were turned to good account practically. In well-managed distilleries,
the custom of aerating the wort and the juices, to render them more
adapted to fermentation, has been introduced. The molasses, mixed with
water, is permitted to run in thin threads through the air at the moment
when the yeast is added. Manufactories have been erected, in which the
manufacture of yeast is almost exclusively carried on. The saccharine
worts, after the addition of yeast, are left to themselves, in contact
with air, in shallow vats of large superficial area, realizing thus on
an immense scale the conditions of the experiments which we undertook in
1861, and which we have already described in determining the rapid and
easy multiplication of yeast in contact with air.
The next experiment attempted was to determine the volume of oxygen
absorbed by a known quantity of yeast, the yeast living in contact with
air, and under such conditions that the absorption of air was
comparatively easy and abundant.
[Illustration: Fig. 65.]
With this object we repeated the experiment that we performed with the
large-bottomed flask (Fig. 62), employing a vessel shaped like Fig. B.
(Fig. 65), which is, in point of fact, the flask A with its neck drawn
out and closed in a flame, after the introduction of a thin layer of
some saccharine juice impregnated with a trace of pure yeast. The
following are the data and results of an experiment of this kind.
We employed 60 c.c. (about 2 fluid ounces) of yeast-water, sweetened
with 2 per cent. of sugar and impregnated with a trace of yeast. After
having subjected our vessel to a temperature of 25° C. (77° F.) in an
oven for fifteen hours, the drawn-out point was brought under an
inverted jar filled with mercury and the point broken off. A portion of
the gas escaped and was collected in the jar.
For 25 c.c. of this gas we found, after absorption by potash, 20·6, and
after absorption by pyrogallic acid, 17·3. Taking into account the
volume which remained free in the flask, which held 315 c.c., there was
a total absorption of 14·5 c.c. (0·88 cub. in.) of oxygen.[118] The
weight of yeast, in a state of dryness, was 0·035 gramme.
It follows that in the production of 35 milligrammes (0·524 grain) of
yeast there was an absorption of 14 or 15 c.c. (about 7/8 cubic inch) of
oxygen, even supposing that the yeast was formed entirely under the
influence of that gas: this is equivalent to not less than 414 c.c. for
1 gramme of yeast (or about 33 cubic inches for every 20 grains).[119]
Such is the large volume of oxygen necessary for the development of one
gramme of yeast when the plant can assimilate this gas after the manner
of an ordinary fungus.
Let us now return to the first experiment described in this paragraph
(page 238), in which a flask of three litres capacity was filled with
fermentable liquid, which, when caused to ferment, yielded 2·25 grammes
of yeast, under circumstances where it could not obtain a greater supply
of free oxygen than 16·5 c.c. (about one cubic inch). According to what
we have just stated, if this 2·25 grammes (34 grains) of yeast had not
been able to live without oxygen, in other words, if the original cells
had been unable to multiply otherwise than by absorbing free oxygen, the
amount of that gas required could not have been less than 2·25 × 414
c.c., that is, 931·5 c.c. (56·85 cubic inches). The greater part of the
2·25 grammes, therefore, had evidently been produced as the growth of an
anaërobian plant.
Ordinary fungi likewise require large quantities of oxygen for their
development, as we may easily prove by cultivating any mould in a closed
vessel full of air, and then taking the weight of plant formed and
measuring the volume of oxygen absorbed. To do this, we take a flask of
the shape shown in Fig. 66, capable of holding about 300 c.c. (10-½
fluid ounces), and containing a liquid adapted to the life of moulds. We
boil this liquid and seal the drawn-out point, after the steam has
expelled the air wholly or in part; we then open the flask in a garden
or in a room. Should a fungus-spore enter the flask, as will invariably
be the case in a certain number of flasks out of several used in the
experiment, except under special circumstances, it will develop there
and gradually absorb all the oxygen contained in the air of the flask.
Measuring the volume of this air, and weighing, after drying, the amount
of plant formed, we find that for a certain quantity of oxygen absorbed
we have a certain weight of mycelium, or of mycelium together with its
organs of fructification. In an experiment of this kind, in which the
plant was weighed a year after its development, we found for 0·008
gramme (0·123 grain) of _mycelium_, dried at 100° C. (212° F.), an
absorption that amounted to not less than 43 c.c. (1·5 cubic inches) of
oxygen, at 25°. These numbers, however, must vary sensibly with the
nature of the mould employed, and also with the greater or less activity
of its development, because the phenomenon is complicated by the
presence of accessory oxidations, such as we find in the case of
_mycoderma vini_ and _aceti_, to which cause the large absorption of
oxygen in our last experiment may doubtless be attributed.[120]
[Illustration: Fig. 66.]
The conclusions to be drawn from the whole of the preceding facts can
scarcely admit of doubt. As for ourselves, we have no hesitation in
finding in them the foundation of the true theory of fermentation. In
the experiments which we have described, fermentation by yeast, that is
to say, by the type of ferments properly so called, is presented to us,
in a word, as the direct consequence of the processes of nutrition,
assimilation, and life, when these are carried on without the agency of
free oxygen. The heat required in the accomplishment of that work must
necessarily have been borrowed from the decomposition of the fermentable
matter, that is from the saccharine substance which, like other unstable
substances, liberates heat in undergoing decomposition. Fermentation by
means of yeast appears, therefore, to be essentially connected with the
property possessed by this minute cellular plant of performing its
respiratory functions, somehow or other, with oxygen existing combined
in sugar. Its fermentative power—which power must not be confounded with
the fermentative activity or the intensity of decomposition in a given
time—varies considerably between two limits, fixed by the greatest and
least possible access to free oxygen which the plant has in the process
of nutrition. If we supply it with a sufficient quantity of free oxygen
for the necessities of its life, nutrition, and respiratory combustions,
in other words, if we cause it to live after the manner of a mould,
properly so called, it ceases to be a ferment, that is, the ratio
between the weight of the plant developed and that of the sugar
decomposed, which forms its principal food, is similar in amount to that
in the case of fungi.[121] On the other hand, if we deprive the yeast of
air entirely, or cause it to develop in a saccharine medium deprived of
free oxygen, it will multiply just as if air were present, although with
less activity, and under these circumstances its fermentative character
will be most marked; under these circumstances, moreover, we shall find
the greatest disproportion, all other conditions being the same, between
the weight of yeast formed and the weight of sugar decomposed. Lastly,
if free oxygen occurs in varying quantities, the ferment-power of the
yeast may pass through all the degrees comprehended between the two
extreme limits of which we have just spoken. It seems to us that we
could not have a better proof of the direct relation that fermentation
bears to life, carried on in the absence of free oxygen, or with a
quantity of that gas insufficient for all the acts of nutrition and
assimilation.
Another equally striking proof of the truth of this theory is the fact,
demonstrated in Chapter IV., that the ordinary moulds assume the
character of a ferment when compelled to live without air, or with
quantities of air too scant to permit of their organs having around them
as much of that element as is necessary for their life as aërobian
plants. Ferments, therefore, only possess in a higher degree a character
which belongs to many common moulds, if not to all, and which they
share, probably, more or less, with all living cells, namely the power
of living either an aërobian or anaërobian life, according to the
conditions under which they are placed.
It may be readily understood how, in their state of aërobian life, the
alcoholic ferments have failed to attract attention. Those ferments are
only cultivated out of contact with air, at the bottom of liquids which
soon become saturated with carbonic acid gas. Air is only present in the
earlier developments of their germs, and without attracting the
attention of the operator, whilst in their state of anaërobian growth
their life and action are of prolonged duration. We must have recourse
to special experimental apparatus to enable us to demonstrate the mode
of life of alcoholic ferments under the influence of free oxygen; it is
their state of existence apart from air, in the depths of liquids that
attracts all our attention. The results of their action are, however,
marvellous, if we regard the products resulting from them, in the
important industries of which they are the life and soul. In the case of
ordinary moulds, the opposite holds good. What we want to use special
experimental apparatus for with them is to enable us to demonstrate the
possibility of their continuing to live for a time out of contact with
air, and all our attention, in their case, is attracted by the facility
with which they develop under the influence of oxygen. Thus the
decomposition of saccharine liquids, which is the consequence of the
life of fungi without air, is scarcely perceptible, and so is of no
practical importance. Their aerial life, on the other hand, in which
they respire and accomplish their process of oxidation under the
influence of free oxygen, is a normal phenomenon, and one of prolonged
duration which cannot fail to strike the least thoughtful of observers.
We are convinced that a day will come when moulds will be utilized in
certain industrial operations, on account of their power of destroying
organic matter. The conversion of alcohol into vinegar in the process of
acetification, and the production of gallic acid by the action of fungi
on wet gall-nuts, are already connected with this kind of
phenomena.[122] On this last subject, the important work of M. Van
Tieghem (_Annales Scientifiques de l’École Normale_, vol. vi.) may be
consulted.
The possibility of living without oxygen, in the case of ordinary
moulds, is connected with certain morphological modifications which are
more marked in proportion as this faculty is itself more developed.
These changes in the vegetative forms are scarcely perceptible in the
case of _penicillium_ and _mycoderma vini_, but they are very evident in
the case of _aspergillus_, consisting of a marked tendency on the part
of the submerged mycelial filaments to increase in diameter, and to
develop cross partitions at short intervals, so that they sometimes bear
a resemblance to chains of conidia. In _mucor_, again, they are very
marked, the inflated filaments which, closely interwoven, present chains
of cells which fall off and bud, gradually producing a mass of cells. If
we consider the matter carefully, we shall see that yeast presents the
same characteristics. For instance, what can more closely resemble the
mucor of Plates V. and VI. than the _saccharomyces_ of Figs. 33 and 37?
Have we not in each case ramified chains of elongated cells or joints,
more or less narrowed in the middle, and shorter segments or cells
dropping off at the constrictions, and proceeding to bud in the liquid
on their own account? Moreover, the less oxygen there is present, the
more marked is the tendency to the formation of these budding cells,
which isolate themselves and soon drop off. Who could ever imagine, in
examining the ferment of _mucor_ represented in Plate VI., that its
first germ was the ordinary _mucor_ that is found everywhere, with fine
filaments, straight or ramified according to the variety, which send up
aerial _hyphae_, terminating in little round heads bearing spores. So
was it that in the ferment of Plate XI. we could scarcely recognize the
ramified filaments of Figs. 33 and 37.
It is a great presumption in favour of the truth of theoretical ideas
when the results of experiments undertaken on the strength of those
ideas are confirmed by various facts more recently added to science, and
when those ideas force themselves more and more on our minds, in spite
of a _primâ facie_ improbability. This is exactly the character of those
ideas which we have just expounded. We propounded them in 1861, and not
only have they remained unshaken since, but they have served to
foreshadow new facts, so that it is much easier to defend them in the
present day than it was to do so fifteen years ago. We first called
attention to them in various notes, which we read before the Chemical
Society of Paris, notably at its meetings of April 12th and June 28th,
1861, and in papers in the _Comptes rendus de l’Académie des Sciences_.
It may be of some interest to quote here, in its entirety, our
communication of June 28th, 1861, entitled, “Influences of Oxygen on the
Development of Yeast and on Alcoholic Fermentation,” which we extract
from the _Bulletin de la Société Chimique de Paris_:—
“M. Pasteur gives the results of his researches on the fermentation of
sugar and the development of yeast-cells, according as that fermentation
takes place apart from the influence of free oxygen or in contact with
that gas. His experiments, however, have nothing in common with those of
Gay-Lussac, which were performed with the juice of grapes, crushed under
conditions where they would not be affected by air, and then brought in
contact with oxygen.
“Yeast, when perfectly developed, is able to bud and grow in a
saccharine and albuminous liquid, in the complete absence of oxygen or
air. In this case but little yeast is formed, and a comparatively large
quantity of sugar disappears—sixty or eighty parts for one of yeast
formed. Under these conditions fermentation is very sluggish.
“If the experiment is made in contact with the air, and with a great
surface of liquid, fermentation is rapid. For the same quantity of sugar
decomposed much more yeast is formed. The air with which the liquid is
in contact is absorbed by the yeast. The yeast develops very actively,
but its fermentative character tends to disappear under these
conditions; we find, in fact, that for one part of yeast formed, not
more than from four to ten parts of sugar are transformed. The
fermentative character of this yeast, nevertheless, continues, and
produces even increased effects, if it is made to act on sugar apart
from the influence of free oxygen.
“It seems, therefore, natural to admit that when yeast functions as a
ferment by living apart from the influence of air, it derives oxygen
from the sugar, and that this is the origin of its fermentative
character.
“M. Pasteur explains the fact of the immense activity at the
commencement of fermentations by the influence of the oxygen of the air
held in solution in the liquids, at the time when the action commences.
The author has found, moreover, that the yeast of beer sown in an
albuminous liquid, such as yeast-water, still multiplies, even when
there is not a trace of sugar in the liquid, provided always that
atmospheric oxygen is present in large quantities. When deprived of air,
under these conditions, yeast does not germinate at all. The same
experiments may be repeated with albuminous liquid, mixed with a
solution of non-fermentable sugar, such as ordinary crystallized
milk-sugar. The results are precisely the same.
“Yeast formed thus in the absence of sugar does not change its nature;
it is still capable of causing sugar to ferment, if brought to bear upon
that substance apart from air. It must be remarked, however, that the
development of yeast is effected with great difficulty when it has not a
fermentable substance for its food. In short, the yeast of beer acts in
exactly the same manner as an ordinary plant, and the analogy would be
complete if ordinary plants had such an affinity for oxygen as permitted
them to breathe by appropriating this element from unstable compounds,
in which case, according to M. Pasteur, they would appear as ferments
for those substances.
“M. Pasteur declares that he hopes to be able to realize this result,
that is to say, to discover the conditions under which certain inferior
plants may live apart from air in the presence of sugar, causing that
substance to ferment as the yeast of beer would do.”
