionized water constant volume after leaving standstill 15min, and this standard solution contains NO
2 -Amount be 0.016,0.032,0.048,0.064,0.080,0.096,0.112ug/ml, measure absorbance in the 540nm place with spectrophotometer again, with NO
2 -Content (μ g/ml) is drawn calibration curve for horizontal ordinate;
6) draw 2ml with pipettor and filtrate in the volumetric flask of 25ml, add the 5ml developer and fully mix, under room temperature condition, leave standstill 15min, use the deionized water constant volume again, measure its absorbance in the 540nm place, draw NO
2 -Concentration.
Test findings is calculated:
A) W (water cut) %=(M
Wet soil-M
Dry ground)/(M
Wet soil-M
The aluminium box) * 100
In the formula: M
Wet soil---the general assembly (TW) (g) of bright soil and aluminium box before the oven dry
M
Dry ground---the general assembly (TW) (g) of oven dry back dry ground and aluminium box
M
The aluminium box---corresponding aluminium box weight (g)
B) calculate the NO that contains in the soil
2 -Amount
M
Soil=C
Liquid* 25 * (30+5 * W)/2
In the formula: M
Soil---the NO that contains in the corresponding pedotheque
2 -Amount (ug)
C
Liquid---the NO in the corresponding soil filtrating
2 -Concentration (ug/ml)
C) calculate recovery rate
Recovery %=(M
Mark-on-M
Mark-on not)/w * 100
In the formula: M
Contrast---do not add NO
2 -Soil content after the standard culture (ug)
M
Standard---add NO
2 -Soil content after the standard culture (ug)
W---add the standard NO of soil
2 -Amount (ug)
The result of this method mensuration soil recovery is as shown in table 1.From table, can find out that this method recovery is fine, the recovery of measuring 8 samples meets the scientific experiment standard fully all more than 95%.
Table 1 this method recovery result
Embodiment 2: contrast this method and classic method are measured the degree of accuracy and the accuracy of nitrification potentiality
Detailed process is following:
1) gather ecological station, Shenyang pedotheque, at first remove foreign material, fully mix, mixed sample is handled with sieve (2mm);
2) get the pedotheque that sieves in right amount in the aluminium box, put into baking oven after weighing, in 105 ℃ of oven dry 12h down, the quality of the oven dry of weighing afterwards soil;
3) take by weighing the 5g bright soil that sieves and place the 100ml triangular flask, use quantitative charger to add respectively then and contain 1mmol/L (NH
4)
2SO
4PBS solution 20ml, in each triangular flask, add 0.5ml KClO with transfer pipet again
3Solution (0.2mol/L), fully mixing.Seal vinyl film, prick 4-5 aperture, place to shake under the dark condition in the isothermal vibration incubator and cultivate 24h;
4) simultaneously, take by weighing the bright soil of 5g in shake flask, add 20ml and do not contain NH
4SO
4PBS solution and 10mlKCl (2mol/L) solution, shake after 1 hour and to filter, filtrating is kept under-20 ℃, waits for cultivating and measures together after finishing;
5) after cultivation finishes, remove the vinyl film on the triangular flask, add 10ml KCl solution (2mol/L) back concussion 1h respectively; Filter; As can not in time measure, can filtrating be positioned under refrigeration (4 ℃) condition and preserve 1d, as then needing to filtrate freezing preservation (20 ℃) for more time;
6) typical curve: draw standard operation liquid 0,0.2,0.4,0.6,0.8,1.0,1.2ml respectively in the 25ml volumetric flask; And the corresponding KCl solution (2mol/L) that adds 2ml; Add the 5ml developer then; Fully mix, room temperature is used the deionized water constant volume after leaving standstill 15min, and this standard solution contains NO
2 -Amount be 0.016,0.032,0.048,0.064,0.080,0.096,0.112ug/ml, measure absorbance in the 540nm place with spectrophotometer again, with NO
2 -Content (μ g/ml) is drawn calibration curve for horizontal ordinate;
7) filtrate in the volumetric flask of 25ml with pipette, extract 2ml, add the 5ml developer and fully mix, leave standstill 15min at ambient temperature after, use the deionized water constant volume again, its absorbance of mensuration at 540nm place, and draw corresponding nitrite ion concentration.
