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 drymatter:—
Composition of Pfeiffer’s Bacillus (Cramer).
About 65-70 per cent. of the dry matter of bacteria consists of protein.
The biggest constituent of the dry matter of bacteria is therefore carbon. In the soil, bacteria find an abundance oforganic 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 CO2of the soil atmosphere. The sources from which these special groups obtain the necessary energy to accomplish this, we have already considered.
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.
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 intotheir 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 nameClostridium pasteurianum. In 1901 the investigations of Beyerinck, in Holland, led to the important discovery of a group of large aerobic organisms, which he namedAzotobacter. 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,Azotobacterhas attracted the greatest attention of workers. The evidence seems to be consistent with the view thatAzotobacteris of importance in the soil. Thus the distribution ofAzotobacterwould 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 itsAzotobacterflora. Thus Lipman and Waynick[46]found that if soil from Kansas were removed to California, its power to produce a growth ofAzotobacter, 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 byAzotobacterin 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 betweenAzotobacterandClostridium pasteurianum, the former absorbing oxygen from the surroundings, and thus creating a suitable anaerobic environment for theClostridium.
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 thatAzotobacterin mixed cultures fixed more nitrogen than in pure cultures.Granulobacter, an organism which they found to be commonly associated withAzotobacterin 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 ofAzotobacterwere grown together. A symbiosis betweenAzotobacterand green algæ has been described, and will be furtherdiscussedby 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 byAzotobacterin 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 ofAzotobacter, and of the cellulose attackingSpirochæta 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 hisClostridiumassimilated 2-3 mgs. of nitrogen per gram of sugar consumed. Lipman found thatAzotobacterfixed 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.
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.
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 ofAzotobacterin 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 lesswasteful 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,Azotobacterwill 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 ofAzotobactersuggests that these may be a step in the process, but at present the data are too inconclusive to form a basis for theorising.
Azotobacteris very rich in phosphorus, an analysis of the surface growth inAzotobactercultures, made by Stoklasa, giving about 60 per cent. of phosphoric acid in the ash. In cultures it has been found that a considerable amount ofphosphate 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 forAzotobactergrowth.
Azotobacteris 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 thatAzotobacteroccurred in soils having an acidity not greater than PH6·0, andChristensen,[7],[9]in Denmark, has found a close association between the occurrence ofAzotobacterin 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 inChapter 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 ofthese 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. (AfterHutchinsonandBewley.)Motile RodsVacuolated Stage“Swarmers”“Bacteroids”“Pre-swarmers”
Fig. 6.—Bacillus radicicola.Stages in the life cycle. (AfterHutchinsonandBewley.)
Motile RodsVacuolated Stage“Swarmers”“Bacteroids”“Pre-swarmers”
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 ithas beenclaimed[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 thanAzotobacter. The limiting degree of acidity has been found to vary among different varieties of the organism from PH3·15 to PH4·9.
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 infectionis 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 culturesby 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.
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.
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 thenumbers 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 contain1⁄250,000th 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 themethod 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 (seeTable 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 supplementarymethods 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,Nitrosomonasappears 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 compositionis 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 (seeTable 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, takenin two rows 6 feet apart, showed similar fluctuations (seeFig. 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.