ferment. If the medium in which they are sown is suitable for their
nourishment, they will multiply to infinity; but the character most
essential to be observed is, that they may be sown in a liquid which
contains only ammonia and crystallisable substances, together with
the fermentable substances, sugar, lactic acid, gum, &c. The butyric
fermentation manifests itself as these little organisms multiply. Their
weight sensibly increases, though it is always minute in comparison
with the quantity of butyric acid produced; this is found to be the
case in all other fermentations.
This experiment no doubt resembles those made with the alcoholic and
lactic ferments. But it is distinguished from them by one circumstance
eminently worthy of attention. The butyric ferment, by its motions
and by its mode of generation, furnishes the irrefutable proof of its
organisation and of its life. This ferment, moreover, presented to
Pasteur a new and unexpected peculiarity. The vibrios live and multiply
without the smallest supply of air or of free oxygen. Not only, indeed,
do they live without air, but the air destroys them and arrests the
fermentation which they initiate. If a current of pure carbonic acid
is made to pass into the liquid where they are multiplying, their life
and reproduction do not appear to be at all affected by it. If, on the
contrary, instead of the current of carbonic acid we employ one of
atmospheric air for only one or two hours, the vibrios fall without
movement to the bottom of the vessel, and the butyric fermentation
which was dependent on their existence is immediately arrested.
Pasteur designated this new class of organisms by the name of
_anaérobies_; that is to say, beings which can live without air. He
reserves the designation _aérobies_ for all the other microscopic
beings which, like the larger animals, cannot live without free oxygen.
'It matters little,' added Pasteur, 'whether the progress of science
makes of this vibrio a plant or an animal; it is a living organism,
endowed with motion, which is a ferment and which lives without air.'
* * * * *
In meditating upon these facts, and upon the general character of
fermentation, Pasteur soon found himself in a position to approach more
nearly to the essential nature of these mysterious phenomena. In what
way do microscopic organisms provoke the phenomena of fermentation?
The organism eats, if one may say so, one part of the fermentable
matter. But how does this phenomenon of nutrition differ so much from
that of higher beings? In general, for a given weight of nutritive
matter which the animal takes in, it assimilates a quantity of the
same order. In fermentation, on the contrary, the ferment, while
nourishing itself with fermentable matter, decomposes a quantity
great in comparison to its own individual weight. Again, the butyric
ferment lives without free oxygen. Is there not, said Pasteur, a hidden
relation between the property of being a ferment and the faculty of
living without free oxygen? Are not vibrios which imperatively require
for their nutrition and multiplication the presence of oxygen gas those
which will never have the properties of ferments?
Pasteur then contrived a series of experiments with the view of placing
in parallelism these two curious physiological facts: life without air
and the characteristics of ferments.
We know how wine and beer are prepared. The must of grapes and the
must of beer are placed in wooden vats, or in barrels of greater or
less dimensions. Whether the fermentation proceeds from germs taken
from the exterior surface of the grapes, or from a small quantity
of ferment sown in the must under the form of yeast, as in the
fermentation of beer, the life of the ferment, its multiplication,
the augmentation of its weight, are so many vital actions which to a
certainty cannot borrow from the free oxygen of the external air, or
from that originally dissolved in the must, an appreciable quantity of
this gas. All the life of the cells of the ferment which multiplies
itself indefinitely appears then to take place apart from free oxygen
gas. In certain breweries in England the fermenting vats have sometimes
a capacity of several thousands of hectolitres; and the fermentation
liberates pure carbonic acid, a gas much heavier than atmospheric
air, which rests on the surface of the liquid in the vat in a layer
thick enough to protect the liquid underneath from any contact with
the external air. All this liquid mass, then, is inclosed between the
wooden sides of the vat and a deep layer of heavy gas which contains
no trace of free oxygen. In this liquid, nevertheless, the life of
the cells of the ferment and the production of all its constituents
go on for several days with extraordinary activity. Here certainly
we have life without air, and the ferment character expresses itself
in the enormous difference between the weight of the ferment formed
and collected from the vats under the name of yeast, at the end of
the operation, and the weight of the sugar which has fermented,
transforming itself into alcohol, carbonic acid, and various other
products.
