Plant & Cell Physiol., 1, 49—62 (1959)
SYNCHRONOUS CULTURE OF CHLORELLA
I. KINETIC ANALYSIS OF THE LIFE CYCLE OF CHLORELLA
TEMPERATURE AND LIGHT INTENSITY
YUJI MORIMURA
Institute of Applied Microbiology, University of Tokyo,
and Tokugawa Institute for Biological Research, Tokyo
(Received Nov. 7, 1959)
1. Using the technique of synchronous culture, investigations
were made of the effects of temperature and light-intensity on
cellular life cycle of Cfilorella ellipsoidea. Some improvements in the
culture technique for obtaining a good synchrony of algal growth
were described.
2. By following the changes of average cell volume and cell
number occurring during culturing, the rates of the following
processes of life cycle were determined: (i) "growth" (or the
increase in cell mass) occurring from the stage of smaller cells
(D,) to the stage of ripened cell (La), (ii) "ripening" (or process
of formation of "nuclear substances" as estimated from the
average number of daughter cells formed from single mother cell),
and (iii) " maturing and division" which leads to the full maturation of mother cells (L-cells) and their division into separate
daughter cells (D-cells).
3. " Growth" and " ripening" were found to be dependent in
light, " maturing and division" light-independent. The time required
for "growth" and "ripening" (TO) is dependent on temperature
but independent of light intensity, the onset of " maturing and
division" occurring at the same time (TD) of culturing under varied
light intensities. The average cell volume at this stage (Lj),
however, was found to be markedly modified by light intensity;
larger with higher temperatures (see Fig. 4).
4. Changes in incubation temperature (under the condition of
saturating light intensities) were found to affect the life cycle in
the following way: (i) The time of onset of "maturing and division" (TD), varies markedly with culturing temperature; earlier
at higher temperatures, (ii) The average cell volume at this
stage also depends on temperature ; smaller at higher temperatures.
5. The average number of daughter cells («) emerging from
single mother' cells, was found to be uninfluenced by culturing
References p. 61
49
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ELLIPSOIDEA AS AFFECTED BY CHANGES OF
Y. MORIMURA
50
Vol. 1 (1959)
During the past several years we have been studying, using the
technique of synchronous culture, various aspects of the physiological and
biochemical events occurring in the life cycle of Chlorella (1-9). In
earlier phases of our studies, two stages of cells, the "dark cells" and
the "light cells", were distinguished as showing essentially different
metabolic features. Later studies (3-9) have revealed that both in dark
and light cells there are different developmental stages which may be
discriminated from each other according to their physiological and biochemical characteristics. The cyclic
sequence of the changes which we now
picture as constituting the process of
algal growth is represented schematically in Fig. 1. In this scheme, the
white arrows indicate the light processes, while the black arrows show
the processes occurring independently of light. Sizes of circles correspond approximately to the relative
sizes of cells and the extent of darkening in dicates the relative photosynthetic activity. The characteristics
and definitions of the successive dev
elopmental stages are as follows :
Fig. 1. Schematic presentation of
(1) "Nascent dark cells" (Dn),
life cycle of Chlorella. Explanation in
text.
which are the young cells newly
produced from mother light cells.
(2) "Active dark cells" (D»), the most photosynthetically active
cells, which are derived from Dn when illuminated.
(3) Cells of transient stage between dark and light cells (D-L).
(4) Unripened light cells (Li), which are large in size, but not yet
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temperature; (4.0-4.1 under the conditions of the present study).
It was found that the division number n is remarkably varied by
changing the light intensity in the "growth" and "ripening"
phases; 2.0 at 1 kilolui, 3.7 at 5 kilolux, 4.2 at saturating light
intensities (10 and 25 kilolux). This finding was explained by
assuming a light-dependent formation of "nuclear substances"
during the "growth" and "ripening" phases, the quantity of the
substances in the cell at L3 stage determining the division number.
