Plant
Phlvsiol. ('1967) 4/?,
120-124
Carbon Dioxide Production by Dry Grain of Zea mays"
Duane P. Bartholomew2
3
and Walter E. Loomis
Department of Botany and Plant Pathology, Iowa State University, Ames, Iowa 50010
Re cived August 18, 1966.
Summary. Use of the gas chromatograph and a mercury-to-glass sealed respirometer adapted for gas syringe sampling, allowed the rapid, accurate characterization of CO2 evolution rates from live and from dead-sterile Zea ntays L. grain
dried to moisture levels of 12.6 to 1.4 %. The live grain at the lowest moisture
level showed an elevated rate inconsistent with the exponential increase in rate of
CO2 evolution with increasing moisture found for maize with moisture contents
from 4 to 12.6 %. At the lowest moisture level, rates of CO2 evolution from
dead-sterile grain were greater than for live grain. Moisture had no effect on
CO2 evolution from dead-sterile grain. Increasing temperature and increasing
levels of 02 in the storage atmosphere resulted in increased rates of CO., evolution
from both live and dead-sterile maize. CO2 production rates from live and from
dead-sterile grain decreased with increasing storage time, even though respirometer
CO2 concentrations were less than 1% at the end of the experiment. Our results
indicate that CO2 production is not a dependable measture of respiration in dry seeds.
Other experiments indicate that oxygen absorption also is not reliable in maize graiin.
Alost seed respiration investigations have tused
seeds with moistture contents at and above those
(leemed safe for commercial storage. MIilner and
Geddes (7) reviewed the effects of v-ariotus agents,
including moistture and ftungi, oIn seed respiration
and deterioration. Seed respiration rates, as measLured by CO2 production or 0, use, have been
reported to increase exponentially, the rate approximately doubling with each 1 % increase in moisture
content between 14 and about 20 % (10, 11). Mtuch
of the CO, evolved has been attributed to fungal
respiration (9). Little or no attempt has been
made to characterize the effects of environment
and of changing environment with time on CO,
evoluition rates from grain of very low moistture
content. Mayer and Poljakoff-Mayber (6) feel
that it is almost impossible to measure O. uptake
or CO2 evolution in dry seeds, and that it is more
than likely that most of the gas exchange measured
in dry seeds is due to microorganisms. Ching (2)
measured the respiration of forage seeds after 27
and 48 months of storage in air, with seed moisture
contents down to 6 %. Respiratory quotients of
less than 0.5 were found, and the average respiration rates decreased with increasing storage time.
1 Journial Paper No.
5494 of the Iowa Agritultural
and Home Economics Experiment Station, Ames, Iowa,
Project No. 1139.
2 Predoctoral Fellow, National Institutes of Health.
3 Present address: Department of Agronomni
and
Soils, University of Haw-aii, Honolulu, Hawaii.
The seed environment generally does not affect
to the extent that intraseed moisture
content does. Ragai and Loomis (10) fouind that
the respiration of maize grain approximately douibled
with each 100 rise. Carbon dioxide is known to
depress the respiration rate of many plant tissues
(1), and the results of several workers (2, 4, 10)
indicate that CO2 inhibits respiration in seeds also.
Simpson (12) reported reduced CO2 production
rates for seeds stored in N, and higher rates in
pure 03 than in air.
The objectives of this stuidy were to characterize some of the physiological responses of maize
grain stored at moisture contents down to 1.4 %.
Some lots were killed and sterilized to determine
the extent to which nonrespiratory processes may
contribuite to CO2 evolutioIn in low moisture grain.
This paper describes the responses of viable and
dead-sterile maize grain to decreasing moisture,
increasing CO2 and varying oxygen levels at 2
respiration
temperatures.
Materials and Methods
Grain of the single cross WF9 X M14 was
hand harvested and shelled, discarding the kernels
from the butt and tip ends of the ear. The results
of Struve (13) have shown that maize grain may
be safely dried to moisture contents near zero with
little or no loss in viability and seedling vigor.
The method used by Struve and in this study,
consisted of slow air drying of the seed to below
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121
BARTHOLOMEW AND LOOMIS-CO., PRODUCTION BY DRY GRAIN OF ZEA MAYS
20 % moisture, followed by high vacuum drying
at 500 to weights calculated for the desired moisture content. Dry weight basis moisture contents
for each lot were determined by air-oven drying
triplicate 10 g samples at 1050 for 48 hours.
