The Quantitative Relation between
Rate of Photosynthesis and
Chlorophyll Content in
Chlorella pyrenoidosa

THE QUANTITATIVE RELATION BETWEEN RATE OF
PHOTOSYNTHESIS AND CHLOROPHYLL CONTENT IN
CHLORELLA PYRENOIDOSA

The Quantitative Relation between
Rate of Photosynthesis and
Chlorophyll Content in
Chlorella pyrenoidosa

TER VERKRIJGING VAN DEN GRAAD VAN
DOCTOR IN DE WIS- EN NATUURKUNDE AAN
DE RIJKS-UNIVERSITEIT TE UTRECHT, OP
GEZAG VAN DEN RECTOR-MAGNIFICUS Dr. J.
BOEKE, HOOGLEERAAR IN DE FACULTEIT
DER GENEESKUNDE, VOLGENS BESLUIT VAN
DEN SENAAT DER UNIVERSITEIT TEGEN DE
BEDENKINGEN VAN DE FACULTEIT DER
WIS- EN NATUURKUNDE TE VERDEDIGEN
OP MAANDAG 20 JUNI 1938, DES NAMIDDAGS
TE 3 UUR

Nu mijn studietijd door het verschijnen van dit proefschrift
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THE QUANTITATIVE RELATION BETWEEN RATE OF
PHOTOSYNTHESIS AND CHLOROPHYLL CONTENT IN
CHLORELLA PYRENOIDOSA

Influence of Magnesium on the relation between Chlorophyll
content and rate of Photosynthesis. The Experiments
of Fleischer.

Effects of the composition of the nutrient solution on the
development of Chlorella. Effects of Nitrogen supply.

Growth and Photosynthetic activity of Chlorella cells grown
in darkness and in inorganic media.

Ever since the discovery of the principles of plant metabolism
the photosynthesis of the green plants has been the object of a
great number of investigations. The results of the most impor-
tant research work have been summarized by Stiles (1925) and by
Spoehr (1926) in two monographs on photosynthesis and surveys
on the latest investigations have been given in the papers of
Gaffron and Wohl (1936) and of Emerson (1936 and 1937), to
which is referred for a review of the evolution of photosyn-
thetic research.

The photosynthesis in the green plants actually is a reduction
of carbon dioxide by means of light energy. TÎie light energy
is taken up by the chlorophyll. The carbon dioxide reduction,
however, can only be achieved in the living plant tissue;
photosynthesis by chlorophyll in vitro could never be established.
Which factor in the plant, besides the chlorophyll, is concerned
in the photosynthesis cannot be said. It is likely that in the
living chloroplast a special structure of the chlorophyll molecules
is present, which is necessary for the photosynthesis and which
is disturbed by chlorophyll extraction.

If no external factor is limiting, it might be expected that the
rate of photosynthesis is proportional to the chlorophyll content
unless another factor in the plant limits the assimilation process.

It has been tried by several authors to discover a quantitative
relation between the rate of photosynthesis and the chlorophyll
content.

Plester (1912) studied the assimilation activity of the leaves
of green, yellow and variegate varieties of different plants.
Though he observed that the photosynthesis of all leaves with
lower chlorophyll content is less than that of normal leaves, the
lower assimilation rate was not proportionate to the chlorophyll
concentration. Plester's methods of photosynthesis determination
were rather crude, so his results are not conclusive.

In the well known experiments of WillstMtter and Stoll
(1918) the assimilation activity is given as the assimilation
number (= „Assimilations-Zahlquot;) which is the photosynthesis in
grams of carbon dioxide absorbed per hour per gram of chloro-
phyll. In a number of different objects widely divergent assimila-
tion numbers were found. Most striking are the high assimilation
nvimbers in aurea-varieties of Samhucus nigra and Ulmus. Also
the leaves of etiolated plants gave high assimilation numbers.

willstëtter and Stoll explained the differences in assimilation
numbers by assuming that an enzyme is playing a part in the
process and that this enzyme determines the rate of photosyn-
thesis. As the enzyme reaction is the rate determining process,
this theory was in accordance with the fact that the photosyn-
thesis has a temperature sensivity that is much higher than in
photochemical processes. Therefore it is a highly interesting
point in the work of these authors that in leaves with a high
assimilation number the temperature sensitivity decreases. This
points to a determination — in this case — of the rate of photo-
synthesis by the photochemical reaction.

Emerson (1929) repeated the experiments of willstatter and
Stoll with the unicellular alga Chlorella measuring photosyn-
thesis manometrically by the method of Warburg (1919, 1920).
For controlling the chlorophyll content, cells were cultivated in
nutrient media containing different iron concentrations. Emerson
found the assimilation nimiber for Chlorella about constant
within the cultures of one set and concludes that the photosyn-
thesis is a function of the amount of chlorophyll just as it is
of external factors too. The fact that the curves of different series
are not superimposed is considered by Emerson a matter of
minor importance. However, this is an indication that there
must be another factor in the cells limiting the photosynthesis,
or limiting the chlorophyll activity. Moreover, in Emerson's
„shorterquot; curve (his fig. 3) the assimilation numbers are not
constant as the curve does not pass through the origin.

Once more the ratio between photosynthesis and chlorophyll
content in Chlorella was investigated by Fleischer (1934). This
author supposed that the irregularities in Emerson's results
might have been caused by the fact that Emerson did not
regularly measure respiration but applied a uniform correction
for it. Fleischer found an about constant assimilation number in
cultures with normal nutrient solution and in media with graded
amounts of iron or nitrogen. So it seems indeed that the amount
of chlorophyll is a rate determining factor in photosynthesis. In
cells grown in media with a low magnesium concentration very
inconstant assimilation numbers were found.

In the experiments of Emerson and Arnold (1933) on photosyn-
thesis of Chlorella exposed to flashes of light was demonstrated
that th' light reaction and so the chlorophyll, as far as its rôle
in the light reaction is concerned, is not the rate determining
factor, as the dark reaction proved to require much more time
and therefore always limits the rate of photosynthesis. The

authors hold the view that the results indicating a constant
ratio between chlorophyll content and rate of photosynthesis
must be attributed to chance.

The experiments described in this paper have been undertaken
to investigate whether a constant ratio between the rate of
photosynthesis and the chlorophyll content really exists. If this
could be stated it would be an indication that the chlorophyll
plays a part in the limiting process of photosynthesis being the
Blackman reaction.

Furthermore, it was investigated by which factors this ratio
could be affected. The effect of low concentrations of magnesium,
found by Fleischer, seemed an interesting point. The results
obtained with cells cultivated in media with graded amounts of
magnesium have been published in a preliminary note (van Hille
1937) and moreover in Chapter IV. It was supposed that the
external conditions during the growth of the cells might have
an influence, as the results obtained in the sets of cultures not
simultaneously cultivated use to yield divergent results.

Experiments were carried out with the well known experi-
mental object Chlorella pyrenoidosa. A pure strain was secured
from the collection of Prof. Dr. E. G. Pringsheim at Prague.
This strain had been cultived in a -test-tube on a solid substrate
of unknown composition.

The algae were cultivated in Erlenmeyer flasks of 100 cm®,
covered with cotton-wool stoppers. The flasks were filled with
50 cm^ of the nutrient solution. The composition of the nutrient
solutions is always given in the concerned chapter. The nutrient
compounds were dissolved in distilled water. It proved not to
be necessary to distil the water from glass to glass. Cultures
provided with distilled water from the brass still or from the
glass still do not show any difference.

The flasks filled with the nutrient medium were sterilized for
half an hour at 100° at a pressure of 1 atmosphere, because most
of the cultures contained V^ per cent of glucose. This way of
sterilizing always proved to be sufficient. Bacterial infections

Culture media containing no glucose were sterilized at 120°
at a pressure of atmospheres for 20 minutes.

end of which had been bent into an eye with a diameter of
Ln-nbsp;t'nbsp;^ Bunsen flame, was

lasting for such a short time. The greater part of the experi-
ments, moreover, were carried out in winter

Chlorella, when cultivated with glucose, supplies thick sus-
pensions m a short time. The cells, especially in young cultures
are rather heavy, so they settle on the bottom of the culture

flasks. Experimental cultures were stirred daily in order to
distribute the cells and the nutrient compounds evenly in the
culture. Part of the cultures were cultivated under continuous
shaking. Growth was not accelerated by the shaking but the cells
showed a greater mutual conformity (see figiire 1). This had
no influence on the cell metabolism, so the shaking has no
essential effect on later experiments.

The pH of the nutrient solution was determined colorimetri-
cally by means of „Hellige's Microcomparatorquot;. The method
has the advantage that only little test solution is needed for the
pH determination. This was necessary because the same
culture was used for estunating photosynthesis several times.
The method is not accurate. In the pH range, within the reach
of two different indicators not always the same pH values are
found. The relations of the quantities of test solution and some
indicators as given in the manual proved not to be satisfactory.

Photosynthesis was determined by the manometric method
of Warburg (1919). The water-basin was heated electrically
and fitted with two stirring apparatus driven bij an electro-
motor. Temperature was kept constant by a toluene regulator
at 25,5° C.

