fermentation in general consists in the small proportion which
the yeast that forms bears to the sugar that decomposes. In all
other known beings the weight of nutritive matter assimilated
corresponds with the weight of food used up, any difference that
may exist being comparatively small. The life of yeast is
entirely different. For a certain weight of yeast formed, we may
have ten times, twenty times, a hundred times as much sugar, or
even more decomposed, as we shall experimentally prove by-and-
bye; that is to say, that whilst the proportion varies in a
precise manner, according to conditions which we shall have
occasion to specify, it is also greatly out of proportion to the
weight of the yeast. We repeat, the life of no other being, under
its normal physiological conditions, can show anything similar.
The alcoholic ferments, therefore, present themselves to us as
plants which possess at least two singular properties: they can
live without air, that is without oxygen, and they can cause
decomposition to an amount which, though variable, yet, as
estimated by weight of product formed, is out of all proportion
to the weight of their own substance. These are facts of so great
importance, and so intimately connected with the theory of
fermentation, that it is indispensable to endeavour to establish
them experimentally, with all the exactness of which they will
admit.
The question before us is whether yeast is in reality an
anaerobian [Footnote: Capable of living without free oxygen--a
term invented by Pasteur.--En.] plant, and what quantities of
sugar it may cause to ferment, under the various conditions under
which we cause it to act.
The following experiments were undertaken to solve this double
problem:--We took a double-necked flask, of three litres (five
pints) capacity, one of the tubes being curved and forming an
escape for the gas; the other one, on the right hand side (Fig.
1), being furnished with a glass tap. We filled this flask with
pure yeast water, sweetened with 5 per cent, of sugar candy, the
flask being so full that there was not the least trace of air
remaining above the tap or in the escape tube; this artificial
wort had, however, been itself aerated. The curved tube was
plunged in a porcelain vessel full of mercury, resting on a firm
support. In the small cylindrical funnel above the tap, the
capacity of which was from 10 cc. to 15 cc. (about half a fluid
ounce) we caused to ferment, at a temperature of 20 degrees or 25
degrees C. (about 75 degrees F.), five or six cubic centimetres
of the saccharine liquid, by means of a trace of yeast, which
multiplied rapidly, causing fermentation, and forming a slight
deposit of yeast at the bottom of the funnel above the tap. We
then opened the tap, and some of the liquid in the funnel entered
the flask, carrying with it the small deposit of yeast, which was
sufficient to impregnate the saccharine liquid contained in the
flask. In this manner it is possible to introduce as small a
quantity of yeast as we wish, a quantity the weight of which, we
may say, is hardly appreciable. The yeast sown multiplies rapidly
and produces fermentation, the carbonic gas from which is
expelled into the mercury. In less than twelve days all the sugar
had disappeared, and the fermentation had finished. There was a
sensible deposit of yeast adhering to the sides of the flask;
collected and dried it weighed 2.25 grammes (34 grains). It is
evident that in this experiment the total amount of yeast formed,
if it required oxygen to enable it to live, could not have
absorbed, at most, more than the volume which was originally held
in solution in the saccharine liquid, when that was exposed to
the air before being introduced into the flask.
[Illustration with caption: Fig. 1]
Some exact experiments conducted by M. Raulin in our laboratory
have established the fact that saccharine worts, like water, soon
become saturated when shaken briskly with an excess of air, and
also that they always take into solution a little less air than
saturated pure water contains under the same conditions of
temperature and pressure. At a temperature of 25 degrees C. (77
degrees F.), therefore, if we adopt the coefficient of the
solubility of oxygen in water given in Bunsen's tables, we find
that 1 litre (1 3/4 pints) of water saturated with air contains
5.5 cc. (0.3 cubic inch) of oxygen. The three litres of yeast-
water in the flask, supposing it to have been saturated, contains
less than 16.5 cc. (1 cubic inch) of oxygen, or, in weight, less
than 23 milligrammes (0.35 grains). This was the maximum amount
of oxygen, supposing the greatest possible quantity to have been
absorbed, that was required by the yeast formed in the
fermentation of 150 grammes (4.8 Troy ounces) of sugar. We shall
better understand the significance of this result later on. Let
us repeat the foregoing experiment, but under altered conditions.
Let us fill, as before, our flask with sweetened yeast-water, but
let this first be boiled, so as to expel all the air it contains.
To effect this we arrange our apparatus as represented in the
accompanying sketch. (Fig 2.) We place our flask, A, on a tripod
above a gas flame, and in place of the vessel of mercury
substitute a porcelain dish, under which we can put a gas flame,
and Which contains some fermentable, saccharine liquid, similar
to that with which the flask is filled. We boil the liquid in the
flask and that in the basin simultaneously, and then let them
cool down together, so that as the liquid in the flask cools some
of the liquid is sucked from the basin into the flask. From a
trial experiment which we conducted, determining the quantity of
oxygen that remained in solution in the liquid after cooling,
according to M. Schutzenberger's valuable method, by means of
hydrosulphite of soda [Footnote: NaHSO2, now called sodium
hyposulphite.--D.C.R.], we found that the three litres in the
flask, treated as we have described, contained less than one
milligramme (0.015 grain) of oxygen. At the same time we
conducted another experiment, by way of comparison (Fig. 3). We
took a flask, B, of larger capacity than the former one, which we
filled about half with the same volume as before of a saccharine
liquid of identically the same composition. This liquid had been
previously freed from alterative germs by boiling. In the funnel
surmounting A, we put a few cubic centimetres of saccharine
liquid in a state of fermentation, and when this small quantity
of liquid was in full fermentation, and the yeast in it was young
and vigorous, we opened the tap, closing it again immediately, so
that a little of the liquid and yeast still remained in the
funnel. By this means we caused the liquid in A to ferment. We
also impregnated the liquid in B with some yeast taken from the
funnel of A. We then replaced the porcelain dish in which the
curved escape tube of A had been plunged, by a vessel filled with
mercury. The following is a description of two of these
comparative fermentations and the results they gave.
[Illustration with caption: Fig 2]
[Illustration with caption: Fig. 3]
The fermentable liquid was composed of yeast-water sweetened with
5 per cent, of sugar--candy; the ferment employed was
sacchormyces pastorianus.