Prior experimentation had shown that exposing
seeds with greater than 10 % moisture to propylene
oxide vapor for 48 hours was sufficient to render
them sterile and nongerminable. Dead, sterile seeds
were resterilized within the respirometers with
methyl bromide to render both the seeds and their
environment sterile. Sterility was determined by
plating seed halves on peptone-dextrose agar and
incubating the plates at 300.
The effects of different 02 levels on COO evolution were determined by evacuating and refilling
the respirometers several times with 99.98 % N2,
CO, free air, or 100 % 02The respirometer used was the special 1-liter
flask described by Ragai and Loomis (10), modified for gas syringe sampling. The modification
consisted of boring the No. 8 rubber stopper nearly
through from the underside with a 1.27 cm drill
bit. This left a thin septum which was easily penetrated by the syringe needle, allowing numerous
1 ml samples to be taken for CO, determinations.
Grain samples were approximately 200 g.
In sampling, the needle was passed through the
mercury seal and rubber septum, eliminating any
possibility of atmospheric contamination. The CO,
was separated and determined with a gas chromatograph (Beckman GC-2A) utilizing a 63 X 0.63
cm coluimn packed with silica gel and maintained
at 1000. The helium carrier gas flow rate was
100 ml per minute. Carbon dioxide percentages
were determined quantitatively from the peak
heights (5). Respirometer volumes were obtained
from the weight of water required to fill them.
The total volume of CO2 at standard temperature
and pressure in any flask was calculated from the
free volume of the flask and the CO percentage
in the sample. CO, prodtuction rates were calculated as ,ul CO2 per kg dry weight of grain per day.
Results
Moistutre Effects. The effects of increasing
moisture, increasing 02 percentages, and time, on
the CO2 production rates of viable maize grain
at 300 are shown in table I and illustrated for
better comparisons in figure 1. Production of
CO2 increased exponentially with moisture percentages between 2.4 and 12.6, but the rate did not
double with each 1 % of moisture as has been
shown (10, 11) for higher moisture levels. The
unexpected result was the high CO2 production
rate shown by the driest grain (1.4 %). Since
maize grain has been dried in this laboratory to
0.1 to 0.2 % weight loss in 48 hours at 1050, or
within the range of moisture free, without loss of
viability (13), drying to 1.4 % moistture is not a
drastic treatment. The data stuggest that heating
at 500 in a high vacuum produced labile compounds
which broke down rapidly to CO2. This interpretation is supported by the rapid decrease in CO2
production by these samples in the first 3 weeks of
the experiment. Both the 2.4 and 4.4 % sample3
showed similar but smaller responses, consistenit
with less drastic drying treatments.
The effects of moistture, 02 and time on CO.,
production by dead-sterile maize grain are shown
Table I. Respiration of Maize Grain Stored at 300
The grain +-,as placed in storage at the indicated °2 and moisture levels. Each value represents the average of
3 replicates.
Initial
moisture,
Oxygen,
,ul Co2/kg d wt/day at days indicated
10
1.4
2.4
4.4
0
21
100
0
21
100
0
21
100
0
21
12.6
100
0
21
100
90.0
97.4
83.7
305
43.7
34.9
41.0
55.5
41.1
50.5
69.0
81.6
124.3
1385
185.3
20
55.9
51.3
46.0
24.8
32.3
33.1
31.2
43.1
37.2
54.9
60.1
76.2
108.4
129.9
159.2
40
30.9
38.9
39.9
26.3
32.7
35.1
28.9
36.6
44.3
53.2
65.1
91.5
121.0
1567
203.5
60
27.4
33.0
36.9
18.9
24 2
280
23.8
31.2
35 5
43.1
52.2
67.1
93.9
119.4
158.3
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80
100
17.0
23.7
28.2
19.0
28.9
37.7
15.1
21.2
27.9
166
27.0
40.2
37.2
45.6
71.1
82 5
52.1
70.4
882
113.7
156.1
35 9
1250
161.5
122
PLANT PHYSIOLOGY
150F
1-I2.6% H20
130F
0
0
0
110o
a
la
90 _-
R
50
0
-
50
30
,2.4% H20
lo,0
1%
20
40
80
60
DAYS
100
FIG. 1. Chianges in the average CO., l)roduction rate
time for maize graini stored at 300, 21% O.,, anid
Nvary itlg miioistuire l)ercentagzes.
w ith
in table II. No consistent effect of varying moistuire percenitages were shown by this material.