The light of a series of incandescent lamps, placed behind
the window in the backside of the thermostat, was reflected by
a mirror in the thermostat on the bottoms of the vessels con-
taining the cell suspensions. In this way an illumination with
4000 Lux was obtained. This illumination has been used only
in a part of the experiments described in Chapter V. As a
surplus illximination was desirable in the remaining experiments
a light intensity of 12000 Lux was used. This was obtained by
fixing up a watertight tank containing a row of closely spaced
56 watt lamps in the thermostat. The top of the lamps were
at a 3 cm distance from the bottoms of the Warburg vessels.
In the last experiments a waterbasin of another construction was
used. A window was put just under the vessels, so the lamps
could be placed outside the thermostat, which has the advantage
of a more even temperature, as the heat of the lamps is less
directly transmitted to the water in the basin. By this modi-
fication the light intensity remained the same.

has an injurious effect on the cells. Especially in aged cells
and in cells cultivated at a low light intensity the chlorophyll is
destructed and the protoplasm is damaged. (Harder 1933) Most
experiments were not extended over a longer period than
halt an hour, so the injurious light effect could be neglected
Only m the experiments of Chapter VII, carried out with
old cells which were exposed to the light for a long time
and in the experiments described in Chapter VIII with cells
cultivated in darkness the injury was perceptible

In the experiments the conical vessels of Warburg with one
side-bulb and without central well were used. The capacity of
the vessels was about 20 cm».

Before the experiment the culture in question was thoroughly
shaken to get an even distribution of algae in the culture me-
dium A quantity of the cell suspension was poured sterilely in
a calibrated centrifugal tube. This quantity ran from 20 cmquot;
tor the young cultures to 0,5 cm» for cultures forming dense
suspensions. This suspension was centrifuged till the supernatant
culture liquid was quite clear. Of this liquid the pH was deter-
mined.

As a suspending fluid 7 cmquot; of Warburg's carbonate mixture
no 9 was employed. This mixture is composed of 85 parts M/10
sodium bicarbonate plus 15 parts M/10 sodium carbonate. The
Warburg vessels were filled with 7 cm» of the cell suspension
by means of pipettes.

Because the ratio between the rate of photosynthesis and
chlorophyll was investigated, always thin cell suspensions were
used, as otherwise the cells would overshade each other so the
amount of chlorophyll might not show its maximal achievement
This concentration of cells had been determined previously by
examining the photosynthesis of various quantities of algae from
one culture and by determining up to which concentration the
assimilation was proportional to the quantity of algae

When the algae in the vessels had been submerged in the
thermostat they were illuminated and shaken for an adjustment
period of 15 minutes. During this period the stop-cocks were
closed.to allow the water vapor in the gas space to get into
equihbrium with the carbonate mixture. The cell suspension
obtained the temperature of the waterbasin and during this
time the induction (Smith (1937) ) took place. At the end of the
adjustment period the stop-cocks were opened momentarily to
level the Brodie fluid in the manometer; after closing the stop-
cocks the first reading was done. The oxygen production was

followed manometrically at 5 or 10 minute intervals, for half
an hour at least, from which, according to the formula given by
Warburg, the exact amount of liberated oxygen was computed.

The computing of the respiration in all the investigations of
photosynthesis is a source of uncertainty (v. d. Paauw 1932).
When the cells contain a great quantity of assimilation products
the rate of respiration is higher than when the cells have not
been able to photosynthesize for a long time (French, Kohn,
Tang 1934).

It could be demonstrated that the respiration is less before
the determination of the photosynthesis than afterwards. It is
probable that during the photosynthesis the respiration increases.
This increase, however, does not appear from a decreased rate
of photosynthesis (cf. Chapter V). In all the experiments I
determined the respiration for half an hour immediately after
the photosynthesis.

From the moment on which the light is switched off, the
development of oxygen goes on for a short time. Five minutes
sufficed for the establisment of the equilibrum. A decrease of
the respiration during the first half hour after the photosynthesis
was not stated. In young cultures the respiration amounts to
one tenth of the photosynthesis; in older cultures the respiration
increases in proportion to the photosynthesis. When cultivating

cells without glucose the respiration is ^ and less of the photo-
synthesis in young cells.

The rate of respiration thus obtained in half an hour was
applied as a correction on the rate of photosynthesis.

After the determination of the respiration the cell suspension
used in the experiment was poured out in centrifugal tubes of
pyrex glass. The algae were centrifuged, the carbonate mixture
was decanted, the cells were boiled in methanol for a few
seconds and centrifuged again. The young cells after having
been boiled once are completely decolourized; the cell residuum
is white. In older cells, even by repeated boiling, part of the
chlorophyll cannot be extracted. This is, however, easily done
by suspending the cells in one or two drops of water. At the
second extraction with methanol also the chlorophyll of the old

cells was then completely taken up. The extracts were poured
together and methanol was added up to 10 cmquot;.

Of this chlorophyll solution the absorption was determined
at the wavelength of 6600 A by means of the spectral pyrometric
method (Ornstein 1935).

The cuvettes were made of pieces of 5 mm thick plateglass
that had been cut in a U-shape (figure 2). At both sides cover-
glasses of planeparallel glass were pasted with a special cover-
glass lac (Deckglaslack 1 a Dr. G. Grübler and Co Leipzig). For
low chlorophyll concentrations cuvettes of 1 cm gauge were

made by pasting two pieces of U-shaped plateglass together.
The cuvettes have a content of 3 and 6 cm'' respectively.

It has often been stated that a chlorophyll solution follows
the absorption laws of Beer and Lambert unless the concentration
is too high (Hubert 1935).

This was proved also in my experiments. According to these
laws for clear solutions of pigments in uncoloured media

lo = light intensity after passing through the uncoloured medium,
I = light intensity after passing through the pigment solution,
k = absorption coefficient,
c = concentration of the pigment,
d = thickness of the layer.

At the use of cuvettes of constant gauge, the concentration is
proportional to log-^.

the shape of the absorption curve is independent of the concen-
tration of the chlorophyll; so the measured absorption curves
are directly comparable (Weigert 1916).

The chlorophyll extracts of cells of different assimilatory
activity, of different age, or cultivated in different nutrient
media do not show differences beyond the experimental error.
Hence it is allowed to calculate the concentration of the chloro-
phyll from the absorption of one special wave length. It is
evident that for this purpose light of a wave-length has been
chosen of which the absorption by chlorophyll is maximal and
which is not absorbed by the carotenoids.

Of the extracted cell residuum finally the volume was deter-
mined by suspending the cells again in 5 cm' water and centrifu-
ging them during a period of minute at a uniform speed in
Tromsdorff centrifugal tubes. This method involves many ob-
jections.

a.nbsp;The cell volume does not remain constant during the photo-
synthetic experiment, but increases in almost all cases.

b.nbsp;The cell volume that is read after centrifuging is depen-
dent on the rate of settling, consequently of the specific gravity
of the cells. Cells having a high specific gravity will be quickly
centrifuged out, forming a compact centrifugate, while specifically
light cells settle slowly into a loose and voluminous sediment.
The volume of the cells determined by the Tromsdorff method
therefore is to a certain extent a reverse measure of the specific
weight of the cells. The only significance of the volume of the
cells is to have a measure for the amount of substance partici-
pating in the metabolism. To this purpose the cell weight is
hardly a better measure than the cell volume as the quantity of
cell-wall substance and store-starch has a far greater influence
on the weight than the living protoplasm.

The cell volumes are recorded only for the sake of com-
pleteness; in my opinion they do not have much value in the
calculation of the results.

The experiments described in this chapter were done with
pure cultures of Chlorella pyrenoidosa. The nutrient solution
had the following composition:

This medium was used in the experiments of Fleischer (1934)
as normal nutrient solution.

The cultures were cultivated in 100 cm» Erlenmeyer flasks,
which had been sterilized and inoculated as described in Chapter
II. Cultures that had been contaminated by bacterial infections
were left out of consideration. The Erlenmeyer flasks were
placed in a greenhouse. The light intensity was low, because
these experiments were carried out in the months November
and December 1937. In this time the sky was almost heavily
clouded throughout. The temperature in the greenhouse was
about 21°. On a few days when during a short time the sun
was shining it could rise to 28°. Under these circumstances all
cultures showed a rapid and regular growth.

Although the cultures are cultivated under equal conditions
they often show individual differences, but these are not essen-
tial. The results recorded in one table or graph always relate
to one culture.

Photosynthesis, chlorophyll content, cell volume, cm» nutrient
solution, pH, were determined by the methods, reported in
Chapter II.

Photosynthesis : mm» oxygen evolved in the light during half
an hour mm» oxygen absorbed in the dark
during an equal following period of time.

the cell quantity in mm', determined by the
Tromsdorff method, present in 1 cm® nutrient
solution.

the photosynthesis of a sample of cells divided
by its relative chlorophyll content, or in other
words, the photosynthesis of the cells per unit
of chlorophyll. This value consequently is pro-
portionate to the assimilation nxmibers (quot;Assi-
milations-Zahlquot;) according to willstatter and
Stoll (1918).

If in my work is referred to assimilation numbers, the photo-
synthesis per unit of chlorophyll is meant. The absolute value

the assimilation number of willstaxter and Stoll, but must be
multiplied by an unknown constant.

