July 24, 1891.J 



SCIENCE. 



49 



the Finkler-Prior bacillus, the bacillus of typhoid-fever, the 

 anthrax bacillus, and others. Heraeus concludes that all 

 these organisms possess oxidizing powers, since they are thus 

 apparently able to oxidize ammonia to nitrous acid. 



The work of Adametz (Uutersuchungen Uber die niederen 

 Pilze der Ackerkrume. Inaug. Diss., Leipzig, 1886) and 

 Frank (Forschungen auf dem Gebiete der Agriculturphysik. 

 X, 56), on the other hand, did much to offset this positive re- 

 sult reached by Herseus. They found, as other investigators 

 had found before them, that the introduction of a small 

 quantity of garden soil into an ammoniacal solution would 

 produce rapid nitrification. The various species of bacteria, 

 however, which they isolated from this same soil, and intro- 

 duced as pure cultures into sterilized ammoniacal solutions, 

 refused to nitrify. In no case was more than a trace of ni- 

 tric acid observed. Frank was so influenced by his contin- 

 ued negative results that at a later date he went so far as to 

 deny that living organisms had anything whatever to do 

 with nitrification. This sceptical attitude seemed for a time 

 to be fully justified by the experiments of Celli and Zucco. 

 It was soon, however, demonstrated by several skilful inves- 

 tigators that nitrification could not be accounted for by 

 purely chemical influences. There was, nevertheless, no 

 cessation in the publication of negative results. The work 

 of Herseus was extended and elaborated by P. F. Frankland 

 and by Wariugton. Frankland (Jour. Chem. Soc, April, 

 1888, Vol. LIII., No. CCCV., p. 373) failed entirely to ob- 

 tain any evidence of oxidation of nitrogen by individual 

 species of bacteria, and on this point came into direct con- 

 flict with Herseus. To use his own words : — 



"The [ammoniacal] solutions were examined after forty 

 days' growth, but in no case was anything more than a faint 

 indication of nitrous acid obtainable with sulphanilic acid, 

 phenol, and ammonia. 



" It is worthy of notice that Herasus had experimented 

 with three of the micro-organisms which we have had under 

 observation, viz., B. subtilis, B. prodigiosus, and B. ramo- 

 sus. On growing these in sterilized urine, he found that B. 

 subtilis alone gave no nitrous acid reaction, whilst the other 

 two gave distinct reactions for nitrites; from this he con- 

 cludes that B. prodigiosus and B. ramosus possess oxidizing 

 powers, and that B, subtilis does not. My experiments, 

 however, conclusively prove that both B. ramosus and B. 

 prodigiosus exert a reducing action, whilst jB. subtilis does 

 not; and therefore that the nitrous acid reactions which he 

 obtained in the case of the two former organisms must ob- 

 viously have been due to the reduction of the nitrate in the 

 urine, and not to oxidation of ammoniacal nitrogen, as he 

 supposes. That nitric nitrogen is an invariable constituent 

 of human urine has been shown by Warington (Trans. 

 Chem. Soc, 1884, p. 669), and has in fact been long known." 

 Frankland summarizes his results as follows: "8. None of 

 the organisms under examination were found capable of 

 oxidizing ammoniacal nitrogen to nitrous or nitric acids, 

 when introduced into a nutritive solution containing ammo- 

 nium chloride." 



This emphatically negative result with pure cultures of 

 single species was directly confirmed by Warington, who 

 wrote: " It seems to me very clear that not one of the inves- 

 tigators who have experimented with isolated species of bac- 

 teria has obtained in his solutions more than a trace of ni- 

 trous or nitric acid ; no one has obtained an amount that 

 could be determined quantitatively. Another point which 

 generally appears is that every organism tried gives nearly 

 the same result. . . . The statement of Herteus that seven 



of the organisms examined commenced the nitrification of a 

 twenty per cent urine solution in one day is apparently due 

 to a mistake. My own experiments show that a urine solu- 

 tion of that strength cannot be nitrified by soil without the 

 addition of gypsum; the commencement of nitrification in a 

 strong solution is also extremely slow. The nitrous acid 

 which so speedily appeared in his solutions was due to the 

 reduction by the organs of the nitrates naturally present in 

 the urine" (Journ. Chem. Soc, August, 1888, Vol. LIII., p. 

 727). Of his own experiments, he says: "A distinct reac- 

 tion with diphenylamine was in some cases obtained, but 

 this did not appear to grow in amount, although in such 

 cases the examination was specially prolonged. The amount 

 of nitric or nitrous nitrogen in the solutions did not appa- 

 rently in any case exceed one per million, and all of this 

 could not be attributed to the action of the organism, as the 

 urkseeded solutions in the incubator also gave some reaction 

 with diphenylamine. When we have discounted the trace 

 of nitrites probably obtained from the atmosphere, there is 

 clearly very little left that can be attributed to the action of 

 the organism. The question whether any part of the nitrate 

 or nitrite present was produced by the organism, I am una- 

 ble to decide ; but it is quite clear that none of the organisms 

 examined possessed any nitrifying power in any way com- 

 parable with that possessed by soil. An organism which ni- 

 trifies as soil nitrifies has yet to be isolated." 



There are thus several views which are held regarding the 

 action of individual species of bacteria on nitrogenous solu- 

 tions: — 



1. That there is a group of bacteria capable of oxidizing 

 ammonia to nitric acid, and another and separate group able 

 to reduce nitrates to nitrites in the presence of organic mat- 

 ter. Both kinds are widely and abundantly distributed. 

 Attendant circumstances determine whether the reducing or 

 the oxidizing group will gain the upper hand (Herseus). 



2. That all kinds of bacteria, under favorable circum- 

 stances, are capable of producing nitric acid, and that the 

 same organisms in the presence of organic matter are capable 

 of reducing nitrates (Celli and Zucco, Leone). 



8. (a) That different species of bacteria vary greatly in 

 their ability to reduce nitrates; and (6) that there is no re- 

 liable evidence that any individual species is able to oxidize 

 ammonia either to nitric or nitrous acid (Warington, Frank- 

 land). 



Such is a brief sketch of the divergent opinions upon ni- 

 trification which were held at the time we began our work 

 in the autumn of 1888. It seemed to us important to ap- 

 proach the subject from all sides, and vire have worked ac- 

 cordingly not only with pure cultivations of bacteria, but 

 also with various sands, soils, and waters containing mix- 

 tures of several kinds. We have considered it of funda- 

 mental importance to determine the distribution of the nitri- 

 fying organism, and, if possible, to ascertain the relative 

 frequency with which it occurs over a wide area. The ques- 

 tion, for instance, naturally arose, is the nitrifying organism 

 present in the Boston city water as delivered from the tap in 

 the laboratories of the Massachusetts Institute of Technology, 

 since this is the water used in making up our solutions. To 

 this question we are able to give a decided affirmative. Am- 

 moniacal solutions carefully made with tap water always 

 nitrify. Moreover, ammoniacal solutions which have been 

 sterilized and then inoculated with a cubic centimetre of 

 fresh tap water always nitrify. Repeated experiments show 

 that the nitrifying organism is invariably present in this 

 water. When, however, ammoniacal solutions were iuocu- 