Treinds 'were (lowiward with time andI the 7.8 %
saample vas generally lower thani the other 2 moistuire lots. The inoteworthy poilnt in these resuilts
is the high CO., pro(duictioni showln b) grain, each
lot of which was tested to insuire nongerminabilitv
and( externial and iinternal sterility. At the lowest
moistuire levels the average Co., pro(ductioni by the
dead-sterile grain was twice that of the check after
20 (laxs, whereas it was somewhat less thani the
clheck at the 12.6 to 13.0 %, moistuire level. This
response of the dead-sterile seed was certainly not
niormal respiration, an(l the failu1re of CO., pro(Ilictioln to increase with the moistuire percentage
siuggests that it was not diue to enizynmes remainlitng
after the gas treatment.
Jiffects of Oxygen. Three levels of 0., were
0, 21, and(
ilsed in each moistuire X time test
100 %. Jn general, the effect of 0., was small in
the clheck lots with less thani 8.7 % moistuire, bht
it ten(le(l to increase somewhat Nwith time. WN'ith
more moistuire the grain showe(d more CO, pro(Itctioni with 0O, as wouild be expecte(I in a respiratioln experimenit. The pictuire is complicated,
howev,er, hy increased CO, from dea(l-sterile grain
(table 11) with 0., eveen at the lowest moistiure
level, and, as stated previouisly, with no consistent
responise to moistiure. At 20 (lays the dead-sterile
seed showed more response to 21 % 0., at all
moistuire levels than the check. The respoinse to
100 % 0O. was
' higher in the check, and(l was maintainie(l with time whereas CO. prouciletion (lecrease(l
with time in the dead-sterile grain.
The results are consistent with the assuimption
that CO, produlction in dead-sterile grain was due
to auitooxidation of a relatively limited sutpply of
labile compoulnds, and that it was probably indepeni(lent of enzvme action within the grain.
Tepe;prature Effects. Temperatilre increases
resuiltedl in greatly increased rates of CO., productioni at all moistilre levels and also inicreased the
effect of moistulre on the rate. As is shown ini
table 1, the rate increased almost 3 times between
8.7 and(I 12.6 % moistuire after 100l days of storage
at 302 and(l 21 % O2. At 150 the rate increasecl
less thani 1.2 times over the same moistuire range
after 100 (lays of storage at 21 % O._ The CO..
evolultioni from dea(l-sterile graini also was fouind to
be temperatuire (lependclenit. Temlperatuire coefficielnts (Ql,) were calcuilate(d for the 150 interval,
tusilng the relationship foulnid by Ragai alnd Loomis
(10). The temperatuire coefficienits for the CO..
pro(luictioll rates of viable a(ll of dead-sterile samples of comparable moistuire per-cenitage are pre-
Tale II. C(O., Evolution fromn Dead-Sterile Maize Grain Stored at 30'
The g-ain was placed in storage at the indicated oxvygen and nioistiire levels. Eaclh valuie represents the average
of 3 replicates.
Moistire,
Oxygen,
,u CO/kg cd wt/dav at days inldicated
40
80
84.5
27.8
1 3)().
64.3)
53.4
20
1.
21
-
ct
1(X)
7.8
Lv3()
21
1 (0
13.0)
0
21
100
118.2
89.4
95.3,
72.7
44.7
85.0
53.1
29.8
44.2
79.7
65.2
45.2
76.5
142.3
117.5
37.6
55.7
61.3
28.4
55.5
59.1
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123
BARTHOLONIEW AND LOOMIS-CO. PRODUCTION BY DRY GRAIN OF ZEA MAYS
Table III. Temperature Coefficients (Qlo) for the CO2 Production of Maize Griain
The Q,0 values were calculated for viable and dead-sterile grain stored at 150 and 300 and 21 %
Moisture,
10
20
1.58
1.83
1.75
2 46
2.4
4.4
87
12.6
1.55
1.72
1.58
2.16
1.6
7.8
13.0
...
2.15
1.94
...
2.14
...
