SEPTElZBEH, 1935 INDUSTRIAL AND E U C l Y E E R l N G CHEAIISTRY 1035

three-arm rackerc: which nil1 deliver up to two hundred forty
half-barrel< per hour n ithout foaming or other loss oi beer.
The washing and cleaning of the kegs are an important part
of the finiihing operatiom. Theae containers are &ject to
all kind< of contamination, particularly those packages which
hare been used for shipping trade. Recently great progress
has been inade in developing more efficient and automatically
operating keg-washing machine.. These function on the
.pray principle whereby streams of hot and cold water a t
preq-ures up to 60 pounds per square inch are injected into
the package to dislodge and wash out dried beer remain.: and
other accumulated foreign substanceb. A sound, clean trade
package i i just as important as the maintenance of sanitary
conditions in other parts of the brewery.
Economic lspects
Unmalted cereals, and products derived therefrom, furnisli
to the brewers' wort colids or extract in amount.: which are
almost equal to the laboratory or theoretical yield. The
brewery yield obtained from barley malt, the main in-
gredient of beer, varieq between 62 and 72 per cent for malt<
produced from American six-rowed barley, depending upon
the quality of this brewing material and the nature and con-
dition of the brewhouse equipment. With the conventional
combination ma\h and lauter tub, the brewhouqe yield from
malt rnay be 4 to 8 per cent below the laboratory figure. With
the separate mash and lauter tubs, laboratory yield may be
approached more closcly, and with a mash filter i t is often
exceeded.
The iecond econoinic aspect is presented by process shrink-
age during cellar operations. Beer is lost by being admixed
with yeast crops in tank or cask iedinients, by entrainment,
and by admixture with witer used to flush out conduits, such
as pipe and hose lines, and in filters. Process shrinkage to
rome extent depends upon the size of units coi1,qtituting the
hrewery equipment, and therefore is smaller in large brew-
eries and greater in the small plants. I n general, a well-
conducted brewery will turn out about 97 barrels of beer per
100 barrels of wort brought into the starting tub, whereas in
smaller plants a final yield of 88 to 90 barrels generally is ob-
tained.
d third economic Triewpoint involve,? the engineering ser-
vices required for brewery operations-that is, production of
steam, electric power, refrigeration, and the maintenance of
wat'er and air supplies. Much has been done during the last
few years to correlate and coordinate the development of
t,hese services with breming procedure, but the so-called en-
gineering departments of breweries still offer a splendid field
for the mechanical and chemical engineer. As in any factory
using process steam, the brewery offers many opportunities
for steam to do considerable work before and after i t has been
used in the actual brewing processes. Many mechanical
improvements can be introduced into existing breweries to
reduce operating costs without in any way reducing quality
of finished product.
Acknowledgment
Acknowledgment is made of the helpful assistance of S.
Laufer and A. R. Erda in preparing material for this paper.
RECEIVED
.ipril 26, 1935.

Chemistry of Alcoholic Fermentation

LEONOR MICHAELIS
The Rockefeller Institute for Medical Research, New York, N. Y.

LCOHOLIC fermentation has h e n known catalytic reaction* could easily he separated from the living
and utilized from times immeniorial, but cells simply by extraction with water.
I
amroaches to the understanding of the
.

chemical and biological processes underlying it are of rather ~i~~~~~~~of a Cell-Free Fermenting .iigent
aecent date. I t is unnecessarv to dwell uDon the stages
represented by such names as Schwann, Gay-Lussac, Liebig,
~- Bucliner ( I ) dixovered in 1897 that by special methods t'he
Wohler, Pasteur. The results of their works may be sum- fermenting agent of the yeast cell can also be separated froin
iiiarized as follows: Fermentation is caused by a living or- the living cell. This discovery made it clear that alcoholic
ganism, the yeast cell, and con- fermentation, like ot'her enzy-
sists mainly in the conversion matic reactions, is caused by a
of sugar into a l c o h o l and car- c h e m i c a l substance or rather
bon dioxide although other end A brief reviewis given Of what is known at by a complicated s y s t e m of
product,s may also be forined present about the chemical processes in- chemical substances which can
in smaller q u a n t i t i e s . T h e valved in alcoholic fermentation. The by no means be c a l l e d l i v i n g
essential difficulty in a further large field ofa~coho~ic fermentation is With- m a t t e r . The achievement' of
the living cell is to produce these
to the
approach consisted
problem the
in theOf fact out boundary and is interlinked with all the Hut once
that no chemical agent endowed adjacent territories of chemistry and bi- produced, these agents act in a
v-ith the faculty of causing fer- ology. The alluring feature is the fact that purely chemical way, even in t,he
mentation could be isolated from any progress in its exploration has always absence of the living material by
the living yeast cell. This was meant a simultaneous progress in the field xhich they were produced.