The impregnation took place on January 20th. The flasks were
placed in an oven at 25 degrees (77 degrees F.).
FLASK A, WITHOUT AIR.
January 21st.--Fermentation commenced; a little frothy liquid
issued from the escape tube and covered the mercury.
The following days, fermentation was active. Examining the yeast
mixed with the froth that was expelled into the mercury by the
evolution of carbonic acid gas, we find that it was very fine,
young, and actively budding.
February 3rd.--Fermentation still continued, showing itself by a
number of little bubbles rising from the bottom of the liquid,
which had settled bright. The yeast was at the bottom in the form
of a deposit.
February 7th.--Fermentation still continued, but very languidly.
February 9th.--A very languid fermentation still went on,
discernible in little bubbles rising from the bottom of the
flask.
FLASK B, WITH AIR.
January 21st.--A sensible development of yeast.
The following days, fermentation was active, and there was an
abundant froth on the surface of the liquid.
February 1st.--All symptoms of fermentation had ceased.
As the fermentation in A would have continued a long time, being
so very languid, and as that in B had been finished for several
days, we brought to a close our two experiments on February 9th.
To do this we poured off the liquids in A and B, collecting the
yeasts on tared filters. Filtration was an easy matter, more
especially in the case of A. Examining the yeasts under the
microscope, immediately after decantation, we found that both of
them remained very pure. The yeast in A was in little clusters,
the globules of which were collected together, and appeared by
their well-defined borders to be ready for an easy revival in
contact with air.
As might have been expected, the liquid in flask B did not
contain the least trace of sugar; that in the flask A still
contained some, as was evident from the non-completion of
fermentation, but not more than 4.6 grammes (71 grains). Now, as
each flask originally contained three litres of liquid holding in
solution 5 per cent of sugar, it follows that 150 grammes (2,310
grains) of sugar had fermented in the flask B, and 145.4 grammes
(2,239.2 grains) in the flask A. The weights of yeast after
drying at 100 degrees C. (212 degrees F.) were--
For the flask B, with air. ... ..1,970 grammes (30.4 grains). For
the flask A, without air ... 1,368 grammes [Footnote: This appears
to be a misprint for 1.638 grammes=25.3 grains.--D. C. R.].
The proportions were 1 of yeast to 76 of fermented sugar in the
first case, and 1 of yeast to 89 of fermented sugar in the
second.
From these facts the following consequences may be deduced:
1. The fermentable liquid (flask B), which since it had been in
contact with air, necessarily held air in solution, although not
to the point of saturation, inasmuch as it had been once boiled
to free it from all foreign germs, furnished a weight of yeast
sensibly greater than that yielded by the liquid which contained
no air at all (flask A) or, at least, which could only have
contained an exceedingly minute quantity.
2. This same slightly aerated fermentable liquid fermented much
more rapidly than the other. In eight or ten days it contained no
more sugar; while the other, after twenty days, still contained
an appreciable quantity.
Is this last fact to be explained by the greater quantity of
yeast formed in B? By no means. At first, when the air has access
to the liquid, much yeast is formed and little sugar disappears,
as we shall prove immediately; nevertheless the yeast formed in
contact with the air is more active than the other. Fermentation
is correlative first to the development of the globules, and then
to the continued life of those globules once formed. The more
oxygen these last globules have at their disposal during their
formation, the more vigorous, transparent, and turgescent, and,
as a consequence of this last quality, the more active they are
in decomposing sugar. We shall hereafter revert to these facts.
3. In the airless flask the proportion of yeast to sugar was
1/59; it was only 1/79 in the flask which had air at first.
The proportion that the weight of yeast bears to the weight of
the sugar is, therefore, variable, and this variation depends, to
a certain extent, upon the presence of air and the possibility of
oxygen being absorbed by the yeast. We shall presently show that
yeast possesses the power of absorbing that gas and emitting
carbonic acid, like ordinary fungi, that even oxygen may be
reckoned amongst the number of food-stuffs that may be
assimilated by this plant, and that this fixation of oxygen in
yeast, as well as the oxidations resulting from it, have the most
marked effect on the life of yeast, on the multiplication of its
cells, and on their activity as ferments acting upon sugar,
whether immediately or afterwards, apart from supplies of oxygen
or air.
In the preceding experiment, conducted without the presence of
air, there is one circumstance particularly worthy of notice.
This experiment succeeds, that is to say, the yeast sown in the
medium deprived of oxygen develops, only when this yeast is in a
state of great vigour. We have already explained the meaning of
this last expression. But we wish now to call attention to a very
evident fact in connection with this point. We impregnate a
fermentable liquid; yeast develops and fermentation appears. This
lasts for several days and then ceases. Let us suppose that, from
the day when fermentation first appears in the production of a
minute froth, which gradually increases until it whitens the
surface of the liquid, we take, every twenty-four hours, or at
longer intervals, a trace of the yeast deposited on the bottom of
the vessel and use it for starting fresh fermentations.
Conducting these fermentations all under precisely the same
conditions of temperature, character and volume of liquid, let us
continue this for a prolonged time, even after the original
fermentation is finished. We shall have no difficulty in seeing
that the first signs of action in each of our series of second
fermentations appear always later and later in proportion to the
length of time that has elapsed from the commencement of the
original fermentation. In other words, the time necessary for the
development of the germs and the production of that amount of
yeast sufficient to cause the first appearance of fermentation
varies with the state of the impregnating cells, and is longer in
proportion as the cells are further removed from the period of
their formation. It is essential, in experiments of this kind,
that the quantities of yeast successively taken should be as
nearly as possible equal in weight or volume, since, celeris
paribus, fermentations manifest themselves more quickly the
larger the quantity of yeast employed in impregnation.
If we compare under the microscope the appearance and character
of the successive quantities of yeast taken, we shall see plainly
that the structure of the cells undergoes a progressive change.
The first sample which we take, quite at the beginning of the
original fermentation, generally gives us cells rather larger
than those later on, and possessing a remarkable tenderness.
