The micro-organisms of the soil

In the preceding chapter an outline has been given of the development of the study of soil protozoa, with especial reference to its qualitative aspects.

Here it is proposed to deal with the quantitative methods which have been devised for studying these organisms and the results obtained.

From the beginning great difficulty has been encountered in finding means for counting protozoa; and most of the early results have been obtained by the use of one of the following methods: (1) direct counts in a known volume of soil suspension by means of a microscope; (2) dilution method as used for counting bacteria, and suggested by Rahn, who made dilutions of the soil and determined, by examination at periodic intervals, the one above which protozoa did not grow; (3) Agar plating as used by Killer; (4) counting per standard loop of suspension as devised by Müller. Of these the two last have been little used, and for various reasons are now discarded by most workers. Direct methods have been used extensively in the United States by Koch[13] and others,[16] who claim to have got satisfactory results; they are, however, highly inaccurate and should be discontinued. The present writer[3] has shown that there exists a surface energy relationship between the soil particles and the protozoa, so that the two are always in intimate contact; thus rendering it impossible to count under the microscope the number of organisms in a given weight of soil suspension (Fig. 9). Further, in a clay soil, such as is found at Rothamsted, the clay particles alone make it very difficult to use such methods.

The demonstration of this surface energy relationship affords an effective rejoinder to the criticism made against Russell and Hutchinson’s hypothesis, viz., that soil protozoa must be very few in numbers, since it was impossible to see them on examining soil under the microscope.

The second or dilution method is the one, therefore, that has been most extensively developed.

Cunningham obtained concordant results in this way, and his method, modified by L. M. Crump, was as follows: 10 grams of soil were added to 125 c.c. of sterile tap-water and shaken for three minutes. This gives a 1 in 12·5 dilution. From it further dilutions were made until a sufficiently high one was obtained. Petri dishes, containing nutrient agar, were inoculated with 1 c.c. of each of the dilutions and incubated. At intervals covering 28 days the plates were examined and the presence or absence of protozoa on each recorded. In this way the approximate number of organisms per gram of soil could be found.

By methods essentially similar to this numerous counts have been made of the bacteria and protozoa in field soil and in partially sterilized soils. They were, however, inconclusive; thus, on the one hand, Goodey and several American observers, found no correlation between the numbers of protozoa and bacteria, while Miss Crump and Cunningham obtained evidence pointing to the reverse conclusion.

Such divergence of opinion was probably mainly due to two causes: firstly, that the time elapsing between the successive counts was too long, for it has been shown recently that the number of bacteria and protozoa fluctuate very rapidly; and secondly, the method was not completely satisfactory since only the total numbers of protozoa were considered, no means having been found of differentiating between the cystic and active forms. This was a particularly serious source of error for it is possible for soil to contain large numbers of bacteria and protozoa, of which a high percentage of the latter are in the form of cysts. A count made on such a soil would give results apparently opposed to the theory that protozoa act as depressors of bacteria.

This difficulty has, however, been overcome by a further modification of the dilution method, and it is now possible in any soil sample to count both the numbers of cysts and active forms. Also a further advance in technique has made it possible to recognise and enumerate the common species of protozoa, instead of simply grouping them as Ciliates, Flagellates, and Amœbæ, as was done in the past.[7]

Briefly the method consists in dividing the soil sample into equal portions (usually 10 grams each) one of which is counted, thus giving the total numbers of protozoa (active + cystic) present. The second portion is treated over-night with 2 per cent. hydrochloric acid, the HCl used being B.P. pure 31·8 per cent. Previous experiments have shown that such acid kills all the active protozoa, leaving viable the cysts. The number of cysts is therefore found by counting this treated sample, and the number obtained subtracted from the total gives the active number.[F]

The discovery of this method at once puts into the hands of the investigator a much more efficient instrument for studying the activities of the soil micro-population, especially since at a slightly later date Thornton’s method for counting bacteria was devised.

Early in 1920 Cutler and Crump[6] decided to make a preliminary survey of the protozoon and bacterial populations of one of the Rothamsted field soils (Broadbalk dunged plot). The investigation was continued for 28 days, daily soil samples being taken. The results so obtained showed that an extended investigation of the micro-population of field soil would yield interesting and important results, especially as it was evident that certain views held by soil biologists required modification.