in c o n t r a s t t o t h e f a c t that of general metabolism both of animals and Buchner's
grinding
method cells
consisted
with
many other agents, now called
e n z y m e s , which bring about Plants, and vice versa. sand and Kieselguhr and, after
o t h e r biologically i m p o r t a n t coniplet'e crushing of the cellh J
1038 INDUSTRIAL AND EYGINEERIKG CHEMISTRY
pressing the mass with a hydraulic press. Thus a press juice
is obtained which is a turbid but otherwise homogeneous
liquid with the faculty of fermenting sugar. The press
juice contains no living cell and exhibits fermentation even
in the presence of drastic antiseptics, such as toluene. Rleth-
ods other than Buchner's have been since described. Lebe-
dew (11) prepared another active extract from yeast, which
is now even more frequently used than Buchner's. H e dries
the yeast a t 25" C. for several days and grinds the dry mass
in a mill to a very fine powder. A suspension of this powder
in water is kept for 2 hours a t 37" C. and then centrifuged.
The supernatant fluid is a turbid solution with the faculty of
fermenting. From this solution a durable dry preparation
may be obtained by precipitation with acetone. The liquid
Lebedew juice itself, very active in the beginning, gradually
becomes inactive on standing, probably because of the pro-
teolytic enzymes which are also present in such a juice and
gradually decompose the fermenting agent.
The discovery of a cell-free fermenting agent made pos-
sible a deeper insight both into the nature of the enzymes
and the chemical processes underlying fermentation. On
working with living yeast cells, all chemical processes take
place &hin the celi. Sugar diffuses into the cell across
the cell wall; the end products of fermentation diffuse out of the
cell into the medium, and no insight can be gained into the
series of intracellular chemical processes which finally lead to
the end products. This is different when the fermentation is
brought about by cell-free extracts. Here i t is possible to
discover stages of the chemical processes intermediate be-
tween sugar and alcohol. On describing these various inter-
mediate steps, it is advisable to follow their natural order and
not the order in which they have been discovered. It is
characteristic of the advancement of knowledge that the de-
scription of the processes now can be made according to their
natural order instead of a historical one.
Combination of Sugar and Phosphoric Acid
The first chemical process is, astonishingly, a synthetic one
and not a breakdown. I t consists in the combination of sugar
with phosphoric acid to form various hexose phosphate esters.
S o fermentation takes place in the absence of phosphates.
Harden and Young (9) discovered that during the firbt stage
of fermentation the inorganic phosphate is diminished and
organic phosphate compounds are formed. They have been
recognized by Harden and Young (7) as phosphate esters of
hexoses. A series of such phosphate esters has been dis-
covered. The first one described by Harden and Young (IO)
is a hexose diphosphate which, according to our prebent
knowledge, is combined with one phosphoric acid molecule
a t carbon atom 1, and another a t 6. The hexose of this eater
appears to be fructose even when the sugar from which it
generated is pure glucose. Furthermore, hexose mono-
phosphate esters have been isolated. I n the first place,
there is the so-called Robison ester (8), which contains only
one phosphoric acid molecule a t carbon atom 6. This prepa-
ration appears to be a mixture of a glucose ester and a fruc-
tose ester. There is, besides, a trehalose monophosphate
(24). Another hexose monophosphate ester, the Neuberg
ester (ZO), has been prepared also by partially hydrolyzing
Harden's diphosphate ester. This ester seems to be identical
with the fructose part of Robison's ester. We have not yet
reached a complete insight into the possible varieties of these
esters. It is difficult to obtain them in crystallized form.
Two hexose monophosphate esters have also been synthesized
by Levene and Raymond (12); one is probably identical with
Robison's but has not yet been crystallized. Recently War-
burg (27) succeeded in obtaining the Robison ester in the
form of its calcium salt in a microcrystalline form. I n the
VOL. 27, NO. 9
Rockefeller Institute laboratory this salt has been perfectly
crystallized.
The theory suggests itself that these phosphate esters are
intermediate products in the fermentation. There is a
criterion as to whether a substance is a n intermediate prod-
uct. When instead of sugar the supposedly intermediate
product is mixed with yeast juice, the fermentation should
proceed at least a t the same rate as, or even a t a higher rate
than, the fermentation of sugar. The most plausible theory
would be that first a monophosphate is formed and then a di-
phosphate, and then that further steps of the development
will follow. If this is true, diphosphate should be a t least as
rapidly fermented as sugar. However, diphosphate is con-
siderably more slowly fermented. Monophosphate i., in
general, more rapidly fermented than diphosphate. The
crystallized monophosphate is fermented a t a rate comparable
to glucose but only in the initial stage of fermentation. The
role of the phosphate esters may be better understood by the
following experiment: When yeast juice is mixed with
glucoqe, a certain time elapses before fermentation begins.
