Nitrogen by Azotobader and the Growth of the Organism . 873 
fixation upon this medium were made, for failure to obtain any growth 
at all occurred so frequently as to prove that this solution was far from 
favourable for the development of the particular strain of Azotobacters con- 
cerned in this investigation. No good results could be obtained in any 
medium which did not contain an excess of calcium or magnesium car- 
bonate ; and, this having been decided, the chief remaining point to be 
considered in this connexion was the question as to whether the addition 
of sodium, either as the hydrate or in any other form, is beneficial to the 
organism ; the fact that Azotobacter will grow and fix nitrogen without 
it proving that it is not necessary. Since the isolation of the organism 
from the soil in 1901 by Beijerinck, many workers have devoted a con- 
siderable amount of time to a determination of the conditions under which 
the most luxuriant growth of vigorous Azotobacters can be obtained ; for the 
importance, from a practical, agricultural point of view, of a precise know- 
ledge of these conditions cannot be over-estimated, and the more simple the 
culture solution which can be used, the better. As regards the elements 
supplied, apart from the sodium and the neutralizing agents, the solutions 
used, except for the recent work above mentioned, are all practically 
similar, so that the liquid medium hitherto used by Professor Bottomley in 
the Botanical Laboratory at King’s College was tested as a type of those 
without sodium, against Ashby’s solution representing those containing 
sodium salts. At the suggestion of Professor Bottomley, an investigation 
was also made to obtain some idea of the efficiency of basic slag as a sub- 
stitute for calcium carbonate, of which further details will be given later. 
When the questions of the relative merits of Bottomley’s and Ashby’s 
solutions, and of calcium carbonate and basic slag as neutralizing agents, 
were settled, there arose the problem as to what is the best quantity of 
nutrient solution to supply to ensure maximum fixation. The practical 
worker requires a maximum yield of combined nitrogen for the minimum 
expenditure of carbohydrate ; and it has been suggested that the activity of 
the organism varies inversely, to a certain extent, with the quantity of food 
material supplied, i. e. that when provided with a very small portion of food, 
the Azotobacter economizes, using the carbohydrate to the best advantage ; 
so that under these conditions fixation is relatively greater than in a liberal 
supply of food material. Closely connected with this is the question as to 
whether the organism is able to utilize the last traces of carbohydrate 
in the medium as a source of energy for the fixation of nitrogen, or whether, 
as has also been suggested by various workers, after a certain period of 
time, while some of the carbohydrate originally supplied is still present 
in the solution, a process of auto-digestion sets in, nitrogen-fixation coming 
to an end for the time being. In the experiments described by different 
authors, amounts of nutrient solution ranging from 10 to 1,000 c c. have 
been employed, with concentrations of carbohydrate ranging from oa to 2 