After the inoculation, the nutrient solution was still quite
clear. The first observation was made when the first green
colouring was visible.

A perusal of table 1, column 3 and figure 3a shows that the
photosynthesis makes a great progress in the beginning, reaches
a maximum and then decreases.

Photosynthesis
cm^ nutr. sol.

Chlorophyll
Chlorophyll

cmquot; nutr. sol.

Cell volume
cmquot; nutr. sol.

Photosynthesis

■quot;ig- ■'»o- Chlorophyll course of time of culture with normal nutrient
solution, cultivated in diffuse daylight.

The chlorophyll content (table 1 column 4) increases first,
reaches a maximum some days later than the photosynthetic
maximum and then decreases too (for discussion see Chapter
VIII, page 748).

The cellvolume increases continuously during the time of
observation; the increase is greatest in the beginning.

As table 1, column 6 and figure 3b show, the assimilation
numbers are not constant, but continuously decrease. This means
that the chlorophyll content increases faster than the photo-
synthesis. Even during the time of unchecked development in
the culture, the assimilation numbers become lower.

As in the first experiment the highest assimilation number is
found, it is possible that a still higher assimilation number might
have been found if an experiment could have been done earlier.
From the shape of the curve it cannot be concluded where the
maximal value of the assimilation number lies.

It was examined which factors in the nutrient solution might
unfavourably affect the photosynthesis in order to explain the
decrease of the assimilation nxmibers.

It must be born in mind that the photosynthesis of the cells
was not determined in the nutrient medium but in the carbonate
mixture, at a pH of 8,5. If the pH in the nutrient solution
should have an injurious influence on the photosynthesis a
recovery of photosynthesis in the carbonate mixture must be
expected. The photosynthesis, however, does not recover in the
carbonate mixture, but remains constant during many hours.

Emerson and Green (1938) observed that the photosynthesis
of Chlorella pyrenoidosa was not influenced by media of a pH
between 5 and 8,5. It is, therefore, improbable that the pH would

II.nbsp;During the growth of the culture, the inorganic substances
and glucose are consumed, so the concentration of the nutrient
solution decreases. Because rapid growth takes place in the be-
ginning, the concentration of the nutrient compounds must also
decrease sharply in the beginning. This is in accordance with
the fact that during the first experiments the assimilation num-
bers show the greatest decline. If the decrease of the assimilation
nimibers should be a direct reaction on the concentration of
the nutrient compounds, higher assimilation nxmibers ought to be
found in a more concentrated nutrient solution, respectively lower
assimilation numbers in a less concentrated nutrient solution.

4 b. Chlorophyll course of tune of culture with 2 X concen-
tration of the nutrient solution, cultivated in diffuse daylight.

This was tested as follows: the nutrient components were given
in the same mutual proportion, but to twice and to half the
concentration respectively. The results of the measurements of
two cultures that were provided with these modified nutrient
solutions aïe reproduced in table 2 and 3, moreover in figure
4a and 4b, and figure 5a and 5b.

These cultures have been inoculated on the same day as the
culture of table 1 and have grown under the same conditions
of light intensity and temperature.

From the graphs it is evident that the photosynthesis and the
chlorophyll content are appreciably influenced by the concen-
tration of the nutrient solution. The maximal values attained
are proportionate to the concentration of the nutrient solution.

The maximal values of the assimilation nimibers (figure 4b
and 5b), however, are not affected by the concentration of the
nutrient solution; the curves of figure 3b, 4b and 5b are almost
identical. The maximal values of all three cultures are almost
on the same level.

So, the level of the assimilation numbers is not directly in-
fluenced by the concentration of the nutrient medium. Otherwise,
the highest assimilation number of the culture of table 3 should
agree with the assimilation number of the culture from table 2,
when this one had exhausted % of its nutrient solution.

From this, it appears that the rising part of the curves coincide;
consequently the photosynthesis in the first period of growth
of the cells in the culture is independent of the concentration of
the nutrient solution. Pearsall and Loose (1937) demonstrate,
in their ample investigations on the growth of Chlorella vulgaris
in pure culture, that in the first period after inoculation the
growth gets on according to an exponential curve.

This is also the case for the rapid growth of Chlorella pyrenoi-
dosa, if photosynthesis is taken as a standard for growth.

From figure 6 appears that the exponent is not influenced by
the concentration of the nutrient solution, until the nutrient
solution (or one of its components) becomes limiting factor.

During the exponential growth, the chlorophyll curves too
almost coincide. Therefore, as the concentration of the nutrient
solution proves not to affect the rate of the chlorophyll for-
mation, nor the rate of photosynthesis, but since in course of
time, it appears as limiting factor, it is not to be expected, that
the assimilation numbers, being the quotient of photosynthesis
and chlorophyll content, will be influenced by the concentration
of the nutrient solution, during the exponential growth.

Because the nutrient solution does affect the duration of the
period of the exponential growth, the curve of the assimilation
numbers must drop off less sharply in the higher concentrations
than in the lower concentrations. This is apparent from com-
parison of figure 3b, 4b and 5b.

As the highest assimilation numbers were found in the ex-
periments with the youngest cultures, I tried to accelerate the
growth, in order to be able to carry out the first experiment
within fewer days after inoculation.

In order to secure faster growing cultures, the cells were
cultivated at a temperature of 27° and in the constant light of
a Philips 500 Watt bulb, at a distance of 90 cm from the cul-
tures. To obtain a regular growth the cultures were continuously
shaken. The frequency of shaking amounted to 90 to the minute
with a amplitude of 1,5 cm.

The results obtained with three cultures provided with three
different concentrations of nutrient solution are recorded in
table 4, 5 and 6, moreover illustrated by the graphs in figure
7, 8 and 9.

Under these different conditions, however, it is impossible
to carry out the first experiment earlier after the inoculation.

When the culture had reached this stage of development the
growth was more rapid than in the cultures in the greenhouse
so the maximal values of chlorophyll and photosynthesis were
attamed within fewer days.

Comparing the data of figure 3 and figure 7, respectively
tablée 1 and table 4 it appears that the constant hight and the
higher temperature do not have an unfavourable influence on

the formation of the chlorophyll, as the maximal chlorophyll
content of the culture is even higher under these circumstances.

The photosynthesis, however, is unfavourably affected. The
maximal photosynthetic value is lower and the decrease is
sharper than in the cultures in the greenhouse.

Also the assimilation numbers are lower. The maximal values
of the assimilation numbers, found in the first experiment with
each culture, are ± 50 per cent of the maximal assimilation
nimibers of the cultures in the greenhouse.

Further it is found again that the maximal values of the
assimilation numbers cannot be influenced by the concentration
of the nutrient solution.

Since the assimilation numbers decrease from the moment
that the first greening is visible, it can be understood why the
authors, who investigated the relation between photosynthesis
and chlorophyll content, always found irregular values.
WiLLSTäTTER and Stoll (1918) too found the highest assimi-
lation numbers with leaves of higher plants when these were
just unfolded.

Emerson (1929) found smaller assimilation numbers with higher
chlorophyll concentrations in Chlorella vulgaris and moreover
a great difference between the assimilation numbers of two
different series. This is understandable, when the different series
were not cultivated under the same circumstances or when
cultures of different age were used.

Also Fleischer (1934) found that the assimilation numbers
of the cultures of one series gave a closer agreement than those
of different series. The irregularities in the magnesium series
in the experiments of Fleischer will be discussed in Chapter IV.

So, the highest photosynthetic activity both in higher plants
and in Chlorella is found with very young organisms. There-
fore, if Chlorella cultures are used for experiments when they
have formed dark green and thick cell suspensions, the assimi-
lation numbers have already decreased to the half or less!

It may be that each of these processes must be devided
into part processes. Since, the various theories on photosynthesis
do not agree on the character of these processes, and the ex-

periments are not conclusive, I will stick to the general division,
mentioned above.

A.nbsp;The light reaction falls off, so an amount of the chlo-
rophyll becomes inactive.

B.nbsp;The dark chemical or Blackman reaction declines, so du-
ring the growth of the culture an ever smaller part of the
radiant energy absorbed in the light reaction is converted further
in the Blackman reaction.

ad A. Several theories exist on the function of the chlorophyll
in the photosynthetic process. These theories can be classified
in two groups.

The chlorophyll absorbs the energy of the light and transfers
it upon some other substance.

2.nbsp;Chlorophyll is photochemical agent and enters into reaction
with carbon dioxide or an hydrated form of carbon dioxide.

If the decline of the assimilation numbers should be ascribed
to the function of chlorophyll, so it must be assumed that in
the first case, chlorophyll is unable to transfer the absorbed
energy. If chlorophyll is photo-inactive, there is no fluorescence
for instance in colloidal solution. Photo-active chlorophyll ever
yields a part of its energy as fluorescence. Though no quantitative
fluorescence measurements were done, the fluorescence was
still clearly visible in aged cultures. This appears to me to be
an argument that it is improbable, that chlorophyll would not
be able to transfer the absorbed light energy.