6. The experimental data were analyzed reaction kinetically,
the rate constants and other characteristics of the reactions constituting the processes of life cycle were determined, and values
for the apparent activation energy for each reaction were computed.
The reactions were discussed with special reference to their relationship with photosynthetic process was discussed.
SYNCHRONOUS CULTURE OF CHLORELLA
I
51
METHODS AND GENERAL REMARKS
The experimental organism used was Chlorella ellipsoidea. The culture methods, which were, in principle, the same as those reported previously, have been improved in some points to obtain a better synchrony
of cell growth. To obtain the starting D»-cells of as uniform in size and
properties as possible, their pieculture has to be effected under constant
conditions, using inocula having as constant a pre-history as possible.
The culture was therefore performed in three steps, namely: (i) culturing of inoculum for the pre-culture, (ii) pre-culturing of D.-cells, and
(iii) the synchronous culture of the main experiment. For all these a
culture medium of the following composition was used: per liter, 5.0g
KNOs; 2.5 g MgSO*-7H2O; 1.25 g KH2PO1; 0.0028 g FeSCVTEbO; and l m l
of ARNON'S As-solution (containing: B, Mn, Zn, Cu and Mo) {10).
Culture of Inoculum for Pre-culture
Culture solution was dispensed in 50-ml-quantities in small, oblong
flat flasks {11), and after being sterilized by autoclaving, inoculated with
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ripe enough to perform cell division when incubated in the dark.
(5) Half-ripened light cells (La), which can only partially divide when
kept in the dark.
(6) Ripened light cells (Ls), which can divide completely when kept
in the dark.
(7) Fully mattered light cells fU), which are at the stage immediately
prior to cell division.
For technical reasons, most of our experiments of synchronous culture are started from the Da-cell stage. It should be noted that, when
the life cycle is in operation under continuous illumination, D n -cells only
rarely emerge as such from the mother cells; instead, they grow inside
the mother cells to some extent and emerge from the latter in the form
of D»-cells, or some of them even in the form of D-L-cells. Whether
inside or outside the mother cells, increase in the cell mass occurs from
the D n -cell stage and ceases somewhere between the stages of L2 and L3
cells. The phase from D a to L2 (see Fig. 1) may, therefore, be called the
"growing phase". The process of ripening, which begins somewhat
later than the growth process, continues, gradually increasing in speed,
until the Lt-cell stage. Since the change from L3 to L< occurs independently of light (3, 5), it seems pertinent to designate it by a specific
term, the "maturing phase", in distinction to the light-requiring "ripening phase". After the completion of maturity (Li-stage), the cells divide
into a number of D-cells, which process may be called the '' dividing phase''.
The purpose of the present study is to investigate whether and in
what manner these phases of cell development are affected by changes of
temperature and light intensity.
52
Y. MORIMURA
Vol. 1 (1959)
a pure algal culture taken from a 2- to 4-week old slant culture which
had been preserved at 20°-25° C under illumination with daylight fluorescent lamps, about 5 kilolux in intensity. The cultures in these flasks
were kept at 25° C under illumination with daylight fluorescent lamps of
10 kilolux in intensity, and with constant aeration of air containing 5%"
CO2.
Synchronous Culture
The D.,-cells obtained as above were suspended in 500 ml each of culture solution which were contained in medium-sized oblong flasks. The
initial cell concentration was about 7x10* cells or 0.15 ml packed cell
volume per liter. The flasks were placed in a water-thermostat and illuminated with incandescent lamps with constant bubbling of C02-enriqhed air. through the cell suspension.. At intervals, measurements were
made of the cell number (using a hemacytometer), and of the statistical
distribution of cell diameter (using an ocular micrometer which permitted detection of a 0.3 fi difference in cell diameter). For each sample of
growing algal suspension the diameters of 200 individual cells were
measured, and the average cell volume (v) was calculated according to
the formula V=0.524XIW*^I 3 /2' J J, in which ni means the number of cells
having a diameter of dt microns.