Pasteur has studied experimentally that which takes place when, without
otherwise changing the conditions of these phenomena, the arrangement
is so modified as to permit the introduction of the free oxygen of the
atmosphere. It sufficed for this purpose to provoke a fermentation of
the must of beer, or the must of grapes, upon shallow glass dishes
presenting a large surface, or in a flat-bottomed wooden trough with
sides a few centimeters in height, instead of in deep vats as before.
In these new conditions the fermentation manifests an activity even
more extraordinary than it did in the deep vats. The life of the
ferment is itself singularly enhanced, but the proportion of the weight
of the decomposed sugar to that of the yeast formed is absolutely
different in the two cases. While, for example, in the deep vats, a
kilogram of ferment sometimes decomposes seventy, eighty, one hundred,
or even one hundred and fifty kilograms of sugar, in the shallow
troughs one kilogram of the ferment will be found to correspond to only
five or six kilograms of decomposed sugar. These proportions between
the weight of the sugar which ferments and the weight of the ferment
produced, constitute the measure of what one might call the ferment's
character--of that character which distinguishes its mode of life from
that of all other existences, great or small, in which the weight of
the organising matter and the assimilated alimentary matter are about
equal. In other words, the more free oxygen the yeast ferment consumes,
the less is its power as a ferment. Such is the case in the shallow
troughs where the extended surface is exposed to the contact of the
oxygen of the air. The more, on the contrary, the life of the ferment
is carried on without the presence of free oxygen, the greater is its
power of decomposing and of fermenting the saccharine matter. This is
the case in deep casks. The intimate co-relation then between life
without air and fermentation appears complete.
The unexpected light which these facts threw upon the cause of the
phenomena of fermentation made a forcible impression upon all thinking
minds. 'In these infinitely small organisms,' M. Dumas said one day
to M. Pasteur before the Academy of Sciences, 'you have discovered
a third kingdom--the kingdom to which those organisms belong which,
with all the prerogatives of animal life, do not require air for their
existence, and which find the heat that is necessary for them in the
chemical decompositions which they set up around them.'
The work of Pasteur, demonstrating that fermentation was always
dependent on the life of a microscopic organism, continued without
interruption. One of the most remarkable of his researches is that
which relates to the fermentation of the tartrate of lime. The
demonstration of life and of fermentation without free oxygen is in
this paper carried to the utmost limits of experimental rigour and
precision.
III.
But there is still another class of chemical phenomena where the life
without air of microscopic organisms is fully shown. Pasteur proved
that in the special fermentation which bears the name of putrefaction
the _primum movens_ of the putrefaction resides in microscopic vibrios
of absolutely the same order as those which compose the butyric
ferment. The fermentation of sugar, of mannite, of gums, of lactate
of lime, by the butyric vibrio, so closely resembles the phenomena of
putrefaction, that one might call these fermentations the putrefaction
of sugar and of the other products.
If it has been thought right to call the fermentation of animal matters
putrefaction, it is because at the moment of the decomposition of
fibrine, of albumen, of blood, of gelatine, of the substance of the
tendons, &c., the sulphur, and even the phosphorus, which enter into
their composition give rise to putrid odours, due to the evil-smelling
gases of sulphur and phosphorus.
The phenomena of putrefaction being then simply fermentations,
differing only in regard to the chemical composition of the fermenting
matters, Liebig naturally included them in his general theory of
the decomposition of organic matters after death. At a period long
antecedent to Pasteur's labours it had been established that there
existed in putrefying matters fungi or microscopic animalculæ, and the
idea had taken shape that these creatures might have an influence in
the phenomena. The proofs were wanting, but the notion of a possible
relation remained. We may read in his 'Lessons on Chemistry' with what
disdain Liebig mentioned these hypothetical opinions.
