FIXATION OF NITROGEN 47 



containing mannitol, isolated the two aerobes Azotohacter 

 chroococcutn and Azotohacter agilis (extremely motile) [4]. 

 Apart from Az. indiciwi, Azotohacter spp. in general do not 

 fix Ng in an acidic environment, consequently their isolation 

 is facilitated by the use of a neutral or slightly alkaline 

 medium, e.g. one containing a buffer or CaCOg. Further- 

 more, the incorporation of a small amount of sodium 

 molybdate (5x10"® per cent) is frequently advantageous 

 since molybdenum appears to be of especial significance 

 in organisms which fix Ng . 



Organic compounds, other than those which serve as 

 sources of carbon and energy, retard the growth of Azoto- 

 hacter but not Rhizohium. It is, however, unlikely that the 

 concentration of organic matter in the soil is ever great 

 enough to affect the fixation of Ng by Azotohacter in its 

 natural habitat. Of the thirty-five compounds tested only 

 aspartic acid, asparagine, glutamic acid, urea and adenine 

 replaced molecular Ng as a source of N for Azotohacter [20]. 

 On the other hand, the growth of Rhizohium spp. was sup- 

 ported by any one of thirty-two organic nitrogen compounds 

 and was luxuriant in rich organic media [30]. The optimal 

 growth of fast-growing, but not slow-growing, strains of 

 Wiizohium is dependent on an exogenous supply of a sub- 

 stance originally named coenzyme R and later identified as 

 biotin [cf. 40]. 



Detection of N ^-fixation 



Although from time to time the power to fix N 2 has been 

 attributed to many other organisms, the majority of these 

 claims must now be discounted on the grounds of faulty 

 experimentation. It is very difficult, if not impossible, to 

 eliminate all nitrogenous compounds from the materials 

 used to make media, consequently growth in what is believed 

 to be, apart from molecular Ng , a N-free medium is not a 

 sufficient criterion for stating that an organism can fix 

 nitrogen and, indeed, there is often no mention of control 

 cultures incubated in the absence of molecular Ng . It is 

 therefore relevant to consider the techniques used in the 

 detection and quantitative study of Ng-fixation. Apart from 



