molecular structure of protein matter is analyzed into amino compounds of simpler and simpler composition, until nitrogen finally appears in the form of ammonia. We know little of the chemistry of the early stages of protein decomposition. The process seems hopelessly complicated from the intricate structure of the molecule. Eventually from the seething caldron of molecular disintegration there appear simpler substances, such as proteoses, peptone, ptomains, amins, leucin, and tyrosin, and other amino substances, as well as organic acids, indol, skatol, phenol, and finally sulphuretted hydrogen, mercaptan, carbonic acid, and ammonia. One of the final products of the process is carbon dioxid, part of which passes into the atmosphere and part of which is retained in the soil as carbonates of alkalies or alkaline bases. The

FIG. 93. THE NITROGEN CYCLE IN DIAGRAMMATIC VERTICAL SECTION.

ammonia, as such, cannot be used by plants. Some of it may escape into the atmosphere, but for the most part it is retained in the soil as ammonium chlorid or ammonium carbonate. In the soil the ammonia is oxidized by the action of nitrifying bacteria into nitrates. This nitrifying action of bacteria, elucidated by Winogradski in 1888, was one of the brilliant discoveries in bacteriology. Through his work and that of later workers, it is now known that this process is usually accomplished in two distinct steps. In the first stage the ammonia is oxidized to nitrous acid. This is done by the nitrosobacteria. These nitrous or nitrite bacteria were called by Winogradski nitrosomonas and nitrosococcus. It is now known that a large number of microorganisms belong to this group. The nitrites exist in the soil probably as salts of potassium and sodium. They remain as the lower oxid a very short time and, therefore, never accumulate, and are never found in any large

amount for they are unstable and readily oxidized to nitrates. The special nitric or nitrate bacteria (nitrobacter) were first accurately described by Winogradski. The nitrates are stable and represent the final stage of the mineralization of nitrogenous matter. In certain arid parts of the world large deposits of nitrates (KNO3, saltpeter) are found as the result of the nitrification of bird excrement (guano), which is rich in available nitrogen. These collections do not occur in places. where there is enough rain to carry away the readily soluble nitrates.

Ordinarily the nitrates go into solution in the soil moisture and are either taken up by the roots of plants or are washed away in the ground water. In a sanitary analysis of water taken from the soil the presence of nitrates and nitrites, therefore, has a special significance. If nitrites are found in soil water it indicates pollution and signifies active bacterial action and the presence of organic matter. Nitrates in soil water, without nitrites, are an index of past pollution (see Water Analysis).

In 1886 Gayon and Dupetit described two organisms, B. denitrificans a and 6, capable of completely reducing nitrates. Many bacteria have this power of denitrification, a sort of reversible process by which nitrates are reduced to ammonia. This is characteristic of very many of the well-known microorganisms, such as the colon group, pyocyaneus, subtilis, and other soil bacteria. Denitrification, however, does not occur to any notable extent in a well-ventilated soil.

In plant metabolism the nitrates are used to build up new protein. Certain plants get some of their nitrogen through the bacterial tubercles on their roots, which have the power of fixing the free nitrogen of the air. These small nodules are abundant on the roots of various leguminous plants (peas, clover, etc.). Pure cultures of the legume or nitrogen fixing bacteria, such as Bacillus radicicola of Beyerinck, may be obtained from these root tubercles.

It should be noted also that certain bacteria (azobacter) have the ability to fix the free nitrogen of the air independently of plant life and may grow under either aerobic or anaerobic conditions. One of the first known of this group was an anaerobe described by Winogradski in 1895 and named by him Clostridium pasteurianus.

It will be noted that in the nitrogen cycle all the essential steps from proteolysis to mineralization of the organic matter, nitrification, oxidation, and reduction, as well as the fixation of free nitrogen from the atmosphere, are all the result of bacterial action. Each stage of the complex process is specific, in the sense that it requires a particular species or group of bacteria to affect the result, and also specific in the sense that special conditions of environment are necessary for its action to take place.

It is important to remember that practically the entire cycle takes place upon the surface and in the upper layers of the soil. A few

feet below the

surface of an undisturbed area the soil contains few or no bacteria. Carcasses buried deep, or sewage placed too far below the surface, do not profit by the nitrogen cycle in its entirety, and under such circumstances incomplete nitrification takes place. Nature's method of disposing of dead wastes is thereby defeated, and pollution of the soil and infection of the ground water may result.

The Carbon Cycle.-Carbohydrates, such as cellulose, starch, sugars, and similar constituents of vegetable and animal matter, are fermented, with the formation of carbon dioxid, alcohol, and various organic acids. The carbon in carbohydrates passes through a series of changes, which may be regarded as the carbon cycle. The carbon dioxid resulting from fermentation unites with water in the plant life, and under the action of chlorophyll and sunlight is again synthetized to starch and sugars.