'Those who pretend to explain the putrefaction of animal substances
by the presence of animalculæ,' he wrote, 'reason very much like a
child who would explain the rapidity of the Rhine by attributing it
to the violent motions imparted to it in the direction of Bingen
by the numerous wheels of the mills of Mayence. Is it possible to
consider plants and animals as the causes of the destruction of
other organisms when their own elements are condemned to undergo
the same decompositions as the creatures which have preceded them?
If the fungus is the cause of the destruction of the oak, if the
microscopic animalcula is the cause of the putrefaction of the dead
elephant, I would ask in my turn what is the cause which determines the
putrefaction of the fungus or of the microscopic animalcula when life
is withdrawn from these two organisms?'
Thirty-two years later, and after Pasteur had accumulated, during more
than twenty years, proof upon proof that the theory of Liebig would not
stand examination, a physician of Paris, M. Bouillaud, asked, with the
insistent voice of a querulous octogenarian: 'Let M. Pasteur then tell
us here, in presence of the Académie de Médecine, what are the ferments
of the ferments.'
Before replying to this argument, which Liebig and M. Bouillaud
believed to be irrefutable, Pasteur, wishing to mark all the phases
of the phenomena, expounded in a short preamble the part played by
atmospheric oxygen in the destruction of animal and vegetable matters
after death. It is easy to understand, indeed, that fermentation and
putrefaction only represent the first phase of the return to the
atmosphere and to the soil of all that has lived. Fermentations and
putrefactions give rise to substances which are still very complex,
although they represent the products of decomposition of fermentable
matters. When sugar ferments, a large proportion of it becomes gas;
but alongside of the carbonic acid gas which is formed, and which
is, indeed, a partial return of the sugar to the atmosphere, new
substances, such as alcohol, succinic acid, glycerine, and materials
of yeast, are produced. When the flesh of animals putrifies, certain
products of decomposition, also very complex, are formed with the
vapour of water and the other gases of putrefaction. Where, then, does
nature find the agents of destruction of these secondary products?
The great fact of the destruction of animal and vegetable matters
is accomplished by slow combustion, through the appropriation of
atmospheric oxygen. Here, again, one must banish from science the
preconceived views which assumed that the oxygen seized directly on the
organic matter after death, and that this matter was consumed by purely
chemical processes. It is life that presides over this work of death.
If fermentation and putrefaction are principally the work of
microscopic anaérobies, living without free oxygen, the slow combustion
is found very largely, if not exclusively, to depend upon a class of
infinitely small aérobies. It is these last which have the property
of consuming the oxygen of the air. It is these lower organisms which
are the powerful agents in the return to the atmosphere of all which
has lived. Mildew, mould, bacteria, which we have already noticed,
monads, two thousand of which would go to make up a millimeter,
all these microscopic organisms are charged with the great work of
re-establishing the equilibrium of life by giving back to it all that
it has formed.
To demonstrate the important part played everywhere by these
microscopic organisms, Pasteur made two experiments. He first
introduced into vessels air deprived of all dust. This process we shall
have occasion to examine in all its details, in connection with the
researches on spontaneous generation. In these vessels, containing pure
air, were placed the water of yeast with sugar dissolved in it, milk,
sawdust--all of which had been deprived by heat of the germs of the
lower organisms. The vessels and their contents were then subjected to
a temperature of twenty-five to thirty-five degrees Centigrade. In a
series of parallel experiments, made under the same conditions and at
the same temperature, Pasteur took no steps to prevent the germination
of the little seeds of mould suspended in the air, or associated with
the substances contained in the vessels, neither did he avoid other
infinitely small germs of the class aérobies.
After some time the air of all the vessels of the two series was
submitted to analysis, when, behold, a very interesting fact! In the
vessels where life had been withdrawn from the organic matters--that
is to say, where there were no germs--the air still contained a
large proportion of oxygen. In the vessels, on the contrary, where
the microscopic organisms had been allowed to develop, the oxygen
was totally absent, having been replaced by carbonic acid gas. And,
further, for this absorption and total consumption of the oxygen gas a
few days had sufficed; while in the vessels without microscopic life
there remained, after several years, a considerable quantity of oxygen
in a free state, so weak is the proportion of oxygen that the organic
matters consume directly and chemically when the infinitely small
organisms are absent.
