_BACTERIA AND FERMENTATION_
It was Pasteur who in 1857 first propounded the true cause and process
of fermentation. The breaking down of sugar into alcohol and carbonic
acid gas had been known, of course, for a long period. Since the time
of Spallanzani (1776) the putrefactive changes in liquids and organic
matter had been prevented by boiling and subsequently sealing the flask
or vessel containing the fluid. Moreover, this successful preventive
practice had been in some measure correctly interpreted as due to the
exclusion of the atmosphere, but wrongly credited to the exclusion of
the oxygen of the air. It was not until the beginning of the present
century that authorities modified their view and declared in favour
of yeast cells as the agents in the production of fermentation. That
this process was due to oxygen _per se_ was disproved by Schwann, who
showed that so long as the oxygen admitted to the flask of fermentative
fluid was sterilised no fermentation occurred. It was thus obvious that
it was not the atmosphere or the oxygen of the atmosphere, but some
fermenting agent borne into the flask by the admission of unsterilised
air. It was but a step to further establish this hypothesis by adding
unsterilised air plus some antiseptic substance which would destroy the
fermenting agent. Arsenic was found by Schwann to have this germicidal
faculty. Hence Schwann supported Latour's theory that fermentation was
due to something borne in by the air, and that this something was
yeast. Passing over a number of counter-experiments of Helmholtz and
others, we come to the work of Liebig. He viewed the transformation
of sugar into alcohol and carbonic acid gas simply and solely as a
non-vital chemical process, depending upon the dead yeast communicating
its own decomposition to surrounding elements in contact with it.
Liebig insisted that all albuminoid bodies were unstable, and if left
to themselves would fall to pieces--_i. e._, ferment--without the aid
of living organisms, or any initiative force greater than dead yeast
cells. It was at this juncture that Pasteur intervened to dispel the
obscurities and contradictory theories which had been propounded.
As in all the conclusions arrived at by Pasteur, so in those relating
to fermentation, there were a number of different experiments which
were performed by him to elucidate the same point. We will choose one
of many in relation to fermentation. If a sugary solution of carbonate
of lime is left to itself, after a time it begins to effervesce,
carbonic acid is evolved, and lactic acid is formed; and this latter
decomposes the carbonate of lime to form lactate of lime. This lactic
acid is formed, so to speak, at the expense of the sugar, which
little by little disappears. Pasteur demonstrated the cause of this
transformation of sugar into lactic acid to be a thin layer of organic
matter consisting of extremely small moving organisms. If these be
withheld or destroyed in the fermenting fluid, fermentation will cease.
If a trace of this grey material be introduced into sterile milk or
sterile solution of sugar, the same process is set up, and lactic acid
fermentation occurs.
Pasteur examined the elements of this organic layer by aid of the
microscope, and found it to consist of small short rods of protoplasm
quite distinct from the yeast cells which previous investigators had
detected in alcoholic fermentation. One series of experiments was
accomplished with yeast cells and these bacteria, a second series with
living yeast cells only, a third series with bacteria only, and the
conclusions which Pasteur arrived at as the result of these labours
were as follows:
"As for the interpretation of the group of new facts which
I have met with in the course of these researches, I am
confident that whoever shall judge them with impartiality will
recognise that the alcoholic fermentation is an act correlated
to the life and to the organisation of these corpuscles,
and not to their death or their putrefaction, any more than
it will appear as a case of contact action in which the
transformation of the sugar is accomplished in the presence of
the ferment without the latter giving or taking anything from
it."
Pasteur occupied six years (1857-1863) with further elucidation of
his wonderful discovery of the potency of these hitherto unrecognised
agents, and the establishment of the fact that "organic liquids do not
alter until a living germ is introduced into them, and living germs
exist everywhere."
It must not be supposed that to Pasteur is due the whole credit of the
knowledge acquired respecting the cause of fermentation. He did not
first discover these living organisms; he did not first study them and
describe them; he was not even the first to suggest that they were the
cause of the processes of fermentation or disease. But, nevertheless,
it was Pasteur who "first placed the subject upon a firm foundation by
proving with rigid experiment some of the suggestions made by others."
Thus it has ever been in the times of new learning and discovery: many
contributors have added their quota to the mass of knowledge, even
though one man appearing at the right moment has drawn the conclusions
and proved the theory to be fact.
In order that no confusion may arise in the mind of the reader, we
may here say that, although fermentation is always due to a living
agent, as proved by Pasteur, the process is conveniently divided into
two kinds.[30] (1) When the action is direct, and the chemical changes
involved in the process occur only in the presence of the cell, the
latter is spoken of as an _organised ferment_; (2) when the action is
indirect, and the changes are the result of the presence of a soluble
material secreted by the cell, acting apart from the cell, this soluble
substance is termed an _unorganised soluble ferment_, or _enzyme_.
The organised ferments are bacteria or vegetable cells allied to the
bacteria; the unorganised ferments, or enzymes, are ferments found in
the secretions of specialised cells of the higher plants and animals.
