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THE ROTHAMSTED MONOGRAPHS ON
AGRICULTURAL SCIENCE
EDITED BY
Sir E. J. RUSSELL, D.Sc. (Lond.), F.R.S.

THE MICRO-ORGANISMS OF THE SOIL

THE ROTHAMSTED MONOGRAPHS ON
AGRICULTURAL SCIENCE.
Edited by Sir E. JOHN RUSSELL, D.Sc., F.R.S.

During the past ten years there have been marked developments in knowledge of the relations between the soil and the growing plant. The subject involves physical, biological, and chemical considerations, and its ramifications are now so wide that they cannot be satisfactorily dealt with in detail in any one book. These monographs collectively cover the whole ground. In “Soil Conditions and Plant Growth” the general outlines are presented: in the monographs the various divisions are fully and critically dealt with by the Heads of the Departments concerned at Rothamsted. A homogeneous treatment is thus secured that will, it is hoped, much facilitate the use of the series.

SOIL CONDITIONS AND PLANT GROWTH, Fourth Edition.
By Sir E. John Russell, F.R.S. 16s. net.

The following volumes are in preparation:—

LONGMANS, GREEN AND CO.,
LONDON, NEW YORK, TORONTO, BOMBAY, CALCUTTA, AND MADRAS.

THE MICRO-ORGANISMS
OF THE SOIL

BY
Sir E. JOHN RUSSELL, F.R.S.
AND
MEMBERS OF THE BIOLOGICAL STAFF OF THE
ROTHAMSTED EXPERIMENTAL STATION

WITH DIAGRAMS

LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON, E.C. 4
NEW YORK, TORONTO
BOMBAY, CALCUTTA and MADRAS
1923

Made in Great Britain

INTRODUCTION.

The purpose of this volume is to give the broad outlines of our present knowledge of the relationships of the population of living organisms in the soil to one another and to the surface vegetation. It is shown that there is a close relationship with vegetation, the soil population being dependent almost entirely on the growing plant for energy material, while the plant is equally dependent on the activities of the soil population for removing the residues of previous generations of plants and for the continued production in the soil of simple materials, such as nitrates, which are necessary to its growth. It is also shown, however, that the soil population takes toll of the plant nutrients and that some of its members may directly injure the growing plant.

The soil population is so complex that it manifestly cannot be dealt with as a whole in any detail by any one person, and at the same time it plays so important a part in the soil economy that it must be seriously studied. Team work therefore becomes indispensable, and fortunately this has been rendered possible at Rothamsted.

Each group of organisms is here dealt with by the person primarily responsible for that particular section of the work. The plan of the book has been carefully discussed by all the authors, and the subject matter has already been presented in a course of lectures given at University College, London, under the auspices of the Botanical Board of Studies of the London University. The interest shown in these lectures leads us to hope that the subject may appeal to a wider public, and above all to some of the younger investigators in biological science. They will find it bristling with big scientific problems, and those who pursue it have the satisfaction, which increases as the years pass by, of knowing that their work is not only of interest to themselves, but of great importance in ministering to the intellectual and material needs of the whole community.

CONTENTS.

CHAPTER I.
THE DEVELOPMENT OF THE IDEA OF A SOIL POPULATION.

From the earliest times agriculturists have been familiar with the idea that decomposition of vegetable and animal matter takes place in the soil, and that the process is intimately connected with soil fertility.

By the middle of the nineteenth century three different ways were known in which the decomposition occurred. One had been since early times specially associated with soil fertility, in that it gave rise to humus, the black sticky substance in farmyard manure or in soil—which was supposed up to 1840 to be the special food of plants. No good account of the process or of the conditions in which it occurred is, however, given by the older writers.

A second resulted in the formation of nitrates. This process became known as nitrification: it was described by Georgius Agricola (1494-1555) in his book “De Re Metallica,” and it was of great importance in the seventeenth and eighteenth centuries, because it was used for the manufacture of gunpowder in the great wars of that period. The conditions for the making of successful nitre beds were so thoroughly investigated that little fresh knowledge was added to that of 1770[A] until quite recently. This process, however, was not usually associated with soil fertility, although both Glauber (1656) and Mayow (1674) had insisted on the connection.

[A] See the remarkable collection of papers entitled “Instructions sur l’établissement des nitrières,” publié par les Régisseurs-généraux des Poudres et Salpêtre. Paris, 1777.

A third type of decomposition was brought into prominence by Liebig in 1840.[7] [B] Reviewing the decomposition of organic matter in the light of the newer chemistry, he concluded that the process was a slow chemical oxidation, to which he gave the name “Eremacausis.” He recognised that humus was formed, but he regarded it only as an intermediate product, and emphatically denied its importance in soil fertility. The true fertility agents, in his view, were the final products—CO₂, potassium and other alkaline salts, phosphates, silicates, etc. He went on to argue brilliantly that instead of applying farmyard or similar manures to the soil it was altogether quicker and better to apply these mineral compounds obtained from other sources than to wait for the slow process of liberation as the result of decomposition. For some reason, difficult to understand, he overlooked nitrification and the part that nitrates might play in soil fertility. Lawes and Gilbert[6] were much attracted by this new idea; they showed that it was incomplete because it took no account of the necessity for supplying nitrogen compounds to the crop. When ammonium salts were added to Liebig’s ash constituents the resulting mixture had almost as good a fertilising effect as farmyard manure. Lawes at once saw the enormous practical importance of this discovery, and set up a factory for the manufacture of artificial fertilisers. He did not, however, follow it up more closely on the scientific side.

[B] The numbers refer to the short bibliography on [p. 18].

Both Lawes and Gilbert were in constant touch with the idea of decomposition in the soil, and they attached so much importance to nitrogen compounds in plant nutrition that it is not easy to understand how they missed the connection with nitrification. But they did so, and like other English and German workers of the day, considered that plant roots assimilated their nitrogen as ammonia. For the first ten years of the history of Rothamsted only few experiments with nitrates were made, and not till thirty-five years had elapsed were they systematically studied.

It was by Boussingault[2] and in France that the connection between nitrification and soil fertility was first recognised. The news came to England, but it was not accepted, although Way, one of the most brilliant agricultural chemists of his time, showed that nitrates were formed in soils to which nitrogenous fertilisers were added, and that they were comparable in their fertiliser effects with ammonium salts.[12] “The French chemists,” he wrote in 1856, “are going further, several of them now advocating the view that it is in the form of nitric acid that plants make use of compounds of nitrogen. With this view I do not at present coincide, and it is sufficient here to admit that nitric acid in the form of nitrates has at least a very high value as manure.” Indeed, Kuhlmann went so far as to argue that the nitrates found in the soil were there reduced to ammonia before assimilation by plants could take place. The water-culture work of the plant physiologists of the ’sixties finally showed the correctness of the French view.

Even when the importance of nitrification was realised its mechanism was not understood: some thought it was chemical, some physical. Again the explanation came from France. Pasteur in 1862 had expressed the view that nitrification would probably be a biological action, since purely chemical oxidation of organic matter was of very limited occurrence. “Pénétrés de ces idées,” as Schloesing tells us, he and Müntz in a memorable investigation cleared up the whole problem, and in 1877 opened the way to a most fruitful field of research.[10] The formal description is given in his papers in the “Comptes Rendus,” but a more lively account is given in his lectures before the École d’application des Manufacteurs de l’état, which, though not printed, were collected and issued in script by his distinguished son, and a copy of this work is among the treasures of the Rothamsted Library.

He had been asked to study the purification of sewage, and he and Müntz showed that it was bound up with nitrification. The process was slow in starting, then it proceeded rapidly. Why, they asked, was the delay? There should be none if the process were physical or chemical, and the fact that it occurred strongly suggested biological action. The process was stopped by chloroform vapour, but could be restarted after the removal of the vapour by the addition of a little fresh soil.

The importance of this work in connection with soil fertility was immediately realised by Warington, who had recently come to Rothamsted.[11] He quickly confirmed the result, and made the valuable discovery that two stages were involved—the conversion of ammonia to a nitrite by one organism, and of the nitrite to nitrate by another. He made long and persistent attempts to isolate the organisms from the soil, using the best technique of his time, but though he found many bacteria none of them could nitrify ammonium salts; yet the soil did it easily. For years he continued his efforts to find the nitrifying organism, but always failed. His health was not good, his life at Rothamsted was not happy owing to disagreements with Gilbert, and although his other research work was succeeding, this investigation on which he had set his heart was not coming out; bacterial technique was not yet sufficiently far advanced. Ten bitter, disappointing years passed, and the crown of disappointment came when Winogradsky, a young bacteriologist in Paris, changed the technique and succeeded at once in isolating both the nitrite and the nitrate-forming organisms.[13]

The numerous bacteria found by Warington in the soil suggested the presence of a soil population, and this idea was greatly strengthened by another line of investigation which was being followed up in France. Boussingault had shown that soils absorb oxygen and give out carbon dioxide; Schloesing extended this discovery, as also did Wollny. It was concluded that oxidation was the result of the activities of the soil organisms in decomposing the organic matter of the soil, and thus preparing the way for the nitrifying organisms.

A third important function of soil bacteria was revealed by Berthelot.[1] It was known that considerable loss of nitrogen from the soil took place as the result of the conversion of nitrogen compounds into nitrates, which were subsequently washed out in the drainage water. It followed inevitably that the stock of nitrogen compounds in the soil must long ago have become exhausted had there been no addition of nitrogen compounds to the soil. Berthelot argued that there must be fixation of atmospheric nitrogen, and, following the prevailing trend of thought in France, he attributed it to bacteria. He confirmed the anticipation by exposing soil to air in such conditions that dust, rain, etc., were excluded, and he found an increase in the percentage of nitrogen.

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.

At first sight it might be thought comparatively easy to settle a question of this kind by examining soil under a microscope or by sterilising it and introducing successively bacteria and known types of protozoa. Unfortunately neither method is simple in practice. It is impossible to look into the soil with a microscope, and methods of teasing-out small pieces of soil on a slide under the high, or even the low power, give no information, because the particles of soil have the remarkable power of attracting and firmly retaining protozoa, and no doubt bacteria as well; indeed, for protozoa (which have been the more fully investigated) there seems to be something not unlike a saturation capacity (see [Fig. 9], p. 78). Further, complete sterilisation of soil cannot be effected without at the same time altering its chemical and physical properties, and changing it as a habitat for micro-organisms. Cutler has, however, overcome the difficulties and shown that the introduction of protozoa into soils sterilised and then reinfected with bacteria considerably reduces the numbers of these organisms.

The method adopted, therefore, is to take a census of population and of production. Counting methods are elaborated, and estimates as accurate as possible are made of the numbers of the various organisms in a natural field soil at stated intervals. Simultaneously, wherever possible some measure is taken of the work done. The details of the census are finally arranged in consultation with the Statistical Department, to ensure that the data shall possess adequate statistical value. From the results it is possible to adduce information of great value as to the life of the population, the influence of external conditions, etc.

The most important investigation of this kind carried out at Rothamsted was organised by Mr. Cutler.[3] A team of six workers was assembled, and for 365 days without a break they counted every day the ciliates, the amœbæ, the flagellates, and the bacteria in a plot of arable ground, distinguishing no less than seventeen different kinds of protozoa. The conclusions arrived at were carefully tested by the Statistical Department.