Their walls are exceedingly thin, the consistency and softness of
their protoplasm is akin to fluidity, and their granular contents
appear in the form of scarcely visible spots. The borders of the
cells soon become more marked, a proof that their walls undergo a
thickening; their protoplasm also becomes denser, and the
granulations more distinct. Cells of the same organ, in the
states of infancy and old age, should not differ more than the
cells of which we are speaking, taken in their extreme states.
The progressive changes in the cells, after they have acquired
their normal form and volume, clearly demonstrate the existence
of a chemical work of a remarkable intensity, during which their
weight increases, although in volume they undergo no sensible
change, a fact that we have often characterized as "the continued
life of cells already formed." We may call this work a process of
maturation on the part of the cells, almost the same that we see
going on in the case of adult beings in general, which continue
to live for a long time, even after they have become incapable of
reproduction, and long after their volume has become permanently
fixed.
This being so, it is evident, we repeat, that, to multiply in a
fermentable medium, quite out of contact with oxygen, the cells
of yeast must be extremely young, full of life and health, and
still under the influence of the vital activity which they owe to
the free oxygen which has served to form them, and which they
have perhaps stored up for a time. When older, they reproduce
themselves with much difficulty when deprived of air, and
gradually become more languid; and if they do multiply, it is in
strange and monstrous forms. A little older still, they remain
absolutely inert in a medium deprived of free oxygen. This is not
because they are dead; for in general they may be revived in a
marvellous manner in the same liquid if it has been first aerated
before they are sown. It would not surprise us to learn that at
this point certain preconceived ideas suggest themselves to the
mind of an attentive reader on the subject of the causes that may
serve to account for such strange phenomena in the life of these
beings which our ignorance hides under the expressions of YOUTH
and AGE; this, however, is a subject which we cannot pause to
consider here.
At this point we must observe--for it is a matter of great
importance--that in the operations of the brewer there is always
a time when the yeasts are in this state of vigorous youth of
which we have been speaking, acquired under the influence of free
oxygen, since all the worts and the yeasts of commerce are
necessarily manipulated in contact with air, and so impregnated
more or less with oxygen. The yeast immediately seizes upon this
gas and acquires a state of freshness and activity, which permits
it to live afterwards out of contact with air, and to act as a
ferment. Thus, in ordinary brewery practice, we find the yeast
already formed in abundance even before the earliest external
signs of fermentation have made their appearance. In this first
phase of its existence, yeast lives chiefly like an ordinary
fungus.
From the same circumstances it is clear that the brewer's
fermentations may, speaking quite strictly, last for an
indefinite time, in consequence of the unceasing supply of fresh
wort, and from the fact, moreover, that the exterior air is
constantly being introduced during the work, and that the air
contained in the fresh worts keeps up the vital activity of the
yeast, as the act of breathing keeps up the vigour and life of
cells in all living beings. If the air could not renew itself in
any way, the vital activity which the cells originally received,
under its influence, would become more and more exhausted, and
the fermentation eventually come to an end.
We may recount one of the results obtained in other experiments
similar to the last, in which, however, we employed yeast which
was still older than that used for our experiment with flask A
(Fig. 2), and moreover took still greater precautions to prevent
the presence of air. Instead of leaving the flask, as well as the
dish, to cool slowly, after having expelled all air by boiling,
we permitted the liquid in the dish to continue boiling whilst
the flask was being cooled by artificial means; the end of the
escape tube was then taken out of the still boiling dish and
plunged into the mercury trough. In impregnating the liquid,
instead of employing the contents of the small cylindrical funnel
whilst still in a state of fermentation, we waited until this was
finished. Under these conditions, fermentation was still going on
in our flask, after a lapse of three months. We stopped it and
found that 0.255 gramme (3.9 grains) of yeast had been formed,
and that 45 grammes (693 grains) of sugar had fermented, the
ratio between the weights of yeast and sugar being thus 0.255
divided by 45 = 1 divided by 176. In this experiment the yeast
developed with much difficulty, by reason of the conditions to
which it had been subjected. In appearance the cells varied much,
some were to be found large, elongated, and of tubular aspect,
some seemed very old and were extremely granular, whilst others
were more transparent. All of them might be considered abnormal
cells.
In such experiments we encounter another difficulty. If the yeast
sown in the non-aerated fermentable liquid is in the least degree
impure, especially if we use sweetened yeast-water, we may be
sure that alcoholic fermentation will soon cease, if, indeed, it
ever commences, and that accessory fermentations will go on. The
vibrios of butyric fermentation, for instance, will propagate
with remarkable facility under these circumstances. Clearly then,
the purity of the yeast at the moment of impregnation, and the
purity of the liquid in the funnel, are conditions indispensable
to success.
To secure the latter of these conditions, we close the funnel, as
shown in FIG. 2, by means of a cork pierced with two holes,
through one of which a short tube passes, to which a short length
of india-rubber tubing provided with a glass stopper is attached;
through the other hole a thin curved tube is passed. Thus fitted,
the funnel can answer the same purposes as our double-necked
flasks. A few cubic centimetres of sweetened yeast-water are put
in it and boiled, so that the steam may destroy any germs
adhering to the sides; and when cold the liquid is impregnated by
means of a trace of pure yeast, introduced through the glass-
stoppered tube. If these precautions are neglected, it is
scarcely possible to secure a successful fermentation in our
flasks, because the yeast sown is immediately held in check by a
development of anaerobian vibrios. For greater security, we may
add to the fermentable liquid, at the moment when it is prepared,
a very small quantity of tartaric acid, which will prevent the
development of butyric vibrios.
[Illustration with caption: Fig. 4.]
The variation of the ratio between the weight of the yeast and
that of the sugar decomposed by it now claims special attention.
Side by side with the experiments which we have just described,
we conducted a third lot by means of the flask C (Fig. 4),
holding 4.7 litres (8 1/2 pints), and fitted up like the usual
two-necked flasks, with the object of freeing the fermentable
liquid from foreign germs, by boiling it to begin with, so that
we might carry on our work under conditions of purity. The volume
of yeast-water (containing 5 per cent. of sugar) was only 200 cc.