Q10 value after storage time indicated
60
40
1.69
1.69
1.93
1.80
2.23
2.16
3.26
3.25
Dead-sterile see(d
sente(l in table III. The values for the viable
grainis ranige(I from 1.55 to 4.00 and were found to
vary with moisture, as was reported by Ragai andI
Loomis (10) for maize grain at moisture levels
greater than 14 %. Temperature coefficients also
showed a tendency to increase with increasing
storage time. The Q1o values obtained fall within
the range of those expected for enzymatically controlled reactions. The Q10 values for dead-sterile
grains raiiged from 1.70 to 3.11 and indicate that
CO., prodluctioin processes in nonviable grain were
temperature dependent.
Storage Tinme. The effect of increasing storage
time was a continuiing decline in the rates of CO,
evolution (tables I, II, and fig 1). The very rapid
(lecline in the 1.4 % moistture sample (fig 1) is
considlered to have involved the depletion of a
CO., precursor accuimulated during the 500, high
vacutum (Irying of the grain. Some of the same
reactioln was shown in the 2.4 and 4.4 % samples.
The slow, relatively tuniform decrease in CO.
prodtuction at 8.7 and 12.6 % moisture may have
been dutie to a decrease in the respiratory substrate
(gltlcose) as well as other changes. In the work
of Ragai and Loomis (10) the exhaustioni of the
O2 and accumtulation of CO2 was an obviouis factor
in the decrease of CO2 produiction. In the experiments reported here, CO2 accuimulatioln did not
excee(l 0.4 % and 02 reduction may be expected
to have been on the same order or somewhat
greater. It is doulbtfull if such chalnges were responsible for the CO., produlction (leclines observed.
Some aginig effect is assunme(d to have beeln a factor
in the resuilts.
Discussion
Inl genleral, the CO. p)rod(lictiom rates obtained
with maize grain samples of 13 % moistulre contenit
andl below were very low. The resuilts show that
low moisture maize grain andcI its associate(d microorganisms had a moistuire-dependent, CO, production rate which increased exponentially from 4
to 12.6 % moistuire. The rate, however, did not
double with each 1 % increase in moistuire content,
as was found for maize grain stored at moisture
1.70
2.04
2.01
80
2,00
2.21
2.34
3.86
O,
100
2.00
2.46
2.25
4.00
1.86
2.54
3.11
contents greater than 14 % (10). The drying time
at 500 reqluired to reduce the see(l moisture to
The elevatedl
1 % was in the range of 65 days.
CO2 prodluc.tion obtained from see(d at 1.4 % moistilre suggests seed changes due to the prolonged
high vacuum drying at the high temperatulre, althotugh the results of Struve (13) and Green (3)
in(licated oily slight reductions in germination and
vigor when maize was (Iried to near zero percent
moisture. Moisture had no effect on CO., production from dlead-sterile grain, as might be expecte(l
if the CO.) was produced by spontaineotus decarboxylation processes. It was anticipated that information
obtained on the CO. production of dead-sterile seeds
woould aid in evaluating the anmotulnt of CO.. pro(luced by nonrespiratory processes in viable seeds.
A comparison of the rates at 1.4 % moistulre in
table I with the rates at 1.6 % in table II after 20 or
40 (lays of storage at 300, shows that dea(l-sterile
grain evolved CO, at a greater rate than the live
grain. At moisture contents near 8 andl 13 % after
40.or 80 days of storage at 1.5, the rate of C()
evoltution .from dead-sterile graiin was greater than
50 % of the viable rate. At 300, the rate from
dead-sterile grain was greater than 30 % of the
viable rate. The results of 'Milthorpe and Robertson (8) indicate that some of the CO., evolved
from live seeds is produced by nonirespiratory processes. Our results in(licate that something less than
70 % of the CO., evolvedl from viable, low moistulre
maize grain may be (Inie to respirationl. The balance
is attributed to nonrespiratory processes.
The effects of temperature are shown in table
III.
The Q,0 values were slightly greater thani 2.0
after 100 days of storage in 21 % O., in(licatinig the
CO. produlction rate increase(d exponenitially with
temperature. The (lata show that the rate of CO.,
evoluitioni from dea(d-sterile see(ds also ilerease(i
exponientially with inicreasinlg temiiperature.
