EXPERIMENT STATION BULLETINS. 479 



is DO such "autotoxic" metabolic product. This puts the entire ani- 

 inonification exi>erimonts upon a new basis. 



Since the ammonia must be considered a metabolic product of sec- 

 ondary importance, it was thought possible to explain its formation 

 as the remainder of an oxidiziug process of the peptone molecule. The 

 Tiitrogcn atoms in the peptone molecule are doubtless present in various 

 combinations. There are very probably amino-groups, imino-groups, 

 acid-amid-groups and substituted amids. It is not unreasonable to 

 assume that some of these groups can be split by hydrolysis liberating 

 ammonia, while others cannot. It is worth while to call attention to 

 the experiments of Shibata* with the "amidase," an ammonia forming 

 cndo-enzyme of Aspergillus niger. This amidase produced ammonia 

 from urea, biuret and certain acid-amids, but not from mono-amino- 

 acids. The enzyme is evidently hydrolytic, and is able to hydrolize only 

 when the NH3 group and oxygen are at the same carbon atom, as is 

 the case in acid-amids, urea and biuret, but not in amino-acids. 

 R — CONH, -f HOH = RCOOH + NH3. 



It seem then that the nitrogen atoms of the amino and imino-groups 

 are not ammonified by hydrolysis. If this be not possible, ammonia can 

 be liberated only through the destruction of the entire molecule by 

 oxidation, as mav be illustrated with amino-acetic acid: 



NHoCH^CO.H + 30 = NH3 + H,0 + 2 CO, 



Such oxidation will take place much more freely in a sand culture 

 than in a solution, and it is quite easily understood that a more com- 

 plete oxidation of the peptone will cause a more complete ammoni- 

 fication of the nitrogen. The ammonia would then be an incidental 

 product, nothing but an indigestible piece of an otherwise digestible 

 molecule. 



This explanation of the ''high endpoint" of ammonia formation can 

 be considered proved, if the increase of ammonia can be shown to be 

 accompanied by an increased production of COj. This was done in 

 the following experiment which was carried out by Mr. H. K. Wright. 



The cultures were kept in flasks with ground glass stoppers which 

 were perforated by two glass tubes, one opening near the bottom of the 

 flask, the other one just allowing gases to escape. The cultures were 

 grown in 25 cc. of a 5% peptone solution, and in 50 g. of medium sand 

 + 5 cc. of peptone solution. Every 24 hours, free air was drawn 

 through these flasks for 20 minutes, and the air, after passing a small 

 wash bottle with n/10 H„SO, and a CaCl, tube, left the CO., in^i potash- 

 bulb, which was weighed daily. On the twelfth day, sulfuric acid was 

 put into the cultures to liberate the COo which was chemically bound to 

 the ammonia; after this, the ammonia was determined. The results of 

 Table XIII prove the theory. 



About 15% of the total ammonia of the sand cultures was found in 

 the small wash bottles. 



*Hoffmeisters Beitrage Band 5"(1904) p 384. 