With the former this book deals in an elementary fashion; with the
latter we have little concern. It will be sufficient to illustrate the
enzymes by a few of the more familiar examples. They form, for example,
the digestive agents in human assimilation. This function is performed,
in some cases, by the enzyme combining with the substance on which
it is acting and then by decomposition yielding the new "digested"
substance and regenerating the enzyme; in other cases, the enzyme, by
its molecular movement, sets up molecular movement in the substance it
is digesting, and thus changes its condition. These digestive enzymes
are as follows: in the saliva, _ptyalin_, which changes starch into
sugar; in the gastric juice of the stomach, _pepsin_, which digests the
proteids of the food and changes them into absorptive peptones; the
pancreatic ferments, _amylopsin_, _trypsin_, and _steapsin_, capable
of attacking all three classes of food stuffs; and the intestinal
ferments, which have not yet been separated in purer condition. In
addition to these, there are ferments in bitter almonds, mustard, etc.
Concerning these unorganised ferments we have nothing further to say.
Perhaps the commonest of them all is _diastase_, which occurs in malt,
and to which some reference will be made later.
Its function is to convert the starch which occurs in barley into
sugar. These unorganised ferments act most rapidly at about 75° C.
(167° F.).[31]
We may now return to the work of Pasteur and the question of _organised
ferments_. Let us preface further remark with an axiom with which
Professor Frankland sums up the vitalistic theory of fermentation,
which was supported by the researches of Pasteur: "_No fermentation
without organisms, in every fermentation a particular organism_." From
these words we gather that there is no one particular organism or
vegetable cell to be designated the micro-organism of fermentation,
but that there are a number of fermentations each started by some
specific form of agent. It is true that the chemical changes induced
by organised ferments depend on the life processes of micro-organisms
which feed upon the sugar or other substance in solution, and excrete
the product of the fermentation. Fermentation nearly always consists
of a process of breaking down of complex bodies, like sugar, into
simpler ones, like alcohol and carbonic acid. Of such fermentation
we may mention at least five: the _alcoholic_, by which alcohol is
produced; the _acetous_, by which wine absorbs oxygen from the air and
becomes vinegar; the _lactic_, which sours milk; the _butyric_, which
out of various sugars and organic acids produces butyric acid; and
_ammoniacal_, which is the putrefactive breaking down of compounds of
nitrogen into ammonia. We have already referred at some length to this
process when considering denitrifying organisms in the soil.
There are four chief conditions common to all these five kinds of
organised fermentation. They are as follows:--
1. The presence of the special living agent or organism of the
particular fermentation under consideration. This, as Pasteur pointed
out, differs in each case.
2. A sufficiency of pabulum (nutriment) and moisture to favour the
growth of the micro-organism.
3. A temperature at or about blood-heat (35-38° C., 98.5° F.).
4. The absence from the solution or substance of any obnoxious or
inimical substances which would destroy or retard the action of the
living organism and agent. Many of the products of fermentation are
themselves antiseptics, as in the case of alcohol; hence alcoholic
fermentation always arrests itself at a certain point.
We are now in a position to consider particular fermentations and
their causal micro-organisms. These latter are of various kinds,
belonging, according to botanical classification, to various different
subdivisions of the non-flowering portion of the vegetable kingdom.
A large part of fermentation is based upon the growth of a class of
microscopic plants termed _yeasts_. These differ from the bacteria in
but few particulars, mainly in their method of reproduction by budding
(instead of dividing or sporulating, like the bacteria). Their chemical
action is closely allied to that of the bacteria. Secondly, there are
special fermentations and modifications of yeast fermentation due
to _bacteria_. Thirdly, a group of somewhat more highly specialised
vegetable cells, known as _moulds_, make a perceptible contribution in
this direction. According to Hansen, these latter, so far as they are
really alcoholic ferments, induce fermentation, not only in solutions
of dextrose and invert sugar, but also in solutions of maltose. _Mucor
racemosus_ is the only member that is capable of inverting a cane-sugar
solution; _M. erectus_ is the most active fermenter, yielding eight
per cent. by volume of alcohol in ordinary beer wort. Each of these
will be referred to as they occur in considering the five important
fermentations already mentioned.
[Illustration: SACCHAROMYCES CEREVISIÆ]
The general microscopic appearance of yeast cells may be shortly
stated as follows: they are round or oval cells, and by budding become
daughter yeasts. Each consists of a membrane and clear homogeneous
contents. As they perform their function of fermentation, vacuoles,
fat-globules, and other granules make their appearance in the enclosed
plasma. As in many vegetable cells a _nucleus_ was detected by Schmitz
by means of special methods of staining, Hansen has found the nucleus
in old yeast cells from "films" without any special staining.
1. _Alcoholic Fermentation._
Cause, yeast; medium, sugar solutions; result, alcohol and
carbonic acid.
