Soil Organisms concerned in Nitrogen Cycle. 513 



extract all the brown soluble matter. Part of this crude sodium humate 

 was set aside for use in the culture media. The remainder was acidified 

 with dilute hydrochloric acid, the precipitate filtered off, washed thoroughly, 

 and redissolved in just sufficient sodium carbonate solution. Part of 

 this purer sodium humate was preserved for experiment, and part was 

 treated with a little calcium chloride solution, the precipitate of calcium 

 humate formed being thoroughly washed. A crude ammonium humate was 

 also prepared by extracting raw peat with a slight excess of ammonium 

 hydrate solution, filtering the extract, and removing the excess of ammonia 

 by evaporation on the water-bath. A solution of artificial sodium humate 

 was obtained by dissolving artificial humic acid, prepared by boiling sucrose 

 with dilute hydrochloric acid in the usual way, in the requisite amount of 

 sodium carbonate solution. All of these preparations were neutral when 

 added to the culture media. 



A pure culture of Azotobacter chroococcum was isolated from soil and 

 cultivated in a solution consisting of 100 c.c. distilled water, 1 grm. mannite, 

 0-2 grm. K 2 HP0 4 , 0-02 grm. MgS0 4 , and 0'2 grm. CaC0 3 . A number of 

 flasks each containing 100 c.c. of this medium were divided into series of six 

 each. To the various series the additions shown in the Table below were 

 made, and all were inoculated with 1 c.c. of a uniform suspension of 

 Azotobacter. Two flasks of each series were then sterilised in an autoclave at 

 135° C. to serve as controls, and the whole set was incubated for ten days 

 at 26° C. Analysis by the Kjeldahl method for the nitrogen content then 

 gave the following results : — 



Table IV. 



Flask. 



Contents of each flask in each series. 



Nitrogen 

 content 



after 

 10 days. 



Gain in 

 nitrogen. 



1 





mgrm. 



0-4 lo-4 

 0-4/ u * 



mg 



rm. 



2 









' 3 





5-6 



5-2^ 





4 





5-4 



5-0 



»5 -0 



5 





5 3 



4-9 



6 





5-4 



5-0^ 





7 









8 











9 



100 c.c. mannite solution + crude sodium humate from 



11 -1 



8-6^ 





10 



- 25 grm. peat 



>> >> j> j) 



11 5 



9 





11 



>> >> >> >> 



11 -4 



89 





12 



>> >> )> )» 



11 -7 



9 2 J 



VOL. LXXXIX. — B. 2 X 



