CN102732466A - Method for culturing denitrifying bacterium and determining water body nitrate nitrogen isotope composition - Google Patents
Method for culturing denitrifying bacterium and determining water body nitrate nitrogen isotope composition Download PDFInfo
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- CN102732466A CN102732466A CN2012102289750A CN201210228975A CN102732466A CN 102732466 A CN102732466 A CN 102732466A CN 2012102289750 A CN2012102289750 A CN 2012102289750A CN 201210228975 A CN201210228975 A CN 201210228975A CN 102732466 A CN102732466 A CN 102732466A
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Abstract
The invention relates to the technical field of water body nitrate pollution treatment and control, and is a method for culturing a denitrifying bacterium and using the bacterium to determine water body nitrate nitrogen isotopic composition. The method comprises the following steps of: (1) carrying out enrichment culture of the denitrifying bacteria, (2) detecting whether the denitrifying bacteria completely converse NO3- in a culture fluid, (3) concentrating of the denitrifying bacteria, (4) adding the concentrated cell culture to a sample bottle, purifying an upper space and removing N2O in the denitrifying bacterium liquid, (5) conversing NO3- in the sample to N2O, (6) determining the N2O nitrogen isotope, and (7) correcting the nitrogen isotope determination result to complete the test. The positive effects of the invention are as follows: the culture of the denitrifying bacteria can be completed by using only a common saline bottle or a triangular flask, the detection of sulfanilamide color development improves the reliability of the determination result, and the determination cost is reduced. For the current situation that severe water body nitrate pollution is urgently needed to be treated, the method of the invention has the advantages of scientificalness, simplicity, and high maneuverability, and has a broad popularization prospect.
Description
Technical field
The present invention relates to a kind of cultivation denitrifying bacterium and distinguish the nitric nitrogen isotopics, belong to water body nitrate Pollution abatement and control techniques field with this bacterium.
Background technology
Underground water is China's economy and social development and necessary, the irreplaceable valuable source of people's lives.The whole world mainly relies on underground water as tap water above 1,500,000,000 population.China part city and vast Rural areas, the resource of water supply that underground water is unique often.In the last few years, the nitric nitrogen polluted underground water had been question of common concern in the world.All there is the phenomenon of Groundwater Nitrate-nitrogen content severe overweight in American-European many countries and regions, and the many areas of China are also in the pollution that has received nitric nitrogen in varying degrees, and especially nitric nitrogen pollution in Rural areas is very general, and serious day by day trend is arranged.Do not endanger though nitric nitrogen itself has directly human body, be reduced to diseases such as to bring out methemoglobinemia, cancer in digestive system behind the nitrite nitrogen and threaten HUMAN HEALTH.Therefore,, effectively administer contaminated body of groundwater, confirm that the source that nitric nitrogen pollutes in the underground water is extremely important for guaranteeing water supply security.
Under the effect of human activity; The nitric nitrogen pollution source is complicated; Existing natural source has artificial source again, it is generally acknowledged that nitric nitrogens a large amount of in the underground water is mainly derived from resident living sewage and form garbage and dejection, chemical fertilizer, trade effluent, atmospheric nitrogen oxygen compound dried wet deposition and sewage irrigation etc.Judge underground water NO
3-The method of N source of pollution is a lot, and its simple and the most traditional method is the land use type of zone of pollution by inquiry and combines the ground water chemistry signature analysis to distinguish source of pollution.But the result that this method obtains is comparatively coarse.This mainly is because the N of different sources forms the NO that is prone to migration through the nitrogen transformation effect
3 -Flow in surface water and the underground water.Different sources NO
3 -Chemical species all be NO
3 -, there is not any difference, be difficult to distinguish its definite source from chemical species merely.But, on the isotropic substance level, NO
3 -Can further divide into
15NO
3 -With
14NO
3 -, N
18O
- 3And N
16O
- 3, the NO of different sources
3 -Have different
15N/
14N with
18O/
16The O ratio feature; These ratios can accurately be measured through isotope mass spectrometer; Therefore, the difference that the nitrogen oxygen isotope is formed in the nitrate salt provides direct approach for the pollution source of identification nitrate salt, and measuring method commonly used at present mainly contains high-temperature combustion method and denitrifying bacterium method; And the denitrifying bacterium method has advantages such as low cost, simple, the required sample size of pre-treatment are few, has become advanced nitrate nitrogen oxygen isotope testing method.Present domestic denitrifying bacterium method test nitric nitrogen isotropic substance is used also less, and also there is following drawback in existing literature method: the special culturing bottle of cultivation needs of (1) denitrifying bacterium, and this is difficult to find in common lab; Whether (2) nitrate salt in the nutrient solution is transformed fully after the denitrifying bacterium enrichment culture can N
2O does not measure and can not influence subsequent sample, and this index detection method lacks at present; (3) N that produces with sample
2O gas can not directly be analyzed, need be with N
2Analyze again after the disposable whole injections of O gas are connected the sample bottle on the mass spectrograph, need sample size big.Analysis-by-synthesis is at present domestic not to form the isotopic method of a kind of detailed exercisable denitrifying bacterium method test water body nitrate nitrogen, thereby has limited the application of this method in the water body nitrate pollution is traced to the source and administered.