But can these microscopic organisms, after having decomposed or burnt
up all these secondary products, be in their turn decomposed?
How, cried M. Bouillaud, repeating his question, can they be destroyed
or decomposed? How can their materials, which are of the same order
as those of all the living creatures of the earth, be gasified and
caused to return to the atmosphere? After having been charged with the
transformation of others, whose business will it be to transform them?
A ferment which has finished its work, replied Pasteur, and which
for want of aliment cannot continue it, becomes in its turn an
accumulation, so to speak, of dead organic matters. Such, for example,
would be an accumulation of yeast exposed to the air. Leave this
mass to itself in summer temperature, and you will see appear in the
interior of the mass anaérobic vibrios and the putrefactions associated
with their life when protected from contact with the air. At the same
time, on the surface of the entire mass--that is to say, that which
finds itself in immediate contact with the oxygen of the air--the germs
of bacteria, the seeds of mould will grow, and, by fixing the oxygen,
determine the slow combustions which gasify the mass. The ferments
of ferments are simply ferments. As long as the aérobic ferments of
the surface have at their disposal free oxygen, they will multiply
and continue their work of destruction. The anaérobic vibrios perish
for want of new matter to decompose, and they form, in their turn, a
mass of organic matter which, by and by, becomes the prey of aérobies.
The portion of the aérobies which has lived becomes the prey either
of new aérobies of different species, or of individuals of their own
species, so that from putrefaction to putrefaction, and from combustion
to combustion, the organic mass with which we started finds itself
reduced to an assemblage of anaérobic and aérobic germs--of those same
germs which were mixed up in the original primitive organic substances.
Though a collection of germs becomes again in its turn a collection
of organic matter, subject to the double action of the phenomena of
putrefaction and of combustion, there need be no anxiety as to their
ultimate destruction; in the final analysis they represent life under
its eternal form, for life is the germ, and the germ is life.
* * * * *
Thus in the destruction of that which has lived, all reduces
itself to the simultaneous action of these three great natural
phenomena--fermentation, putrefaction, and slow combustion. A living
organism dies--animal, or plant, or the remains of one or the other.
It is exposed to the contact of the air. To the life which has quitted
it succeeds life under other forms. In the superficial parts, which
the air can reach, the germs of the infinitely small aérobies hatch
and multiply themselves. The carbon, the hydrogen, and the nitrogen
of the organic matters are transformed by the oxygen of the air, and
under the influence of the life of these aérobies, into carbonic acid,
vapour of water, and ammonia gas. As long as organic matter and air
are present, these combustions will continue. While these superficial
combustions are going on, fermentation and putrefaction are doing
their work in the interior of the mass by the developed germs of the
anaérobies, which not only do not require oxygen for their life, but
which oxygen actually kills. Little by little, at length, by this work
of fermentation and slow combustion, the phenomenon is accomplished.
Whether in the free atmosphere, or under the earth, which is always
more or less impregnated with air, all animal and vegetable matters
end by disappearing. To arrest these phenomena an extremely low
temperature is required. It is thus that in the ice of the Polar
regions antediluvian elephants have been found perfectly intact. The
microscopic organisms could not live in so cold a temperature. These
facts still further strengthen all the new ideas as to the important
part performed by these infinitely small organisms, which are, in fact,
the masters of the world. If we could suppress their work, which is
always going on, the surface of the globe, encumbered with organic
matters, would soon become uninhabitable.
FOOTNOTE:
[8] [A millimeter is 1/25th of an inch.]
_ACETIC FERMENTATION._
THE MANUFACTURE OF VINEGAR.