This summary and the preconceived views that it set forth have lost
nothing of their exactness; on the contrary, time has strengthened them.
The surmises of the last two paragraphs have received a valuable
confirmation from recent observations made by Messrs. Lechartier and
Bellamy, as well as by ourselves, an account of which we must put before
our readers. It is necessary, however, before touching upon this curious
feature in connection with fermentations to insist on the accuracy of a
passage in the preceding summary, the statement, namely, that yeast
could multiply in an albuminous liquid, in which it found a
non-fermentable sugar, milk-sugar for example. The following is an
experiment on this point:—On August 15th, 1875, we sowed a trace of
yeast in 150 c.c. (rather more than 5 fluid ounces) of yeast-water,
containing 2-½ per cent. of milk-sugar. The solution was prepared in one
of our double-necked flasks, with the necessary precautions to secure
absence of germs, and the yeast sown was itself perfectly pure. Three
months afterwards, November 15th, 1875, we examined the liquid for
alcohol; it contained only the smallest trace; as for the yeast, which
had sensibly developed, collected and dried on a filter paper, it
weighed 0·050 gramme (0·76 grain). In this case we have the yeast
multiplying without giving rise to the least fermentation, like a
fungoid growth, absorbing oxygen, and evolving carbonic acid, and there
is no doubt that the cessation of its development in this experiment was
due to the progressive deprivation of oxygen that occurred. As soon as
the gaseous mixture in the flask consisted entirely of carbonic acid and
nitrogen, the vitality of the yeast was dependent on, and in proportion
to, the quantity of air which entered the flask in consequence of
variations of temperature. The question now arose, was this yeast, which
had developed wholly as an ordinary fungus, still capable of manifesting
the character of a ferment? To settle this point we had taken the
precaution, on August 15th, 1875, of preparing another flask, exactly
similar to the preceding one in every respect, and which gave results
identical with those described. We decanted this on November 15th,
pouring some wort on the deposit of the plant, which remained in the
flask. In less than five hours from the time when we placed it in the
oven, the plant had started fermentation in the wort, as we could see by
the bubbles of gas rising to form patches on the surface of the liquid.
We may add that yeast in the medium which we have been discussing will
not develop at all without air.
The importance of these results can escape no one; they prove clearly
that the fermentative character is not an invariable phenomenon of
yeast-life, they show that yeast is a plant which does not differ from
ordinary plants, and which manifests its fermentative power solely in
consequence of particular conditions under which it is compelled to
live. It may carry on its life as a ferment or not, and after having
lived without manifesting the slightest symptom of fermentative
character, it is quite ready to manifest that character when brought
under suitable conditions. The fermentative property, therefore, is not
a power peculiar to cells of a special nature. It is not a permanent
character of a particular structure, like, for instance, the property of
acidity or alkalinity. It is a peculiarity dependent on external
circumstances and on the nutritive conditions of the organism.
§ II.—Fermentation in Saccharine Fruits Immersed in Carbonic Acid Gas.
The theory which we have, step by step, evolved, on the subject of the
causes of the chemical phenomena of fermentation, may claim a character
of simplicity and generality that is well worthy of attention.
Fermentation is no longer one of those isolated and mysterious phenomena
which do not admit of explanation. It is the consequence of a peculiar
vital process of nutrition which occurs under certain conditions,
differing from those which characterize the life of all ordinary beings,
animal or vegetable, but by which the latter may be affected, more or
less, in a way which brings them, to some extent. within the class of
ferments, properly so called. We can even conceive that the fermentative
character may belong to every organized form, to every animal or
vegetable cell, on the sole condition that the chemico-vital acts of
assimilation and excretion must be capable of taking place in that cell
for a brief period, longer or shorter it may be, without the necessity
for recourse to supplies of atmospheric oxygen; in other words, the cell
must be able to derive its needful heat from the decomposition of some
body which yields a surplus of heat in the process.
As a consequence of these conclusions it should be an easy matter to
show, in the majority of living beings, the manifestation of the
phenomena of fermentation; for there are, probably, none in which all
chemical action entirely disappears, upon the sudden cessation of life.
One day, when we were expressing these views in our laboratory, in the
presence of M. Dumas, who seemed inclined to admit their truth, we
added: “We would make a wager that if we were to plunge a bunch of
grapes into carbonic acid gas, there would be immediately produced
alcohol and carbonic acid, in consequence of a renewed action starting
in the interior cells of the grapes, in such a way that these cells
would assume the function of yeast-cells. We will make the experiment,
and when you come to-morrow—it was our good fortune to have M. Dumas
working in our laboratory at that time—we will give you an account of
the result.” Our predictions were realized. We then endeavoured to find,
in the presence of M. Dumas, who assisted us in our endeavour, cells of
yeast in the grapes; but it was quite impossible to discover any.[123]
Encouraged by this result, we undertook fresh experiments on grapes, on
a melon, on oranges, on plums, and on rhubarb leaves, gathered in the
garden of the _École Normale_, and, in every case, our substance when
immersed in carbonic acid gas, gave rise to the production of alcohol
and carbonic acid. We obtained the following surprising results from
some _prunes de Monsieur_[124]:—On July 31st, 1872, we placed
twenty-four of these plums under a glass cylinder, which we immediately
filled with carbonic acid gas. The plums had been gathered on the
previous day. By the side of the cylinder we placed other twenty-four
plums, which were left there uncovered. Eight days afterwards, in the
course of which time there had been a considerable evolution of carbonic
acid from the cylinder, we withdrew the plums and compared them with
those which had been left exposed to the air. The difference was
striking, almost incredible. Whilst the plums which had been surrounded
with air (the experiments of Bérard have long since taught us that,
under this latter condition, fruits absorb oxygen from the air and emit
carbonic acid gas in almost equal volume) had become very soft and
watery and sweet, the plums taken from under the jar had remained very
firm and hard, the flesh was by no means watery, but they had lost much
sugar. Lastly, when submitted to distillation, after crushing, they
yielded 6·5 grammes (99·7 grains) of alcohol, more than 1 per cent. of
the total weight of the plums. What better proof could we have than
these facts of the existence of a considerable chemical action in the
interior of fruit, an action which derives the heat necessary for its
manifestation from the decomposition of the sugar present in the cells?
Moreover, and this circumstance is especially worthy of our attention,
in all these experiments we found that there was a liberation of heat,
of which the fruits and other organs were the seat, as soon as they were
plunged in the carbonic acid gas. This heat is so considerable that it
may at times be detected by the hand, if the two sides of the cylinder,
one of which is in contact with the objects, are touched alternately. It
also makes itself evident in the formation of vapour, which condenses in
little drops on those parts of the bell which are less directly exposed
to the influence of the heat resulting from the decomposition of the
sugar of the cells.[125]
In short, fermentation is a very general phenomenon. It is life without
air, or life without free oxygen, or, more generally still, it is the
result of a chemical process accomplished on a fermentable substance,
_i.e._ a substance capable of producing heat by its decomposition, in
which process the entire heat used up is derived from a part of the heat
that the decomposition of the fermentable substance sets free. The class
of fermentations, properly so called, is, however, restricted by the
small number of substances capable of decomposing with the production of
heat, and at the same time of serving for the nourishment of lower forms
of life, when deprived of the presence and action of air. This, again,
is a consequence of our theory, which is well worthy of notice.
The facts that we have just mentioned in reference to the formation of
alcohol and carbonic acid in the substance of ripe fruits, under certain
special conditions, and apart from the action of ferment, are already
known to science. They were discovered in 1869 by M. Lechartier,
formerly a pupil in the _École Normale Supérieure_, and his coadjutor,
M. Bellamy.[126] In 1821, in a very remarkable work, especially when we
consider the period when it appeared, Bérard demonstrated several
important propositions in connection with the maturation of fruits:—
I. All fruits, even those that are still green, and likewise even those
that are exposed to the sun, absorb oxygen and set free an almost equal
volume of carbonic acid gas. This is a condition of their proper
ripening.
II. Ripe fruits placed in a limited atmosphere, after having absorbed
all the oxygen and set free an almost equal volume of carbonic acid,
continue to emit that gas in notable quantity, even when no bruise is to
be seen—“as though by a kind of fermentation,” as Bérard actually
observes—and lose their saccharine particles, a circumstance which
causes the fruits to appear more acid, although the actual weight of
their acid may undergo no augmentation whatever.
In this beautiful work, and in all subsequent ones of which the ripening
of fruits has been the subject, two facts of great theoretical value
have escaped the notice of the authors; these are the two facts which
Messrs. Lechartier and Bellamy pointed out, for the first time, namely,
the production of alcohol and the absence of cells of ferments. It is
worthy of remark that these two facts, as we have shown above, were
actually foreshadowed in the theory of fermentation that we advocated as
far back as 1861, and we are happy to add that Messrs. Lechartier and
Bellamy, who, at first, had prudently drawn no theoretical conclusions
from their work, now entirely agree with the theory we have
advanced.[127] Their mode of reasoning is very different from that of
the savants with whom we discussed the subject before the Academy, on
the occasion when the communication which we addressed to the Academy,
in October, 1872, attracted attention once more to the remarkable
observations of Messrs. Lechartier and Bellamy.[128] M. Fremy, in
particular, was desirous of finding in those observations a confirmation
of his views on the subject of _hemi-organism_, and a condemnation of
ours, notwithstanding the fact that the preceding explanations and, more
particularly our Note of 1861, which we have quoted word for word in the
last paragraph, furnish the most conclusive evidence in favour of those
ideas which we advocate. Indeed, as far back as 1861 we pointed out very
clearly that if we could find plants able to live when deprived of air,
in the presence of sugar, they would bring about a fermentation of that
substance, in the same manner as yeast does. Such is the case with the
fungi already studied in Chapter IV.; such, too, is the case with the
fruits employed in the experiments of Messrs. Lechartier and Bellamy,
and in our own experiments, the results of which not only confirm those
obtained by these gentlemen, but even extend them, in so far as we have
shown that fruits, when surrounded with carbonic acid gas, immediately
produce alcohol. When surrounded with air, they live in their aërobian
state, and we have no ferment-action; immersed immediately afterwards in
carbonic acid gas, they now assume their anaërobian state, and at once
begin to act upon the sugar in the manner of ferments, and emit heat. As
for seeing in these facts anything like a confirmation of the theory of
hemi-organism, imagined by M. Fremy, the idea of such a thing is absurd.
The following, for instance, is the theory of the fermentation of the
vintage, according to M. Fremy.[129]
“To speak here of alcoholic fermentation alone,”[130] our author says,
“I hold that in the production of wine it is the juice of the fruit
itself that, in contact with air, produces grains of ferment, by the
transformation of the albuminous matter; M. Pasteur, on the other hand,
maintains that the fermentation is produced by germs existing outside
the skin of the grapes.”
Now what bearing on this purely imaginary theory can the fact have, that
a whole fruit, immersed in carbonic acid gas, immediately produces
alcohol and carbonic acid? In the preceding passage, which we have
borrowed from M. Fremy, an indispensable condition of the transformation
of the albuminous matter is the contact with air and the crushing of the
grapes. Here, however, we are dealing with _uninjured fruits in contact
with carbonic acid gas_. Our theory, on the other hand, which, we may
repeat, we have advocated since 1861, maintains that all cells become
fermentative when their vital action is protracted in the absence of
air, which are precisely the conditions that hold in the experiment on
fruits immersed in carbonic acid gas. The vital energy is not
immediately suspended in their cells, and the latter are deprived of
air. Consequently, fermentation must result. Moreover, we may add, if we
destroy the fruit, or crush it before immersing it in the gas, it no
longer produces alcohol or fermentation of any kind, a circumstance that
may be attributed to the fact of the destruction of vital action in the
crushed fruit. On the other hand, in what way ought this crushing to
affect the hypothesis of hemi-organism? The crushed fruit ought to act
quite as well, or even better than that which is uncrushed. In short,
nothing can be more directly opposed to the theory of the mode of
manifestation of that hidden force to which the name of hemi-organism
has been given, than the discovery of the production of these phenomena
of fermentation in fruits surrounded with carbonic acid gas; whilst the
theory, which sees in fermentation a consequence of vital energy in
absence of air, finds in these facts the strictest confirmation of an
express prediction, which from the first formed an integral part of its
statement.
We should not be justified in devoting further time to opinions which
are not supported by any serious experiment. Abroad, as well as in
France, the theory of the transformation of albuminous substances into
organized ferments had been advocated long before it was taken up by M.
Fremy. It no longer commands the slightest credit, nor do any observers
of note any longer give it the least attention; it might even be said
that it has become a subject of ridicule.
An attempt has also been made to prove that we have contradicted
ourselves, inasmuch as in 1860 we published our opinion that alcoholic
fermentation can never occur without a simultaneous occurrence of
organization, development, and multiplication of globules; or continued
life, carried on from globules already formed.[131] Nothing, however,
can be truer than that opinion, and at the present moment, after fifteen
years of study devoted to the subject, since the publication to which we
have referred, we need no longer say “we think,” but instead, “we
affirm” that it is correct. It is, as a matter of fact, to alcoholic
fermentation, properly so called, that the charge to which we have
referred relates—to that fermentation which yields, besides alcohol,
carbonic acid, succinic acid, glycerine, volatile acids, and other
products. This fermentation undoubtedly requires the presence of
yeast-cells, under the conditions that we have named. Those who have
contradicted us have fallen into the error of supposing that the
fermentation of fruits is an ordinary alcoholic fermentation, identical
with that produced by beer-yeast, and that, consequently, the cells of
that yeast must, according to our own theory, be always present. There
is not the least authority for such a supposition. When we come to exact
quantitative estimations—and these are to be found in the figures
supplied by Messrs. Lechartier and Bellamy—it will be seen that the
proportions of alcohol and carbonic acid gas produced in the
fermentation of fruits differ widely from those that we find in
alcoholic fermentations, properly so called, as must necessarily be the
case, since, in the former, the ferment-action is effected by the cells
of a fruit, but in the latter by cells of ordinary alcoholic ferment.
