CN102732466B - 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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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 nitric nitrogen isotopics with this bacterium, belonging to water body nitrate Pollution abatement and control techniques field.
Background technology
Underground water is China's economy and social development and necessary, the irreplaceable valuable source of people's lives.The whole world exceedes 1,500,000,000 population and mainly relies on underground water as tap water.China urban and vast Rural areas, underground water unique resource of water supply often.In the last few years, nitrate-N pollution 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, the many areas of China are also in the pollution that has been subject in varying degrees nitric nitrogen, and especially Rural areas nitrate-N pollution is very general, and has day by day serious trend.Though nitric nitrogen itself, is reduced to and can brings out the disease such as methemoglobinemia, cancer in digestive system after nitrite nitrogen and threaten HUMAN HEALTH without directly endangering human body.Therefore, be assure feed water safety, effectively administer contaminated body of groundwater, determine that in underground water, the source of nitrate-N pollution is extremely important.
Under the effect of human activity, nitrate-N pollution source is complicated, existing natural source, has again artificial source, it is generally acknowledged that nitric nitrogens a large amount of in 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 the land use type that its simple and the most traditional method is zone of pollution by inquiry also descends Chemical characteristic analysis to distinguish source of pollution in combination.But the result that this method obtains is comparatively coarse.This is mainly because the N of different sources forms the NO of easy migration by nitrogen transformation effect
3 -flow in surface water and groundwater.Different sources NO
3 -chemical species be all NO
3 -, there is no any difference, be difficult to distinguish it from chemical species merely and definitely originate.But, in 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 and
18o/
16o ratio feature, these ratios can pass through isotope mass spectrometer Accurate Measurement, therefore, in nitrate, the difference of nitrogen oxygen isotope composition provides direct approach for the pollution source of identification nitrate, at present conventional measuring method mainly contains high-temperature combustion method and denitrifying bacterium method, and denitrifying bacterium method has the advantages such as low cost, simple, the required sample size of pre-treatment are few, become advanced nitrate nitrogen oxygen isotope testing method.The application of current domestic denitrifying bacterium method test nitric nitrogen isotropic substance is also less, and existing literature method also exists following drawback: the cultivation of (1) denitrifying bacterium needs special culturing bottle, and this is difficult to find in common lab; (2) whether after denitrifying bacterium enrichment culture, the nitrate in nutrient solution is transformed completely can N
2o, measures and can not affect subsequent sample, and this index detection method lacks at present; (3) with sample produce N
2o gas can not directly be analyzed, need to be by N
2after the disposable whole injections of O gas are connected to the sample bottle on mass spectrograph, analyze again, need sample size large.Comprehensive analysis at present domesticly do not form a kind of isotopic method of detailed exercisable denitrifying bacterium method test water body nitrate nitrogen, thus limited this method water body nitrate pollute trace to the source with administer in application.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, the cultivation of a kind of denitrifying bacterium is provided and utilizes this denitrifying bacterium to measure the method for water body nitrate nitrogen isotopic composition.
For achieving the above object, the technical solution used in the present invention is:
The cultivation of denitrifying bacterium and a mensuration water body nitrate nitrogen isotope method, is characterized in that, comprises the following steps:
(1) enrichment culture of denitrifying bacterium
To lack N
2the denitrifying bacterium inoculation of O reductase activity contains NO
3 -tSB sterile medium (26 DEG C, 180rpm) on shaking table cultivate, carry out the enrichment culture of denitrifying bacterium;
(2) whether detect denitrifying bacterium by NO in nutrient solution
3 -transform completely
Get step (1) gained bacterium liquid and sulfanilamide (SN) nitrite ion and carry out color reaction, if bacterium liquid becomes pink, illustrate that denitrifying bacterium is not by NO in nutrient solution
3 -be converted into N completely
2o, but rest on NO
2 -in the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid, can carry out subsequent experimental;
(3) denitrifying bacterium is concentrated
By step (1) and by step (2) detect bacterium liquid at ultra-high speed whizzer 30000rpm, 18 DEG C centrifugal 20 minutes, pour out supernatant liquid, residue thalline use without NO
3 -tSB nutrient solution suspend, be condensed into the bacterium liquid of 2 times;
(4) in sample bottle, add concentrating cells culture, the N on purifying in sheaf space and removal denitrifying bacterium bacterium liquid
2o
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is 12mL test tube, more than high-purity helium for each test tube (flow velocity is 10～20mL/min) purges 3h;
(5) NO in sample
3 -be converted into N
2o
In the test tube of step (4) with syringe inject be less than 4mL water sample (total nitric nitrogen amount be 0.5 μ g), sample turns upside down fully to mix after injecting; Test tube is inverted into the thermostat container 3h-24h that arranges 26 DEG C; After incubation time, inject the NaOH that 0.10mL concentration is 10mol/L, termination reaction after dissipation bacterium absorbs the CO producing simultaneously
2;
(6) N
2o nitrogen isotope is measured
The N that step (5) gained test tube is produced
2o gas utilizes trace gas analysis instrument (TraceGas)-isotope ratio mass spectrometer (IRMS) to measure, and obtains N
2o nitrogen isotopic composition measured value.