Soon afterwards Pasteur came upon a most curious illustration of
the 'fixation' of atmospheric oxygen by a microscopic organism--the
transformation of wine into vinegar. As its name indicates, vinegar is
nothing else than wine turned sour. Everybody has remarked that wine,
left to itself, in circumstances which occur daily, is frequently
transformed into vinegar. This is noticed more particularly when
bottles, having been uncorked, are left in a half-empty condition.
Sometimes, however, wine turns sour even in corked bottles. In this
case we may be sure that the bottles have been standing upright, and
that corks more or less defective have permitted the air to penetrate
into the wine. The presence of air, in fact, is indispensable to the
chemical act of transforming wine into vinegar. How does this air
intervene? And what is the little microscopic creature which, in
conjunction with the air, becomes the agent of this fermentation?
In a celebrated lecture given at Orleans at the request of the
manufacturers of vinegar in that town, Pasteur, after having stated the
two foregoing scientific questions, proceeded to examine the difference
between wine and vinegar. What takes place in the fermentation of the
juice of the grape which yields the wine? The sugar of this juice
disappears, giving place to carbonic acid gas, which is exhaled during
fermentation, and to alcohol, which remains in the fermented liquid.
Formerly, chemists gave the name of 'spirit' to all volatile matters
which could be collected from distillation. Now, when we distil wine
and condense the vapour in a worm surrounded by cold water, we collect
the spirit of wine at the extremity of the worm--this, when the water
with which it is mixed during distillation is withdrawn from it, we
designate by the name of alcohol. Vinegar contains no alcohol. When
distilled it yields water and a spirit. But this spirit is acid, with a
very pungent odour, and not inflammable like spirit of wine. Separated
from the water which had accompanied it during the distillation, this
spirit takes the name of acetic acid. This is the form in which it is
used in smelling bottles--in those bottles of English salts the vapour
of which is so penetrating.
In the formation of vinegar in contact with air the alcohol disappears,
and is replaced by acetic acid. The air has thus given up something to
the wine. Atmospheric air every one knows to be a mixture of nitrogen
and oxygen, the nitrogen in the proportion of four-fifths of the total
volume, and the oxygen of one-fifth. Well, in the transformation of
wine into vinegar the nitrogen remains inactive. It is the oxygen alone
which enters into combination with the alcohol. You ask for the proof
of this? Take a bottle of wine turned sour, a bottle which at the same
time is stopped hermetically; if the oxygen of the air contained in the
bottle has combined with the alcohol, then, instead of air, there will
be nothing in the bottle but nitrogen gas. Turn the bottle upside down
and open it in a basin of water. The water of the basin will rush into
the bottle to fill the partial vacuum created by the disappearance of
the oxygen. The volume of water which enters the bottle is precisely
equal to a fifth part of the total original volume of the air which the
bottle contained at the time when it was closed. Moreover, it is easy
to show that the gas which remains in the bottle has the properties of
nitrogen gas. A lighted match is extinguished in it as if plunged into
water, and a bird dies immediately in it of asphyxia.
If we confine our knowledge to what has gone before, it would seem that
alcohol diluted with water and exposed to the air ought to furnish
acetic acid. It is not so, however. Pure water alcoholised to the
degree of ordinary wines may remain for whole years in contact with
the air, without the least acetification. In this difference between
natural wine and pure water alcoholised, and exposed to contact with
air, we touch upon a vital point in the phenomena of fermentation. The
celebrated theory of Liebig, which Pasteur was destined to overthrow,
might be thus summed up:--If pure alcoholised water cannot become sour
in contact with air, as is the case with wine, it is because the pure
alcoholised water lacks the albuminoid substance which exists in the
wine in a state of chemical alteration, and which is a ferment capable
of causing the oxygen of the air to combine with the alcohol. And the
proof, according to Liebig, that things act rigorously thus is, that if
you add to the mixture of water and alcohol a little flour, or a little
meat-juice, or even a minute quantity of any vegetable juice, the
acetic fermentation arises, as if by compulsion. In other words, by the
addition of a small quantity of any nitrogenised substance in process
of alteration, you cause the union of the oxygen of the air with the
alcohol.