Indeed we have a strong conviction that each fruit would be found to
give rise to a special action, the chemical equation of which would be
different from that in the case of other fruits. As for the circumstance
that the cells of these fruits cause fermentation, without multiplying,
this comes under the kind of activity, which we have already
distinguished by the expression _continuous life in cells already
formed_.
We will conclude this paragraph with a few remarks on the subject of the
equations of fermentations, which have been suggested to us principally
in attempts to explain the results derived from the fermentation of
fruits immersed in carbonic acid gas.
Originally, when fermentations were put amongst the class of
decompositions by contact-action, it seemed probable, and, in fact, was
believed, that every fermentation had its own well-defined equation,
which never varied. In the present day, on the contrary, it must be
borne in mind that the equation of a fermentation varies essentially
with the conditions under which that fermentation is accomplished, and
that a statement of this equation is a problem no less complicated than
that in the case of the nutrition of a living being. To every
fermentation may be assigned an equation in a general sort of way, an
equation, however, which, in numerous points of detail, is liable to the
thousand variations connected with the phenomena of life. Moreover,
there will be as many distinct fermentations brought about by one
ferment as there are fermentable substances capable of supplying the
carbon element of the food of that same ferment, in the same way that
the equation of the nutrition of an animal will vary with the nature of
the food which it consumes. As regards fermentation producing alcohol,
which may be effected by several different ferments, there will be, in
the case of a given sugar, as many general equations as there are
ferments, whether they be ferment-cells, properly so called, or cells of
the organs of living beings functioning as ferments. In the same way the
equation of nutrition varies in the case of different animals nourished
on the same food. And it is from the same reason that ordinary wort
produces such a variety of beers when treated with the numerous
alcoholic ferments which we have described. These remarks are applicable
to all ferments alike; for instance, butyric ferment is capable of
producing a host of distinct fermentations, in consequence of its
ability to derive the carbonaceous part of its food from very different
substances, from sugar, or lactic acid, or glycerine, or mannite, and
many others.
When we say that every fermentation has its own peculiar ferment, it
must be understood that we are speaking of the fermentation considered
as a whole, including all the accessory products. We do not mean to
imply that the ferment in question is not capable of acting on some
other fermentable substance and giving rise to fermentation of a very
different kind. Moreover, it is quite erroneous to suppose that the
presence of a single one of the products of a fermentation implies the
co-existence of a particular ferment. If, for example, we find alcohol
among the products of a fermentation, or even alcohol and carbonic acid
gas together, this does not prove that the ferment must be an alcoholic
ferment, belonging to alcoholic fermentations, in the strict sense of
the term. Nor, again, does the mere presence of lactic acid necessarily
imply the presence of lactic ferment. As a matter of fact, different
fermentations may give rise to one or even several identical products.
We could not say with certainty, from a purely chemical point of view,
that we were dealing, for example, with an alcoholic fermentation,
properly so called, and that the yeast of beer must be present in it, if
we had not first determined the presence of all the numerous products of
that particular fermentation, and that they were present in those
proportions, characteristic of that fermentation under conditions
similar to those under which the fermentation in question had occurred.
In works on fermentation, the reader will often find those confusions
against which we are now attempting to guard him. It is precisely in
consequence of not having had their attention drawn to such observations
that some have imagined that the fermentation in fruits, immersed in
carbonic acid gas, is in contradiction to the assertion which we
originally made in our Memoir on alcoholic fermentation, published in
1860, the exact words of which we may here repeat:—“The chemical
phenomena of fermentation are related essentially to a vital activity,
beginning and ending with the latter; we believe that alcoholic
fermentation never occurs”—we were discussing the question of ordinary
alcoholic fermentation produced by the yeast of beer—“without the
simultaneous occurrence of organization, development, and multiplication
of globules, or continued life, carried on by means of globules already
formed. The general results of the present Memoir seem to us to be in
direct opposition to the opinions of MM. Liebig and Berzelius.” These
conclusions, we repeat, are as true now as they ever were, and are as
applicable to the fermentation of fruits, of which nothing was known in
1860, as they are to the fermentation produced by means of yeast. Only,
in the case of fruits, it is the cells of the parenchyma that function
as ferment, _by a continuation of their vital activity in carbonic acid
gas_, whilst in the other case the ferment consists of the cells of
yeast.
There should be nothing very surprising in the fact that fermentation
can originate in fruits and form alcohol, without the presence of yeast,
if the fermentation of fruits were not confounded completely with
ordinary alcoholic fermentation, yielding the same products and in the
same proportions. It is through the misuse of words that the
fermentation of fruits has been termed _alcoholic_, in a way which has
misled many persons.[132] In this fermentation, neither alcohol nor
carbonic acid gas exists in those proportions in which they are found in
fermentations produced by yeast; and although we may determine in it the
presence of succinic acid, glycerine, and a small quantity of volatile
acids,[133] the relative proportions of these substances will be
different from what they are in the case of alcoholic fermentation.
§ III.—Reply to certain Critical Observations of the German Naturalists,
Oscar Brefeld and Moritz Traube.
The essential point of the theory of fermentation, which we have been
concerned in proving in preceding paragraphs, may be briefly put in the
statement that ferments, properly so called, constitute a class of
beings possessing the faculty of living out of contact with free oxygen;
or, more concisely still, we may say, fermentation is a result of life
without air.
If our affirmation were inexact, if ferment-cells did require for their
growth or for their increase in number or weight, as all other vegetable
cells do, the presence of oxygen, whether gaseous or held in solution in
liquids, this new theory would lose all value, its very _raison d’être_
would be gone, at least as far as the most important part of
fermentations is concerned. This is precisely what M. Oscar Brefeld has
endeavoured to prove, in a Memoir read to the Physico-Medical Society of
Wurzburg, on July 26th, 1873, in which, although we have ample evidence
of the great experimental skill of its author, he has, nevertheless, in
our opinion, arrived at conclusions entirely opposed to fact.
“From the experiments which I have just described,” he says, “it
follows, in the most indisputable manner, that _a ferment cannot
increase without free oxygen_. Pasteur’s supposition that a ferment,
unlike all other living organisms, can live and increase at the expense
of oxygen held in combination, is, consequently, altogether wanting in
any solid basis of experimental proof. Moreover, since, according to the
theory of Pasteur, it is precisely this faculty of living and increasing
at the expense of the oxygen held in combination that constitutes the
phenomenon of fermentation, it follows that the whole theory, commanding
though it does such general assent, is shown to be untenable; it is
simply inaccurate.”
The experiments to which Dr. Brefeld alludes, consisted in keeping under
continued study with the microscope, in a room specially prepared for
the purpose, one or more cells of ferment in wort, in an atmosphere of
carbonic acid gas, free from the least traces of free oxygen. We have,
however, recognized the fact that the increase of a ferment out of
contact with air is only possible in the case of a very young specimen;
but our author employed brewer’s yeast taken after fermentation, and to
this fact we may attribute the non-success of his growths. Dr. Brefeld,
without knowing it, operated on yeast in one of the states in which it
requires gaseous oxygen to enable it to germinate again. A perusal of
what we have previously written on the subject of the revival of yeast,
according to its age, will show how widely the time required for such
revival may vary in different cases. What may be perfectly true of the
state of a yeast to-day may not be so to-morrow, since yeast is
continually undergoing modifications. We have already shown the energy
and activity with which a ferment can vegetate in the presence of free
oxygen, and we have pointed out the great extent to which a very small
quantity of oxygen held in solution in fermenting liquids can operate at
the beginning of fermentation. It is this oxygen that produces revival
in the cells of the ferment and enables them to resume the faculty of
germinating and continuing their life, and of multiplying when deprived
of air.
In our opinion, a simple reflection should have guarded Dr. Brefeld
against the interpretation which he has attached to his observations. If
a cell of ferment cannot bud or increase without absorbing oxygen,
either free or held in solution in the liquid, the ratio between the
weight of ferment formed during fermentation and that of oxygen used up
must be constant. We had, however, clearly established, as far back as
1861, the fact that this ratio is extremely variable, a fact, moreover,
which is placed beyond doubt by the experiments described in the
preceding paragraph. Though but small quantities of oxygen are absorbed,
a considerable weight of ferment may be generated; whilst if the ferment
has abundance of oxygen at its disposal, it will absorb much, and the
weight of yeast formed will be still greater. The ratio between the
weight of ferment formed and that of sugar decomposed may pass through
all stages between certain very wide limits, the variations depending on
the greater or less absorption of free oxygen. And in this fact, we
believe, lies one of the most essential supports of the theory which we
advocate. In denouncing the impossibility, as he considered it, of a
ferment living without air or oxygen, and so acting in defiance of that
law which governs all living beings, animal or vegetable, Dr. Brefeld
ought also to have borne in mind the fact which we have pointed out,
that alcoholic yeast is not the only organized ferment which lives in an
anaërobian state. It is really a small matter that one more ferment
should be placed in a list of exceptions to the generality of living
beings, for whom there is a rigid law in their vital economy which
requires for continued life a continuous respiration, a continuous
supply of free oxygen. Why, for instance, has Dr. Brefeld omitted the
facts bearing on the life of the vibrios of butyric fermentation?
Doubtless he thought we were equally mistaken in these: a few actual
experiments would have put him right.
These remarks on the criticisms of Dr. Brefeld are also applicable to
certain observations of M. Moritz Traube’s, although, as regards the
principal object of Dr. Brefeld’s attack, we are indebted to M. Traube
for our defence. This gentleman maintained the exactness of our results
before the Chemical Society of Berlin, proving by fresh experiments that
yeast is able to live and multiply without the intervention of oxygen.
“My researches,” he said, “confirm in an indisputable manner M.
Pasteur’s assertion that the multiplication of yeast can take place in
media which contain no trace of free oxygen.... M. Brefeld’s assertion
to the contrary is erroneous.” But, immediately afterwards, M. Traube
adds: “Have we here a confirmation of Pasteur’s theory? By no means. The
results of my experiments demonstrate, on the contrary, that this theory
has no sure foundation.” What were these results? Whilst proving that
yeast could live without air, M. Traube, as we ourselves did, found that
it had great difficulty in living under these conditions; indeed he
never succeeded in obtaining more than the first stages of true
fermentation. This was doubtless for the two following reasons—first, in
consequence of the accidental production of secondary and diseased
fermentations, which frequently prevent the propagation of alcoholic
ferment; and, secondly, in consequence of the original exhausted
condition of the yeast employed. As long ago as 1861 we pointed out the
slowness and difficulty of the vital action of yeast when deprived of
air, and a little way back, in the preceding paragraph, we have called
attention to certain fermentations that cannot be completed under such
conditions without going into the causes of these peculiarities. M.
Traube expresses himself thus: “Pasteur’s conclusion, that yeast in the
absence of air is able to derive the oxygen necessary for its
development from sugar, is erroneous; its increase is arrested, even
when the greater part of the sugar still remains undecomposed. _It is in
a mixture of albuminous substances that yeast, when deprived of air,
finds the materials for its development._” This last assertion of M.
Traube’s is entirely disproved by those fermentation experiments in
which, after suppressing the presence of albuminous substances, the
action, nevertheless, went on in a purely inorganic medium, out of
contact with air, a fact of which we shall give irrefutable proofs.[134]
§ IV.—Fermentation of Dextro-Tartrate of Lime.[135]
Tartrate of lime, in spite of its insolubility in water, is capable of
complete fermentation in a mineral medium.
If we put some pure tartrate of lime, in the form of a granulated,
crystalline powder, into pure water, together with some sulphate of
ammonia and phosphates of potassium and magnesium, in very small
proportions, a spontaneous fermentation will take place in the deposit
in the course of a few days, although no germs of ferment have been
added. A living, organized ferment, of the vibrionic type, filiform,
with tortuous motions, and often of immense length, forms spontaneously
by the development of some germs derived in some way from the inevitable
particles of dust floating in the air or resting on the surface of the
vessels or materials which we employ. The germs of the vibrios concerned
in putrefaction are diffused around us on every side, and, in all
probability, it is one or more of these germs that develop in the medium
in question. In this way they effect the decomposition of the tartrate,
from which they must necessarily obtain the carbon of their food,
without which they cannot exist, while the nitrogen is furnished by the
ammonia of the ammoniacal salt, the mineral principles by the phosphate
of potassium and magnesium, and the sulphur by the sulphate of ammonia.
How strange to see organization, life, and motion originating under such
conditions! Stranger still to think that this organization, life, and
motion are effected without the participation of free oxygen. Once the
germ gets a primary impulse on its living career by access of oxygen, it
goes on reproducing indefinitely, absolutely without atmospheric air.
Here then we have a fact which it is important to establish beyond the
possibility of doubt, that we may prove that yeast is not the only
organized ferment able to live and multiply when out of the influence of
free oxygen.
Into a flask, like that represented in Fig. 67, of 2·5 litres (about
four pints) in capacity, we put:—
Pure, crystallized, neutral tartrate of 100 grammes.
lime
Phosphate of ammonia 1 gramme.
Phosphate of magnesium 1 gramme.
Phosphate of potassium 0·5 gramme.
Sulphate of ammonia 0·5 gramme.
(1 gramme=15·43 grains.)
To this we added pure distilled water, so as to entirely fill the flask.
In order to expel all the air dissolved in the water and adhering to the
solid substances, we first placed our flask in a bath of chloride of
calcium, in a large cylindrical white iron pot, set over a flame. The
exit-tube of the flask was plunged in a test-tube of Bohemian glass
three-quarters full of distilled water, and also heated by a flame. We
boiled the liquids in the flask and test-tube for a sufficient time to
expel all the air contained in them. We then withdrew the heat from
under the test-tube, and immediately afterwards covered the water which
it contained with a layer of oil, and then permitted the whole apparatus
to cool down.