This is called the induction time. This induction time is
abolished when, besides glucose. a small amount of hexose
phosphate ester'is added: I n this case fermentation starts
immediately. Thus it appears that the phosphate esters are
not only intermediate products, but that some interaction be-
tween the free sugar and the phosphate ester takes place. The
monophosphate seems to be the more important, or better, the
more active one, and the accumulation of diphosphate during
fermentation seems to be something like a retarding reaction.
Probably the diphosphate is a reserve from which the more
active monophosphate is to be rebuilt during t h e further
stages of fermentation. This problem, however, is not yet
definitely settled.
Breakdown of the 6-Carbon Chain
The next step is the breakdown of the 6-carbon chain of
glucose or the hexose phosphate esters into two molecules,
each containing a 3-carbon chain. This type of splitting had
been surmised for a long time, but only recently has experi-
mental evidence been given to support it. The scheme for the
appearance and further fermentation of these 3-carbon phos-
phoric esters was the last achievement of Embden (4) be-
fore his premature death, and was not only confirmed but
also supplemented in a rather surprising way by Meyerhof
and Lohmann (18). As matters stand according to present
developments this splitting process is as follows: Hexose
diphosphate is split by an enzyme to give two molecules of
a triose monophosphate. There are two trioses, dioxyace-
tone and glycerol aldehyde-the one a ketose, the other a n
aldose. Meyerhof and Lohmann stated that the triose phos-
phate developed to be the ester of dioxyacetone. The split-
ting of the hexose into the two trioses does not go on to com-
pletion but leads to a n equilibrium. Hexose diphosphate is
split by this enzyme to a certain extent. On the other hand,
triose esters are synthesized by the same enzyme to form hex-
ose diphosphate. The equilibrium reached is the same,
whether we start from the one side or the other. The equi-
librium is brought about in less than a minute. The situa-
tion of this equilibrium depends largely on temperature.
Surprisingly, t h e synthesis is favored by increasing the tem-
perature.
Before proceeding farther, we may hint a t a weak point in
our present knowledge. This breakdown of the 6-carbon
chain has been demonstrated as yet only for hexose diphos-
phate, not for monophosphate. As stated above, the hexose
monophosphate is more likely to be a faster reacting inter-
mediate product than the diphosphate. Perhaps the dis-
crepancy may be solved by a future discovery which would
SEPTEMBER, 1935 INDUSTRIAL AND ENGINEERING CHEMISTRY
not be quite unexpected-namely, that a comparable split-
ting process is also possible for hexose monophosphate.
Dismutations
The dioxyacetone phosphate formed from hexose diphos-
phate immediately undergoes further changes. If we as-
sume that dioxyacetone phosphate is in equilibrium with its
isomer, glycerol aldehyde phosphate, we may expect that a
reaction common to aldehydes occurs; that is, two molecules
react upon each other in such a way that one is reduced by the
other which, in turn, is oxidized. Such a process is called a
Cannizzaro reaction or a dismutation, and other examples
will be encountered later on in the further stages I-If fermenta-
tion. This prqcess converts two molecules of dioxyacetone
phosphate into one molecule of glycerophosphoric acid and
one molecule of what is called phosphoglyceric acid.
Phosphoglyceric acid undergoes a remarkable further en-
zymatic alteration discovered by Embden. Free phosphoric
acid is split out, not hydrolytically by adding a molecule of
water in the way an ester is usually split by water, but in such
a way that the hydrogen atom which is necessary to supple-
ment the phosphoric acid residue to phosphoric acid is with-
drawn from the organic part of the molecule. What is left
is then pyruvic acid. Quite recently, Lohmann and hIeyer-
hof (16), studying this process in the metabolism of frog
muscle, discovered that this reaction is not direct but goes
through the intermediate state of a phosphate ester of pyru-
vic acid. The process is as follows: Phosphoglyceric acid is
converted by an enzyme to phosphopyruvic acid, and the
latter is hydrolysed in the presence of a coenzyme to pyruvic
acid and phosphoric acid. The fate of pyruvic acid ha3 been
known for many years by Neuberg's discovery (22) that,
because of a sDecial enzvme called carboxvlase., Iilvruvic
" chains has been moved s t r i c t l y e x p e r i m e n t a l l y by
acid is split inio carbon dioxide and acetaldkhyde. Acet-
aldehyde as an intermediate product of fermentation had
been surmised for a long time and has been definitely
proved by Neuburg (23) to arise during fermentation.
When fermentation takes plgce in the presence of sodium
sulfite or calcium sulfite, the aldehyde combines with this
reagent to form the well-known aldehyde bisulfite compound.
