CN1074547C - Method for biological fixed nitrogen detection - Google Patents
Method for biological fixed nitrogen detection Download PDFInfo
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- CN1074547C CN1074547C CN 94103368 CN94103368A CN1074547C CN 1074547 C CN1074547 C CN 1074547C CN 94103368 CN94103368 CN 94103368 CN 94103368 A CN94103368 A CN 94103368A CN 1074547 C CN1074547 C CN 1074547C
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- nitrogen
- nitrogen fixation
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Abstract
The present invention relates to a method for detecting biological nitrogen fixation. The method is characterized in that a detected sample of nitrogen fixation is placed in a culture bottle with an amount of volume; an amount of carbohydrate with less than ten carbon atoms and sterilized water are added, and the culture bottle is sealed by a rubber plug; then, the detected sample of nitrogen fixation is cultured for a certain time at proper temperature, and mixed gas is taken out from the culture bottle and is detected by a gas chromatograph. The method has the advantages of accuracy, reliability, simplicity, practicality, low cost, etc. The method is widely applied to the detection of nitrogen fixation activity and nitrogen fixation amount. The method can be accurately qualitative, and can accurately detect the nitrogen fixation amount.
Description
The present invention relates to the method that a kind of biological nitrogen fixation detects, especially a kind of research that is applied to the fixed nitrogen science, the discriminating of nitrogen-fixing bacteria, azotobacterin and the method for measuring of microorganism fixed nitrogen fertilizer quality.
The research of fixed nitrogen science is heat subject in the world, and from reported in literature as can be known, this research is still classified as the great main direction of 21 century scientific research task by developed countries such as the U.S., Japan.In the research of this science, need nitrogen-fixing microorganism, nitrogen-fixing plants are differentiated, expression to the DNA of nitrogen-fixing microorganism reorganization performance, the decision analysis of azotobacterin, microbial manure nitrogenase activity, amount of nitrogen fixation is all needed a kind of highly sensitive, method accurately and reliably.The method of Ying Yonging is " Kjeldahl " the earliest, and this method has its accuracy and high reliability features, but sensitivity is too low, and the faint mensuration of some amount of nitrogen fixation can't be used.What grow up is thereupon "
15The N trace method ", this method " Kjeldahl " has relatively improved 1,000 times, but this method need contain
15The salt of N or other chemicals cost an arm and a leg, and need to use large-scale instrument " isotope mass spectrometer ", are difficult to widely use.Invented a kind of " acetylene reducing process " in recent years again, this method is relative again "
15" sensitivity has improved 1,000 times to the N trace method, and just " Kjeldahl " improved 10 relatively
6Doubly.Therefore scientists has been isolated nearly more than 70 nitrogen-fixing microorganisms that belong to kind more than 400.Though this method has highly sensitive characteristic, it is a kind ofly can reduce the theoretical foundation of three key compounds according to azotase and set up.That is to say that azotase can reduce N ≡ N and also can reduce CH ≡ CH.Because this method is not directly to measure nitrogen but the acetylene measured generates the amount of ethene, just be equivalent to reduce a N
2Generate 2NH
3There are many different versions of a story for scientists in practical application, can't be quantitative so only can be used for qualitative analysis.
The purpose of this invention is to provide and a kind ofly can determine nitrogenase activity, can accurately determine amount of nitrogen fixation again, the method that low, the simple and easy to do biological nitrogen fixation of cost detects.
The concrete solution of the present invention is: the method that a kind of biological nitrogen fixation detects, it is characterized in that: the tested sample of fixed nitrogen is put in the culture flask of an amount of volume of people, and add an amount of following hydrate of ten carbon and sterilized water, seal with plug, then under the proper temperature condition, cultivate certain hour, take out combination gas gas chromatograph for determination in the culture flask.Example 1: the tested sample of fixed nitrogen is: Tianlibao microorganism chemical fertilizer, the following carbohydrates of ten carbon is a sucrose.Example 2: the tested sample of fixed nitrogen is: the active combination azotobacter of rhizosphere, the following carbohydrates of ten carbon is a glucose.Example 3: the tested sample of fixed nitrogen is: nodule azotobacter and microbial inoculum and the fertilizer made with them, the following carbohydrates of ten carbon is a glucose.Example 4: the tested sample of fixed nitrogen is: azotobacter and with they made microbial inoculum and fertilizer, the following carbohydrates of ten carbon is a glucose.
