Microbes & toxins

Front matter

MICROBES AND

TOXINS

THE PROLONGATION OF LIFE.

Chapter I

THE GENERAL FUNCTIONS OF MICROBES — THE TRANSFORMATION CYCLES OF CARBON AND NITROGEN

The circulation of matter ; anabolism and catabolism — Views of Lavoisier — The transformation cycles of carbon and nitrogen — The role of micro¬ organisms : I. Maturation of the soil and formation of arable land. II. Fermentation of vegetable matter — Decomposition of starch and manure — Hypotheses on the formation of coal. III. Putrefaction of albuminous materials. IV. Nitrification and denitrification : in agricul¬ ture ; in the biological purification of sewage. V. Fixation of atmos¬ pheric nitrogen in the soil : by bacteria alone ; by bacteria in association with algoe ; by the nodule bacteria of Leguminosoe — Some ideas about the useful micro-organisms : Fermentations in con¬ nection with food production and in various industries.

Almost all living matter is made up of water, that is to say, of oxygen and hydrogen, and of compounds of carbon and nitrogen. Other elements may enter into the tissues of animals and vegetables, for example, sulphur, iron, arsenic, boron ; but by following the circulation of carbon and nitrogen, it is possible to have a general view of the movements of exchange between matter and life.

The living creature restores to nature what it has absorbed, and eventually its own body, by its excretions during life and by its decomposition after death. The elements set free are recombined into organic bodies, and these exchanges and this circulation form the essence of life. It is an abuse of our subjective attitude to consider as two opposites life and death. At most, life is the opposite of inertia. Death is a special kind of accident, life being the all-embracing phenomenon. The list of the work ers in the great cycle of life are the microbes,

B and the decompositions and re-combinations of living matter | depend entirely upon them.

Life without microbes is not conceivable at the present day. That does not mean that they were the first living beings to appear on the surface of the earth. It is the more difficult to get an idea of their origin, since in all probability they have undergone evolution, and have not always had the appearance they have at present. It is possible that under forms that we can hardly guess at, life appeared long before the existence of microbial forms ; but microbes have been the chief agents in : the spread and extension of life throughout the world.

In the world of to-day, the building up and breaking down of organic substances are functions of cells, conducted by an ; infinite number of diastases. These cells include the cells! built up into animal and vegetable tissues, and the isolated cells, the microbes. It would not be right to contrast too; much the microbial cells with the cells of tissues, merely ; because the former are separate individuals, whereas the latter are arranged in systems. In nature, micro-organisms can . exist as solitary individuals, but are seldom actively at work in this condition. For example, in the soil, the bacteria which fix nitrogen act rather like a wide-spread tissue. The active: agents are in any case invariably cells.

To describe in a few words the cycle which organic matter follows, one cannot do better than quote the celebrated page of Lavoisier, “ Plants draw from the circumambient air, from water and in general from the mineral kingdom, the substances necessary for their own organisation. Animals feed either on plants or on other animals, which have themselves fed on plants, so that eventually the matter building them up is always derived either from the air or from the mineral; kingdom.

“Finally, fermentation, putrefaction and combustion are! continually restoring to the atmosphere and to the mineral kingdom the elements which plants and animals have borrowed.

“By what rocesses does nature effect this wonderful circulation between the two kingdoms ? How does nature succeed in producing substances which are combustible, putrescible and capable of fermentation from combinations which have none of these properties ? Here are impenetrable mysteries. One may perceive, however, that since combustion and putrefaction are the means which nature employs to return to the mineral kingdom what has been drawn from it in the building up of plants and animals, the latter process must be the converse of the former.”

Green plants get their carbon from the carbonic acid of the air. In virtue of their chlorophyll activity they build up this ! carbon into starch, cellulose, sugars and fats. This , carbohydrate synthesis then represents the accumulation of energy. A green plant kept in the dark burns up its hydrocarbons, returning them to the atmosphere. A dead plant returns its hydrocarbons after a series of decompositions, in the form of carbonic acid and water. A plant which has been eaten by an animal supplies the animal with glycogen and fats which are consumed in the course of muscular work and respiration. Dying animals like dead plants return their hydrocarbons to nature. Plants without chlorophyll and all animals spend and dissipate the energy accumulated by the chlorophyllous plants, energy derived entirely from the rays of the sun.