In the second case it might be assumed that chlorpohyll
turns inactive by a chemical reaction. I tried to demonstrate
a chemical transformation of the chlorophyll by determination
of the absorption spectra of chlorophyll extracted from algae
of very divergent photosynthetic activity. This failed. The
spectra all agree within the experimental error.

Of some cultures of different photosynthetic activity the tem-
-perature sensitivity was determined. As appears from the follow-
ing data the temperature sensitivity remains fairly constant in
spite of differences in cultivation and age. The temperature
sensitivity is given as the Qj (20,5° C.—25,5° C.) being the
quotient of the rates of photosynthesis at 25,5° C. and 20,5° C.

From these data it is obvious that the photosynthesis is not
limited in older cells by:

as the cell walls of the older cells become thicker (cf. Pearsall
and Loose 1937),

2 : by the photochemical process (which would be the case
if the chlorophyll became inactive and so the quantity of photo-
active chlorophyll would limit the rate of photosynthesis),
for the temperature sensitivity of diffusion is considerably
lower and that of photochemical processes = 1.

Moreover, it will be proved in the experiments described in
Chapter VIII that the opinion that chlorophyll is inactivated by
photosynthesis itself caimot be sustained.

Consequently there is no reason to suppose that chlorophyll
is the cause of the decline of the assimilation numbers.

ad B. The decline of the assimilation numbers, therefore, must
originate from limiting of the photosynthesis by the Blackman
reaction.

Usually, the Blackman reaction is considered as an enzymatic
reaction, on account of its temperature sensitivity.

So the decrease of the assimilation numbers could be explained
by presuming that the formation of the Blackman enzyme is a
process, that is more sensitive to the circumstances in the
culture during the growth of the algae than the chlorophyll for-
mation. Assuming this theory there must exist, besides the
enzyme formation, an inactivation of the enzyme. This is apparent

of the photosynthetic optimum. Hence it is also possible that
the enzyme is formed in a constant ratio to the chlorophyll but
that during the exponential growth of the algae it already turns
inactive.

As a consequence the assimilation numbers must not be con-
sidered as a measure for the chlorophyll activity, but as a
measure for the Blackman reaction, for the assimilation number
indicates which part of the absorbed light energy is converted
further into chemical energy in the Blackman reaction.

by which the photosynthetic activity can be measured, to prefer
to the photosynthesis per cell volume, per cell number, per dry
weight or per fresh weight.

How the decline of the assimilation numbers must be ex-
plained will be reported in the subsequent chapters.

Influence of Magnesium on the relation between CUorophyll
content and rate of Photosynthesis, The Experiments of Fleischer.

Fleischer (1934) cultivated chlorotic Chlorella cells by defi-
ciency of iron, nitrogen or magnesium, in order to investigate the
ratio photosynthesis-chlorophyll. With cells chlorotic by defi-
ciency of iron or nitrogen, Fleischer found the rate of photo-
synthesis proportional to the chlorophyll content.

The behaviour of the Chlorella cells in nutrient solution with
graded quantities of magnesium, however, was quite different.
No definite proportionality between the chlorophyll amount and
rate of photosynthesis was found. Fleischer describes his results:

quot;At low concentrations of magnesium the rate of photosynthesis
is relatively independent of the chlorophyll content. As the
magnesium concentration is increased, the rate of photosynthesis
rises rapidly and during the rise is relatively independent of
the chlorophyll content. Eventually the rate of photosynthesis
reaches the value indicated by full nutrient determinations
and at this point the relation between the rate of photo-
synthesis and chlorophyll content is comparable to the relation
existing in iron and nitrogen graphs for similar values.quot;

Fleischer explains the magnesium effect by assimiing that
magnesium plays a part in the process of photosynthesis in
addition to its effect upon the chlorophyll content.

Though this may be true, it is not a sufficient explanation of
the phenomenon, because the same may be said of nitrogen.
It would be more probable that lack of iron, which is no con-
stituent part of the chlorophyll molecule, would disturb the pro-
portionality of photosynthesis and chlorophyll.

Fleischer found cells being highly chlorotic but having an
almost normal assimilation number. Also he found cells with

a normal chlorophyll content having an abnormal low assi-
milation nimiber. However, Fleischer gives no data of the
magnesium concentrations which gave rise to these phenomena.

In the nutrient solution, given in Chapter III, 1,42 g sodium
sulfate was substituted for 2,46 g magnesium sulfate.

The magnesiimi standard solution contained 1 mg magnesium
per cm®. I used magnesium sulfate in the standard solution,
because magnesium chloride, used by Fleischer, on account
of its hygroscopical quality is less suited for quantitative work.

Varying amounts of the magnesium standard solution were
added to Erlenmeyer flasks of 100 cm», containing 50 cm'
nutrient solution. Fleischer added varying amounts of the
standard solution to the cultures so as to give concentrations
ranging from 0,02 to 2,0 parts per million, being in corres-
pondence with 0,1 cm» — 0,001 cm» of the standard solution
to 50 cm» of the nutrient solution.

The normal nutrient solution (cf. page 691) contains 2,46 g
magnesium sulfate per litre, or 25 mg magnesium per 50 cm».

As in Fleischer's experiments the cultures were grown in
constant light. Growth must have been a little more rapid in
Fleischer's experiments than in mine. With his cultures it
was possible to carry out experiments three days after inoculation
already. My first series were inoculated as described in Chapter
II. With these series experiments could not be done before
the sixth day. In order to acquire a more rapid growth, I
inoculated new series with greater quantities of algae. For
that purpose, a mature culture was no longer shaken but put
aside. So a thick layer of cells was formed on the bottom. The
nutrient solution was decanted and replaced by sterile water.
With 2 cm» of this cellsuspension the cultures were inoculated
by means of sterile pipettes. On this way too, it was impossible
to carry out experiments earlier than after 4 or 5 days.
Fleischer does his experiments 3 to 6 days after inoculation.

From the experiments of Chapter III, it is apparent that
within 3 days the ratio photosynthesis-chlorophyll of one and
the same culture can shift appreciably. This difference will be
the greater in different cultures which moreover are supplied
with a various composition of nutrient components.

That is why, as much as possible, I carried out my experiments
within one series on the same day.

It appeared that Chlorella needs only little magnesium. Even
if no magnesium at all was added to the nutrient solution
there was some development of the algae, though distilled water
and pure salts were used.

From the results tabulated in table 7, it is evident that after
6 or 7 days the assimilation numbers of the different cultures
are fairly constant.

Cultures with less magnesium than 0,004 of the magnesixim
standard solution did not supply enough cells to carry out
accurate photosynthetic determinations.

Because the ratio chlorophyll — cell volume too was rather
constant, there cannot be question of Chlorose here, though the
lack of magnesium appeared clearly from the slight development
of the cultures. It is, however, possible that this disagreement
with Fleischer's results was caused by the different way of
determining the cell volume.

Fdeischer notes that the cells chlorotic by magnesium
deficiency were larger than cells which were green and abun-
dantly supplied with magnesium. In his experiments the cell
volimie was determined by measuring and counting of cells.
The cell volume in my experiments has been determined by
centrifuging the cells after the chlorophyll had been extracted.
If the cells from the cultures with magnesium deficiency
contain more water than the normal cells, it is possible that the
difference in chlorophyll amount per cell volume is lost by
extraction of the cells with methanol.

4
6
9
12
16
17

4
7
9
12
16

4
7
9
12
16
17

4
7
9
12
16

6
9
12
16

4
6
9
12

By the other way of inoculating it was possible with another
series to carry out the first experiments after four days. Though
it is not with certainty to discriminate, it does not seem
probable that this way of inoculating with great quantities agrees
with the inoculation as done by Fleischer. The results of this
series are recorded in table 8.

By this way of inoculating, the culture contains already in
the first period of growth so many cells that they form a
light green suspension. These cells are dividing slowly during
the first days after inoculation. So the aged cells are still
appreciably influencing the results when an experiment is
done four days after inoculation, because within this time only
few new cells have been formed. The culture, from which was
inoculated, had on the day of inoculation an assimilation num-
ber of 0,450.

In this way too, Fleischer's experiments could not be affirmed.
After four days the assimilation nximbers of all cultures were
constant and about three times as high as in the original
culture. Difference became visible in the cultures by continuing
the experiments. In the cultures with little magnesium no further
growth appeared and the assimilation numbers became lower.
In the cultures with more magnesium the suspension of algae
became denser and in the first days the asshnilation numbers
increased. The maximum of the assimilation number was never
reached later than on the seventh day and then declined too
in the same way as in the culture provided with normal
nutrient solution.

No more the results of Fleischer could be confirmed with
cultures of Chlorella vulgaris. These too give results completely
corresponding with those of Chlorella pyrenoidosa, only the
photosynthetic activity is somewhat lower.

In Chapter III has been demonstrated that during the growth
of the cultures the photosynthetic activity (expressed as the
assimilation number) becomes ever lower.

In the cultures cultivated in continuous light, the photo-
synthesis can go on unremittently. In these cultures more

photosynthetic products can be formed than in cultures growa
in diffuse daylight.

So, it might be possible, that the photosynthesis would be
inhibited by accumulation of photosynthetic products and that
for this reason the first mentioned cultures show lower assi-
milation numbers than the latter.