Summary of the invention
The present invention is directed to the deficiency of prior art, the cultivation of a kind of denitrifying bacterium is provided and utilizes this denitrifying bacterium to measure the method that the water body nitrate nitrogen isotope is formed.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
Cultivation of a kind of denitrifying bacterium and mensuration water body nitrate nitrogen isotope method is characterized in that, may further comprise the steps:
(1) enrichment culture of denitrifying bacterium
To lack N
2The denitrifying bacterium inoculation of O reductase activity contains NO
3 -The TSB sterile medium on shaking table (26 ℃, 180rpm) cultivate, carry out the enrichment culture of denitrifying bacterium;
(2) whether detect denitrifying bacterium with NO in the nutrient solution
3 -Transform fully
Get step (1) gained bacterium liquid and sulfanilamide (SN) colour developing liquid and carry out coupling reaction,, explain that denitrifying bacterium is not with NO in the nutrient solution if bacterium liquid becomes pink
3 -Be converted into N fully
2O, but rest on NO
2 -In the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid can be carried out subsequent experimental;
(3) denitrifying bacterium concentrates
The bacterium liquid that detects with step (1) and through step (2) is at ultra-high speed whizzer 30000rpm, 18 ℃ centrifugal 20 minutes, pour out supernatant liquid, the residue thalline is with no NO
3 -The TSB nutrient solution suspend, be condensed into 2 times bacterium liquid;
(4) in sample bottle, add the concentrating cells culture, the N on the purifying in sheaf space and the removal denitrifying bacterium bacterium liquid
2O
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is the 12mL test tube, and (flow velocity is more than 10～20mL/min) the purging 3h to each test tube with high-purity helium;
(5) NO in the sample
3 -Be converted into N
2O
In the test tube of step (4), inject the water sample (total nitric nitrogen amount is 0.5 μ g) less than 4mL with syringe, sample turns upside down with abundant mixing after injecting; Test tube is inverted into the thermostat container 3h-24h that is provided with 26 ℃; Injecting 0.10mL concentration after incubation time finishes is the NaOH of 10mol/L, and termination reaction behind the dissipation bacterium absorbs the CO that produces simultaneously
2
(6) N
2The O nitrogen isotope is measured
N with the generation of step (5) gained test tube
2O gas utilizes trace gas analysis appearance (TraceGas)-isotope ratio mass spectrometer (IRMS) to measure, and obtains N
2O nitrogen isotope composition measuring value.
(7) correction of nitrogen isotope test result
δ
15The standard of N adopts δ
15 NUSGS34=-1.8 ‰, δ
15N
USGS32=+180 ‰;
The nitrogen isotope correction equation is following :+180 ‰=m * δ
15N
USGS32-meas+ b 1.
-1.8‰＝m×δ
15N
USGS34-meas+b②，
Actual value (T) and measured value (M) according to these two standard models obtain m and b, thereby obtain δ
15The correction equation of N.
Again cultivate denitrifying bacterium then, carry out the mensuration of next batch sample.
The said TSB nutrient solution of step (1) (contains NO
3 -) compound method is: 30g pancreas peptone soybean broth agar (TSB), 1.0g KNO
3, 0.5g (NH
4)
2SO
4, 6.15g K
3PO
43H
2O, the 1000mL deionized water divides to install to 500mL saline bottle mesohigh sterilization 60min.
The said saline bottle of step (1) is that cubic capacity is hospital's common infusion fluid bottle of 580mL, and bottle cap adopts rubber plug, 460mL-480mL is housed in the saline bottle has NO
3 -The TSB nutrient solution, 121 ℃ of sterilizations 60 minutes after the denitrifying bacterium switching, with sealing film phonograph seal, prevent the air inerchange with the external world with bottle stopper.
