STUDIES IN DENITRIFICATION. 



209 



mum of nitrate or organic matter, some of the flasks being retained unin- 

 oculated for controls. The cultures were kept in a darkened room at 

 an average temperature of twenty-five degrees C. In all the work with 

 mineral solutions bouillon tubes were inoculated with small portions 

 from the culture and control flasks to verify the vitality of the cultures 

 and sterility of the controls. Determinations of nitrite and free ammonia 

 were made at the time of inoculation and at intervals thereafter. 



TABLE I. 



Nitrites expressed in grains per litre of N^ 03. 

 Ammonia expressed in grams per litre of NHi. 



B. subtilis 



B. megaterium 



B. mycoides 



Uninoculated Control 



At 

 two days. 



N.. O3 NH3 



None .0099 



None trace 



.033 .016 



None None 



At 

 sixty days. 



N2 O3 NH3 



.264 .0064 



.4198 .0064 



.0882 .0128 



None None 



The theoretical amount of No O, per litre of the original culture solu- 

 tion from KNO. equals 3. 702 grams. The amount of NH3 per litre from 

 the KNO3 of the culture solution equals .841 grams. The results show 

 a steady increase in the amount of nitrite as the cultures become older. 

 The amount of ammonia shows a decrease at the time of the last test, 

 a phenomenon which may be accounted for by the gradual degradation of 

 the ammonia compounds formed, and the liberation of free nitrogen, a 

 question which at present remains unsettled. 



A. Do the Nitrites and Free Ammonia Result from the Breaking 

 Down of the Nitrate or the Peptone? 



Upon examination of the results of Table I., the question arose whether 

 at least a portion of the nitrite and free ammonia might not be products 

 of the analytical destruction of the peptone. To settle the point, a solu- 

 tion was prepared which was identical with the previous one with the 

 exception that no nitrate was used, and the cultures were handled in 

 exactly the same manner. The results are shown in Table II. 



TABLE II. 

 Nitrites expressed in grams per litre of N^ O3. 

 Ammonia expressed in grams per litre of NH^. 



B. subtilis 



B. megaterium 



B. mycoides 



Uninoculated Control 



At 

 inoculation. 



N2 O3 NH3 



None None 



None None 



None None 



None None 



At 

 two days. 



NoO^i 



NH3 



None .0121 



None None 



None .0048 



None None 



At 

 seven days. 



N2 O3 NH3 



None .0194 



None .0242 



None .0291 



None None 



At 

 sixty days. 



N2 O3 NH3 



Trace .0048 

 Trace .0291 

 .011 .0194 



Trace None 



From a comparison of the tables the fact becomes evident that most 

 of the ammonia in the nitrate cultures results from the degradation of 



