FIXATION OF NITROGEN 49 



proportional to the mass of cytoplasm present in the 

 system. With Azotohacter, the Ng uptake is insignificant 

 compared with the O2 uptake, and is therefore neglected in 

 calculating the results. Direct proof that such assumptions 

 are justified was provided by Burk and Meyerhof, the 

 originators of this technique [29, 7]. In their experimental 

 conditions, irrespective of whether growth was followed by 

 nephelometry, dry-weight measurements or total cell counts, 

 the grov^h curves were all reasonably superimposable on 

 the graph relating respiration rate with age of culture. 

 Manometry has since been applied to the study of Na-fixa- 

 tion by anaerobic bacteria, such as Clostridia, Desulpho- 

 vibrio and photosynthetic organisms [34]. 



Unequivocal evidence as to whether a system can fix 

 N2 is provided by the use of N2 enriched with the non- 

 radioactive isotope N^^, a technique first introduced by 

 Burris [9] and which later proved to be of value in the 

 elucidation of the route of N2-fixation. The requisite control 

 experiments showed that non-enzymic exchange reactions 

 between molecular N2^^ and compounds containing nitrogen 

 were insignificant, and that in vivo there was no preferential 

 uptake of one isotope over another, i.e. the ratio of the 

 isotopes one to another in the gas-phase did not change 

 throughout the course of the experiment. To detect Ng- 

 fixation, the experimental material is placed in a closed 

 system through which N2 enriched with N^^ is circulated by 

 a Urey pump. The nitrogen-containing compounds of the 

 experimental material may be fractionated prior to being 

 converted into N2 for analysis in the mass spectrograph. 

 The technique is very sensitive and an increase of o-oi atom 

 per cent N^^ above the normal distribution of 0-36 is 

 regarded as being significant, and in certain conditions it is 

 possible to detect the fixation of o-ooi //g. N [9, 10]. 



Organisms fixing N2 



In addition to CI. pasteurianuni and Azotobacter spp., there 

 is now adequate evidence, in many cases based on experi- 

 ments with N^^, that Ng-fixation is a property of several 

 other species of Clostridia [32], various photosynthetic 