A typical example of the change in cell size occurring during the
synchronous culture is illustrated in Fig. 2, in which the data obtained
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Pre-culture and Separatum of D
After 7-8 days, 25 ml lots of inoculum culture were taken out with a
sterilized pipette and poured into a medium-sized oblong flasks each containing 500 ml of the sterilized culture solution [11). The culture was
kept at 25° C under illumination with an incandescent lamp and with
constant aeration of CO2-enriched air. For the first 4-5 days the light
intensity was kept at 10 kilolux, and then reduced to 0.8 kilolux. Culturing under the lower light intensity lasted for 3-4 days, by which time the
algal population density attained the level of about 10 ml packed cell
volume per liter, and more than 90% of the total population were found
to be in the form of D»-cells having diameters ranging from 2.5 to 3.5
microns. This population was subjected to fractional centrifugation to
select the D.-cells of uniform size. For this purpose, the culture was
diluted 10-20-fold with fresh culture solution, and then centrifuged at
1200 # for 5-6 minutes to discard larger and heavier cells. The supernatant cell suspension was further centrifuged at 1,200 # for 12-15
minutes; this time to remove the smaller cells remaining in the supernatant. The precipitated cells, of which more than 80 per cent were
3.0 microns in diameter (the size range of the total population 2.7 to 3.3
microns in diameter), were used as the starting material of the synchronous
culture.
SYNCHRONOUS CULTURE OF CHLORELLA
f
3
4
I
53
3
40
ft
r\
33
Jl .
30
fl
-
LU
I
I-
28
40
a.
O
a.
Z
•o o
K
g
12
40
0
Z
1
3
4
5
6
**"
7
CELL DIAMETER IN U.
Fig. 2. Distribution of cell diameter
showing transformation of cells in
life cycle. (Temperature: 22°C,
light intensity: 10 kilolux)
Fig. 3. Changes in average cell volume
and cell number through life cycle
(data from the same experiment as
presented in Fig. 2).
at several representative stages of cell development are reproduced. Such
measurements were made successively at different stages during synchronous culture, and the average cell volume at each stage was calculated
on the basis of the results obtained. In Fig. 3 are reproduced the course
of changes in average cell volume (v) and cell number which were
measured in parallel. In comparing the data of Fig. 3 with the scheme
of the life cycle presented in Fig. 1, the following points must be borne
in mind. As is apparent from the data shown in Fig. 2, the polygons
representing the statistical distribution of cell size remain single-peaked
during the period from the starting D,-cells to the stage of La-cells,
where the v-curve attains its maximum (see Fig. 3). After that, however,
the polygons split into two, one representing the light cells (Ls and/or L.),
and the other the daughter D.-cells. The polygons become single again
when all L-cells complete their division into D-cells. As may be seen
from Fig. 3, the cell number remains unchanged during the period from
the starting D.-cells to the stage of Ls-cells, but increases thereafter to
attain eventually a definite level when cell division is completed. Experimentally, the time of beginning of cell division (at Ls) and the time of
its completion (at D&- and Dn-stages in the light and dark, respectively;
see below) can be accurately determined by following the distribution of
Rifsrencss p. 61
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a.
54
Y. MORIMURA
Vol. 1 (1959)
RESULTS
Effect of Light Intensity on Life Cycle {at a Constant Temperature, 16° C)
Keeping the temperature constant at 16° C, the effect of light intensity upon the process of the life io«
cycle was investigated. The reCELL NUMBER
PER LITER
sults obtained at four different
light intensities (10, 5, 2.5, 1.0
*
and 0.4 kilolux) are reproduced
in Fig. 4. (Experiments were
"
oTtBT. A " "
also performed with 25 kilolux
light, which gave results quite
_i'
AVERAGE
identical with those for 10 kilolux
'« CELL
VOLUME
light.) In Table I are listed
,„
the data obtained from this experiment.
The following interesting
"
facts emerge from these results :
«
(1) As was expected, the
K
initial rate of growth was higher
c
at higher light intensities, but
H
irrespective of the difference in
Fig. 4. Effect of varied light intensities
the growth rate, the incipient cell
O n life cycle (at 16° C).