It is often cited in literature that the accumulation of photo-
synthetic products causes a decreased rate of photosynthesis.
The first notice of this theory has been given by Boussingault
(1868) on account of decreased photosynthesis of leaves re-
moved from the plant. Further evidence has been given by
investigations of Saposchnikoff (1893).

In many experiments, as well with detached leaves as with
leaves attached to the plant a decline of photosynthesis was
found after a prolonged illumination. It should be expected
that this effect, if it must be attributed to the accumulation of
the photosynthetic products, should make its appearance earlier
in detached leaves than in leaves attached to the entire plant,
as the latter are capable to translocate synthesized carbo-
hydrates to other parts of the plant.

Emerson (1937), however, remarks that a more constant rate
of photosynthesis is obtained in experiments with excised leaves.
So it seems probable that the unaccountably fluctuating rates
must be ascribed to the methods of determination of photo-
synthesis, making higher demands upon keeping constant the
external factors in the case of experimental work with entire
plants.

Harder (1933) in his experiments with Fontinalis found that
definite fluctuations in prolonged experiments on photosynthesis
arise when the light intensity during the growth of the experi-
mental plants varies from the light intensity used during the
photosynthetic determination. If these light intensities correspond
within certain limits there is no decrease of photosynthetic rate.
After a dark period of twelve hours Fontinalis when illuminated
shows a slowly increasing photosynthesis, which reaches its
maximal activity in the couise of hours.

Kostytschew, Bazyrina and Tsci^esnokov (1928) working
with plants in their natural stand found great fluctuations of
the assimilation rate. In their experiments with leaves of
several higher plants an assimilation was found being high
in the morning, declining and even stopping at the midday. In
many cases it was observed that carbon dioxide was evolved
in much greater quantities than during the determinations of

respiration. In the afternoon several plants showed a second
assimilation maximum. However, the curves of the photo-
synthetic activity of different plants on different days are so
divergent that a definite conclusion cannot be given.

The authors emphasize that the stopping of the assimilation
cannot be ascribed to an exposure to too intense light or too
high temperature because also on rainy and cloudy days this
phenomenon was observed. Therefore it was supposed that
these irregularities were due to the accumulation of carbo-
hydrates. After the criticism by Montfort (1929) Kostytschew
(1931) recants this idea. In a further investigation Bazyrina and
Tschesnokov (1930) found a very regular day-assimilation curve,
independent from the fact whether the assimilation products
during the photosynthesis are translocated (Pisum) or accumu-
lated (Solanum).

A number of other authors working with plants in different
climates (Henrici (1921), Guttenberg and Buhr (1935), Mönch
(1937) ) ascribe the decline of the rate of photosynthesis wholly
or partly to the accumulation of photosynthetic products. Gutten-
berg and Buhr especially marked an inhibitory effect of starch.
Kurssanow (1933) found with several higher plants a day-
assimilation curve with two maxima (cf. Kostytschew) and
remarked that the photosynthesis was decreased the more
when the translocation of carbohydrates was inhibited. In his
experiments with Cladophora he obtained the same results.
This is important because by working with a waterplant he
could eliminate the influence of air humidity and the behaviour
of the stomates. Besides he foimd that the photosynthetic
activity decreased by cultivating the algae in a 1 per cent invert-
sugar solution. This too is imputed to the accimiulation of carbo-
hydrates, as the invertsugar is taken up and assimilated.

Investigations carried out in the laboratory in artificial light
in several cases proved to give a constant rate of photo-
synthesis during a long time.

Boysen Jensen and Muller (1928) could not find an influence
of the photosynthetic products in Fraxinus excelsior. Neither
could KjäR (1937) with seedlings of Sinapis alba. This author,
however, stated that the assimilation products were rapidly
transported, so an intense staining with iodine could never be
found.

Very constant results were obtained in the experiments by
Mitchell (1936). For seven hours at a stretch an intensive and
constant rate of photosynthesis was found. Even the air himiidity

As in a unicellular organism a translocation of assimilation
products is impossible it was to be expected, that the accumulation
of the photosynthetic products in Chlorella would have a
greater influence on the photosynthesis than in higher plants.

For these experiments Chlorella cells were used which at
the start of the experiments were free of photosynthetic products.
To that purpose cultures were grown in the normal nutrient
solution (cf. page 691) but glucose was omitted, for Chlorella,
like higher plants, is able to assimilate glucose in the dark
and to convert it into starch (Böhm (1883), Genevois (1928,
1929) ).

Growth without glucose supply is much slower (cf. Chapter
VIII). The cells contain more chlorophyll, but the rate of
photosynthesis, calculated per unit of chlorophyll (= assimilation
number), is not greater than in cultures cultivated in an
organic medium.

Before the experiment was carried out the culture was put
in the dark for twelve hours. After chlorophyll extraction no
staining with iodine was produced than.

As under conditions of continued exposure the light intensity
of 12000 Lux used in the other experiments has an injurious
effect on the chlorophyll, a light intensity of 4000 Lux was
used in these experiments. In this illumination the photo-
synthesis of the cells amounts to 80 per cent of the assimilation
in the light intensity of 12000 Lux.

In these experiments dilute cell suspensions were used in
order that the prolonged photosynthesis would have little influ-
ence on the composition of the carbonate mixture.

Only little growth took place during this time because the
cells were deprived from all nutrient salts.

Table 9 gives the intervals during which the cells were
illuminated and the observed photosynthesis and respiration per
half hour. A perusal of the table shows that the circumstances
in course of the experiment were not unfavourable for the
cells, because the rate of photosynthesis is not lower at the
end of the experiment than at the start.

With iodine treatment on the cells that were subjected to
the experiment in the parallel vessels the depth and degree
ot the stammg was followed. At the start of the experiment
no staining by iodine was found.

After half an hour of photosynthetic activity only a verv faint
reaction with iodine is visible.

After 21^ hours illumination a distinct iodine reaction is
found in a part of the cells. In other cells small dark points
or no staining are observed.

^ter 25 hours of photosynthetic activity the greatest part
ot the cells IS stained intensively with iodine. Other cells show
no reaction.

The I ate of photosynthesis after 25 hours continual illumination
does not decrease but still increases. So nothing is perceivable
of an inhibition by accimiulation of synthesized carbohydrates.
When the cells have been in darkness for a long time, the rate
of photosynthesis is not greater than after a light period. So
the disappearing of products of photosynthesis has no stimulating
influence on the assimilatory process. On the contrary the rate
of photosynthesis continuously increases during the illimiination.

The influence of a preceding light period on the respiration
is described by Van der Paauw (1932) for Hormidium and by
French, Kohn and Tang (1934) for Chlorella pyrenoidosa. The
results of my experiments with Chlorella are in agreement with
the results of the latter authors showing a gradual decrease
after a light period to a constant value.

Of two cultures (see table 10) of the same age, containing
about an equal amount of chlorophyll, one culture had been
cultivated in constant light and the other in diffuse daylight
(cf. Chapter III). It was thought, that the photosynthesis would
have been higher in constant light so that if photosynthesis
were inhibited by accumulation or carbohydrates, it should be
the case here.

Before the photosynthetic experiment the amount of starch of
the cells from both cultures was determined with the iodine test.
It appealed that exactly the cultures, cultivated in diffuse
daylight, contained more starch.

It is apparent from this that also in this case no inhibition
of photosynthesis is found in cells containing more photo-
synthetic products.

It is often observed in more aged cultures that in a standing
culture a portion of the cells does not settle but remains sus-
pended in the nutrient solution. The greater amount of store
starch makes the specific gravity of the cells increase. Cells,
which show a deep starch staining with iodine can much faster

be concentrated by centrifuging than other cells. I applied
this fact to make cells photosynthesize, which were centrifuged
out in short time, apart from cells that were only centrifuged
after a very long time.

Before the experiment was done, it was ascertained that
the former contained more store products than the latter.

It appears from table 11 that the photosynthesis per unit of
chlorophyll is highest in cells with the highest amount of
reserve carbohydrates, which also in this case have no inhibiting
effect on the photosynthesis.

The last column of table 11 shows that the rate of respiration
is higher in the presence of reserve carbohydrates.

It is obvious from the experiments of this Chapter, that an
inhibition of photosynthesis by accumulation of carbohydrates
never could be demonstrated. Cells containing a large amoimt
of synthesized carbohydrates in contrary show a higher photo-
synthetic activity in comparison with cells containing few
assimilatory products.

From the last experiment it appears that one culture may
contain cells of different metabolic capacity. The results of
photosynthetic experiments with Chlorella are always an average
of the rate of photosynthesis of cells in different states present
in the suspension. It is evident from this last experiment that
the calculation of the rate of photosynthesis per cell volume
must give appreciable irregularities. The cells which have the
lowest specific gravity, will take the greatest volume with the
Tromsdorff method, while their assimilatory activity is slighter.

Nor is the respiration rate in experiments with Chlorella a
useful standard for the quantity of organic matter concerned in
metabolism, as assumed van den Honert (1930) for Hormidium.
When accepting this idea the assimilation activity in the last

mentioned experiment would be considered to be the highest
in the cells with the less photosynthesized products while, in
my opinion, it is more correct to say that in these cells the
respiration is low. That the assimilation activity is lower than
in the other cells (though to a less degree than the respiration)
appears from the calculation of the photosynthesis per chloro-
phyll content (assimilation number).

