Methodology for Nitrogen Fixation

Hydrogen evolution

Nitrogenase catalyses not only the reduction of dinitrogen to ammonia but also donates electrons to protons to evolve H2 which is considered as an unnecessary expenditure of cellular energy in legume nodules whereas the same is considered as a fuel synthesizing process in organisms like cyanobacteria.

Hydrogen evolution by nodules can be measured by mass spectrometry, gas chromatography and amperometry. The gas chromatography technique is described below.


Hydrogen evolved by a known quantity of nodules is measured directly in a gas chromatograph.

» Serum Bottles (Penicillin vials, 21 or 69mL capacity), with caps
» Air-tight Syringes
» Gas Chromatograph
Detector                             Thermal conductivity
Detector temp  90°C
Column                             Molecular sieve 5A 60-80 mesh
Carrier gas        Argon; 15mL/min
Oven temp        Isothermal 65°C
» Retention time H2    0.45 min
O2    0.80 min
N2    1.55 min

Place the nodules (fresh), still attached to the root into the serum bottles and seal with the rubber cap.
At periodic intervals (1h to 3h) remove aliquots of gas sample (0.5 to 1mL) with an air-tight syringe, and inject into the preconditioned GLC.
Look for the H2 peak and measure the peak height. Note down the attenuation.
Record the dry weight of the detached nodules.
As a standard, inject 100 to 500mL(Z) of pure H2 gas into an empty sealed serum-bottle (volume of the bottle should be the same as that for the sample). Shake well. Remove 0.5mL or 1mL (as in the case of sample) and inject into GLC.
More than one volume (Z) may be used as standard and can be verified for linearity.

Standard amount of H2 mmol =
0.0446 x Z mL
Peak height in mm x attenuation

Amount of H2 mmol evolved
= E x H2peak height of sample in mm x attenuation
Express H2 evolved mmol per unit weight of nodules.


Sufficient quantities of nodules should be placed in the bottles to produce adequate H2 for accurate measurement in a reasonable time.
To compare the nodules for uptake hydrogenase (Hup), carry out an experiment in sealed serum bottles with nodules after displacing the air in the bottle with argon and oxygen. After a period of incubation, analyze gas samples in GLC. Measure the H2 evolved. If little or no hydrogen is evolved, it shows high uptake hydrogenase activity in the sample. If the H2 evolution is high as that evolved in the previous experiment, when incubated in the presence of air, it indicates low or nil uptake hydrogenase activity in the sample.
If argon is not available, N2 can be used as carrier gas in GLC. The sensitivity will be somewhat low. Analysis of samples will be fast due to the absence of N2 peak.


1. Turner, G L and Gibson, A H (1980) In: Methods for Evaluating Biological Nitrogen Fixation (Ed Bergersen F J) John Wiley and Sons NY p 111.
2. Hanus, F J, Kevin, R C and Evans, H J (1980) In: Methods in Enzymology (Ed Anthony San Pietro) Academic Press New York 69 p 731.