It was Caignard-Latour who first demonstrated that yeast cells, by
their growth and multiplication, set up a chemical change in sugar
solutions which resulted in the transference of the oxygen from the
hydrogen in the sugar compound to the carbon atoms, that is to say, in
the evolution of carbonic acid gas and the production, as a result, of
alcohol. If we were to express this in a chemical formula, it would
read as follows:
C_{6}H_{12}O_{6} (plus the yeast) = 2 C_{2}H_{6}O + 2 CO_{2}.
A natural sugar, like grape-sugar, present in the fruit of the vine,
is thus fermented. The alcohol remains in the liquid; the carbonic
acid escapes as bubbles of gas into the surrounding air. It is thus
that brandy and wines are made. If we go a step further back, to
cane-sugar (which possesses the same elements as grape-sugar, but in
different proportions), dissolve it in water, and mix it with yeast,
we get exactly the same result, except that the first stage of the
fermentation would be the changing of the cane-sugar into grape-sugar,
which is accomplished by a soluble ferment secreted by the yeast cells
themselves. If now we go yet one step further back, to starch, the same
sort of action occurs. When starch is boiled with a dilute acid it
is changed into a gum-like substance named dextrin, and subsequently
into a sugar named maltose, which latter, when mixed with these living
yeast cells, is fermented, and results in the evolution of carbonic
acid gas and the production of alcohol. In the manufacture of fermented
drinks from cereal grains containing starch there is therefore a double
chemical process: first the change of starch into sugar by means of
_conversion_,[32] and secondly the change of the sugar into alcohol and
carbonic acid gas by the process of _fermentation_, an organic change
brought about by the living yeast cells.
In all these three forms of alcoholic fermentation the principal
features are the same, viz., the sugar disappears; the carbonic acid
gas escapes into the air; the alcohol remains behind. Though it is
true that the sugar disappears, it would be truer still to say that it
reappears as alcohol. Sugar and alcohol are built up of precisely the
same elements: carbon, hydrogen, and oxygen. They differ from each
other in the proportion of these elements. It is obvious, therefore,
that fermentation is really only a change of _position_, a breaking
down of one compound into two simpler compounds. This redistribution of
the molecules of the compound results in the production of some heat.
Thus we must add heat to the results of the work of the yeasts.
When alcohol is pure and contains no water it is termed _absolute
alcohol_. If, however, it is mixed with 16 per cent. of water, it is
called _rectified spirit_, and when mixed with more than half its
volume of water (56.8 per cent.) it is known as _proof spirit_.
We shall have to consider elsewhere a remarkable faculty which some
bacteria possess of producing products inimical to their own growth.
In some degree this is true of the yeasts, for when they have set up
fermentation in a saccharine fluid there comes a time when the presence
of the resulting alcohol is injurious to further action on their part.
It has become indeed a poison, and, as we have already mentioned, a
necessary condition for the action of a ferment is the absence of
poisonous substances. This limit of fermentation is reached when the
fermenting fluid contains 13 or 14 per cent. of alcohol.
Having discussed shortly the "medium" and the results, we may now turn
to the bacteriology of the matter, and enumerate some of the chief
forms of the yeast plant. Professor Crookshank[33] gives more than a
score of different members of this family of _Saccharomycetes_. Before
dwelling upon some of the chief of these, it will be desirable to
consider a number of properties common to the genus.
The yeast cell is a round or oval body of the nature of a fungus,
composed of granular protoplasm surrounded by a definite envelope, or
_capsule_. It reproduces itself by budding, or, as it is sometimes
termed, _gemmation_. At one end of the cell a slight swelling or
protuberance appears, which slowly enlarges. Ultimately there is
a constriction, and the bud becomes partly and at last completely
separated from the parent cell. In many cases the capsules of the
daughter cell and the parent cell adhere, thus forming a chain of
budding cells. The character of the cell and its method of reproduction
do not depend merely upon the particular species alone, but are also
dependent upon external circumstances. There are differences in the
behaviour of species towards different media at various temperatures,
towards the carbohydrates (especially maltose), and in the chemical
changes which they bring about in nutrient liquids. In connection with
this Professor Hansen has pointed out that, whilst some species can be
made use of in fermentation industries, others cannot, and some even
produce diseases in beer.[34]
[Illustration: ASCOSPORE FORMATION]
One of the most remarkable evidences of the adaptability of the yeasts
to their surroundings and a specific characteristic occurs in what
is sometimes called _ascospore_ formation. If a yeast cell finds
itself lacking nourishment or in an unfavourable medium, it reproduces
itself not by budding, but by forming spores out of its own intrinsic
substance, and within its own capsule. To obtain this kind of spore
formation Hansen used some gypsum blocks as medium on which to grow
his yeast cells. Well-baked plaster of Paris is mixed with distilled
water, and made into a liquid paste. Small moulds are made by pouring
this paste into cardboard dishes, where it hardens again. The mould is
sterilised by heat, and a small portion of yeast is placed on its upper
surface, and then the whole is floated in a small vessel of water and
covered with a bell-jar. Under these conditions of limited pabulum the
cell undergoes the following changes: it increases in size, loses much
of its granularity, and becomes homogeneous, and about thirty hours
after being sown on the gypsum there appear several refractile cells
inside the parent cell. These are the ascospores. In addition to the
gypsum, it is necessary to have a plentiful supply of oxygen, some
moisture (gained from the vessel of water in which the gypsum floats),
a certain temperature, and a young condition of the protoplasm of the
parent yeast cells. Hansen found that the lowest temperature at which
these ascospores were produced was .5-3° C., and at the other extreme
up to 37° C., which is blood-heat. The rapidity of formation also
varies with the temperature, the favourable degree of warmth being
about 22-25° C.