(7 fl. oz.), and consequently, taking into account the capacity
of the flask, It formed but a very thin layer at the bottom. On
the day after impregnation the deposit of yeast was already
considerable, and forty-eight hours afterwards the fermentation
was completed. On the third day we collected the yeast after
having analyzed the gas contained in the flask. This analysis was
easily accomplished by placing the flask in a hot-water bath,
whilst the end of the curved tube was plunged under a cylinder of
mercury. The gas contained 41.4 per cent. of carbonic acid, and,
after the absorption, the remaining air contained:--
Oxygen . ... . ... . ... . ... . ... . ... . ... ... 19.7
Nitrogen . ... . ... . ... . ... . ... . ... . ... . 80.3
100.0
Taking into consideration the volume of this flask, this shows a
minimum of 50 cc. (3.05 cub. in.) of oxygen to have been absorbed
by the yeast. The liquid contained no more sugar, and the weight
of the yeast, dried at a temperature of 100 degrees C (212
degrees F.), was 0.44 grammes. The ratio between the weights of
yeast and sugar is 0.44/10=1/22.7 [Footnote: 200 cc. of liquid
were used, which, as containing 3 per cent., had in solution 10
grammes of sugar.--D.C.R.]. On this occasion, where we had
increased the quantity of oxygen held in solution, so as to yield
itself for assimilation at the beginning and during the earlier
developments of the yeast, we found instead of the previous ratio
of 1/76 that of 1/23.
[Illustration with caption: Fig. 5]
The next experiment was to increase the proportion of oxygen to a
still greater extent, by rendering the diffusion of gas a more
easy matter than in a flask, the air in which is in a state of
perfect quiescence. Such a state of matters hinders the supply of
oxygen, inasmuch as the carbonic acid, as soon as it is
liberated, at once forms an immovable layer on the surface of the
liquid, and so separates off the oxygen. To effect the purpose of
our present experiment, we used flat basins having glass bottoms
and low sides, also of glass, in which the depth of the liquid is
not more than a few millimetres (less than 1/4 inch) (Fig. 5). The
following is one of our experiments so conducted:--On April 16th,
1860, we sowed a trace of beer yeast ("high" yeast) in 200 cc. (7
fl. oz.) of a saccharine liquid containing 1.720 grammes (26.2
grains) of sugar-candy. From April 18th our yeast was in good
condition and well developed. We collected it, after having added
to the liquid a few drops of concentrated sulphuric acid, with
the object of checking the fermentation to a great extent, and
facilitating filtration. The sugar remaining in the filtered
liquid, determined by Fehling's solution, showed that 1.04
grammes (16 grains) of sugar had disappeared. The weight of the
yeast, dried at 100 degrees C. (212 degrees F.), was 0.127 gramme
(2 grains), which gives us the ratio between the weight of the
yeast and that of the fermented sugar 0.123/1.04=1/8.1, which is
considerably higher than the preceding ones.
We may still further increase this ratio by making our estimation
as soon as possible after the impregnation, or the addition of
the ferment. It will be readily understood why yeast, which is
composed of cells that bud and subsequently detach themselves
from one another, soon forms a deposit at the bottom of the
vessels. In consequence of this habit of growth, the cells
constantly covering each other prevents the lower layers from
having access to the oxygen held in solution in the liquid, which
is absorbed by the upper ones. Hence, these which are covered and
deprived of this gas act on the sugar without deriving any vital
benefit from the oxygen--a circumstance which must tend to
diminish the ratio of which we are speaking. Once more repeating
the preceding experiment, but stopping it as soon as we think
that the weight of yeast formed may be determined by the balance
(we find that this may be done twenty-four hours after
impregnation with an inappreciable quantity of yeast), in this
case the ratio between the weights of yeast and sugar is gr/024
yeast/0 gr. 09 sugar=1/4. This is the highest ratio we have been
able to obtain.
Under these conditions the fermentation of sugar is extremely
languid: the ratio obtained is very nearly the same that ordinary
fungoid growths would give. The carbonic acid evolved is
principally formed by the decompositions which result from the
assimilation of atmospheric oxygen. The yeast, therefore, lives
and performs its functions after the manner of ordinary fungi: so
far it is no longer a ferment, so to say; moreover, we might
expect to find it to cease to be a ferment at all if we could
only surround each cell separately with all the air that it
required. This is what the preceding phenomena teach us; we shall
have occasion to compare them later on with others which relate
to the vital action exercised on yeast by the sugar of milk.
We may here be permitted to make a digression.
In his work on fermentations, which M. Schutzenberger has
recently published, the author criticises the deductions that we
have drawn from the preceding experiments, and combats the
explanation which we have given of the phenomena of fermentation.
[Footnote: International Science Series, vol. xx, pp. 179-182.
London, 1876.--D. C. R.] It is an easy matter to show the weak
point of M. Schutzenberger's reasoning. We determined the power
of the ferment by the relation of the weight of sugar decomposed
to the weight of the yeast produced. M. Schutzenberger asserts
that in doing this we lay down a doubtful hypothesis, and he
thinks that this power, which he terms FERMENTATIVE ENERGY, may
be estimated more correctly by the quantity of sugar decomposed
by the unit-weight of yeast in unit-time; moreover, since our
experiments show that yeast is very vigorous when it has a
sufficient supply of oxygen, and that, in such a case, it can
decompose much sugar in a little time, M. Schutzenberger
concludes that it must then have great power as a ferment, even
greater than when it performs its functions without the aid of
air, since under this condition it decomposes sugar very slowly.
In short, he is disposed to draw from our observations the very
opposite conclusion to that which we arrived at.
M, Schutzenberger has failed to notice that the power of a
ferment is independent of the time during which it performs its
functions. We placed a trace of yeast in one litre of saccharine
wort; it propagated, and all the sugar was decomposed. Now,
whether the chemical action involved in this decomposition of
sugar had required for its completion one day, or one month, or
one year, such a factor was of no more importance in this matter
than the mechanical labour required to raise a ton of materials
from the ground to the top of a house would be affected by the
fact that it had taken twelve hours instead of one. The notion of
time has nothing to do with the definition of work. M.