Because of this reaction, acetaldehyde escapes further al-
terations by yeast and can be recovered quantitatively.
Under ordinary conditions, without sulfite, acetaldehyde un-
dergoes a further change. We recall now the above-men-
tioned glycerophosphoric acid which is the other moiety of
the splitting process of the 6-carbon chain. This substance
is oxidized by acetaldehyde to form a phosphate ester of
glycerol aldehyde, and the acetaldehyde in its turn is reduced
to ethyl alcohol. Here we come t o the desired end product
of fermentation. The other end product, carbon dioxide, has
been accounted for already; it is generated from the splitting
of pyruvic acid.
We now come to the phosphate ester of glycerol aldehyde.
By another process of dismutation this ester is again converted
into equal parts of glycerophosphoric acid and phospho-
glyceric acid. A fresh portion of phosphoglyceric acid is
thus produced which undergoes the changes already de-
scribed. It furnishes a new portion of alcohol and the re-
mainder undergoes the cycle described above. Thus the
cycle is started over again until all sugar is finally converted
into alcohol and carbon dioxide.
Sources of Knowledge of Chemical Processes
The methods by which the present knowledge of the chemi-
cal processes during fermentation has been reached can be
only briefly mentioned here. The foundation of all experi-
mentation is, as stated above, the cell-free press juice or
1039
maceration juice. On working with living yeast, the ap-
pearance of the phosphate esters and most of the other in-
termediate products escapes detection because everything
occurs inside the cell. Hexose phosphates, added to living
yeast, are not fermented, or, at best, are very sluggishly
fermented. These esters cannot penetrate the cell wall and
are not accessible to the enzymes within the cell. But even
on working with the cell-free extract, obtaining the interme-
diate products is not an easy matter. The hexose phos-
phates can be prepared by interrupting the fermentation
a t a suitable stage. The system is fixed with trichloroacetic
acid, and the filtrate is treated with lead acetate; from this
precipitate the phosphate esters can be isolated by compli-
cated processes in the form of their barium or calcium salts.
Certain poisons were especially useful to obtain the other
intermediate products. Sodium fluoride has a specific in-
hibiting action on the enzyme which splits phosphoglyceric
acid. When a triose phosphoric acid ib added to the enzyme
containing also sodium fluoride, the dismutation into glyc-
erophosphoric acid and phosphoglyceric acid takes place,
but further processes are inhibited. In this way Meyerhof
succeeded in accumulating these two otherwise transitory
substances. Addition of sodium sulfite prevents the acetal-
dehyde from further chemical alteration as stated above and
so permits its detection. Pyruvic acid cannot be obtained
in the same way, although pyruvic acid also forms a bisulfite
compound. Unfortunately this compound is just as easily
fermented as pyruvic acid itself, whereas the acetaldehyde-
bisulfite compound is not attacked by yeast. The best evi-
dence that pyruvic acid is an intermediate product of fer-
mentation is given by Seuberg's discovery that pyruvic acid
is fermented by yeast a t least as fast as glycose itself.
The splitting of the 6-carbon chain into two 3-carbon
Embden and by *Meyerhof and -Lohmann f o r m u s c Ik
extract. From analogy, we transfer this idea also to
yeast fermentation. Later on we shall d e m o n s t r a t e
the justification of drawing a n a 1o gi e4 f r o m muwle
metabolism to yeast metabolism.
Enzymes
All of the chemical processes discusbed to this point are
caused by enzymes. More than enough enzymes in yeast are
known to account for the many chemical reactions discussed.
h'one of these enzymes has been prepared in a pure condition.
But a great many of them can be separated and isolated from
each other in such a may as to ascertain the hypothesis that
we have to deal with a great number of different enzyme-.
It is certain that there are many enzymes each with a special
function and not a single enzyme that would exhibit different
functions under different conditions. There are even more
enzymes than those concerned with the chemical reaction-
already mentioned. Some of these enzymes are not con-
cerned a t all with fermentation, such as the enzymes capable
of splitting polypeptides. We shall pass over the complicated
enzyme system involved in respiration. Other enzymes are
linked with fermentation only in a preparatory way. Thus,
invertase hydrolyzes sucrose, and maltase hydrolyzes maltose
to a simple, fermentable hexose, although it is almost certain
that these disaccharides are accessible to fermentation to a
certain extent, even without this preparatory hydrolysis.