References p. 61
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cell size and the cell number.
Let us denote in the following,
by TO : the time elapsing from the start of synchronous culture to
the stage of Ls-cells (the maximum of the v-curve),
and
by TD : the time elapsing from the stage of L3-cells to the stage of
completion of cell division.
By and large, TO may be regarded as the time of "growth" and
"ripening", while TD represents the time required for "maturing and
division" (see Fig. 1), the latter two processes being difficult to separate
by the experimental arrangements adopted in the present study.
Measurement of the increase in cell number during the period of TD
allows us to determine the average number of daughter cells derived
from single L-cells. This number, which has been called the "division
number" and denoted by n (3, 5), is important since it has been shown
to bear a close proportionality to the quantity of "nuclear substances"
formed during the maturing process of L-cells (see below). As will be
shown later, well synchronized cultures usually give an w-value of 4
when culturing is run under saturating light intensity, indicating that
under such conditions, the content of "nuclear substances" in matured
L-cells is 4 times that in D-cells.
SYNCHRONOUS CULTURE OF CHLORELLA
I
55
TABLE I
Effect of Light Intensity on Life Cycle
Temperature : 16° C; average cell volume and cell number at start of
culturing: 15.2 /*' and 6.12 xlO 9 per liter, respectively.
25
10
5
2.5
1.0
Average cell
volume (/j»)
at L8
126
126
98
77
43
Time (hr) from
stage D» to Ls
TO
43
;,
//
II
II
Time (hr) from
stage Lg to D»
TD
24
;/
;/
//
//
Division
number
n
4.2
4.2
3.7
3.1
2.0
division (Li) occurred simultaneously at all light intensities except at 0.4
kilolux, in which case the cells continued to grow very slowly without
dividing during the whole course of the experiment. The value of TO was
thus independent of light intensity, while the volume of Ls-cells at the
stage of incipient cell division was greater at higher light intensities.
(2) The value of TD was also found to be independent of the light
intensity, indicating that the process of " m a t u r i n g and division" is a
light independent process.
(3) The division number (n) was largely dependent on the light intensity, being larger at higher light intensities.
Effect of Temperature on Life Cycle {under Saturating Light Intensity)
Entirely different phenomena were observed when the temperature
was varied, keeping the light intensity constant at a saturating level (10
kilolux). The temperatures applied were 25°, 16° and 9° C, and the results
obtained a r e presented in Fig. 5 and Table II.
TABLE II
Effect of Temperature on Lifa Cycle under the Condition of Light-Saturation
Light intensity applied: 10 kilolux, average cell volume and cell number
at start of culturing: 15.7 p.3 and 6.98x109 per liter, respectively.
Tom , r , t , , r .
(Q
25°
16°
9°
Average cell
volume (^S)
at Ls
Time (hr) from
stage D, to L s
Time (hr) from
stage L 3 to D»
TO
TD
Division
number
n
88
115
137
26
56
195
10
19
132
4.1
4.1
4.0
The facts revealed by this experiment may be summarized as follows :
(1) Both the rate of growth and the rate of "maturing and division"
were strongly temperature-dependent, both being higher at higher temperatures.
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Light
intensity
(klux)
56
Y. MORIMURA
40
60
80
" MO
MO
110
2O0
220 "lOO
300
320
3*0
HOURS
Fig. 5. Effect of temperature on life cycle (under the condition
of light saturation 10 kilolux).
(2) The higher the temperature, the earlier was the onset of the
division of L-cells; however, the average size of the cells at this stage
was smaller at higher temperatures.
(3) Despite the marked temperature effect on TO and TD, the division
number remained constant (4.0-4.1) at different temperatures.
From the observations made in the two sets of experiments described
above, we learn that the size of the cells does not necessarily reflect the
degree of ripening, and that process of division can occur, to some extent
independent of the degree of maturity, as reflected in the variation of
the n-value observed at different light intensities.