(7) correction of nitrogen isotope test result
δ
15the standard of N adopts δ
15 nUSGS34=-1.8 ‰, δ
15n
uSGS32=+180 ‰;
Nitrogen isotope correction equation is as follows :+180 ‰=m × δ
15n
uSGS32-meas+ b 1.
-1.8‰＝m×δ
15N
USGS34-meas+b②，
According to the actual value of these two standard models (T) and measured value (M), obtain m and b, thereby obtain δ
15the correction equation of N.
Then again cultivate denitrifying bacterium, carry out the mensuration of next batch sample.
The said TSB nutrient solution of step (1) is (containing 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, 1000mL deionized water, divides and installs to 500mL saline bottle mesohigh sterilizing 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 with NO in saline bottle
3 -tSB nutrient solution, 121 DEG C of sterilizings 60 minutes, after denitrifying bacterium switching, seal bottle stopper with sealed membrane, prevent and extraneous air inerchange.
The said sulfanilamide (SN) nitrite ion of step (2) compound method is: 4g sulfanilamide (SN), and 10mL high-quality pure 85% phosphoric acid, 0.1g N-1-naphthyl ethylenediamine hydrochloride, constant volume, to 100mL, should be colourless, just can not use if pulverize is red again.
Step (3) is said without NO
3 -tSB nutrient solution compound method be: 30g TSB, 0.5g (NH
4)
2sO
4, 4.9g KH
2pO
4, 2000mL deionized water, divides and installs in the serum bottle of 100mL, and autoclaving 60min is for 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 helium purge instrument, and test tube stands upside down and is inserted on helium pin, inserts in addition the syringe needle of a long 25gauge of 8cm, by the N in the air in test tube and bacterium liquid in silicagel pad
2o goes out with helium replacement.
Step (5), if said water sample add-on is greater than 4mL, just need to add an exhaust needle excessive to prevent pressure on test tube pad.
The said trace gas analysis instrument of step (6) (TraceGas)-isotope ratio mass spectrometer (IRMS), TracsGas is furnished with Gilson automatic sampler, 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, by Gilson automatic sampler by N
2o gas is delivered to TraceGas, extracts purifying and trapping N through TraceGas
2o gas, finally measures nitrogen isotopic composition by isotope ratio mass spectrometer.
With respect to prior art, the present invention has following excellent results:
1, adopt the ordinary salt water bottle easily obtaining to cultivate denitrifying bacterium, reduced a series of loaded down with trivial details formalities such as searching, customized production of special culturing bottle, reduced the cultivation cost of denitrifying bacterium simultaneously;
2, whether the present invention adopts in sulfanilamide (SN) nitrite ion detection denitrifying bacterium culturing process and the nitrate of nutrient solution itself is exhausted, simple to operate, has got rid of the impact of the inner nitrate of nutrient solution on sample nitrate, has improved the confidence level of measurement result.
3, it is time saving and energy saving that method of the present invention is measured 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 is for the seriously polluted present situation of needing improvement badly of the current 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 described in detail; but protection scope of the present invention is not limited to following enforcement introduction; all equivalent variation or accommodations of doing according to method of the present invention, all should be considered as the category that the present invention protects.