There is also a phosphatase which is generally able to hy-
drolyze phosphate esters, and probably the same enzyme can
also, according to conditions, bring about the synthesis of
phosphate ester. Furthermore, there is the enzyme that
splits phosphoglyceric acid into phosphoric acid and pyruvic
acid, and another enzyme (Neuberg's carboxylase) that splits
carbon dioxide from pyruvic acid. There is also an enzyme
1040 INDUSTKL4L AND ENGINEERING CHEMISTRY
that converts methylglyoxal into lactic acid, discovered by
Dakin ( 8 ) and by Neuberg (19). The presence of such an
enzyme conveyed the idea that methylglyoxal is an interne-
diate product of fermentation, although lactic acid is not an
end product, or a t least not a conspicuous end product of
fermentation by yeast. Methylglyoxal was thought of as
that 3-carbon compound which originates from the breaking
of the hexose in two halves. This idea has a t present been
abandoned, and the process described above adopted in-
stead. It is hard to imagine that the occurrence of methyl-
glyoxalase is quite fortuitous and of no purpoqe, and it is
possible that some day methylglyoxal will be again given a
part to play in the drama of fermentation.
Some of these enzymes are in a certain sense simple, since
they need no coenzyme for their action. Among the enzymes
concerned with the processes proper of fermentation, car-
boxylase may be mentioned. Other enzymes need home
supplementary agent to develop their faculties. Rlethyl-
glyoxalase requires the presence of glutathione, as Loh-
mann (13)
. , has shown. The enzymes concerned with the
first stages of fermentation are of a still more complicated
nature. When yeast juice is dialyzed, neither the remaining
juice nor the dialyzate is capable of fermenting. A mixture of
both is again active. Thus the enzyme consists of two constitu-
ents, one of a more colloidal nature, not dialyzable, the other
of obviously smaller molecular weight and easily diffusible.
They are distinguished as zymase and cozymase. Zymase is
very sensitive to heat, whereas cozymase is rather resistant to
heat, almost up to the boiling point. I t has been further shown
by Lohmann (14) that, besides these two constituents of the
enzyme system, a small amount of a magnesium salt is indis-
pensable. S o t much purification of the zymase has been
attained as yet. The cozymase has been prepared by Euler
(6)in a much purer state, but not yet nearly pure enough to
be designated a definable chemical compound. Carboxylase
can easily be separated from most of the other enzymes. An-
other auxiliary substance has been found by Euler (6)which
he called the a-factor. It is very heat-resistant (much more
than cozymasej and increases considerably the ferment’ation
with living yeast cells, but it has no effect on the fermentation
with yeast juice. 4 great number of other substances of a
more or less enzymatic nature can find no place in this paper
because they are not involved in the fermentation of carbo-
hydrates-namely, the vitamins of yeast.
Origin of Secondary End Products
Returning to the chemical processes in fermentation, we
have accounted for what may be called the normal end prod-
ucts of fermentation-ethyl alcohol and carbon dioxide. The
origin of the other end products, which may occur sometimes
only in small amounts, sometimes to such an extent as to ex-
ceed the normal end products in quantity, will now be dis-
cussed.
Glycerol in small quantities is usually found among the end
products of fermentation. The normally developed small
amount of glycerol is probably formed from phosphoglyceric
acid, which in part escapes the cyclic process described above
and is hydrolyzed instead by an enzyme called phosphatase
into glycerol and phosphoric acid. Under special conditions,
however, the course of fermentation can be artificially modi-
fied in such a may as to furnish glycerol as a main product of
fermentation. When fermentation occurs in the presence of
sodium sulfite, acetaldehyde is thrown out of the reaction and
can no longer be reduced to alcohol. Instead, other com-
pounds are reduced. The trioses, when subjected to this re-
duction which normally takes place with acetaldehyde, will
give glycerol even in the absence of sodium sulfite when the
fermentation takes place in a n alkaline medium. Sormally
VOL. 27, NO. 9
yeast becomes acid on fermentation. When artificially the
fermentation is forced on an alkaline medium, glycerol and
also acetic acid are formed instead of alcohol and carbon
dioxide, as Seuberg (91) discovered.
Other important by-products of fermentation are the fusel
oils and the higher alcohols, especially isoamyl and d-amyl.
The origin of fusel oil has been discovered by Ehrlich (3).
The parent substance is not a carbohydrate but consists of
amino acids, although the presence of a fermentable sugar is
requisite for the action of yeast on amino acids. The source
of these acids is the protein of the fermenting mixture. T h e
amino acid is first converted into a keto acid. The latter,
like pyruvic acid, loses carbon dioxide, and the remaining
material is an aldehyde. Just as acetaldehyde is reduced to
ethyl alcohol, these aldehydes are reduced to their correspond-
ing alcohols. From isoleucin d-amyl alcohol is formed, and
from leucin isoamyl alcohol is formed. If the amino
arid happens to be dicarboxylic, such as aspartic acid,
it is first converted into the keto acid. The latter loses
only one molecule of carbon dioxide, and the resulting
aldehyde-carbonic acid is, instead of being reduced to
hydroxy acid, oxidized to a dicarboxylic acid with one car-
a
bon atom less than the original amino acid. From aspartic
acid succinic acid is developed, which has been known for a
long time to be one of the minor end products of alcoholic
fermentation. Other amino acids furnish in the same way a
part of those substances responsible for the various flavors of
fermented beverages.
