CHAPTER II.SOIL BACTERIA.A.Occurrence and Methods of Study.
To understand the development of our knowledge of soil bacteria, it must be remembered that bacteriology is under the disadvantage that it started as an applied science. Although bacteria were first seen by Leeuwenhoeck about the middle of the seventeenth century, and some of their forms described by microscopists of the eighteenth and early nineteenth centuries, it was only with the work of Pasteur on fermentation, and of Duvaine, Pasteur, and their contemporaries on disease bacteria, that bacteriology may be said to have started. From the outset, therefore, attention has been directed mainly to the bacteria in their specialised relationship to disease or to fermentation and similar processes. As a result, little research was done on the pure biology of the bacteria, so that even now many of the most fundamental and elementary problems concerning them are quite unsolved.
In their work on fermentations and disease bacteria, the earlier workers were assisted by the fact that under both sets of conditions the causative bacteria exist, as a rule, either in practically pure culture, or else in preponderating numbers. The study and elucidation of such a mixed micro-population as exists in the soil, became possible only when methods had been devised for isolating the different kinds of bacteria, and thus studying them apart from each other. It was the development of the gelatine plate method of isolating pure cultures by Koch[36]in 1881 that made the study of the soil bacteria practicable. The plating methodopened 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 whenit 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.
Culture 15 hours old.Culture 3 days old.
Fig. 1.—Change in appearance, in culture, of a cresol decomposing bacterium.
The inadequacy of mere morphology as a basis for describing bacteria led to the search for diagnostic characters, based on the biochemical changes that they produced in their culture media, and the appearance of their growth in the mass on various media. These characters unfortunately have also proved to be very much influenced by the exact composition of the medium and other conditions of culture.Recently an attempt has been made by the American Society of Bacteriologists to standardise the diagnostic characters used in describing bacteria, and also the media and cultural conditions under which they are grown for the purpose of description. The need for such precautions, however, was not sufficiently realised by the early workers, many of whose descriptions cannot now be referred to any definite organism.
The large number of organisms found in the soil, and the difficulty and labour of adequately describing them, is such that even now we have no comprehensive description of the common soil bacteria that appear on gelatine platings. A careful study based on modern methods of characterisation has been made of certain selected groups of bacteria, and it is hoped that the laborious systematic work of describing the common forms will gradually be completed.
Several attempts have been made to classify the bacteria that appear commonly on gelatine platings. This work was commenced by Hiltner and Stormer in Germany, and continued by Chester, Harding, and Conn in America.Conn[10],[14]found that the common organisms fell into the following maingroups:—
(1) Large spore-forming bacteria, related toBacillus subtilis, which form about 5-10 per cent. of the numbers. He adducedevidence[12],[13]that these organisms exist in the soil mainly as spores, so that they may not form an important part of the active soil population.
(2) Short non-sporing organisms, related toPseudomonas 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 arespecial 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 ofBacillus subtilis,B. amylobacter,B. fluorescens,B. caudatus, andB. 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 theBacillus amylobactergroup, 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-fastB. 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 ofBacillus 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 fixingAzotobacteris 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.