Nitrogen exists in the atmosphere, from which it enters the soil. It exists in the dejecta of living animals and in the bodies of plants and animals rotting on the surface of the earth. It accumulates there in the mould or humus : plants take up the nitrogenous matter of the soil in the form of salts of ammonia and nitrates, and build up from them the vegetable proteins. Animals which eat plants produce from these the animal proteins. Animals and plants in decomposition, and the excretions of animals in general, return their nitrogen to fertilize the soil.

Animals are in a certain sense parasites of plants, since they are unable to build up by themselves, starting from mineral elements, their hydrocarbons and proteins. Even in plants

B 2 the formation of the protein compounds is dependent on the chlorophyll activity ; without this it would be impossible for them to build up the carbohydrates, and these are indispensable in the employment and elaboration of the ( nitrogenous elements of the soil. The chlorophyll activity ( is itself dependent on the sunlight, so that life is one great i paean to the sun.

In the transformation of carbon and nitrogen all the operations are not carried out by microbes, for these latter do not count at all in chlorophyll assimilation nor in animal digestion. But it is the bacteria which keep up the supply ofj organic matter which forms the source of animal and ; vegetable life ; it is the bacteria which restore to circulation those elements which were for a moment arrested in the bodies of animals and plants: they restore to life matter which had; ceased to live. It is they also which prepare the soil for vegetation and cultivation : they accumulate in the soil the nitrogen which we are to eat in the form of cereals.

It is they which carry on the impenetrable mystery referred; to by Lavoisier.

They prepare the primary material of life. Their activity'! is as universal as that of water, as that of light.

The discoveries of Pasteur have not only revolutionised; medicine, they have filled with new life the science andi practice of agriculture and husbandry.

I. — Maturation of the soil and formation of arable land.

It was not the bacteria which shattered, splintered and powdered the rocks which formed the first crust of the cooling globe. But as soon as in this chaos there appeared water and! alkaline phosphates, algae and bacteria could establish; themselves and with the help of carbonic acid continue the! first formation of soil.

In those rocks known as “ rotten rock,” Mimtz found nitrifying microbes : the nitrifying ferment has also attacked the Faulhorn, a mountain of the Bernese Oberland near

Grindelwald, which is composed of a calcareous schist, black, friable, and crumbling down. There is therefore a sort of putrefaction of rocks (alkalis and alkaline earths). According to Fausto Sestini the carbonic acid produced in the respira¬ tion of plant roots hastens the breaking down of feldspars.

Mosses, lichens, algae and bacteria prepare the way for the highest plants. The more there is growth of plants, the more there is decomposition. In this way, soil or humus has little by little spread over all the earth.

II. — Fermentation of Carbohydrates.

These fermentations restore to circulation the hydrocarbons of animals and plants.

For the decomposition of sugars, starches, fats, glucosides, and celluloses, several series of fermentations are necessary, each 'supplementing another in the work. The anaerobic organisms break down the large organic molecules, whereas the aerobes carry out, in particular, oxidations. When the bacteria are insufficient, the moulds continue the work. Finally, nothing is left but carbonic acid and water. All the operations which in our laboratory analyses we distinguish and conceive in terms of abstract formulae, are combined and intermingled in nature, each succeeding and limiting the other. The yeasts transform sugars into carbonic acid and alcohol. Alcohol attacked by the acetic ferments is turned into acetic acid. Finally, the Bacterium aceti can split the acetic acid of vinegar into carbonic acid and water. The Mycoderma vim destroys and oxidises both alcohol and acetic acid, producing again carbonic acid and water. The sugar contained in Raulin’s fluid (v. infra), for example, is attacked by Aspergillus niger , the products including oxalic acid. Various moulds and bacteria turn starch into sugar ; it is the moulds in particular which complete the oxidations, producing carbonic acid and water.

Milk left to itself ferments, i.e., produces lactic acid, which, meeting with an alkaline carbonate, furnishes calcium lactate.

Pasteur discovered the transformation of the lactate into the butyrate by the butyric vibrio ; the butyrate can be completely consumed in its turn by the moulds.

The glucosides (compounds of sugar with an organic body, an alcohol or phenol) are decomposed into their two elements. A diastase, tannase, secreted by the Aspergillus , splits tannin into two molecules of gallic acid, from which other moulds produce again carbonic acid and water.