The mechanism by which a carbohydrate breaks down by
the metabolism of yeast appears a t first glance to be very
different from the may a carbohydrate is utilized by the cell\
of higher living organisms. Here the end products of the
normal aerobic metabolism are carbon dioxide and water.
Under anaerobic conditions however, the main end product i5
lactic acid. This production of lactic acid is comparable to
that of alcohol by yeast and is often also referred to as fermen-
tation, in distinction to respiration which takes place in the
presence of oxygen. The eqergy evolved from the respira-
tion of one gram of sugar is about twenty times the energy
evolved by fermentation. Here two important problems
ariqe: I n which way is the lactic acid production by higher
cells correlated with the alcohol production by yeast; and
what explanation can be given for the difference of metabo-
lism in the presence and in the absence of oxygen in higher
cells, and is there such a difference also in the metabolism of
yeast?
Correlation of Lactic Acid and Alcohol
As regards the correlation between lactic acid and alcohol,
it bas been shown that relatively slight changes of the enzyme
system are able to bring about this difference in the end prod-
uct. On the whole, the intermediate products of fermenta-
tion in higher organisms and yeast are rather similar. In
either case, sugar is subjected to phosphorylation. The two
cases are not comparable in all details, however. I n the
metabolism of muscle there is a chain of phosphorylations in
which not only carbohydrates but also creatinine and ade-
nylic acid are involved. Otherwise, the similarity is striking
(17) until we arrive a t the stage where pyruvic acid is formed.
We have seen how this acid leads to formation of alcohol in
yeast, mainly because of the action of carboxylase, which
splits out carbon dioxide from pyruvic acid. I n higher or-
ganisms this process does not take place, but pyruvic acid i;
transformed into lactic acid by a simple reduction. In the
presence of oxygen, according to Neyerhof’s discovery, only
about one-fifth of the lactic acid is oxidized to carbon dioxide
plus water, and the remainder is resynthesized to form a car-
bohydrate, glycogen, which now can serve as a further sub-
 SEPTEMBER, 1935 INDUSTRIAL AND ENGINEERING CHEMISTRY 1041
d r a t e for fermentation and respiration. The energy evolved Thus it i i obvious that the ~netabolibinof yeaht ia intere.t-
hy the combustion of that small fraction of lactic iicid is suffi- ing on it. own account, either from the purely scientific or
cient not only for the energy required for the life of the cell from the industrial point of view. It is no exaggeration to
but also for t'he resynthesis of the remainder of lactic acid to assert that any progre+ in the knowledge of yea\t fernienta-
glycogen. The problem arises as to whether a comparable tion immediately brings about an advancement in the under-
change of metabolisni according to the presence or absence of itanding of the metaboliTm of all other living organi5nii, and
oxygen occurs also in yeast. Such a problem has heen clearly \?ice versa.
stated already by Pasteur. He advanced the idea that fer-
inentation is & saris air; that is, the energy evolved in the Some Compounds Involved in Fermentation
absence of air by fermentation is the .source of energy for the
cell in the same way that the energy evolved from respira-
tion is the source of energy for respiring cell..
Effect of Air on Fermentation
CHqOH CHOH CHiOH G13cerol
This ingenious idea led Pasteur to the postulate that a cell CHiOH CHOH CHO GI>cerolaldehyde
CHzOH CO CH20H Dioxyatetone
capable of fermenting sugar should do so only in the absence CHzOH CHOH COOH a-Glycerlc acid
of air and that the same cell in the presence of air should burn
up the carbohydrate to carbon dioxide and water. However,
he did not succeed in proving this postulate satisfactorily by
experiment. Yeast ferments sugar both in the absence and in
the presence of oxygen. But in the presence of oxygen, be- 1 2 3 4 5 6
sides fermentation there is also combustion t o carbon dioxide CHOH CHOH CHOH CHOH CH CHiOH Aldose (glucose)
L- 0 _1
and water-in other words, respiration. When this respira- 1 2 5
3 6 4
tion takes place, the extent of the fermentation is somewhat CHzOH CH CHOH CHOH CHOH CHIOH Ketose ( h i tose)
diminished. This is a certain approach to his postulate, but L
- 02
-
1 2 3 4 5 6
it does not justify t'he claim that in the presence of oxygen CHOH CHOH CHOH CH CHOH CH O(POaH9) Hexoee monophos-
-~ 0 phoric acid
respiration entirely replace,? the fermentation which occurs CH>O(POBHI)
CH CHOH CHOH COH CH?O(PO?Hg) Hexose diphos-
without oxygen. -- 0-- phorrc acid
The experimental test for the soundness of Pasteur's idea
was demonstrated niuch later by lleyerhof (16). Instead of C H: hTPak?I
I 1
working with cultivated strains of yeast, such as are used in CHO'_H_- - __ I +
manufacturing beer or mine, he studied naturally occurring JOOH
wild yeast varieties and found the variety Torula utilis

'
Phosphoglycerir acid
especially suitable. This variety complies with Pasteur's
hypothesis. It ferments in the absence of oxygen, hut fails CH2 CH3
to ferment a1moi;t entirely in the presence of oxygen and re- O
H
<!!( or CO
I
+ HsPOi
.spires instead. =llso this behavior is quit'e analogous to what
happens in inost higher organisms; they produce lactic acid
COOH COOH 1
(enolic form) (ketonic form)
in the absence of oxygen but none or very little lactic acid in Pyruvic acid
the presence of oxygen, in which case they respire instead.