[Illustration: Fig. 67.]
Next day we applied a finger to the open extremity of the exit-tube,
which we then plunged in a vessel of mercury. In this particular
experiment which we are describing, we permitted the flask to remain in
this state for a fortnight. It might have remained for a century without
ever manifesting the least sign of fermentation, the fermentation of the
tartrate being a consequence of life, and life after the boiling no
longer existed in the flask. When it was evident that the contents of
the flask were perfectly inert, we impregnated them rapidly, as
follows:—All the liquid contained in the exit-tube was removed by means
of a fine caoutchouc tube, and replaced by about 1 c.c. (about 17
minims) of liquid and deposit from another flask, similar to the one we
have described, but which had been fermenting spontaneously for twelve
days; we lost no time in refilling completely the exit-tube with water
which had been first boiled and then cooled down in carbonic acid gas.
This operation lasted only a few minutes. The exit-tube was again
plunged under mercury. Subsequently the tube was not moved from under
the mercury, and as it formed part of the flask, and there was neither
cork nor india-rubber, any introduction of air was consequently
impossible. The small quantity of air introduced during the impregnation
was insignificant, and it might even be shown that it injured rather
than assisted the growth of the organisms, inasmuch as these consisted
of adult individuals which had lived without air and might be liable to
be damaged or even destroyed by it. Be this as it may, in a subsequent
experiment we shall find the possibility removed of any aeration taking
place in this way, however infinitesimal, so that no doubt may linger on
this subject.
The following days the organisms multiplied, the deposit of tartrate
gradually disappeared, and a sensible ferment action was manifest on the
surface, and throughout the bulk of the liquid. The deposit seemed
lifted up in places, and was covered with a layer of a dark-grey colour,
puffed up, and having an organic and gelatinous appearance. For several
days, in spite of this action in the deposit, we detected no
disengagement of gas, except when the flask was slightly shaken, in
which case rather large bubbles adhering to the deposit rose, carrying
with them some solid particles, which quickly fell back again, whilst
the bubbles diminished in size as they rose, from being partially taken
into solution, in consequence of the liquid not being saturated. The
smallest bubbles had even time to dissolve completely before they could
reach the surface of the liquid. In course of time the liquid was
saturated, and the tartrate was gradually displaced by mammillated
crusts, or clear, transparent crystals of carbonate of lime at the
bottom and on the sides of the vessel.
The impregnation took place on February 10th, and on March 15th the
liquid was nearly saturated. The bubbles then began to lodge in the bent
part of the exit-tube, at the top of the flask. A glass measuring-tube
containing mercury was now placed with its open end over the point of
the exit-tube under the mercury in the trough, so that no bubble might
escape. A steady evolution of gas went on from the 17th to the 18th,
17·4 c.c. (1·06 cubic inches) having been collected. This was proved to
be nearly absolutely pure carbonic acid, as indeed might have been
suspected from the fact that the evolution did not begin before a
distinct saturation of the liquid was observed.[136]
The liquid, which was turbid on the day after its impregnation, had, in
spite of the liberation of gas, again become so transparent that we
could read our handwriting through the body of the flask.
Notwithstanding this, there was still a very active operation going on
in the deposit, but it was confined to that spot. Indeed, the swarming
vibrios were bound to remain there, the tartrate of lime being still
more insoluble in water saturated with carbonate of lime than it is in
pure water. A supply of carbonaceous food, at all events, was absolutely
wanting in the bulk of the liquid. Every day we continued to collect and
analyze the total amount of gas disengaged. To the very last, it was
composed of pure carbonic acid gas. Only during the first few days did
the absorption by the concentrated potash leave a very minute residue.
By April 26th all liberation of gas had ceased, the last bubbles having
risen in the course of April 23rd. The flask had been all the time in
the oven, at a temperature between 25° C. and 28° C. (77° F. and 83°
F.). The total volume of gas collected was 2·135 litres (130·2 cubic
inches). To obtain the whole volume of gas formed we had to add to this
what was held in the liquid in the state of acid carbonate of lime. To
determine this we poured a portion of the liquid from the flask into
another flask of similar shape, but smaller, up to a gauge-mark on the
neck.[137] This smaller flask had been previously filled with carbonic
acid. The carbonic acid of the fermented liquid was then expelled by
means of heat, and collected over mercury. In this way we found a volume
of 8·322 litres (508 cubic inches) of gas in solution, which, added to
2·135 litres, gave a total of 10·457 litres (638·2 cubic inches) at 20°
and 760, which calculated to 0° C. and 760 mm. atmospheric pressure (32°
F. and 30 inches) gave a weight of 19·70 grammes (302·2 grains) of
carbonic acid.
Exactly half of the lime of the tartrate employed got used up in the
soluble salts formed during fermentation; the other half was partly
precipitated in the form of carbonate of lime, partly dissolved in the
liquid by the carbonic acid. The soluble salts seemed to us to be a
mixture or combination of 1 equivalent of metacetate of lime, with 2
equivalents of the acetate, for every 10 equivalents of carbonic acid
produced, the whole corresponding to the fermentation of 3 equivalents
of neutral tartrate of lime.[138] This point, however, is worthy of
being studied with greater care: the present statement of the nature of
the products formed is given with all reserve. For our point, indeed,
the matter is of little importance, since the equation of the
fermentation does not concern us.
[Illustration: Fig. 68.]
After the completion of fermentation there was not a trace of tartrate
of lime remaining at the bottom of the vessel: it had disappeared
gradually as it got broken up into the different products of
fermentation, and its place was taken by some crystallized carbonate of
lime—the excess, namely, which had been unable to dissolve by the action
of the carbonic acid. Associated, moreover, with this carbonate of lime
there was a quantity of some kind of animal matter, which, under the
microscope, appeared to be composed of masses of granules mixed with
very fine filaments of varying lengths, studded with minute dots, and
presenting all the characteristics of a nitrogenous organic
substance.[139] That this was really the ferment is evident enough from
all that we have already said. To convince ourselves more thoroughly of
the fact, and at the same time to enable us to observe the mode of
activity of the organism, we instituted the following supplementary
observation. Side by side with the experiment just described, we
conducted a similar one, which we intermitted after the fermentation was
somewhat advanced, and about half of the tartrate dissolved. Breaking
off, with a file, the exit tube at the point where the neck began to
narrow off, we took some of the deposit from the bottom by means of a
long, straight piece of tubing, in order to bring it under microscopical
examination. We found it to consist of a host of long filaments of
extreme tenuity, their diameter being about 1/1000th of a millimetre
(0·000039 in.); their length varied, in some cases being as much as
1/20th of a millimetre (0·0019 in.). A crowd of these long vibrios were
to be seen creeping slowly along, with a sinuous movement, showing
three, four, or even five flexures. The filaments that were at rest had
the same aspect as these last, with the exception that they appeared
punctate, as though composed of a series of granules arranged in
irregular order. No doubt these were vibrios in which vital action had
ceased, exhausted specimens which we may compare with the old granular
ferment of beer, whilst those in motion may be compared with young and
vigorous yeast. The absence of movement in the former seems to prove
that this view is correct. Both kinds showed a tendency to form
clusters, the compactness of which impeded the movements of those which
were in motion. Moreover, it was noticeable that the masses of these
latter rested on tartrate not yet dissolved, whilst the granular
clusters of the others rested directly on the glass, at the bottom of
the flask, as if, having decomposed the tartrate, the only carbonaceous
food at their disposal, they had then died at the spot where we captured
them from inability to escape, precisely in consequence of that state of
entanglement which they combined to form, during the period of their
active development. Besides these we observed vibrios of the same
diameter, but of much smaller length, whirling round with great
rapidity, and darting backwards and forwards; these were probably
identical with the longer ones, and possessed greater freedom of
movement, no doubt in consequence of their greater shortness. Not one of
these vibrios could be found throughout the mass of the liquid.
We may remark that as there was a somewhat putrid odour from the deposit
in which the vibrios swarmed, the action must have been one of
reduction, and no doubt to this fact was due the greyish coloration of
the deposit. We suppose that the substances employed, however pure,
always contain some trace of iron, which becomes converted into the
sulphide, the black colour of which would modify the originally white
deposit of insoluble tartrate and phosphate.
But what is the nature of these vibrios? We have already said that we
believe that they are nothing but the ordinary vibrios of putrefaction,
reduced to a state of extreme tenuity by the special conditions of
nutrition involved in the fermentable medium used; in a word, we think
that the fermentation in question might be called putrefaction of
tartrate of lime. It would be easy enough to determine this point by
growing the vibrios of such a fermentation in media adapted to the
production of the ordinary forms of vibrio; but this is an experiment
which we have not ourselves tried.
One word more on the subject of these curious beings. In a great many of
them there appears to be something like a clear spot, a kind of bead, at
one of their extremities. This is an illusion arising from the fact that
the extremity of these vibrios is curved, hanging downwards, thus
causing a greater refraction at that particular point, and leading us to
think that the diameter is greater at that extremity. We may easily
undeceive ourselves if we watch the movements of the vibrio, when we
will readily recognize the bend, especially as it is brought into the
vertical plane passing over the rest of the filament. In this way we
will see the bright spot, the head disappear, and then reappear.
The chief inference that it concerns us to draw from the preceding facts
is one which cannot admit of doubt, and which we need not insist on any
further—namely, that vibrios, as met with in the fermentation of neutral
tartrate of lime, are able to live and multiply when entirely deprived
of air.
§ V.—Another Example of Life Without Air—Fermentation of Lactate of
Lime.
As another example of life without air, accompanied by fermentation
properly so called, we may lastly cite the fermentation of lactate of
lime in a mineral medium.
In the experiment described in the last paragraph, it will be remembered
that the ferment-liquid and the germs employed in its impregnation came
in contact with air, although only for a very brief time. Now,
notwithstanding that we possess exact observations which prove that the
diffusion of oxygen and nitrogen in a liquid absolutely deprived of air,
so far from taking place rapidly, is, on the contrary, a very slow
process indeed; yet we were anxious to guard the experiment that we are
about to describe from the slightest possible trace of oxygen at the
moment of impregnation.
We employed a liquid prepared as follows: Into from 9 to 10 litres
(somewhat over 2 gallons) of pure water the following salts[140] were
introduced successively, viz:—
Pure lactate of lime 225 grammes
Phosphate of ammonia 0·75 grammes
Phosphate of potassium 0·4 grammes
Sulphate of magnesium 0·4 grammes
Sulphate of ammonia 0·2 grammes
[1 gramme=15·43 grains.]
[Illustration: Fig. 69.]
On March 23rd, 1875, we filled a 6 litre (about 11 pints) flask, of the
shape represented in Fig. 69, and placed it over a heater. Another flame
was placed below a vessel containing the same liquid, into which the
curved tube of the flask was plunged. The liquids in the flask and in
the basin were raised to boiling together, and kept in this condition
for more than half-an-hour, so as to expel all the air held in solution.
The liquid was several times forced out of the flask by the steam, and
sucked back again; but the portion which re-entered the flask was always
boiling. On the following day, when the flask had cooled, we transferred
the end of the delivery tube to a vessel full of mercury and placed the
whole apparatus in an oven at a temperature varying between 25° C. and
30° C. (77° F. and 86° F.); then, after having refilled the small
cylindrical tap-funnel with carbonic acid, we passed into it with all
necessary precautions 10 c.c. (0·35 fl. oz.) of a liquid similar to that
described, which had been already in active fermentation for several
days out of contact with air and now swarmed with vibrios. We then
turned the tap of the funnel, until only a small quantity of liquid was
left, just enough to prevent the access of air. In this way the
impregnation was accomplished without either the ferment-liquid or the
ferment-germs having been brought in contact, even for the shortest
space, with the external air. The fermentation, the occurrence of which
at an earlier or later period depends for the most part on the condition
of the impregnating germs, and the number introduced in the act, in this
case began to manifest itself by the appearance of minute bubbles from
March 29th. But not till April 9th did we observe bubbles of larger size
rise to the surface. From that date onward they continued to come in
increasing number, from certain points at the bottom of the flask, where
a deposit of earthy phosphates existed; and at the same time the liquid,
which for the first few days remained perfectly clear, began to grow
turbid in consequence of the development of vibrios. It was on the same
day that we first observed a deposit on the sides of carbonate of lime
in crystals.
It is a matter of some interest to notice here that, in the mode of
procedure adopted, everything combined to prevent the interference of
air. A portion of the liquid expelled at the beginning of the
experiment, partly because of the increased temperature in the oven and
partly also by the force of the gas, as it began to be evolved from the
fermentative action, reached the surface of the mercury, where, being
the most suitable medium we know for the growth of bacteria, it speedily
swarmed with these organisms.[141] In this way any passage of air, if
such a thing were possible, between the mercury and the sides of the
delivery-tube was altogether prevented, since the bacteria would consume
every trace of oxygen which might be dissolved in the liquid lying on
the surface of the mercury. Hence it is impossible to imagine that the
slightest trace of oxygen could have got into the liquid in the flask.
Before passing on we may remark that in this ready absorption of oxygen
by bacteria we have a means of depriving fermentable liquids of every
trace of that gas with a facility and success equal or even greater than
by the method of preliminary boiling. Such a solution as we have
described, if kept at summer heat, without any previous boiling, becomes
turbid in the course of twenty-four hours from a _spontaneous_
development of bacteria; and it is easy to prove that they absorb all
the oxygen held in solution.[142] If we completely fill a flask of a few
litres capacity (about a gallon) (Fig. 67) with the liquid described,
taking care to have the delivery-tube also filled, and its opening
plunged under mercury, and, forty-eight hours afterwards, by means of a
chloride of calcium bath, expel from the liquid on the surface of the
mercury all the gas which it holds in solution, this gas, when analyzed,
will be found to be composed of a mixture of nitrogen and carbonic acid
gas, _without the least trace of oxygen_. Here, then, we have an
excellent means of depriving the fermentable liquid of air; we have
simply to completely fill a flask with the liquid, and place it in the
oven, merely avoiding any addition of butyric vibrios before the lapse
of two or three days. We may wait even longer; and then, if the liquid
does not become impregnated spontaneously with vibrio germs, the liquid,
which at first was turbid from the presence of bacteria, will become
bright again, since the bacteria when deprived of life, or, at least, of
the power of moving, after they have exhausted all the oxygen in
solution, will fall inert to the bottom of the vessel. On several
occasions, we have determined this interesting fact, which tends to
prove that the butyric vibrios cannot be regarded as another form of
bacteria, inasmuch as, on the hypothesis of an original relation between
the two productions, butyric fermentation ought in every case to follow
the growth of bacteria.
