CHAPTER X.
PHYSIOLOGICAL ACTIVITIES (Continued).
PUTREFACTION OF PROTEINS.
The word “putrefaction” is now restricted to the action of bacteria on the complex nitrogen-containing substances, proteins, and their immediate derivatives. The process is usually accompanied by the development of foul odors.
Bacteria make use of proteins chiefly as a source of nitrogen, but also as a source of carbon and other elements. Proteins contain nitrogen, carbon, hydrogen, oxygen, sulphur and frequently phosphorus. Some of the metals—potassium, sodium, calcium, magnesium, iron and manganese and the non-metal chlorine—are nearly always associated with them more or less intimately. Since these bodies are the most complex of natural chemical substances it follows that the breaking up of the molecule to secure a part of the nitrogen gives rise to a great variety of products.
There are marked differences among bacteria in their ability to attack this class of compounds. Some can break up the most complex natural proteins such as albumins, globulins, glyco-, chromo-, and nucleoproteins, nucleins and albuminoid derivatives like gelatin. The term saprogenic (σαπρος = rotten) is sometimes applied to bacteria which have this power. These proteins are large-moleculed and not diffusible, so that the first splitting up that they undergo must occur outside the bacterial cell. The products of this first splitting may diffuse into the cell and be utilized there. The bacteria of this class attack not only these proteins in the natural state or in solution, but also in the coagulated state. The coagulum becomes softened and finally changed into a liquid condition. The process when applied to the casein of milk is usually called “digestion,” also when coagulated blood serum is acted on. In the latter case the serum is more commonly said to be “liquefied” as is the case when gelatin is the substance changed. Most of these bacteria have also the property of coagulating or curdling milk in an alkaline medium, and then digesting the curd. A second class of bacteria has no effect on the complex proteins just mentioned but readily attacks the products of their first splitting, i.e., the proteoses, peptones, polypeptids and amino-acids. They are sometimes called saprophilic bacteria.
Other bacteria derive their nitrogen from some of the products of the first two groups, and still further break down the complex protein molecule. Under normal conditions these various kinds of bacteria all occur together and thus mutually assist one another in what is equivalent to a symbiosis or rather a metabiosis, a “successive existence,” one set living on the products of the other. The result is the complete splitting up of the complete protein molecule. A part of the nitrogen is built up into the bodies of the bacteria which are using it as food. A part is finally liberated as free nitrogen or as ammonia after having undergone a series of transformations many of which are still undetermined.
One class of compounds formed received at one time much attention because they were supposed to be responsible for a great deal of illness. These are the “ptomaines,” basic nitrogen compounds of definite composition—amines—some few of which are poisonous, most of them not. The basic character of ptomaines may be understood if they be regarded as made up of one or more molecules of ammonia in which the hydrogen has been replaced by alkyl or other radicals. Thus ammonia (NH3) may be represented as . The simplest ptomaine is , in which one H is replaced by methyl, methylamine, a gaseous ptomaine. With two hydrogens replaced by methyl, , dimethylamine, also a gas at ordinary temperature, is formed. Trimethylamine, , a liquid, results when three hydrogens are similarly replaced. All three of these occur in herring brine and are responsible for the characteristic odor of this material. Putrescin and cadaverin—tetramethylene—diamine, and pentamethylenediamine respectively—occur generally in decomposing flesh, hence the names. They are only slightly poisonous. One of the highly poisonous ptomaines is neurin C5H13NO or C2H3N(CH3)3OH = trimethyl-vinyl ammonium hydroxide. This is a stronger base than ammonia, liberating it from its salts. Numerous other ptomaines have been isolated and described. These bodies were considered for a long time to be the cause of various kinds of “meat poisoning,” “ice cream poisoning,” “cheese poisoning,” etc. It is true that they may sometimes cause these conditions, but they are very much rarer than the laity generally believe. Most of the “meat poisonings” in America are due, not to ptomaines, but to infections with certain bacilli of the Bacterium enteritidis group. Occasionally a case of poisoning by the true toxin (see Chapter XII) of Clostridium botulinum occurs, and in recent years has become entirely too common due to insufficient heating of canned goods. The boiling of such material will destroy this toxin. The safest rule to follow is not to eat any canned material that shows any departure from the normal in flavor, taste or consistency.
As ptomaines result from the putrefaction of proteins, so they are still further decomposed by bacteria and eventually the nitrogen is liberated either as free nitrogen or as ammonia.
Another series of products are the so-called aromatic compounds—phenol (carbolic acid), various cresols, also indol and skatol or methyl indol (these two are largely responsible for the characteristic odor of human feces). All of these nitrogen compounds are attacked by bacteria and the nitrogen is eventually liberated, so far as it is not locked up in the bodies of the bacteria, as free nitrogen or as ammonia.
