312 FLORIDA STATE HORTICULTURAL SOCIETY, 1963

Discussion and Summary LITERATURE CITED

1. Anderson, D. L. and P. F. Shaffer. Improved methods

Florida sweet corn is harvested with 2 to 6 of trimming produce in retail food stores. U. S. Dept. Agr.
Mktg. Res. Rept. 192. 1957.
inches of shank and several flag leaves attached 2. Bartholomew, E. T. Internal decline of lemons. III.
to the ear. Long shanks and fresh husks have Water deficient in lemon fruits caused by excessive leaf
evaporation. Amer. Jour. Bot. 13:102-117. 1926.
been considered indexes of good quality. Storage 3. Chandler, W. H. Sap studies with horticultural plants.
Mo. Agr. Exp. Sta. Res. Bui. 14. 1914.
tests with 15 lots of sweet corn showed that 4. Comin, Donald, and William Junnila. Water loss
ears with long shanks had dented kernels much from vegetables in storage. Ohio Agr. Exp. Sta. Bimonthly
Bui. 243. 1946.
sooner than ears with short shanks. However, 5. Hardenburg, Robert E. Further studies on moisture
losses of vegetables packaged in transparent films and their
husks remained fresh longer on ears with long effect on shelf life. Proc. Amer. Soc. Hort. Sci. 57:277-284.
shanks. An indication was found that non-trim 1951.
6 . Prepackaging carrots in different types
med ears with long flags lost more water of consumer bags. Proc. Amer. Soc. Hort. Sci. 61:404-412.
1953.
during storage than similar ears with short 7. Kramer, Paul J. Plant and soil water relationships.
flags. McGraw-Hill Book Company. New York. pp. 266-294. 1949.
8. Lewis, William E. Maintaining produce quality in
As the shanks were trimmed to shorter retail stores. U. S. Dept. Agr. Handbook 117. 1957.
9. Lutz, J. M., J. Kaufman, and H. W. Hruschka.
lengths, greater numbers of outer husks were Shelf life of pre-packaged radishes in relation to type of
severed at their attachment to the shank. These film, temperature, and amount of trimming. Pre-Pack-Age
8:13-16. 1954.
cut husks wilted and discolored much more 10. McCrory, S. A. Heat and drought tolerance of sweet
corn as influenced by tillers. Proc. Amer. Soc. Hort. Sci.
rapidly than those still attached. The fact that 40:424-428. 1942.
ears with severed outer husks lost less weight 11. Parsons, C. S. and R. C. Wright. Effects of tempera
ture, trimming and packaging methods on lettuce deteriora
and their kernels did not dent, further indicated tion. Proc. Amer. Soc. Hort. Sci. 68:283-287. 1956.
12. Platenius, Hans. Physiological and chemical changes
that water was not moving from kernels to husks in carrots during growth and storage. Cornell Agr. Exp.
Sta. Mem. 161. 1934.
of these ears.
13 ,—. Films for produce—their physical
No moisture was lost from kernels on closely characteristics and requirements. Modern Packaging. 20:
139-143. Oct., 1946.
trimmed ears with wilted husks, compared with 14. Showalter, R. K., W. S. Greig, C. S. Parsons, and
a constant decrease in moisture from kernels on K. D. Demaree. Quality of Florida sweet corn as affected
by marketing practices. Proc. Fla. State Hort. Soc. 68:
non-trimmed ears with fresh husks. Movement 182-185. 1955.

of water from kernels to husks was indicated on

ears without closely trimmed shanks.

A SIMPLE METHOD FOR CO DETERMINATION IN FRUIT

AND VEGETABLE RESPIRATION STUDIES

H. M. Vines1 and M. F. Oberbacher2

alkaline solution. In this method the CO2 was
Florida Citrus Commission precipitated as barium carbonate with barium
In Cooperation with the University of Florida chloride. The amount of CO2 evolved from a
Citrus Experiment Station given weight of fruit for a given time was calcu
lated by filtering, drying and weighing the barium
Lake Alfred carbonate precipitated. This method is laborious,
time consuming and possesses certain inherent
Introduction inaccuracies. A titrimetric method sometimes
employed measures the reduction in alkalinity
A rapid method for determining the rate of
following a given period of CO2 absorption. This
respiration of fruit is desirable where numerous
is also time consuming.
measurements are to be made simultaneously.
A number of instruments are available which
Either a measure of oxygen (O2) uptake or
are useful in measuring CO2 and O2. A gas
carbon dioxide (CO2) evolution is an acceptable
chromatographic apparatus, which measures
indication of the rate of respiration.
CO2, O2 and N2 in low concentrations (Fisher
In the past, the method generally employed
Gas Partitioner) has been described by the
involved the absorption of evolved CO2 in an
authors (3). Other instruments available in
clude the infrared CO2 analyzer, the paramag
l Associate Biochemist.
2Associate Plant Physiologist.
netic O2 analyzer and more recently the polari-
 VINES AND OBERBACHER: VEGETABLE RESPIRATION 313

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 -

should be stored in pyrex in the dark.