We may also call attention to another striking experiment, well suited
to show the effect of differences in the composition of the medium upon
the propagation of microscopic beings. The fermentation which we last
described commenced on March 27th and continued until May 10th; that to
which we are now to refer, however, was completed in four days, the
liquid employed being similar in composition and quantity to that
employed in the former experiment. On April 23rd, 1875, we filled a
flask of the same shape as that represented in Fig. 69, and of similar
capacity, viz., 6 litres, with a liquid composed as described at page
293. This liquid had been previously left to itself for five days in
large open flasks, in consequence of which it had developed an abundant
growth of bacteria. On the fifth day a few bubbles, rising from the
bottom of the vessels, at long intervals, betokened the commencement of
butyric fermentation, a fact, moreover, confirmed by the microscope, in
the appearance of the vibrios of this fermentation in specimens of the
liquid taken from the bottom of the vessels, the middle of its mass, and
even in the layer on the surface that was swarming with bacteria. We
transferred the liquid so prepared to the 6-litre flask arranged over
the mercury. By evening a tolerably active fermentation had begun to
manifest itself. On the 24th this fermentation was proceeding with
astonishing rapidity, which continued during the 25th and 26th. During
the evening of the 26th it slackened, and on the 27th all signs of
fermentation had ceased. This was not, as might be supposed, a sudden
stoppage, due to some unknown cause; the fermentation was actually
completed, for when we examined the fermented liquid on the 28th we
could not find the smallest quantity of lactate of lime. If the needs of
industry should ever require the production of large quantities of
butyric acid, there would, beyond doubt, be found in the preceding fact
valuable information in devising an easy method of preparing that
product in abundance.[143]
Before we go any further, let us devote some attention to the vibrios of
the preceding fermentations.
On May 27th, 1862, we completely filled a flask, capable of holding
2·780 litres (about five pints), with the solution of lactate and
phosphates.[144] We refrained from impregnating it with any germs. The
liquid became turbid from a development of bacteria, and then underwent
butyric fermentation. By June 9th the fermentation had become
sufficiently active to enable us to collect in the course of twenty-four
hours, over mercury, as in all our experiments, about 100 cc. (about 6
cubic inches) of gas. By June 11th, judging from the volume of gas
liberated in the course of twenty-four hours, the activity of the
fermentation had doubled. We examined a drop of the turbid liquid. Here
are the notes accompanying the sketch (Fig. 70) as they stand in our
note-book:—“A swarm of vibrios, so active in their movements that the
eye has great difficulty in following them. They may be seen in pairs
throughout the field, apparently making efforts to separate from each
other. The connection would seem to be by some invisible, gelatinous
thread, which yields so far to their efforts that they succeed in
breaking away from actual contact, but yet are, for a while, so far
restrained that the movements of one have a visible effect on those of
the other. By and by, however, we see a complete separation effected,
and each moves on its separate way with an activity still greater than
it had before.”
[Illustration: Fig. 70.]
One of the best methods that can be employed for the microscopical
examination of these vibrios, quite out of contact with air, is the
following:—After butyric fermentation has been going on for several days
in a flask, A (Fig. 71), we connect this flask by an india-rubber tube
with one of the flattened bulbs previously described, page 156 (Fig.
31), which we then place on the stage of the microscope (Fig. 71). When
we wish to make an observation we close, under the mercury, at the point
_b_, the end of the drawn-out and bent delivery-tube. The continued
evolution of gas soon exerts such a pressure within the flask, that when
we open the tap _r_, the liquid is driven into the bulb _l l_, until it
becomes quite full and the liquid flows over into the glass V. In this
manner we may bring the vibrios under observation without their coming
into contact with the least trace of air, and with as much success as if
the bulb, which takes the place of an object glass, had been plunged
into the very centre of the flask. The movements and fissiparous
multiplication of the vibrios may thus be seen in all their beauty, and
it is indeed a most interesting sight. The movements do not immediately
cease when the temperature is suddenly lowered, even to a considerable
extent, 15° C. (59° F.) for example; they are only slackened.
Nevertheless, it is better to observe them at the temperatures most
favourable to fermentation, even in the oven where the vessels employed
in the experiment are kept at a temperature between 25° C. and 30° C.
(77° F. and 86° F.).
[Illustration: Fig. 71.]
We may now continue our account of the fermentation which we were
studying when we made this last digression. On June 17th that
fermentation produced three times as much gas as it did on June 11th,
when the residue of hydrogen, after absorption by potash, was 72·6 per
cent.; whilst on the 17th it was only 49·2 per cent. Let us again
discuss the microscopic aspect of the turbid liquid at this stage.
Appended is the sketch we made (Fig. 72) and our notes on it:—“A most
beautiful object: vibrios all in motion, advancing or undulating. They
have grown considerably in bulk and length since the 11th; many of them
are joined together into long sinuous chains, very mobile at the
articulations, visibly less active and more wavering in proportion to
the number that go to form the chain, or the length of the individuals.”
This description is applicable to the majority of the vibrios which
occur in cylindrical rods and are homogeneous in aspect. There are
others, of rare occurrence in chains, which have a clear corpuscle, that
is to say, a portion more refractive than the other parts of the
segments, at one of their extremities. Sometimes the foremost segment
has the corpuscle at one end, sometimes at the other. The long segments
of the commoner kind attain a length of from 10 to 30 and even 45
thousandths of a millimetre. Their diameter is from 1-½ to 2, very
rarely 3, thousandths of a millimetre.[145]
[Illustration: Fig. 72.]
[Illustration: Fig. 73.]
On June 28th, fermentation was quite finished; there was no longer any
trace of gas, nor any lactate in solution. All the infusoria were lying
motionless at the bottom of the flask. The liquid clarified by degrees,
and in the course of a few days became quite bright. Here we may
inquire, were these motionless infusoria, which from complete exhaustion
of the lactate, the source of the carbonaceous part of their food, were
now lying inert at the bottom of the fermenting vessel—were they dead
beyond power of revival?[146] The following experiment leads us to
believe that they were not perfectly lifeless, and that they behave in
the same manner as the yeast of beer, which, after it has decomposed all
the sugar in a fermentable liquid, is ready to revive and multiply in a
fresh saccharine medium. On April 22nd, 1875, we left in the oven, at a
temperature of 25° C. (77° F.), a fermentation of lactate of lime that
had been completed. The delivery tube of the flask, A, (Fig. 73) in
which it had taken place had never been withdrawn from under the
mercury. We kept the liquid under observation daily, and saw it
gradually become brighter; this went on for fifteen days. We then filled
a similar flask, B, with the solution of lactate, which we boiled, not
only to kill the germs of vibrios which the liquid might contain, but
also to expel the air that it held in solution. When the flask, B, had
cooled, we connected the two flasks, avoiding the introduction of
air[147], after having slightly shaken the flask, A, to stir up the
deposit at the bottom. There was then a pressure, due to carbonic acid
at the end of the delivery tube of this latter flask, at the point _a_,
so that on opening the taps _r_ and _s_, the deposit at the bottom of
flask A was driven over into flask B, which in consequence was
impregnated with the deposit of a fermentation that had been completed
fifteen days before. Two days after impregnation, the flask B began to
show signs of fermentation. It follows, that the deposit of vibrios of a
completed butyric fermentation may be kept, at least for a certain time,
without losing the power of causing fermentation. It furnishes a butyric
ferment, capable of revival and action in a suitable, fresh, fermentable
medium.
The reader who has attentively studied the facts which we have placed
before him cannot, in our opinion, entertain the least doubt on the
subject of the possible multiplication of the vibrios of a fermentation
of lactate of lime out of contact with atmospheric oxygen. If fresh
proofs of this important proposition were necessary, they might be found
in the following observations, from which it may be inferred that
atmospheric oxygen is capable of suddenly checking a fermentation
produced by butyric vibrios, and rendering them absolutely motionless,
so that it cannot be necessary to enable them to live. On May 7th, 1862,
we placed in the oven a flask holding 2·580 litres (4-½ pints), and
filled with the solution of lactate of lime and phosphates, which we had
impregnated on the 9th with two drops of a liquid in butyric
fermentation. In the course of a few days fermentation declared itself:
on the 16th it was in progress, but feebly; on the 18th it was active;
on the 30th it was very active. On June 1st it yielded hourly 35 c.c.
(2·3 cubic inches) of gas, containing ten per cent. of hydrogen. On the
2nd we began the study of the action of air on the vibrios of this
fermentation. To do this we cut off the delivery-tube on a level with
its point of junction to the flask, then with a 50 c.c. pipette we took
out that quantity (1-3/4 fl. oz.) of liquid which was, of course,
replaced at once by air. We then reversed the flask with the opening
under the mercury, and shook it every ten minutes for more than an hour.
Wishing to make sure, to begin with, that the oxygen had been absorbed,
we connected under the mercury the beak of the flask by means of a thin
india-rubber tube filled with water, with a small flask, the neck of
which had been drawn out, and was filled with water; we then raised the
large flask with the smaller kept above it. A Mohr’s clip, which closed
the india-rubber tube, and which we then opened, permitted the water
contained in the small flask to pass into the large one, whilst the gas,
on the contrary, passed upwards from the large flask into the small one.
We analyzed the gas immediately, and found that, allowing for carbonic
acid and hydrogen, it did not contain more than 14·2 per cent. of
oxygen, which corresponds to an absorption of 6·6 c.c., or of 3·3 c.c.
(0·2 cubic inch) of oxygen for the 50 c.c. (3·05 cubic inches) of air
employed. Lastly, we again established connection by an india-rubber
tube between the flasks, after having seen by microscopical examination
that the movements of the vibrios were very languid. Fermentation had
become less vigorous without having actually ceased, no doubt because
some portions of the liquid had not been brought into contact with the
atmospheric oxygen, in spite of the prolonged shaking that the flask had
undergone after the introduction of the air. Whatever the cause might
have been, the significance of the phenomenon is not doubtful. To assure
ourselves further of the effect of air on the vibrios, we half filled
two test tubes with the fermenting liquid taken from another
fermentation which had also attained its maximum of intensity, into one
of which we passed a current of air, into the other carbonic acid gas.
In the course of half an hour, all the vibrios in the aerated tube were
dead, or at least motionless, and fermentation had ceased. In the other
tube, after three hours’ exposure to the effects of the carbonic acid
gas, the vibrios were still very active, and fermentation was going on.
There is a most simple method of observing the deadly effect of
atmospheric air upon vibrios. We have seen in the microscopical
examination made by means of the apparatus represented in Fig. 71, how
remarkable were the movements of the vibrios when absolutely deprived of
air, and how easy it was to discern them. We will repeat this
observation, and at the same time make a comparative study of the same
liquid, under the microscope, in the ordinary way, that is to say, by
placing a drop of the liquid on an object-glass, and covering it with a
thin glass slip, a method which must necessarily bring the drop into
contact with air, if only for a moment. It is surprising what a
remarkable difference is observed immediately between the movements of
the vibrios in the bulb and of those under the glass. In the case of the
latter we generally see all movement at once cease near the edges of the
glass, where the drop of liquid is in direct contact with the air; the
movements continue for a longer or shorter time about the centre, in
proportion as the air is more or less intercepted by the vibrios at the
circumference of the liquid. It does not require much skill in
experiments of this kind to enable one to see plainly that immediately
after the glass has been placed on the drop, which has been affected all
over by atmospheric air, the whole of the vibrios seem to languish and
to manifest symptoms of illness—we can think of no better expression to
explain what we see taking place—and that they gradually recover their
activity about the centre, in proportion as they find themselves in a
part of the medium that is less affected by the presence of oxygen.
Some most curious facts are to be found in connection with an
observation, the correlative and inverse of the foregoing, on the
ordinary aërobian bacteria. If we examine below the microscope a drop of
liquid full of these organisms under a coverslip, we very soon observe a
cessation of motion in all the bacteria which lie in the central portion
of the liquid, where the oxygen rapidly disappears to supply the
necessities of the bacteria existing there; whilst, on the other hand,
near the edges of the cover-glass the movements are very active, in
consequence of the constant supply of air. In spite of the speedy death
of the bacteria beneath the centre of the glass, we see life prolonged
there if by chance a bubble of air has been enclosed. All round this
bubble a vast number of bacteria collect in a thick, moving circle, but
as soon as all the oxygen of the bubble has been absorbed they fall
apparently lifeless, and are scattered by the movement of the
liquid.[148]
We may here be permitted to add, as a purely historical matter, that it
was these two observations just described, made successively one day in
1861, on vibrios and bacteria, that first suggested to us the idea of
the possibility of life without air, and caused us to think that the
vibrios which we met so frequently in our lactic fermentations must be
the true butyric ferment.
We may pause a moment to consider an interesting question in reference
to the two characters under which vibrios appear in butyric
fermentations. What is the reason that some vibrios exhibit refractive
corpuscles, generally of a lenticular form, such as we see in Fig. 72?
