The first experiments of Berthelot date from 1885. Their object was the fixation of nitrogen by denuded soils, leaving out, consequently, all idea of vegetation. The soils used for the purpose were chosen from among the poorest in nitrogen. They were sandy clays taken from Meudon or from Sevres, below the level of the quarries, or, again, porcelain earths, crude kaolins not yet crushed in the mills. These soils, four in number, were submitted to five series of experiments. They were left to themselves in glazed pots, either within a well-closed room or in the open air in a meadow, either without shelter or under a little glass roof, merely to protect them from vertical rains, or on the top of a tower 29 meters above the ground and without any shelter, or finally, in corked flasks, so as to exclude all possibility of absorption of ammoniacal or nitric vapors. In the fifth series of experiments the same soils had first been exposed to a temperature of 100°, so as to destroy from the first all the organic germs that they might contain. The quantity of nitrogen, determined with great precision in each of the samples at the very beginning of the experiment, was again analyzed after two months, and again after remaining five months under the conditions indicated above, allowance being made for exterior additions attributable to air and to the rains when the pots were not sheltered.

The results obtained did not leave the slightest doubt. In every case in which the earth had been left in its normal state it had become enriched, and sometimes to a very great extent more than doubling the quantity of the initial nitrogen; when, on the contrary, the soil had been sterilized by heat, it became constantly more impoverished. In a word, then, poor clayey soils are able to absorb atmospheric nitrogen directly. This absorption is not accompanied by any increase in the previous proportions of ammonia or of nitric acid; it is, then, due to the formation of complex organic substances. Finally, it is the work of a micro-organism, since it ceases to be produced as soon as the soil has been sterilized.

To what sum per hectare does such a fertilization correspond? Berthelot estimates at 20 or 30 kilograms for a thickness of one decimeter of soil. Hence for a thickness of 0.35 meter it would suffice to compensate for the losses inherent to drainage and cultivation; but before going further it is well to remark that the experiments which we have just described relate to particularly poor soils, which are therefore of a nature to enrich themselves. In truly arable soils, averaging from 1 to 2 grams of nitrogen per kilogram, Berthelot has also observed a perceptible fixing of nitrogen, which, however, is relatively less than in sandy clays, and it is probable that this phenomenon would cease to be apparent after a certain limit, which, doubtless, is not very high.

The conditions which, according to Berthelot, apear the most favorable to the fixing of nitrogen by the naked soil are:

1. The presence of a quantity of water comprised between 3 and 15 per cent of total saturation;

2. A sufficient porosity to assure the free penetration of air throughout the whole mass of earth;

3. A temperature of between 10° and 40° C.

These conditions define the microbe which secretes or fixes the nitrogen as an aerobic organism (i. e., one that feeds on the atmosphere or is aerobiotic).

Except under the conditions previously pointed out, the phenomenon is no longer seen, and, in general, it is limited by the inverse action that is to say, by a continual dissipation of nitrogen or ammonia into the gaseous state.

Whatever may fix this limit, the fact observed by Berthelot is of the first importance. It is the first time, in fact, that we see the fixation of nitrogen in naked soils clearly stated; especially is it the first time that we see a cause experimentally defined and demonstrated without any reasonable doubt stand forth in the midst of such complex phenomena. This cause, as we have seen, is no other than the development of inferior organisms whose nature it remains for us to define more precisely.

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This was an entirely new idea and one which could not fail to produce its fruits. We shall therefore see researches rapidly multiply and lead their authors to more and more definite conclusions.

A. Gautier and Drouin verified first, in artificial soils, the principal results stated by Berthelot; they employed a mixture of siliceous sand, pure limestone, kaolin, and neutral phosphate of potash, to which they added, in particular cases, humus, humic acid or humates, or oxide of iron. This mixture, with the addition of a little nitrate of potassium, seems to be very favorable to the development of leguminous plants.

Under these conditions Gautier and Drouin recognized that the fixation of nitrogen always takes place in mixtures which have received organic matter; in its absence, on the contrary, there is always a loss. Organic matter appears, then, to be an important factor in this great natural phenomenon. It acts, doubtless, by promoting the nutrition of the microbe which fixes the nitrogen.

I will now indicate other experiments, repeated by Ville and Boussingault, in which we shall see the effect of the intervention of vegetation.

Berthelot first undertook a series of cultivations of leguminous plants in large pots which were left in the open air, either with or without shelter, or kept under a glass cover, care being taken to supply the plants with the carbonic acid necessary to their growth.