Table I summarizes the effects of increasinlg
O., on the respiration of (lry maize graini. \With
100 days of storage, the respirattion rate increased
with increasing 0, at all moisture levels. The
review of Carr (1) indicates that high levels of
WNTithin the
02
are toxic to some plant tissues.
storage period of this stuldy, an increase in O2 renear
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124
I'LANT PHYSIOLOGY
sulte(l in anl enihainced rate of CO2 evoluition and(l
conifirmedI the fiindings of Simpson (12). Carboni
dioxide was also produce(d in moderate quantities
by anaerolbic processes, since the rates of evoluitioni
in purifiedI N., were only slightly lower than those
in the presenice of oxygen. Increasing oxygen restilted in aln inlcrease in the rate of CO, evoltitioni
from dead-sterile maize (table II). This wouild be
the expected resuilt if the CO., were produiced by
oxidlative mechanlisms. This, however, leaves CO,
evolution from the dead-sterile graiin stored in puire
N., ulnexplainied.
Increasinig storage time generally resutlted in a
decrease in the rate of CO., evolution from both
viable anid dead-sterile maize. The very low moisture samples of viable graini showed a rapid (lecline
in rate (table I) for the first 40 (lays, after which
the rate leveled off. The higher moistuire samples
(2.4 % H2O anid above) showed a slower, steady
declinie. The rapi(d initial (lecline of CO, production in the samples (lrie(l to 1.4 % miistulre suiggests
the decarboxylationi of labile compouinds formed
duirinig drying. The steady rate of decline in all
samples may- have been the resuLlt of some aging
effect. The rate of CO., evoluttioin from (lead grain,
however, also (lecreased with time. Since the
mechanism of CO., produiction in this material is
unknowni, any discutssion of the effect of time on
rate would be speculation.
These results argule against the uise of CO., as
an indicator of respiration in lowv moistuire seeds.
Chiing (2) has commente(l that O., measurements
may yield better seed respiratioln data. The results
of others (3, 7) show that carotenoids and lipids
are readily oxidized and couild easily accouint for
the relatively greater amouints of oxygen taken up
than the CO2 evolved. The very low respiratory
qtlotienits indicated in ouir earlier experiments appear to be more easilv explained by excess 0,
absorption than by CO.2 resorption. Ouir data indicate that neither O. inor CO. proviide anl adlequiate
inidicator of respiratioil in very dry seeds.
Literature Cited
1. CARR, D. J. 1961. Chemical influen1ce of the en-
2.
3.
4.
5.
vironment. In: Handbuch der Pflanzenphlvsiologie.
Georg Melchers, ed. Springer-Verlag, Berlin,
Germany. Band 16: 737-94.
CHING, T. M. 1961. Respiration of forage seed
in sealed cans. Agron. J. 53: 6-8.
GREEN, J. G. 1961. High temperature storage of
dry maize. Plh D. Thesis, Iowa State University,
Ames, Iowa.
HYDE, M. D. AND T. A. OXLEY. 1960. Airtiglit
storage c f damp grain. Ann. Appl. Biol. 48:
687-710
LYsYj, I. 1962. A parallel dual column system for
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Brenner, J. E. Callen, and M. D. Weiss, eds.
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6. MAYER, A. M. AND A. POLJAKOFF-MAYBER. 1963.
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7. MILNER, M. AND W. F. GEDDES. 1954. Respiration and heating. In: Storage of Cereal Grains
and Their Products. J. A. A.nderson and A. W.
Alcock, eds. American Assoc. of Cereal Chemists,
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8. MILTHORPE, J. AND R. N. ROBERTSON. 1948. Heating in stored wheat. I. Respiration of dry grain,
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9. OXLEY, T. A. AND J. D. JONES. 1944,. Apparenit
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fungal mnvceliuim beneath the epiidermis. Nature
154: 826-27.
10. RAGAI, H. AND W. E. Loomis. 1954. Respiration
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respiration and storage behavior of soybeans.
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12. SIMPSON, D. M. 1953. Cottonseed storage in various gases under controlled temperature and moisture. Tenn. Univ. Agr. Expt. Sta. Bull. 228.
13. STRUVE. W. M. 1958. Drying and germinabilitv
of maize. Ph1.D. Thesis, ToN-a State U'iiversitv,
A,lmes, Iowa.
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