Of the protozoa the flagellates were found to be the most numerous, the amœbæ came next, and the ciliates were by far the fewest. The numbers of each organism varied from day to day in a way that showed conclusively the essentially trophic nature of the protozoan population. The numbers of amœbæ—especially Dimastigamœba and of a species called α—were sharply related to the numbers of bacteria: when the amœbae were numerous the bacteria were few, and vice versa. Detailed examination showed that the amœbæ were probably the cause of the fluctuations in the bacterial numbers, but Mr. Cutler has not yet been able to find why the amœbæ fluctuated; it does not appear that temperature, moisture content, air supply or food supply were determining causes. The flagellates and ciliates also showed large fluctuations, amounting in one case—Oicomonas—to a definite periodicity, apparently, however, not related to bacterial numbers, or, so far as can be seen, to external conditions of moisture, temperature and food supply, and showing no agreement with the fluctuations of the amœbæ. However, one cannot be certain that lack of agreement between curves expressing protozoan numbers and physical factors implies absence of causal relationships: the observations (though the best that can yet be made) are admittedly not complete. If we saw only the end of the bough of a tree, and could see no connection with a trunk, we might have much difficulty in finding relationships between its motion and the wind; whatever the direction of the wind it would move backwards and forwards in much the same way, and even when the wind was blowing along the plane of its motion it would just as often move against the wind as with it.

Meanwhile evidence was obtained that the twenty-four hour interval adopted by the protozoological staff was too long for bacteria, and accordingly the Bacteriological Department, under Mr. Thornton, refined the method still further. Bacterial counts were made every two hours, day and night, for several periods of sixty or eighty hours without a break. The shape of the curve suggests that two hours is probably close enough, and for the present counts at shorter intervals are not contemplated. But there is at least one maximum and one minimum in the day, although the bacterial day does not apparently correspond with ours, nor can any relationship be traced with the diurnal temperature curve.

The nitrate content of the soil was simultaneously determined by Mr. Page and found to vary from hour to hour, but the variations did not sharply correspond with the bacterial numbers; this, however, would not necessarily be expected. The production of nitrate involves various stages, and any lag would throw the nitrate and bacterial curves out of agreement. There is a suggestion of a lag, but more counts are necessary before it can be regarded as established.

Examination of these and other nitrate curves obtained at Rothamsted has brought out another remarkable phenomenon. No crop is growing on these plots, and no rain fell during the eighty hours, yet nitrate is disappearing for a considerable part of the time. Where is it going to? At present the simplest explanation seems to be that it is taken up by micro-organisms. A similar conclusion had to be drawn from a study of the nitrogen exhaustion of the soil. The whole of the nitrate theoretically obtainable from the organic matter of the soil is not obtained in the course of hours or even days; in one of our experiments at Rothamsted nitrification is still going on, and is far from complete, even after a lapse of fifty-three years. The explanation at present offered is that part of the nitrate is constantly being absorbed by micro-organisms and regenerated later on.

Now what organisms could be supposed to absorb nitrates from the soil? Certain bacteria and fungi are known to utilise nitrates, and one naturally thinks of algæ as possible agents also. Dr. Muriel Bristol was therefore invited to study the algæ of the soil. Her account is given in [Chapter VI.] She has found them not only on the surface, but scattered throughout the body of the soil, even in the darkness of 4 inches, 5 inches, or 6 inches depth, where no light can ever penetrate, and where photosynthesis as we understand it could not possibly take place. Some modification in their mode of life is clearly necessary, and it may well happen that they are living saprophytically. Dr. Bristol has not yet, however, been able to count the algæ in the soil with any certainty, although she has made some estimates of the numbers.

The quantitative work on the soil population indicates other possibilities which are being investigated. There is not only a daily fluctuation in the numbers, but so far as measurements have gone, a seasonal one also. There seems to be some considerable uplift in numbers of bacteria, protozoa, and possibly algæ and fungi in the spring-time, followed by a fall in summer, a rise in autumn, and a fall again in winter. At present we are unable to account for the phenomenon, nor can we be sure that it is general until many more data are accumulated.

In the cases of the protozoa and the algæ, there was a definite reason for seeking them in the soil.

Another section of the population, the fungi, was simply found, and at present we have only limited views as to their function. The older workers considered that they predominated in acid soils, while bacteria predominated in neutral soils. Present-day workers have shown that fungi, including actinomycetes, are normal inhabitants of all soils. The attempts at quantitative estimations are seriously complicated by the fact that during the manipulations a single piece of mycelium may break into fragments, each of which would count as one, while a single cluster of spores might be counted as thousands. Little progress has therefore been made on the quantitative lines which have been so fruitful with protozoa. Dr. Brierley gives, in [Chapters VII.] and [VIII.], a critical account of the work done on fungi.

In addition to the organisms already considered there are others of larger size. The nematodes are almost visible to the unaided eye, most of them are free living and probably help in the disintegration of plant residues, though a few are parasitic on living plants and do much injury to clover, oats, and less frequently to onions, bulbs, and potatoes. Further, there are insects, myriapods and others, the effects of which in the soil are not fully known. Special importance attaches to the earthworms, not only because they are the largest in size and in aggregate weight of the soil population, but because of the great part they play in aerating the soil, gradually turning it over and bringing about an intimate admixture with dead plant residues, as first demonstrated by Darwin. Earthworms are the great distributors of energy material to the microscopic population. Systematic quantitative work on these larger forms is only of recent date, and Dr. Imms, in [Chapter IX.], discusses our present knowledge.

TABLE I.
Soil Population, Rothamsted, 1922.

(The figures for algæ and fungi are first approximations only, and have considerably less value than those for bacteria and protozoa.)

Are there any other members of the soil population that are of importance? As already shown, the method of investigating the soil population in use at Rothamsted is to find by chemical methods the changes going on in the soil; to find by biological methods what organisms are capable of bringing about these changes; and then to complete the chain of evidence by tracing the relationships between the numbers or activities of these organisms and the amount of change produced. The list as we know it to-day is given in [Table I.]

The method, however, does not indicate whether the account is fairly complete, or whether there are other organisms to be found. We might, of course, trust to empirical hunting for organisms, or to chance discoveries such as led Goodey to find the mysterious Proteomyxan Rhizopods, which cannot yet be cultured with certainty, so that they are rarely found by soil workers. It is possible that there are many such organisms, and it is even conceivable that these unknown forms far outnumber the known. The defect of the present method is that it always leaves us in doubt as to the completeness of the list, and so we may have to devise another.

Reverting to [Table I.], it obviously serves no purpose to add the numbers of all the organisms together. We can add up the weights of living organisms, of their dry matter or nitrogen, so as to form some idea of the proportion of living to non-living organic matter, and this helps us to visualise the different groups and place them according to their respective masses. But a much better basis for comparing the activities of the different groups would be afforded by the respective amounts of energy they transform, if these could be determined. It is proposed to attempt such measurements at Rothamsted. The results when added would give the sum of the energy changes effected by the soil population as we know it: the figure could be compared with the total energy change in the soil itself as determined in a calorimeter. If the two figures are of the same order of magnitude, we shall know that our list is fairly well complete; if they are widely different, search must be made for the missing energy transformers. There are, of course, serious experimental difficulties to be overcome, but we believe the energy relationships will afford the best basis for further work on the soil population.

Finally, it is necessary to refer to the physical conditions obtaining in the soil. These make it a much better habitat for organisms than one might expect. At first sight one thinks of the soil as a purely mineral mass. This view is entirely incorrect. Soil contains a considerable amount of plant residues, rich in energy, and of air and water. The usual method of stating the composition of the soil is by weight, but this is misleading to the biologist because the mineral matter has a density some two and a half times that of water and three times that of the organic matter. For biological purposes composition by volume is much more useful, and when stated in this way the figures are very different from those ordinarily given. [Table II.] gives the results for two Broadbalk arable plots, one unmanured and the other dunged; it includes also a pasture soil.

The first requirement of the soil population is a supply of energy, without which it cannot live at all. All our evidence shows that the magnitude of the population is limited by the quantity of energy available. The percentage by weight of the organic matter is about two to four or five, and the percentage by volume runs about four to twelve. Not all of this, however, is of equal value as source of energy. About one-half is fairly easily soluble in alkalis, and may or may not be of special value, but about one-quarter is probably too stable to be of use to soil organisms.

A second requirement is water with which in this country the soil is usually tolerably well provided. Even in prolonged dry weather the soil is moist at a depth of 3 inches below the surface. It is not uncommon to find 10 per cent. or 20 per cent. by volume of water present, spread in a thin film over all the particles, and completely saturating the soil atmosphere.

TABLE II.
Volume of Air, Water and Organic Matter in 100 Volumes of Rothamsted Soil.

(1) Arable, no manure applied to soil. (2) Arable, dung applied to soil. (3) Pasture.

The air supply is usually adequate owing to the rapidity with which diffusion takes place. Except when the soil is water-logged, the atmosphere differs but little from that of the one we breathe. There is more CO₂, but only a little less oxygen.[8] The mean temperature is higher than one would expect, being distinctly above that of the air, while the fluctuations in temperature are less.[5]

The reaction in normal soils is neutral to faintly alkaline; pH values of nearly 8 are not uncommon. Results from certain English soils are shown on [p. 18].

The soil reaction is not easily altered. A considerable amount of acid must accumulate before any marked increase in intensity of pH value occurs; in other words, the soil is well buffered. The same can be said of temperature, of water, and of energy supply. Like the reaction, they alter but slowly, so that organisms have considerable time in which to adapt themselves to the change.

Hydrogen Ion Concentration and Soil Fertility.

A SELECTED BIBLIOGRAPHY.

[1] Berthelot, Marcellin, “Fixation directe de l’azote atmosphérique libre par certains terrains argileux,” Compt. Rend., 1885, ci., 775-84.

[2] Boussingault, J. B., and Léwy, “Sur la composition de l’air confiné dans la terre végétale,” Ann. Chim. Phys., 1853, xxxvii., 5-50.

[3] Cutler, D. W., Crump, L. M., and Sandon, H., “A Quantitative Investigation of the Bacterial and Protozoan Population of the Soil, with an Account of the Protozoan Fauna,” Phil. Trans. Roy. Soc., Series B, 1922, ccxi., 317-50.

[4] Hellriegel, H., and Wilfarth, H., “Untersuchungen über die Stickstoffnahrung der Gramineen und Leguminosen,” Zeitsch. des Vereins f. d. Rübenzucker-Industrie, 1888.

[5] Keen, B. A., and Russell, E. J., “The Factors determining Soil Temperature,” Journ. Agric. Sci., 1921, xi., 211-37.

[6] Lawes, J. B., and Gilbert, J. H., “On Agricultural Chemistry, Especially in Relation to the Mineral Theory of Baron Liebig,” Journ. Roy. Agric. Soc., 1851, xii., 1-40.

[7] Liebig, Justus, “Chemistry in its Application to Agriculture and Physiology,” 1st and 2nd editions (1840 and 1841), 3rd and 4th editions (1843 and 1847); “Natural Laws of Husbandry,” 1863.