This phenomenon that an anaerobic fermentation is quantit'a- Literature Cited
tively shifted to respiration by the presence of oxygen has
Buchner, E., Ber., 30,117, 1110 (1897).
been termed by Warburg ( 2 5 , 26) the Pasteur-Meyerhof re- Dakin, H . D . , and Dudley, H. \T., J . B i d . Chern., 14, 155, 423;
action to show that t'he occurrence of this phenomenon was 15, 127 (1913).
predicted by Pasteur and experimentally confirmed by Meyer- Ehrlich, F., Jahr. I'ersuchs- I A m n s t a l t BraiLerie Berlin, 10,515
hof. This Pasteur-lleyerhof reaction is not necessarily corn- (1907); Ehrlich, F., and Jarohsen, K. A,, Ber.. 44,888 (1911);
Ehrlich, F., and Piotschimuka, P., Ibid.,45,1006 (1912).
plete in all types of cells. Sometimes the various enzyme Embden, G. H . , Deuticke, J., and Graff, G., Klin. Woehschr., 12,
systems of a cell may not be entirely adapted to the purpose 213 (1933).
of shifting anaerobic fermentation quantitatively to aerobic Euler, H . von, and Myrbkck, K., 2. physiol. Chem., 131, 179
respiration. This may be thought of as a kind of insuffi- (1923); 133,260; 136,107; 139,15, 287 (1924).
Ihid., 141,297 (1924).
ciency of the chemical structure or coordination of the organ- Harden, A., ".llcoholic Fermentation," hlonographs in Bio-
ism. I n such a case a smaller or greater percentage of the chemistry, 4th ed., London, 1932.
fermentation may persist even during the aerobic respiration. Harden, d., and Robison, R., Proc. C'hern. Soc., 30,10 (1914).
The industrially used cultivated yeasts show this phenomenon Harden, -1.. and Young, W.,J., Ihid., 21,189 (1905).
Harden, A,, and Young, IT. ,J., Proc. Rou. Soc. ( L o ~ ~ d o i i ) ,
to a particularly high extent. They are cultivated for t,he B80,299 (1908).
purpose of fermentation and possess this faculty in so high a Lebedew, A. Ton, Conapt. rend., 152,49, 1129 (1911).
degree that aerobic respiration cannot cope with the enormous Lerene, P. A., and Raymond, A. L., J . B i d . Chert&.,89, 479
amount of fermentation products. Thus Pasteur happened (1930); 92,765 (1931).
to meet in his cultivated yeast a somewhat unnatural, do- Lohmann, K.,Biochem.Z.,254,332 (193%).
Lohmann, K., Saturwi'ssei~schaften,19,180 (1931).
mesticated organism. Later, Warburg (25) demonstrated Lohmann, K., and Meyerhof, O., Biochem. Z., 272,60 (1934).
that certain tissues in higher animals also show what we may Meyerhof, O., Ihid., 162,43 (1925) ; .\'uturwissensc/iuften, 13,
call an incomplete Pasteur reaction. They produce lactic 980 (1925).
acid even when respiring in oxygen, although to a smaller Meyerhof, O., "Die cheniischcn Vorginge in1 Muskel," Berlin,
1930.
extent than in absence of oxygen. He showed this to be Meyerhof, O., and Kiessling, H., Biochem. Z., 264,40; 267,318
true especially for embryonic cells and, what is most, interest- (1933).
ing, for the cells of malignant tumors. These cells, like do- Neuberg, C., Ibid., 49,6 0 2 ; 51,484 (1913).
mesticated yeast, cause such a high fermentation that res- Ibid., 88,432 (1818).
Neuberg, C., and F i r b e r , E., Biochem. Z., 78,238 (1917); Neu-
piration is incapable of burning u p all the products of fer- berg, C., and Hirsch, J., Ibid., 98,141 (1919).
mentation. Neuberg, O., and Hildesheimer, A , , I h i d . , 31,170 (1911).