The said sulfanilamide (SN) colour developing of step (2) liquid making method is: 4g sulfanilamide (SN), and 10mL high-quality pure 85% phosphoric acid, 0.1g N-1-naphthyl ethylenediamine hydrochloride, constant volume should be colourless to 100mL, just can not use if pulverize is red again.
The said no NO of step (3)
3 -TSB nutrient solution compound method be: 30g TSB, 0.5g (NH
4)
2SO
4, 4.9g KH
2PO
4, the 2000mL deionized water divides to install in the serum bottle of 100mL, and autoclaving 60min is subsequent use.
The said test tube of step (4) is the test tube of the high 100mm of internal diameter 10mm, adopts Teflon silicagel pad and screw-cap.
The said high-purity helium purge of step (4) adopts the helium purge appearance, and test tube stands upside down and is inserted on the helium pin, on silicagel pad, inserts the syringe needle of a long 25gauge of 8cm in addition, with the N in air in the test tube and the bacterium liquid
2O goes out with helium replacement.
Step (5) if said water sample add-on greater than 4mL, it is excessive to prevent pressure just need on the test tube pad, to add an exhaust needle.
The said trace gas analysis appearance of step (6) (TraceGas)-isotope ratio mass spectrometer (IRMS), TracsGas is furnished with the Gilson automatic sampler, and high-purity helium is carrier gas, and flow is 50-60mL/min, and reference standard gas is corrected 99.9%N
2O gas.Step (5) gained test tube is positioned on the test-tube stand of automatic sampler configuration, through the Gilson automatic sampler with N
2The O gas delivery is extracted purifying and is captured N through TraceGas to TraceGas
2O gas is measured nitrogen isotope by isotope ratio mass spectrometer at last and is formed.
With respect to prior art, the present invention has following excellent results:
1, adopts the ordinary salt water bottle that is prone to obtain to cultivate denitrifying bacterium, reduced a series of loaded down with trivial details formalities such as the searching of special culturing bottle, customized production, reduced the cultivation cost of denitrifying bacterium simultaneously;
2, the present invention's nitrate salt of adopting sulfanilamide (SN) colour developing liquid whether to detect in the denitrifying bacterium culturing process nutrient solution itself is exhausted, and is simple to operate, got rid of the influence of the inner nitrate salt of nutrient solution to sample nitrate salt, improved mensuration result's confidence level.
3, it is time saving and energy saving that method of the present invention is measured the nitrate nitrogen isotopics, reduced testing cost, for the azotate pollution work of tracing to the source provides important research means.
4, method of the present invention has been directed against the seriously polluted present situation of needing improvement badly of the present water body nitrate of China, has science, simplicity, workable advantage, and promotion prospect is wide.
Embodiment
Below provide a kind of denitrifying bacterium of the present invention and cultivate and measure the embodiment of the method for water body nitric nitrogen isotopics; So that method of the present invention is carried out detailed explanation; But protection scope of the present invention is not limited to following enforcement introduction; The variation or the accommodation of all equivalences of doing according to method of the present invention all should be regarded as the category of the present invention's protection.
