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graphic determination of O2. Due to the com A commercial source of 1-2% C02 and 100%
plexity and cost of these instruments, a more N2 in gas cylinders are checked to verify the
simple method is described for measuring CO2 acutal CO2 in both cylinders. This can be done
evolution from slowly respiring fruit. by absorbing CO2 on ascorite and weighing or by
any other convenient means. These two gasses
Description of Method
can be mixed to give an air stream of known
This method is an adaptation of the Claypool CO2 content. This air stream is equilibrated with
and Keefer method (1). The principle is based the BTB indicator solution and the per cent trans
on a change in pH brought about as CO2 in an air mission at 617 m^ recorded. The data are plotted
stream is bubbled through a buffered sodium to give a standard curve as seen in Figure 1. A
carbonate solution until equilibrium is attained. table can be prepared from the standard curve
In practice, a suitable indicator is added to the which simplifies the conversion of per cent trans
bicarbonate solution and very small changes in mission to percent CO2.
pH can be measured colorimetrically. If enough Respirometer.—The schematic drawing shown
bicarbonate is present in the indicator solution in Figure 2 appears somewhat complicated but
so that the changes in [HCO3]- are negligible, is necessary for maintaining a constant pressure
then the change in [H] + occurs thus a change of of air on the system. It was constructed of rela
color reflects the quantity of CO2 in the air tively inexpensive glass and rubber tubing which
stream. was readily available.
Bromthymol Blue Indicator Solution (BTB). The respiration chambers (J) were wide-
—A stock solution (10 x concentration) was pre mouth glass jars with screw-type metal lids. A
pared by dissolving 10.000 gm. sodium bicar %-inch soft rubber gasket was glued securely to
bonate (reagent grade) and 0.500 gm. bromthy the inside of the lid to insure a gas-tight seal.
mol blue (dibromthymolsulfonphthalein) in ca Each lid had two 1% x % inch copper tubes in
500 ml. of water first before being brought to serted as inlet and outlet. The inlet tube had a
10 liters volume. piece of flexible tubing attached which reached
Indicator solution was made by diluting one to the bottom of the inside of the jar to insure
liter of the above stock solution 10 times with the proper mixing of the gases. The fruit were
distilled water and adjusting as follows: placed in the jar and the container closed gas
Combine 50 ml. of the above indicator solution tight.
with 5 ml. of 0.1 N NaOH and bring to 100 ml. The liquid reserve jar (O) acts as a high
This test solution should have a 55% transmis pressure arm to all the air manometers attached
sion. If the reading is less than 55, additions to manifold (M) and causes the solution to rise
of 0.1 N NaHCO3 to the total diluted BTB solu
tion is made and the above procedure repeated
until the desired reading is obtained. The BTB
indicator solution is now ready for use and 20 -
Figure 2.—Schematic drawing of respirometer. C.—Capillary tubes for maintaining flow rates. J.—Respiration chamber.
L.—Pressure regulating tower. M.—Manifold. O.—Liquid reserve jar. P.—Manometer tube. T.—Colorimeter tubes.
in manometer tubes (P). The height of this rise has been passed over a given weight of fruit or
is controlled by adjusting the depth of the pres vegetable.
sure-regulating tube in the pressure-regulating Thus:
tower (L). The height (h) must remain constant. I. % CO2 X air flow/min X 60 = CO2 (ml) /hr.
The capillary tubes (C) for regulating air flow "TOO
rates were calibrated on a selected height (10 Then the conversion of volume (ml) to weight
in.). This head, in inches, is maintained in nor (mg) must be made with allowance for tempera
mal operation and during the time readings are ture. Table 2 shows the conversion factor for
being taken. The resultant constant air pressure various temperatures.
flows through the previously calibrated capillary
Thus:
tube (C) and into the jar (J) and, during read
II. CO2 (ml) /hr. X conv. factor = CO2 (mg) /hr.
ings, into tube (T) which contains 5 ml of the
(from I) (Table 2) ("F")
BTB indicator solution. During readings a small
This constant "F" is entered in the data sheet
bore glass tube is immersed in fresh indicator
(Table 1) and remains the same for the spe
solution. An adjustment of the pressure-regulat
cific air flow in all experiments.
ing tube is necessary to maintain the constant air
Then divide CO2 (mg) /hr. ["F" from II] by
pressure during the readings.
weight (kg) of the test material.
Twelve respiration chambers were attached
III. CO2 (mg)/hr. = CO2 (mg)/kg FW/hr.
by means of 30-inch lengths of rubber tubing to
FW (kg) ("J")
a 12 outlet board (Figure 2).
The measurements were taken by attaching This jar constant "J" is entered in the data
rubber tubes of equal length from the outlet sheet (Table 1) and remains the same for a
tube in each lid to a small bore glass tube in given weight of fruit or vegetable.
serted in the test tube (T) containing the indi For conversion of daily percentage CO2 read
cator solution. In the interest of efficiency and ings to mg CO2/kg FW/hr. the jar constant "J"
accuracy, a setup of 12 rubber tubes each with is multiplied by the per cent C02; i.e. line "C"
small bore glass tubes was constructed in a times constant "J" is recorded on line "D" as
mg CO2/kg FW/hr. in Table 1.
holder to fit the rack of 12 test tubes. This al
lowed the 12 tubes to be inserted and removed at Operation:
one time following the ten-minute equilibrium 1. Weigh the fruit or vegetable and record in
time. Table 1 in kg.
