March 29, 1894] 



NATURE 



513 



compounds, but only if the algal growth is freely exposed to 

 the atmosphere in the closed chambers employed. These 

 statements confirm earlier, but less definite, experimental 

 results by Frank ; and the latter has recently expressly stated 

 that certain fungi — e.g. Peiiicillium cladosporioides — can flourish 

 in a medium to which no nitrogen but that of the atmosphere 

 has access. 



Berthelot goes further, and claims to have established that 

 several species of soil-bacteria and fungi, including the fungoid 

 organism of the leguminous tubercles cultivated separately, 

 can " fix " free nitrogen ; and if the analyses of the small quanti- 

 ties of materials in his flasks survive the criticism of the chemists, 

 it seems difficult to refuse credence to the views he puts forward ; 

 but, as in most of these cases, it is the enormous difficulties of 

 analyses which lie at the root of the matter. 



Moreover, different observers differ considerably on this 

 question. Beyerinck, while regarding it as probable that the 

 nodule-organisms "fix" atmospheric nitrogen, admits that he 

 does not prove it ; and in Laurent's special investigation into 

 this question, he left it also uncertain ; while Immendorf failed 

 to satisfy' himself that these organisms can flourish without 

 organic compounds of nitrogen ; and Frank insists that they do 

 not thrive at all without organic nitrogenous food-materials. 

 Moreover, it must not be overlooked that other observers, e.g. 

 Gautier and Drouin, have given evidence pointing to possible 

 phenomena of " fixation " of nitrogen by compounds of iron and 

 other substances clinging to particles of the sand employed, 

 which may interfere with the accuracy of conclusions drawn 

 •firom experiments where sterilised soil in the open air is 

 concerned. 



When we reflect how very minute these organisms are, and 

 what excessively small quantities of nitrogen they need for their 

 life-purposes, we cannot be surprised at the difficulties met with 

 in these investigations. But, however far from proved we may 

 regard the question of fixation of- free nitrogen by soil organ- 

 isms, it is perfectly clear that here is a most pressing question 

 for further experimental research, and agricultural and forest 

 practice are alike keenly interested in having the question 

 definitely answered. 



The third possible view — that the leguminosse are able 

 to force free nitrogen into combination with other elements, 

 owing to the energetic action of their protoplasmic machinery 

 stimulated by the symbiotic fungoid organism — deserves 

 more consideration than may at first sight appear, especially 

 to those who are not familiarised with the remarkable pheno- 

 mena of symbiosis generally. 



In the first place, the fact that leguminous plants amply 

 provided with the root-nodules do " fix " the atmospheric 

 nitrogen, under conditions in which the same plants devoid of 

 the nodules fail to increase their supplies of nitrogen, is far 

 better established than any of the other cases discussed, and 

 must now be accepted as proved by the experiments of Frank, 

 Hellriegel, myself, Lawes and Gilbert, and especially by the 

 recent splendid investigations of Laurent and Schloesing. 



It is true that Frank says the symbiosis is not absolutely 

 necessary for the fixation to proceed, but even he declares that 

 the leguminosae are stimulated to greater powers of nitrogen- 

 fixation by the nodule-organisms. 



A curious and significant confirmation of the symbiosis theory 

 comes from the experiments of Nobbe, Schmid, Hiltner, and 

 Hotter, who find that ELzagmts plants, the roots of which 

 develop nodules due to the invasion of a fungus totally different 

 from the one causing the leguminous nodules, also "fix" and 

 assimilate the free nitrogen of the atmosphere, as shown by 

 their growing and flourishing much better and more rapidly 

 than Elaagims plants side by side with them, but not infected 

 with the root organism. It will be interesting to see if further 

 research shows similar results with any of the physiologically 

 similar root-outgrowths, due to very different fungi, met with 

 in Taxodium, Podocarptis, Alnus, Jtmcus, and many other 

 plants, including some vascular Cryptogams. 



Now comes the question, in what part of the leguminous 

 plant does the actual " fixation " of the free nitrogen occur? 

 Frank stands practically alone in claiming the leaves to be the 

 organs concerned. Nearly all other observers regard the roots 

 as the region, and the nodules themselves as the actual seat of 

 fixation. 