Just as there is not one starch but several, so there are several celluloses, which resemble starches but are more stable : (C6H10O5)w. They form the walls of vegetable cells, and make up one-third of the weight of the straw, which is the principal component of farmyard manure. If the celluloses

Fig. i. — The microbe which ferments cellulose, described by Omeliansky : i

bacilli with spores.

were not decomposed and restored to circulation, the earth ; would soon be cumbered with useless refuse material. But from i the beginning moulds establish themselves on the outer skin of the living plant ; when it dies they invade its tissues, attacking | first the sugar and then the cellulose, the latter being hydrolysed, transformed into sugars and consumed. The B. Amylobacter of Van Tieghem, an anaerobe, produces from cellulose hydrogen, carbonic acid and butyric acid. Omeliansky has demonstrated two methods of anaerobic fermentation in cellulose, one with production of hydrogen, the other with production of methane. Those ferments which in a tube in i the laboratory decompose the cellulose of Berzelius’ paper, : act in precisely the same way in manure heaps. The aerobic | fermentation of cellulose is carried on by moulds, by fungi !

more highly organised, and by the nitrifying and denitrifying bacteria.

Pectose is a hydrocarbon associated with cellulose in the membranes and interstices of plant cells ; the rust which attacks hemp and linen is a fermentation of the pectose, transforming it into pectic acid, then to sugar, by the B. amylobacter and by the Granulobacter of Fribes and Winograd¬ sky. In the manure heap, aerobic fermentation proceeds at the surface and raises the temperature up to nearly 8o° C. In the depths of the heap the temperature is low, and there the anaerobic ferments attack the cellulose. The bubbles which rise and burst on the surfaces of ponds are signs of the ■ anaerobic fermentation which is decomposing organic debris at the bottom. Manure kept in a latticed box with free access of air heats up without there being destruction of the cellulose ; loss of nitrogen takes place. Manure heaped in a closed box or kept corked in a large carboy, liberates methane from the decomposition of the cellulose. It is possible to collect the gas and by means of an exit tube to make it furnish a light.

It was a natural step to attribute to the ferments of cellulose the formation of peat, lignite and coal. Microbes certainly are at work in the decomposition of vegetable matter in the peat-bogs, and coal is supposed to be the product of more complete fermentations, two varieties at least of bacteria being in activity in succession ; the first dissolves the central membranes of the cell walls, the others attacking the cellulose, more or less pure, which constitutes the thick parts of the wall. “ The bacterial activity,” says the most convinced defender of this theory, “ produced a de-hydrogenation and de-oxidation, the final result being the production of carbon. We do not know if the final limit of this process has ever been reached, but figures show that the more geologically ancient the fuel, the more carbon it contains ” (Bernard Renault). Fuel of less age, lignite, and peat, for example, contains besides bacteria, amoebae and infusoria. In the lignites, the cannel coals, and the boghead coals, we find fungi and algae, which do not occur in coal proper. Bacteria alone exist in all the fuels ; they are much less altered than the structures which surround them : therefore it is supposed that they must have survived tissues which they destroyed.

The presence of bacteria in coals derived from organic matter should not astonish us. But it appears difficult to believe that bacteria have been the only agents in the formation of coal. The study of their form is extremely difficult ; fine particles of iron pyrites and little crystals often imitate bacterial forms in the thin sections of coal, so difficult to prepare and examine by transmitted light under the microscope. On the other hand the fermentation of cellulose does not explain the frequent impregnation of the debris by a blackish, bituminous material, nor perhaps does it really explain the enrichment in carbon of the deposits ; further, there have been produced from the fermentation of fatty bodies, resins and similar volatile substances, phenol bodies like those found in coal (E. Duclaux).

There is nothing surprising in the presence of algae in the Bogheads , for these are precisely the coals derived from algae and often named algal coals. Their formation was due to luxuriant algae growths rapidly developed on the surface of stagnant pools, which then sank to the bottom carried down by a coagulum in the peaty water ; this mass contained bituminous substanceswhich must have come from elsewhere, for there is no indication that they took their origin on the spot by an alteration of the algae. It is these bitumens which have produced the carbon enrichment of the mass, so that on the whole instead of destruction a preservation process has taken place. In other cases, instead of this enormous growth of algae, clouds of pollen and spores from the primeval forests have been deposited : these “ rains of sulphur ” also sank in the peaty water and became saturated with bitumen, hence the so-called spore and pollen coals.

