114 Perspectives in Microbiology 



Using this equipment, we studied the effect of PCO2, PO2, 

 and pN2 on the rate of nitrogen fixation. 



This apparatus was admittedly crude, but it had one 

 virtue — it required considerable volumes of gases, so when 

 a gas was required to fill out a particular mixture being 

 studied, H2 was the natural choice because it was the 

 cheapest. This choice of diluent gas was a happy one, for 

 which we cannot claim any credit, as it was forced upon 

 us by the method used. In one trial, the tank of H2 became 

 empty just as the experiment was begun; for several days, 

 it was necessarily continued without the use of a diluent 

 gas until a new tank was delivered. Fortunately, this was 

 delayed, and since the response of the plants appeared to 

 be different without the Ho, the experiment was completed 

 without diluent gas. The results were unequivocal — the 

 plants fixed nitrogen better in the absence of H2. Through 

 the courtesy of Professor Roebuck in the physics depart- 

 ment we were given a tank of helium, which was difficult 

 to obtain at that time. The necessary crucial experiment 

 in which Ho and He were compared as the diluent gas 

 demonstrated that H2, far from being an inert gas, defi- 

 nitely inhibited nitrogen fixation in red clover plants. 

 Further studies provided evidence that this inhibition was 

 competitive (15). 



Once again, fullest exploitation of this new tool of re- 

 search necessitated the forging of a sharper instrument; 

 this was accomplished when the finding was extended to 

 fixation of nitrogen by Azotobacter (20). Then, studies on 

 the enzyme system employing H2 as a specific inhibitor 

 could be made with the more precise microrespirometer 

 technique. Not only has this particular tool been valuable 

 for exploring the enzymatic system responsible for fixa- 

 tion of nitrogen, but also it has led to investigations that 

 have made significant contributions to our knowledge of 

 the enzyme hydrogenase (3, 5, 11). Finally, it has been di- 

 rectly concerned with uncovering several new agents of 