Kossowitsch has even attempted the heroic task of deciding 

 between leaves and roots, by enclosing the former or the latter 

 respectively in air-tight receptacles, shut off from the non-enclosed 



NO. 1274, VOL. 49] 



parts, in which gases devoid of nitrogen were circulated. He 

 could not always keep the apparatus perfectly gas-tight, how- 

 ever, and this and other failures met with in these exceedingly 

 difficult experiments, undoubtedly weakens the force of his 

 conclusions that it is in the roots and not in the leaves that the 

 process occurs, though it does look as if the balance of evidence 

 obtained fairly support his conclusion so far as it goes. 



There are facts, however, to be gathered from the microscopic 

 analyses of the root-nodules, as furnished by myself and others, 

 which have been in great part overlooked in the discussions on 

 this subject, and which, although not conclusive, seem to sup- 

 port the view that the seat of fixation may be in the nodules 

 themselves. For instance, the nodules are supplied with a 

 regular system of conducting vascular bundles, communicating 

 with those of the roots ; then their cells, during the period of 

 incubation of the symbiotic organism, are abundantly supplied 

 with starch ; further, the cells in which the fungoid organism is 

 vigorously flourishing are evidently exceedingly active, as may 

 be deduced from their large size, brilliant nuclei, protoplasm, 

 and sap-vacuole, all of which show signs of intense metabolic 

 activity, lasting for considerable periods. The fact that the sap 

 expressed from these active tissues is alkaline, has been inter- 

 preted as in accordance with Loew's suggestion that the living 

 protoplasm, in presence of an alkali and free nitrogen, can 

 build up ammonium nitrite, or some similar body. Be this as it 

 may, there can be no question as regards the infected nodule- 

 cells being centres where intense physiological activity is going 

 on ; and it seems impossible to avoid the conclusion that the 

 vascular supplies from the roots into the nodules bring to these 

 cells water in which various salts, carbo-hydrates, &c. are dis- 

 solved, and carry off from them the soluble products of 

 metabolism. 



Presumably these products of metabolism include nitrogenous 

 bodies. 



In the ordinary course of events, theory teaches that these 

 nitrogenous bodies — e.g. amides, preceded by simpler com- 

 pounds — are built up by the machinery of the ordinary living 

 cell-protoplasm from carbo-hydrates and nitrates, the energy 

 necessary for the metabolism being derived chiefly (if not en- 

 tirely) by the oxidation of part of the carbo-hydrates supplied. 



This constructive metabolic work of the protoplasm is an act 

 which we cannot explain in detail. We can only dimly perceive 

 that it must be due to some remarkable power the protoplasm 

 possesses — and in virtue of which it is an illimitable machine 

 much more economical in its actions than any apparatus we can 

 construct — of so placing the atoms and molecules of the nitrate, 

 carbo-hydrate, water, &c. with which it works, that they are 

 enabled to undergo movements into which we cannot as yet force 

 them in the laboratory. 



The whole matter seems to depend on some peculiar mode of 

 presentment of the atoms and molecules concerned ; and we can 

 see no further than that this can be done in the living cell, be- 

 cause the protoplasm is a suitable engine for thus bringing the 

 combining elements into the necessary positions in space. 



Now, if this is so, there seems no exclusion of the possibility, 

 at any rate, that the cell-machinery may be so stimulated into 

 greater activity that it can even force the notorious-ly inert 

 nitrogen molecules, properly presented, into combinations with 

 other molecules, resulting in the production of nitrites, amides, 

 or similar bodies in ascending order. 



The whole matter no doubt resolves itself into some such 

 question of a properly adapted engine sufficiently supplied with 

 energy. The matter seems capable of explanation, in some de- 

 gree, if we remember that carbo-hydrates and oxygen are 

 present in abundance ; the real difficulty is with the machinery, 

 for we cannot as yet picture the exact construction or working 

 of such an engine, as physiology nevertheless impels us to 

 suppose the cell-protoplasm must be. 



It may be remarked, by the way, that the likeness of the 

 living protoplasm to an engine, in the sense implied, may hold 

 good whether the former is an " emulsion,'' in the sense of the 

 defenders of that hypothesis, or a " structure,'"' in the sense of 

 those who refuse the emulsion hypothesis. 



The fourth of the possible views as to the means by which 

 free nitrogen becomes available to the leguminous plant, how- 

 ever, reminds us that, although the evidence points to the 

 stimulated leguminous plant as the best established example of 

 one capable of doing this work, there are other possibilities. 



Berthelot's recent insistance that certain soil-bacteria can fix 

 free nitrogen, taken with Frank's, Laurent and Schlcesing's, and 