We ought not to be too ready to reject the bacterial theories of the formation of the various coals, especially should it turn out to be impossible to explain without microbes the formation of the bitumen which impregnates them. The small lower algae are rich in fats and capable of yielding petroleum bodies by distillation. The problem of the origin of coal has therefore some relation to the problem of the origin of petroleum. The decomposition of fatty substances commences with their saponification, which splits them into fatty acids and glycerine, and glycerine is a good food medium for various bacteria.

The moulds decompose fatty acids into carbonic acid and water. Fats resist decomposition longer than other carbo¬ hydrates, and longer than nitrogenous substances ; it is from this cause that the proportion of fat increases in a cheese which has been kept, or in a dead body which is decomposing. The ‘ adipocere' is the final stage in an animal body left entirely to nature.

III. Putrefaction of albuminous materials.

Putrefaction returns to the soil the nitrogen which makes up 15 per cent, of the proteins of animal tissues.

Since plants draw their nitrogen from the soil in the form of nitrates and salts of ammonia, i protein molecule to be broken

Fig. 2. — Ammoniacal fermentation of urine : Urocococcus of Pasteur.

is necessary for the complex down so as to supply finally

Fig. 3. — Urobacillus of Duclaux.

nitrogen in the form of ammonia and nitric acid. Later green plants and finally animals, raise this mineral nitrogen to the level of organic nitrogen.

Already during life animals discharge nitrogen with their excretions in the form of urea, uric acid and hippuric acid. The Urobacteria (there are about a hundred species known)

transform the urea, by means of a urease which they secrete, into carbonate of ammonia. Hippuric acid is transformed into benzoic acid and glycocoll, and finally into ammonia.

The putrefactions of albuminous materials in nature are never simple, that is to say carbohydrates almost always accompany the proteins ; even meat contains a little sugar. Putrefactions therefore are almost always mixed fermentations.

Pasteur thought that all putrefaction was the work of anaerobes. He discovered the vibrion septujue , an anaerobic bacillus capable of decomposing proteins. Later, the anaerobes were neglected and it was thought that various aerobes, in particular Proteus , were the principal agents of putrefaction. But after the study of fetid suppurations had drawn attention to the presence of anaerobes (Veillon), the idea rose again that they too must play a part in putrefaction, and the methodical study of this subject was recommenced, chemical analysis going hand in hand with bacteriological examination.

In their experiments, Tissier and Martelly followed for months the events which took place in flasks into which meat had been put and allowed to putrefy either with or without access of air. Meat taken from the slaughter-house as fresh as possible contains all the germs necessary and sufficient for putrefaction, and, as it contains carbohydrates, it is a mixed putrefaction which occurs.

There are two phases. In the first the sugar and the proteins are attacked by mixed ferments, that is to say, by microbes which decompose at the same time both proteins and sugar, proteolytic and saccharolytic — (peptolytic is the term applied to the ferments which attack protein only after its reduction to peptone). In the second phase the protein and its products are attacked by ferments which are proteolytic or peptolytic pure and simple, not saccharolytic.

But between these two phases a critical turning-point occurs: the decomposition of the sugars produces an acidity sufficient to stop putrefaction. The “ antagonistic force ” which Bienstock observed in his investigations on a putrefactive bacterium the B. putrificus, is nothing but this acidity. A piece of meat, as is well known, keeps excellently in unboiled milk, because the milk on fermenting produces lactic acid, which protects the proteins of the meat from putrefaction. That is why the housewife when she salts down meat, does not forget to add some vinegar, i.e., acid. It would seem then that putrefaction ought to cease after the action of the mixed ferments.

If it does not stop, it is because there is not sufficient sugar. The limit of acidity for the pure proteolytic ferments has not been reached. Further, owing to the decomposition of the albumins already begun, there appears a base, ammonia, which neutralises the acidity, and the pure proteolytic ferments can begin their action. It is the anaerobes which break down the protein molecule and produce putrefaction ; but they cannot do without the auxiliary aerobes ; thanks to the aerobes which hasten the production of ammonia and the alkalinisation of the medium, putrefaction passes successfully through the crisis of the antagonistic acidity.

