Looking back over the work, it is difficult to understand the result. The fixation of nitrogen is a process that absorbs energy, and should have necessitated some source of energy, which apparently was not supplied. But in spite of this drawback the investigation was immediately fruitful in that it gave the key to another problem which had long puzzled agriculturists.
It had long been known that the growth of leguminous crops, unlike that of others, enriched the ground,[C] and Lawes and Gilbert had shown that this was due to an increase of soil nitrogen. But no explanation could be found till Hellriegel and Wilfarth solved the problem.[4] In studying the nitrogen nutrition of gramineous and leguminous crops, they discovered that the gramineous plants died in absence of nitrate, and in its presence made growth which increased regularly with nitrate supply; while leguminous plants sometimes died and sometimes flourished in absence of nitrate, and behaved equally erratically with increasing nitrate supply. When the plants flourished nodules were invariably present on the roots, but not otherwise. They concluded, therefore, that the nitrogen nutrition of leguminous plants differed from that of the gramineæ, and depended on some factor which sometimes came into their experiments and sometimes did not, and, in any case, was associated with the nodule. Knowing that the nodules on the roots of leguminous plants contained bacteria-like bodies, and remembering Berthelot’s results, they explored the possibility of bacterial fixation. They sterilised the sand and found that peas invariably failed to develop nodules and died, but after adding a little garden soil nodules were found and vigorous growth was obtained.
[C] “Of the leguminous plants the bean best reinvigorates the ground ... because the plant is of loose growth and rots easily, wherefore the people of Macedonia and Thessaly turn over the ground when it is in flower” (i.e. dig it into the ground if the soil is poor). Theophrastus, “Enquiry into Plants,” bk. viii. 2, and bk. ix. I. This book is of profound interest to agriculturists and botanists. An excellent translation by Sir Arthur Hort is now available. (Loeb’s Classical Library.)
Chemical analysis showed considerable fixation of gaseous nitrogen, which Hellriegel associated with the nodule organism. This has proved to be correct, and the fixation of nitrogen by bacteria is now a well-recognised process, the conditions of which are being thoroughly worked out. Two types of organisms are known—those associated with leguminous plants, and those living in a free and independent state in the soil. Of the latter the Clostridium, isolated by Winogradsky, is anaerobic, and the Azotobacter of Beijerinck is aerobic. The essential conditions are that a source of energy must be supplied—usually given as sugar—that the medium must not be acid, and that sufficient phosphate must be present.
All this brilliant work had been accomplished in the short space of the ten years 1880 to 1890. The inspiration had in each instance come from France, and is traceable direct to Pasteur, although coming long after his own work on bacteriology. It is impossible for us now to realise the thrill of wonder and astonishment with which students, teachers, and writers of those days learned that the nutrition of plants, and therefore the growth of crops and the feeding of themselves, was largely the result of the activity of bacteria in the dark recesses of the soil. It is not surprising that the ideas were pushed somewhat too far, that the soil population was regarded as solely bacterial, and that important chemical and physical changes were sometimes overlooked.
Gradually there came the inevitable reaction and a somewhat changed outlook. Continued examination showed the presence in soil of almost every kind of bacteria for which search was made. Some of them were almost certainly in the resting condition as spores, and the new generation of workers had an uneasy feeling that the case for the overwhelming importance of bacteria in the economy of the soil was not too well founded. It was shown that the decomposition of nitrogen compounds to form ammonia would take place without micro-organisms if, as presumably would happen, the plant enzymes continued to act after they got into the soil. Even the oxidation of ammonia to nitrate—the great stronghold of the biological school—was accomplished by chemical agents. The fixation of nitrogen in soil conditions was beyond the power of chemists to achieve, and here it was universally agreed that bacteria were the active agents. And finally, chemists were themselves bringing into prominence a set of bodies—the colloids—whose remarkable properties seemed indefinitely expansible, and were in addition sufficiently incomprehensible to the ordinary student to attain much of the magnificence of the unknown.
All the time, however, a faithful body of workers was busy exploring the ground already won, improving the technique, making counts of the numbers of bacteria in the soil, and trying to measure the amount of bacterial activity. Much of the value of this work was limited by the circumstance that the bacteria were regarded as more or less constant in numbers and activities, so that a single determination was supposed to characterise the position in a given soil.
This was the condition of the subject when it was seriously taken up at Rothamsted. Before turning to agriculture, the writer had been studying the mechanism of certain slow chemical oxidations, and one of his first experiments in agriculture was to examine the phenomena of oxidation in soil. The results accorded with the biological explanation of Schloesing: when the soil was completely sterilised oxidation almost ceased. But the striking discovery was made, as the result of an accident to an autoclave, that partial sterilisation increased the rate of oxidation, and therefore presumably the bacterial activity. This remarkable phenomenon had, however, already been observed, and it had been shown that both bacterial numbers and soil fertility were increased thereby. A full investigation was started in 1907 by Dr. Hutchinson and the writer.[9] From the outset the phenomena were recognised as dynamic and not static, and the rates of change were always determined: thus the bacterial numbers, the nitrate and ammonia present were estimated after the several periods. Close study of the curves showed that the chemical and bacterial changes were sufficiently alike to justify the view that bacteria were in the main the causes of the production of ammonia and of nitrate; although non-biological chemical action was not excluded, there was no evidence that it played any great part. Thus the importance of micro-organisms in the soil was demonstrated.
The factor causing the increased bacterial numbers after partial sterilisation was studied by finding out what agents would, and what would not, allow the numbers to increase, e.g. it was found that the bacterial increases became possible when soil had been heated at 56° C., but not at 40° C. Again, it was shown that the high numbers in partially sterilised soils rose for a time even higher if a little fresh untreated soil were incorporated into the partially sterilised soil, but afterwards they fell considerably. Putting all the results together, it appeared that some biological cause was at work depressing the numbers of bacteria in normal soils, but not—or not so much—in the partially sterilised soils. Studied in detail, the data suggested protozoa as the agent keeping down bacterial numbers, and they were found in the untreated, but not in the treated, soils. The hypothesis was therefore put forward that bacteria are not the only members of the soil population, but that protozoa are also present keeping them in check, and therefore adversely affecting the production of plant food.
This conclusion aroused considerable controversy. It was maintained that protozoa were not normal inhabitants of the soil, but only occasional visitants, and, in any case, they were only there as cysts; the soil conditions, it was urged, were not suitable to large organisms like protozoa. The objection was not to be treated lightly, but, on the other hand, the experiments seemed quite sound. As neither Dr. Hutchinson nor the writer were protozoologists, Dr. T. Goodey and (after he left) Mr. Kenneth R. Lewin were invited to try and find out, quite independently of the partial sterilisation investigation, whether protozoa are normal inhabitants of the soil, and if so, whether they are in a trophic condition, and what is their mode of life and their relation to soil bacteria. Had it turned out that protozoa had nothing to do with the matter, search would have been made for some other organism. Goodey showed that the ciliates were not particularly important; Lewin soon demonstrated the existence of trophic amœbæ and flagellates. Unfortunately he was killed in the war before he had got far with the work. After the Armistice, Mr. Cutler accepted charge of the work: he will himself relate in [Chapters IV.] and [V.] what he has done.