We are strongly inclined to believe that these corpuscles have to do
with a special mode of reproduction in the vibrios, common alike to the
anaërobian forms which we are studying, and the ordinary aërobian forms
in which also the corpuscles of which we are speaking may occur. The
explanation of the phenomenon, from our point of view, would be that,
after a certain number of fissiparous generations, and under the
influence of variations in the composition of the medium, which is
constantly changing through fermentation as well as through the active
life of the vibrios themselves, cysts, which are simply the refractive
corpuscles, form along them at different points. From these gemmules we
have ultimately produced vibrios, ready to reproduce others by the
process of transverse division for a certain time, to be themselves
encysted later on. Various observations incline us to believe that, in
their ordinary form of minute, soft, exuberant rods, the vibrios perish
when submitted to desiccation, but when they occur in the corpuscular or
encysted form they possess unusual powers of resistance, and may be
brought to the state of dry dust and be wafted about by winds. None of
the matter which surrounds the corpuscle or cyst seems to take part in
the preservation of the germ, when the cyst is formed, for it is all
re-absorbed, gradually leaving the cyst bare. The cysts appear as masses
of corpuscles, in which the most practised eye cannot detect anything of
an organic nature, or anything to remind one of the vibrios which
produced them; nevertheless, these minute bodies are endowed with a
latent vital action, and only await favourable conditions to develop
long rods of vibrios. We are not, it is true, in a position to adduce
any very forcible proofs in support of these opinions. They have been
suggested to us by experiments, none of which, however, have been
absolutely decisive in their favour. We may cite one of our observations
on this subject.
In a fermentation of glycerine in a mineral medium—the glycerine was
fermenting under the influence of butyric vibrios—after we had
determined the, we may say, exclusive presence of lenticular vibrios,
with refractive corpuscles, we observed the fermentation, which, for
some unknown reason, had been very languid, suddenly become extremely
active, but now through the influence of ordinary vibrios. The gemmules
with brilliant corpuscles had almost disappeared; we could see but very
few, and those now consisted of the refractive bodies alone, the bulk of
the vibrios accompanying them having undergone some process of
re-absorption.
Another observation which still more closely accords with this
hypothesis is given in our work on the silkworm disease (vol. i., page
256). We there demonstrate that, when we place in water some of the dust
formed of desiccated vibrios, containing a host of these refractive
corpuscles, in the course of a very few hours large vibrios appear,
well-developed rods fully grown, in which the brilliant points are
absent; whilst in the water no process of development from smaller
vibrios is to be discerned, a fact which seems to show that the former
had issued fully grown from the refractive corpuscles, just as we see
_colpoda_ issue with their adult aspect from the dust of their cysts.
This observation, we may remark, furnishes one of the best proofs that
can be adduced against the spontaneous generation of vibrios or
bacteria, since it is probable that the same observation applies to
bacteria. It is true that we cannot say of mere points of dust, examined
under the microscope, that one particular germ belongs to vibrio,
another to bacterium; but how is it possible to doubt that the vibrios
issue, as we see them, from an ovum of some kind, a cyst, or germ, of
determinate character, when, after having placed some of these
indeterminate motes of dust into clean water, we suddenly see, after an
interval of not more than one or two hours, an adult vibrio crossing the
field of the microscope, without our having been able to detect any
intermediate state between its birth and adolescence?
It is a question whether differences in the aspect and nature of
vibrios, which depend upon their more or less advanced age, or are
occasioned by the influence of certain conditions of the medium in which
they propagate, do not bring about corresponding changes in the course
of the fermentation and the nature of its products. Judging at least
from the variations in the proportions of hydrogen and carbonic acid gas
produced in butyric fermentations, we are inclined to think that this
must be the case; nay, more, we find that hydrogen is not even a
constant product in these fermentations. We have met with butyric
fermentations of lactate of lime which did not yield the minutest trace
of hydrogen, or anything besides carbonic acid. Fig. 74 represents the
vibrios which we observed in a fermentation of this kind. They present
no special features. Butyl alcohol is, according to our observations, an
ordinary product, although it varies and is by no means a necessary
concomitant of these fermentations. It might be supposed, since butylic
alcohol may be produced, and hydrogen be in deficit, that the proportion
of the former of these products would attain its maximum when the latter
assumed a minimum. This, however, is by no means the case; even in those
few fermentations that we have met with in which hydrogen was absent,
there was no formation of butylic alcohol.
[Illustration: Fig. 74.]
From a consideration of all the facts detailed in this paragraph we can
have no hesitation in concluding that, on the one hand, in cases of
butyric fermentation, the vibrios which abound in them and constitute
their ferment, live without air or free oxygen; and that, on the other
hand, the presence of gaseous oxygen operates prejudicially against the
movements and activity of those vibrios. But now does it follow that the
presence of minute quantities of air brought into contact with a liquid
undergoing butyric fermentation would prevent the continuance of that
fermentation, or even exercise any check upon it? We have not made any
direct experiments upon this subject; but we should not be surprised to
find that, so far from hindering, air may, under such circumstances,
facilitate the propagation of the vibrios and accelerate fermentation.
This is exactly what happens in the case of yeast. But how could we
reconcile this, supposing it were proved to be the case, with the fact
just insisted on as to the danger of bringing the butyric vibrios into
contact with air? It may be possible that _life without air_ results
from habit, whilst _death through air_ may be brought about by a sudden
change in the conditions of the existence of the vibrios. The following
remarkable experiment is well known: A bird is placed in a glass jar of
one or two litres (60 to 120 cubic inches) in capacity, which is then
closed. After a time the creature exhibits every sign of intense
uneasiness and asphyxia long before it dies; a similar bird of the same
size is introduced into the jar; the death of the latter takes place
instantaneously, whilst the life of the former may still be prolonged
under these conditions for a considerable time, and there is no
difficulty even in restoring the bird to perfect health by taking it out
of the jar. It seems impossible to deny that we have here a case of the
adaptation of an organism to the gradual contamination of the medium;
and so it may likewise happen that the anaërobian vibrios of a butyric
fermentation, which develop and multiply absolutely without free oxygen,
perish immediately when suddenly taken out of their airless medium, and
that the result might be different if they had been gradually brought
under the action of air in small quantities at a time.
We are compelled here to admit that vibrios frequently abound in liquids
exposed to the air, and that they appropriate the atmospheric oxygen,
and could not withstand a sudden removal from its influence. Must we,
then, believe that such vibrios are absolutely different from those of
butyric fermentations? It would, perhaps, be more natural to admit that
in the one case there is an adaptation to life with air, and in the
other case an adaptation to life without air; each of these varieties
perishing when suddenly transferred from its habitual condition to that
of the other, whilst by a series of progressive changes one might be
modified into the other.[149] We know that in the case of alcoholic
ferments, although these can actually live without air, propagation is
wonderfully assisted by the presence of minute quantities of air; and
certain experiments, which we have not yet published, lead us to believe
that, after having lived without air, they cannot be suddenly exposed
with impunity to the influence of large quantities of oxygen.
We must not forget, however, that aërobian torulæ and anaërobian
ferments present an example of organisms apparently identical, in which,
however, we have not yet been able to discover any ties of a common
origin. Hence we were forced to regard them as distinct species; and so
it is possible that there may likewise be aërobian and anaërobian
vibrios without any transformation of the one into the other.
The question has been raised whether vibrios, especially those which we
have shown to be the ferment of butyric and many other fermentations,
are, in their nature, animal or vegetable. M. Ch. Robin attaches great
importance to the solution of this question, of which he speaks as
follows[150]:—“The determination of the nature, whether animal or
vegetable, of organisms, either as a whole or in respect to their
anatomical parts, assimilative or reproductive, is a problem which has
been capable of solution for a quarter of a century. The method has been
brought to a state of remarkable precision, experimentally, as well as
in its theoretical aspects, since those who devote their attention to
the organic sciences consider it indispensable in every observation and
experiment to determine accurately, before anything else, whether the
object of their study is animal or vegetable in its nature, whether
adult or otherwise. To neglect this is as serious an omission for such
students, as for chemists would be the neglecting to determine whether
it is nitrogen or hydrogen, urea or stearine that has been extracted
from a tissue, or which it is whose combinations they are studying in
this or that chemical operation. Now, scarcely any one of those who
study fermentations, properly so called, and putrefactions, ever pay
attention to the preceding data.... Among the observers to whom I allude
even M. Pasteur is to be found, who, even in his most recent
communications, omits to state definitely what is the nature of many of
the ferments which he has studied, with the exception, however, of those
which belong to the cryptogamic group called _torulaceæ_. Various
passages in his works seem to show that he considers the cryptogamic
organisms called _bacteria_, as well as those known as _vibrios_, as
belonging to the animal kingdom (see _Bulletin de l’Académie de
Médecine_, Paris, 1875, pp. 249, 251, especially 256, 266, 267, 289, and
290). These would be very different, at least physiologically, the
former being _aërobian_, whilst the vibrios are _anaërobian_, that is to
say, requiring no air to enable them to live, and being killed by
oxygen, should it be dissolved in the liquid to any considerable
extent.”
We are unable to see the matter in the same light as our learned
colleague does; to our thinking, we should be labouring under a great
delusion were we to suppose “that it is quite as serious an omission not
to determine the animal or vegetable nature of a ferment as it would be
to confound nitrogen with hydrogen, or urea with stearine.” The
importance of the solutions of disputed questions often depends upon the
point of view from which these are regarded. As far as the result of our
labours is concerned, we devoted our attention to these two questions
exclusively:—1. Is the ferment, in every fermentation properly so
called, an organized being? 2. Can this organized being live without
air? Now, what bearing can the question of the animal or vegetable
nature of the ferment, of the organized being, have upon the
investigation of these two problems? In studying butyric fermentation,
for example, we endeavoured to establish these two fundamental
points:—1. _The butyric ferment is a vibrio._ 2. _This vibrio may
dispense with air in its life, and, as a matter of fact, does dispense
with it in the act of producing butyric fermentation._ We did not
consider it at all necessary to pronounce any opinion as to the animal
or vegetable nature of this organism, and, even up to the present
moment, the idea that vibrio is an animal and not a plant is, in our
minds, a matter of sentiment rather than of conviction.
M. Robin, however, would have no difficulty in determining the limits of
the two kingdoms. According to him, “every variety of cellulose is, we
may say, insoluble in ammonia, as also are the reproductive elements of
plants, whether male or female. Whatever phase of evolution the elements
which reproduce a new individual may have reached, treatment with this
reagent, either cold or raised to boiling, leaves them absolutely intact
under the eyes of the observer, except that their contents, from being
partially dissolved, become more transparent. Every vegetable, whether
microscopic or not, every mycelium, and every spore thus preserves in
its entirety its special characteristics of form, volume, and structural
arrangements; whilst in the case of microscopic animals, or the ova and
microscopic embryos of different members of the animal kingdom, the very
opposite is the case.”
We should be glad to learn that the employment of a drop of ammonia
would enable us to pronounce an opinion, with this degree of confidence,
on the nature of the lowest microscopic beings; but is M. Robin
absolutely correct in his assumptions? That gentleman himself remarks
that spermatozoa, which belong to animal organisms, are insoluble in
ammonia, the effect of which is merely to make them paler. If a
difference of action in certain reagents, in ammonia, for example, were
sufficient to determine the limits of the animal and vegetable kingdoms,
might we not argue that there must be a very great and natural
difference between moulds and bacteria, inasmuch as the presence of a
small quantity of acid in the nutritive medium facilitates the growth
and propagation of the former, whilst it is able to prevent the life of
bacteria and vibrios? Although, as is well known, movement is not an
exclusive characteristic of animals, yet we have always been inclined to
regard vibrios as animals, on account of the peculiar character of their
movements. How greatly they differ in this respect from the diatomacæ,
for example! When the vibrio encounters an obstacle it turns, or after
having assured itself by some visual effort or other that it cannot
overcome it, it retraces its steps. The colpoda—undoubted
infusoria—behave in an exactly similar manner. It is true one may argue
that the zoospores of certain cryptogamia exhibit similar movements; but
do not these zoospores possess as much of an animal nature as do the
spermatozoa? As far as bacteria are concerned, when, as already
remarked, we see them crowd round a bubble of air in a liquid to prolong
their life, oxygen having failed them everywhere else, how can we avoid
believing that they are animated by an instinct for life, of the same
kind as that which we find in animals. M. Robin seems to us to be wrong
in supposing that it is possible to draw any absolute line of separation
between the animal and vegetable kingdoms. The settlement of this line,
however, we repeat again, no matter what it may be, has no serious
bearing upon the questions that have been the subject of our researches.
In like manner the difficulty which M. Robin has raised in objecting to
the employment of the word _germ_, when we cannot specify whether the
nature of that germ is animal or vegetable, is in many respects an
unnecessary one. In all the questions which we have discussed, whether
we were speaking of fermentation or spontaneous generation, the word
_germ_ has been used in the sense of _origin of living organism_. If
Liebig, for example, said of an albuminous substance that it gave birth
to ferment, could we contradict him more plainly than by replying: “No;
ferment is an organized being, the germ of which is always present, and
the albuminous substance merely serves by its occurrence to nourish the
germ and its successive generations.”
In our Memoir of 1862, on so-called _spontaneous_ generations, would it
not have been an entire mistake to have attempted to assign specific
names to the microscopic organisms which we met with in the course of
our observations? Not only would we have met with extreme difficulty in
the attempt, arising from the state of extreme confusion which even in
the present day exists in the classification and nomenclature of these
microscopic organisms, but we should have been forced to sacrifice
clearness in our work besides; at all events, we should have wandered
from our principal object, which was the determination of the presence
or absence of life in general, and had nothing to do with the
manifestation of a particular kind of life in this or that species,
animal or vegetable. Thus we have systematically employed the vaguest
nomenclature, such as _mucors_, _torulæ_, _bacteria_, and _vibrios_.