[Illustration: GYPSUM BLOCK]
Hansen pointed out that it was possible by means of sporulation to
differentiate species of yeasts. For it happens that different species
show slight differences in spore formation, _e. g._:
(_a_) The spores of _Saccharomyces cerevisiæ_ expand during the first
stage of germination, and produce partition walls, making a compound
cell with several chambers. Budding can occur at any point on the
surface of the swollen spores. To this group belong _S. pastorianus_
and _S. ellipsoideus_.
(_b_) The spores of _Saccharomyces Ludwigii_ fuse in the first stage,
and afterwards grow out into a promycelium, which produces yeast cells.
(_c_) The spores of _Saccharomyces anomalus_ are different in shape
from the others in that they possess a projecting rim round the base.
Another point in the cultivation of yeasts has been elucidated by a
number of workers, chief among whom perhaps is Hansen, namely, methods
of obtaining _pure cultures_. We know, generally speaking, what this
term means, and there is no difference in its meaning here to what is
understood as its meaning with regard to bacteria. There is, however,
some difference in the mode of securing it. It is only by starting
with one individual cell that we can hope to secure a pure culture of
yeasts. For the study of the morphology of yeasts under the microscope
the problem was not a difficult one. It was comparatively easy to keep
out foreign germs from a cover-glass preparation enough to perceive
germination of spores and growth of mycelium. But when we require pure
cultures for various physiological purposes, then a different standard
and method are necessary.
[Illustration: YEAST (SACCHAROMYCES CEREVISIÆ)
× 1000]
[Illustration: ASCOSPORE FORMATION IN YEAST
(The capsule of the parent cell around the spores is invisible)
× 1000]
[Illustration: NITROGEN FIXING BACTERIA FROM ROOTLET-NODULES
(SUBCULTURE)
× 1000]
[Illustration: BACILLUS OF TETANUS
(From broth culture, showing spore formation)
× 1000
_By permission of the Scientific Press, Limited_]
Pasteur and Cohn adopted a practice based upon the fact that when
organisms find themselves in a favourable medium they multiply to the
exclusion of others to which the medium is less favourable. Hence if
an impure mixture be placed under such circumstances there comes a
time when those organisms for which the circumstances are favourable
multiply to such an extent that they form an almost pure culture. The
method is open to fallacy, and will rarely result in a really pure
culture; and even if that be secured, it is quite possible that it
will be to the exclusion of the desired culture. Hansen has devised
a much improved process for securing a pure culture of yeast which
depends upon dilution. We believe Lister was one of the first who,
in the seventies, introduced some such plan as this. Hansen employed
dilution with water in the following manner:
Yeast is diluted with a certain amount of sterilised water. A drop is
carefully examined under the microscope, a single cell of yeast is
taken, and a cultivation made upon wort. When it has grown abundantly
a quantity of sterilised water is added. From this, again, a single
drop is taken and added, to, say 20 cc. of sterilised water in a fresh
flask. This flask will contain we will suppose ten cells. It is now
vigorously shaken, and the contents are divided into twenty portions
of 1 cc. each, and added to twenty tubes of sterilised water. It is
highly probable that half of those tubes have received one cell each.
In the course of a few days it can be seen how far a culture is pure.
If only one colony is present, the culture is a pure one, and as this
grows we obtain an absolutely pure culture in necessary quantity.
Even when the gelatine-plate method is used it is desirable to start
with a single cell (Hansen). The advantage of Hansen's yeast method
over Koch's bacterial-plate method is that it has a certain definite
starting-point. This is obviously impossible when dealing with such
microscopic particles as the bacteria proper.
A third matter in the differentiation of yeast species is the question
of _films_. Hansen set to work, after having obtained pure cultures
and ascospores, to examine films appearing on the surface of liquids
undergoing fermentation. The object of this was to ascertain whether
all yeasts produced the same mycelial growth on the surface of the
fermenting fluid. To produce these films the process is as follows:
Drop on to the surface of sterilised wort in a flask a very small
quantity of a pure culture of yeast; secure the flask from movement,
and protect it, not from air, which is necessary, but from falling
particles in the air. In a short time small colonies appear, which
coalesce and form patches, then a film or membrane which covers
the liquid and attaches itself to the sides of the flask. By the
differences in the films and the temperatures at which they form it is
possible to obtain something of a basis for classification. The further
advances in a yeast culture and in our knowledge of the agencies of
fermentation have, however, tended to show that no strict dividing
lines can be drawn. Hansen's researches have, notwithstanding, been
of the greatest moment to the whole industry of fermentation. What
has been found true in bacteriology has also been demonstrated in
fermentation, namely, that though many yeasts differ but little in
structure and behaviour, they may produce very different products
and possess very different properties. Industrial cultivation of
these finer differences in fermentative action has to a large extent
revolutionised the brewing industry.