Schutzenberger has not perceived that in introducing the
consideration of time into the definition of the power of a
ferment, he must introduce at the same time, that of the vital
activity of the cells which is independent of their character as
a ferment. Apart from the consideration of the relation existing
between the weight of fermentable substance decomposed and that
of ferment produced, there is no occasion to speak of
fermentations or of ferments. The phenomena of fermentation and
of ferments have been placed apart from others, precisely
because, in certain chemical actions, that ratio has been out of
proportion; but the time that these phenomena require for their
accomplishment has nothing to do with either their existence
proper, or with their power. The cells of a ferment may, under
some circumstances, require eight days for revival and
propagation, whilst, under other conditions, only a few hours are
necessary; so that, if we introduce the notion of time into our
estimate of their power of decomposition, we may be led to
conclude that in the first case that power was entirely wanting,
and that in the second case it was considerable, although all the
time we are dealing with the same organism--the identical
ferment.
M. Schutzenberger is astonished that fermentation can take place
in the presence of free oxygen, if, as we suppose, the
decomposition of the sugar is the consequence of the nutrition of
the yeast, at the expense of the combined oxygen, which yields
itself to the ferment. At all events, he argues, fermentation
ought to be slower in the presence of free oxygen. But why should
it be slower? We have proved that in the presence of oxygen the
vital activity of the cells increases, so that, as far as
rapidity of action is concerned, its power cannot be diminished.
It might, nevertheless, be weakened as a ferment, and this is
precisely what happens. Free oxygen imparts to the yeast a vital
activity, but at the same time impairs its power as yeast--qua
yeast, inasmuch as under this condition it approaches the state
in which it can carry on its vital processes after the manner of
an ordinary fungus; the mode of life, that is, in which the ratio
between the weight of sugar decomposed and the weight of the new
cells produced will be the same as holds generally among
organisms which are not ferments. In short, varying our form of
expression a little, we may conclude with perfect truth, from the
sum total of observed facts, that the yeast which lives in the
presence of oxygen and can assimilate as much of that gas as is
necessary to its perfect nutrition, ceases absolutely to be a
ferment at all. Nevertheless, yeast formed under these conditions
and subsequently brought into the presence of sugar, OUT OF THE
INFLUENCE OF AIR, would decompose more IN A GIVEN TIME than in
any other of its states. The reason is that yeast which has
formed in contact with air, having the maximum of free oxygen
that it can assimilate is fresher and possessed of greater vital
activity than that which has been formed without air or with an
insufficiency of air. M. Schutzenberger would associate this
activity with the notion of time in estimating the power of the
ferment; but he forgets to notice that yeast can only manifest
this maximum of energy under a radical change of its life
conditions; by having no more air at its disposal and breathing
no more free oxygen. In other words, when its respiratory power
becomes null, its fermentative power is at its greatest. M.
Schutzenberger asserts exactly the opposite (p. 151 of his work--
Paris, 1875) [Footnote: Page 182, English edition], and so
gratuitously places himself in opposition to facts.
In presence of abundant air supply, yeast vegetates with
extraordinary activity. We see this in the weight of new yeast,
comparatively large, that may be formed in the course of a few
hours. The microscope still more clearly shows this activity in
the rapidity of budding, and the fresh and active appearance of
all the cells. Fig. 6 represents the yeast of our last experiment
at the moment when we stopped the fermentation. Nothing has been
taken from imagination, all the groups have been faithfully
sketched as they were. [Footnote: This figure is on a scale of
300 diameters, most of the figures in this work being of 400
diameters].
[Illustration with caption: Fig. 6]
In passing it is of interest to note how promptly the preceding
results 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 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 with caption: Fig. 7]
With this object we repeated the experiment that we performed
with the large-bottomed flask (Fig. 4), employing a vessel shaped
like Fig. B (Fig. 7), 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 cc. (about 2 fluid ounces) of yeast-water,
sweetened with two percent. of sugar and impregnated with a trace
of yeast. After having subjected our vessel to a temperature of
25 degrees C. (77 degrees 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 cc. 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 cc., there was a total
absorption of 14.5 cc. (0.83 cub. in.) of oxygen. [Footnote: It
may be useful for the non-scientific reader to put it thus: that
the 25 cc. which escaped, being a fair sample of the whole gas in
the flask, and containing (1) 25-20.6=4.4 cc., absorbed by potash
and therefore due to carbonic acid, and (2) 20.6-17.3=3.3 cc.,
absorbed by pyrogallate, and therefore due to oxygen, and the
remaining 17.3 cc. being nitrogen, the whole gas in the flask,
which has a capacity of 312 cc., will contain oxygen in the above
portion and therefore its amount may be determined provided we
know the total gas in the flask before opening. On the other hand
we know that air normally contains approximately, 1-5 its volume
of oxygen, the rest being nitrogen, so that, by ascertaining the
diminution of the proportion in the flask, we can find how many
cubic centimeters have been absorbed by the yeast. The author,
however, has not given all the data necessary for accurate
calculation.--D.C.R.] The weight of the 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 cc. (about
7/8 cub. in.) of oxygen, even supposing that the yeast was formed
entirely under the influence of that gas: this is equivalent to
not less than 414 cc. for 1 gramme of yeast (or about 33 cubic
inches for every 20 grains). [Footnote: This number is probably
too small; it is scarcely possible that the increase of weight in
the yeast, even under the exceptional conditions of the
experiment described, was not to some extent at least due to
oxidation apart from free oxygen, inasmuch as some of the cells
were covered by others. The increased weight of the yeast is
always due to the action of two distant modes of vital energy--
activity, namely, in presence and activity in absence of air. We
might endeavor to shorten the duration of the experiment still
further, in which case we would still more assimilate the life of
the yeast to that of ordinary moulds.]
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 the
paragraph on page 292 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 cc.
(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 X
4l4 cc., that is, 931.5 cc. (56.85 cubic inches). The greater
part of the 2.25 grammes, therefore, had evidently been produced
as the growth of an anaerobian plant.
Ordinary fungi likewise require large quantities of oxygen for
their development, as we may readily 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. 8, capable of
holding about 300 cc. (10 1/2 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 gram) of MYCELIUM, dried at 100 degrees C. (212
degrees F.), an absorption that amounted to not less than 43 cc.
