A MANOMETRIC METHOD FOR THE DETERMINATION

OF GAS IN FERMENTATIONS.
BY ALBERT L. RAYMOND.
(From the Laboratories of The Rockefeller Institute for Medical Research,
New York.)

(Received for publication, June 15, 1929.)

The method previously described’ for determining carbon diox-

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ide during fermentation is specific for that substance and may
not be used for other gaseous products. The method has also
the disadvantage that volatile substances such as toluene or
acetaldehyde are removed from the fermenting mixtures and their
useis thus excluded. To obviate these difficulties a new apparatus
has been devised, based on Slator’ method of measuring the
change in pressure at constant volume. In addition to these
improvements a shaking device has been designed which greatly
facilitates the introduction and removal of the tubes. A year’s
experience with the new apparatus has shown it to be so satis-
factory that a description seems warranted.
The fermentations are performed in tubes which are immersed
in a small thermostat and are actively shaken except when read-
ings are being made. The tubes are partially evacuated at the
start and the increase in pressure is measured at constant volume
on a modified Van Slyke type manometer.3
The manometer is shown in Fig. 1. In place of the usual
leveling bulb, a closed mercury reservoir has been substituted.
The reservoir consists of a stoppered Erlenmeyer flask into which
two side tubes have been sealed. The lower of these is connected
by heavy rubber tubing to the bottom outlet of the manometer
while the upper side tube, which is well above the mercury level?
is attached to a 3-way stop-cock, A. One branch of this stop-cock
1 Raymond, A. L., and Winegarden, H. M., J. Biol. Chem., 74,189 (1927).
2 Slator, A., J. Chem. Sot., 89, 128 (1906).
3 Van Slyke, D. D., J. Biol. Chem., 73,121 (1927).
611
612 Determination of Gas in Fermentation

is open to the atmosphere and the other is connected to a 2 liter

bottle in which a considerably diminished pressure is constantly
maintained by a water aspirator. By turning the stop-cock one
way or the other, the pressure above the mercury may be made

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FIG. 1. Fermentation apparatus. Front view showing mercury reser-

voir and manometer connections.

atmospheric or less as desired. This is equivalent to raising or

lowering the usual leveling bulb but is both faster and more
convenient.
In order further to speed up the determinations, the bottom
stop-cock, B, of the regular Van Slyke manometer is by-passed
 A. L. Raymond 613
with a branch which carries another smaller stop-cock, C, and,
just below it, a very fine const,riction. The constrict,ion is so
chosen that the mercury flows through it only very slowly. The
rough adjustments are performed with Stop-cock B while the final
settings are made by using Stop-cock C. Although these changes
may appear to complicate the manipulation, actually they result
in a considerable time-saving.

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FIG. 2. Fermentation apparatus. Top view showing shaker and method

of connecting tubes.

In Fig. 2 are shown the thermostat and shaking device and the
manifold for connecting the tubes to the manometer. The mani-
fold is of capillary tubing and consists of three a-way stop-cocks
so arranged that any one of the six tubes may be selected.4 Sealed
to the manifold there is another 3-way stop-cock, D, one branch
of which leads to the manometer while the other is open. Through
this open branch the tubes may be evacuated at the beginning or
during a run, as desired. A vacuum source is provided for this
4 The apparatus was arbitrarily designed for six tubes.
614 Determination of Gas in Fermentation
purpose by placing another outlet from the vacuum reservoir
near at hand (the 2-way Stop-cock E in Fig. 2).
The tubes which we use are of the two types shown in Fig. 3.
The lower is a closed tube, sealed at one end, while the upper is
designed to permit the introduction of solutions through an inlet
equipped with a stop-cock. The support arms are furnished with
copper sleeves into which are soldered small brass rods bent to a
right angle. The sleeves are attached to the glass arms with
sealing wax or with De Khotinsky cement.

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FIG. 3. Fermentation tubes. The lower is a closed tube, sealed at one

end; the upper has an inlet equipped with a stop-cock.

Referring again to Fig. 2, the method of operation is seen.

The tubes are attached at one end to the manifold with rubber
connections, and at the other end rest in slots cut in a Bakelite
support. The brass arms fit into corresponding slots in another
Bakelite bar which is oscillated horizontally by the motor-driven
eccentric seen at the right. The eccentricity is small, the pin
being set only 2.5 mm. off center, and this permits a shaking of high
frequency and small amplitude. The motor has a variable series
resistance to afford speed adjustment.
 A. L. Raymond 615
At high speeds the shaking arms tend to rise out of the slots and
to avoid this a hinged bar is attached to the back of the Bakelite
support. To the bar are soldered thin flat strips of spring steel
which press lightly on the copper sleeves and hold the arms in the
slots.
The thermostat needs little comment. A copper tank, placed
in a wooden box and packed with hair-felt to avoid excessive heat
loss was found satisfactory. A toluene-mercury thermoregulator
operates a 60 or 100 watt lamp through a relay. The regulator