[8] Russell, E. J., and Appleyard, A., “The Composition of the Soil Atmosphere,” Journ. Agric. Sci., 1915, vii., 1-48; 1917, viii., 385-417.

[9] Russell, E. J., and Hutchinson, H. B., “The Effect of Partial Sterilisation of Soil on the Production of Plant Food,” Journ. Agric. Sci., 1909, iii., 111-14; Part II., Journ. Agric. Sci., 1913, v., 152-221.

[10] Schloesing, Th., and Müntz, A., “Sur la Nitrification par les ferments organisés,” Compt. Rend., 1877, lxxxiv., 301-3; 1877, lxxxv., 1018-20; and 1878, lxxxvi., 892-5. “Leçons de chimie agricole,” 1883.

[11] Warington, R., “On Nitrification,” Part I., Journ. Chem. Soc., 1878, xxxiii., 44-51; Part II, Journ. Chem. Soc., 1879, xxxv., 429-56; Part III., Journ. Chem. Soc., 1884, xlv., 637-72; Part IV., Journ. Chem. Soc., 1891, lix., 484-529.

[12] Way, J. T., “On the Composition of the Waters of Land Drainage and of Rain,” Journ. Roy. Agric. Soc., 1856, xvii., 123-62.

[13] Winogradsky, S., “Recherches sur les organismes de la nitrification,” Ann. de l’Inst. Pasteur, 1890, iv., 1^e Mémoire, 213-31; 2^e Mémoire, 257-75; 3^e Mémoire, 760-71.

“Recherches sur l’assimilation de l’azote libre de l’atmosphère par les microbes.” Arch. des Sci. Biolog. St. Petersburg, 1895, iii, 297-352.

For further details and fuller bibliography, see E. J. Russell, “Soil Conditions and Plant Growth,” Longmans, Green & Co.

CHAPTER II.
SOIL BACTERIA.

A. Occurrence and Methods of Study.

To understand the development of our knowledge of soil bacteria, it must be remembered that bacteriology is under the disadvantage that it started as an applied science. Although bacteria were first seen by Leeuwenhoeck about the middle of the seventeenth century, and some of their forms described by microscopists of the eighteenth and early nineteenth centuries, it was only with the work of Pasteur on fermentation, and of Duvaine, Pasteur, and their contemporaries on disease bacteria, that bacteriology may be said to have started. From the outset, therefore, attention has been directed mainly to the bacteria in their specialised relationship to disease or to fermentation and similar processes. As a result, little research was done on the pure biology of the bacteria, so that even now many of the most fundamental and elementary problems concerning them are quite unsolved.

In their work on fermentations and disease bacteria, the earlier workers were assisted by the fact that under both sets of conditions the causative bacteria exist, as a rule, either in practically pure culture, or else in preponderating numbers. The study and elucidation of such a mixed micro-population as exists in the soil, became possible only when methods had been devised for isolating the different kinds of bacteria, and thus studying them apart from each other. It was the development of the gelatine plate method of isolating pure cultures by Koch[36] in 1881 that made the study of the soil bacteria practicable. The plating method opened up two lines of research. In the first place, it provided a simple means of isolating organisms from the mixed population of the soil, and thus enabled a qualitative study to be made of each organism in pure culture, and, in the second place, from it was developed a counting technique for estimating differences in bacterial numbers between samples of soil, from which has sprung much of our knowledge of the quantitative side.

The earliest studies of the soil bacteria consisted of such estimations of numbers, and showed that the soil contained a very numerous population of bacteria. About 20,000,000 bacteria per gram of soil is now considered a fair average number. The number and variety of bacteria existing in the soil is so enormous that the method of separating out all the different forms, and of discovering their characters and functions, has proved impracticable. In practice, therefore, the problem has been approached from the biochemical standpoint. That is to say, the special chemical changes that the bacteria produce in the soil have first been investigated, and this has been followed by the isolation and study of the various groups of bacteria that bring about the changes under investigation.

The method commonly employed in isolating the organisms that produce a given chemical change in the soil is called the “elective” method. The soil is inoculated into a culture medium that will especially favour the group of bacteria to be isolated, to the exclusion of others. For example, if it is desired to isolate the organisms that attack cellulose, a medium is made up containing no other organic carbon compounds except cellulose. Such a selective medium encourages the growth of the group of organisms to be investigated, so that after several transfers to fresh medium a culture is obtained containing only two or three different types of organisms. These are separated by plating and pure cultures obtained.

Another difficulty which has not yet been completely overcome is that of adequately describing an organism when it is isolated. The morphology of bacteria is not the constant thing that is seen in the more stable higher organisms. In many cases the appearance of a single strain is entirely different on different media, and may be quite altered by such conditions as changes in acidity of the medium or temperature of incubation. Even on a single medium remarkable changes in morphology occur, at any rate, in some bacteria. This is well seen in a cresol-decomposing organism under investigation at Rothamsted. In cultures a few days old this organism develops as bent and branching rods; these rods then break up into chains of cocci and short rods, which separate, and in old cultures all the organisms may be in the coccoid form ([Fig. 1]). It is claimed by Löhnis[47b] that the possession of a complex life-cycle of changing forms is a universal character in the bacteria. The instability of shape in many bacteria makes it necessary to standardise very carefully the cultural conditions under which they are kept when their appearance is described.

Culture 15 hours old. Culture 3 days old.

Fig. 1.—Change in appearance, in culture, of a cresol decomposing bacterium.

The inadequacy of mere morphology as a basis for describing bacteria led to the search for diagnostic characters, based on the biochemical changes that they produced in their culture media, and the appearance of their growth in the mass on various media. These characters unfortunately have also proved to be very much influenced by the exact composition of the medium and other conditions of culture. Recently an attempt has been made by the American Society of Bacteriologists to standardise the diagnostic characters used in describing bacteria, and also the media and cultural conditions under which they are grown for the purpose of description. The need for such precautions, however, was not sufficiently realised by the early workers, many of whose descriptions cannot now be referred to any definite organism.

The large number of organisms found in the soil, and the difficulty and labour of adequately describing them, is such that even now we have no comprehensive description of the common soil bacteria that appear on gelatine platings. A careful study based on modern methods of characterisation has been made of certain selected groups of bacteria, and it is hoped that the laborious systematic work of describing the common forms will gradually be completed.

Several attempts have been made to classify the bacteria that appear commonly on gelatine platings. This work was commenced by Hiltner and Stormer in Germany, and continued by Chester, Harding, and Conn in America. Conn[10]^, [14] found that the common organisms fell into the following main groups:—

(1) Large spore-forming bacteria, related to Bacillus subtilis, which form about 5-10 per cent. of the numbers. He adduced evidence[12]^, [13] that these organisms exist in the soil mainly as spores, so that they may not form an important part of the active soil population.

(2) Short non-sporing organisms, related to Pseudomonas fluorescens, that are rapid gelatine liquefiers. These form another 10 per cent. of the numbers.

(3) Short rod forms that liquefy gelatine slowly or not at all, and develop colonies very slowly. These form 40-75 per cent. of the numbers, and may therefore be of considerable importance in the soil.

(4) A few micrococci also occur.

These groups comprise the larger portion of the bacterial flora of the soil, but, in addition to these organisms, that develop on the media commonly used for plating, there are special and important groups that appear only on special media, either owing to their being unable to grow on ordinary media or because they get swamped by other forms. Examples of such groups are the ammonia and nitrite oxidising bacteria, the nitrogen fixing groups, the cellulose decomposing organisms, and the sulphur bacteria.

In order that we may apply the results of the study of a definite organism to other localities, a knowledge of the geographical distribution of the soil bacteria is clearly needed. We have, unfortunately, very little knowledge of the distribution of soil organisms. The common spore-forming groups appear to be universally distributed. Thus Barthel, in a study of the bacterial flora of soils from Greenland and the island of Disko, obtained soil organisms belonging to the groups of Bacillus subtilis, B. amylobacter, B. fluorescens, B. caudatus, and B. Zopfii, which are common groups in European soil, indicating that the general constitution of the bacterial flora of the soil in arctic regions is not widely different from that of Western Europe. Bredemann, who made an extensive study of the Bacillus amylobacter group, obtained soil samples from widely scattered localities, and found these organisms in soil from Germany, Holstein, Norway, Italy, Morocco, Teneriffe, Russia, Japan, China, the East Indies, Samoa, Illinois, Arizona, German East Africa, and the Cameroons. Some soil organisms, on the other hand, are apparently absent from certain districts. This may be due to the conditions, such as climatic environment, being unfavourable to them. A study has recently been made at Rothamsted of the distribution over Great Britain of a group of bacteria that are capable of decomposing phenol and cresol. One of these organisms, apparently related to the acid-fast B. phlœi, has an interesting distribution. It has been found in 50 per cent. of the soils samples examined from the drier region, where the annual rainfall is less than 30 inches, but in only 20 per cent. of the samples in the wetter parts of Britain. Another example of limited distribution is found in the case of Bacillus radicicola, the organism that produces tubercles on the roots of leguminous plants. The distribution of the varieties of this organism follows that of the host plants with which they are associated, so that when a new leguminous crop is introduced into a country, nodules may not appear on the roots unless the soil be specially inoculated with the right variety of organism. In cases where a group of soil organisms is widely distributed over the globe, it may yet be absent from many soils owing to the soil conditions not suiting it. Thus, phenol decomposing bacteria, though abundant in the neighbourhood of Rothamsted, are yet absent from field plots that have been unmanured for a considerable period. The occurrence of the nitrifying organisms and the nitrogen fixing Azotobacter is also very dependent on the soil conditions.

Owing to the method by which our knowledge of soil bacteria has been acquired, by studying first the chemical changes in the soil and then the bacteria that produce them, it is natural for us to divide them into physiological groups according to the chemical changes that they bring about. This grouping is the more reasonable since so little is known as to the true relationships of the different groups of bacteria that a classification based on morphology is well-nigh impossible. In considering the activities of bacteria in the soil, it is convenient to group the changes which they bring about into the two divisions into which they naturally fall in the economy of the organisms.

In the first place, there are the changes that result in a release of energy, which the bacteria utilise for their vital processes.

In the second place, there are the processes by which the bacteria build up the material of their bodies. These building up processes involve an intake of energy for their accomplishment.

It will be convenient to deal first with the release of energy for their own use by bacteria, and its consequences.

B. Activities Connected with the Acquirement of Energy.

Unlike the green plants, most bacteria are unable to obtain the energy that is required for their metabolism from sunlight. They must, therefore, make use of such chemical changes as will involve the release of energy.

As an example of the acquirement of energy in this way may be taken the oxidation of methane by B. methanicus. This organism, described by Söhngen, obtains its energy supply by the conversion of methane into CO₂ and H₂O.

CH₄ + 2O₂ = CO₂ + 2H₂O 220 Cal.

A further example is the acetic organism that obtains its energy through the oxidation of alcohol to acetic acid.

C₂H₆O + O₂ = C₂H₄O₂ + H₂O 115 Cal.

The decomposition processes brought about by micro-organisms in obtaining energy are usually oxidations, but this is not necessarily so, as can be seen in case of the fermentation of sugar into alcohol.[E]

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂ 50 Cal.

[E] These examples are from Orla-Jensen (Centralblatt f. Bakt., II., Bd. 22, p. 305).

