The micro-organisms of the soil

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

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

CH₄ + 2O₂ = CO₂ + 2H₂O 220 Cal.

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

C₂H₆O + O₂ = C₂H₄O₂ + H₂O 115 Cal.

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

C₆H₁₂O₆ = 2C₂H₆O + 2CO₂ 50 Cal.

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

(1) Decomposition of Non-nitrogenous Compounds.

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

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

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

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.

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

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

(2) Ammonia Production.

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

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

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

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

(3) Nitrate Production.

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

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

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

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

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

KNO₂ + O = KNO₃ + 22 Cals.

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

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

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

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

S + 3O + H₂O = H₂SO₄ + 141 Cals.

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

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

(4) Anaerobic Respiration.

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

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

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

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