Method for culturing denitrifying bacterium and determining water body nitrate nitrogen isotope composition

A technology of denitrifying bacteria and nitrogen isotopes, applied in bacteria, measuring devices, instruments, etc., can solve the problems that the gas cannot be directly analyzed, lack of detection methods, and difficult to find, so as to reduce the cost of testing, reduce the cost of cultivation, and save composition time-saving effect

Active Publication Date: 2014-07-02
INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application of denitrifying bacteria to test nitrate nitrogen isotopes in China is still relatively small, and the existing literature methods also have the following disadvantages: (1) The cultivation of denitrifying bacteria requires special culture bottles, which are difficult to find in ordinary laboratories; (2) Whether the nitrate in the culture solution is completely converted to N after enrichment culture of denitrifying bacteria 2 O, without affecting follow-up sample determination, this index detection method is lacking at present; (3) N produced with sample 2 O gas cannot be analyzed directly, and N 2 O gas is injected into the sample bottle connected to the mass spectrometer at one time and then analyzed, which requires a large sample volume
Comprehensive analysis At present, there is no detailed and operable denitrifying bacteria method to test the nitrate nitrogen isotope in water, which limits the application of this method in the traceability and treatment of nitrate pollution in water

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A method for culturing denitrifying bacteria and determining the composition of nitrate nitrogen isotopes in water, comprising the following steps:

[0041] (1) Enrichment culture of denitrifying bacteria

[0042] Add 30 g Tryptone Soy Broth Agar (TSB), 1.0 g KNO per liter of deionized water 3 , 0.5g (NH 4 ) 2 SO 4 , 6.15g K 3 PO 4 ·7H 2 O, respectively take 480mL and put them into two saline bottles, and take another 2 pieces of 5mL and put them into test tubes, seal with rubber stoppers, and autoclave for 60min to form a denitrifying bacteria enrichment culture solution; 2 O-reductase activity of denitrifying bacteria caused by Pseudomonas aureus P. aureofaciens was inoculated into 5mL with NO 3 - In a test tube of TSB sterilized culture medium, culture it on a shaker (26 °C, 180 rpm) for one day, and transfer the 5 mL of the bacterial solution to an NO-containing medium the next day. 3 - 480mL of TSB sterilized culture solution in a saline bottle and cultur...

Embodiment 2

[0060] A method for culturing denitrifying bacteria and measuring the composition of nitrate nitrogen isotopes in water, the specific operation steps are:

[0061] Steps (1) to (3) are the same as in Example 1.

[0062] (4) Add concentrated cell culture to the sample bottle, purify the upper space and remove N in the denitrifying bacterial solution 2 O

[0063] Take 3mL of the concentrated bacterial solution obtained in step (3) and inject it into a 20mL crimp headspace sample bottle, and each sample bottle is purged with high-purity helium gas (the flow rate is 10-20mL / min) for more than 3h;

[0064] (5) NO in the sample 3 - convert to N 2 O

[0065] In the test tube of step (4), inject 0.5 mL of groundwater sample (the nitrate nitrogen content is 25.75 mg / L) with a syringe. After the sample is injected, turn it upside down to fully mix; put the test tube upside down and put it into an incubator at 26°C for overnight incubation. The next day, 0.10 mL of NaOH with a conc...

Embodiment 3

[0071] A method for culturing denitrifying bacteria and measuring the composition of nitrate nitrogen isotopes in water, the specific operation steps are:

[0072] (1) Enrichment culture of denitrifying bacteria

[0073] Add 30 g Tryptone Soy Broth Agar (TSB), 1.0 g KNO per liter of deionized water 3 , 0.5g (NH 4 ) 2 SO 4 , 6.15g K 3 PO 4 ·7H 2 O, respectively take 520mL and put them into two 500-mL conical flasks, and take another 2 pieces of 5mL and put them into test tubes, seal them with rubber stoppers, and sterilize them by high pressure for 60min to form a denitrifying bacteria enrichment culture solution; the denitrifying bacteria strains are used as Pseudomonas chlororaphis, the enrichment culture steps are the same as those in Example 2(1);

[0074] Step (2) is with embodiment 2 to step (4);

[0075] (5) NO in the sample 3 - convert to N 2 O

[0076]Use a syringe to inject 5 mL of rainwater sample (with nitrate nitrogen content of 0.21 mg / L) into the crim...

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Abstract

The invention relates to the technical field of treatment and control of nitrate pollution in water bodies, and is a method for cultivating a denitrifying bacterium and using the bacterium to determine the nitrogen isotope composition of nitrate in water. Whether the denitrifying bacteria can completely convert NO3- in the culture solution, ③ concentration of denitrifying bacteria, ④ adding concentrated cell culture in the sample bottle, purifying the upper space and removing N2O in the denitrifying bacteria liquid, ⑤ NO3- in the sample Converted to N2O, 6. N2O nitrogen isotope determination, 7. the correction of nitrogen isotope test results, this test is completed; the positive effect of the present invention is: only adopt common brine bottle or triangular flask just can complete the cultivation of denitrifying bacteria, the sulfonamide color development The detection improves the reliability of the measurement results and reduces the cost of the test; and it is aimed at the current situation of serious water nitrate pollution in my country that urgently needs to be treated. It has the advantages of scientificity, simplicity, and strong operability, and has broad prospects for promotion.

Description

technical field [0001] The invention relates to cultivating denitrifying bacteria and using the bacteria to distinguish the isotope composition of nitrate nitrogen, belonging to the technical field of nitrate pollution treatment and control of water body. Background technique [0002] Groundwater is an irreplaceable important resource necessary for my country's economic and social development and people's life. More than 1.5 billion people worldwide rely primarily on groundwater for drinking water. In some cities and vast rural areas in China, groundwater is often the only source of water supply. In recent years, the pollution of groundwater by nitrate nitrogen has become a common concern in the world. In many countries and regions in Europe and the United States, the content of nitrate nitrogen in groundwater has seriously exceeded the standard. Many areas in my country have also been polluted by nitrate nitrogen to varying degrees, especially in rural areas. The pollutio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20G01N27/62
Inventor 徐春英李玉中郝卫平孙东宝李巧珍董一威
Owner INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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