By far the greater part of the decomposition of organic matter is brought about by bacteria in the process of acquiring energy. In the soil, nearly the whole of the material utilised by bacteria as a source of energy is derived ultimately from green plants. The energy materials left in the soil by the plant fall into two groups, the non-nitrogenous compounds, which are mainly carbohydrates and their derivatives, and the nitrogenous compounds, principally derived from proteins.

(1) Decomposition of Non-nitrogenous Compounds.

The simpler carbohydrates and starches are attacked and decomposed by a large variety of bacteria. The addition of such substances to soil causes a rapid increase in bacterial numbers. In nature the sugars are in all probability among the first plant constituents to be destroyed during the decay processes.

A large proportion of plant tissues consist of cellulose and its derivatives. These compounds are consequently of great importance in the soil. Unfortunately our knowledge of the processes by which cellulose is broken down in the soil is very inadequate. The early experimental study of cellulose decomposition, such as that of Tappeiner[60] and Hoppe-Seyler,[33] was mostly carried out under conditions of inadequate aeration, and the products of decomposition were found to include methane and CO₂, and sometimes fatty acids and hydrogen. The bacteriology of this anaerobic decomposition was studied by Omelianski,[54] who described two spore-bearing organisms, one of which attacked cellulose with the production of hydrogen, and the other with the production of methane. Both species also produce fatty acids and CO₂. It is probable that these organisms operate in the soil under conditions of inadequate aeration. In swamp soils, in which rice is grown, it has been shown that methane, hydrogen, and CO₂ are evolved in the lower layers. In these soils, however, the methane and hydrogen are oxidised when they reach the surface layers. This oxidation is also effected by micro-organisms. Bacteria capable of deriving energy by the oxidation of hydrogen gas have been isolated and studied by Kaserer,[37] and by Nabokich and Lebedeff,[52] while Söhngen[57] has isolated an organism which he named Bacillus methanicus, that was capable of oxidising methane.

Under normal conditions in cultivated soils, however, the decomposition of cellulose takes place in the presence of an adequate air supply, and so follows a different course from that studied by Omelianski. Our knowledge of this aerobic decomposition is very scanty. A number of bacteria, capable of decomposing cellulose aerobically, are known. A remarkable organism was investigated by Hutchinson and Clayton,[30] who named it Spirochæta cytophaga. This organism, which they isolated from Rothamsted soil, though placed among the Spirochætoidea, is of doubtful affinities. During the active condition it exists for the most part as thin flexible rods tapered at the extremities. This form passes into a spherical cyst-like stage, at first thought to be a distinct organism ([Fig. 2]). Spirochæta cytophaga is very aerobic, working actively, only at the surface of the culture medium. It is very selective in its action. It appears unable to derive energy from any carbohydrate other than cellulose. Indeed, many of the simple carbohydrates, especially the reducing sugars, are toxic to the organism in pure culture. An extensive study of aerobic cellulose decomposition by bacteria was made by McBeth and Scales,[50] who isolated fifteen bacteria having this power. Five of these were spore-forming organisms. Unlike Spirochæta cytophaga, they are all able to develop on ordinary media such as beef agar or gelatine, and are thus not nearly so selective in their food requirements.

Fig. 2.—Spirochæta cytophaga. Changes occurring in culture. (After Hutchinson and Clayton.)

We are at present ignorant as to which organisms are most effective in decomposing cellulose in the soil under field conditions, or what are the conditions best suited to their activity. It is possible that fungi also help in the decomposition of cellulose to a great extent. This subject of the decomposition of cellulose offers one of the most promising fields of research in soil bacteriology. The difficulty of the subject is further increased by our present ignorance of the chemical aspect of cellulose decomposition. It has been supposed that the early decomposition products are simpler sugars, but these are not found under conditions in which cellulose is being decomposed by pure cultures of the bacteria mentioned above. Hutchinson and Clayton found that their organism produced volatile acids, mucilage, and a carotin-like pigment. The organisms isolated by McBeth and Scales also produce acids, and in some cases yellow pigments. It is known, however, that the decomposition products of cellulose can be utilised as energy supply for other organisms, such as nitrogen fixing bacteria.

When plant remains decompose in the soil there are ultimately produced brown colloidal bodies collectively known as humus. The processes by which this humus is produced are not yet properly understood. Humus is of great importance in the soil, in rendering the soil suitable for the growth of crops. It affects the physical properties of the soil to a great extent. In the first place, it improves the texture of the soil, making heavy clay soils more friable, and loose sandy soils more coherent. Secondly, it has great water-retaining powers, so that soils rich in organic matter suffer comparatively little during periods of drought. And lastly, it exerts a strong buffering effect against soil acids. Now, it is one of the problems of present-day farming that soil is becoming depleted of its humus. This is due to the increasing scarcity of farmyard manure in many districts, and the consequent use of mineral fertilisers to supply nitrogen, potash, and phosphate to the crop. A need has therefore arisen for a substitute for farmyard manure, by means of which the humus content of soils may be kept up in districts where natural manure is scarce.

Fig. 3.—Cellulose decomposed by S. cytophaga in media with increasing amounts of nitrogen. (After Hutchinson and Clayton.)

X-axis: Milligrams of nitrogen supplied as sodium-ammonium phosphate.

Y-axis: Milligrams of cellulose decomposed in 21 days.

It is well known that if fresh unrotted manure or straw be added to the soil, it often produces harmful effects on the succeeding crop. The problem, therefore, was to develop a method by which fresh straw, before application to the soil, could be made to rot down to a mixture of humus compounds such as occur in well-rotted farmyard manure. The solution of this problem came as a result of an investigation by Hutchinson and Richards,[30b] at Rothamsted, into food requirements of the cellulose decomposing bacteria. They realised that since more than 10 per cent. of the dry weight of bacteria consists of nitrogen, it would be necessary to supply the cellulose decomposing bacteria with a supply of nitrogen, in order that they should attain their greatest activity. Experiments with cultures of Spirochæta cytophaga showed that the amount of cellulose decomposed depended upon an adequate supply of nitrogen for the organism ([Fig. 3]). Similarly, materials such as straw will scarcely decompose at all if wetted with pure water. An adequate supply of nitrogen compounds is needed to enable decomposition to take place. Hutchinson and Richards tested the effect of ammonium sulphate, and discovered experimentally the proportion of ammonia to straw that produced the most rapid decomposition. They found that if a straw heap was treated with the correct proportion of ammonia, it decomposed into a brown substance having the appearance of well-rotted manure. This has resulted in the development of a commercial process for making synthetic farmyard manure from straw. The method of manufacture is as follows: A straw stack is made and thoroughly wetted with water. The correct amount of ammonium sulphate is then sprinkled on the top and wetted, so that the solution percolates through the straw. The cellulose bacteria attack the straw, breaking it down and assimilating the ammonia. This ammonia is not wasted, as it is converted into bacterial protoplasm that eventually decays in the soil. Field trials of this synthetic manure show that it produces an effect closely similar to that of natural farmyard manure.

While cellulose and related carbohydrates are by far the most important non-nitrogenous compounds left in the soil by plants, there are other compounds whose destruction by bacteria is of special interest. Such, for example, is the case of phenol. This compound is produced by bacterial action as a decomposition product of certain amino-acids. It occurs in appreciable amounts in cow urine. It is probable that it forms a common decomposition product in soil and also in farmyard manure. If this phenol were to persist in the soil, it would eventually reach a concentration harmful to plant growth. It does not, however, accumulate in the soil; indeed, if pure phenol or cresol be added to ordinary arable soil, a rapid disappearance occurs. This disappearance is of some practical importance, since it limits the commercial use of these compounds as soil sterilising agents. The cause of the disappearance has been to some extent elucidated at Rothamsted,[58] where it was found to be in part a purely chemical reaction with certain soil constituents, and partly due to the activity of bacteria capable of decomposing it. A large number of soil bacteria have now been isolated that can decompose phenol, meta-, para-, and ortho-cresol, and are able to use these substances as the sole sources of energy for their life processes. These organisms have a wide distribution, having been found in soil samples taken from all over Great Britain, from Norway, the Tyrol, Gough Island, Tristan da Cunha and South Georgia. Soil bacteria have also been isolated that are able to decompose and derive their energy from naphthalene and from toluene. The ability of the bacteria to break up the naphthalene is very remarkable, and all the more so since they can hardly have come across this compound in the state of nature. The naphthalene organisms have a distribution as world-wide as the phenol group.

(2) Ammonia Production.

The second main group of products left in the soil by higher plants are the nitrogen-containing compounds, such as the proteins and amino-acids. Plant remains are not the only source of organic nitrogen compounds available to soil bacteria. There are, in addition, the dead bodies of other soil organisms, such as protozoa and algæ. The relative importance of these sources of nitrogen is not known, but almost certainly varies greatly with the state of activity of the various groups of the soil population. Bacteria are able to utilise organic nitrogen compounds as energy sources, as can be exemplified in the oxidation of a simple amino-acid:—

It will be seen that, in the acquirement of energy from such a compound, ammonia is released as a by-product. It is not certainly known what is the exact course of the reactions brought about by bacteria in soil during the breaking-down of organic nitrogen compounds, but they result in the splitting off of most of the nitrogen as ammonia. Herein lies the great importance of the process, for the production of ammonia is an essential stage in the formation of nitrate in the soil, and on the supply of nitrate the growth of most crops largely depends.

Fig. 4.—Quantities of ammonia produced by pure cultures from 5 grams of casein in the presence of varying quantities of dextrose. (After Doryland.)

X-axis: Percentage of dextrose added.

Y-axis: Milligrams of NH₃ produced.

It is very important to note that the production of this ammonia is only a by-product in the economy of the bacteria, the benefit that they derive from the reactions being due to the release of energy involved in the decomposition. The common ammonia-producing bacteria in the soil have been found equally capable of deriving their energy by the oxidation of sugars and similar non-nitrogenous compounds. [Fig. 4] shows an experiment by Doryland,[17] in which cultures of common soil bacteria were grown in peptone solution, to which increasing quantities of sugar were added. One can see that, as the amount of sugar is increased, the production of ammonia is lowered, since the bacteria are obtaining energy from the sugar instead of from the nitrogen compound, peptone. Consequently, if soil contains a quantity of easily decomposible carbohydrate material, bacteria will derive their energy from this source, and the production of ammonia and nitrate will be lowered. Thus the addition of sugar or unrotted straw to the soil often lowers the nitrate production, and consequently reduces the crop yield. If the soil is sufficiently rich in carbohydrate material, the bacteria may multiply until the supply of organic nitrogen is used up, and then will actually assimilate some of the ammonia and nitrate already existing. There is thus a balance of conditions in the soil due to varying proportions of nitrogenous and non-nitrogenous energy material. When nitrogen compounds are the predominant energy source, the bacteria utilise them, and ammonia is released. When a non-nitrogenous energy source predominates, this is utilised and little or no ammonia is released, and in extreme cases ammonia may be assimilated.