The soil, the seeds, the gathered plants, the drainage water and rain water were all analyzed with the greatest care in order that an exact comparison might be established between the initial and the final nitrogen.

Under the glass cover the fixation of nitrogen was very weak, because the plant, under these circumstances, did not reach its normal development, but in the open air the quantity of nitrogen fixed was, in every case, superior to that fixed by the soil alone.

For example, the tare tripled this quantity; the crop furnished by a mixture of kidney-vetch and Medicago lupulina contained ten times more nitrogen than was contained in the seed bed; a crop of lucerne grass contained sixteen times more, and this excess of nitrogen was always found more abundantly in the roots than in the leafy parts of the plant.

The soil enriched itself, but in a less degree than plant and soil together; therefore active vegetation promotes in an enormous degree

@Aerobies: Micro-organisms which live in contact with the air and require oxygen for their growth. Anaerobies: Micro-organisms which do not require oxygen, but are killed by it.

the assimilation of free nitrogen by the earth, a fact which is in conformity with all observations made in extensive farming operations. The distribution of this nitrogen in the plant shows that it enters through the roots, doubtless in consequence of microbic intervention. Finally, if we sum up the excess of nitrogen thus found in the crop and in the soil, together with the drainage water, we should find, according to Berthelot, 300, 500, and even 700 kilograms per hectare, a part of which evidently remains in the ground as roots, if we are contented to gather only the portion of the crop which is above ground, as is generally done in practical agriculture.

Thus it is that there results the progressive enriching of arable soils under the ameliorating or improving action of leguminous plants; thus also results the possibility of continuous cultivation of certain crops, such as meadow grass or forest trees, without fertilizers and without the earth becoming impoverished.

Joulie arrives at very similar conclusions from experiments of the same kind. The cultivation of buckwheat and of hay on a piece of land in the department of Dombes showed in two years a fixation of nitrogen equal to more than 1,000 kilograms per hectare. The mean of twelve experiments, one only of which showed a loss of 0.0136 gram per 1.5 kilograms of soil, showed a fixation of about 500 kilograms of nitrogen per hectare in a space of two years.

A little later Messrs. Gautier and Drouin also found, under the influence of the cultivation of common beans, an enrichment of their artificial soils which, as they estimated, corresponded to 185 kilograms per hectare for a single crop only.

Finally Pagnoul, after having recognized that the soil alone is capable of directly fixing the nitrogen of the air, found like the preceding authorities that the enrichment of the soil took place to a considerable extent even with a simple crop of grass or clover. For the latter he found fixations amounting to 500 and 900 kilograms of nitrogen per hectare.

We see that all these results are in absolute accord with each other, and, what is worthy of remark, they are of the same order of magnitude in experiments made by several different persons. Nothing is wanting to them but the direct control to be obtained by a change in the composition of the gases in which the plants grow.

From this point of view the experiment is particularly difficult to carry out. The plants must be kept constantly in closed vases in a confined atmosphere, consequently in the presence of vapor of water at its maximum intensity, which seems to be an eminently unfavorable condition; besides, it is necessary to be able to measure the volumes of the gas contained in the apparatus, to analyze them with scrupulous exactitude, and, finally, to promote the chylophyllic nutrition by regular additions of carbonic acid without allowing the proportion of oxygen to vary too greatly. Schloesing, jr., and Laurent have triumphantly overcome all these difficulties. In a memoir published in 1890 these clever experimentalists state that in the space of three months three seeds of dwarf peas sown in a soil destitute of nitrogen, but prepared in such a manner that the absorption of nitrogen could easily take place, absorbed from 26 to 29 cubic centimeters of nitrogen, weighing 32.5 milligrams and 36.5 milligrams, respectively. This nitrogen, measured volumetrically, was found again (with all the precision requisite in so delicate a research) partly in the soil,

which was enriched on an average to 12 milligrams, partly in the plants, which had gained 20 to 30 milligrams, although, owing to the narrow space in which they were confined, they were not able to attain their full development.

This last proof appears to have finally closed the discussion formerly inaugurated by Boussingault and which had not been completely closed by the analytic results explained above.

Thus a few years have sufficed to definitely decide this theory of a direct assimilation of nitrogen by plants, first enunciated by Ville. What, now, is the mechanism or modus operandi of this assimilation? We have just seen how Berthelot was led, by certain peculiarities of his experiments, and, above all, by the complete cessation of all fixation of nitrogen in soils that had been subjected to a temperature of 100°, to admit that nitrogen is assimilated directly by certain inferior organisms which force it into organic combination; but we have also seen that the fixation of nitrogen by naked soils is always weak and generally insufficient for the necessities of a normal vegetation.