The calculating of test findings:
A) calculating of soil moisture content
W (water cut) %=(M
Wet soil-M
Dry ground)/(M
Wet soil-M
The aluminium box) * 100
In the formula: M
Wet soil---the general assembly (TW) (g) of bright soil and aluminium box before the oven dry
M
Dry ground---the general assembly (TW) (g) of oven dry back dry ground and aluminium box
M
The aluminium box---corresponding aluminium box weight (g)
B) calculate the NO that contains in the soil
2 -Amount
M
Soil=C
Liquid* 25 * (30+ (1-m))/2
In the formula: M
Soil---the NO that contains in the corresponding pedotheque
2 -Amount (ug)
C
Liquid---corresponding soil filtrating is gone up machine and is measured concentration (ug/ml)
The bright soil oven dry of m---5g dry weight (g)
C) calculating of nitrification potentiality
N=(M
Compost-M
Original soil)/m
In the formula: N---nitrification potentiality (ug/g24h)
M
Original soil---NO in the original soil
2 -Amount (ug)
M
Compost---NO in the corresponding compost
2 -Amount (ug)
The bright soil oven dry of m---5g dry weight (g)
It is following that classic method is measured detailed process:
1) measure water cut: the pedotheque of getting in right amount sieve (2mm) is put into baking oven after weighing in the aluminium box, dries 12h down at 105 ℃, the quality of the oven dry of weighing afterwards soil.
2) cultivate: get be equivalent to the 20g dry ground sieve (2mm) fresh soil in the 250mL triangular flask, in every bottle by the ratio adding (NH of 100g dry ground 25mg N
4)
2SO
4, regulate soil moisture to water capacity 60% then.Be provided with simultaneously and do not add (NH
4)
2SO
4Control treatment, build bottleneck with vinyl film immediately then, 30 ℃ of constant temperature culture 5d.
3) NO
3 -Assay: cultivate and finish the back, filter behind the concussion 1h with 100ml KCl solution (2mol/L) lixiviate soil sample; Get filtrating 50ml and add in the cucurbit, distill after adding 1g MgO, use that to contain 20ml concentration be that 2% boric acid absorbs, abandon; Will be with adding 0.25g Vad alloy in a residue distillate, other gets a boric acid absorption liquid (20g/L), and absorbing redistillation is the ammonia that produces; With the ammonia amount in watery hydrochloric acid (0.053mol/L) the titration absorption liquid; At last according to NO before and after cultivating
3 -Calculate soil nitrification intensity.
Result of calculation:
A) W=(M
Before the oven dry-M
After the oven dry)/(M
After the oven dry-M
The aluminium box) * 100
In the formula: W---soil moisture content (%)
M
Before the oven dry---the general assembly (TW) (g) of bright soil and aluminium box before the oven dry
M
After the oven dry---the general assembly (TW) (g) of oven dry back dry ground and aluminium box
M
The aluminium box---corresponding aluminium box weight (g)
b)M
N＝C
HCl×(V-Vo)×62×1000×(100+30×W)/50
In the formula: M
N---the NO in the soil
3 -Content (ug)
C
HCl---the concentration (mmol/ml) of salt standard acid solution
The volume (ml) of V---sample titration hydrochloric acid standard solution
The volume (ml) of Vo---blank titration hydrochloric acid standard solution
W---soil moisture content (%)
C) calculating of nitrification
N=(M
Cultivate-M
Contrast)/M
In the formula: N---the soil nitrification potentiality
M
Cultivate---cultivate NO in the soil of back
3 -Content (ug)
M
Contrast---NO in the control soil
3 -Content (ug)
The bright soil oven dry of M---5g dry weight (g)
It is as shown in table 2 to use this method and classic method to measure ecological station pedotheque nitrification potentiality result.Can find out by the result, the result that the result that conventional method is measured measures apparently higher than this method, this is the result for nitrification and nitrosification stack who measures because of conventional method, and this method is represented actual nitrification; The standard deviation of this method measurement result is little than conventional method, and it is more accurate reliable to illustrate that this method is measured the soil nitrification potentiality.