Embodiment 1:
The method of water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium, may further comprise the steps:
(1) enrichment culture of denitrifying bacterium
In every liter of deionized water, add 30g pancreas peptone soybean broth agar (TSB), 1.0g KNO
3, 0.5g (NH
4)
2SO
4, 6.15g K
3PO
47H
2O gets 480mL respectively and installs in two saline bottles, gets 2 5mL in addition and installs in the test tube, and plug seals, and autoclaving 60min constitutes denitrifying bacterium enrichment culture liquid; To lack N
2The denitrifying bacterium Pseudomonas aureofaciens P.aureofaciens of O reductase activity is inoculated into 5mL has NO
3 -The test tube of TSB sterile medium in, on shaking table (26 ℃, 180rpm) cultivated one day, this 5mL bacterium liquid was transferred in second day and contains NO
3 -The saline bottle of 480mLTSB sterile medium in and on shaking table (26 ℃, 180rpm) cultivate a week, carry out the denitrifying bacterium enrichment culture;
(2) whether detect denitrifying bacterium with NO in the nutrient solution
3 -Transform fully
Step (1) gained bacterium liquid is got 1mL, adds 80 μ L sulfanilamide (SN) colour developing liquid, if bacterium liquid becomes pink, explains that denitrifying bacterium is not with NO in the nutrient solution
3 -Be converted into N fully
2O, but rest on NO
2 -In the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid can be carried out subsequent experimental;
(3) denitrifying bacterium concentrates
Step (1) is reached the bacterium liquid 200mL that detects through step (2) to be poured in the 200-mL centrifugal bottle; At ultra-high speed whizzer 30,000rpm, 18 ℃ centrifugal 20 minutes (bacterium should become bulk at the bottom of bottle); Discard supernatant liquid (noting not confusing bacterial mass), do not have NO from a 100-mL
3 -Substratum in take out 10ml respectively and be added in each bottle, fully concussion is transferred to the bacterium of suspension again in the beaker of a 200-mL, with remaining no NO with the bacterium that suspends again
3 -Substratum cleans centrifugal bottle, all is incorporated into them in the beaker of 200-mL, adds 10 antifoam B (available from Sigma company);
(4) in sample bottle, add the concentrating cells culture, the N on the purifying in sheaf space and the removal denitrifying bacterium bacterium liquid
2O
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is the 12mL test tube, and test tube is the test tube of the high 100mm of internal diameter 15mm, adopts Teflon silicagel pad and screw-cap, and (flow velocity is more than 10～20mL/min) the purging 3h to each test tube with high-purity helium;
(5) NO in the sample
3 -Be converted into N
2O
In the test tube of step (4), inject 0.8mL river sample (nitrate nitrogen content is 1.58mg/L) with syringe, sample turns upside down with abundant mixing after injecting; Test tube is inverted into 26 ℃ of incubated overnight of thermostat container.Second day injects 0.10mL concentration is the NaOH of 10mol/L, and termination reaction behind the dissipation bacterium absorbs the CO that produces simultaneously
2
(6) N
2The O nitrogen isotope is measured
N with the generation of step (5) gained test tube
2O gas utilizes trace gas analysis appearance (TraceGas)-isotope ratio mass spectrometer (IRMS) to measure, and obtains measured value δ
15N=-2.35 ‰.
(7) correction of nitrogen isotope test result
δ
15The standard of N adopts δ
15N
USGS34=-1.8 ‰, δ
15N
USGS32=+180 ‰;
The nitrogen isotope correction equation is following :+180 ‰=m * δ
15N
USGS32-meas+ b 1.
-1.8‰＝m×δ
15N
USGS34-meas+b②，
Measured value and actual value according to USGS34 and USGS32 obtain δ
15The correction equation of N is T=1.07*M+5.56, thereby obtains the nitric nitrogen δ of this river sample
15N=3.03, according to document, this value is traced to the source and is shown that this river azotate pollution comes from chemical fertilizer.
This shows, be fully feasible with method test river nitric nitrogen of the present invention isotopics.
Embodiment 2:
The method of water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium, and its concrete operations step is:
Step (1) arrives (3) with embodiment 1.
(4) in sample bottle, add the concentrating cells culture, the N on the purifying in sheaf space and the removal denitrifying bacterium bacterium liquid
2O
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is 20mL jaw headspace sample bottle, and (flow velocity is more than 10～20mL/min) the purging 3h to each sample bottle with high-purity helium;
(5) NO in the sample
3 -Be converted into N
2O
In the test tube of step (4), inject 0.5mL underground water sample (nitrate nitrogen content is 25.75mg/L) with syringe, sample turns upside down with abundant mixing after injecting; Test tube is inverted into 26 ℃ of incubated overnight of thermostat container.Second day injects 0.10mL concentration is the NaOH of 10mol/L, and termination reaction behind the dissipation bacterium absorbs the CO that produces simultaneously
2
(6) N
2The O nitrogen isotope is measured
N with each headspace sample bottle generation of step (5)
2O gas extracts 2mL with the resistance to air loss syringe and injects the test tube that vacuumizes in advance, through the Gilson automatic sampler with N
2The O gas delivery is extracted purifying and is captured N through TraceGas to TraceGas
2O gas is measured nitrogen isotope by isotope ratio mass spectrometer at last and is formed, and obtains measured value δ
15N=6.20 ‰.
Step (7) obtains the nitric nitrogen δ of this underground water sample with embodiment 1
15The N actual value is 12.21, and according to document, this value shows that azotate pollution mainly comes from muck in this underground water sample.