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and lamp support are seen at the left in Fig. 2.
For satisfactory operation of the apparatus it is necessary to
have perfect vacuum-tight rubber connections between the tubes
and manifold. After trying several grades of tubing and a number
of lubricants without success, it was finally found that heavy red
stethescope tubing, impregnated with Vaseline, made very satis-
factory joints. In preparing the tubing it is cut to short lengths,
placed in a wide-mouth flask, covered with melted Vaseline, and
heated under reduced pressure at about 65” until nearly gas-free.
After pouring off the liquid Vaseline and wiping off the excess, the
pieces are stored in a stoppered bottle until needed. Just before
use they are lightly lubricated with Vaseline and will then slip
easily over the glass parts. The glass-rubber attachment is so
firm, however, that it is necessary to cut the connections off with
a sharp knife or razor blade at the end of a run.
For the glass joints, any good grade of lubricant may be used;
in our own case we employ Lubriseal (A. H. Thomas) for the
stop-cocks and Vacuum Wax (11028 Central Scientific Company)
for sealing the ground joints of the fermentation tubes.
The mercury must not adhere to the capillary manifold or the
readings are incorrect. Chromic acid cleaning mixture may be
used, followed by distilled water and by acetone, and the latter then
removed by sucking clean dry air through the manifold till dry.
The motor used for the shaker is a Westinghouse sewing machine
motor, but for satisfactory operation at low speeds it is essential
that the commutator and brushes be kept scrupulously clean.
To this end it is desirable to use little or no lubricant at the
commutator end of the motor.
The technique of operation as now used may be best illustrated
by describing a typical run. The thermostat is brought to tem-
616 Determination of Gas in Fermentation
perature by using a bent glass rod in place of one of the tubes as
a stirrer to attain uniform temperature throughout the bath.
The tube stoppers are attached to the manifold with the vaselined
rubber connections as described. The ground joints of the fer-
mentation tubes are greased with Vacuum Wax (not too heavily)
and the tubes are then filled with the fermentation mixtures.
The spring-bar is unscrewed and the tubes are placed in position
one by one, the ground joints being well seated.
The spring-bar is then put back in place. Stop-cock D is

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turned to the open branch which is then attached to the vacuum
reservoir. The manifold stop-cocks are turned so as to evacuate
each fermentation tube in turn. The reservoir pressure should
not be too low, or it may cause the mixtures to froth. Stop-cock
D is next shut (as in Fig. 2) and the ground joints of the fermen-
tation tubes are reseated to assure perfect seals. The shaker may
then be started, its speed being such as to break up the liquid
surface as much as possible. It will be found that several ‘lsyn-
chronization speeds” exist at which the liquid remains relatively
quiet and these are to be avoided.
For making pressure readings the following procedure has been
found satisfactory. The shaker is stopped and with Stop-cock D
and all the manifold stop-cocks shut, Stopcock A is turned to
connect with the vacuum source. Stop-cock Bis carefully opened
until the pressure recorded on the manometer is a litt,le less than
that existing in the tube which is to be read. Stop-cock B is
shut, Stop-cock A is turned t.o allow air to enter the reservoir, the
manifold stop-cock is opened to the particular tube desired, and
Stop-cock D is turned to connect the manifold with the manometer.
The mercury flows into the manometer but the bulb will allow for
all reasonable differences in pressure. Stop-cock B is now cauti-
ously opened and the mercury rises and enters the manifold.
Stop-cock B is shut and Stop-cock C is operated until the mercury
approaches to within 4 to 5 mm. of the manifold stop-cock.
Stop-cocks C and D and the manifold stop-cock are then shut in
this sequence and the pressure is read on the manometer. This is
repeated for each tube.
One.familiar with the apparatus can read six tubes in about 2
minutes (except for the first set of readings) and during this time
most of the gas (if CO,) which is formed will remain dissolved in the
 A. L. Raymond 617
liquid if the shaker is not running. The time of reading may there-
fore be taken at the moment of stopping the shaker and will be
the same for all six tubes.
One further word may be added about the manometer tube
itself. This is a simple tube, completely filled with mercury and
then shut off with a stop-cock which is mercury-sealed. In the
Van Slyke model the tube is graduated, but for our purposes such
high accuracy was not required and a meter stick alongside was
found quite satisfactory. It may be mentioned that the tube

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when graduated is scaled in millimeters and not in volume.
For calculating the volume of gas evolved the following formula
obtains
av =
[
(P* - pdV2 _ (PI - PdVl
T2 Tl 1 c

where AV is the volume of gas evolved;

P is the manometer reading;
p is the vapor pressure of the fermenting mixture;
V is the volume of the gas space;
T is the absolute temperature of the gas;
C is a conversion constant;
and where the subscripts refer to a first and second reading.

However when the temperature is kept constant and there is

no significant change in the liquid, then the formula reduces to

AV = (P, - PI) ; C

If PI and Pz are expressed in mm., V in cc., and AV in CC. of

gas under standard conditions (760 mm. and 0’) and T is t,he ab-
solute temperature, then
C = 0.359

V may be calculated with sufficient accuracy by subtracting the

volume of the fermenting mixture from the total volume of the
tube and stopper, plus the added value of the gas space in the
manifold and manifold stop-cock. This last correction is small;
only about 0.2 cc. in the particular machines which we are using,
and is practically the samefor each connection. For convenience,
we number each tube and its corresponding stopper and det.er-
mine the volume of the two by weighing, filled and empty. TO
618 Determination of Gas in Fermentat’ion
this is added the 0.2 cc. correction for the gas space of the mani-
fold, and this corrected volume is tabulated against the tube
number. These volumes are used in making the calculations.
With tubes of the size illustrated and containing about 4 cc. of
liquid, an increase in pressure of 1 mm. corresponds to t’he pro-
duction of about 0.02 cc. of gas (under standard conditions) or
about lo-* millimols. The sensitivity may be increased if de-
sired, by using smaller tubes or by filling them with more liquid.

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SUMMARY.

An apparatus is illustrated for determining the gas production

in fermenting mixtures. It is based on Slator’s method of measur-
ing the increase in pressure at constant volume but has been
designed for easy and rapid determination of several simultaneous
samples.
The details of construction and manipulation are described.
A MANOMETRIC METHOD FOR THE
DETERMINATION OF GAS IN
FERMENTATIONS
Albert L. Raymond
J. Biol. Chem. 1929, 83:611-618.

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