Although a large number of the common organisms in the soil produce ammonia in culture media containing peptone, the relative importance of these in the soil has yet to be decided. It was supposed that the spore-forming organisms related to Bacillus mycoides were of chief importance. This supposition dates from the work of Marchal,[49] who studied the production of ammonia by an organism of this group in culture solution, and found it to be a very active ammonifier. As already mentioned, however, there is some doubt as to whether the large spore-forming organisms are very active under soil conditions.[12]^, [13] The existence of rapid fluctuations in nitrate content, found to exist in soil, may in the future indicate which are the most active of the common bacteria in the soil itself by enabling us to observe which types increase during periods of rapid ammonia and nitrate formation.

(3) Nitrate Production.

The ammonia produced in the soil under normal field conditions is rapidly oxidised successively to nitrite and to nitrate, a process known as nitrification. The process of nitrification is more rapid than that of ammonia production, with the consequence that no more than traces of ammonia are able to accumulate. The rate at which nitrate is formed in the soil is consequently set by the slower process of ammonia production.

The work of Schloesing and of Warington showed that the oxidation of ammonia was the work of living organisms. It is, however, to Winogradsky’s isolation and study of the causative organisms that we owe our present knowledge of the biology of the process. By a new and ingenious technique, he isolated from soil two remarkable groups of bacteria that bring about nitrification. The first group oxidises ammonium carbonate to nitrite, and was divided by Winogradsky into the two genera, Nitrosomonas, a very short rod-like organism bearing a single flagellum, and Nitrosococcus, a non-motile form found in South America. The second group oxidises nitrites to nitrates. They are minute pear-shaped rods to which he gave the name Nitrobacter.

Winogradsky found that the first, or nitrite-producing group, would live in a culture solution containing:—

Nitrobacter would grow in a similar medium containing sodium nitrite instead of ammonium sulphate. There being no organic carbon in these media, the organisms had no source of carbon for their nutrition, except the CO₂ of the air, or possibly that of bicarbonate in solution. It therefore followed that the organisms must obtain their carbon supply from one of these sources. Unlike green plants, the nitrous and nitric organisms are able to carry on this carbon assimilation in the dark, and must therefore obtain the energy needed for the process from some chemical reaction. The only sources of energy in Winogradsky’s solutions were the nitrogen compounds, and it consequently followed that the organisms must derive their energy supply by the oxidation of ammonia and nitrite respectively. The release of energy obtained by these two reactions has been calculated by Orla-Jensen to be as follows:—

(NH₄)₂CO₃ + 3O₂ = 2HNO₂ + CO₂ + 3H₂O + 148 Cals.

KNO₂ + O = KNO₃ + 22 Cals.

The exact process by which ammonium carbonate is converted into nitrite is not at present known. The two groups of organisms are extremely selective in their source of energy. The nitrous organisms can derive their energy only by the oxidation of ammonia to nitrite, and the nitric organisms only by the oxidation of nitrite to nitrate. In culture media they are, indeed, inhibited by soluble organic compounds such as sugars. Under natural conditions, however, they appear to be less sensitive, since ammonium carbonate is readily nitrified in substrata rich in organic matter. The rapid nitrification that takes place during the purification of sewage is an example of this. The conditions in culture, with regard to aeration and the removal of metabolic products from the neighbourhood of the organisms, are very different from those in the soil, and perhaps account for the discrepancies found.

The oxidation of ammonium carbonate by nitrosomonas results in the formation of nitrous acid. The organisms are very sensitive to acidity, and can only operate if the nitrous acid produced is neutralised by an available base. In normal soils calcium carbonate supplies this base, and in acid soils the formation of nitrite is, as a rule, increased by the addition of lime, or of calcium or magnesium carbonate. There is evidence that in the absence of calcium carbonate, other compounds can be used as a base. It was found by Hopkins and Whiting[32] that in culture solution the nitrifying organisms could use insoluble rock phosphate as a base, producing therefrom the soluble acid phosphate. There is evidence, however, that in ordinary soil containing calcium carbonate very little solution of phosphate takes place in this way. The further oxidation of nitrite to nitrate by Nitrobacter does not produce acid, and requires no further neutralising base.

The nitrate produced in this way is the main source of nitrogen supply to plants under normal conditions. Experiments have shown that a number of plants are capable of utilising ammonia as a source of nitrogen, and Hesselmann[34] has found forest soils in Sweden where no nitrification was proceeding, and where, therefore, plants would presumably obtain their nitrogen in this way, but such cases must be regarded as exceptional.

Another group of bacteria capable of deriving their energy from an inorganic source exists in the soil. This comprises the sulphur bacteria, which are able to derive energy by the oxidation of sulphur, sulphides, or thiosulphates to sulphuric acid:—

S + 3O + H₂O = H₂SO₄ + 141 Cals.

One organism studied by Waksman and Joffe[63] is able to live in inorganic solution, deriving its carbon from carbon dioxide. The sulphur bacteria have recently come into prominence in America owing to their faculty for producing acid. Thus Thiospirillum will increase the acidity of its medium to a reaction of P_H 1·0 before growth ceases. The potato scab disease in America is now treated by composting with sulphur. This treatment depends on the production of sulphuric acid by the sulphur oxidising bacteria, which renders the soil too acid for the parasite. There is some evidence also that acid thus produced can be used to render insoluble phosphatic manures more available in the soil.

Analogous to the sulphur organisms are certain bacteria isolated from sheep dig tanks in South Africa by Green,[28b] which can derive energy by the oxidation of sodium arsenite to arsenate.

(4) Anaerobic Respiration.

As is seen in the examples mentioned, energy is commonly obtained by bacteria through an oxidation process in which free oxygen is utilised. In water-logged soil, however, or in soil overloaded with organic matter, anaerobic bacteria may develop, which obtain their oxygen from oxidised compounds. Thus there are soil organisms described by Beijerinck[2] and others which can obtain oxygen by reducing sulphates to sulphides.

A more important source of oxygen under these conditions is nitrate, which can supply oxygen to a larger number of bacteria. The stage to which the reduction can be carried varies according to the organism. A very large number of bacteria are capable of reducing nitrates to nitrites. Many can reduce nitrate to ammonia, and some can produce an evolution of nitrogen gas from nitrate. The effects of nitrate reduction, therefore, appear under water-logged conditions in soils. For example, in swamp soils in which rice is grown, it has been found by Nagaoka,[53] in Japan, that treatment with nitrate of soda depresses the yield, probably owing to the formation of poisonous nitrites by reduction.

Under normal conditions of well aerated soil, however, it is unlikely that the reduction of nitrate is of great importance. In such soils the activities through which bacteria acquire their energy are, as we have seen, of vital importance to the plant, resulting in the disintegration of plant tissues, with the ultimate formation of humus, and in the production of nitrate.

In their activities connected with the building up of their protoplasm, bacteria may, on the other hand, compete with the plant. These activities and their consequences will be reviewed in the following chapter.

CHAPTER III.
SOIL BACTERIA.

C. Activities Connected with the Building-up of Bacterial Protoplasm.

(1) Composition of Bacteria.

The activities of the soil bacteria that we have yet to consider are those connected with the building-up from simpler materials of the protoplasm of the bacterial cell. It is important to bear in mind that this process is one requiring an expenditure of energy on the part of the organism. The sources of energy we have already considered.

The bodies of bacteria contain the same elements common to other living matter. Analyses of various bacteria have been made by a number of workers. About 85 per cent. of their weight is made up of water. This analysis of Pfeiffer’s Bacillus by Cramer[15] shows the typical percentages of carbon, nitrogen, hydrogen, and ash in the dry matter:—

Composition of Pfeiffer’s Bacillus (Cramer).

About 65-70 per cent. of the dry matter of bacteria consists of protein.

(2) Sources of Carbon.

The biggest constituent of the dry matter of bacteria is therefore carbon. In the soil, bacteria find an abundance of organic matter from which they may derive their carbon supply. A special case, however, is furnished by the nitrifying organisms, certain sulphur oxidising bacteria, and others that derive their carbon from the CO₂ of the soil atmosphere. The sources from which these special groups obtain the necessary energy to accomplish this, we have already considered.

(3) Assimilation of Nitrogen Compounds.

Of chief importance in its consequences are the means adopted by bacteria to obtain their nitrogen supply.

There is some reason to believe that soil bacteria do not take up protein and peptones as such, but must first break down these bodies into simpler compounds. When a sufficient amount of easily decomposable organic nitrogen is present in the soil, the ammonifying bacteria use such compounds as sources of energy, and in this case have a nitrogen supply exceeding their requirements.

But where there is an excess of carbohydrate or other non-nitrogenous source of energy available in the soil, the case is different. Here the organisms have a supply of energy which enables them to multiply rapidly until the organic nitrogen is insufficient for their needs. Hence they turn to the ammonia and nitrate present in the soil, and build up their proteins from this source. Doryland[17] has shown that many common soil ammonifiers assimilate ammonia and nitrate when supplied with carbohydrate. There may thus be a temporary loss of nitrate from soil when sugar, starch, straw, or such materials are added to it.

(4) Fixation of Free Nitrogen.

The bacteria that we have so far considered take up their nitrogen directly from compounds containing this element. There remain, however, a comparatively small but very important group of bacteria possessing the power of causing elemental nitrogen to combine, and of building it up into their proteins. This fixation of nitrogen by micro-organisms is a vital step in the economy of nature. Losses of nitrogen from the land are continually occurring through the washing-out of nitrates by rain, and through the evolution of gaseous nitrogen during the processes of decay. To maintain the supply of combined nitrogen which is essential to living organisms, there must therefore be a compensating process by which the supply of nitrogen compounds in the soil is kept up.

It was discovered in the middle of the nineteenth century that if soil were kept moist and exposed to the air, there was an increase in the amount of nitrogen compounds present. Berthelot, in 1893, studied the nitrogen relationships of soil, and recognised that this fixation of nitrogen in soil was the work of micro-organisms.

Winogradsky followed up his work and isolated from soil a large anaerobic spore-forming organism, capable of fixing nitrogen, to which he gave the name Clostridium pasteurianum. In 1901 the investigations of Beyerinck, in Holland, led to the important discovery of a group of large aerobic organisms, which he named Azotobacter. These were found to be very active in fixing free nitrogen. More recently, a number of other nitrogen-fixing bacteria have been described, and the property has been found to exist to a small extent in several previously well-known organisms.

It becomes important to determine which are the groups of bacteria whose nitrogen-fixing powers are of chief importance in the soil.

On account of its energetic fixation of nitrogen in culture media, Azotobacter has attracted the greatest attention of workers. The evidence seems to be consistent with the view that Azotobacter is of importance in the soil. Thus the distribution of Azotobacter would appear to be world-wide. It is found all over Western Europe and the United States. Lipman and Burgess[45] found it in soils collected from Italy and Spain, Smyrna, Cairo, the Fayum, the Deccan in India, Tahiti, Hawaii, Mexico, Guatemala, and Canada. C. M. Hutchinson[29] found it to be distributed throughout India. It was found by Omelianski[55] to be widely distributed in European and Asiatic Russia, and by Groenewege[28] in Java. Ashby[1] at Rothamsted, isolated it from soils from the Transvaal, East Africa, and Egypt. Also, an association has sometimes been found between the ability of a soil to fix nitrogen and the occurrence and vigour of its Azotobacter flora. Thus Lipman and Waynick[46] found that if soil from Kansas were removed to California, its power to produce a growth of Azotobacter, when inoculated into a suitable medium, was lost, and, at the same time, its nitrogen-fixing power was greatly reduced. Moreover, it is known that conditions favourable to the fixation of nitrogen by Azotobacter in cultures on the whole favour nitrogen fixation in soils. The conditions that favour other aerobic nitrogen-fixing bacteria are, however, not sufficiently distinct to make such evidence of great value.