In July of the same year, therefore, it was decided to start an extended investigation of the soil protozoa and bacteria. The method adopted was to make counts of the numbers of bacteria and of six[G] species of protozoa in soil samples taken daily direct from the field (Barnfield dunged plot) and by statistical methods to correlate these counts one with another and with the data for external conditions. Observations at shorter periods than 24 hours could not be made, but it was found possible to continue the research for 365 days.[7]

The number of all the organisms showed large fluctuations of two kinds, daily and seasonal. The size of the changes that took place within so short a period as 24 hours was, perhaps, the most surprising fact that the experiment revealed. Thus three consecutive samples gave 58·0, 14·25 and 26·25 millions of bacteria per gram respectively; and the changes exhibited by any of the species of protozoa were at times even larger. This fact is of extreme importance, since in the past it has always been assumed that the number of bacteria remained fairly constant from day to day, and investigators have not hesitated to separate the taking of soil samples by long periods. It is now obvious that such a procedure is of little use for comparative purposes (Fig. 10).

It has usually been assumed that the changes in the external conditions markedly affect the density of the soil population. To test this the environmental conditions—temperature, moisture content and rainfall were examined; but contrary to all expectation no connection could be traced between any of these and the daily changes in numbers of any of the organisms investigated, and moreover the species of protozoa appeared in the main to be living independently of one another.

It is difficult to believe that external conditions are as inoperative as appears from the above; and in view of the known complexity of the soil it is possible that further research will show that certain combinations of external conditions are important agents in effecting the changes.

In the case of the bacteria, however, the agent causing the fluctuations is mainly the active amœbæ. This was well shown during the year’s count, for with only 14 per cent. of exceptions, 10 per cent. of which can be explained as due to rapid excystation or encystation, a definite inverse relationship was established between the active numbers of amœbæ and the number of bacteria (Figs. 11 and 12). Thus a rise from one day to the next in the amœbic population was correlated with a fall in the numbers of bacteria and vice versa. It must not be supposed that the flagellates are of no account in this process; some species, known to eat bacteria, undoubtedly induce slight depressions, but, owing to their small size, any effect is masked by the greater one of the amœbæ.

These experiments seem to admit of no doubt that in field soil the active protozoa are instrumental in keeping down, below the level they might otherwise have attained, the numbers of bacteria; but a further proof of this contention ought to be obtained by inoculation experiments. It should be possible, by inoculating sterile soil with bacteria alone and with bacteria plus protozoa, to demonstrate fluctuations in bacterial numbers in the latter, while those of the former remained constant. This admittedly crucial test has often been tried, but owing to difficulties in technique, etc., has always failed. Recently, however, by using new methods confirmatory results have been obtained.[5]

Ordinary field soil was sterilised by heat at 100° C. for 1 hour on four successive days; it was then divided into equal portions, one of which was inoculated with three known species of bacteria, and the other inoculated with the same number of bacteria plus the cysts of the common soil amœba Nægleria gruberi. The numbers of bacteria in each soil were counted daily for the first eight days and then daily from the 15th to the 21st day after the experiment started. The results are given in Table VII. and Fig. 13.

TABLE VII.

It will be noted that the numbers of bacteria in each soil rose steadily until a maximum was reached 6-8 days after inoculation. This is in accordance with expectation, since the reproductive rate of bacteria is much greater than that of the amœbæ, which, until their active forms are numerous, will not exert any appreciable influence on the bacterial population. Further, since the protozoa were inoculated as cysts an appreciable time would elapse before excystation took place. The last seven days of the experiment are of particular interest. During this period the amœbæ were known to be active in the soil, and were depressing the bacterial numbers, for in the control (protozoa-free) soil the variation in numbers was within experimental error, while in the other soil the variations were considerable and well outside experimental error. In fact the variations were comparable with those found from day to day in untreated field soils. Finally, the experiment shows that the bacteria in protozoa-free soil are able to maintain high numbers for a longer period than those living in association with protozoa.

Seasonal Changes.

Superimposed on the daily variations in numbers there are seasonal changes, as is clearly shown when fourteen day averages are made of the numbers for each species. Bacteria have long been known to show autumn and spring rises, but recent research has demonstrated that the protozoan population also rises to a maximum at the end of November, with a less marked spring rise at the end of March and beginning of April (Figs. 14 and 15).

