Measurement of Respiration Rate of Stored Sugar Beet

Measurement of Respiration Rate of Stored Sugar BeetAuthor(s): J. I. Burke, B. Rice and V. A. DoddSource: Irish Journal of Agricultural Research, Vol. 18, No. 3 (Dec., 1979), pp. 305-313Published by: TEAGASC-Agriculture and Food Development AuthorityStable URL: http://www.jstor.org/stable/25555955 .

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Ir. J. agric. Res. 18: 305-313, 1979

MEASUREMENT OF RESPIRATION RATE OF STORED SUGAR BEET

J. I. Burke, B. Rice

An Foras Ta I tint a is, Oak Park Research Centre, Car low

V. A. Dodd

Department of Agricultural Engineering, University College, Dublin 2

ABSTRACT

Methods based on the measurement of C02 production have been widely used to determine the

respiration rates of living tissue. When applied to sugar beet there is a possibility that because such

systems involve the initial removal of all C02 from the air, they may affect the respiration rate. This was

demonstrated using the Warburg respirometer by employing three techniques involving 0, 1 and 5% C02

concentrations in the respiring flasks. Differences were measured with the three methods: respiration rates with the Pardee (1% C02)

and indirect (5% C02) techniques were 19 and 84% respectively greater than with the direct system (0% C02).

An alternative method of respiration measurement based on 02

consumption rather than C02 production was developed. It was verified by comparing the calculated

with the measured heat production.

INTRODUCTION

Respiration is the main cause of sucrose loss in the storage of sugar beet (1, 2, 3).

The respiration reaction proceeds as follows:

C]2H22On + 12 02+ 74ADP + 74 Pi - 12 C02 + 11H20 + 74 ATP + 3175.5

kJ/g mole The gross energy value of sucrose is 5671 kJ/g mole. Since the cell is only 44%

efficient in utilising this energy (4), the heat released is 3175.5 kJ/g mole (9.285 kJ/g).

A respiration measurement technique should provide the following:

i) It should permit the use of a reasonable number of whole roots,

ii) It should maintain a normal storage environment.

iii) It should permit continuous measurement of respiration rate throughout the

storage period.

305



306 IRISH JOURNAL OF AGRICULTURAL RESEARCH, VOL. 18, NO. 3, 1979

Three procedures have been widely used for the measurement of respiration rate:

a) The Warburg constant volume respirometer has been used in respiration studies

of plant and animal tissue (5). Samples 50 mm square, 30 pm thick are placed in a

sealed container, and the carbon dioxide released during respiration is absorbed

by an alkali solution. The change in the pressure of gas in the sealed container is

measured at constant volume, and the C02 production is calculated. The small

samples used in the Warburg respirometer render it unsuitable for most sugar

beet storage investigations, except those involving either in-root or between-root

studies in which comparative rather than absolute values are adequate. The effect

of factors such as crowning, leaf regrowth or root damage cannot be investigated,

and because of the tissue damage caused during preparation of the sample, the

measured respiration rate bears little relationship to that which would occur with

whole roots.

b) Some workers on sugar beet storage have adopted the Warburg principle

(absorption of C02 in alkali solution within a sealed container) but have provided sealed containers large enough to hold several whole roots (2, 6, 7).

c) One of the more widely used procedures is to meter C02-

free air through the

respiring beet, and measure the C02

content of the exhaust air (8, 9, 10, 11, 12).

All of these methods have one common feature, i.e., the removal of C02

from the

air, either in or before entry into the beet container.

Fixation of C02 coupled with respiration, occurs in the dark, and is referred to by

Steward as the dark fixation reaction (13). In this reaction a four carbon keto-acid is

formed when pyruvic acid (CH3COCOOH) combines with C02 to produce oxalo acetic acid (HOOCCOCH2COOH). It is possible that removal of C02 from the air

might restrict the occurrence of the dark fixation reaction. In view of the possible

effects of C02

on respiration rate, it was decided to investigate the effect of C02

removal on respiration using the Warburg respirometer. The results obtained led to

the development of a respiration measurement technique satisfying the conditions as

outlined in i), ii) and iii) above, in which air was metered through whole roots and the depletion of

02 from the air measured. The technique developed was then used

to examine field factors such as crowning, leaf regrowth and root damage. This

paper describes the technique and the results of measurements to verify its accuracy.

