METABOLISM OF AZOTOBACTER 267 



Treatment. Nitrogen fixed. 



per cent. 



No manure .100 



5 tons of manure per acre 103 



10 tons of manure per acre ... .... 110 



1 5 tons of manure per acre . . 105 



20 tons of manure per acre . . 103 



25 tons of manure per acre 101 



These results indicate clearly that stable manure has a beneficial 

 effect upon the nitrogen-fixing powers of the soil, but if used in 

 large quantities the benefit is not so pronounced as if used in smaller 

 quantities. 



This decrease in nitrogen-fixation with increased additions of 

 manure must be considered as due to its physical effect upon the 

 soil, for Richards found that Azotobacter grow and fix nitrogen in 

 horse manure when it is well aerated and contains sufficient moisture 

 and calcium carbonate. There is, too, a close connection between 

 the diet and the effect. Horses fed on oats gave feces which induced 

 the greatest fixation; horses on grass next; cattle receiving oatmeal 

 cake third ; but the feces from cattle fed on grass proved unsuitable. 



Manures often contain nitrogen-fixing organisms of considerable 

 activity. Their activity appears to be greatest in fermenting ma- 

 nures mixed with straw which serves as a source of energy. 



Although Fulmer and Fred were unable to find Azotobacter in any 

 of the samples of manure examined, they did obtain many nitrogen- 

 fixing bacteria from it. One of these organisms, for which they 

 suggested the name of B. azophile, is as efficient in fixing nitrogen 

 as is Azotobacter. This would make it appear that manure may often 

 carry to the soil nitrogen-fixing organisms. 



Metabolism of Azotobacter. Much time has been given to a study 

 of the metabolism of Azotobacter, yet our knowledge of this subject 

 is far from satisfactory. It is well known that the organisms oxidize 

 the various carbohydrates and with the energy thus obtained build 

 up complex nitrogen compounds. Berthelot early recognized that 

 the nitrogen so fixed is insoluble in water, thus indicating its protein 

 nature. Lipman found that there was a small but appreciable 

 quantity of nitrogen in both young and old cultures of A. vinelandii 

 not precipitated by lead acetate and a large proportion not precipi- 

 tated by phosphotungstic or by tannic acid. Further work indicated 

 that the substances were either amino-acids or comparatively simple 

 peptids. He considered that one of the early substances synthesized 

 by these organisms is alanin. An analysis of the Azotobacter mem- 

 brane gave the following results: 



Nitrogen as Non-Basic Nitrogen in MgO Total per cent. 



Ammonia Basic Nitrogen Nitrogen Precipitate Nitrogen 



per cent. per cent. per cent. per cent. per cent. 



0.98 2.76 6.39 0.42 10.45 