Advantage of the present invention and good effect are as follows: 1. and highly sensitive, accurately and reliably, can be used for the mensuration of multiple fixed nitrogen magnitude.2. simple and easy to do, cost is low, and intuitive is strong.3. be applied to the detection of nitrogenase activity and amount of nitrogen fixation, outside decapacitation is accurately qualitative, also can accurately determine amount of nitrogen fixation.
Provide specific embodiment of the present invention below.
(nitrogen-fixing microorganism is purebred to get an amount of sample that will test, nitrogen-fixing microorganism suspension, azotobacterin or nitrogen-fixing microorganism goods), place in the culture flask of suitable volume, it is an amount of to add corresponding nutrient culture media, seal with rubber plug or other closed materials, after vacuumizing then, injection contains O
220%, N
280% normal mixture body (or not vacuumizing the direct natural air of using), making in the bottle is 1 atmospheric pressure.The culture flask for preparing placed under corresponding temperature (general 10～40 ℃) condition cultivates, cultivate certain hour according to different nitrogen-fixing microorganisms or product requirements after, detect N with the standard sampling container sampling
2The variation of percentage amounts, thus obtain N
2Reduction, according to N
2Reduction, extrapolate the amount of nitrogen fixation of every gram nitrogen-fixing microorganism and goods thereof.
Embodiment one: 1. get an amount of Tianlibao microbial manure, be ground into greater than 60 purpose pulvis with mortar.2. the pulvis in 1 being got same amount, to place capacity be 100 milliliters culture flask, and every bottle 0.5～1.0 gram adds 0.25～1.0 gram glucose or sucrose then, and injects 2～10 milliliters of sterilized waters, uses anti-chewing-gum plug plug good then.3. the culture flask in 2 is evacuated, injects then and contain O
220%, N
280% mixed gas is 1 atmospheric pressure, approximately will inject the above-mentioned mixed gas about 90～98 milliliters.Take out combination gas gas chromatograph for determination in the bottle with standard sampling container immediately, calculate bottle outlet and include nitrogen (N
2) be V
1Mole places under 28 ℃ ± 1 ℃ condition after cultivating in 2～10 days, gets combination gas in the bottle again and measures, and calculates bottle outlet and includes N
2Be V
2Mole.4. use with quadrat method and make blank, measure N in the bottle before cultivating
2Molar weight be M
1, cultivate the interior N of bottle after 1～6 day
2Molar weight be M
2, (general M
1With M
2Between change very faint, in 1～2% scope).5. calculate N
2Reduction, and the actual fixed amount after the blank natural reduction of deduction is V
m
V
m=[V
1-V
2-(m
1-m
2)]/0.5～1.0
Can fix nitrogen more than 100 milligrams by calculating general every gram Tianlibao, but reach more than 560 milligrams if be converted into every consumption 1 gram sugar amount of nitrogen fixation.
Embodiment two: 1. get active combination azotobacter 0.1～1 gram of an amount of rhizosphere, placing the sterilization volume is 5～20 milliliters culture flask, adds glucose 0.1～1 gram, adds 2～10 milliliters of sterilized waters then, good with the plug plug then.2. vacuumize back notes people then and contain O
220% contains N
280% mixed gas or do not vacuumize and use natural air, making pressure of the inside of a bottle is 1 atmospheric pressure.Get bottle interior combination gas 10～100 microlitre injection gas chromatographies with sampler immediately and detect a bottle N
2Volume be V
1Mole.Place cultivate 6～24 hours under 15～40 ℃ of conditions after, get in 10～100 microlitre bottles combination gas again and measure, bottle includes N
2Be V
2Mole.3. use with quadrat method and make blank, measure and cultivate N in the preceding bottle
2Molar weight be M
1, cultivate the interior N of bottle after 6～24 hours
2Molar weight be M
24. calculate N
2Reduction, and the actual fixed amount of having deducted after the blank natural reduction is V
m
V
m=[V
1-V
2-(m
1-m
2)]/0.1～1 gram
Said method also is applicable to nodule azotobacter, azotobacter and with they made microbial inoculum and fertilizer.