EXPERIMENTAL

Experiment 1: Effect of Carbon Dioxide on Respiration To study the effect of

C02 on respiration rate a Warburg VI66 apparatus shaken at

170 strokes per minute of amplitude 11cm was used. The temperature of the

experiment was maintained constant at 20?C. Fourteen conical flasks of volume 13

to 14 ml were used. The rim of the centre well was greased to prevent creeping of the

alkali and one piece of filter paper was cut to project 5 cm above the rim and folded



BURKE ET AL: RESPIRATION RATE IN SUGAR BEET 307

to increase the surface area of the alkali in the centre cup. Each treatment was

replicated five times while the four remaining manometers were used as thermo

barometers, which recorded any pressure changes not related to respiration. The

results obtained were corrected to allow for these changes. Three methods as follows

were employed to assess the effect of C02 concentration on respiration rate of the

beet:

a) Warburg's Direct Method (0% C02) (5) b) Method of Pardee (1% C02) (14) c) Warburg's Indirect Method (5% C02) (5)

a) Warburg *s direct method In this method, 02 uptake by living tissues was measured by absorbing the C02 continuously in 0.2 ml of a 20% solution of KOH contained in the centre well (5). The respiring tissue was immersed in 3 ml of a 7.6 x 10-3m bicarbonate buffer solution (pH 7.0). The desired pH was obtained by bubbling C02 through the bicarbonate solution prior to the experiment. The

C02 liberated during respiration

was measured by the use of an extra flask similar to the one used above, except that

no alkali was used in the centre well. After equilibration at 20?C, readings were

taken at 15 minute intervals for 60 minutes. The basis of this method is that the 02 uptake in the absence of C02 is measured in one flask; the change in uptake that should have taken place in the second flask if no C02 were produced is then calculated. The observed uptake was always less than the calculated figure, the

difference being due to C02 liberation.

b) Method of Pardee In this method (14) measurement of 02 uptake by respiring cells takes place in the

presence of C02

as in Warburg's indirect method. However, the principle employed

here differs from that used by Warburg. The presence of C02 in the respiring flask is achieved by adding to the centre well 0.6 ml of a buffer solution made up from 6 ml

diethanolamine (CH2CH2OH)2NH), 15 mg thiourea (H2N.CS.NH2), 3 g of KHC03, 2.2 ml 6 NHC1 and 6.8 ml H20, which was shaken and stoppered overnight. This solution allows a 1 % concentration of C02 to be maintained in the respiring flask as

represented by the following equation:

NH(CH2CH2OH)2 + C02 + H2O^Z?HCO^ + "NH2 (CH2CH2OH)2

Any C02 formed by metabolism is removed by the reaction proceeding from left to

right: any C02 utilisation in dark fixation is replaced by the reverse reaction.

c) Warburg ys indirect method

This method permits the determination of 02 uptake and C02 evolution in the

presence of adequate supplies of C02 (5). A comparison witht the direct method enables the effect of

C02 on respiration to be calculated.



308 IRISH JOURNAL OF AGRICULTURAL RESEARCH, VOL 18, NO 3, 1979

^^^i?i?i Hi I ft U _. : H

I-~

EH BI' 10 Fig. 2: Schematic drawing of system developed for respiration measurement.

Beet tissues were placed in each of two Warburg conical flasks, and 2 and 5 ml of

a 7.6 x 10~3m bicarbonate buffer solution (pH 7.0) were added respectively. After

equilibration at 20?C, readings were taken at 10 minute intervals for 50 minutes.

The experiments were conducted at a pH of 7.0. At this pH the solubility of C02

can

be significant as outlined by the Henderson-Hasselbach equation (5). This was

prevented by using 5% C02 concentration in the respiring flasks together with a 7.6 x 10-3m bicarbonate solution which maintained a pH of 7.0 at 20?C (5). The gas

mixture of 5% C02/95% Oz was supplied from a steel cylinder and was vented

through the sidearm of the manometer. This method is based upon the principle that

changes in the volume of two gases of markedly different solubility (e.g., C02 and

02) can be measured simultaneously by following the manometric changes occurring

over identical reaction mixtures in the two flasks of different fluid volumes.