Calculations.—This method provides us with 2. Place in respirometer and close gas tight
the percentage of CO2 in an air stream which and attach to respiration board.
VINES AND OBERBACHER: VEGETABLE RESPIRATION 315
Table t. Conversion of O>2 (ml) to C02 (mg). 5. Care should be employed to maintain the
10-inch head pressure (h, Figure 2) at all times.
Conversion Factor
Adjustment is necessary when the equilibration
Temperat ure
°C. °F. mg C02/ml C02
is started and again when it is complete.
0 32 .977 6. A check for leaks in the system should be
2.5 37 L.959
5.0 41 L.941 made daily. The check is made by closing off
45 L.924
7.5
10.0 50 1.901 the outlet hole of the individual respiratory
1.890
12.5 55
1.874
chamber with thumb pressure and observing
15.0 59
L.842
20.0 68
1.811
the pressure build-up as seen by a slow depres
25.0 77
30.0 86 . 1.781
sing of the 10-in water head. No depression in
35.0 95 1.752
dicates a leak in the system.
The above conversion factors are obtained according to the 7. The length of all sets of tubing should be
following formula:
the same and the length of the small bore glass
273 X 44,000 " conversion factor
273 + t 22,257 tubing used to bubble the CO2 laden air into the
where 273 in "Kelvin - 0° C. test tube (T) should be kept constant.
t o temp. In degrees C.
44,000 o molecular weight in mg of CO . 8. The small bore glass tubing should be
22,257 « vol. in ml of 1 mole of C02 at 0° C.
wiped immediately before being inserted into the
3. Take percentage transmission as specified indicator solution to avoid contamination that
in another section and record in line "A" may have an acid or alkaline effect.
Table 1, along with date and hour. 9. The V2 inch light path cuvettes were origi
4. Convert percentage transmission to per nally used for measuring color change in the
centage CO2 by reference to standard curve B & L Spectronic 20 spectrophotometer. It was
or chart and record in line "B" Table 1. found that transferring the indicator solution
5. Subtract either 0.03% CO2 normal concen from the test tube into the cuvette exposed the
tration in air or the reading of an empty solution to air and changed the equilibrium, thus
jar and record net CO2 in line "C" Table 1. resulting in an error. To overcome this error,
6. Multiply net % CO2 by jar constant "J" test tubes were selected and adjusted to 100 per
and record in line "D." cent transmission and the position marked on
Precautions.—Certain daily variations occur the tube. Readings are now taken by reading
in measuring CO2 concentration by this method. the tubes in which equilibration was established
Some are real and some are inherent in the which are corked with waxed stoppers immediate
method or the operator. Therefore, certain pre ly after being equilibrated.
cautions should be observed. Discussion of Respirometer.—For universal
1. The temperature of the indicator solution comparison of data on fruit respiration the gen
should be maintained constant. In this case the erally accepted basis is milligrams of CO2 per
10L container of BTB indicator solution from kilogram of fruit per hour (mg CO2/kg FW/hr.).
which the solution was drawn for each measure The milligram basis was preferred over the cubic
ment was kept in the 70° room. A 70° P. water centimeter of CO2 because it removes the variable
bath was used to maintain indicator solution temperature factor thus permitting respiration
temperature when respiration studies were being measurements at different temperatures to be
conducted at a different temperature. compared directly. Some research workers still
2. A minimum of 10 minutes bubbling time is use ml instead of mg; however, this conversion
advisable in order that equilibration is always can be made.
reached. It is advisable to main an air flow sufficiently
3. A constant temperature of the respiratory rapid to keep the CO2 level at 0.5 per cent or
chambers is a necessity. Precautions should be less in order that its accumulation will not in
taken to prevent the refrigeration fan from blow fluence metabolic processes in the fruit under
ing directly on the respiration chamber. A con study. This flow rate will also prevent accumu
tinuous temperature recorder should be kept in lation of physiologically active volatiles such as
the temperature controlled room. ethylene.
4. A constant voltage supply to the spectro- This method was specifically designed for
photometer to prevent line voltage variation is the study of citrus fruit respiration, a relatively
desirable. It is advisable to check the zero setting slowly respiring fruit. For studies on faster re
with the water blank after every third reading. spiring fruit or at higher temperatures, less than
316 FLORIDA STATE HORTICULTURAL SOCIETY, 1963
Table 2.
A = % Transmission
Exp. No._ BOARD B = % C02
C = Net % CO2
D * Mg CO2/hr/kg fruit
Harvested Remarks
Pretreatment
Jar no.
1 2 3 4 5 6 7 8 9 10 11 12
Flow - ml/min.
n-pM
Wt. lost
Date A
Time B
Date A
Time B
Date A
Time B
Date A
Time B
D
VINES AND OBERBACHER: VEGETABLE RESPIRATION 317
the air can be measured by flowing air through total CO2 evolved from this given fruit weight
an empty jar and the exact amount subtracted as it is completely oxidized therefore remains
relatively constant. The rate of oxidation (mg
from the respiring jar readings to give a true
measure of fruit respiration. It is possible to CO2/ kg FW/hr) under adverse conditions as
install a CO2 scrubber between the air compres high temperature will be higher and the fruit
sor and the respirometer but this adds to an will break down quicker since there is just so
stall a humidifier in the air line. This is more respiration often before it can be seen by inspec
important if compressed air is used than when a tion. Again the total CO2 evolution remains
small air pump is used. A standard laboratory constant but the rate is increased many times
sintered glass aerator filled with water and a and the breakdown is rapid.