100
Air Flow Regulation.—With this method, a -

known volume of air flow per minute must be 90

maintained. This is provided with capillary tub

80
ing under constant air pressure. With a constant
pressure, the volume of air is dependent on the 70
- /
■/
bore and length of tubing. Appropriate lengths
are cut and calibrated in ml/min for known air 60

flow by water displacement or soap bubble tech

50
nique at the constant pressure which will be
used in normal operation. Each capillary tube 40 /
should be calibrated because of bore size varia
30
bility, marked and stored in a dust-free container O 0.2 0.4 0.6 OB LO 12 14
%CO2
when not in use.
Standard Curve.—The standard curve is made
by plotting the per cent transmission of the BTB
indicator solution which has been equilibrated
Figure 1. Bromthymol blue standard curve of per cent carbon
with an air stream of known CO2 content. dioxide versus per cent transmission at 617 ntyx.
314 FLORIDA STATE HORTICULTURAL SOCIETY, 1963

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
holder to fit the rack of 12 test tubes. This al
lowed the 12 tubes to be inserted and removed at
one time following the ten-minute equilibrium
time.
Calculations.—This method provides us with
the percentage of CO2 in an air stream which
times constant "J" is recorded on line "D" as
mg CO2/kg FW/hr. in Table 1.
Operation:
1. Weigh the fruit or vegetable and record in
Table 1 in kg.
2. Place in respirometer and close gas tight
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

Variety__ Source Date set up_

Harvested Remarks

Pretreatment

Jar no.
1 2 3 4 5 6 7 8 9 10 11 12

No. fruit per jar

Cap. tube no.

Flow - ml/min.

n-pM

Jar constant - "J"

Wt. fruit gms. - init.

Wt. fruit gms. - fin.

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

Table 3. evil. An over simplification of respiration is the

reaction of one molecule of sugar with six mole
cules of O2 to yield six molecules of CO2, six
Mg CO2/kg F.W./hr. at 70° F
molecules of H2O and 680 kilocalories of energy
25 - 60 per mole.
Oranges
The rate of respiration which is an indirect
Lemons 15 - 30
measure of the storage life or rate of breakdown
40 - 100
Peaches varies with the specific product, storage tempera

Pears 25 - 70 ture, handling, packaging and many other factors

both known and unknown. Therefore, a measure
Cherries 80 - 120
of the rate of CO2 evolution as described here
60 - 100
Strawberries can be used as a means of comparing different
Bananas
50 - 80 plant material, different treatments of the same
plant material and the effect of various storage
Apples 15 - 30
temperatures. An average expected rate of res
piration for some fruit is listed in Table 3. These
data are averages of rates obtained from dif
a 1,000 gm. sample or larger flow rates should
ferent varieties by the authors and those cited
be used.
by Biale (1).
Air is generally considered to contain ap
In general, a given fruit weight contains a
proximately 0.03 per cent CO2 but varies with
the time and place. The actual per cent CO2 in relatively constant amount of stored food. The

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

already complicated setup.

much available stored food to supply CO2. Simi
In order to prevent dessication of fruit and larly, infection by fungi or decay organisms can

especially leafy vegetables it is advisable to in be detected by a serious increase in the rate of

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.

small amount of butyl alcohol to break up the

BIBLIOGRAPHY
air into bubbles is installed in the air line.
1. Biale, Jacob B. 1960. Respiration of fruits. Hand-
buch Der Pflanzenphysiologie, Vol. XII: 536-592.
Discussion and Conclusion 2. Claypool, L. L. and R. M. Keefer. 1942. A colori-
metric method for CO2 determination in respiration studies.
In general terms, fruit respiration is the Amer Soc. Hort. Sci. Proc. 40: 177-186.
3. Vines, H. M. and M. F. Oberbacher. 1961. Changes
oxidation of stored foods present in the plant in carbon dioxide concentrations within fruit and containers
material and has been referred to as a necessary during storage. Fla. State Hort. Soc. 74: 243-246.