Experiment 2: Respiration measurement based on oxygen uptake

In view of the fact that the removal of C02 may have an effect on the respiration

rate, the development of a system employing 02 consumption was considered best.

It was decided to use a metered-flow system rather than sealed containers, as it was

easier to control the degree of 02 depletion during long runs. Wyse (15) showed that when the oxygen content of the air dropped below 15%, respiration was reduced. In

a metered-flow system the level of 02 depletion could be controlled by selecting a

suitable air-flow rate.

In the equipment developed (Figs 1 and 2) air was supplied from a compressed air

system (A) through a pressure regulator (B) and the moisture content was kept near

saturation by passing the air through a cylinder of water (D). A secondary regulator

(E) was used to maintain a constant head of 150 mm of water. The air supply was

then passed into a conical flask where water condensing in the line was trapped (F).



F/g. // Equipment used for respiration experiment




The air was then passed to a manifold (G) and fed to eight individual rotameter flow-meters (H) with needle valves, which were used to adjust the air flow before

passing through the sugar beet roots in each respiration vessel (I) to a sampling valve

(J) in which a polarographic 02 electrode was placed. The decrease in the percentage

02 in the exhaust air was indicated on an I.L.504 analyser. In preparing the beets to

be used in respiration experiments, each lot was carefully washed, dried and

uniformly topped. Samples were then selected to give uniformity of size, weight and

numbers of roots. Temperatures of the beet and of inlet and exhaust air were

recorded on a Philips PM 8235 12-point temperature recorder using copper -

constantan thermocouples. The temperature of the beet was maintained at a chosen

value by placing the respiration vessels in an insulated duct through which air was recirculated. The temperature of the air was controlled by passing it over the

evaporator of a refrigeration unit.

Preliminary trials were carried out to establish the optimum air-flow rate through

the respiring beet. High flow rates led to changes in 02content so small that they were hard to measure accurately. Low flow rates caused 02 depletion to a level that

might interfere with respiration. It was considered that the 02

content of the exhaust

air should be maintained between 17.5 and 20%; the air flows needed to achieve this were found to lie between 1.5 and 5.0 x 10_6mVs, depending on the respiration rate

of the beet.

To establish the validity of this method of measuring respiration rate, the heat

production calculated from the measured 02 depletion using the respiration

equation was compared with the measured heat production. A 50-litre insulated bin

was filled with 20.95 kg of beet and ventilated at 2.5 and 5.0 x 10-6mVs in successive experiments. Sufficient insulation was required to ensure that the heat

loss through the insulation was insignificant, compared with the heat removed by the air flow through the beet. The bins were insulated with a 230-mm layer of

polystyrene granules at which the estimated heat loss was 0.10 J/m2s ?C. The

temperature difference between the room and the respiration vessel was maintained

at less than 1?C by a 2 kW fan heater controlled by a differential thermostat. At both flow rates the temperature and relative humidity of inlet and exhaust air and

the beet temperature were recorded continuously and the 02 concentration of

exhaust air was measured at hourly intervals. The average respiration rate and rate

of increase of temperature over a 20 hour period was used to calculate the

respiration heat produced and the sensible heat taken up by the beet.

RESULTS AND DISCUSSION

Experiment 1

The respiration rate measured by the direct method, i.e., in the absence of C02,

was

less than half that measured by the indirect method, in the presence of adequate supplies of CO, (Table 1).




oxygen exchange 800

|--i 228 8

V Direct 0% / 700 -

0/ / "

200 2 o Pardee 1 /o / , ^ / x

& Indirect 5 /o / -

E 600 - / - 171 6 -

? / * D /

?E 500 r / - U3 0"oT

5 / y E

w 400 - / X J^,114 4

^ / X X\ z / >r ,/ < / ^ /^ I / X yX S 300r / x y^

"858 < / XX

200 - / / X*^ - 57 2

100 -

//yX

' 28 6

e_-1-1-1 0 15 30 45 60

TIME mm

Fig. 3: Effect of C02 concentration on respiration rate

The method of Pardee which measures only 02 uptake, gave a smaller (18%) difference when compared with Warburg's direct method (Fig. 3). This was

probably due to the differences in the concentration of C02 in the respiring flasks. The solution of diethanolamine in the centre well of Pardee's method maintained a chosen concentration of

C02 (1%) throughout the experiment, whereas in

Warburg's indirect system the level of C02 was fixed at 5%.