1042 INDUSTRIAL AND ENGINEERING CHEMISTRY
(23) Neuberg, O., and Reinfurth, E., Ibid., 89,365 (1918)
I (26) Warburg, O., “Uber die katalytischen Wirkungen der leben-
(24) Robison, R., and Morgan, W. T. J., B i o c h a . J., 22, 1277
(1928).
(25) Warburg, O., “Uber den Stoffwechsel der Tumoren,” Berlin,
1926.
Application of Oxidation-Reduction
Potential to Brewing Control
The theory of the oxidation-reduction
systems and their potentials has recently
been applied to brewing control work and
the preliminary results appear to justify a
. more intense study.
Certain factors influencing the flavor and
keeping quality of beer, which have been
little understood, may thereby find an ex-
planation. Outstanding are the influence
of aeration during the various stages of the
brewing process, the propagation of micro-
organisms, and the effect of oxidizing and
reducing reactions on beer flavor.
The oxidation-reduction potential is
URING the past F. P. SIEBEL, JR., ARD E. SINGRUENJ’
five decades the Siebel Institute of Technology,
brewing industry
has more and more turned to science
for assistance in the solution of its
practical problems. The advancements in physical, colloidal,
and biochemistry, in particular, have contributed a great
deal to the better understanding of purely empirical experi-
ence and rule-of-thumb methods. I n many cases brewing
scientists have traced the causes of frequent disturbances
and, based on this knowledge, have been successful in devising
means to remedy faulty conditions and eliminate them for the
future.
The application of the theory of hydrogen-ion concentra-
tion to malting and brewing has become a well-known, com-
paratively simple, and effective method for controlling proc-
essing methods. Sumerous other problems of physico-
chemical character, however, have remained unsolved. It
is only during the past year that European brewing scientists
have put another modern theory-that of the oxidation-re-
duction systems-to work, to study into some of the hereto-
fore least understood factors influencing the flavor and the
keeping quality of the beer. These include all oxidation and
reduction reactions which take place during beer production.
Outstanding in this respect are the influence of aeration, the
propagation of microorganisms, and the effect of oxidation
and reduction on the beer flavor.
Other industries are already making practical use of the oxi-
dation-reduction potential. I n the dairy industry the de-
gree and, to a certain extent, the nature of milk infections are
determined by means of suitable oxidation-reduction indica-
tors; it is also claimed that the suitability of milk for certain
types of cheese can be predicted by this method.
1 Present address, 542 Arlington Place, Chicago, Ill.
VOL. 27, NO. 9
digen Substanz,” Berlin, 1928.
(27) Warburg, O., and Christian, W., Biochem. Z.,254, 438 (1932).
RECEIVED
April 27, 1935.
generally expressed by the symbol, rH (the
negative logarithm of the pressure of the re-
ducing hydrogen present in the solution).
The oxidizing power of a solution is the
greater the higher its potential, and the re-
ducing power is greater the lower its poten-
tial. An electrometric and a colorimetric
method are available for the determination
of rH, providing means for a methodical
study of the oxidation factor wherever it
occurs during the entire brewing process.
Its application is described in the study of
aeration, of the so-called light-taste, and of
yeast turbidities.
French wineries recognize the effect
of the oxygen in the air on the wine
aroma and are now investigating the
Chicago, Ill. oxidation-reduction potential as a
possible means of controlling aera-
tion. It also finds application in sewage treatment and in the
study of the biological condition of soils.
De Clerck in France ( 1 ) and iMendlik in Holland (2) were
the first to study the merits of the oxidation-reduction poten-
tial for brewing control work. Although their investigations
are still in the experimental stage, the preliminary results a p
pear to be deserving of attention.
Theory of Oxidation-Reduction Systems
Under oxidation, originally all those reactions were classi-
fied in which oxygen combined with another substance to
form a new compound. I n the meantime, however, a better
understanding of intermolecular arrangement has taught us
that atoms consist of an inner, positively charged nucleus
(protons) around which particles with negative charges
(electrons) revolve. Atoms which lose a n electron in the
course of a chemical reaction gain a positive charge.
For instance, an increase in valence constitutes a gain of a
positive charge-in other words, an oxidation. Since no
electron can exist by itself, it will be bound by another sub-
stance which is thereby reduced. It is evident, therefore,
that neither oxidation nor reduction can take place alone but
that both reactions always are concurrent and consist in the
transfer of electrons from the oxidized to the reduced sub-
stance, If we consider the gain of a positive electric charge
as oxidation, although no oxygen may be involved at all in the
reaction, and the gain of a negative electric charge as reduc-
tion, the modern definition of oxidation would be “loss of
electrons.”