Embodiment 1:
A method for water body nitric nitrogen isotopics is cultivated and measured to denitrifying bacterium, comprises the following 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 respectively 480mL and installs in two saline bottles, and get in addition 2 5mL and install in test tube, plug sealing, autoclaving 60min, forms denitrifying bacterium enrichment culture liquid; To lack N
2the denitrifying bacterium Pseudomonas aureofaciens P.aureofaciens of O reductase activity is inoculated into 5mL NO
3 -the test tube of TSB sterile medium in, on shaking table, (26 DEG C, 180rpm) are cultivated one day, this 5mL bacterium liquid are transferred to containing NO in second day
3 -the saline bottle of 480mLTSB sterile medium in and on shaking table (26 DEG C, 180rpm) cultivate one week, carry out denitrifying bacterium enrichment culture;
(2) whether detect denitrifying bacterium by NO in nutrient solution
3 -transform completely
Step (1) gained bacterium liquid is got 1mL, adds 80 μ L sulfanilamide (SN) nitrite ions, if bacterium liquid becomes pink, illustrates that denitrifying bacterium is not by NO in nutrient solution
3 -be converted into N completely
2o, but rest on NO
2 -in the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid, can carry out subsequent experimental;
(3) denitrifying bacterium is concentrated
The bacterium liquid 200mL detecting by step (1) and by step (2) pours in 200-mL centrifugal bottle, at ultra-high speed whizzer 30,000rpm, 18 DEG C centrifugal 20 minutes (bacterium should become bulk at the bottom of bottle), discard supernatant liquid (note do not confuse bacterial mass), from a 100-mL without NO
3 -substratum in take out respectively 10ml and be added in each bottle, fully concussion, with Eddy diffusion bacterium, is transferred to the bacterium of Eddy diffusion in the beaker of a 200-mL, with remaining without NO
3 -substratum cleans centrifugal bottle, and they are all incorporated in the beaker of 200-mL, adds 10 antifoam B (purchased from Sigma company);
(4) in sample bottle, add concentrating cells culture, the N on purifying in sheaf space and removal denitrifying bacterium bacterium liquid
2o
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is 12mL test tube, test tube is the test tube of the high 100mm of internal diameter 15mm, adopt Teflon silicagel pad and screw-cap, more than high-purity helium for each test tube (flow velocity is 10～20mL/min) purges 3h;
(5) NO in 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 fully to mix after injecting; Test tube is inverted into 26 DEG C of incubated overnight of thermostat container.Within second day, inject the NaOH that 0.10mL concentration is 10mol/L, termination reaction after dissipation bacterium absorbs the CO producing simultaneously
2;
(6) N
2o nitrogen isotope is measured
The N that step (5) gained test tube is produced
2o gas utilizes trace gas analysis instrument (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 ‰;
Nitrogen isotope correction equation is as follows :+180 ‰=m × δ
15n
uSGS32-meas+ b 1.
-1.8‰＝m×δ
15N
USGS34-meas+b②，
According to the measured value of USGS34 and USGS32 and actual value, 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.
As can be seen here, be feasible with method test river nitric nitrogen of the present invention isotopics.
Embodiment 2:
A method for water body nitric nitrogen isotopics is cultivated and measured to denitrifying bacterium, and its concrete operation step is:
Step (1) arrives (3) with embodiment 1.
(4) in sample bottle, add concentrating cells culture, the N on purifying in sheaf space and removal denitrifying bacterium bacterium liquid
2o
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is 20mL jaw headspace sample bottle, more than high-purity helium for each sample bottle (flow velocity is 10～20mL/min) purges 3h;
(5) NO in 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 fully to mix after injecting; Test tube is inverted into 26 DEG C of incubated overnight of thermostat container.Within second day, inject the NaOH that 0.10mL concentration is 10mol/L, termination reaction after dissipation bacterium absorbs the CO producing simultaneously
2;
(6) N
2o nitrogen isotope is measured
The N that each step (5) headspace sample bottle is produced
2o gas with resistance to air loss syringe extract 2mL inject the test tube vacuumizing in advance, by Gilson automatic sampler by N
2o gas is delivered to TraceGas, extracts purifying and trapping N through TraceGas
2o gas, finally measures nitrogen isotopic composition by isotope ratio mass spectrometer, obtains measured value δ
15n=6.20 ‰.