This shows, be fully feasible with method test Groundwater Nitrate-nitrogen of the present invention isotopics.
Embodiment 3:
The method of water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium, and its concrete operations step is:
(1) enrichment culture of denitrifying bacterium
In every liter of deionized water, add 30g pancreas peptone soybean broth agar (TSB), 1.0g KNO
3, 0.5g (NH
4)
2SO
4, 6.15g K
3PO
47H
2O gets 520mL respectively and installs in two 500-mL triangular flasks, gets 2 5mL in addition and installs in the test tube, and plug seals, and autoclaving 60min constitutes denitrifying bacterium enrichment culture liquid; Adopting the denitrifying bacterium bacterial strain is Pseudomonas chlororaphis P.chlororaphis, and the enrichment culture step is with embodiment 2 (1);
Step (2) arrives step (4) with embodiment 2;
(5) NO in the sample
3 -Be converted into N
2O
In the jaw head space bottle of step (4), inject 5mL rainwater sample (nitrate nitrogen content is 0.21mg/L) with syringe; After sample injects; Turn upside down with abundant mixing, and on bottle holder, add an exhaust needle, be inverted to be placed on and put into 26 ℃ of incubated overnight of thermostat container on the test-tube stand.Second day injects 0.10mL concentration is the NaOH of 10mol/L, and termination reaction behind the dissipation bacterium absorbs the CO that produces simultaneously
2
Step (6)-(7) obtain the nitric nitrogen δ of this rainwater sample with embodiment 2
15The N actual value is 0.16, according to document, this value show in this rainwater the nitrate salt source mainly with air in nitrogen deposition and ground chemical fertilizer ammonia volatilization relevant.
This shows, be fully feasible with method test rainwater nitric nitrogen of the present invention isotopics.
Claims (8)
1. cultivation of a denitrifying bacterium and mensuration water body nitrate nitrogen isotope method is characterized in that, may further comprise the steps:
(1) enrichment culture of denitrifying bacterium
To lack N
2The denitrifying bacterium inoculation of O reductase activity contains NO
3 -The TSB sterile medium on shaking table (26 ℃, 180rpm) cultivate, carry out the enrichment culture of denitrifying bacterium;
(2) whether detect denitrifying bacterium with NO in the nutrient solution
3 -Transform fully
Get step (1) gained bacterium liquid and sulfanilamide (SN) colour developing liquid and carry out coupling reaction,, explain that denitrifying bacterium is not with NO in the nutrient solution if bacterium liquid becomes pink
3 -Be converted into N fully
2O, but rest on NO
2 -In the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid can be carried out subsequent experimental;
(3) denitrifying bacterium concentrates
The bacterium liquid that detects with step (1) and through step (2) is at ultra-high speed whizzer 30,000rpm, 18 ℃ centrifugal 20 minutes, pour out supernatant liquid, the residue thalline is with no NO
3 -The TSB nutrient solution suspend, be condensed into 2 times bacterium liquid;
(4) in sample bottle, add the concentrating cells culture, the N on the purifying in sheaf space and the removal denitrifying bacterium bacterium liquid
2O
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is the 12mL test tube, and (flow velocity is more than 10～20mL/min) the purging 3h to each test tube with high-purity helium;
(5) NO in the sample
3 -Be converted into N
2O
In the test tube of step (4), inject the water sample (total nitric nitrogen amount is 0.5 μ g) less than 4mL with syringe, sample turns upside down with abundant mixing after injecting; Test tube is inverted into 26 ℃ of incubated overnight of thermostat container.Second day injects 0.10mL concentration is the NaOH of 10mol/L, and termination reaction behind the dissipation bacterium absorbs the CO that produces simultaneously
2
(6) N
2The O nitrogen isotope is measured
N with the generation of step (5) gained test tube
2O gas utilizes trace gas analysis appearance (TraceGas)-isotope ratio mass spectrometer (IRMS) to measure, and obtains N
2O nitrogen isotope composition measuring value.
(7) correction of nitrogen isotope test result
δ
15The standard of N adopts δ
15N
USGS34=-1.8 ‰, δ
15N
USGS32=+180 ‰;
The nitrogen isotope correction equation is following :+180 ‰=m * δ
15N
USGS32-meas+ b 1.
-1.8‰＝m×δ
15N
USGS34-meas+b②，
Actual value (T) and measured value (M) according to these two standard models obtain m and b, thereby obtain δ
15The correction equation of N.
