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[Vol. XVIII. No. 442 



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INVESTIGATIONS- UPON NITRIFICATION AND THE 

 NITRIFYING ORGANISM/ 



The nitrogen of organic substances is, for the most part, 

 liberated during decay in the form of ammonia or ammo- 

 niacal compounds; and these substances yield, by oxidation, 

 nitrous acid and finally nitric acid, which, in turn, in the 

 form of nitrates, feeds the living plant, and thus begins again 

 the cycle of transformation. 



The oxidation of the nitrogen of ammonia, and its ulti- 

 mate conversion into nitric acid, is called nitrification. This 

 change is especially active in soils near the surface, where 

 nitrates are formed abundantly from percolating waters 

 which contain much nitrogenous matter. 



This phase of nitrification, the formation of nitrates in 

 porous soil, has been attentively studied: but less attention 

 has been given to the process of nitrification as it goes on in 

 surface waters, such as streams and ponds; and it is to this 

 side of the question, namely, nitrificatioa as it occurs in 

 natural waters, that our study has been chiefly directed. 



Some eighty samples of water, selected from the two hun- 

 dred and forty coming each month to the laboratory of the 

 State Board of Health, were examined at intervals of from 

 two to seven days for ammonia, nitrites, and nitrates. These 

 samples were received from all parts of the State, and in- 

 cluded all classes of surface water, rivers, ponds, and reser- 

 voirs. They were examined repeatedly during the months 

 of June, July, and August, 1888. 



The results may be briefly stated as follows. The organic 

 matter in suspension decays in about seven days, as is shown 

 by the increase in " free ammonia." In about fourteen days 

 this " free ammonia " has disappeared, and nitrite has taken 

 its place, reaching a maximum in about twenty-one days. 

 Later the nitrite too disappears, and in twenty-eight days or 



1 Edwin O. Jordan and Ellen H. Ricliards, in report on water supply and 

 sewerage to the State Board of Health of Massachusetts. (The series of ex- 

 periments detailed in this paper were planned and carried out jointly by the 

 authors, the bacteriological portion of the work being done by Mr. Jordan, 

 and the chemical portion by Mrs. Richards.) 



more all the nitrogen has been converted into the form of 

 nitrate. When the svispended matter is removed by filtra- 

 tion through paper, or by precipitation with alumina, no 

 change occurs unless free ammonia were present at the out- 

 set. 



These changes were so universal, and so independent of 

 the character of the water and of its condition of aeration, 

 that it seemed important to avail ourselves of the unusual 

 opportunity offered by the close proximity of the chemical 

 and biological laboratories of the State Board of Health, to 

 carry on a series of chemical and bacteriological investi- 

 gations on solutions of known composition. Accordingly, 

 we began a series of experiments covering a period of nearly 

 two years, in which the daily and weekly changes caused by 

 the growth of bacteria were watched from both the chemical 

 and the bacteriological standpoint, in order to determine the 

 sequence and rate of such changes. Other points came up 

 in the course of the work, as will appear from the following 

 pages. 



It has long been known that the first step — the decompo- 

 sition of nitrogenous matter, and consequent production of 

 ammonia — is due to the vital activity of bacteria. The 

 early experiments of Scliwann and Schultze (1839), and the 

 later and thoroughly coaclusive work of Pasteur, showed 

 that putrefaction of organic matter is brought' about solely 

 by the small vegetable organisms known as bacteria. Even 

 after this fact became generally known, it was some time- 

 before the importance of the complete range of this discovery 

 was suspected. It was still maintained that the process of 

 nitrification proper — the oxidation of ammonia to nitric 

 acid — was of a purely chemical nature, although the bur- 

 den of proof was soon thrown on those who upheld this 

 view. The close dependence of nitrification upon a rather 

 narrow range of temperature, the cessation of the process on 

 the addition of antiseptics, the operation of "seeding" one 

 solution with another, the impossibility of effecting rapid 

 nitrification by chemicals, the analogous phenomena of pu- 

 trefaction, — all pointed clearly to the fact that nitrification 

 depends on the presence of living organisms. 



The first conclusive proof that such was the case, however, 

 came from the work of Schloesing and Muntz in 1877 

 (Comptes Rendus, 1877, Tome 84, p. 301). The work of 

 these observers rendered it practically certain that living or- 

 ganisms of some kind are the true agents of nitrification. 

 " It now remains for us," they said, ■' to discover and isolate 

 the nitrifying organisms." Schloesing and Muntz, in their 

 subsequent investigations, believed that they had succeeded 

 in making this discovery; but, in view of the facts of mod- 

 ern bacteriology, we are unfortunately unable to assign 

 much value to this part of their work. It is not easy to sat- 

 isfy one's self that Schloesing and Muntz ever worked with 

 really " pure cultures " of isolated species. While the work 

 of these investigators established beyond all question the fact 

 that nitrification, like the analogous phenomena of fermenta- 

 tion and putrefaction, is caused by living organisms, it left 

 entirely open the precise nature of these organisms. 



The first experiments with species of bacteria isolated by 

 modern methods, and therefore undoubtedly pure cultiva- 

 tions, are those recorded by Heraus (Zeitschr. fllr Hygiene,. 

 I., 1886, p. 193). Herseus exjierimented with fourteen well- 

 known species of bacteria, and with about as many others 

 freshly isolated by himself from water and soil. He culti- 

 vated these in an ammoniacal solution, and obtained in the 

 case of several familiar species good qualitative tests for ni- 

 trous acid. Among these species were -Baci'ZZMSjjrodiQ'iostts, 



