CHAPTER XI.
PHYSIOLOGICAL ACTIVITIES (Continued).
PRODUCTION OF ACIDS.
The production of organic acids has been sufficiently discussed in preceding chapters. It should be noted that not only these in great variety are produced by bacteria but that under certain conditions mineral acids, such as nitric, sulphuric and phosphoric may be formed (see Oxidation, p. 114). Acid production is of great value in the identification of bacteria in dairy and soil work and in connection with certain types of pathogenic bacteria.
GAS PRODUCTION.
It will be sufficient merely to enumerate collectively the various gases mentioned in preceding paragraphs and to state that those commonly observed in the study of pathogenic bacteria are the first six mentioned. Most of them come in in dairy work either in the study of bacteria causing milk and cheese “failures” or as affecting the flavors of butter or cheese. In the study of soil organisms, any or all of them are liable to be of importance. The gases are: CO2, H, CH4, N, NH3, H2S, gaseous mercaptans, gaseous ptomaines, volatile fatty acids, ethereal salts or esters and others, both of pleasant and of foul odor, but of unknown composition.
PRODUCTION OF ESTERS.
The production of esters, as mentioned in Chapters IX and X, of various alcohols and aldehydes are activities which are sometimes of value in the study of bacteria, but need not be further discussed.
PRODUCTION OF “AROMATIC” COMPOUNDS.
These have been mentioned in discussing the putrefaction of proteins, as indol, skatol, phenol and various cresols. Of these only the first is ordinarily tested for in the study of bacteria, though others of the group become of value in certain special cases.
PHOSPHORESCENCE OR PHOTOGENESIS.
This is a most interesting phenomenon associated with the growth of some bacteria. The “fox fire” frequently seen on decaying wood which is covered with a slimy deposit is most commonly due to bacteria, though also to other fungi. Phosphorescent bacteria are very common in sea water, hence they are frequently found on various sea foods, especially when these are allowed to decompose, such as fish, oysters, clams, etc. The light is due to the conversion of the energy of unknown easily oxidizable compounds directly into visible radiant energy through oxidation without appreciable quantities of heat. The light produced may be sufficient to tell the time on a watch in absolute darkness, and also to photograph the growths with their own light, but only after several hours’ exposure (Fig. 72). None of the phosphorescent bacteria so far discovered produce disease in the higher animals or man.
PRODUCTION OF PIGMENT OR CHROMOGENESIS.
One of the most striking results of bacterial activity is this phenomenon. The particular color which results may be almost any one throughout the range of the spectrum, though shades of yellow and of red are of more frequent occurrence.
In the red sulphur bacteria the “bacteriopurpurin” which they contain appears to serve as a true respiratory pigment in a manner similar to the chlorophyl in green plants, except that these bacteria oxidize H2S in the light as a source of energy instead of splitting up CO2. The red pigment produced by certain bacteria has been shown to have a capacity for combining with O resembling that of hemoglobin, and some investigators have believed that such bacteria do store O in this way for use when the supply is diminished. With these few exceptions the pigments seem to be merely by-products of cell activity which are colored and have no known function.
The red sulphur bacteria above mentioned and one or two other kinds retain the pigments formed within the cell. Such bacteria are called chromophoric as distinguished from the chromoparic bacteria whose pigment lies outside the cell.
The chemical composition of no bacterial pigment has been determined up to the present. Some are soluble in water, as shown by the discoloration of the substances on which they grow. Others are not soluble in water but are in alcohol, or in some of the fat solvents as ether, chloroform, benzol, etc. These latter are probably closely related to the lipochromes or “fat colors” of higher plants and animals. Attempts have been made to render the production of pigments a still more reliable means of identification of species of bacteria through a careful examination of the spectra of their solutions, but such study has not as yet led to any valuable practical results.
The production of pigment depends on the same general factors which determine the growth of the organism but does not necessarily run parallel with these. It is especially influenced by the oxygen supply (only a very few organisms are known which produce pigment anaërobically—Spirillum rubrum is one); by the presence of certain food substances (starch, as in potato, for many bacteria producing yellow and red colors; certain mineral salts, as phosphates and sulphates, for others); by the temperature (many bacteria cease to produce color at all if grown at body temperature, 37°—Erythrobacillus prodigiosus—or if grown for a longer time at temperatures a few degrees higher).