Again cultivate denitrifying bacterium then, carry out the mensuration of next batch sample.
2. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1, it is characterized in that the said TSB nutrient solution of step (1) (contains NO
3 -) compound method is: 30g pancreas peptone soybean broth agar (TSB), 1.0g KNO
3, 0.5g (NH
4)
2SO
4, 6.15g K
3PO
43H
2O, the 1000mL deionized water divides to install to 500mL saline bottle mesohigh sterilization 60min.
3. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1; It is characterized in that; The said saline bottle of step (1) is that cubic capacity is hospital's common infusion fluid bottle of 580mL, and bottle cap adopts rubber plug, 460mL-480mL is housed in the saline bottle has NO
3 -The TSB nutrient solution, 121 ℃ of sterilizations 60 minutes after the denitrifying bacterium switching, with sealing film phonograph seal, prevent the air inerchange with the external world with bottle stopper.
4. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1; It is characterized in that the said sulfanilamide (SN) colour developing of step (2) liquid making method is: 4g sulfanilamide (SN), 10mL high-quality pure 85% phosphoric acid; 0.1g N-1-naphthyl ethylenediamine hydrochloride; Constant volume should be colourless to 100mL, just can not use if pulverize is red again.
5. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1, it is characterized in that the said no NO of step (3)
3 -TSB nutrient solution compound method be: 30g TSB, 0.5g (NH
4)
2SO
4, 4.9gKH
2PO
4, the 2000mL deionized water divides to install in the serum bottle of 100mL, and autoclaving 60min is subsequent use.
6. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1, it is characterized in that the said test tube of step (4) is the test tube of the high 100mm of internal diameter 10mm, adopts Teflon silicagel pad and screw-cap.
7. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1; It is characterized in that; The said high-purity helium purge of step (4) adopts the helium purge appearance; Test tube stands upside down and is inserted on the helium pin, on silicagel pad, inserts the syringe needle of a long 25gauge of 8cm in addition, with the N in air in the test tube and the bacterium liquid
2O goes out with helium replacement.
8. the method for water body nitric nitrogen isotopics is cultivated and measured to a kind of denitrifying bacterium according to claim 1; It is characterized in that; The said trace gas analysis appearance of step (6) (TraceGas)-isotope ratio mass spectrometer (IRMS), TracsGas is furnished with the Gilson automatic sampler, and high-purity helium is carrier gas; Flow is 50-60mL/min, and reference standard gas is corrected 99.9%N
2O gas; Step (5) gained test tube is positioned on the test-tube stand of automatic sampler configuration, through the Gilson automatic sampler with N
2The O gas delivery is extracted purifying and is captured N through TraceGas to TraceGas
2O gas is measured nitrogen isotope by isotope ratio mass spectrometer at last and is formed.
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| CN103822963A (en) * | 2014-03-19 | 2014-05-28 | 哈尔滨工业大学 | Method for identifying whether organic matters are utilized by ANAMMOX (anaerobic ammonium oxidation) bacteria in ANAMMOX reactor sludge |
| CN103940645A (en) * | 2014-02-24 | 2014-07-23 | 中国科学院南京地理与湖泊研究所 | Pretreatment method used for measuring water body nitrogen and oxygen isotopes via chemical conversion |
| CN104422729A (en) * | 2013-08-22 | 2015-03-18 | 华东师范大学 | Membrane entrance mass spectrometer based method for analyzing content of dissolved nitrogen isotope |
| CN104630053A (en) * | 2015-01-23 | 2015-05-20 | 长江大学 | Production method of denitrifying bacteria test bottle |
| CN110988106A (en) * | 2020-03-04 | 2020-04-10 | 中国农业科学院农业环境与可持续发展研究所 | Nitrous oxide isotope delta15N correction method |
| CN112320941A (en) * | 2020-10-21 | 2021-02-05 | 西安建筑科技大学 | Domestication product N by using separation membrane filler2Device and method for O denitrifying bacteria |
| CN113933372A (en) * | 2021-11-11 | 2022-01-14 | 广西大学 | Method for quantitatively identifying river entering load and river entering coefficient of river basin nitrate nitrogen source |
| CN115014916A (en) * | 2022-08-10 | 2022-09-06 | 中国地质大学（北京） | Nitrate nitrogen isotope enrichment and purification device and method |
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