CHAPTER II.. Soil Bacteria.

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 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, 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, 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.

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂ 50 Cal.

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 and Hoppe-Seyler, 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, 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, and by Nabokich and Lebedeff, while Söhngen 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, 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, 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.

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.

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, 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:--

H O | // H--C--C + 3O = 2CO₂ + H₂O + NH₃ + 152 Cal. | \ NH₂ OH

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.

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, 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, 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., 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:--

2·25 grams ammonium sulphate, 2·0 „ sodium chloride, 1·0 „ magnesium carbonate, to the litre of well water.

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 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 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 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 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, 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.