( 70) 



Now follows a table of some analyses 1 ) which give an idea of the 

 amount of nitrogen fixed through Azotobacter, when organic salts 

 are used as carbon food. (See table). 



These numbers show that the amount of nitrogen which can be 

 fixed in the crude culture is at most 4.9 and 2,8 m.g. per gram of 

 oxidised calciumsalt, obtained respectively with calciumpropionate 

 and calciumacetate (experiment 10 and 11), while, per gram of 

 calciummalate was fixed about 2,6 m.g. (experiment 2), and per 

 grain of lactate 1,8 m.g. (experiment 9). It seems that the fixation 

 goes on more rapidly at the beginning than later in the course of 

 the experiment, whence it follows that when little of the organic 

 salt is used proportionately more nitrogen is fixed than by larger 

 amounts. This should be taken into consideration in judging the 

 favourable results obtained with propionate and acetate, for then 

 solutions were used with only 1% of the salt. As to these salts, they 

 have proved to be in general an unfavourable source of carbon for 

 Azotobacter if the rapidity of the growth is taken as indicator of the 

 process, and only then to be able to give good results, when for the 

 inoculation, cultures in malate solutions are used, in which a certain 

 variety of our species is present. But also then, as observed above, 

 already at the first passage from acetate into acetate the growth stops 

 almost entirely. Pure cultures of Azotobacter develop hardly at all ') 

 in solutions of calciumacetate and natriumacetate, whatever may 

 have been the conditions to which these cultures were previously sub- 

 jected. Propionates and lactates still require a nearer investigation. 



Of calciummalate, on the other hand, it has decidedly been proved 

 that not only the crude cultures succeed very well and fix much nitrogen 

 even at repeated passages in the same medium, but that this also 

 holds good with regard to the pure cultures of Azotobacter. This is 

 the first case in which I got the certainty that no other microbes 

 are wanted, neither in the medium nor in the infection materials, 

 but Azotobacter alone to cause the said phenomena. Various authors 

 surely have repeatedly described the fixation of free nitrogen in pure 

 cultures of Azotobacter, among others of late with respect to the acetates, 

 but never had I been able to confirm the accuracy of these state- 

 ments until I made a systematic investigation with calciummalate, 

 a salt which had never before been used to this end, although I had 



i) I owe to Mr. D. C. J. Minkman, assistant to my laboratory the determinations 

 here referred to. 



8 ) The different varieties behave, however differently and some will begin to 

 grow but the growth soon ceases. 



