We are intending to calculate the atmospheric nitrogen fixing ability of diazotrophs through Kjeldhal's method as we don't have the facility of Gas Chromatograph at our lab. Anyone to guide the full procedure of the same please.
Biological nitrogen fixation, which is the reduction of atmospheric nitrogen (N2) to two molecules of ammonia, is the second most important biological process on earth after photosynthesis. Rice plants are generally associated with several diazotrophs, and the Azospirillum, Azoarcus, Enterobacter, Herbaspirillum, Burkholderia and Corynebacteria genera are commonly associated with the rice rhizosphere.
With the development of new techniques for detecting the ability to fix nitrogen such as Kjeldahl, 15N and acetylene (C2H2) reduction methods, an increased number of species and systems able to fix nitrogen have been identified.
The Kjeldahl method for determination of nitrogen content was introduced in 1883 and was the only available method until the 1940’s when the 15N method was adopted to assess nitrogen-fixation in Azotobacter. Subsequently, different methods were developed to ascertain the ability of an organism to fix nitrogen.
In spite of its limitations the Kjeldahl method is still very useful for assessing the response to inoculation, especially in laboratories lacking the specialized equipment required for other methods, although it cannot be used to present conclusive evidence of the capacity of an organism to fix nitrogen.
The method includes:
1. Incubation of the test material in the presence of N2 enriched with 15N.
2. Conversion of all N in the sample to ammonia by Kjeldahl digestion.
3. isolation of the ammonia by distillation.
4. Oxidation of the ammonia to N2 .
5. Mass analysis of N2 in a mass spectrometer. Rennie and Fried, 1978. Use of isotopes in biological nitrogen-fixation).
Any 15N 2 incorporated by the test material is detected as an increase in the abundance of mass 15 above the background value of 0.364% due to natural abundance of 15N. This increase is expressed as % 15N excess. Samples giving an atom % 15N excess of more than 0.015 could be accepted as positive evidence for N2 fixation.
For detail please find attached herewith article.
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