Claims (5)
1. the method that detects of a biological nitrogen fixation is characterized in that by following step:
(1) the tested sample of fixed nitrogen is inserted in the culture flask, add ten carbon following carbohydrates and sterilized water, the sealing of blended rubber plug vacuumizes then;
(2) injection contains the mixed gas that volume ratio is 20% oxygen and 80% nitrogen, adopts combination gas in the gas chromatograph for determination bottle immediately, calculates nitrogen content;
(3) then the above-mentioned bottle that contains mixed gas is placed under the 10-40 ℃ of temperature and cultivates;
(4) use the quantity of measuring nitrogen in the bottle with quadrat method, calculate the reduction of nitrogen, extrapolate the amount of nitrogen fixation of tested sample then according to the reduction of nitrogen.
2. a kind of biological nitrogen fixation detection method according to claim 1 is characterized in that: the tested sample of fixed nitrogen is: the Tianlibao microbial manure, the following carbohydrates of ten carbon is a sucrose.
3. a kind of biological nitrogen fixation detection method according to claim 1 is characterized in that: the tested sample of fixed nitrogen is: root is marked active combination azotobacter, and the following carbohydrates of ten carbon is a glucose.
4. a kind of biological nitrogen fixation detection method according to claim 1 is characterized in that: the tested sample of fixed nitrogen is: nodule azotobacter and microbial inoculum and the fertilizer made with them, the following carbohydrates of ten carbon is a glucose.
5. a kind of biological nitrogen fixation detection method according to claim 1 is characterized in that: the tested sample of fixed nitrogen is: azotobacter and with they made microbial inoculum and fertilizer, the following carbohydrates of ten carbon is a glucose.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94103368 CN1074547C (en) | 1994-03-31 | 1994-03-31 | Method for biological fixed nitrogen detection |
| PCT/US1994/008846 WO1995004814A1 (en) | 1993-08-06 | 1994-08-05 | Recombinant microbial fertilizer and methods for its production |
| AU76301/94A AU7630194A (en) | 1993-08-06 | 1994-08-05 | Recombinant microbial fertilizer and methods for its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 94103368 CN1074547C (en) | 1994-03-31 | 1994-03-31 | Method for biological fixed nitrogen detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1109595A CN1109595A (en) | 1995-10-04 |
| CN1074547C true CN1074547C (en) | 2001-11-07 |
Family
ID=5031026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 94103368 Expired - Fee Related CN1074547C (en) | 1993-08-06 | 1994-03-31 | Method for biological fixed nitrogen detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1074547C (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6416982B1 (en) | 2000-09-05 | 2002-07-09 | Ultra Biotech Ltd. | Biological fertilizer based on yeasts |
| WO2002020431A1 (en) | 2000-09-05 | 2002-03-14 | Ultra Biotech Limited | A biological fertilizer based on yeasts |
| US6761886B2 (en) | 2001-03-01 | 2004-07-13 | Ultra Biotech Limited | Biological fertilizer compositions comprising cattle manure |
| US6596273B2 (en) | 2001-03-01 | 2003-07-22 | Ultra Biotech Limited | Biological fertilizer compositions comprising swine manure |
| US6596272B2 (en) | 2001-03-01 | 2003-07-22 | Ultra Biotech Limited | Biological fertilizer compositions comprising poultry manure |
| US6800466B2 (en) | 2001-03-01 | 2004-10-05 | Ultra Biotech Limited | Biological fertilizer compositions comprising sludge |
| US6828132B2 (en) | 2001-03-01 | 2004-12-07 | Ultra Biotech Limited | Biological fertilizer compositions comprising garbage |
| CN105866275A (en) * | 2016-04-05 | 2016-08-17 | 济宁神农画圆生态系统模式推广有限公司 | Method and device for quantitative analysis on nitrogen-oxygen absorption capacity of nitrogen-fixing bacteria in sealed container |
-
1994
- 1994-03-31 CN CN 94103368 patent/CN1074547C/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| CN1109595A (en) | 1995-10-04 |
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Granted publication date: 20011107 |