It has sometimes been claimed that the numbers of soil organisms are closely linked with the soil moisture, but no support for this view was found during the course of the experiment. Similarly, as in the case of the daily variations, no connection could be traced between the seasonal changes and any of the external conditions considered.

It is interesting to note, however, that the seasonal variations in the numbers of soil organisms is very similar to those recorded for many aquatic organisms. Miss Delf,[8] for instance, found that in ponds at Hampstead the algæ are most numerous in spring and again in the autumn, and like changes are recorded in British lakes by West and West[25] and in the Illinois river by Kofoid.[14]

It is difficult to resist the conclusion that these annual variations are produced by similar causes, from which it follows that the increase in the numbers of protozoa in the soil is not wholly conditioned by an increased food supply—the bacteria—for the algæ are not dependent on such a form of nourishment. This is substantiated by the fact that the numbers of protozoa, except those of Oicomonas, rose during March, whereas the corresponding increase in the bacteria was delayed till the early part of April.

Owing to the variations in the numbers of both protozoa and bacteria, little reliance can be placed on figures obtained from an isolated count, since on one day the total numbers of flagellates may be nearly 2,000,000 per gram and drop by more than half this figure in 24 days. It is certain, however, that the numbers recorded in the past are much too low, since the total flagellate and amœbæ species were lumped together in two groups. Some idea of the size of the soil population can be obtained, nevertheless, by using the fourteen-day averages mentioned above. In Table VIII. are tabulated the average total numbers of flagellates, and amœbæ for the two periods of the year when the population was at its maximum and minimum respectively. An endeavour has also been made to strike a rough balance sheet as to the amount of protoplasm represented by protozoa and bacteria in a ton of soil. For this purpose it has been assumed that the organisms have a specific gravity of 1·0 and are spheres of diameters, 6μ for the flagellates, 10μ for the amœbæ, and 1μ for the bacteria; and that they are uniformly distributed through the top nine inches of soil. The top nine inches of soil is taken as weighing 1000 tons.

TABLE VIII.

It must be remembered that the above figures are minimum ones, as many species of bacteria and protozoa, known to occur in the soil, are not included in the statement owing to their not appearing on the media used for counting purposes.

Before leaving the discussion of daily variations in numbers of protozoa, reference must be made to the flagellate species. As already mentioned, their active numbers fluctuate rapidly, and for the most part entirely irregularly. One species, however, Oicomonas termo, is characterised by possessing a periodic change; high active numbers on one day being succeeded by low, which are again followed by high on the third day. This rhythm was maintained, with few exceptions, for 365 days (Fig. 16), and has been shown to take place in artificial culture kept under controlled laboratory conditions (Fig. 17).

It was thought that an explanation of this phenomenon might be found in alternate excystation and encystation, since the latter is a constituent part of the animals’ life history (see p. 73). This, however, does not hold, for the cyst curve is not the inverse of that of the active; and, moreover, statistical treatment demonstrated that cyst formation is wholly unperiodic in character.

An explanation must therefore be sought in the changes in the organisms during the active period of their life, and the deduction can be drawn that, increased active numbers tend to be followed by death, conjugation, or both, while decreases in the active numbers are followed by rises in total numbers, i.e., reproduction, and this rhythmically.

This somewhat surprising conclusion appears to hold, in a lesser degree, for other soil protozoa, and is of sufficient importance to warrant further research. The direction in which this is being pursued is by a study of the reproductive rates of pure cultures of certain ciliates and flagellates under varying external conditions. Space does not admit of adequate discussion of this problem, but the results already obtained justify the view that such lines of work will elucidate some of the baffling problems of soil micro-biology.

Soil Reaction.

The development of the artificial fertiliser industry has in many ways revolutionised farm practice, with the inevitable result that new problems have arisen, not the least of which are biological in character.

If, as seems to be indubitable, the micro-organisms of the soil are of importance to soil fertility, it is necessary for us to know in what way this population is affected by the application of fertilisers, and a start has been made by investigating the effects of hydrogen ion concentration on soil protozoa. Much has already been written concerning this question, but almost entirely on results obtained in artificial cultures. It is always dangerous to argue from the artificial to the natural environment of organisms and particularly so in respect to the soil. Also, as Collett has shown, the toxic effects of acids are probably not entirely a function of the hydrogen ion concentration, but that the molecules of certain acids are in themselves toxic, an action which can, however, be diminished by the antagonistic powers of many substances such as NaCl.