Table 2 this method and classic method are measured the pedotheque nitrification potentiality result contrast of ecological station
Claims (3)
1. soil nitrification potentiality method for measuring is characterized in that:
Adopt the soil supension cultivation, and add quantitative KClO
3And NaClO
3Suppress NO
2 -Be oxidized to NO
3 -, cultivate behind the 24h with the NO in the KCl lixiviate soil
2 -After the filtration, adopt the NO in the spectrophotometry filtrating
2 -Content is at last with NO before and after cultivating
2 -The content difference is represented the soil nitrification potentiality.
2. according to the described method of claim 1, it is characterized in that:
Concrete steps are following:
1) takes by weighing fresh native 5g, place pocket, add 20ml and contain 1mM (NH
4)
2SO
4Phosphate buffer solution and the 0.2-0.4mol/L KClO of 1ml
3, mixing seals, and sealing part is provided with aperture, is placed on then and cultivates 24h in the isothermal vibration incubator;
2) take by weighing pedotheque simultaneously, carry out the mensuration of soil moisture according to the requirement of GB7172;
3) after cultivation finishes,, filter behind the concussion 1h with the 2mol/L KCl liquid lixiviate soil of 10ml;
4) typical curve preparation: with one group of 5 above 25ml volumetric flask; The nitrite titer that adds different amounts respectively, and the corresponding 2mol/L KCl solution that adds 2ml add the 5ml developer in each volumetric flask; Fully mix, leave standstill 15min at ambient temperature; Use spectrophotometer to be 540nm place colorimetric, NO at wavelength
2 -Content (μ g) is drawn calibration curve for horizontal ordinate;
5) get filtrating 2ml and place the volumetric flask of 25ml, add the 5ml developer and fully mix, leave standstill 15min at ambient temperature after, with spectrophotometer at its NO of 540nm place colorimetric estimation
2 -Concentration;
6) get fresh soil in addition, promptly original soil 5g adopts the 2mol/L KCl liquid lixiviate soil of 10ml, filters behind the concussion 1h; Get the volumetric flask that filtrating 2ml places 25ml; Add the 5ml developer and fully mix, leave standstill 15min at ambient temperature after, with spectrophotometer at its NO of 540nm place colorimetric estimation
2 -Concentration.
Test findings is calculated:
N=(C
Original soil-C
Compost)/(5 * (1-W))
In the formula: N---soil nitrification potentiality (ug/g24h)
C
Original soil---NO in the original soil
2 -Amount (ug)
C
Compost---NO in the corresponding compost
2 -Amount (ug)
W---the water cut (g) of bright soil.
3. according to the described method of claim 1, it is characterized in that:
The developer preparation:
Solution 1: take by weighing 0.4g sulfanilamide (SN) (C
6H
8N
2O
2S), put into the beaker that fills about 50ml water, add 40ml hydrochloric acid then, stirring and dissolving is transferred to and is also used the deionized water constant volume in the 200ml volumetric flask;
Solution 2: take by weighing how ethylenediamine-hydrochloride (C of 0.1g
12H
16N
2C
122HCl), put into fully stirring and dissolving of beaker, transfer to the brown volumetric flask of 100ml again and use the deionized water constant volume;
Chromophoric solution: draw 80ml solution 1 respectively and place the brown volumetric flask of 100ml, fully mix with 20ml solution 2.
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| 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 |
| CN110988308A (en) * | 2019-12-30 | 2020-04-10 | 中国科学院城市环境研究所 | Method for simultaneously determining different types of nitrification potentials in soil |
| CN111474127A (en) * | 2020-04-27 | 2020-07-31 | 济南大学 | Improved nitrite reductase assay |
| CN112858449A (en) * | 2021-01-12 | 2021-05-28 | 中国科学院南京土壤研究所 | Method for determining nitrogen fixation potential of rice field soil organisms |
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Application publication date: 20120530 |