DISCUSSION
To explain these phenomena we have to assume at least four processes occurring—more or less independently of each other—in the life
cycle of Chlorella.
(1) First, there is a process, by which the cell volume (v) increases.
This process, which no doubt reflects the increase in cell material and
hence; involves the photosynthetic process as its basis, can be measured
by following the increase in average cell volume occurring from the
starting D.-stage to the Ls-stage1. Let us denote the average cell volumes
1
It has been shown that the packed cell volume (in ml) is in a ratio of 1 : 0.256
to the dry weight (in g) of cells. This ratio varied only in a narrow range between
0.252 and 0.263 without showing any systematic change according to the stage of
cell development ( i ) .
References p. 61
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*O
Vol. 1 (1959)
SYNCHRONOUS CULTURE OF CHLORELLA
I
.
57
at these two stages by vo and vc, respectively. Apparently, this process
occurs in the manner of an exponential function. If the rate constant
of this process is denoted by kT, we may write:
In—=k T t
1)
Vo
In—=k T r 0
vo
or
kT = —In—
Tc
2)
Vo
(2) Secondly, there is the process of "ripening", by which preparation is gradually made in the cells for the formation of daughter
cells. The essential substances formed during this phase has been shown
to be "nuclear substances" (reported by deoxyribonucleic acid, DNA)
as revealed by chemical analysis (3), as well as by the staining technique (9).
At will be natural to assume that the quantity of these substances
accumulated in the cell at the end of the maturing stage will determine
the division number n, which can be readily estimated through microscopic observation. If we denote the quantity (per cell) of the "nuclear
substances" at the D»- and Ls-stage by No and Nc, respectively, we
may write :
Assuming an exponential formation of these substances, we may
write :
ln-|p = k»t
N
A
and
or
1
N
4)
1
ln-r—-=k a ro
No
kn = — I n n
5)
Tc
where N is the quantity of the "nuclear substances"; kn is the rate
constant.
(3) Thirdly, it is further assumed that there is light-dependent
formation of some substance (s), inducing the onset of division (Ls- LiDa), or switching the process of "ripening" to "maturing and division".
This principle will be named "switching factor" and denoted as S,
although its nature is still obscure. Assuming again an exponential
formation of this substance, and denoting its concentration at the D»and Ls-stages by So and Sc, respectively, we have :
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and
58
Y. MORIMURA
Vol. 1 (1959)
ln-f-=k.t
and
So
1
1
or
6)
=k,Te
,
S
<=
7)
ko ==
8)
Since vo, v., n, TO and TD are known quantities, we can determine
the values of kT, kn and kD from Eqs. (2), (5) and (8), respectively.
From Eqs. (5) and (7), we have
9)
assuming that Sc/So is a constant independent of temperature and light
intensity, we can investigate the trend of change of k,/kn under different conditions, since n is a quantity readily estimated by microscopic
counting.
By substituting the values given in Table I in the above equations,
we obtain values which are summarized in Table HI.
TABLE III
Effect of Light Intensity on Rates of Various Processes of Life
Cycle, as Calculated from Data Given in Table I
Temperature : 16° C
Light
intensity
(klux)
25
10
5.0
2.5
1.0
Growth
Ripening
kT
kn
(1/hr)
(1/hr)
0.049
0.049
0.044
0.038
0.024
0.033
0.033
0.030
0.026
0.016
Maturing &
division
k D (1/hr)
0.042
n
n
a
a
Formation
of inducer
kn
kT
0.67
0.67
0.68
0.68
0.67
kn
k.*
const.
:,
increases
* Assumption is made in this calculation that S C /SQ is independent of light intensity.
References p. 61
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k.=—\n~
Te
S>0
where kg is the rate constant. Although we have no means of determining the value of Sc/so, it must be reasonable to assume that this ratio,
which represents the critical level of S inducing the "maturing and
division", is independent of both temperature and light intensity.