REDUCING ACTIONS.
Reduction of nitrates to nitrites or to ammonia or even to free nitrogen is brought about by a great many different kinds of bacteria. In many instances this phenomenon is due to a lack of free oxygen, which is obtained by the bacteria from these easily reducible salts. In other cases a portion of the nitrogen is removed to be used as food material in the building up of new protein in the bacterial cell. This latter use of the nitrogen of nitrates by bacteria might theoretically result in considerable loss of “available nitrogen” in the soil as has actually been shown in a few experiments. The reduction of nitrates as above mentioned would also diminish this supply, but probably neither of these results has any very great practical effect on soil fertility. The building up of protein from these mineral salts by bacteria in the intestines of herbivorous animals has been suggested by Armsby as a considerable source of nitrogenous food, and this suggestion appears possible.
The liberation of nitrogen from nitrates or nitrites, either as free nitrogen or as ammonia, is spoken of as “dentrification,” though this term was formerly applied to such liberations, from compounds of nitrogen generally even from proteins.
Certain bacteria may also reduce sulphates and other sulphur compounds to H2S, a phenomenon frequently observed in sewage and likewise of importance in the soil. It is possible that phosphates may be similarly reduced.14 Further and more careful study of the reducing actions of bacteria is needed.
OXIDATION.
As has been stated in discussing the respiration of bacteria (Chapter VIII) most of these organisms gain their energy through the oxidation of carbon in various forms, chiefly organic, so that CO2 is a product of the activity of nearly all bacteria. Some few oxidize CO to CO2, others CH4 and other paraffins to CO2 for this purpose. One class of bacteria even oxidizes H in small amounts for its energy and uses the carbon dioxide of the air or traces of organic carbon in the air as a source of carbon for “building” purposes.
One of the familiar oxidations of organic carbon is that of the acetic acid bacteria in the making of vinegar. These oxidize the alcohol which results from the action of yeast to acetic acid according to the formula CH3CH2OH + O2 = CH3COOH + H2O (see Fig. 67).
Of the various phenomena of oxidation due to bacteria, the formation of nitrites and nitrates has the greatest practical importance, since it is by this means that the ammonia which results from the decomposition of animal and vegetable tissue and waste products is again rendered available to green plants as food in the form of nitrates. Practically all the nitrates found in nature, sometimes in large quantities, are formed in this way. There are two distinct kinds of bacteria involved. One, the nitrous bacteria, oxidizes the ammonia to nitrous acid which forms nitrites with bases, and the other, the nitric bacteria, oxidizes the nitrous to nitric acid, giving nitrates with bases. A striking peculiarity of these two classes of organisms is that they may live entirely on inorganic food materials, are proto-autotrophic, prototrophic for oxygen (aërobic) and autotrophic for the other elements. Their carbon is derived from CO2 or carbonates. The importance of such organisms in keeping up the supply of nitrates in the soil can scarcely be overestimated.
The oxidation of the H2S, which is formed in the putrefaction of proteins, to free S by the sulphur bacteria and the further oxidation of this free S to sulphuric acid, and of the phosphorus, so characteristic of the nucleins, to phosphoric acid have been referred to. These activities of bacteria are of great value in the soil. Doubtless the commercial “phosphate rock” owes its origin to similar bacterial action in ages past.
The oxidation of H2S to free S may be an explanation of the origin of the great deposits of sulphur which are found in Louisiana and along the Gulf coast. These deposits occur in the same general regions as natural gas and oil. The sulphur might have been derived from the same organic material carried down by the Mississippi which yielded the oil and gas.15
A purposeful utilization of the oxidizing power of bacteria is in “contact beds,” “sprinkling filters” and “aërated sludge tanks” in sewage disposal works. In these instances the sewage is thoroughly mixed with air and brought in contact with large amounts of porous material so as to expose an extensive surface for oxidation (Fig. 73).
PRODUCTION OF HEAT.
A direct result of the oxidizing action of bacteria is the production of heat. Under most conditions of bacterial growth this heat is not appreciable. It may become well marked. The “heating” of manure is one of the commonest illustrations. The temperature in such cases may reach 70°. The heating of hay and other green materials is due chiefly to bacterial action. This heating may lead to “spontaneous combustion.” The high temperatures (60° to 70°) favor the growth of thermophil bacteria which cause a still further rise. The heat dries out the material, portions of which are in a state of very fine division due to the disintegrating action of the organisms. The hot, dry, finely divided material oxidizes so rapidly on contact with the air that it ignites.