In the putrefaction of milk, two analogous phases succeed each other. Milk being richer in sugar than meat, the acidity developed in the first phase is greater and the crisis more difficult to surmount. To surmount it, more powerful fer¬ ments are required than the aerobes which produce the ammonia in the putrefaction of meat : these are the fungi ( Oidiuni lactis , Rhizopus nigricans ) and the yeasts, which destroy the acids, consume the milk-sugar, and attack the casein. The fungi prepare the way for the pure ferments, which then carry out the second phase of putrefaction.

When an animal dies, all the microbes necessary for its putrefaction are present in its intestine. They invade the tissues and carry out on a larger scale what we see on a small scale in the experimental flasks. The worm of the grave is an old poetical image long out of date. The real destroyer is the microbe.

It is a fact well established to-day, that in a normal intestine, the bacteria of putrefaction are capable of vegetative existence, and that our digestion is accompanied by a commencement of putrefaction, — and of intoxication. The aerobic bacteria of the Proteus group can produce putrefaction there, but it is chiefly the anaerobes, as was Pasteur’s opinion, which do this. There have been found in the human intestine all the most important of these, the B. putrificus , the B. sfiorogenes, and the B. perfringens. The anaerobic flora of the intestine does not differ much from the anaerobic flora of the putrefying meat in Tissier’s experiments. These microbes produce poisons which are the true source of auto-intoxications (Metchnikoff). For life in general, putrefaction is necesssary to permit of the circulation of nitrogen in nature : but our own particular interest demands that putrefaction should not begin too soon, i.e.j in our intestine, and mask itself under an appearance of perfect health.

To combat the intestinal putrefaction, it is necessary to adopt a diet capable of producing in us that limit of acidity which induces the crisis separating the two phases of putre¬ faction of meat or milk, and of arresting decomposition in our bodies at the end of the first phase. Therefore, we ought to eat carbohydrates and sugars, and so alter the conditions in our intestine as to favour the lactic ferments.

IV. — Nitrification and Denitrification .

Nitrates represent the form of nitrogen preferred bv the plants, and it has long been known that the ammonia set free in putrefaction becomes oxidised in the soil, the ammonium salts being transformed into nitrates. This is the process of nitrification and is carried on by bacteria.

It was long thought that the ammoniacal salts were oxidised in contact with the soil by the direct action of atmospheric oxygen, in the same way that certain chemical combinations can take place on contact with porous substances. But chalk and sand, which ought to act as porous substances, cannot take the place of earth. Pasteur perceived with his peculiar intuition that the lower plant must play a part in nitrification.

That nitrification is the work of living creatures has been proved by the celebrated experiment of Schlcesing and Miintz. If a cylinder is filled with cultivated soil, ammonia poured on the top appears at the bottom as nitrate of lime. Tut this transformation no longer occurs if the earth is previously heated to ioo° C., or if it is impregnated with the vapour of chloroform or of carbon bisulphide, i.e., if the living organisms that it contains are killed or paralysed. The soil recovers its activity when the paralysing vapours have been removed by passing through a current of air.

Nitrification is a process of two stages, and is carried out by two species of bacteria, each with its own function. In the first phase, the ammoniacal salts are transformed into nitrites by the microbes known as the nitrous ferment, e.g. Nitrosomonas of Europe, Nitrosomonas of Java, and the Nitrosococcus of America (Mexico and Brazil). In the second phase the nitrites are turned into nitrates by the nitro-bacterium or nitric ferment, the Nitrobacter of Winogradsky (figs. 4-6).

Neither the nitrous nor the nitric ferments develop in presence of organic matter. The activity of the nitrous ferment is arrested by 0*3 per cent, of glucose, peptone or asparagin. The nitric ferment, less sensitive, is stopped by o-3 per cent, of glucose, i-2 5 per cent, of peptone or 1 per cent, of asparagin. The former is inhibited by 1*5 per cent., the latter by 3 per cent, of sodium acetate.

Now we are accustomed to the idea that bacteria live on organic matter. Yet the bacteria of nitrification must, as we have seen, get their nourishment elsewhere. They differ from other bacteria in being capable of taking up carbon by decom¬ posing carbonates, in being aerobic, and in behaving like chlorophyllous plants. They are not saprophytes in the same sense as the majority of bacteria.