It is usually found that nitrogen fixation is most active in well-aerated soil. Thus Ashby,[1] at Rothamsted, found the nitrogen-fixing power of a soil to decrease rapidly with depth. Similar results were obtained in Utah by Greaves. This suggests, at first sight, that anaerobic nitrogen fixers are unimportant under normal soil conditions. It is, however, quite possible that they may assume an importance when acting in conjunction with aerobic organisms. Thus Omelianski and Salunskov[55] found that beneficial association, or symbiosis, could occur between Azotobacter and Clostridium pasteurianum, the former absorbing oxygen from the surroundings, and thus creating a suitable anaerobic environment for the Clostridium.

The question of symbiosis of nitrogen-fixing bacteria with each other and with other organisms offers an inviting field for research. There is evidence that this factor may have considerable importance. Beijerinck and Van Delden[3] early recognised that Azotobacter in mixed cultures fixed more nitrogen than in pure cultures. Granulobacter, an organism which they found to be commonly associated with Azotobacter in crude cultures, appears to increase its nitrogen-fixing powers (Krzeminiewski).[41] It was also found by Hanzawa[31] that a greater fixation of nitrogen was obtained when two strains of Azotobacter were grown together. A symbiosis between Azotobacter and green algæ has been described, and will be further [discussed] by Dr. Bristol. It is likely that this association may be of importance under suitable conditions on the soil surface where the algæ are exposed to light.

The combination of elemental nitrogen is an endothermic process which requires a very considerable amount of energy for its accomplishment. This fact is well illustrated by the various commercial processes in use for fixation of atmospheric nitrogen. The nitrogen-fixing bacteria obtain this energy from the carbon compounds in the soil. A number of compounds were compared as sources of energy by Löhnis and Pillai,[47] who tested their effect on the amounts of nitrogen fixed by Azotobacter in culture. It was found that mannitol and the simpler sugars give the best results as sources of energy, but that other organic compounds can also be used. Mockeridge[51] has adduced evidence that ethylene glycol, methyl-, ethyl-, and propyl-alcohol, lactic, malic, succinic, and glycocollic acids could also be utilised. Since so large a part of the organic matter added to soil is in the form of celluloses, it is of great importance to ascertain how far these compounds and their decomposition products can be utilised in nitrogen fixation. Stubble, corn-stalks and roots, oak leaves, lupine and lucerne tops, maple leaves, and pine needles may all serve as useful sources of energy to nitrogen-fixing organisms in the soil. Pure cellulose cannot apparently be used as a source of energy, but when acted upon by cellulose decomposing organisms, it becomes available as a source of energy. Hutchinson and Clayton, at Rothamsted, found that a fixation of nitrogen could be brought about by mixed cultures of Azotobacter, and of the cellulose attacking Spirochæta cytophaga, when grown in cultures containing pure cellulose. It is not known how far cellulose decomposition must proceed to produce an effective source of energy, nor what are the substances thus produced that are utilised. This point will not be decided until something more is known of the course of changes in the breaking-down of cellulose in the soil.

The amount of nitrogen fixed per unit of energy material decomposed varies greatly, according to the organism and the conditions. Winogradsky found that his Clostridium assimilated 2-3 mgs. of nitrogen per gram of sugar consumed. Lipman found that Azotobacter fixed 15-20 mgs. of nitrogen per gram of mannite consumed.

Fig. 5.

Caption: Azotobacter. Decrease in efficiency in N fixation with age of culture. (Koch & Seydel.)

X-axis: Days.

Y-axis: Milligrams of Nitrogen fixed per gram of dextrose consumed.

It is found, however, that in liquid culture, the ratio of nitrogen fixed to carbohydrates oxidised varies according to the age of the culture, falling off rapidly as the age increases[42] ([Fig. 5]). This decreasing efficiency in cultures may be due to the accumulation of metabolic products such as would not occur under soil conditions. Indeed, the efficiency of Azotobacter in a sand culture has been found by Krainskii[39] to be considerably greater than in solution. It is thus probable that in soil the nitrogen-fixing organisms are less wasteful of energy material than under the usual laboratory conditions. It is to be hoped that future research will indicate what are the conditions that produce the greatest economy of energy material in nitrogen fixation.

The fixation of nitrogen in soil is depressed by the presence of considerable amounts of nitrates. This is, in all probability, due to the fact that nitrogen-fixing organisms are able to utilise compounds of nitrogen where these are available. The energy needed to build up amino-acids and proteins from nitrate or ammonia is, of course, far less than that required to build up these substances from elemental nitrogen. It is, therefore, not surprising that where nitrate is available, Azotobacter will use it in preference to fixing atmospheric nitrogen.[5]

TABLE III.—ASSIMILATION OF NITRATES.
By Azotobacter in Pure Culture—(Bonazzi).

The chemical process by which nitrogen is fixed is quite unknown, although a number of speculative suggestions have been made. The appearance of considerable amounts of amino acids in young cultures of Azotobacter suggests that these may be a step in the process, but at present the data are too inconclusive to form a basis for theorising.

Azotobacter is very rich in phosphorus, an analysis of the surface growth in Azotobacter cultures, made by Stoklasa, giving about 60 per cent. of phosphoric acid in the ash. In cultures it has been found that a considerable amount of phosphate is needed to produce full development. As would be expected, therefore, nitrogen fixation in soil is often greatly stimulated by the addition of phosphates. Christensen has, indeed, found soils where lack of phosphate was the limiting factor for Azotobacter growth.

Azotobacter is very intolerant of an acid medium, and is very dependent on the presence of an available base. In cultures this is usually provided in the form of calcium or magnesium carbonate. Gainey[21] found that Azotobacter occurred in soils having an acidity not greater than P_H 6·0, and Christensen,[7]^, [9] in Denmark, has found a close association between the occurrence of Azotobacter in soils and the presence of an adequate supply of calcium carbonate. So close was this association that he devised a technique based on this fact for detecting a deficiency of lime in a soil sample.

In addition to the groups already discussed, there is a remarkable and important group of nitrogen-fixing bacteria that inhabit and can carry on their functions within the root tissues of higher plants. It has been known at least from classical times that certain leguminous plants would, under suitable conditions, render the soil more productive. On the roots of leguminosæ small tubercles are commonly found. These were noted and figured by Malpighi in the seventeenth century, and for a long time were regarded as root-galls. As was described in [Chapter I.], the true nature of these tubercles was finally elucidated by Hellriegel and Wilfarth in 1886. As the result of a series of pot experiments, they made the very brilliant deduction that the ability to fix nitrogen, possessed by the legumes, was due to bacteria associated with them in the tubercles.

These bacteria were finally isolated and studied in pure culture by Beijerinck. Since then a very great deal of literature has accumulated on the subject of the nodule-producing bacteria, which it is impossible to deal with in a small space. The nodule organism, Bacillus radicicola, when grown on suitable media, passes through a number of different changes in morphology. The most connected account of these changes is given in a paper by Bewley and Hutchinson.[4] In a vigorous culture the commonest type is a rod-shaped bacillus which may or may not be motile. As these get older they often become branched, or irregular in shape, the formation of these branched forms being perhaps due to conditions in the medium. These irregular forms, known as “bacteroids,” are a characteristic type in the nodules. Their production in culture media has been found to be stimulated by sugars and organic acids such as would occur in their environment within the host plant. In the older rods and bacteroids the staining material becomes condensed into granules, and finally the rods disintegrate or break up into coccoid forms. By suitable culture conditions, Bewley and Hutchinson obtained cultures consisting almost entirely of this stage. If such a culture be inoculated into a fresh medium rich in sugar, the swarmer stage appears in great numbers. These swarmers are very minute coccoid rods, ·9 × ·18 in size, that are actively motile. They apparently develop later into the rod stage.

Fig. 6.—Bacillus radicicola. Stages in the life cycle. (After Hutchinson and Bewley.)

Motile Rods

Vacuolated Stage

“Swarmers”

“Bacteroids”

“Pre-swarmers”

Very little is known as to the life of the organism in the soil. It is able, however, to fix nitrogen in cultures, and it has been claimed[35]^, [48] that it can do so in the soil outside the plant, so that it is possible that we must take it into consideration in this connection. More knowledge is needed as to the optimum conditions for the growth of the organism in the soil. It seems to be more tolerant of acid soil conditions than Azotobacter. The limiting degree of acidity has been found to vary among different varieties of the organism from P_H 3·15 to P_H 4·9.

A long controversy has been held as to whether the nodule organisms found in different host-plants all belong to one species, or whether there are a number of separate species, each capable of infecting a small group of host-plants. As the term “species” has at present no exact meaning when applied to bacteria, the discussion in this form is unlikely to reach a conclusion. The evidence seems to show that the nodule organisms form a group that is in a state of divergent specialisation to life in different host-plants, and that this specialisation has reached different degrees with different hosts. Thus the organisms from the nodule of the pea (Pisum sativum) will also produce nodules on vicia, Lathyrus, and Lens, but seem to have lost the ability normally to infect other legumes. On the other hand, the bacteria from the nodules of the Soy Bean (Glycine hispida) have become so specialised that they do not infect any other genus of host-plant, and soy beans are resistant to infection by other varieties of the nodule organism. Burrill and Hansen,[6] after an extensive study, divided the nodule bacteria into eleven groups, within each of which the host-plants are interchangeable. The existence of different groups of nodule organisms has been confirmed by the separate evidence of serological tests (Zipfel, Klimmer, and Kruger).[40] The results of cross-inoculation tests have sometimes been conflicting. It seems, indeed, that the host-plant has a variable power of resisting infection, so that when its resistance is lowered it may be capable of infection by a strange variety of the nodule organism. The question that has thus arisen of the ability of the legume to resist infection is of fundamental importance, and its elucidation should throw light on the relation of plants to bacterial infection as a whole.

The stage of the organism that infects the plant is not at present known. It may be supposed that it is the motile “swarmer.” The entry is normally effected through the root-hairs. The hair is attacked close to the tip, and an enzyme is apparently produced which causes the tip to bend over in a characteristic manner. The organisms multiply within the root hair and pass down it, producing a characteristic gelatinous thread filled with bacteria, in the rod form. This “infection thread” passes down into the cells of the root tissue, where it branches profusely. In young stages of nodule formation the branches can be seen penetrating cells in the pericycle layer. Rapid cell division of these root cells is induced. In the course of this cell division abnormal mitotic figures are sometimes found, such as occur in pathological growths. The cells push outward the root cortical layer, and so form a nodule.

Certain of the cells in the centre of the nodule become greatly enlarged, and in the fully grown nodule are seen to be filled with bacteria. Differences have been described in the morphology of the organisms in different parts of the nodule.[62] Whether the different stages of the organism are equally capable of fixing nitrogen, or what is the significance of these stages within the nodule, is not certainly known. It has been held that it is the irregular bacteroid forms that are chiefly concerned with nitrogen fixation. In older nodules the organisms become irregular and stain faintly, and the bacteroidal tissue breaks down, the nodule finally decaying. In the fixation of nitrogen that occurs in the nodules, the bacteria without doubt derive the necessary energy from the carbohydrates of the host-plant. There is evidence that the plant assists the process of fixation by removing soluble metabolic products from the neighbourhood of the bacteria. Golding[22] was able to obtain a greatly increased fixation of nitrogen in artificial cultures by arranging a filtering device so as to remove the products of metabolism.