It is true that when the aid of a leguminous plant is invoked the fixation becomes more active and may become powerful enough to compensate alone for all the known causes of loss; but how, then, are we to account for the difference in this respect found between the Leguminosa and the Gramineæ? Shall we be forced to admit that the Leguminosa are able, by themselves, to assimilate gaseous nitrogen, by a power possessed by them which is wanting in the other species?

Berthelot has concluded, from his researches upon this subject, that in the development of leguminous plants there comes into play some micro-organism which facilitates the fixation of nitrogen upon the root of the plant, or rather upon the mass formed by the root and the soil, intimately connected one to the other; but this idea could not be definitely adopted unless the existence of such a microbe were proved by experiments. This result is fully demonstrated by a series of very remarkable experiments made by Hellriegel, Wilfarth, Frank, Prazmoffski, and others in Germany, and which have been most successfully verified by Bréal, Schloesing, jr., and Laurent in France, and, finally, by Lawes and Gilbert in England.

Before proceeding to explain these researches I must call attention to a well-established fact which had been well known for a great many years, although no one before Hellriegel and Wilfarth ever thought of seeing in it anything more than a phenomenon of nature. When we examine the roots of a leguminous plant grown in good soil we always see irregularly disposed on them tuberculous enlargements, a kind of nodosity [node, nodule, knot, or knob] formed of a special tissue and apparently quite accidental. Examined with a microscope the interior of these excrescences appears to be filled with corpuscles of varying forms, always animated with the "Brownian " movement, although they have sometimes a movement of their own. These assume various shapes; sometimes they are like simple rods similar in form to certain bacteria; sometimes they have the appearance of vegetable coralloids and take the branched T or Y form more or less ramified.

Botanists have for a long time discussed the nature of these excrescences, but at present it seems to be generally admitted that, morpho

logically considered, they constitute roots modified by the penetration of an exterior organism. Under no circumstances have we a right to consider them as a natural production of the plant, because, as Prazmoffski has shown, plants that are kept protected from all causes of contamination are always free from them; while, on the contrary, their roots become covered with a multitude of nodosities when plunged into a liquid where a tubercle has been crushed or when they are replanted in any sort of soil that is watered with a similar liquid. The artificial infection of the roots of leguminous plants, as enunciated a dozen years ago by Prillieux, has been verified by Hellriegel and Wilfarth, Prazmoffski, Laurent, and Bréal. This latter investigator has even discovered that we may certainly assure the formation of a tubercle by pricking the root of a leguminous plant with a needle which had been previously inserted into a tubercle growing on

another root.

There remains no doubt of this fact: The nodules of the Leguminosæ have a microbian origin. The organism which causes them has received the name Bacillus radicicola; Laurent places it beside the Pasteuria ramosa, between bacteria proper and the lower fungi. Essentially aerobic in its nature, it resists all freezing and drying; but a temperature of 70° C. is sufficient to destroy it. It has been successfully cultivated in bouillons made of peas, or of beans, supplemented with gelatine and asparagine, or even in a solution of phosphate of potash and of sulphate of magnesia, to which is added a little sugar, but without any nitrogenous substance whatever. This organism grows in such liquids, preserving its habitual ramified forms, but without producing any true spores.

As to the tubercles themselves, they have until lately been considered as morbid productions, useless to the plant. Some authors have sought to see in them organs either of reserve or organs for the transformation of the albuminous substances necessary for the nutrition of the plant; others and this is the general opinion at the present time-look upon them as the result of a symbiosis-that is to say, of an extremely intimate association between the root of the plant and the microbe living with it, entirely different, however, from the action of the ordinary parasite.

Hellriegel and Wilfarth were the first to discover a connection between the development of bacteroidal nodosities and the assimilation of gaseous nitrogen by the Leguminosa. After having observed that in a culture of peas the most vigorous plants were always those that possessed the greatest number of tubercles, these investigators carried out many series of systematic experiments in glass jars containing 4 kilograms of quartz sand, to which they added certain of the principal minerals necessary to vegetation, such as phosphoric acid, sulphuric acid, chlorine, potassium, etc., and in certain cases a small quantity of nitrogen in the form of nitrates.

In these jars, which were exposed to the open air, they sowed barley, oats, and peas. The results were exactly the same as those formerly obtained by Ville and Boussingault.

In soils destitute of nitrogen the crop of cereals (barley and oats) is nearly nil, but it increases in approximate proportion to the dose of nitrate added, so that for each added milligram of nitrogen there is an increase of crop equal, on an average, to 95 milligrams of vege