That the glucose has no injurious influence on the assimilation
number appears from the fact that the cultures with inorganic
nutrient solution do not have higher assimilation numbers than
the cultures grown in organic nutrient solution. The cells of
the organic cultures, moreover, have the highest assimilation
numbers in the beginning, viz. when the concentration of the
glucose is still the greatest.

Though the accumulation of carbohydrates may cause a
decrease of the rate of the assimilation in higher plants, in expe-
riments with Chlorella no inhibitory effect could be demonstrated.

In the theory of willstatter and Stoll (1918) the decom-
position of a peroxide would be responsible for the liberation of
oxygen produced in photosynthesis.

In experiments with Chlorella, Warburg and Uyesugi (1924)
found that the decomposition of hydrogen peroxide and the
Blackman reaction showed a certain conformity in sensitivity to
several narcotics. Yabusoe (1924) compared the influence of
temperature on the Blackman reaction and on the decomposition
of added hydrogen peroxide and found that the two processes
were both linear functions of temperature, while the influence
of temperature on other biological processes for instance on the
respiration of Chlorella showed the characteristics of the Van 't
Hoff and Arrhenius' rule.

Warburg and his co-workers held these results to support the
theory of WillstUtter and Stoll and explained such differences
as were found between the two reactions by the fact that in
the case of the Blackman reaction it was not hydrogen peroxide
which played the part of substrate of catalase activity, but some
organic peroxide.

In several theories on the mechanism of photosynthesis the
formation of H2O2 (Franck 1935) or some other compound (WiLL-
STaTTER 1933, Gapfron and Wohl 1936) is considered to be
the reaction preceding the liberation of oxygen in photosynthesis.

Other authors have pointed out that the coinciding occurrence
ot catalase activity and the Blackman reaction in green plants
IS merely casual. Catalase is a very unspecific enzyme and occurs
m numerous organisms, which show no Blackman reaction or
even never evolve oxygen in their metabolism
French (1934) demonstrated that the splittmg of HoOo could be

The latter is the case with yeast as French concluded from the
data of SoHNGEN and Smith (1924) on the temperature sensitivity
of the decomposition of H2O2, which deviates from the tempe-
rature sensitivity of the splitting activity of Chlorella cells decom-
posing hydrogen peroxide by an enzyme.

The influence of temperature on the catalase activity as found
noofr^'^'' ® correspond with the results of Yabusoe
(1924). Emerson and Green (1937) showed that though there is
a remote resemblance between the two curves for Chlorella
pyrenoidosa they are not identical and quot;surely insufficient to
serve as evidence for a relationship between the two processes.
Between the two curves for Chlorella vulgaris there seems to
be no resemblance whateverquot;. These authors, moreover, were
able to reduce the Blackman reaction and nevertheless found
an undiminished capacity to split added hydrogen peroxide.

The conclusion of their paper is that the hypothesis that the
Klackman reaction involves the decomposition of some peroxide
by catalase is deprived of experimental support but that it
cannot be said that the Blackman reaction is not a peroxide
decomposition.

As the decline of the assimilation described in Chapter III
was attributed to a reduced capacity of the Blackman reaction
I mvestigated the catalytic power of the catalase reaction in
the Chlorella cells during the growth of the culture

It the catalase activity should give a similar curve as the
photosynthetic activity, this would be an experimental support
tor the hypothesis that the Blackman reaction involves the de
composition of some peroxide by catalase.

For the determination of the catalase activity I followed the
methods ot Warburg and Uyesugi (1924).

Of a sample of cells, centrifuged from their nutrient solution
and suspended in 7 cm' of Warburg's carbonate mixture no. 9
the rate of assimilation and respiration was determined. Here-
after 2 cm' hydrogen peroxide solution was put into the side
bulb and shaken for ten minutes, in order to obtain the tempe-
rature of the waterbath and subsequently mixed up with the
cell suspension. The level of the manometer was read immediately.

peroxide had been mixed up with the cell suspension. Warburg
and Uyesugi used the same concentration. The concentration of
the hydrogen peroxide was determined by titrating with a
KMn04 solution of known concentration after acidifying with
sulphuric acid (cf. Treadwell 1927). The strain of Chlorella
pyrenoidosa used in my experiments had a very high catalase
activity. With readings of 5 minutes it was impossible to get
constant values for the hydrogen peroxide splitting with the

concentration of ^ N. In order to obtain a more constant
rate of catalytic decomposition a concentration of hydrogen per-
oxide of ^ N was used in all experiments described in this

chapter. Readings were made every minute. Maximal values for
the rate of hydrogen peroxide decomposition were reached at
the second or the tkird reading. The maximal peroxide splitting
was computed as the average of the five successive readings at
which the highest catalytic power was found.

The concentrations of algae in the Warburg vessel in all ex-
periments have been chosen so as to secure a constant rate of
peroxide splitting for at least five minutes.

The peroxide decomposition in my experiments is higher as
compared to the Blackman reaction than in the experiments of
Warburg and Uyesugi (1924) and of Emerson and Green (1937).
As the rate of the Blackman reaction matches with the values
found by these authors, the catalase activity of the Chlorella
strain, used by me, must be higher. Moreover a more concen-
trated peroxide solution was used.

Because the amount of oxygen liberated during the peroxide
decomposition was notably higher than during the photosynthesis,
the splitting of hydrogen peroxide has been calculated per ten
minutes, while the assimilation has been given per half hour.

Otherwise, it would be impossible to combine the results of the
rates of photosynthesis and of hydrogen peroxide decomposition
in one graph.

Like Warburg and Uyesugi (1924) I made no correction for
the respiration during the peroxide decomposition.

The experiments on the rate of splitting added hydrogen
psrcxide have been carried out with the cultures of which the
assimilatory activity has been described in Chapter III.

Of four cultures, which were differently cultivated and pro-
vided with different concentrations of the nutrient solution, the
results are recorded in table 12, 13, 14, 15 and illustrated in
figure 10, 11, 12, 13.

18.4

94.0

79.1
44,6

29.5
16,3

Fig. 13. See table 15. N.B. scale of Peroxide splitting reduced to 50% of
that of the figures 10, 11, 12.

Though the results of the determinations of the amounts of
splitted peroxide are rather fluctuating, it is obvious that the
curves of photosynthesis and hydrogen peroxide decomposition
ghow no correlating course in any of these cases.

As has already been disciissed in Chapter III the curves o£
photosynthetic activity all show a distinct maximum. The peroxide
splitting, on the contrary, increases in the first period of growth
in the culture and subsequently remains approximately constant.

It seems highly probable that the enzyme of the Blackman
reaction is inactivated during the growth of the culture, whUe
the catalase still survives.

The method of cultivation has a widely different influence
on the Blackman reaction and on the hydrogen peroxide de-
composition. This is very striking when comparing the values
given in table 15 and figure 13.

The rate of photosynthesis in these cultures reaches its
maximal value early and, in spite of the high concentration of
the nutrient compounds, the Blackman reaction decreases quickly
at the constant illumination and the high temperature in the
incubating room.

The peroxide decomposition, however, still increases apprecia-
bly thanks to the high concentration of the nutrient solution and
is much higher in the last determination than during the time
when the assimilatory activity is maximal.

That the peroxide decomposition is strongly dependent on the
concentration of the nutrient solution is also apparent from the
rates in the cultures of table 14 and figure 12. The catalase
effect here is on the main highly decreased and is even lower
in the last experiment.

The capacity to split hydrogen peroxide and that of the Black-
man reaction do not show similarity in the course of their activity
during the growth of Chlorella pyrenoidosa in pure culture. Both
are differently affected by the external conditions prevailing in
the cultivation of the cells. It is therefore unlikely that the
Blackman reaction — the name given to the rate determining
process, when photosynthesis has been saturated both with light
and with carbon dioxide — should proceed by means of the
same mechanism as the peroxide decomposition.

In Chapter III the experiments with cultures of Chlorella,
supplied with various concentrations of the normal nutrient
solution have been described. It appeared from the determina-
tions of the photosynthesis that the growth of Chlorella
proceeds at a rate, which is independent of the concentration of
the nutrient solution, but that the time during which the
growth continues is proportional to this concentration (cf.
figure 6).

Consequently the maxima of photosynthesis, chlorophyll con-
tent and cell volume are also proportionate to the concentration
of the nutrient medium. So this means that the nutrient supply
limits the growth of the Chlorella cells in culture.

would limit the growth, but that one of the compounds would
act as limiting factor.

In order to investigate which compound of the nutrient
medium was the limiting factor, nutrient media were made,
varying in such a way as to give one of the compounds in
double, respectively half the concentration, whereas the other
compounds were given in the normal dose.