The great practical importance of leguminous crops in agriculture has led to numerous attempts being made to increase their growth, and the fixation of nitrogen in them, by inoculating the seed or the soil with suitable nodule-bacteria. This inoculation can be effected either with soil in which the host-plant has been successfully grown, and which should consequently contain the organism in fair numbers, or else pure cultures of the organisms isolated from nodules may be used. Very varying results have been obtained with inoculation trials.

In farm practice a leguminous crop has often been introduced into a new area where it has never previously grown. In such soil it is very probable that varieties of the nodule organism capable of infecting the roots may not exist. In such cases inoculation with the right organism or with infected soil often produces good results.

The more difficult case, however, is that in which the legume crop has been grown for a long time in the locality, and where the soil is already infected with right organisms. This, the more fundamental problem, applies especially to this country. Here it would seem that inoculation with a culture of the organism will benefit the plant only (1) if the naturally occurring organisms are present in very small numbers; or (2) if the organisms in the culture added are more virulent than those already in the soil. The problem of successful inoculation would therefore seem to be bound up with that of grading up the infective virulence of the organism to a higher level.

Successful nodule development in a legume crop is also dependent to a large degree on the soil conditions. The effects of soil conditions on nodule development have been studied by numerous workers. Moisture has been found very greatly to affect the nodule development. Certain salts have a very definite effect on nodule formation.[64] Their effect on the number of nodules developing has been studied, but the reason for this effect is unusually difficult to decide. The action is usually a complex one. Thus phosphates are known to stimulate nodule formation. They probably act in several ways. In the first place, they may cause the nodule organisms to multiply in the soil; in the second place, they produce a greater root development in the plant, thus increasing the chances of infection; and in the third place, Bewley and Hutchinson[4] have found that phosphates cause the appearance of the motile stage of the organism in cultures. A real understanding of the influence of environment on nodule production will produce great improvements in our methods of legume cropping.

D. The Relation of Bacterial Activities to Soil Fertility.

The various activities of the soil bacteria have a vital importance to the growth of higher plants, which are dependent for their existence on certain of these processes. In the first place, as we have seen, bacteria decompose the tissues of higher plants and produce humus materials, which are essential to the maintenance of good physical properties in the soil. Then the nitrate supply on which most higher plants depend is produced by the decomposition of organic nitrogen compounds by bacteria in their search for energy. The depletion of the total nitrogen content of the soil through rain and through the removal of nitrogen in the crops, is to some extent compensated by the fixation of atmospheric nitrogen by certain bacteria. On the other hand, in the assimilation of nitrogen compounds to build up protein, the bacteria are competing with higher plants for one of their essential food constituents, and their action may, under certain conditions, cause a temporary nitrogen starvation. One must remember, however, that large quantities of nitrate are lost from field soils by washing-out through rain action, especially in winter. The assimilation of nitrate and ammonia by micro-organisms keeps some of this nitrogen in the soil, and at certain periods may thus be beneficial.

There is another important respect in which soil bacteria influence plant growth. Their activities result in the release of inorganic salts, such as potash and phosphates, in a form available for the use of plants. The release of phosphorus and potassium compounds takes place in two ways. In the first place, organic matter containing phosphorus and potassium, in an insoluble form, is attacked by bacteria, resulting in these elements being set free as inorganic salts available to the higher plant. Secondly, much of the phosphorus supplied to the soil from rock minerals is present as insoluble phosphates, such as apatite and iron phosphate. Much of the potassium, too, is derived from insoluble silicate minerals. In both cases the conversion of the insoluble minerals into soluble phosphates and potassium compounds is brought about to a large extent by solution in water containing carbonic and other acids. These acids are largely produced by micro-organisms, which, in addition to carbonic acid, produce organic acids, and in specialised cases, sulphuric and nitrous acids. It has been found, for example, that in a compost of soil with sulphur and insoluble phosphate, sufficient sulphuric acid may be produced by the oxidation of the sulphur by bacteria to convert an appreciable amount of phosphate into a soluble form. When we consider the functions performed by soil bacteria, therefore, it is not surprising to find that high bacterial activity in the soil is associated, as a rule, with fertility.

E. Changes in Bacterial Numbers and Activities, and their Relation to External Factors.

The object of soil bacteriologists is to discover means of favouring the activity of soil bacteria, especially those activities that are useful to the higher plant. Knowledge is therefore needed of the changes in numbers and activities of the soil bacteria, and of the influence of soil conditions on them. The necessity of studying these changes has required the development of a quantitative technique by which the numbers of bacteria in the soil and their activities can be estimated.

The method commonly used in counting bacteria in soil is a modification of the plating method of Koch. In counting bacteria two difficulties have to be overcome—their immense numbers and their small size. The numbers of bacteria in soil are so large that the bacterial population of a gram of soil could not, of course, be counted directly. The method adopted, therefore, is to make a suspension of soil in sterile salt solution, and to dilute this suspension to a convenient and known extent, which will depend on the numbers of bacteria expected. In ordinary field soils it is found convenient, for example, to dilute the soil suspension so that one cubic centimeter of the diluted suspension will contain ¹⁄_250,000th of a gram of soil. Such a volume will commonly contain a number of bacteria sufficiently small to count. The second difficulty is that the organisms are microscopic, and yet cannot be readily counted under the microscope owing to the presence of soil particles in the suspension. Hence recourse is had to plating. One cubic centimeter of diluted suspension is placed in a petri dish and mixed with a suitable nutrient agar medium, melted, and cooled to about 40° C. The medium sets, and after a few days’ incubation the organisms multiply and produce colonies visible to the naked eye. By counting these colonies we obtain an estimate of the number of bacteria in the one cubic centimeter of suspension, it being assumed that every organism has developed into one colony, and by multiplying this number by the degree of dilution we obtain the numbers per gram of soil. In practice a number of parallel platings are made from one cubic centimeter portions of the diluted suspension and the mean number of colonies per plate is taken. By this means the error due to the random distribution of bacteria in the suspension is reduced, because of the greater number of organisms counted.

In drawing conclusions from bacterial count data, it is necessary to distinguish between the indication which the method gives of the absolute numbers of bacteria in the soil and the accuracy with which it enables the numbers of two soil samples to be compared. The method cannot be used for the former purpose at present. We do not know how far the figures obtained by this counting method fall short of the actual number of bacteria in the soil. One reason for this is the difficulty of effecting a complete separation of the clumps of bacteria into discrete individuals in the suspension. Then again, there is no known medium upon which all the physiological groups of bacteria will develop and produce colonies. And even on a suitable medium some of the individuals may fail to multiply.

In comparing the bacterial numbers in two soil samples, however, the case is different. Within each bacterial group investigated the plate method should give counts proportional to the bacterial numbers in the soil. Thus, by the method one should be able to tell whether the bacterial numbers are increasing or decreasing over a period of time, or whether a certain soil treatment produces an increase or a decrease. With this end in view the technique of the method has been improved by recent workers. It was found that, when carefully standardised, the process of dilution of the soil could be carried out without significant variation in result ([Table IV.]), and that the accuracy of the method is limited mainly by the variation in colony numbers on parallel platings, due in part to random distribution of bacteria throughout the final suspension, and partly to the uneven development of colonies on the medium. The question of the medium was therefore taken up with a view to improving the uniformity of results obtained with it. Lipman, Conn, and others effected an improvement by using chemical compounds as nutrient ingredients, thus making their media more closely reproducible. On most agar media, an important disturbing factor is the growth of spreading colonies, which prevent the development of some of the other colonies. A medium has been devised at Rothamsted on which these spreading organisms are largely restricted.[61] A statistical examination[19] has shown that on this medium errors due to the uneven development of colonies, except in special cases, are prevented, so that in fact the variation in colony numbers between parallel plates is found to be that produced merely by random distribution of bacteria in the diluted suspension (see [Table IV.]). In this case the accuracy of the counts of the bacteria in the diluted suspension depend directly on the number of colonies counted, and can be known with precision.

TABLE IV.—BACTERIAL COUNTS OF A SOIL SAMPLE.
Parallel Plate Counts from Four Sets of Dilutions made by Different Workers.

The knowledge obtained from counts of soil bacteria is subject to another serious limitation. We do not know which of the bacteria counted are the most effective in bringing about the various changes that take place in the soil. It is not even known which of them are active in the soil and which are in a resting condition. It is thus possible to have two soils containing equal numbers of bacteria but showing widely different biochemical activity, if one soil contains organisms of a higher efficiency. Moreover, as has been pointed out, many important groups of soil bacteria do not develop on the plating media, and so are not counted. These considerations led to the development of supplementary methods by which it was hoped to estimate the actual biochemical activity of the soil microflora. The first of these methods was developed by Remy, who attempted to study the biochemical activity of a soil by placing weighed amounts into sterile solutions of suitable and known composition, keeping them under standard conditions for a definite time and then estimating the amount of the chemical change that was being studied. Thus, to test the activity of the organisms that produce ammonia from organic nitrogen compounds, he inoculated soil into 1 per cent. peptone solution and measured the amount of ammonia produced in a given time. By similar methods the power of a soil to oxidise ammonia to nitrate, to reduce nitrate, or to fix atmospheric nitrogen, is tested. This method has been extensively used and developed by more recent workers. It suffers, however, from the same serious disadvantage that it was designed to avoid, for we cannot be certain that those bacteria that develop in the nutrient solution are the types that are active in the soil, and, moreover, even where the same types do function in the two conditions, we do not know that the degree of their activity is the same in soil and in solution cultures. For instance, Nitrosomonas appears to show very different degrees of activity in soil and in culture.

Another method, therefore, of studying the activity of soil micro-organisms is the obvious one of estimating the chemical changes that they produce in the soil itself. This method has obvious advantages over the unnatural methods developed from Remy’s, but it has a number of limitations that make its actual application difficult. In the first place, we cannot always tell whether changes found to occur in soil are due to the activity of micro-organisms, or are purely chemical reactions unassisted by biological agencies. Then, if we succeed in showing that the changes are due to micro-organisms, it is very difficult to determine which organisms are effecting them. This cannot be definitely tested by isolating suspected organisms and testing their activity in sterile soil, because in sterilising soil its nature and composition is altered. In spite of these difficulties, however, the study of the chemical changes that take place in the soil has produced valuable knowledge, when it has been combined with a study of the changes in the number and variety of the micro-organisms that accompany these reactions. This method of investigation is well illustrated by the work of Russell and Hutchinson on the effects of heat and volatile antiseptics on soil, where a study of the chemical changes such as ammonia production, that occurred in these treated soils, combined with a study of the changes in bacterial numbers, led to the realisation that the soil micro-population was a complex one, containing active protozoa.

