STUDIES IN DENITRIFICATION. 211 



ducts. In only one of the solutions, Asparagin, were satisfactory results 

 obtained. There was jjractically no formation of nitrite or ammonia, 

 and upon plating in ordinary gelatine it was found that while the micro- 

 organisms still possessed vitality, there was no vigor of growth. The 

 cultures in Asparagin developed vigorously, and there was a very marked 

 production of ammonia, the controls yielding no reaction for ammonia. 

 A comparison of Hie r(.^sults obtained in these strictly mineral solutions 

 with those obtained from solutions containing a small amount of organic 

 matter, leads to the conclusion that the presence of the latter is impera- 

 tive in order to secure abundant denitrification. What would be the 

 effect of large amounts of organic matter is still an unsettled question. 



5. The Effect Upon Denitrification of an Abundant Supply of Air, 



In a study of this question, Deherain in 1897, upon growing micro- 

 organisms froui straw and the solid excrement of animals, found that 

 the reduction was much more ra])id in closed than in o])en flasks, although 

 he does not state whether the conditions Avere strictly anaerobic. Maassen 

 states, that while denitrification is not inhibited by anaerobic conditions, 

 it proceeds best in a limited supply of air. I'feiffer in 1897, found that 

 in a culture of B. denitrificans the admitting of pure air did not 

 lessen the amount of denitrification. In 1898, Ampola and IHpiana iso- 

 lated from soil a micro-organism which grew well both in the presence 

 and absence of air. 



In our work, one purpose was to so construct a piece of apparatus 

 that air might be freely drawn through the culture, and yet that the 

 danger of contaminations might be eliminated. Five seventy-five c. cm. 

 Erlenmeyer flasks were tightly fitted with two-hole rubber stoppers and 

 connected with U tubes, one arm of which extended to the bottoni of the 

 flasks. Two each of the end flasks contained normal-fifth sulphuric acid, 

 the middle one holding the culture solution. Thus when air was drawn 

 through the apjjaratus, the sulphuric acid in the first two flasks would 

 absorb any ammonia from the air ])assing through, as well as any micro- 

 organisms which might be drawn in witli the current. The other two 

 acid flasks were to catch any ammonia that might be given off from the 

 culture drawn over upon aeration. 



Furthermore, one series each of cultures sealed, and simply ])lugged 

 with cotton wool were used, uninoculated controls being used in each 

 case; also cultures and controls were placed in an atmosphere entirely 

 devoid of oxygen. The culture solution was prepared similarly to the 

 previous ones of mineral salts and containing a small amount of pe]»tone. 



After eleven days growth determinations Avere made of nitrites and 

 free ammonia in the cultures and controls. The acid flasks of the aera- 

 tion api)aratus were titrated against normal-fifth sodium hydroxide, but 

 showed no indication of absor])tion of ammonia. 