There was nothing arbitrary in our doing this, whereas there is much
that is arbitrary in adopting a definite system of nomenclature, and
applying it to organisms but imperfectly known, the differences or
resemblances between which are only recognizable through certain
characteristics, the true signification of which is obscure. Take, for
example, the extensive array of widely different systems that have been
invented during the last few years for the species of the genera
bacterium and vibrio in the works of Cohn, H. Hoffmann, Hallier, and
Billroth. The confusion which prevails here is very great, although we
do not of course by any means place these different works on the same
footing as regards their respective merits.
M. Robin is, however, right in recognizing the impossibility of
maintaining in the present day, as he formerly did, “that fermentation
is an exterior phenomenon, going on outside cryptogamic cells, a
phenomenon of contact. It is probably,” he adds, “an interior and
molecular action at work in the inmost recesses of the substance of each
cell.” From the day when we first proved that it is possible for all
organized ferments, properly so called, to spring up and multiply from
their respective germs, sown, whether consciously or by accident, in a
mineral medium free from organic and nitrogenous matters other than
ammonia, in which medium the fermentable matter alone is adapted to
provide the ferment with whatever carbon enters into its composition,
from that time forward the theories of Liebig, as well as that of
Berzelius, which M. Robin formerly defended, have had to give place to
others more in harmony with facts. We trust that the day will come when
M. Robin will likewise acknowledge that he has been in error on the
subject of the doctrine of spontaneous generation, which he continues to
affirm, without adducing any direct proofs in support of it, at the end
of the article to which we have been here replying.
We have devoted the greater part of this chapter to the establishing
with all possible exactness the extremely important physiological fact
of life without air, and its correlation to the phenomena of
fermentations properly so called—that is to say, of those which are due
to the presence of microscopic cellular organisms. This is the chief
basis of the new theory that we propose for the explanation of these
phenomena. The details into which we have entered were indispensable on
account of the novelty of the subject no less than on account of the
necessity we were under of combating the criticisms of the two German
naturalists, Drs. Oscar Brefeld and Traube, whose works had cast some
doubts on the correctness of the facts upon which we had based the
preceding propositions. We have much pleasure in adding that at the very
moment when we were revising the proofs of this chapter, we received
from M. Brefeld an essay, dated from Berlin, January, 1876, in which,
after describing his later experimental researches, he owns with
praiseworthy frankness that Dr. Traube and he were both of them
mistaken. Life without air is now a proposition which he accepts as
perfectly demonstrated. He has witnessed it in the case of _mucor
racemosus_, and has also verified it in the case of yeast. “If,” he
says, “after the results of my previous researches, which I conducted
with all possible exactness, I was inclined to consider Pasteur’s
assertions as inaccurate, and to attack them, I have no hesitation now
in recognizing them as true, and in proclaiming the service which
Pasteur has rendered to science in being the first to indicate the exact
relation of things in the phenomenon of fermentation.” In his later
researches. Dr. Brefeld has adopted the method which we have long
employed for demonstrating the life and multiplication of butyric
vibrios in the entire absence of air, as well as the method of
conducting growths in mineral media associated with the fermentable
substance. We need not pause to consider certain other secondary
criticisms of Dr. Brefeld. A perusal of the present work will, we trust,
convince him that they are based on no surer foundation than were his
former criticisms.
To bring one’s self to believe in a truth that has just dawned upon one
is the first step towards progress; to persuade others is the second.
There is a third step, less useful perhaps, but highly gratifying
nevertheless, which is, to convince one’s opponents.
We, therefore, have experienced great satisfaction in learning that we
have won over to our ideas an observer of singular ability, on a subject
which is of the utmost importance to the physiology of cells.
§ VI.—Reply to the Critical Observations of Liebig, Published in
1870.[151]
In the Memoir which we published, in 1860, on alcoholic fermentation,
and in several subsequent works, we were led to a different conclusion
on the causes of this very remarkable phenomenon from that which Liebig
had adopted. The opinions of Mitscherlich and Berzelius had ceased to be
tenable in the presence of the new facts which we had brought to light.
From that time we felt sure that the celebrated chemist of Munich had
adopted our conclusions, from the fact that he remained silent on this
question for a long time, although it had been until then the constant
subject of his study, as is shown by all his works. Suddenly there
appeared in the _Annales de Chimie et de Physique_ a long essay,
reproduced from a lecture delivered by him before the Academy of Bavaria
in 1868 and 1869. In this Liebig again maintained, not, however, without
certain modifications, the views which he had expressed in his former
publications, and disputed the correctness of the principal facts
enunciated in our Memoir of 1860, on which were based the arguments
against his theory.
“I had admitted,” he says, “that the resolution of fermentable matter
into compounds of a simpler kind must be traced to some process of
decomposition taking place in the ferment, and that the action of this
same ferment on the fermentable matter must continue or cease according
to the prolongation or cessation of the alteration produced in the
ferment. The molecular change in the sugar would, consequently, be
brought about by the destruction or modification of one or more of the
component parts of the ferment, and could only take place through the
contact of the two substances. M. Pasteur regards fermentation in the
following light:—The chemical action of fermentation is essentially a
phenomenon correlative with a vital action, beginning and ending with
it. He believes that alcoholic fermentation can never occur without the
simultaneous occurrence of organization, development, and multiplication
of globules, or continuous life, carried on from globules already
formed. But the idea that the decomposition of sugar during fermentation
is due to the development of the cellules of the ferment, is in
contradiction with the fact that the ferment is able to bring about the
fermentation of a pure solution of sugar. The greater part of the
ferment is composed of a substance that is rich in nitrogen and contains
sulphur. It contains, moreover, an appreciable quantity of phosphates,
hence it is difficult to conceive how, in the absence of these elements
in a pure solution of sugar undergoing fermentation, the number of cells
is capable of any increase.”
Notwithstanding Liebig’s belief to the contrary, the idea that the
decomposition of sugar during fermentation is intimately connected with
a development of the cellules of the ferment, or a prolongation of the
life of cellules already formed, is in no way opposed to the fact that
the ferment is capable of bringing about the fermentation of a pure
solution of sugar. It is manifest to any one who has studied such
fermentation with the microscope, even in those cases where the
sweetened water has been absolutely pure, that ferment-cells do
multiply, the reason being that the cells carry with them all the
food-supplies necessary for the life of the ferment. They may be
observed budding, at least many of them, and there can be no doubt that
those which do not bud still continue to live; life has other ways of
manifesting itself besides development and cell-proliferation.
If we refer to the figures on page 81 of our Memoir of 1860, Experiments
D, E, F, G, H, I, we shall see that the weight of yeast, in the case of
the fermentation of a pure solution of sugar, undergoes a considerable
increase, even without taking into account the fact that the sugared
water gains from the yeast certain soluble parts, since, in the
experiments just mentioned, the weights of solid yeast, washed and dried
at 100° C. (212° F.), are much greater than those of the raw yeast
employed, dried at the same temperature.
In these experiments we employed the following weights of yeast,
expressed in grammes (1 gramme = 15·43 grains)—
2·313
2·626
1·198
0·699
0·326
0·476
which became after fermentation, we repeat, without taking into account
the matters which the sugared water gained from the yeast—
grammes. grains.
2·486 [Increase 0·173 = 2·65
2·963 “ 0·337 = 5·16
1·700 ” 0·502 = 7·7
0·712 “ 0·013 = 0·2
0·335 ” 0·009 = 0·14
0·590 “ 0·114 = 1·75
Have we not in this marked increase in weight a proof of life, or, to
adopt an expression which may be preferred, a proof of a profound
chemical work of nutrition and assimilation?
We may cite on this subject one of our earlier experiments, which is to
be found in the _Comptes rendus de l’Académie_ for the year 1857, and
which clearly shows the great influence exerted on fermentation by the
soluble portion that the sugared water takes up from the globules of
ferment:—
“We take two equal quantities of fresh yeast that have been washed very
freely. One of these we cause to ferment in water containing nothing but
sugar, and, after removing from the other all its soluble particles—by
boiling it in an excess of water and then filtering it to separate the
globules—we add to the filtered liquid as much sugar as was used in the
first case along with a mere trace of fresh yeast, insufficient, as far
as its weight is concerned, to affect the results of our experiment. The
globules which we have sown bud, the liquid becomes turbid, a deposit of
yeast gradually forms, and, side by side with these appearances, the
decomposition of the sugar is effected, and in the course of a few hours
manifests itself clearly. These results are such as we might have
anticipated. The following fact, however, is of importance. In effecting
by these means the organization into globules of the soluble part of the
yeast that we used in the second case, we find that a considerable
quantity of sugar is decomposed. The following are the results of our
experiment: 5 grammes of yeast caused the fermentation of 12·9 grammes
of sugar in six days, at the end of which time it was exhausted. The
soluble portion of a like quantity of 5 grammes of the same yeast caused
the fermentation of 10 grammes of sugar in nine days, after which the
yeast developed by the sowing was likewise exhausted.”
How is it possible to maintain that, in the fermentation of water
containing nothing but sugar, the soluble portion of the yeast does not
act, either in the production of new globules or the perfection of old
ones, when we see, in the preceding experiment, that after this
nitrogenous and mineral portion has been removed by boiling, it
immediately serves for the production of new globules, which, under the
influence of the sowing of a mere trace of globules, causes the
fermentation of much sugar?[152]
In short, Liebig is not justified in saying that the solution of pure
sugar, caused to ferment by means of yeast, contains none of the
elements needed for the growth of yeast, neither nitrogen, sulphur, nor
phosphorus, and that, consequently, it should not be possible, by our
theory, for the sugar to ferment. On the contrary, the solution does
contain all these elements, as a consequence of the introduction and
presence of the yeast.
Let us proceed with our examination of Liebig’s criticisms:—
“To this,” he goes on to say, “must be added the decomposing action
which yeast exercises on a great number of substances, and which
resembles that which sugar undergoes. I have shown that malate of lime
ferments readily enough through the action of yeast, and that it splits
up into three other calcareous salts, namely the acetate, the carbonate,
and the succinate. If the action of yeast consists in its increase and
multiplication, it is difficult to conceive this action in the case of
malate of lime and other calcareous salts of vegetable acids.”
This statement, with all due deference to the opinion of our illustrious
critic, is by no means correct. Yeast has no action on malate of lime,
or on other calcareous salts formed by vegetable acids. Liebig had
previously, much to his own satisfaction, brought forward urea as being
capable of transformation into carbonate of ammonia during alcoholic
fermentation in contact with yeast. This has been proved by us to be
erroneous. It is an error of the same kind that Liebig again brings
forward here. In the fermentation of which he speaks (that of malate of
lime), certain spontaneous ferments are produced, the germs of which are
associated with the yeast, and develop in the mixture of yeast and
malate. The yeast merely serves as a source of food for these new
ferments without taking any direct part in the fermentations of which we
are speaking. Our researches leave no doubt on this point, as is evident
from the observations on the fermentations of tartrate of lime
previously given.
It is true that there are circumstances under which yeast brings about
modifications in different substances. Doebereiner and Mitscherlich,
more especially, have shown that yeast imparts to water a soluble
material, which liquefies cane-sugar and produces inversion in it by
causing it to take up the elements of water, just as diastase behaves to
starch or emulsin to amygdalin.
M. Berthelot also has shown that this substance may be isolated by
precipitating it with alcohol, in the same way as diastase is
precipitated from its solutions.[153] These are remarkable facts, which
are, however, at present but vaguely connected with the alcoholic
fermentation of sugar by means of yeast. The researches in which we have
proved the existence of special forms of living ferments in many
fermentations, which one might have supposed to have been produced by
simple contact action, had established beyond doubt the existence of
profound differences between those fermentations, which we have
distinguished as fermentations proper, and the phenomena connected with
soluble substances. The more we advance, the more clearly we are able to
detect these differences. M. Dumas has insisted on the fact that the
ferments of fermentation proper multiply and reproduce themselves in the
process, whilst the others are destroyed.[154] Still more recently M.
Müntz has shown that chloroform prevents fermentations proper, but does
not interfere with the action of diastase (_Comptes rendus_, 1875.) M.
Bouchardat had already established the fact that “hydrocyanic acid,
salts of mercury, ether, alcohol, creosote, and the oils of turpentine,
lemon, cloves, and mustard destroy or check alcoholic fermentation,
whilst in no way interfering with the glucoside fermentations” (_Annales
de Chimie et de Physique_, 3rd series, t. xiv., 1845.) We may add, in
praise of M. Bouchardat’s sagacity, that that skilful observer has
always considered these results as a proof that alcoholic fermentation
is dependent on the life of the yeast-cell, and that a distinction
should be made between the two orders of fermentation.
M. Paul Bert, in his remarkable studies on the influence of barometric
pressure on the phenomena of life, has recognized the fact that
compressed oxygen is fatal to certain ferments, whilst under similar
conditions it does not interfere with the action of those substances
classed under the name of _soluble ferments_, such as diastase (the
ferment which inverts cane sugar), emulsin, and others. During their
stay in compressed air, ferments proper ceased their activity, nor did
they resume it, even after exposure to air at ordinary pressures,
provided the access of germs was prevented.
We now come to Liebig’s principal objection, with which he concludes his
ingenious argument, and to which no less than eight or nine pages of the
_Annales_ are devoted.
Our author takes up the question of the possibility of causing yeast to
grow in sweetened water, to which a salt of ammonia and some yeast-ash
have been added—a fact which is evidently incompatible with his theory
that a ferment is always an albuminous substance on its way to
decomposition. In this case the albuminous substance does not exist; we
have only the mineral substances which will serve to produce it. We know
that Liebig regarded yeast, and, generally speaking, any ferment
whatever, as being a nitrogenous, albuminous substance which, in the
same way as emulsin, for example, possesses the power of bringing about
certain chemical decompositions. He connected fermentation with the easy
decomposition of that albuminous substance, and imagined that the
phenomenon occurred in the following manner:—“The albuminous substance
on its way to decomposition possesses the power of communicating to
certain other bodies that same state of mobility by which its own atoms
are already affected; and through its contact with other bodies it
imparts to them the power of decomposing or of entering into other
combinations.” Here Liebig failed to perceive that the ferment, in its
capacity of a living organism, had anything to do with the fermentation.