Wyse (15) also found that as the C02 content in the respiration vessel was raised, the respiration rate was increased. The results, therefore, suggest that respiration

studies carried out with sugar beet in which the effect of C02 is neglected may be

inaccurate, and may not be a true reflection of actual respiration rates occurring

under natural storage conditions. This effect may, however, be of greater

importance, when using the Warburg constant volume respirometer, due to the

greatly increased respiration rates obtained therein. The damage caused during the

preparation of tissues, coupled with the immersion in a buffer solution, resulted in



BURKE ETAL: RESPIRATION RATE IN SUGAR BEET 311

respiration rates much greater, than those obtained with whole roots (Table 1, Fig.

1).

TABLE 1: Effect of C02

concentration on respiration rate

C02 Respiration rate Method concentration

/i02/mg h (dry weight) SE

Direct 0 398.42 ?12.69 Pardee 1 438.30 ?10.24

Indirect 5 673.93 ?18.03

Experiment 2:

The sensible heat gained by the beet (Hs), sensible heat removed by ventilation (Q), the latent heat of vaporization (HL) and respiration heat were calculated as follows

at a flow rate of 2.5 x lO-^Vs:

Sensible heat H = (Mb) (Sb) (Tb) J/s

where Mb

= mass of beet = 20.95 kg

Sb =

specific heat of beet = 3893.7 J/kg?C (16)

Tb = beet temperature change, ?C/s

H = (20.95 (3893.7) (13.8 x 10~6) J/s = 1.13 J/s

The sensible heat removal by ventilation

Q = (M) (S) (AT) J/s where M = mass air flow rate, kg/s

(density of air = 1.2 kg/m3) S = specific heat of humid air (1.02 kJ/kg ?C AT =

temperature difference between inlet and exhaust air, ?C

Q = (2.5 x 10-6) (1.2) (1.02) (2.0) x 103 J/s = 6.12 x 10-3 J/s

The heat lost to vaporization

HL = (M) (G) (V) J/s

where M = mass air flow rate of air, kg/s = 2.5 X 10-6 kg/s

G = change in moisture content, kg/kg dry air V = Latent heat of vaporization

= 2465 kJ/kg

HL = (2.5 x 10-6) (1.2) (1.5 x 10"3)(2465 x 103) J/s = 11.09 x 10-3 J/s

Hs + Q + HL = 1.15 J/s




Respiration rate = 22.39 mg 02/kg h = 19.92 mg sucrose/kg h = 0.417 x 10-3 kg/h for 20.95 kg beet

Respiration heat R = 0.417 x 9.285 - 3.87 kJ/h = 1.075 J/s

(H + Q + HL) ?R = 1.15 ? 1.075 = 0.075 J/s

At a flow rate of 5.0 x 10_6m3/s the results obtained were as follows:

Hs = 1.428 J/s

Q = 1.29 x 10-3 J/s

HL = 14.8 x 10-3 J/s

R = 1.323 J/s

(Q + HL + H) ?R = 1.444 ? 1.323 = 0.121 J/s

The calculations show that the heat production calculated from the 02 depletion

was close to the figures obtained from the heat balance. The small discrepancy

obtained can be explained by the uncertainty of the cell's efficiency which was taken

in this instance as 44% under ideal conditions; this can range from 40% to 44%, as once the energy rich phosphate bonds have been utilized, further heat is given off.

The results establish that the respiration rate as measured by the system outlined in

this paper is reasonably accurate.

Oldfield et al (11), when estimating the heat production from sugar beet, used a

gross energy value of 16.5 kJ/g of sucrose. However, when sucrose is utilized in the

body's cell only 56% is converted to heat, and not 100%.

Eight bins were filled with beet selected from the same field plot to enable the

degree of random error associated with the system to be assessed. Respiration rate

was recorded daily for 14 days. It reached a maximum value of 10.13 mg 02/kg of

beet on the day after harvesting, and at that stage the standard error of the eight

daily readings was 0.26. After 10 days the respiration had stabilized at an average

value of 5.58 mg02/kg. The average respiration rate for the duration of the trial was

5.07 mg02kg

with a standard error of 0.10.