Starting from the nutrient solution of Chapter III page 691,
the nutrient media were prepared by changing in the „normalquot;
solution the quantities of the successive elements as follows:

0,06 FeS04 and 2,- Na-citrate gram p. litre
0,015 FeS04 and 0,5 Na-citrate „ „ „

2,52 KNOg
0,63 KNO3
4,92 MgS04
1,23 MgS04
2,44 KH2PO4
0,61 KH2PO4
30,- Glucose
7,5 Glucose

In 100 cm^ Erlenmeyer flasks 50 cm' nutrient solution was
pipetted. Sterilization and inoculation were done as usually.
All cultures were cultivated in the greenhouse.

It is not necessary to give the complete growth curves of
all these cultures. In none of the cases they show a fundamental
deviation from the curves given in Chapter III. Hence only
some characteristic values of the growth curves are recorded in
table 16. As such the following values have been chosen:

3.nbsp;Time, in number of days after inoculation, on which the
maximal rate of photosynthesis is reached.

The analogous data of the cultures of which the growth is
described in Chapter III and which were cultivated under about
the same environmental conditions in the greenhouse are
reproduced for comparison at the head of table 16.

Day on which

Photosynthesii
cm' nutr. sol,
is reached.

(5)
(12)

(30)

(7)

(9)
(7)

(31)
(4)

(7)

(10)

(7)

(10)
(9)
C7)

(8)

(9)
(10)
(8)

11
18

12
20
12

38
10

11
20
11

17
15
11
15

20
21
15

0,180
0,400

0,850

0,561
0,567
0,309

1,060
0,258

0,424
0,474
0,500

0,518
0,537
0,495
0,435

0,487
0,506
0,505

78,4
132

245

158
156
83,7

179
101

121

110,3

124

143,5
140,8
120
122

115

128,5

134,2

There is much variability as to the time (number of days) on
which the maximal rate of photosynthesis in the cultures was
reached. This is partly caused by the fact that the cultures
were inoculated with cells from different cultures. This, how-
ever, is necessary because the cultures used for the inoculation
during these manipulations have a great chance to get con-
taminated as has already been alluded to in Chapter II. If inocu-
lation from such a culture is continued then, infection may
appear in all subsequently inoculated media. To avoid this,
several cultures must be used for the inoculation of one series.
These cultures differed in density of suspension and in age,
by which the first growth of the new culture is appreciably
affected. On this account it is a better standard to calculate
the number of days between the first visible greening and
the attairraient of the maximal assimilation. These data are
presented in brackets in the last column of table 16 and prove
to be rather regular.

The maximal rate of photosynthesis of all cultures could not
be determined with the same accuracy as it was not possible
to cairy out the experiments on every day with all the
cultures of so large a series. For this reason it may be that the
data on the maximal photosynthetic rate of the different cul-
tures as given in the table differ more than in reality. The assi-
milation curves in fact show a sharp maximum, and not always
the determinations will have been done at this very moment.

As the course of the curve of the chlorophyll content shows
a broad top, the maximum of the chlorophyll amount is rather
equal in the different cultures, except in the case of low iron
supply and in the case of nitrogen plus and -minus media. The
data on cultures at a low level of iron supply, however, are not
reliable because the cultures deteriorated by bacterial infections
before the maxima were rcached.

With a high iron supply a somewhat higher photosynthetic
maximum is reached. The curve, however, shows a sharp opti-
mum (fig. 14) ; r.iter reaching tha maximum ofnbsp;the

rate of photosynthesis decreases rapidly which points to the
deficiency of another factor. This factor undoubtedly is the
nitrogen supply, as will be shown below.

found in none of the other nutrient media; in all other cultures
the rate of photosynthesis declines soon after having passed its
maximal value.

from the curve of the culture supplied with the nutrient solution
in double concentration only in the lower value of the maximum
of the assimilation. This must be caused by the difference in
concentration of the other compounds of the nutrient solution
and on account of the investigations it must be the concentration
of the iron, which prevents the assimilation to surpass a certain
maximum.

to be proportional to the nitrogen content. That the values of
the chlorophyll content of the cultures described in Chapter III
are lower than the values of this last series must be ascribed
to the fact that the series were not cultivated simultaneously
and so they were not exposed to the same external conditions
during the growth.

If one of the other compounds of the nutrient solution, for
instance magnesiimi, phosphorus, glucose are varied, no differ-

ence is found, neither in the attained maxima nor in the slope
of the curves of assimilation and of chlorophyll content. So in
the composition of the applied quot;normalquot; solution the concen-
tration of these elements are actually not limiting the growth
of the Chlorella cells in culture.

A large amount of nitrogen enables the cells to sustain the
maximal assimilation for a long time. The photosynthesis in
the N-minus cultures and also in the cultures suppHed with the
normal nitrogen amount must decline by nitrogen deficiency.
During the growth the amount of nitrate decreases. The nitrate
is converted into a form unable to keep the assimilatory process
going on.

The rôle of the iron is of quite a different character. Though
an increase of the iron concentration enables to a higher photo-
synthetic maximum (see Fe plus cultures) the photosynthesis
in case of the normal iron amount does not decrease until another
compound (in this case nitrogen) makes the rate of carbon
fixation decline. If iron plays a part in the assimilatory process,
which must be accepted on account of the investigations in-
dicating that in assimilation a catalysis by a heavy metal plays
a part (Wurmser 1921; Warburg 1919, 1920, 1921), the above
experiments allow to accept that the iron is not converted into
an inactive form.

As far as the other elements of the nutrient solution are
concerned it is not yet possible to conclude on their rôle in the
assimilatory process on account of the mentioned experiments,
but this would be possible by varying their concentrations to
such an extent that they would limit the growth of Chlorella
cells in the culture.

The influence of nitrogen on photosynthesis was investigated
more in detail by adding potassium nitrate to cells with a low
photosynthetic activity.

these cultures did not show their maximal rate of photosynthesis
per unity of chlorophyll (= assimilation number) since a long
time, because this ratio decreases in the cultures from the very
start of the growth. It was ascertained first that the cells were
still alive.

After the rate of photosynthesis of these cultures had been
determined in the carbonate mixture nr. 9 (Warburg) under the
normal conditions, from the side-bulb 2 cm' of a KNO3 solution
were added in such a concentration that in the Warburg vessels
the KNO3 concentration of the nitrogen-plus cultures was
obtained. This concentration amounts to 2,52 g/1 KNO3 = 0,025 N.
In the control vessel 2 cm' of distilled water were added to the
cells.

In table 17 and figure 15 the results of the photosynthetic
measurements of two equal samples of cells from the same culttiïe
are recorded. In this experiment cells of a nitrogen-minus
culture were used. The experiment was done on the 27th day
after the inoculation. The photosynthetic maxitnxmi, stated on
the tenth day, has been passed already for a considerable time.

The assimilatory activity of the cells was low:
Photosynthesis _ ^q
cm' nutr. sol. ~
Photosynthesis _ ^ g^g
quot; Chlorophyll

After having ascertained that the photosynthesis of the samples
differed less than 5 per cent, the one sample was supplied with
2 cm' of KNO3 solution and the other with 2 cm' of distilled
water. The results of the respiratory and assimilatory measure-
ments per half hour are given in table 17 and figure 15.

The entire experiment extended over 34 hours, of which the
cells were exposed to the light for 834 hours.

Table 17 and the graph 15 show that by adding KNO3 to the
cells the rate cA photosynthesis increases gradually in the course
of the experifiient. The oxygen output rises from 22,3 to 199,8
mm' per half an hour, which means an increase to 900 per cent
of the original rate. The control cells also show a slight increase
in photosynthetic rate, reaching at the end of the experiment an
oxygen emission amounting to 152 per cent of the initial rate
per half an hour.

/ o

° /
/

o /
/

Light
Dark

Light

Dark

Light
ff
9i

Dark

Light
»

Dark

Light
»

Dark
»

Light

When at the end of the experiment the chlorophyll amount
of the used cells was determined, it appeared that it had not
remained constant. At the beginning of the experiment an
equal sample of cells as used in the experiment contained a
chlorophyll concentration of 0,312. At the end of the experiment
the chlorophyll content of the cells supplied with nitrate has
risen to 0,494 = 159 per cent of the original concentration. It
is evident from this that the increase of photosynthesis cannot
be ascribed to the new formation of chlorophyll alone, as the
quantity of newly formed chlorophyll is much smaller

proportion than the photosynthetic increase. As will be seen
from the following experiments the formation of new chlorophyll
is not necessary for the increase of the photosynthesis. In many
cases a marked increase of photosynthesis was found without
any increase of the chlorophyll content. In some cases it could
be stated that the chlorophyll increase appeared later than the
increase of the assimilation.

Also the respiration rate of cells supplied with KNOg increases.
This increase of the respiration rate can already be observed in
dark period, directly following the addition of KNO3. This
increased respiration is a phenomenon for itself. In the suc-
ceeding dark periods the respiration is increased not only by
the application of the nitrate, but also by the photosynthetic
products accumulated in the preceding periods of higher rate of
carbon fixation (cf. Chapter V).