(4) Fourthly, the process of "maturing and division" ensues. The
rate of this process (kD), may be defined by the rate constant kD : where
TD is the time from Ls to D».
59
SYNCHRONOUS CULTURE OF CHLORELLA I
It is apparent that both kT and kn are markedly light-dependent,
while kD and k, are not affected by changes in light intensity. Of interest is the fact that the ratio kn/kT remains constant at different light
intensities. Fig. 6 shows values of kT
and kn as a function of light intensity.
For comparison, the values for the
rate of photosynthesis (kp) obtained
in an earlier experiment (I) are also
given in the same figure.2 As may be
seen, the three k-values show a similar
dependency on light intensity, all
being saturated at 10 kilolux (at 16° C).
KILOLUX
These results suggest that "growth"
(kT) and "ripening" (kn) are photoFig. 6. Dependency upon light intensity of "growth" and " ripenchemically related to the process of
i n g " as compared with that of
photosynthesis.
photosynthesis.
(Temperature:
The light dependency in these
16° C).
cases may be expressed by the following relations:
1O,-,»TH[|I8
kT=-
al+kp
akyl
al+kv
kn =
where I is the light intensity, kp, kv, and kN (with capital subscripts)
are the light-saturated values of kP> kT and kn, respectively, and a the
value of dk/dl at sufficiently low light intensities.
The value of a seems to be the same for all the above equations,
while kP, kv and kN, which represent the rate constants of the "dark
reactions" involved in respective processes, are apparently different
from each other.
We shall now turn to a consideration of the temperature-dependency
of the rates of these "dark reactions" as well as of kD and k. which
have been shown to be also light-independent (dark reactions). The
data presented in Fig. 5 and Table II are those obtained under the cona These values were calculated from the results of our earlier experiments (i)
according to the formula: k p = t
where Wi and W, are the weights of
a
— ti
Wj
organic photosynthate (CHOH) formed at the times t, and t a , respectively, measured
in terms of hours.
References p. 61
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kP=
(t,)
60
Vol. 1 (1959)
Y. MORIMURA
dition of light-saturation (10 kilolux). By applying these data to the
equations describe above, we calculated various k-values at different
temperatures. The results obtained are summarized in Table IV.
TABLE IV
Effect of Temperature on Rates of Various Dark Reactions in Life
Cycle, as Calculated from Data Given in Table II
Growth
k,
(1/hr)
25°
16°
9°
0.066
0.036
0.011
5
Ripening Maturing & Formation of
division
inducer*
kN
(1/hr)
kD (1/hr)
ks (1/hr)
0.054
0.025
0.007
0.10
0.053
0.008
I
kN
kT
ks*
kN
0.82
0.71
0.64
const.
decreases
In this calculation the assumption is made that Sc/So is independent of temperature.
These results clearly show that all the rate constants (kv, kN, kD
and kB) are temperature-dependent, being smaller at lower temperatures. The fact that the ratio kN/kv changes with temperature indicates
that the process of ripening, although photochemically related to the
process of growth as mentioned above, involves certain dark reactions
not involved in the latter process. On the other hand, the ratio
ks/kN is independent of temperature. This fact may be interpreted
as indicating that the formation (kg) of the "switching factor" may be
a process which is, in some way,
related to the dark reactions of
the ripening process.
To compare the temperaturedependency of kv, kN and kD, the
logarithms of these values are
-I.O.
plotted against the reciprocals of
absolute temperature (Fig. 7).
For comparison, the values (kp)
for the photosynthesis, deter-2.0
mined in our earlier work are
also plotted in the same figure.
The figures given on the curves
33
34
35
3*
indicate the apparent activation
IO/T
energies in kcal calculated from
Fig. 7. Temperature dependency of photothe inclination of the curves at
synthesis, "growth" and "ripening",
the indicated temperature spans
(after light saturation) and of a "maturcompared with the dark reaction
ing and division". Figures given on
of photosynthesis. The reactions
the curves indicate apparent activation
energies in kcal calculated for respective
of growth, "ripening" and
temperature spans.