A practical use of heat production by bacteria is in the making of “hot beds” for forcing vegetables (Fig. 74).
ABSORPTION OF FREE NITROGEN.
This is likewise one of the most important practical activities of certain types of bacteria present in the soil. The ability of plants of the legume family to enrich the soil has been known and taken advantage of for centuries, but it is only about thirty years since it was demonstrated that this property is due to bacteria. These plants, and several other kinds as well, have on their roots larger or smaller nodules (Fig. 75) spoken of as “root tubercles” which are at certain stages filled with bacteria. When conditions are favorable, these bacteria live in symbiotic relationship with the plant tissues, receiving carbonaceous and other food material from them and in return furnishing nitrogenous compounds to the plant. This nitrogenous material is built up from free nitrogen absorbed from the air by the bacteria. The utilization of this peculiar property through the proper cultivation of clover, alfalfa, soy beans and other legumes is one of the best ways of building up and maintaining soil fertility in so far as the nitrogen is concerned. The technical name of these bacteria is Rhizobium leguminosarum.
There are also types of “free-living,” as distinguished from these symbiotic, bacteria which absorb the free nitrogen of the air and aid materially in keeping up this supply under natural conditions. One of the most important of these types is the aërobic “Azotobacter” (Fig. 76), while another is the anaërobic Clostridium pasteurianum. The nitrogen which is absorbed is built up into the protein material of the cell body and this latter must in all probability be “worked over” by various types of decomposition bacteria and by the nitrous and nitric organisms and be converted into utilizable nitrates just as other protein material is, as has been discussed in Chapter X. At any rate there is as yet no definite knowledge of any other method of transformation. Up to the present no intentional practical utilization of this valuable property of these free-living forms has been made.
Nitrogen Nutrition of Green Plants.—It is the belief of botanists that green plants obtain their nitrogen chiefly in the form of nitrates, though ammonium salts may be utilized to some extent by certain plants at least. Exceptions to this general rule are those plants provided with root tubercles (and the bog plants and others which have mycorrhiza?). These plants obtain their nitrogen in the form of organic compounds made for them by the bacteria growing in the tubercles. That nitrogen circulates throughout the structure of plants in organic combination is certain. There does not appear to be any reason why similar compounds which are soluble and diffusible (amino-acids?) should not be taken up through the roots of plants and utilized as such. It seems to the author that this is very probably the case. Arguments in favor of this view are: (1) The nitrogen nutrition of leguminous and other plants with root nodules. (2) The close symbiosis between “Azotobacter” and similar nitrogen-absorbing bacteria and many species of algæ in sea water at least. (3) The vigorous growth of plants in soils very rich in organic matter, which inhibits the production of nitrates by the nitrous-nitric bacteria when grown in culture, and possibly (?) in the soil, so that nitrates may not account for the vigorous growth. (4) The effect of nitrate fertilizers is to add an amount of nitrogen to the crop much in excess of the amount added as nitrate. (5) The most fertile soils contain the largest numbers of bacteria. The doctrine that nitrates furnish the only nitrogen to plants was established before the activities of bacteria in the soil were suspected, and, so far as the author is aware, has not been supported by experiments under conditions rigidly controlled as to sterility.
It would seem that one of the chief functions of soil bacteria is to prepare soluble organic compounds of nitrogen for the use of green plants and thus to make a “short cut” in the nitrogen cycle (p. 107), as now believed in, direct from the “decomposition bacteria” to green plants.
Experiments have been made by different observers in growing seedling plants of various kinds in water culture with one or in some cases several of the amino-acids as sources of nitrogen. Most of these experiments were disappointing. Plant proteins are not so different from animal proteins, or plant protoplasm (apart from the chlorophyl portions of plants) from animal protoplasm as to lead one to suppose that it could be built up from one or two amino-acids any more than animal protoplasm can. The author is strongly convinced that this subject should be thoroughly investigated. It will require careful experimentation and perhaps rather large funds to provide the amounts of amino-acids that would probably be needed, but might result in a decided change in our ideas of soil fertility, and especially in the use of nitrogen fertilizers.