The fermentation of the carbohydrates is also due to the action of microorganisms. In a mixture containing both carbohydrates and protein, as a rule, the bacteria act upon the carbohydrates first. In other words, the putrefaction of protein is delayed or hindered by the presence of fermentable carbohydrates. For this reason the disposal of sewage containing wastes from breweries is difficult.

Fats are also attacked by bacteria, with the consequent production of acids. The hydrocarbons are broken down with more difficulty than either the carbohydrates or protein. An excessive amount of fat in sewage always gives trouble on a filter. For instance, the drainage from a wool-scouring mill containing lanolin and the discharges from slaughter houses and the wastes from creameries and cheese factories containing animal fat present special problems in sewage disposal.

CHAPTER II

THE SOIL AND ITS RELATION TO DISEASE

Bacteria in Soil.-Countless millions of bacteria occur in the upper few inches of the soil. The enormous overgrowth of bacteria in the upper layers of the soil gives it the sticky, moist feeling which rich soils possess. The odor of the soil, such as that which is particularly noticed after a rainstorm, is due in large part to Cladothrix odorifera and other organisms which are commonly found in the soil. Few bacteria are found in an undisturbed soil below a depth of 4 to 6 feet. A sand bed used for filtering sewage shows a similar vertical distribution of bacteria. Below six feet the statement is made that the soil is usually sterile. This is not strictly true, but the numbers are much diminished and bacterial activity has practically ceased. As a rule, living bacteria are not obtained from samples of soil obtained 10 to 12 feet below the surface, except in soils with large pores or crevices, or in cases where the bacteria have been carried by burrowing animals. It is exceedingly difficult to determine the number of bacteria in the soil, as so many of them are anaerobes and vast hordes belong to the nitrifying groups, which grow only upon selective media. The soil is also the home of other species, requiring special conditions for growth in artificial culture media.

Of the ordinary bacteria that grow upon the usual laboratory media Houston found an average of 100,000 per gram in an uncultivated sandy soil, 1,500,000 per gram in a garden soil, and 115,000,000 per gram in a sewage soil. Peaty soils have smaller numbers. The actual numbers must be vastly greater, for many microorganisms in the soil do not grow upon the common media. In fact, the soil is the home of the greatest number and variety of bacteria found anywhere. It is the bacteria in the upper layers of the soil that make it resemble a living gland. Each particle of earth is coated with a zoogleal envelope. The sand and mineral particles form the supporting structures, the coating of bacteria corresponds to the glandular epithelium, and the interspaces between the particles are the capillary and lymph channels.

Most of the bacteria in the soil are saprophytes. The microorganisms pathogenic for man do not find conditions favorable for growth. and development in the soil. For the most part the tempera

ture is too low; further, they are crowded out by the overgrowth of the saprophytes. Koch has demonstrated that anthrax and other pathogenic bacteria may be grown in sterile soil, but cannot be grown in unsterilized soil, that is, in living soil. They die in the struggle for existence. Experiments have shown that the soil of graveyards contains no more bacteria than the corresponding soil in the same locality, and is noticeable by the absence of pathogenic microorganisms. The soil often contains the bacteria (or their spores) of certain wound infections, such as malignant edema, anthrax, B. aerogenes capsulatus, and tetanus. The relation of the soil to typhoid, cholera, dysentery, hookworm disease, Cochin-China diarrhea, and other infections will be discussed presently.

The function of the bacteria in the soil may best be understood by studying the fate of organic matter polluting the soil and the processes which accomplish its purification (see Nitrogen Cycle, page 676).

Pollution of the Soil.-The soil is capable of disposing of great quantities of organic matter. However, if it is overburdened it remains polluted and may endanger health through contamination of the drinking water and in other ways. It is not only the amount but the kind of pollution, and also the manner of its disposal, that plays a very important part. It must first of all be remembered that the purifying action of the soil is largely dependent upon bacteria, and that this action takes place almost solely in the upper layers. If carcasses are buried deeply, or if sewage is allowed to enter the soil at several or more feet below the surface, the process of purification is long delayed or checked. A leaky cesspool or broken drain which discharges its contents into the soil at a depth of 5 feet or more may seriously pollute the ground water, whereas the same material placed upon or just beneath the surface may be entirely mineralized and all infection destroyed before it reaches the depth of 5 feet. Vegetable matter in a water-logged soil undergoes a partial and unusual decomposition into muck or peat. Trees buried deeply, where bacterial action is practically absent, remain for many hundreds of years practically unchanged. Many factors retard the purifying action of the soil. Among these the temperature and moisture and absence of oxygen predominate.

When organic matter falls upon the soil it is consumed and digested by the hungry earth. Without this property the surface of the earth would long ago have become clogged with vegetable and animal matter. Albuminous substances are dissolved by the action of the proteolytic bacteria, and converted into simpler chemical compounds. The intermediate products of protein putrefaction are exceedingly complex. For our present purposes it is sufficient to know that ultimately the nitrogen is largely converted into ammonia and the carbon into carbon dioxid. The ammonia is then oxidized by the action of nitrifying