208 MICHIGAN ACADEMY OF SCIENCE. 



STUDIES IN DENITRIFICATION. 



S. F. EDWARDS. 



Denitrification is, as the term implies, a reducing process in which 

 the nitrogen of the soil is eliminated in one form or another by the 

 breaking down of the nitrate or complex nitrogenous compounds, there- 

 by becoming lost to plant growth. It is still to a great degree an open 

 question in what form the nitrogen is lost, whether as oxides of nitro- 

 gen, free nitrogen or free ammonia, or in more than one of these forms. 



The denitrification phenomenon was first noticed in cultivated soil 

 in 1862 by Goppelsroeder, and for a long time it was regarded as a 

 purely chemical process. The first reference to the agency of bacteria 

 in the changes produced was made in 1875 by C. Meusel, and the earliest 

 pure cultures of such micro-organisms were made in 1882. by U. Gayon 

 and G. Dupetit. In 1886 W. Heraeus made known several species of the 

 denitrification class and two years later Percy Frankland and R. War- 

 ington found a large percentage of soil bacteria capable of reducing 

 nitrates to nitrites although they do not state whether their cultures 

 possess the power of further reduction. 



In later years scores of workers have entered the field, and, working 

 upon different phases of the subject have secured some very valuable 

 results. 



The object of our investigation was to contribute something toward a 

 solution of the problem by a detailed study of the denitrifying proper- 

 ties of three germs found commonly at a depth of two to six inches in 

 different soils upon the farm at Michigan Agricultural College, viz. : 

 Bacillus subtilis, B. megaterium, and B. mycoides. 



A brief outline of the investigation pursued follows: 



1. Action upon nitrate. 



2. Action upon nitrite. 



3. Action upon ammonium salt. 



4. Effect of the presence of organic matter. 



5. Influence of abundant oxygen supply. 

 1. Action upon Nitrate. 



As has been stated, in 1888 Franklin and >yarrington found a large 

 number of soil bacteria capable of reducing nitrate to nitrite, but leave no 

 record of further products. In our work an endeavor was made to deter- 

 mine quantitatively the reduction of nitrate to nitrite, and whether any 

 further products resulted. The culture solution used contained, besides 

 the potassium nitrate which formed its basis, the other elements neces- 

 sary for plant growth in various salts, and a small amount of organic 

 matter in the form of peptone. 



Seventy-five c. cm. Erlenmeyer flasks, plugged and sterilized in dry 

 heat, received twenty-five c. cm. of the culture solution and were steril- 

 ized intermittently in moist heat. The inoculations were made with .5 

 c. cm. of cultures of the germs in a mineral solution containing a mini- 