The carbon which occurs in proteins accompanies the nitrogen in many of the above products, but also appears in nitrogen-free organic acids, aldehydes and alcohols which are all eventually split up, so that the carbon is changed to carbon dioxide or in the absence of oxygen partly to marsh gas.
The intermediate changes which the sulphur in proteins undergoes are not known, but it is liberated as sulphuretted hydrogen (H2S) or as various mercaptans (all foul-smelling), or is partially oxidized to sulphuric acid. Some of the H2S and the sulphur of the mercaptans are oxidized by the sulphur bacteria to free sulphur and finally to sulphuric acid.
Phosphorus is present especially in the nucleoproteins and nucleins. Just what the intermediate stages are, on whether there are any, so far as the phosphorus is concerned, in the splitting up of nucleic acid by bacterial action is not determined. The phosphorus may occur as phosphoric acid in such decompositions, or when the conditions are anaërobic, as phosphine (PH3), which burns spontaneously in the air to phosphorus pentoxide (P2O5), and water.13
The hydrogen in proteins appears in the forms above indicated: H4C, H3N, H3P, H2S, H2O and as free H. The oxygen as CO2 and H2O.
In the breaking down of the complex protein molecule even by a single kind of bacterium there is not a perfect descending scale of complexity as might be supposed from the statement that there result proteoses, peptones, polypeptids, amino-acids. These substances do result, but at the time of their formation simpler ones are formed also, even CO2, NH3 and H2S. It appears that the entire molecule is shattered in such a way that less complex proteins are formed from the major part, while a minor portion breaks up completely to the simplest combinations possible. A more complete knowledge of these decompositions will aid in the further unravelling of the structure of proteins. The presence or absence of free oxygen makes a difference in the end-products, as has been indicated. There are bacteria which oxidize the ammonia to nitric acid and the H2S to sulphuric acid. (See Oxidation, Chapter XI.) Bacteria which directly oxidize phosphorus compounds to phosphoric acid have not been described. It does not seem that such are necessary since this is either split off from nucleic acid or results from the spontaneous oxidation of phosphine when this is formed under anaërobic conditions.
Not only are proteins decomposed as above outlined, but also their waste products, that is, the form in which their nitrogen leaves the animal body. This is largely urea in mammals, with much hippuric acid in herbivorous animals and uric acid in birds and reptiles. These substances yield NH3, CO2 and H2O with a variety of organic acids as intermediate products in some cases. The strong odor of ammonia in stables and about manure piles is the everyday evidence of this decomposition.
Where the putrefaction of proteins occurs in the soil with moderate amounts of moisture and free access of air a large part of the products is retained in the soil. Thus the ammonia and carbon dioxide in the presence of water form ammonium carbonate; the nitric, sulphuric and phosphoric acids unite with some of the metals which are always present to form salts. Some of the gases do escape and most where the oxygen supply is least, since they are not oxidized.
The protein-splitting reactions afford valuable tests in aiding in the recognition of bacteria. In the study of pathogenic bacteria the coagulation and digestion of milk, the digestion or liquefaction of blood serum, the liquefaction of gelatin and the production of indol and H2S are those usually tested for. In dairy bacteriology the coagulation of milk and the digestion of the casein are common phenomena. Most bacteria which liquefy gelatin also digest blood serum and coagulate and digest milk, though there are exceptions. In soil bacteriology the whole range of protein changes is of the greatest importance.
The three physiological activities already discussed explain how bacteria break down the chief complex, energy-rich substances—carbohydrates, fats and proteins which constitute the bulk of the organic material in the bodies of plants and animals, as well as the waste products of the latter—into energy-free compounds like carbon dioxide, water, ammonia, nitric, sulphuric and phosphoric acids—mineralize them, as is frequently said. By so doing the bacteria act as the great scavengers of nature removing the dead animal and vegetable matter of all kinds which but for this action would accumulate to such an extent that all life, both on land and in the water, must cease. It is further to be noted that not only is all this dead organic matter removed; but it is converted into forms which are again available for plant growth. Carbon dioxide forms the source of the carbon in all green plants, hence in all animals; the sulphates and phosphates are likewise taken up by green plants and built up again into protein compounds; the ammonia is not directly available to green plants to any large extent but is converted by the nitrifying bacteria (Chapter XI) into nitrates which is the form in which nitrogen is assimilated by these higher types. Even the free nitrogen of the air is taken up by several kinds of bacteria, the symbiotic “root-tubercle bacteria” of leguminous and other plants, and some free-living forms, and made available. Hence bacteria are indispensable in nature, especially in keeping up the circulation of nitrogen. They are also of great service in the circulation of carbon, sulphur and phosphorus. Though some few kinds cause disease in man and animals, if it were not for the saprophytic bacteria above outlined, there could be no animals and higher plants to acquire these diseases.