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from acetate into acetate the growth stops almost entirely. Pure cultures of Azoto- 

 bacter develop hardly at all 2 ) in solutions of calicumacetate and natriumacetate, 

 whatever may have been the conditions to which these cultures were previously 

 subjected. 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 pass- 

 ages in the same medium, but that this also holds good with regard to the pure cultures 

 of Asotobacter. This is the first case in which I got ithe certainty that other microbes 

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

 alone to cause the said phenomena. Various authors surely have repeatedly described 

 the fixation of free nitrogen in pure cultures of Asotobacter, among others of late 

 with respect to the acetates, but never had I been able to confirm the accuracy of these 

 statements until I made a systematic investigation with calciummalate, a salt which 

 had never before been used to this end, although I had already called attention to it 

 as an excellent source of carbon for Asotobacter in my papers of 1902. 



It must be allowed that the amount of fixed nitrogen in these pure cultures is not 

 considerable, about 1.5 m.g. for each gram of oxidised malate, but perhaps here too, 

 will be observed a greater production if only the very young cultures are examined; 

 then, however, only little of the salt be oxidised and the absolute quantities will of 

 course be small. 



It seems not superfluous here to call to mind that it is by no means the same 

 whether a known amount of calciummalate be absorbed from a dilute solution or 

 from a more concentrated one. In the latter case the malate will be more easily assi- 

 milable for the Asotobacter cells, which will induce a stronger oxidation and thus 

 an increased oxygen assimilation in equal times, so that the tension of the oxygen in 

 the liquid will be less than in the less concentrated solutions. As the growth of 

 Asotobacter seems favoured by this lower tension, and in any case, a rather strong 

 concentration of the carbon food proves favourable to the process of nitrogen fix- 

 ation in absolute quantity this circumstance has been taken into consideration in all 

 the experiments. Further, we did not always wait for the moment at which the 

 malate had disappeared from the medium, but commonly it was much earlier sub- 

 jected to the analysis for the reason mentioned above. 



The observation that calciummalate can, glucose, cane-sugar and mannite on 

 the other hand, cannot form the starting point for nitrogen fixation in liquid pure 

 cultures, while yet the said carbon hydrates are in the crude cultures much more 

 productive and may even give gains of nitrogen of 7 m.g. per gram of decomposed 

 sugar, gives rise to the supposition that these carbon hydrates must previously be 

 changed by other bacteria into organic acids and that these, at the moment of their 

 production, serve as carbon food for Asotobacter and primarily cause the fixation 

 of the nitrogen. 



Of course it cannot be malic acid which hereby originates from the sugar; but 

 the important growth of Asotobacter to which also the acetates, the propionates and 

 lactates may give rise, suggest the question whether perhaps the acids of these salts 

 may be first produced from the carbon hydrates and then govern the nitrogen fixation. 



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

 but the growth soon ceases. 