The formation of films is not a peculiarity of certain species, but
must be regarded as a phenomenon occurring somewhat commonly amongst
yeasts. The requisites are a suitable medium, a yeast cell, a free,
still surface, direct access of air, and a favourable temperature. The
wort loses colour, and becomes pale yellow. Microscopic differences
soon appear between the sedimentary yeast and the film yeast of the
same species, the latter growing out into long mycelial forms, the
character of which depends in part upon the temperature. This often
varies from 3° to 38° C.
A fourth point helpful in diagnosis is the temperature which proves to
be the thermal death-point. _Saccharomyces cerevisiæ_ is killed by an
exposure to 54° C. for five minutes, and 62° C. kills the spores. As a
rule, yeasts can resist a considerably higher temperature when in a dry
state than in the presence of moisture.
Lastly, yeasts may be cultivated on solid media. Hansen employed
wort-gelatine (5 per cent. gelatine), and found that at 25° C. in a
fortnight the growths which develop show such microscopic differences
as to aid materially in diagnosis. _Saccharomyces ellipsoideus I._
exhibits a characteristic network which readily distinguishes it.
There is one other point to which reference must be made. The process
of fermentation may be set up by a "high" or a "low" yeast. These
terms apply to the temperature at which the process commences. "High"
yeasts rise to the surface as the action proceeds, accomplish their
work rapidly, and at a comparatively high temperature, say about 16°
C.; "low" yeasts, on the contrary, sink in the fermenting fluid,
act slowly, and only at the low temperature of 4° or 5° C. This is
maintainable by floating ice in the fluid. Formerly all beer was
made by the "high" mode, but on the continent of Europe "low" yeast
is mostly used, while the "high" is in vogue in England. This latter
method is more conducive to the development of extraneous organisms,
and therefore risky in all but well-ordered brewing establishments.
Whether high and low yeasts consist of one or several species is not
known.
Before proceeding to mention shortly some of the commoner forms of
yeast we must again emphasise Hansen's method of analysis in separating
a species. The shape, size, and appearance of cells are not sufficient
for differentiation, because it is found that the same species when
exposed to different external conditions can occur in very different
forms. Hence Hansen established the analytical method of observing
(1) the microscopic appearance, (2) the formation of ascospores, and
(3) the formation of films. In addition, the temperature limits,
cultivation on solid media, and behaviour towards carbohydrates,
are characters which aid in the separation of yeasts. By basing
differentiation of species upon these features, the following can be
distinguished:
_Saccharomyces Cerevisiæ._ Oval or ellipsoidal cells; reproduction by
budding; ascospores, rapidly at 30° C., slowly at 12° C., not formed
at all at lower temperatures; film formation, seven to ten days at 22°
C.; an active alcoholic ferment, producing in a fortnight in beer wort
from 4 to 6 per cent. by volume of alcohol (Jörgensen). This species
is a typical English "high" yeast, possessing the power of "inverting"
cane-sugar previous to producing alcohol and carbonic acid. It is said
to have no action on milk-sugar.
[Illustration: S. ELLIPSOIDEUS]
[Illustration: S. PASTORIANUS]
_Saccharomyces Ellipsoideus I._ Round, oval, or sausage-shaped cells,
single or in chains; ascospores in twenty-four hours at 25° C. (not
above 30° C., not below 4° C.). Grown on the surface of wort-gelatine,
a network is produced by which they can be recognised (in eight to
twelve days at 33° C.). At 13-15° C. a characteristic branching mass
is produced. It is an alcoholic ferment as active as _S. cerevisiæ_.
_S. Ellipsoideus II._ Round and oval, rarely elongated, a widely
distributed yeast, causing "muddiness" in beer and a bitter taste. It
is essentially a "low" yeast.
_Saccharomyces Conglomeratus_ is a round cell, often united in
clusters, and occurring in rotting grapes, and at the commencement of
fermentation.
_Saccharomyces Pastorianus I._ Oval or club-shaped cells, occurring
in after-fermentation of wine, etc., and producing a bitter taste,
unpleasant odour, and turbidity. The spores frequently occur in the air
of breweries.
_S. Pastor. II._ Elongated cells, possessing an invertose ferment. They
do not, like _S. pastor I._, produce disease in beer.
_S. Pastor. III._ Oval or elongated cells, producing turbidity in beer.
Grown on yeast-water gelatine, the colonies show after sixteen days
crenated hairy edges.