In the laboratory, the two phases of nitrification can be demonstrated separately by pure culture. But in nature they are simultaneous. Under laboratory conditions ammonia exerts an inhibiting action on the nitric ferment, whereas in nature both the actions can occur in presence of quantities of ammonia frequently very considerable. Schlcesing has shown that in the soil ammonia does not prevent the nitric ferment from acting. These apparent contradictions between theory and practice are capable of explanation. Ammonia prevents the development of the nitric ferment but scarcely acts at all on the same ferment in the adult state. The soil in nature being populated by the adult ferment, the inhibiting action of ammonia is much more limited than in an experimental flask (Boullanger and Massol).

The nitrous ferment will stand neither an excess of ammonia nor an excess of its own product, the nitrite of magnesium. The nitric ferment ceases to act when too much nitrite has

Fig. 5. — Nitric ferment : the nitro-bacterium from Quito (after Kayser).

Fig. 4. — Nitrous ferment from Gennevilliers.

been produced around it and when it has itself developed a certain quantity of nitrates. Thus each demands suitable proportions both of the primordial material and of the products. It is, no doubt, to maintain these favourable conditions that we have the denitrifying microbes which partially undo the labour of the nitrifying organisms. They return the nitrates and nitrites to the condition of ammonia, liberating protoxide of nitrogen, dioxide of nitrogen, or simply nitrogen. Wheat straw, the straw of maize and of lucerne and oilcakes contain denitrifying bacteria. Animal excrement also contains them, for soil to which cow dung is added loses part of its nitrates. When soil is treated with nitrate of soda too soon after receiving farmyard manure it loses nitrogen. The denitrifying organisms act best in presence of the excess of organic matter which is so prejudicial to nitrification. Probably the two functions balance and regulate each other. The nitrogen liberated by the denitrifying bacteria is, however, not lost ; it may be taken up by the bacteria which fix nitrogen. It is nevertheless true that farmers ought to be on their guard against the denitrifying organisms and avoid putting on the soil farmyard manure, especially if fresh, along with nitrates.

Formerly in the manufacture of saltpetre cultivated soil was

Fig. 6. — Nitrifying bacteria (after Winogradsky).

A. Nitrous ferment from Zurich.

B. The same in the motile form with flagella.

C. Nitrous ferment from Kazan.

D. Nitrous ferment from Java, motile cells and groups of cells.

freely sprinkled with urine. If the deposits of nitrate in Chili and Peru should some day be exhausted, it would not be impossible to make it with the help of bacteria (not to speak of the electrical methods which have already been employed). Saltpetre is produced everywhere when suitable conditions of moisture and organic matter are given. The saltpetre of cellars is nitrate of lime which has risen from the soil through the walls by capillarity and has undergone evaporation.

Long ago Muntz prepared artificial nitre beds which furnished eight grams of nitrate daily ; in recent experiments he and Laine have obtained such a yield that they find themselves capable of preparing by means of nitrifying organisms all the saltpetre for the powder necessary to defend the nation in war. Distributed over beds of peat of two metres thickness on the top of a layer of clinker, the nitrifying bacteria are capable of producing, for each 25 acres of area, 1,500 tons of nitrate per day, after a starting period of one month at most to get the beds into working order; in five days, that is, 7,500 tons, or the annual requirement of powder for the army. It is easy to calculate what 750,000 acres of peat bogs in France could produce if necessary.

The purification of sewage is one of the greatest tasks which burden the hygienists of large towns. Broad irrigation followed by cultivation demands much land and is not quite safe except when employed solely for the growth of forage, not for market¬ gardening. Hence it is gradually giving place to the biological method of purification, an intensive process carried on in small space, and here again by bacteria.

We shall not enter here upon the details of its application. In principle, complete biological purification goes on in two phases : a phase of anaerobic fermentation in the septic tank and a phase of aerobic fermentation in the bacterial beds.

In the septic tank, into which the sewage must be run gently — so as not to carry in the air which would favour aerobic fermentation — in which it must be allowed to circulate quietly, and to stay at least twenty-four hours, the disintegration takes place both of hydrocarbons and proteins under the agency of legions of bacteria which secrete all sorts of diastases. The sludge dissolves and the resultant product can be sub¬ mitted to the action of the nitrifying agents.