(2.5 cubic inches) of oxygen at 25 degrees. 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 phenomena 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. [Footnote: In
these experiments, in which the moulds remain for a long time in
contact with a saccharine wort out of contact with oxygen--the
oxygen being promptly absorbed by the vital action of the plant
(see our Memoire sur les Generations dites Spontanees, p. 54.
note)--there is no doubt that an appreciable quantity of alcohol
is formed because the plant does not immediately lose vital
activity after the absorption of oxygen.
A 300 cc. (10-oz.) flask, containing 100 cc. of must, after the
air in it had been expelled by boiling, was open and immediately
re-closed on August 15th, 1873. A fungoid growth--a unique one,
of greenish-grey colour--developed from spontaneous impregnation,
and decolourized the liquid, which originally was of a yellowish-
brown. Some large crystals, sparkling like diamonds, of neutral
tartrate of lime, were precipitated, about a year afterwards,
long after the death of the plant, we examined this liquid. It
contained 0.3 gramme (4.6 grains) of alcohol, and 0.053 gramme
(0.8 grain) of vegetable matter, dried at 100 degrees C. (212
degrees F.). We ascertained that the spores of the fungus were
dead at the moment when the flask was opened. When sown, they did
not develop in the least degree.]
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 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. [Footnote: We find in M. Raulin's note that
"the minimum ratio between the weight of sugar and the weight of
organized matter, that is, the weight of fungoid growth which it
helps to form, may be expressed as 10/3.2=3.1." JULES RAULIN,
Etudes chimiques sur la vegetation. Recherches sur le
developpement d'une mucedinee dans un milieu artificiel, p. 192,
Paris, 1870. We have seen in the case of yeast that this ratio
may be as low as [Proofers note: unreadable symbol]] 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 previously demonstrated 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 aerobian 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 aerobian
or anaerobian life, according to the conditions under which they
are placed.
It may be readily understood how, in their state of aerobian
life, the alcoholic ferments have failed to attract attention.
These ferments are only cultivated out of contract 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 anaerobian 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 utilised in
certain industrial operations, on account of their power in
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. [Footnote: We shall show, some day,
that the processes of oxidation due to growth of fungi cause, in
certain decompositions, liberation of ammonia to a considerable
extent, and that by regulating their action we might cause them
to extract the nitrogen from a host of organic debris, as also,
by checking the production of such organisms, we might
considerably increase the proportion of nitrates in the
artificial nitrogenous substances. By cultivating the various
moulds on the surface of damp bread in a current of air we have
obtained an abundance of ammonia, derived from the decomposition
of the albuminoids effected by the fungoid life. The
decomposition of asparagus and several other animal or vegetable
substances has similar results.] On this last subject, the
important work of M. Van Tieghem (Annales Scientifiques de
l'Ecole 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. * * * *
It is a great presumption in favor 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 prima facie improbability. This is
exactly the character of those ideas which we have just
expounded. We pronounced 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 Comtes rendus de
l'Academie 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 Societe Chimique de Paris:--
"M. Pasteur gives the result 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 into 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 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 cc. (rather more than 5 fluid ounces) of yeast--
water, containing 2 1/2 per cent, of milk-sugar. The solution was
prepared in one of our double-necked flasks, with the necessary
precautions to secure the 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
November 15th, pouring some wort on the deposit of the plant,
which remained in the flask. In less than five hours from the
time we placed it in the oven, the plant 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 cause 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
tinder 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 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 should like to 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 gas, in consequence of a renewed action starting in
the interior cells of the grapes, in such a way that these cells
would assume the functions 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. [Footnote:
To determine the absence of cells of ferment in fruits that have
been immersed in carbonic acid gas, we must first of all
carefully raise the pellicle of the fruit, taking care that the
subjacent parenchyma does not touch the surface of the pellicle,
since the organized corpuscles existing on the exterior of the
fruit might introduce an error into our miscroscopical
observations. Experiments on grapes have given us an explanation
of a fact generally known, the cause of which, however, had
hitherto escaped our knowledge. We all know that the taste and
aroma of the vintage, that is, of the grapes stripped from the
bunches and thrown into tubs, where they get soaked in the juice
that issues from the wounded specimens, are very different from
the taste and aroma of an uninjured bunch. Now grapes that have
been immersed in an atmosphere of carbonic acid gas have exactly
the flavour and smell of the vintage; the reason is that, in the
vintage tub, the grapes are immediately surrounded by an
atmosphere of carbonic acid gas, and undergo, in consequence, the
fermentation peculiar to grapes that have been plunged into this
gas. These facts deserve to be studied from a practical point of
view. It would be interesting, for example, to learn what
difference there would be in the quality of two wines, the grapes
of which, in the once case, had been perfectly crushed, so as to
cause as great a separation of the cells of the parenchyma as
possible; in the other case, left, for the most part, whole, as
in the case in the ordinary vintage. The first wine would be
deprived of those fixed and fragrant principles produced by the
fermentation of which we have just spoken, when the grapes are
immersed in carbonic acid gas, by such a comparison as that which
we suggest we should be able to form a priori judgment on the
merits of the new system, which had not been carefully studied,
although already widely adopted, of milled, cylindrical crushers,
for pressing the vintage.]
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 Ecole 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:[Footnote: We have sometimes found small quantities of
alcohol in fruits and other vegetable organs, surrounded with
ordinary air, but always in small proportion, and in a manner
which suggested its accidental character. It is east to
understand how, in the thickness of certain fruits, certain parts
of those fruits might be deprived of air, under which
circumstances they would have been acting under conditions
similar to those under which fruits act when wholly immersed in
the carbonic acid gas. Moreover, it would be useful to determine
whether alcohol is not a normal product of vegatation.]--On July
21, 1872, we placed twenty-four of these plums under a glass
bell, which we immediately filled with carbonic acid gas. The
plums had been gathered on the previous day. By the side of the
bell 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
bell, 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 Berard 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 than these facts could we have 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 bell, one of which is in contact
with the objects, are touched alternately. It also makes itself
evident in the formation of 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.