A great difficulty in applying quantitative methods to bacteria in the field is the great variation in the density of the bacterial population over a plot of field soil, which may be so great that a bacterial count from a single sample is quite valueless. For example, the distribution of bacterial numbers over a plot of arable soil near Northampton was studied by taking sixteen samples distributed over an area about 12 feet square. The result showed that in some cases the bacterial numbers in samples taken 6 inches apart differed by nearly 100 per cent. Fortunately, under favourable conditions, a remarkably uniform distribution of bacterial numbers over a plot of soil can be found.

On such a plot it is possible to investigate the rapidity with which the numbers of the soil micro-organisms alter in point of time. For example, on the dunged plot of Barnfield, Rothamsted, which has been cropped with mangolds for forty-seven successive years, the area distribution of bacteria has been found to be so uniform that if a number of samples of soil are taken from the plot at the same time, the difference in bacterial numbers between the samples cannot be detected by means of the counting technique (see [Table V.]). The work of Cutler, Crump, and Sandon[16] on this plot showed that the bacterial numbers vary very greatly from one day to the next, and that these fluctuations took place over the whole plot, since two series of samples, taken in two rows 6 feet apart, showed similar fluctuations (see [Fig. 7]). The discovery of these big daily fluctuations in numbers led to an inquiry as to how quickly bacterial numbers change, and samples from Barnfield, taken at two-hourly intervals, showed that significant changes in numbers took place even at such short intervals.

TABLE V.—BACTERIAL COUNTS OF FOUR SOIL SAMPLES.
From Barnfield, Taken Simultaneously.

Fig. 7.

X-axis (top): Days.

Y-axis (left): (Series A) Bacteria—millions per gramme of soil.

Y-axis (right): (Series B) Bacteria—millions per gramme.

Caption: Daily changes in bacterial numbers in field soil.
Counts from two series of soil samples taken 6 feet apart.
(After Cutler.)

Since the bacteria involved in this fluctuation are of great importance to the crops, being for the most part ammonia producing types, further knowledge as to the cause of this fluctuation and of its effect on the ammonia and nitrate in the soil is of fundamental importance. There is evidence, which will be discussed later, that the cause is connected with the changing activities of certain soil protozoa, since the daily changes in the numbers of active amœbæ in the soil have been found to be in the reverse direction to those of the bacterial numbers. It appears, therefore, that we are dealing with an equilibrium between the various members of the soil population, the point of equilibrium changing at frequent intervals.

In addition to daily changes, it is possible to detect changes in the numbers and activity of the soil population related to the season. There is a well-marked increase in the spring and autumn (see [Figs. 15], [16], pp. 89, 90). This is well seen when the fortnightly averages of the daily bacterial and protozoal counts from Barnfield soil are plotted. These spring and autumn increases comprise both the bacterial and the protozoal population, and therefore differ from the short time fluctuations in being due, not to a disturbance of the bacteria-protozoa equilibrium, but to a general rise in activity of both groups of organisms.

When we consider the action of external conditions on the soil bacteria, the existence of a complex soil population and the interdependence of its members must be borne in mind. Changes in external conditions may affect the different components of the population in different ways or to different degrees, thus upsetting the equilibrium between the various groups. For example, the addition of a mild aromatic antiseptic to the soil apparently affects the protozoa in such a way as to disturb the bacteria-protozoa equilibrium in favour of the bacteria, while in some cases the aromatic compound affords a food supply to special bacteria, causing these to increase, upsetting the equilibrium between the different bacterial groups. When our knowledge of the effect of external factors on the soil population becomes sufficient, it will probably be found that in nearly all cases a change in the soil conditions produces some disturbance in the equilibrium between the components of the soil population, though at present there are only certain examples where this disturbance is a probable explanation of the facts.

Since bacteria are dependent on adequate supplies of energy and food, it is to be expected that additions of organic matter or of inorganic food materials will greatly benefit their activities. The effect of added farmyard manure in increasing bacterial activities has been much studied.[27] Some of the increased bacterial numbers and activities in this case may be due to the addition of bacteria with the manure, but it is thought that this factor is of less importance than the added energy and food supply which the general soil flora obtain from it. Nutritive salts such as phosphates and salts of potassium usually increase the bacterial activities.

The effect of alkali salts on soil bacteria has been especially studied in the Western United States, where the existence of alkali in the soil is a serious problem.[23] Soil bacteria are usually stimulated by small doses of alkali salts that are toxic in higher concentration. As a rule, chlorides are the most toxic salts, the electronegative ion playing an important part in the effect of the salt. Salts affect bacteria both owing to the changes in osmotic pressure which they produce, and through their specific action on the bacterial protoplasm.[26] When equal weights of various salts are added to soil, their toxic action on bacteria shows so little association with their respective osmotic pressures that we must conclude that this factor is the less important. There is reason to suppose that the toxic action of salts on bacteria is often connected with an effect of the specific ions on the permeability of the bacterial cell-wall. This conclusion is based on the changes in electrical conductivity of bacterial suspensions in the presence of various salts.[59]

A definite antagonism between various salts has been found to exist. It is possible that future work in this line may indicate what are the proportions of common electrolytes which will produce a properly “balanced” soil solution so that the harmful excess of one salt may be antagonised.

Certain salts, such as those of arsenic[24] and manganese, seem to exercise a stimulating action on bacterial activities; the causes of this action are not at present understood.

The acidity of the soil has an important effect on the bacterial processes. The acidity of soils may increase to such a point that the decomposition of plant tissues by bacteria is hindered, a peat layer being thus produced. The degree of acidity that is toxic varies very greatly with different soil bacteria, some of them, like Azotobacter and Nitrosomonas being very intolerant of acidity.

The conditions of aeration, water content, and temperature are inter-related in field soil. Ammonifying organisms are not greatly dependent on aeration, but this factor is sometimes a limiting one in the case of the very aerobic nitrifying bacteria. Hence efficient soil cultivation is beneficial to nitrification.

Many attempts have been made to correlate the temperature and moisture of field soils with the bacterial numbers and activities. These attempts have given very discordant results. It is generally agreed that a plentiful moisture supply is beneficial. Thus Greaves, in Utah, found the optimum water content for ammonia and nitrate production to be about 60 per cent. of the water-holding capacity. On the other hand, Prescott[56] found that the summer desiccation of soil in Egypt was followed by increased bacterial activities. Fabricius and Feilitzen,[18] using moor soil, found a direct relationship between soil temperature and bacterial numbers, showing that temperature can be a limiting factor under certain conditions. With normal arable soils, however, no such direct effect of temperature or moisture can be found[16] (see [Fig. 8]). It has even been found by Conn[11] that freezing of the soil may cause a marked increase in bacterial numbers. The erratic effects of temperature and moisture on the soil bacteria probably afford instances of a disturbance of the equilibrium between the bacteria and other components of the soil micro-population. Thus desiccation and freezing, though they harmfully affect the bacteria, may inhibit other micro-organisms to a greater degree, thus freeing the bacteria from competition. It is in the investigation of this equilibrium, and of the factors that can control it to our benefit, that the great advances in soil biology in the future are to be expected.

Fig. 8.—Effect of frost on the bacterial numbers in the soil. (After Conn.)

X-axis: Nov.-May

Y-axis (bottom): Temperature—Degrees C.

Y-axis (top): Bacteria—Millions per Gramme of Soil.

REFERENCES TO CHAPTERS II. AND III.

[1] Ashby, S. F., Journ. Agric. Sci., 1907, vol. ii., p. 35.

[2] Beijerinck, M. W., Centr. f. Bakt., 1900, Abt. II., Bd. 6, p. 1.

[3] Beijerinck, M. W., and Van Delden, A., Centr. f. Bakt., 1902, Abt. II., Bd. 9, p. 3.

[4] Bewley, W. F., and Hutchinson, H. B., Journ. Agric. Sci., 1920, vol. x., p. 144.

[5] Bonazzi, E., Journ. Bact., 1921, vol. vi., p. 331.

[6] Burrill, T. J., and Hansen, R., Illin. Exp. Sta., 1917, Bulletin 202.

[7] Christensen, H. R., Centralblatt. f. Bakt., 1915, Abt. II., Bd. 43, p. 1.

[8] Christensen, H. R., Centralblatt. f. Bakt., 1907, Abt. II., Bd. 17, pp. 109, 161.

[9] Christensen, H. R., and Larsen, O. H., Centralblatt. f. Bakt., 1911, Abt. II., Bd. 29, p. 347.

[10] Conn, H. J., Centralblatt. f. Bakt., 1910, Abt. II., Bd. 28, p. 422.

[11] Conn, H. J., Centralblatt. f. Bakt., 1914, Abt. II., Bd. 42, p. 510.

[12] Conn, H. J., Journ. Bact., 1916, vol. i., p. 187.

[13] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 137.

[14] Conn, H. J., Journ. Bact., 1917, vol. ii., p. 35.

[15] Cramer, E., Arch. f. Hyg., 1893, Bd. 16, p. 151.

[16] Cutler, W., Crump, L. M., and Sandon, H., Phil. Trans. Roy. Soc., 1923, Series B, vol. ccxi., p. 317.

[17] Doryland, C. J. T., N. Dakota Agr. Exp. Sta., 1916, Bulletin 116.

[18] Fabricius, O., and Feilitzen, H., Centr. f. Bakt., 1905, Abt. II., Bd. 14, p. 161.

[19] Fisher, R. A., Thornton, H. G., and Mackenzie, W. A., Ann. Appl. Biol., 1922, vol. ix., p. 325.

[20] Fred, E. B., and Hart, E. B., Wisconsin Agr. Exp. Sta. Research, 1915, Bulletin 35.

[21] Gainey, P. L., Journ. Agric. Research, 1918, vol. xiv., p. 265.

[22] Golding, J., Journ. Agric. Sci., 1905, vol. i., p. 59.

[23] Greaves, J. E., Soil Sci., 1916, vol. ii., p. 443.

[24] Greaves, J. E., Journ. Agric. Res., 1916, vol. vi, p. 389.

[25] Greaves, J. E., Soil Sci., 1920, vol. x., p. 77.

[26] Greaves, J. E., and Lund, Y., Soil Sci., 1921, vol. xii., p. 163.

[27] Greaves, J. E., and Carter, E. G., Journ. Agric. Research, 1916, vol. vi., p. 889.

[28] Groenewege, J., Arch. Suikerindust., 1913, Bd. 21, p. 790.

[28b] Green, H. H., Union of S. Africa Dept. Agr., Rept. of Director Vet. Res., 1918, p. 592.

[29] Hutchinson, C. M., Rept. Agr. Res. Inst. and Col. of Pusa, 1912, p. 85.

[30] Hutchinson, H. B., and Clayton, J., Journ. Agric. Sci., 1919, vol. ix., p. 143.

[30b] Hutchinson, H. B., and Richards, H. H., Journ. Min. Agric., 1921, vol. xxviii., p. 398.

[31] Hanzawa, J., Centr. f. Bakt., 1914, Abt. II., Bd. 41, p. 573.

[32] Hopkins, C. G., and Whiting, A. L., Ill. Agr. Exp. Sta., 1916, Bulletin 190, p. 395.

[33] Hoppe-Seyler, G., Ztschr. Phys. Chem., 1886, vol. x, pp. 201, 401; 1887, vol. xi., p. 561.

[34] Hesselmann, H., Skogsvårdsför. Tidskr., 1917, No. 4, p. 321.