"maturing and division" were
References t>. 61
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Temp.
(C)
SYNCHRONOUS CULTURE OF CHLORELLA
I
61
This work was carried out as a part of the studies on the growth of Chlorella
directed by Prof. HIROSHI TAMIYA, to whom the writer is indebted for his guidance.
Thanks are also due to Dr. KAZUO SHIBATA for his valuable suggestions in the
theoretical part of the paper. This work aided by grants from the Rockefeller
Foundation and the Ministry of Edncation. To these bodies we extend our grateful
thanks.
REFERENCES
1l)
H. T A M I Y A , K. IWAMURA, K.
SHIBATA, E . HASE and
T. NIEHI.
1953.
Correlation between photosynthesis and light-independent metabolism in
the growth of Chlorella. Biochim. Biophys. Ada, 12, 23-40.
( 2 ) T. NIHEI, T. SASA, S. MIYACHI, K. SUZUKI and H. TAMIYA. 1954.
Change of photosynthetic activity of Chlorella cells during the course of
their normal life cycle. Arch. Mikrobiol., 21, 155-164.
( 3 ) T. IWAMURA. E. HASE, Y. MORIMURA and H. TAMIYA. 1955. Life Cycle
of the green alga Chlorella with special reference to the protein and
nucleic acid contents of cell in successive formative stages. Aim. Acad.
Sdsnt. Fenrricae, A. II. 60, 89-102.
(4) T. NlHEI. 1955. A phosphorylative process, accompanied by photochemical
liberation of oxygen, occurring at the stage of nuclear division in Chlorella
cells, I. / Biochem., 42, 245-256.
( 5 ) T. IWAMURA. 1955. Change of nucleic acid content in Chlorella cells
during the course of their life-cycle. / Biochsm., 42, 575-589.
(6) E. HASE, Y. MORIMURA and H. TAMIYA. 1957. Some data on the
growth physiology of Chlorella studied by the technique of synchronous
culture. Arch. Biochem. Biophys., 69, 149-165.
( 7 ) T. NlHEI. 1957. A phosphorylative process, accompanied by photochemical
liberation of oxygen, occuring at the stage of nuclear division in Chlorella cells IT. / Biochem., 44, 389-396.
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found to require greater activation energies, as especially at lower temperatures. Most pronounced was the great activation energy (as high
.as 43kcal) encountered in the process of "maturing and division" tit
lower temperatures. Since the ratio ks/ku has been deduced to be independent of temperature (see Table IV), the activation energy for k s
must be regarded as being the same as that for k*.
In their earlier studies on Chlorella, TAMIYA et al. (i) made a kinetic
analysis of the over-all growth process, assuming that the growth process
is composed of two main steps, a light-dependent step, photosynthesis,
and light-independent formative metabolism, by which the primary photosynthetic products are converted into cell material. They showed that
the light-independent process had an activation energy (49kcal at lower
temperature) markedly greater than that of light-dependent process
(15kcal at lower temperature). It may now be infered that the great
activation energy of 49kcal found on TAMIYA'S experiments pertain to
the process of "maturing and division" as defined in the present study.
62
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Vol. 1 (1959)
T. KANAZAWA. 1958. Synchronous culture of Chlorella with special
reference to the processes of assimilation of potassium, magnesium and
iron. J. Gen. Apfil. MicroUol., A, 102-107.
E. HASE, Y. MORIMURA, S. MIHARA and H. TAMIYA. 1958. The role
of sulfur in the cell division of Chlorella. Arch. AEkrobiol., 32, 87-95.
D. I. ARNON. 1938. Microelements in culture-solution experiments
with higher plants. Amer. Jour. Bat., 25, 322-325.
T. KANAZAWA, C. FUJITA, T. YUHARA and T. SASA. 1958. Mass culture
of unicellular algae using the " open circulation method ". / Gen. Appl.
Microbiol., 4, 135-152.
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Y. MORIMURA