[Footnote: In these studies of plants living immersed in carbonic
acid gas, we have come across a fact which corroborated those
which we have already given in reference to the facility with
which lactic and viscous ferments, and generally speaking, those
which we have termed the disease ferments or beer, develop when
deprived of air, and which shows, consequently, how very marked
their aerobian character is. If we immerse beet-roots or turnips
in carbonic acid gas, we produce well-defined fermentations in
those roots. Their whole surface readily permits the escape of
the highly acid liquids, and they become filled with lactic,
viscous, and other ferments, This shows us the great danger which
may result from the use of pits, in which the beet-roots are
preserved, when the air is not renewed, and that the original
oxygen is expelled by the vital processes of fungi or other
deoxidizing chemical actions. We nave directed the attention of
the manufacturers of beet-root sugar to this point.]
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 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 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 Ecole Normale
Superieure, and his coadjutor, M. Bellamy. [Footnote: Lechartier
and Bellamy, Comptes rendus de l'Academie des Sciences, vol.
lxix., pp., 366 and 466, 1869.] In 1821, in a very remarkable
work, especially when we consider the period when it appeared,
Berard 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 Berard 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 fore-shadowed 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.
[Footnote: Those gentlemen express themselves thus: "In a note
presented to the Academy in November, 1872, we published certain
experiments which showed that carbonic acid and alcohol may be
produced in fruits kept in a closed vessel, out of contact with
atmospheric oxygen, without our being able to discover alcoholic
ferment in the interior of those fruits.
"M. Pasteur, as a logical deduction from the principle which he
has established in connection with the theory of fermentation,
considers that THE FORMATION OF ALCOHOL MAY BE ATTRIBUTED TO THE
FACT THAT THE PHYSICAL AND CHEMICAL PRECESSES OF LIFE IN THE
CELLS OF FRUIT CONTINUE UNDER NEW CONDITIONS, IN A MANNER SIMILAR
TO THOSE OF THE CELLS OF FERMENT. Experiments, continued during
1872, 1873, and 1874, on different fruits have furnished results
all of which seem to us to harmonize with this proposition, and
to establish it on a firm basis of proof."--Comptes rendus, t.
lxxix., p. 949, 1874.] 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. [Footnote: PASTEUR, Faites nouveaux pour servir a la
connaissance de la theorie des fermentations proprement dites.
(Comptes rendus de l'Academie des Sciences, t. lxxv., p. 784.)
See in the same volume the discussion that followed; also,
PASTEUR, Note sur la production de l'alcool par les fruits, same
volume, p. 1054, in which we recount the observations anterior to
our own, made by Messrs. Lechartier and Bellamy in 1869.] M.
Fremy, in particular, was desirous of finding in these
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, quoted word for word in the preceding section, furnish
the most conclusive evidence in favor 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 that yeast does. Such is
the case with the fungi already studied; 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 aerobian state and we
have no fermentation; immersed immediately afterwards in carbonic
acid gas, they now assume their anaerobian 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.
[Footnote: Comptes rendus, meeting of January 15th, 1872.]
"To speak here of alcoholic fermentation alone," 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; Pasteur,
on the other hand, maintains that the fermentation is produced by
germs existing outside of the grapes." [Footnote: As a matter of
fact, M. Fremy applies his theory of hemi-organism, not only to
the alcoholic fermentation of grape juice, but to all other
fermentations. The following passage occurs in one of his notes
(Comptes rendus de l'Academie, t. lxxv., p. 979, October 28th,
1872):
"Experiments on Germinated Barley.--The object of these was to
show that when barley, left to itself in sweetened water,
produces in succession alcoholic, lactic, butyric, and acetic
fermentations, these modifications are brought about by ferments
which are produced inside the grains themselves, and not by
atmospheric germs. More than forty different experiments were
devoted to this part of my work."
Need we add that this assertion is based on no substantial
foundation? The cells belonging to the grains of barley, or their
albuminous contents, never do produce cells of alcoholic ferment,
or of lactic ferment, or butyric vibrios. Whenever those ferments
appear, they may be traced to germs of those organisms, diffused
throughout the interior of the grains, or adhering to the
exterior surface, or existing in the water employed, or on the
side of the vessels used. There are many ways of demonstrating
this, of which the following is one: Since the results of our
experiments have shown that sweetened water, phosphates, and
chalk very readily give rise to lactic and butyric fermentations,
what reason is there for supposing that if we substitute grains
of barley for chalk, the lactic and butyric ferments will spring
from those grains, in consequence of a transformation of their
cells and albuminous substances? Surely there is no ground for
maintaining that they are produced by hemi-organism, since a
medium composed of sugar, or chalk, or phosphates of ammonia,
potash, or magnesia contains no albuminous substances. This is an
indirect but irresistible argument against the hemi-organism
theory.]
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
experiments 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 had been 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. [Footnote: PASTEUR, Memoire sur la fermentation
alcoolique, 1860: Annales de Chimie et de Physique. The word
globules is here used for cells. In our researches we have always
endeavoured to prevent any confusion of ideas. We stated at the
beginning of our Memoir of 1860 that: "We apply the term
alcoholic to that fermentation which sugar undergoes under the
influence of the ferment known as BEER YEAST." This is, the
fermentation which produces wine and all alcoholic beverages.
This, too, is regarded as the type for a host of similar
phenomena designated, by general usage, under the generic name of
fermentation, and qualified by the name of one of the essential
products of the special phenomenon under observation. Bearing in
mind this fact in reference to the nomenclature that we have
adopted it will be seen that the expression ALCOHOLIC
FERMENTATION cannot be applied to every phenomenon of
fermentation in which alcohol is produced, inasmuch as there may
be a number of phenomena having this character in common. If we
had not at starting defined that particular one amongst the
number of very distinct phenomena, which, to the exclusion of the
others, should bear the name of alcoholic fermentation, we should
inevitably have given rise to a confusion of language that would
soon pass from words to ideas, and tend to introduce unnecessary
complexity into researches which are already, in themselves,
sufficiently complex to necessitate the adoption of scrupulous
care to prevent their becoming still more involved. It seems to
us that any further doubt as to the meaning of the words
ALCOHOLIC FERMENTATION, and the sense in which they are employed,
is impossible, inasmuch as Lavoisier, Gay-Lussac, and Thenard
have applied this term to the fermentation of sugar by means of
beer yeast. It would be both dangerous and unprofitable to
discard the example set by these illustrious masters, to whom we
are indebted for our earliest knowledge of this subject.]