_Saccharomyces Apiculatus._ Lemon-shaped cells. They give rise to a
feeble alcoholic fermentation, and produce two kinds of spores--round
and oval; they appear at the onset of vinous fermentation, but give way
later on to _S. cerevisiæ_.
_Saccharomyces Mycoderma._ Oval or elliptical cells, often in branching
chains. They form the so-called "mould" on fermented liquids, and
develop on the surface without exciting fermentation. When forced to
grow submerged they produce a little alcohol.
_Saccharomyces Exiguus._ Conical cells, appearing in the
after-fermentation of beer.
_Saccharomyces Pyriformis._ Oval cells, converting sugary solutions
containing ginger into ginger-beer.
_Saccharomyces Illicis_, _Hansenii_, _et Aquifolii_ produce a small
percentage of alcohol.
2. _Acetous Fermentation._
Cause, Mycoderma aceti; medium, wine and other alcoholic
liquids; result, the formation of vinegar.
If alcohol be diluted with water, and the specific ferment mixed
with it and exposed to the air at 22° C., it is rapidly converted
into vinegar. The change is accompanied by the absorption of oxygen,
one atom of which combines with two of hydrogen to form water, and
a substance remains called _aldehyde_, further oxidation of which
produces the acetic acid. We may express it chemically thus:
Alcohol. Aldehyde. Water.
C_{2}H_{6}O (+ oxygen and the ferment) = C_{2}H_{4}O + H_{2}O.
The aldehyde becomes further oxidised:
C_{2}H_{4}O + O = C_{2}H_{4}O_{2} (acetic acid).
Now this method of simply oxidising alcohol to obtain acetic acid may
be carried out chemically without any ferment. If slightly diluted
alcohol be dropped upon _platinum black_, the oxygen condensed in
that substance acts with energy upon the spirit, and union readily
occurring, acetic acid results. Here the whole business of the platinum
sponge is to persuade the oxygen of the air and the hydrogen of the
alcohol to unite. In the ordinary manufacture this is accomplished by
the vegetable cells of _Mycoderma aceti_.
There are two chief methods adopted in the commercial manufacture of
vinegar, both of which depend upon the presence of the _Mycoderma_.
The method in vogue at Orleans when Pasteur (about 1862) commenced
his studies of the vinegar organism was to fill vats nearly to the
brim with a weak mixture of vinegar and wine. Where the process is
proceeding the surface is covered with a fragile pellicle, "the
mother of vinegar," which is produced by and consists of certain
micro-organisms whose function is to convey the oxygen of the air to
the liquor in the vats, thus oxidising the alcohol into vinegar. This
oxidation may be carried on even beyond the stage of acetic acid (when
no more alcohol remains to be oxidised), resulting in carbonic acid
gas, which escapes into the air. But as in the alcoholic, so in the
acetic, fermentation, there comes a time when the presence of an excess
of the acid inhibits the further growth of the organism. This point is
approximately when the acetic acid has reached a percentage as high as
14. But if the acid be removed, and fresh alcohol added, the process
recommences.
The second method, sometimes called by the Germans the "quick vinegar
process," is to pour the weakened alcohol through a tall cylinder
filled with wood-shavings, having first added some warm vinegar to the
shavings. After a number of hours the resulting fluid is charged with
acetic acid. What has occurred? Liebig maintained that a chemical and
mechanical change had brought about the change from the alcohol put
into the cylinder and the vinegar drawn off at the exit tube. It was
reserved for Pasteur to demonstrate by experiment that the addition
of the warm vinegar to the shavings was in reality an addition of a
living micro-organism, which, forming a film upon the shavings, became
"the mother of vinegar," and oxidised the alcohol which passed over it,
inducing it to become aldehyde and then acetic acid.
_Mycoderma Aceti_ (described by Persoon 1822, Kützing 1837, and Pasteur
1864). It must be understood that this term is the name rather of
a family than an individual. Pasteur believed it to be a specific
individual, but Hansen pointed out that it was composed of two
distinctly different species (_Bacterium aceti_ and _B. pasteurianum_),
and subsequently other investigators have added members to the acetic
fermentation group of which _M. aceti_ is the type.
This bacterium is made up of small, slightly elongated cells, with a
transverse diameter of 2 or 3 µ, sometimes united in short chains of
curved rods. They frequently show a central constriction, are motile,
and produce in old cultures involution forms. The way in which the
cells act and are made to perform their function is as follows: A
small quantity, taken from a previous pellicle, is sown on the surface
of an aqueous liquid, containing 2 per cent. of alcohol, 1 per cent.
of vinegar, and traces of alkaline phosphates. Very rapidly indeed
the little isolated colonies spread, and, becoming confluent, form a
membrane or pellicle over the whole area of fluid. When the surface is
covered the alcohol acidifies to vinegar. After this it is necessary
to add each day small quantities of alcohol. When the oxidation is
completed the vinegar is drawn off, and the membrane is collected and
washed, and is then again ready for use. It ought not to remain long
out of fermenting liquid, nor ought it to be allowed to over-perform
its function, for thus having oxidised all the alcohol it will commence
oxidation of the vinegar.
