Another case where the gain of nitrogen to the surface-soil is very striking is in that of leguminous crops, such as clover, beans, peas, &c. This fact has been long recognised--especially with regard to clover--by farmers, and has been largely instrumental in leading to the investigation of the "free" nitrogen question. That a soil bearing a leguminous crop increases in nitrogen at a very striking rate is a problem that requires to be solved. A partial explanation of the phenomenon is found in the extraordinary capacity such a crop as clover has, by means of its multitudinous and ramifying roots, for collecting nitrogen from the subsoil. This, however, would only account for the increase in nitrogen to a certain extent. There must be some other source, and the only other source is the air. That the free nitrogen of the air is, after all, available for the plant's needs, is a supposition which has long seemed extremely probable, and which, within the last few years, has been proved beyond doubt to be a fact in the case of leguminous plants. _The Fixation of "Free" Nitrogen._ The method in which these plants are able to make use of the free nitrogen is still a point requiring much research. So far as the question is at present investigated, it would seem that the fixation is effected by means of micro-organisms present in tubercles or root excrescences found on the roots of leguminous plants.[82] Not merely has this been placed beyond doubt, but attempts have been made to isolate and study the bacteria effecting this fixation. From Nobbe's exceedingly interesting experiments, recently carried out, it would seem that the different kinds of leguminous plants have different bacteria. Thus the bacteria in the tubercle on the pea seems to be of a different order from the bacteria in the tubercles of the lupin, and so on. This discovery is of great importance, it need scarcely be pointed out, as it throws much light on the principles of the rotation of crops. _Influence of Manures in increasing Soil-nitrogen._ It may be doubted, however, if under any other conditions there is a positive gain of soil-nitrogen. In other cases the amount in the soil is only _maintained_ under liberal manuring. In connection with this point a very striking fact has been observed with regard to the effect of continuous large applications of farmyard manure. It has been found at Rothamsted that in such a case, after a while, the manure does not seem to increase the soil-nitrogen, although where the nitrogen goes to remains a mystery. In the case of the application of artificial manures, there does not seem to be almost any appreciable gain to the soil-nitrogen. The soil-nitrogen is only increased by means of the residue of crops. In this way, of course, by increasing the amount of this crop-residue, artificial manures may be said indirectly to increase the soil-nitrogen.[83] SOURCES OF LOSS OF NITROGEN.