In this laboratory S. M. Nasir, by unpublished work, has shown that the limiting value on the acid side for Colpoda cucullus was P_H 3·3; for a flagellate (Heteromita globosus), 3·5; and for an amœba (Nægleria gruberi), 3·9.

Also Mlle. Perey, investigating the numbers of protozoa in one of the Rothamsted grass plots of P_H 3·65, found a total of 13,600 protozoa, of which 90 per cent. were active.

The tolerance, therefore, of these organisms to varying external conditions is greater than has formerly been supposed, a conclusion which is becoming more evident from the researches mentioned in Chapter IV. on soils from different parts of the world.

Protozoa and the Nitrogen Cycle.

In partially-sterilised soil from which protozoa were absent Russell and Hutchinson obtained an increased ammonia production, a result also obtained by Cunningham. Hill, on the other hand, concluded that protozoa have no effect on ammonification, but his technique is open to criticism.

Lipman, Blair, Owen and McLean’s work[17] contains many figures obtained by adding dried blood, tankage, soluble blood flour, cottonseed meal, soy-bean meal, wheat flour, corn meal, etc., to soil. It is difficult to understand how accurate results could be expected when, to an already little understood complex substance, such as soil, is added a series of substances whose effects are practically unknown.

Free nitrogen-fixation in soils is an important process, more especially in soils of a light sandy nature, from which crops are taken year after year without any application of manure. The effect of protozoa on the organisms causing this process has in the past received little attention. Recently, however, Nasir[20] has studied the influence of protozoa on Azotobacter, both in artificial culture and in sand. From a total of 36 experiments done in duplicate or triplicate, 31 showed a decided gain in nitrogen fixation over the control, while only 5 gave negative results.

As might be expected, the fixation figures varied from culture to culture, the highest recorded being 36·04 per cent. above the control and this in a sand culture (Fig. 18). Reference to the details of the experiments shows that the criticisms made against similar work done in the past do not hold here, and one must conclude that Azotobacter is capable of fixing more atmospheric nitrogen in the presence of protozoa than in their absence.

At present it is impossible to say how this occurs, but it is highly improbable that the protozoa are themselves capable of fixing nitrogen. A more likely explanation is that the protozoa, by consuming the Azotobacter, kept down the numbers, and transfer the nitrogen to their own bodies. This will tend to prevent the bacteria from reaching a maximum density, and reproduction, involving high metabolism, will be maintained for a longer period than would have otherwise occurred. This and other possible explanations, are being tested.

Little has been said regarding the application of protozoology to the question of soil partial sterilisation. As already pointed out, in the past much work has been done, but the results were conflicting. In view, however, of our recently acquired knowledge of the life of protozoa in ordinary field soil, most of the early experiments require repeating. A beginning has already been made, but the work is not sufficiently advanced to warrant discussion.

What is urgently needed, however, is to increase our knowledge of the general physiology of these unicellular animals. Until we know what are the inter-relationships between the members of the micro-organic population of normal soil it is almost impossible to hope that means will be devised by which they can be controlled.

At present we are almost entirely ignorant of the simplest of physiological reactions, such as the exact effect of various inorganic salts found in the soil.

Also some experiments in Germany and the States indicate that amœbæ are selective as regards the bacteria they ingest. If this is substantiated it may prove of importance to economic biology.

It has been shown that the flagellates occur in the soil in large numbers, and many of them feed on bacteria. It is probable, however, that certain of them feed saprophytically and must therefore exert some influence on the soil solution, though what this may be is entirely unknown.

Finally, as Nasir has shown, the protozoa play a part in the complicated nitrogen cycle, and work of this type needs extending.

Such, then, are a few of the outstanding problems that confront the soil protozoologist; but he must always remember that the organisms he studies are but a small fraction of the total, and that any influence affecting one part of the complex will be reflected in another. As Prof. Arthur Thomson said in his Gifford Lectures, “No creature lives or dies to itself, there is no insulation. Long nutritive chains often bind a series of organisms together in the very fundamental relation that one kind eats the others.” Such nutritive chains obtain in the soil as markedly as in other haunts of living creatures.

SELECTED BIBLIOGRAPHY.

* Papers giving extensive bibliographies.