In wort-gelatine _Bacterium pasteurianum_ develops round colonies with
a smooth or wavy border, whilst _B. aceti_ has a tendency towards
stellate arrangement. Spores have not been observed, and from a
morphological point of view the two species behave alike. Neither
produces any turbidity in the liquid containing them. In order to
flourish, _B. aceti_ requires a temperature of about 33° C. and a
plentiful supply of oxygen. In a cool store or cellar there is,
therefore, nothing to fear from _B. aceti_. Frankland has isolated a
_Bacillus ethaceticus_, which is a fermentative organism producing
ethyl-alcohol and acetic acid. By oxidation the ethyl-alcohol may be
converted into acetic acid.
3. _Lactic Acid Fermentation._
Cause, Bacillus acidi lactici; medium, milk-sugar, cane-sugar,
glucose, dextrose, etc.; result, lactic acid.
The process set up by the lactic ferment is simply a decomposition, an
exact division of one molecule of sugar into two molecules of lactic
acid, there being neither oxidation nor hydration. The conditions
under which the ferment acts are very similar to those we have already
considered. There is frequently carbonic acid gas formed; there is a
cessation of fermentation when the medium becomes too acid; there
is the same method of starting the process by inoculation of sour
milk or cheese or any substance containing the specific bacillus. It
is probable that such inoculated matter will contain a mixture of
micro-organisms, but if the lactic bacillus is present, it will grow so
vigorously and abundantly that the fermentation will be readily set up.
[Illustration: B. ACIDI LACTICI]
_The Bacillus Acidi Lactici._ Rods about 2 µ long and 4 µ wide,
occurring singly or in chains and threads. It is non-motile. Spore
formation is present, the spores appearing irregularly or at one end of
the rod.
On the surface of gelatine a delicate growth appears along the track of
the needle, with round colonies appearing at the edges of the growth.
It does not liquefy gelatine. It grows best at blood-heat; but much
above that it fails to produce its fermentation, and it ceases to
grow under 10° C. It inverts milk-sugar and changes it to dextrose,
from which it then produces lactic acid. Sugars do, however, differ
considerably in the degrees to which they respond to the influence of
the lactic ferment, and some which are readily changed by the alcoholic
ferment are untouched by the _Bacillus acidi lactici_. It will be
necessary to refer again to this micro-organism when we come to speak
of milk and other dairy products.
Van Laer has described a saccharobacillus which produces lactic acid
amongst other products, and brings about a characteristic disease
in beer, named _tourne_. The liquid gradually loses its brightness
and assumes a bad odour and disagreeable taste. The bacillus is a
facultative anaërobe. A number of workers have separated organisms,
having a lactic acid effect, which diverge considerably from the
orthodox type of lactic acid bacillus. This is but further evidence of
a fact to which reference has been made: that nomenclature restricted
to one individual has now become adapted to a family.
4. _Butyric Acid Fermentation._
Cause, Bacillus butyricus and B. amylobacter; medium, milk,
butter, sugar and starch solutions, glycerine; result, butyric
acid.
When sugars are broken down by the _Bacillus acidi lactici_ the lactic
acid resulting may, under the influence of the butyric ferment,
become converted into butyric acid, carbonic acid, and hydrogen.
Neither butyric acid nor lactic acid is as commonly used as alcohol
or vinegar. Both, like vinegar, can be manufactured chemically, but
this is rarely practised. Butyric acid is a common ingredient in old
milk and butter, and its production by bacteria is historically one
of the first bacterial fermentations understood. Moreover, in its
investigation Pasteur first brought to light the fact that certain
organisms acted only in the absence of oxygen. In studying a drop of
butyric fermenting fluid, it was observed that the organisms at the
edge of the drop were motionless and apparently dead, whilst in the
central portion of the drop the bacilli were executing those active
movements which are characteristic of their vitality. To Pasteur's
mind this at once suggested what he was able later to demonstrate,
namely, that these bacilli were paralysed by contact with oxygen.
When he passed a stream of air through a flask containing a liquid in
butyric fermentation, he observed the process slacken and eventually
cease. So were discovered the _anaërobic_ micro-organisms. The aërobic
ferments give rise to oxidation of certain products of decomposition;
the anaërobic organisms, on the other hand, only commence to grow
when the aërobic have used up all the available oxygen. Thus in such
fermentations certain bodies (carbohydrates, fatty acids, etc.) undergo
decomposition, and by oxidation become carbonic acid gas, and the
remainder is left as a "reduced" product of the whole process. Hence
sometimes this is termed fermentation by reduction. The chemical
formula of this butyric reaction may be expressed thus:--
C_{6}H_{12}O_{6} (by simple decomposition) = 2 C_{3}H_{6}O_{3}.
Glucose, Lactic acid.
which is followed by the fermentation of the lactic acid:--
2 C_{3}H_{6}O_{3} = C_{4}H_{8}O_{2} + 2 CO_{2} + 2 H_{2}.
Lactic acid. Butyric acid. Carbonic Free hydrogen.
acid gas.