CONCLUSIONS

The results of the trials with the Warburg respirometer show that the CO, level can

have a substantial effect on the respiration rate of sugar beet. This at least raises the

possibility that respiration measuring systems based on CO, measurement, in which




all the C02 is removed from the air entering or contained in the respiration chamber,

themselves affect the rate at which the beet respires. This effect may be of greater

importance when using the Warburg apparatus, due to the greatly increased

respiration rate obtained therein.

As well as overcoming the difficulties that might be associated with C02 measurement, the system based on

02 consumption outlined in this paper is both

simple and accurate in use. It permits the use of whole roots, maintains an

environment similar to that occurring in practical sugar beet storage and permits it

to be measured continuously over several weeks.

REFERENCES

1. Stout, M. and Smith, C. H. Studies on the respiration of sugar beets as affected by bruising, mechanical harvesting, severing into top and bottom halves, chemical treatment, nutrition and

variety. /. Am. Soc. Sug. Beet Technol. 6: 670, 1950. 2. McCready, R. M. and Goodwin, G. C. Sugar transformations in stored sugar beets. J. Am. Soc.

Sug. Beet Technol. 14: 197, 1966. 3. Wyse, R. E. and Dexter. S. T. Effect of agronomic and storage practices on raffinose, reducing

sugars and amino acid content of sugar beet varieties. J. Am. Soc. Sug. Beet Technol. 16: 369, 1971. 4. McDonalds, P., Edwards, R. A., and Greenhaigh, J. F. D. "Animal Nutrition". 2nd Ed. Longman

Group Ltd., New York and London, pp. 149-160, 1975. 5. Umbreit, W. W., Burris, R. H. and Stauffer, J. F. "Manometric biochemical techniques". 5th Ed.

Burgess Publ. Co. Ltd., U.S.A., pp. 1-63, 1972. 6. Vagna-Papp, M. Z, Atmungsversuche mit Rheinischen Zuckerruben, Zeitschrift fur die,

Zuckenndustrie LXXXIII: 377, 1958. 7. Nelson, R. T. and Wood, R. R. Respiration and spoilage studies employing a modification of a

method developed by Stout and Fort. J. Am. Soc. Sug. Beet Technol. 6: 660, 1950. 8. Barr, C. A., Mervine, E. M. and Bice, R. A. A preliminary report on the effect of temperature and

beet conditions on respiration and sugar loss from beets in storage. Proc. Am. Soc. Sug. Beet Technol. 1: 52, 1940.

9. Stout, M. Some recent advances in the physiology and biochemistry of respiration. Proc. Am. Soc.

Sug. Beet Technol. 8: 417, 1954. 10. De Villers, P., Lolier, M. and Chartier, J. C. Respiration of sugar beet. Inds aliment, agric. 83: 901,

1966. 11. Oldfield, J. F. T., Dutton, J. V. and Haughton, B. J. Deduction of the optimum conditions of

storage from studies of respiration of beets. Int. Sug. J. 875: p. 326. 876: p.361, 1971. 12. Chartier, J. C, De Villers, P., Lolier, M., Chablay, R., Guyot, J. Y., Mesnard, G. and Wilde De,

D. Evolution des pertes de sucre au cours de la conservation des betteraves. Sucr. fr. Ill: 449, 1971. 13. Steward, F. C "Plants at work". Addison-Wesley Publishing Company, Reading, Massachussetts,

Palo Alto, London, Don Mills, Ontario, pp. 62-82, 1967. 14. Pardee, A. B. Measurement of oxygen uptake under controlled pressures of carbon dioxide. J. biol.

Chem 179: 1085, 1949. 15. Wyse, R. E. Factors influencing the respiration rate of sugar beet roots. I.I.R.B. 33: Report No. 2,

9, 1970. 16 Bakker-Arkema, F. W. and Bickert, W. G. A deed bed computational cooling procedure for

biological products. Trans. Am. Soc. agric. Engrs 9: 834, 1966.

Received September 14, 1978



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Measurement of Respiration Rate of Stored Sugar Beet