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 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
fermentaction 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
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 section with a few remarks on the subject
of 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 has 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 as 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 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 the globules
already formed. The general results of the present Memoir seem to
us to be it 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 the 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 activity in carbonic acid gas
whilst in the other case the ferment consists of 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 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. [Footnote:
See, for example, the communications of MM. Colin and Poggiale,
and the discussion on them. In the Bulletin de l'Academie de
Medecine, March 2d, 9th, and 30th, and February 16th and 23rd,
1875.] In this fermentation, neither alcohol nor carbonic acid
gas exists in those proportions in which they are found in
fermentation produced by yeast; and, although we may determine in
it the presence of succinic acid, glycerine, and a small quantity
of volatile acids [Footnote: We have elsewhere determined the
formation of minute quantities of volatile acids in alcoholic
fermentation. M. Bechamp, who studied these, recognized several
belonging to the series of fatty acids, acetic acid, butyric acid
&c. "The presence of succinic acid is not accidental, but
constant; if we put aside volatile acids that form in quantities
which we may call infinitely small, we may say that succinic acid
is the only normal acid of alcoholic fermentation."--PASTEUR,
Comptes rendus de l' Academie, t. xlvii., P. 224, 1858] 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 the 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
that 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'etre 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 the 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
section. 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 within 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 anaerobian 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
true 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 section,
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. [Footnote: Traube's conceptions
are governed by a theory of fermentation entirely his own, a
hypothetical one, as he admits, of which the following is a brief
summary: "We have no reason to doubt," Traube says, "that the
protoplasm of vegetable cells is itself, or contains within it, a
chemical ferment which causes the alcoholic fermentation of
sugar; its efficacy seems closely connected with the presence of
the cell, inasmuch as, up to the present time, we have discovered
no means of isolating it from the cells with success. In the
presence of air this ferment oxidizes sugar by bringing oxygen to
bear upon it; in the absence of air it decomposes the sugar by
taking away oxygen from one group of atoms of the molecule of
sugar and bringing it to act upon other atoms; on the one hand
yielding a product of alcohol by reduction, on the other hand a
product of carbonic acid gas by oxidation."
Traube supposes that this chemical ferment exists in yeast and in
all sweet fruits, but only when the cells are intact, for he has
proved for himself that thoroughly crushed fruits give rise to no
fermentation whatever in carbonic acid gas. In this respect this
imaginary chemical ferment would differ entirely from those which
we call SOLUBLE FERMENTS, since diastase, emulsine, &c., may be
easily isolated.
For a full account of the views of Brefeld and Traube, and the
discussion which they carried on on the subject of the results of
our experiments, our readers may consult the Journal of the
Chemical Society of Berlin, vii., p. 872. The numbers for
September and December, 1874, in the same volume, contain the
replies of the two authors.]
IV. FERMENTATION OF DEXTRO-TARTRATE OF LIME.
[Footnote: See PASTEUR, Comptes rendus de l'Academie des
Sciences, t. lvi., p. 416.]
Tartrate of lime, in spite of its insolubility in waters 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 material 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. 9, of 2.5 litres
(about four pints) in capacity, we put:
Pure, crystallized, neutral tartrate of lime. .. 100 grammes
Phosphate of ammonia. ... . ... . .. ... . ... 1 grammes
Phosphate of magnesium. ... . ... . ... . ... .. 1 grammes
Phosphate of potassium. ... . ... . ... . .. 0.5 grammes
Sulphate of ammonia. ... . ... . ... . ... .. 0.5 grammes
(1 gramme = 15.43 grains)
To this we added pure distilled water, so as entirely to 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 with caption: Fig. 9]
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 fort-night. It might have
remained there for a century without ever manifesting the least
sign of fermentation, the fermentation of the tartrate being a
consequence of life, and life after 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. (about 17
minims) of liquid and deposit from another flask, similar to the
one we have just 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 doubts 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 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 cc. (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. [Footnote: Carbonic add
being considerably more soluble than other gases possible under
the circumstances.--ED.]
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 degrees C. and 28 degrees C. (77 degrees
F. and 83 degrees 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 the gaugemark on the neck.
[Footnote: We had to avoid filling the small flask completely,
for fear of causing some of the liquid to pass on to the surface
of the mercury in the measuring tube. The liquid condensed by
boiling forms pure water, the solvent affinity of which for
carbonic acid, at the temperature we employ, is well known. 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 the 2.135 litres, gave a total of 10.457 litres
(638.2 cubic inches) at 20 degrees and 760 mm., which, calculated
to 0 degrees, C. and 760 mm. atmospheric pressure (32 degrees F.
and 30 inches) gave a weight of 19.700 grammes (302.2 grains) of
carbonic acid.
Exactly half of the lime in 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.
[Footnote: The following is a curious consequence of these
numbers and of the nature of the products of this fermentation.
The carbonic acid liberated being quite pure, especially when the
liquid has been boiled to expel all air from the flask, and
capable of perfect solution, it follows that the volume of liquid
being sufficient and the weight of tartrate suitably chosen--we
may set aside tartrate of lime in an insoluble, crystalline
powder, alone with phosphates at the bottom of a closed vessel
full of water, and find soon afterwards in their place carbonate
of lime, and in the liquid soluble salts of lime, with a mass of
organic matter at the bottom, without any liberation of gas or
appearance of fermentation ever taking place, except as far as
the vital action and transformation in the tartrate are
concerned. It is easy to calculate that a vessel or flask of five
litres (rather more than a gallon) would be large enough for the
accomplishment of this remarkable and singularly quiet
transformation, in the case of 50 grammes (767 grains) of
tartrate of lime.]. 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.
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. [Footnote: We treated the whole deposit with
dilute hydrochloric acid, which dissolved the carbonate of lime
and the insoluble phosphates of calcium and magnesium; afterwards
filtering the liquid through a weighed filter paper. Dried at 100
degrees C. (212 degrees F.), the weight of the organic matter
thus obtained was 0.54 gramme (8.3 grains), which was rather more
than 1/200 of the weight of fermentable matter.] 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 punctuate, 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 on 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
shortness. Not one of these vibrios could be found throughout the
mass of the liquid.
[Illustration with caption: Figure 10.]
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.