[Illustration: B. BUTYRICUS]
_Bacillus Butyricus._ Long and short rods, generally straight, with
rounded ends, single or in chains, reproducing themselves both by
fission and spores, and sometimes growing out into long threads,
actively motile, anaërobic, and liquefying. The spores are widely
distributed in nature, and grow readily on fleshy roots, old cheese,
etc. The favourable temperature is blood-heat, and on liquid media
they produce a pellicle. The resistant spores are irregularly placed
in the rod, and may cause considerable variations in morphology. The
culture gives off a strong butyric acid odour. It grows most readily at
a temperature of about 40° C.
Although, according to Pasteur's researches, the butyric acid ferment
performs its functions anaërobically, many butyric organisms can act in
the presence of oxygen, and yield somewhat different products.
All of them, however, ferment most actively at a temperature at or
about blood-heat, and the spores are able to withstand boiling for from
three to twenty minutes (Fitz). It will be observed that as in lactic
acid fermentation so in butyric, the results are not due to one species
only.
5. _Ammoniacal Fermentation_ (see under Soil).
_Diseases in Beer._ We have seen how a knowledge of fermentation has
been compiled by a large number of workers. Spallanzani, Schwann,
Pasteur, and Hansen all made epoch-making contributions. In the same
way the investigations of diseases in beers and wines were carried out
by many observers, and were closely connected with those relating to
spontaneous generation and mixed cultures of bacteria in fermentation.
These so-called "diseases" are analogous to the taints occurring in
milk and due to fermentations. Turning (_tourne_), turbidity, ropiness,
bitterness, acidity, mouldiness, are all terms used to describe these
diseases. They are chiefly brought about by four agencies:--
1. Bacteria.
2. Mixed yeasts.
3. "Wild" yeasts.
4. Moulds.
To each species of wild yeast there belongs some taint-producing power
in the fermentations for which it is responsible. _Saccharomyces
ellipsoideus II._ and _S. pastorianus I._, _III._, are such yeasts;
they only produce their diseases when introduced at the commencement of
the fermentation.
_Saccharomyces pastorianus I._ is a low fermentative yeast in elongated
cells, producing a _bitter_ taste to beer and an unpleasant odour. It
can also produce turbidity. _S. pastorianus III._ produces turbidity,
and _S. ellipsoideus II._ has a similar effect.
In 1883 Hansen demonstrated that the much-dreaded turbidity and
disagreeable tastes and smells in beer may be due to mixture of two
yeasts, each of which by itself gives a faultless product.
_Industrial Application of Bacterial Ferments._ From what has been
said we trust it has been made evident that bacteriology has a place
of ever-increasing importance in regard to fermentative processes. Not
only have the causal agents of various fermentations been isolated and
studied, but from their study practical results follow. The question of
pure cultures alone is one of practical importance; the recognition of
the causes of "diseases" of beer is another.
We cannot enter into a full discussion of the rôle of bacteria in
industrial processes, but several of the chief directions may be
pointed out. Without exception, bacteria have a part in them on account
of their powers of fermentation. In securing their food, bacteria
break down material, and bring about chemical and physical change.
The power which organisms have of chemically destroying compounds is
in itself of little importance, but the products which arise as a
result are of an importance in the world which has not hitherto been
recognised. We have used bacteria abundantly in the past, but we have
not perceived that we were doing so. _The maceration industries_ may
be mentioned as illustrative of this use without acknowledgment. The
flax stem is made up of cellular substance, flax fibres, and wood
fibres; the later are of no service in the making of linen, but the
whole is bound together by a gummy, resinous substance. Now this
connective element is got rid of in the process of _retting_. There
is dew-retting and water-retting. The former is practised in Russia,
and consists in spreading the flax on the grass and exposing it to the
influence of dew, rain, air, and light. The result is a soft and silky
fibre. Water-retting is accomplished by means of steeping the flax in
bundles, roots downwards, in tanks or ponds. In ten to fourteen days,
according to the weather, fermentation sets in, and breaks the "shore"
or "shive" from the fibre, and the process is complete. This is always
done by the aid of bacteria, which, under the favourable circumstances,
multiply rapidly, and cause decomposition of the pectin resinous
matter. The same operation occurs in _jute_ and _hemp_. _Sponges_, too,
are cleared in this manner by the rotting of the organic matter in
their interstices. The preparation of _indigo_ from the indigo plant
is brought about by a special bacterium found on the leaves. If the
leaves are sterilised, no fermentation occurs, and no indigo is formed.
_Tobacco-curing_ is also in part due to decomposition bacteria, and
several bacteriologists have experimented independently in fermenting
tobacco leaves by the action of pure cultures obtained from tobacco of
the finest quality.
In all these applications we have advanced only the first stage of
the journey. Nevertheless, here, as in nature on a big scale in the
formation of fertile soils and coal-measures, we find bacteria silently
at work, achieving great ends by co-operating in countless hordes.