Step (7), with embodiment 1, obtains the nitric nitrogen δ of this underground water sample
15n actual value is 12.21, and according to document, this value shows that in this underground water sample, azotate pollution mainly comes from muck.
As can be seen here, be feasible with method test Groundwater Nitrate-nitrogen of the present invention isotopics.
Embodiment 3:
A method for water body nitric nitrogen isotopics is cultivated and measured to denitrifying bacterium, and its concrete operation 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 respectively 520mL and installs in two 500-mL triangular flasks, and get in addition 2 5mL and install in test tube, plug sealing, autoclaving 60min, forms denitrifying bacterium enrichment culture liquid; Adopting denitrifying bacterium bacterial strain is Pseudomonas chlororaphis P.chlororaphis, and enrichment culture step is with embodiment 2 (1);
Step (2) arrives step (4) with embodiment 2;
(5) NO in 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 fully to mix, and on bottle holder, add an exhaust needle, be inverted to be placed on and on a test-tube stand, put into 26 DEG C of incubated overnight of thermostat container.Within second day, inject the NaOH that 0.10mL concentration is 10mol/L, termination reaction after dissipation bacterium absorbs the CO producing simultaneously
2;
Step (6)-(7), with embodiment 2, obtain the nitric nitrogen δ of this rainwater sample
15n actual value is 0.16, and according to document, this value shows that in this rainwater, nitrate source is main relevant with nitrogen deposition in air and ground chemical fertilizer ammonia volatilization.
As can be seen here, be feasible with method test rainwater nitric nitrogen of the present invention isotopics.
Claims (8)
1. cultivate denitrifying bacterium and measure a method for water body nitrate nitrogen isotope, it is characterized in that, comprise the following steps:
(1) enrichment culture of denitrifying bacterium
To lack N
2the denitrifying bacterium inoculation of O reductase activity contains NO
3 -tSB sterile medium on shaking table, cultivate, carry out the enrichment culture of denitrifying bacterium; The condition that described shaking table is cultivated is 26 DEG C, 180rpm;
(2) whether detect denitrifying bacterium by NO in nutrient solution
3 -transform completely
Get step (1) gained bacterium liquid and sulfanilamide (SN) nitrite ion and carry out color reaction, if bacterium liquid becomes pink, illustrate that denitrifying bacterium is not by NO in nutrient solution
3 -be converted into N completely
2o, but rest on NO
2 -in the stage, this bottle is abandoned processing; If the nondiscoloration of bacterium liquid, can carry out subsequent experimental;
(3) denitrifying bacterium is concentrated
By step (1) and by step (2) detect bacterium liquid at ultra-high speed whizzer 30,000rpm, 18 DEG C centrifugal 20 minutes, pour out supernatant liquid, residue thalline use without NO
3 -tSB nutrient solution suspend, be condensed into the bacterium liquid of 2 times;
(4) in test tube, add concentrating cells culture, the N on purifying in sheaf space and removal denitrifying bacterium bacterium liquid
2o
Getting the concentrated bacterium liquid 3mL injection of step (3) gained volume is 12mL test tube, and each test tube is with more than high-purity helium purge 3h; The flow velocity of described high-purity helium is 10～20mL/min;
(5) NO in sample
3 -be converted into N
2o
In the test tube of step (4), inject with syringe the water sample that is less than 4mL, sample turns upside down fully to mix after injecting; Test tube is inverted into 26 DEG C of incubated overnight of thermostat container; Within second day, inject the NaOH that 0.10mL concentration is 10mol/L, termination reaction after dissipation bacterium absorbs the CO producing simultaneously
2; Total nitric nitrogen amount of described water sample is 0.5 μ g;
(6) N
2o nitrogen isotope is measured
The N that step (5) gained test tube is produced
2o gas utilizes trace gas analysis instrument (TraceGas)-isotope ratio mass spectrometer (IRMS) to measure, and obtains N
2o nitrogen isotopic composition measured value;
(7) correction of nitrogen isotope test result
δ
15the standard of N adopts δ
15n
uSGS34=-1.8 ‰, δ
15n
uSGS32=+180 ‰;
Nitrogen isotope correction equation is as follows :+180 ‰=m × δ
15n
uSGS32-meas+ b 1.-1.8 ‰=m × δ
15n
uSGS34-meas+ b 2.,
According to the actual value of these two standard models (T) and measured value (M), obtain m and b, thereby obtain δ
15the correction equation of N;
Then again cultivate denitrifying bacterium, carry out the mensuration of next batch sample.
2. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, is characterized in that the said NO that contains of step (1)
3 -tSB sterile medium compound method be: 30g pancreas peptone soybean broth, 1.0g KNO
3, 0.5g (NH
4)
2sO
4, 6.15g K
3pO
43H
2o, 1000mL deionized water, divides and installs to 500mL saline bottle mesohigh sterilizing 60min.
3. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, it is characterized in that, the enrichment culture of the said denitrifying bacterium of step (1) is to carry out in saline bottle, saline bottle is that cubic capacity is hospital's common infusion fluid bottle of 580mL, bottle cap adopts rubber plug, 460mL-480mL is housed with NO in saline bottle
3 -tSB nutrient solution, 121 DEG C of sterilizings 60 minutes, after denitrifying bacterium switching, seal bottle stopper with sealed membrane, prevent and extraneous air inerchange.
4. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, it is characterized in that, the said sulfanilamide (SN) nitrite ion of step (2) compound method is: 4g sulfanilamide (SN), pure 85% phosphoric acid of 10mL top grade, 0.1g N-1-naphthyl ethylenediamine hydrochloride, constant volume, to 100mL, should be colourless, just can not use if pulverize is red again.
5. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, is characterized in that, step (3) is said without NO
3 -tSB nutrient solution compound method be: 30g TSB, 0.5g (NH
4)
2sO
4, 4.9gKH
2pO
4, 2000mL deionized water, divides and installs in the serum bottle of 100mL, and autoclaving 60min is for subsequent use.
6. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, 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. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, it is characterized in that, the said high-purity helium purge of step (4) adopts helium purge instrument, test tube stands upside down and is inserted on helium pin, in silicagel pad, insert in addition the syringe needle of a long 25gauge of 8cm, by the N in the air in test tube and bacterium liquid
2o goes out with helium replacement.
8. a kind of method of cultivating denitrifying bacterium mensuration water body nitrate nitrogen isotope according to claim 1, it is characterized in that, the said trace gas analysis instrument of step (6) (TraceGas)-isotope ratio mass spectrometer (IRMS), TracsGas is furnished with Gilson automatic sampler, 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, by Gilson automatic sampler by N
2o gas is delivered to TraceGas, extracts purifying and trapping N through TraceGas
2o gas, finally measures nitrogen isotopic composition by isotope ratio mass spectrometer.
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| CN103776674A (en) * | 2014-01-20 | 2014-05-07 | 西华大学 | Nitrogen extracting and purifying device of nitrogen isotope sample in water |
| 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 |
| CN103822963B (en) * | 2014-03-19 | 2016-03-30 | 哈尔滨工业大学 | Whether the ANAMMOX bacterium in qualification ANAMMOX reactor mud utilizes organic method |
| CN104630053A (en) * | 2015-01-23 | 2015-05-20 | 长江大学 | Production method of denitrifying bacteria test bottle |
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| CN112320941B (en) * | 2020-10-21 | 2022-10-11 | 西安建筑科技大学 | Domestication product N by using separation membrane filler 2 Device and method for O denitrifying bacteria |
| CN113933372B (en) * | 2021-11-11 | 2023-11-03 | 广西大学 | Quantitative identification method for river basin nitrate nitrogen source river entering load and river entering coefficient |
| 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 |
| CN115266280B (en) * | 2022-09-28 | 2023-03-21 | 中国农业科学院农业环境与可持续发展研究所 | Method for detecting nitrogen and oxygen isotopes of nitrate |
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