FIXATION OF NITROGEN 5I 



specifically concerned with the fixation mechanism (for 

 discussion and references concerning trace element nutri- 

 tion, see 8, 44, 21, 6). 



Burk was the first to demonstrate that the rate of Ng- 

 fixation by Azotobacter was a function of the partial pressure 

 of Ng (pN2)» the relationship between the two conforming 

 to that expressed by the Michaelis-Menten equation for an 

 enzymic reaction. The pNg at which fixation occurred at 

 half the maximal rate (i.e. K^) was 0-21 atm., an unex- 

 pectedly high value for a gaseous substrate [7]. In order to 

 prepare gas mixtures containing different pNg , Burk had 

 used Hg as a diluent gas since at that time Hg was thought 

 to be inert in biological systems. Later experiments, first 

 with red clover plants [41, 47] and subsequently with 

 Azotobacter [50], revealed that Hg inhibited Ng-fixation and 

 increasing the pHg caused a progressive decrease in the 

 rate of growth: Hg had no effect on growth in the presence 

 of fixed nitrogen, e.g. (NH4)2HP04 . Helium and argon 

 exhibited no such inhibitory action, and in the absence of 

 Hg , fixation by Az. vinelandii was maximal at a pNg of 

 0-15 atm. and half maximal at 0-02 atm. (Fig. 4.1). Hence, 

 Wilson suggested that in natural conditions the pNg is not 

 a factor limiting fixation. By comparing the rate of grov^1:h 

 in air with that in gas phases containing, in addition to Og , 

 either 0-15 atm. Ng or 0-3 atm. Ng , together with different 

 amounts of Hg , Wyss and Wilson deduced that Hg inhibited 

 fixation in a competitive manner. Unlike Azotobacter, Hg is 

 a normal product in fermentations by CI. pasteurianum, and 

 although the pHg did not influence the rate of Ng-fixation 

 by this organism it did affect the amount of Ng fixed and 

 the efficiency of the fixation process (i.e. mg. N fixed/g. 

 glucose fermented) [32]. Wilson has noted that all the free- 

 living organisms shown to fix Ng also possess the enzyme 

 hydrogenase [cf. 42]. He suggests that the latter may be 

 involved in the fixation mechanism, since the hydrogenase 

 activity of Azotobacter is greatly reduced, even in the 

 presence of Hg , w^hen growth is no longer dependent on 

 the fixation of molecular Ng [25]. Hydrogenase has not been 

 detected in Rhizobium either free-living or symbiotic, or in 