This theory dates back as far as 1843. In 1846 Messrs. Boutron and
Fremy, in a Memoir on lactic fermentation, published in the _Annales de
Chimie et de Physique_, strained the conclusions deducible from it to a
most unjustifiable extent. They asserted that one and the same
nitrogenous substance might undergo various modifications in contact
with air, so as to become successively alcoholic, lactic, butyric, and
other ferments. There is nothing more convenient than purely
hypothetical theories, theories which are not the necessary consequences
of facts; when fresh facts which cannot be reconciled with the original
hypothesis are discovered, new hypotheses can be tacked on to the old
ones. This is exactly what Liebig and Fremy have done, each in his turn,
under the pressure of our studies, commenced in 1857. In 1864 Fremy
devised the theory of _hemi-organism_, which meant nothing more than
that he gave up Liebig’s theory of 1843, together with the additions
which Boutron and he had made to it in 1846; in other words, he
abandoned the idea of albuminous substances being ferments, to take up
another idea, that albuminous substances, in contact with air, are
peculiarly adapted to undergo organization into new beings—that is, the
living ferments which we had discovered—and that the ferments of beer
and of the grape have a common origin.
This theory of hemi-organism was word for word the antiquated opinion of
Turpin, as may be readily seen by referring to Chapter IV., section III.
of the present work. The public, especially a certain section of the
public, did not go very deeply into an examination of the subject. It
was the period when the doctrine of spontaneous generation was being
discussed with much warmth. The new word hemi-organism, which was the
only novelty in M. Fremy’s theory, deceived people. It was thought that
M. Fremy had really discovered the solution of the question of the day.
It is true that it was rather difficult to understand the process by
which an albuminous substance could become all at once a living and
budding cell. This difficulty was readily solved by M. Fremy, who
declared that it was the result of some power that was not yet
understood, the power of “organic impulse.”[155]
Liebig, who, as well as M. Fremy, was compelled to renounce his original
opinions concerning the nature of ferments, devised the following
obscure theory (Memoir by Liebig, 1870, already cited):—
“There seems to be no doubt as to the part which the vegetable organism
plays in the phenomenon of fermentation. It is through it alone that an
albuminous substance and sugar are enabled to unite and form this
particular combination, this unstable form under which alone, as a
component part of the mycoderm, they manifest an action on sugar. Should
the mycoderm cease to grow, the bond which unites the constituent parts
of the cellular contents is loosened, and it is through the motion
produced therein that the cells of yeast bring about a disarrangement or
separation of the elements of the sugar into other organic molecules.”
One might easily believe that the translator for the _Annales_ has made
some mistake, so great is the obscurity of this passage.
Whether we take this new form of the theory or the old one, neither can
be reconciled at all with the development of yeast and fermentation in a
saccharine mineral medium, for in the latter experiment fermentation is
correlative to the life of the ferment and to its nutrition, a constant
change going on between the ferment and its food-matters, since all the
carbon assimilated by the ferment is derived from sugar, its nitrogen
from ammonia, and phosphorus from the phosphates in solution. And even
all said, what purpose can be served by the gratuitous hypothesis of
contact-action or communicated motion? The experiment of which we are
speaking is thus a fundamental one; indeed, it is its possibility that
constitutes the most effective point in the controversy. No doubt Liebig
might say, “but it is the motion of life and of nutrition which
constitutes your experiment, and this is the communicated motion that my
theory requires.” Curiously enough, Liebig does endeavour, as a matter
of fact, to say this, but he does so timidly and incidentally: “From a
chemical point of view, which point of view I would not willingly
abandon, a _vital action_ is a phenomenon of motion, and, in this double
sense of _life_ M. Pasteur’s theory agrees with my own, and is not in
contradiction with it (page 6).” This is true. Elsewhere Liebig says:—
“It is possible that the only correlation between the physiological act
and the phenomenon of fermentation is the production, in the living
cell, of the substance which, by some special property analogous to that
by which emulsin exerts a decomposing action on salicin and amygdalin,
may bring about the decomposition of sugar into other organic molecules;
the physiological act, in this view, would be necessary for the
production of this substance, but would have nothing else to do with the
fermentation (page 10).” To this, again, we have no objection to raise.
Liebig, however, does not dwell upon these considerations, which he
merely notices in passing, because he is well aware that, as far as the
defence of his theory is concerned, they would be mere evasions. If he
had insisted on them, or based his opposition solely upon them, our
answer would have been simply this: “If you admit with us that
fermentation is correlated with the life and nutrition of the ferment,
we agree upon the principal point. So agreeing, let us examine, if you
will, the actual cause of fermentation;—this is a second question, quite
distinct from the first. Science is built up of successive solutions
given to questions of ever-increasing subtlety, approaching nearer and
nearer towards the very essence of phenomena. If we proceed to discuss
together the question of how living, organized beings act in decomposing
fermentable substances, we will be found to fall out once more on your
hypothesis of communicated motion, since, according to our ideas, the
actual cause of fermentation is to be sought, in most cases, in the fact
of life without air, which is the characteristic of many ferments.”
Let us briefly see what Liebig thinks of the experiment in which
fermentation is produced by the impregnation of a saccharine mineral
medium, a result so greatly at variance with his mode of viewing the
question.[156] After deep consideration he pronounces this experiment to
be inexact, and the result ill-founded. Liebig, however, was not one to
reject a fact without grave reasons for his doing so, or with the sole
object of evading a troublesome discussion. “I have repeated this
experiment,” he says, “a great number of times, with the greatest
possible care, and have obtained the same results as M. Pasteur,
excepting as regards the formation and increase of the ferment.” It was,
however, the formation and increase of the ferment that constituted the
point of the experiment. Our discussion was, therefore, distinctly
limited to this: Liebig denied that the ferment was capable of
development in a saccharine mineral medium, whilst we asserted that this
development did actually take place, and was comparatively easy to
prove. In 1871 we replied to M. Liebig before the Paris Academy of
Sciences in a Note, in which we offered to prepare in a mineral medium,
in the presence of a commission to be chosen for the purpose, as great a
weight of ferment as Liebig could reasonably demand.[157] We were bolder
than we should, perhaps, have been in 1860; the reason was that our
knowledge of the subject had been strengthened by ten years of renewed
research. Liebig did not accept our proposal, nor did he even reply to
our Note. Up to the time of his death, which took place on April 18th,
1873, he wrote nothing more on the subject.[158]
When we published, in 1860, the details of the experiment in question,
we pointed out at some length the difficulties of conducting it
successfully, and the possible causes of failure. We called attention
particularly to the fact that saccharine mineral media are much more
suited for the nutrition of bacteria, lactic ferment, and other lowly
forms, than they are to that of yeast, and in consequence readily become
filled with various organisms from the spontaneous growth of germs
derived from the particles of dust floating in the atmosphere. The
reason why we do not observe the growth of alcoholic ferments,
especially at the commencement of the experiments, is because of the
unsuitableness of those media for the life of yeast. The latter may,
nevertheless, form in them subsequent to this development of other
organized forms, by reason of the modification produced in the original
mineral medium by the albuminous matters that they introduce into it. It
is interesting to peruse, in our Memoir of 1860, certain facts of the
same kind relating to fermentation by means of albumens—that of the
blood, for example, from which, we may mention incidentally, we were led
to infer the existence of several distinct albumens in the serum, a
conclusion which, since then, has been confirmed by various observers,
notably by M. Béchamp. Now, in his experiments on fermentation in
sweetened water, with yeast-ash and a salt of ammonia, there is no doubt
that Liebig had failed to avoid those difficulties which are entailed by
the spontaneous growth of other organisms than yeast. Moreover, it is
possible that, to have established the certainty of this result, Liebig
should have had recourse to a closer microscopical observation than from
certain passages in his Memoir he seems to have adopted. We have little
doubt that his pupils could tell us that Liebig did not even employ that
instrument without which any exact study of fermentation is not merely
difficult but well-nigh impossible. We ourselves, for the reasons
mentioned, did not obtain a simple alcoholic fermentation any more than
Liebig did. In that particular experiment, the details of which we gave
in our Memoir of 1860, we obtained lactic and alcoholic fermentation
together; an appreciable quantity of lactic acid formed and arrested the
propagation of the lactic and alcoholic ferments, so that more than half
of the sugar remained in the liquid without fermenting. This, however,
in no way detracted from the correctness of the conclusion which we
deduced from the experiment, and from other similar ones; it might even
be said that, from a general and philosophical point of view—which is
the only one of interest here—the result was doubly satisfactory,
inasmuch as we demonstrated that mineral media were adapted to the
simultaneous development of several organized ferments, instead of only
one. The fortuitous association of different ferments could not
invalidate the conclusion that all the nitrogen of the cells of the
alcoholic and lactic ferments was derived from the nitrogen in the
ammoniacal salts, and that all the carbon of those ferments was taken
from the sugar, since, in the medium employed in our experiment, the
sugar was the only substance that contained carbon. Liebig carefully
abstained from noticing this fact, which would have been fatal to the
very groundwork of his criticisms, and thought that he was keeping up
the appearance of a grave contradiction by arguing that we had never
obtained a simple alcoholic fermentation. It would be unprofitable to
dwell longer upon the subject of the difficulties which the propagation
of yeast in a saccharine mineral medium formerly presented. As a matter
of fact, the progress of our studies has imparted to the question an
aspect very different from that which it formerly wore; it was this
circumstance which emboldened us to offer, in our reply to Liebig before
the Academy of Sciences in 1871, to prepare, in a saccharine mineral
medium, in the presence of a commission to be appointed by our opponent,
any quantity of ferment that he might require, and to effect the
fermentation of any weight of sugar whatsoever.
Our knowledge of the facts detailed in the preceding chapters concerning
pure ferments and their manipulation in the presence of pure air,
enables us to completely disregard those causes of embarrassment that
result from the fortuitous occurrence of the germs of organisms,
different in character from the ferments, introduced by the air or from
the sides of vessels, or even by the ferment itself.
Let us once more take one of our double-necked flasks (Fig. 22, p. 110),
which we will suppose is capable of containing three or four litres (six
to eight pints).
Let us put into it the following:—
Pure distilled
water.
Sugar candy 200 grammes.
Bitartrate of 1·0 “
potassium
” “ ammonia 0·5 ”
Sulphate of ammonia 1·5 “
Ash of yeast 1·5 ”
[1 gramme = 15·43
grains.]
Let us boil the mixture, to destroy all germs of organisms that may
exist in the air or liquid or on the sides of the flask, and then permit
it to cool, after having placed, by way of extra precaution, a small
quantity of asbestos in the end of the fine, curved tube. Let us next
introduce a trace of ferment into the liquid, through the other neck,
which, as we described, is terminated by a small piece of india-rubber
tube closed with a glass stopper.
Here are the details of such an experiment:—
On December 9, 1873, we sowed some pure ferment—_saccharomyces
pastorianus_. From December 11, that is, within so short a time as
forty-eight hours after impregnation, we saw a multitude of extremely
minute bubbles rising almost continuously from the bottom, indicating
that at this point the fermentation had commenced. On the following
days, several patches of froth appeared on the surface of the liquid. We
left the flask undisturbed in the oven, at a temperature of 25° C. (77°
F.). On April 24, 1874, we tested some of the liquid, obtained by means
of the straight tube, to see if it still contained any sugar. We found
that it contained less than two grammes, so that 198 grammes (4·2 oz.
Troy) had already disappeared. Some time afterwards the fermentation
came to an end; we carried on the experiment, nevertheless, until April
18, 1875.
There was no development of any organism absolutely foreign to the
ferment, which was itself abundant, a circumstance that, added to the
persistent vitality of the ferment, in spite of the unsuitableness of
the medium for its nutrition, permitted the perfect completion of
fermentation. There was not the minutest quantity of sugar remaining.
The total weight of ferment, after washing and drying at 100° C. (212°
F.), was 2·563 grammes (39·5 grains).
In experiments of this kind, in which the ferment has to be weighed, it
is better not to use any yeast-ash that cannot be dissolved completely,
so as to be capable of easy separation from the ferment formed. Raulin’s
liquid, the composition of which we have already given (p. 89,
footnote), may be used in such cases with success.
All the alcoholic ferments are not capable to the same extent of
development by means of phosphates, ammoniacal salts, and sugar. There
are some whose development is arrested a longer or shorter time before
the transformation of all the sugar. In a series of comparative
experiments, 200 grammes of sugar-candy being used in each case, we
found that whilst _saccharomyces pastorianus_ effected a complete
fermentation of the sugar, the caseous ferment did not decompose more
than two-thirds, and the ferment which we have designated _new “high”
ferment_ not more than one-fifth: and keeping the flasks for a longer
time in the oven had no effect in increasing the proportions of sugar
fermented in these two last cases.
We conducted a great number of fermentations in mineral media, in
consequence of a circumstance which it may be interesting to mention
here. A person who was working in our laboratory asserted that the
success of our experiments depended upon the impurity of the sugar-candy
which we employed, and that if this sugar had been pure—much purer than
was the ordinary, white, commercial sugar-candy, which up to that time
we had always used—the ferment could not have multiplied. The persistent
objections of our friend, and our desire to convince him, caused us to
repeat all our previous experiments on the subject, using sugar of great
purity, which had been specially prepared for us, with the utmost care,
by a skilful confectioner, Seugnot. The result only confirmed our former
conclusions. Even this did not satisfy our obstinate friend, who went to
the trouble of preparing some pure sugar for himself, in little
crystals, by repeated crystallizations of carefully-selected commercial
sugar-candy; he then repeated our experiments himself. This time all his
doubts were overcome. It even happened that the fermentations with the
perfectly pure sugar instead of being slow were very active, when
compared with those which we had conducted with the commercial
sugar-candy.