Investigations on the addition of nitrogen during the photosyn-
thetic experiment were published in a paper of Pirson (1937) du-
ring the course of my experimental work. Pirson used in his in-
vestigations cells of Chlorella vulgaris f. pyrenoidosa, grown in
inorganic media with nitrogen deficiency. His experiments too
were carried out with the Warburg method; the cells, however,
were suspended during the photosynthetic measurements in
their culture medium, not in carbonate mixture. The concen-
tration of the added nitrogen was lower than in my experiments
viz. 0,01 N.nbsp;.Xi-,

In my opinion it is better to work in a medium contaming
no other nutrient compounds than the one which must be added
and of which the effect on the rate of photosynthesis has to be
investigated. In the latter case the cell growth is inhibited by
deficiency of the other nutrient compounds and so the increase
of the rate of assimilation can be ascribed more safely to a
recovery of the assimilatory mechanism.

The age of the cultures used in some of Pirson's experiments
is 12 days. It is not to discriminate whether his cultures had
already reached their maximal development as to their photo-
synthesis and chlorophyll content. Growth in inorganic media in
my experiments is a little slower than that with glucose addition,
but the nitrogen concentration in the nutrient medium used by
Pirson is so low that the maximum, no doubt, must be reached
within short time.

Notwithstanding these different methods the results of Pirson
are in agreement with my findings. In his experiments too the
rate of photosynthesis is markedly affected in a short time by

nitrogen supply and in comparison the amount of newly formed
chlorophyll is small.

As is evident from table 17 and figure 15 the rate of photo-
synthesis rises gradually; it requires some time before the effect
of nitrate gets apparent. Whether this effect is realized in the
dark or in the light is of no significance for the photosynthetic
rate.

ScHiMPER (1888) held the view that nitrate could only be
converted into organic substances in the light by green plants,
and assumed that chlorophyll was necessary for this process.
His theory, however, was refuted by Godlewsky (1903) who
demonstrated in experiments on the chemical composition of
seedlings of Triticum that proteins could be synthesized by
green plants in the dark as well as in the light. For a protein
synthesis in the dark it is, however, necessary that the plants
contain a sufficient amount of carbohydrates. The favourable
effect of the light on the synthesis of proteins must in the first
place be the supply with carbohydrates. Besides Godlewsky
assumes that also the energy, necessary for the nitrate reduction,
can be derived directly from the light.

Hamner (1936) examining the effect of nitrogen supply on the
rate of photosynthesis and respiration of Triticum cultivated at
N-deficiency, finds that addition of nitrogen increases the rate
of photosynthesis but that the effect is dependent on the amount
of carbohydrates in the plant. Hamner's experiments with Triticum
are not quite comparable with the experiments with Chlorella.
The experiments extended over such a long period of time,
that the plants supplied with extra nitrate showed a considerable
growth. At the end of the experiment (after ± 14 days) the
control-plants showed a definite injury, for the lower leaves
were partly or completely dead and the other ones were yellow-
ish green in colour. It stands to reason that the rate of photo-
synthesis of these plants was low and could not serve as a
control for the plants supplied with nitrogen.

With Chlorella I have examined whether the increase of photo-
synthesis by nitrogen addition is dependent on the amount of
reserve carbohydrates in the cells and on the jKJSsibility of
forming new carbohydrates by illumination.

In the culture used in the experiment of table 17 and figure
15 it has been stated that this one contained a large amount of
carbohydrates at the beginning of the experiment. This is un-
derstandable from the slight development of the culture (by the
deficiency of nitrogen) in a nutrient medium abvmdantly contain-

ing glucose (Ij^ per cent). Moreover, it appears from the high
respiration rate at the beginning of the experiment, amounting
to 20 per cent of the rate of photosynthesis.

In the following experiments the influence of carbohydrates
is clearly demonstrated.

In table 18 and figure 16 the results are recorded of an ex-
periment also done with cells cultivated in nitrogen-minus
medium.

The age of the used culture was 57 days. Consequently there
exists a marked N-deficiency. The photosynthetic rate is particu-
larly low; assimilation number = 0,422. The nitrogen in all ves-
sels was supplied at the same time, but two of the vessels were
kept in the dark for different periods of time. It now becomes
obvious, that the longer the dark period, the more the photo-
synthetic rate has made progress. Consequently a sufficient
amount of reserve materials has been present in the cells to
restore the photosynthetic mechanism.

It seems rather curious that the rate of photosynthesis in all
samples with N-supply is not the same at the end of the first
light period. The effect of the nitrate supply on the increase of

the photosynthetic rate seems less in the light than in the dark.
This, however, must be ascribed to the injurious effect of the
prolonged exposure to light of high intensity (see p. 686). By
this the assimilatory system is injured, so the photosynthetic
rate is the higher, the shorter the preceding illumination has
been continued.

The respiration rate during the dark period is equally high
in the three vessels supplied with nitrate. Most likely it is not
allowed to conclude from this that the injurious effect of the
light acts only on the assimilatory system and not on the
respiration. As described before, the respiration rate is increased
by a greater amount of photosynthetic products; where the il-
lumination has been the longest, the most photosynthetic products
will have been accumulated and consequently the respiration
shall be mostly increased in this case. As the respiration rate is
nevertheless equally high in the three cases, this points to an
injurious effect of the light on the respiration as well. That, on
the long run, the injury of the light is rather sweeping, is
evident from the fact that after a dark period of 11 hours a
recovery has not yet appeared. Though the photosynthetic rate
of all three samples has become higher than on the previous
day, the photosynthetic rate is highest in the culture which
has been exposed to illumination for the shortest period.

Quits a different result was obtained in the following experi-
ment (see table 19). The used culture had been cultivated in the
normal nutrient solution with i/^ per cent of glucose instead of the
usual supply of l]/2 per cent. The culture was 36 days old; the last
10 days it had stood in the dark, so the assimilatory products for
the greater part had been dissimilated. This is apparent from the

When potassium nitrate has exerted its effect on the cells
during 24 hours the following data are obtained:

First colvrain : In the control cells, illuminated during 12 hours,
the photosynthetic rate has remained constant. The chlorophyll
content has been decreased by the long intensive illumination.

Second column : These cells, supplied with nitrate and il-
luminated during a long period, have been able to photo-
synthesize and consequently have produced a large amount of
storage material. The photosynthetic rate has increased more
than twice. Also the chlorophyll content has appreciably in-
creased.

Third column : Cells not supplied with nitrogen and illuminated
during a short period of time, have preserved their initial
chlorophyll content. The photosynthetic rate is somewhat higher
than in the control cells mentioned in the first colimin, which
must be ascribed to the fact that these cells have been exposed
for a shorter time to the injurious effect of the light intensity.

luminated for a short period of time have formed some more
chlorophyll, the assimilation rate has not increased in this case.

So these phenomena can be well explained by the theory
of Godlewsky. The photosynthetic rate is only increased if the
cells are able to form new proteins. For this both a suitable
nitrogen source and carbohydrates are necessary. If one of
these two is lacking the photosynthesis does not increase. The
light is only needed for the formation of carbohydrates. If these
are present the improvement of the photosynthesic system takes
place just as well in the dark as in the light. It is not probable
in my experiments that light has a promoting effect on the in-
crease of the activity of the assimilatory process. It is not with
certainty to discriminate because the assimilatory mechanism is
damaged by continued illimiination, which injury is dependent
again on the age and the chemical composition of the cells.

As appears from the described experiments, the rate of
photosynthesis is only increased when the formation of new
proteins is possible and the increase is not dependent on the
chlorophyll content. Once more it appears from this fact that
the process of photosynthesis is limited by a protoplasmic factor.

When by an organism proteins are formed from nitrate taken
up from the medium, the nitrate must be reduced. This reduction
of the nitrate happens in the dark as well as in the light, as
may be concluded from the fact that the photosynthetic rate
is equally raised whether the nitrate has affected the cells in the
dark as in the light.

In the light the photosynthetic rate is measured only by the
production of oxygen. It might be thought that the extra oxygen,
production originated from the nitrate reduction and not from
the photosynthesis, which is a carbon dioxide reduction.

By the investigations of Warburg and Negelein (1920) on the
nitrate reductive power of Chlorella was shown that by illimii-
nating Chlorella cells in a nitrate mixture great quantities of
oxygen were liberated, originating from the nitrate reduction.
By the differences in methods, however, the results of Warburg
and Negelein are not comparable with the experiments described
above. The experiments of these investigators were carried out
with young cells rich in nitrogen. The cells that had been
in a 0,1 N NaNOs solution before the experiment were sus-
pended in a 0,1 N NaNOg, 0,01 N HNO3 mixture during the
experiment. When illuminated the extra oxygen liberation im-
mediately took place, also when the cells were deprived of car-
bon dioxide and so the assimilation was inhibited.

The addition of HNO3 is an essential point in the experiments
of Warburg and Negelein. When the cells are suspended in a
0,1 N NaNOs solution the nitrate reduction was not measurable
with the manometric method.

In my experiments only in cells with nitrogen deficiency an
increase in oxygen liberation appears and even in this case
only when in the cells a sufficient amount of carbohydrates is
present.

It could be decided whether the increase in oxygen producti-
on caused by addition of nitrate originated from nitrate re-
duction or from carbon dioxide reduction by inhibiting the photo-
synthesis in a natural way. Warburg and Negelein showed that,
in this case, this can be done best by lowering the carbon dioxide
concentration in the medium, since when using narcotics no
extra oxygen is emitted in the nitrate reduction either.

In the experiment, the results of which are lecorded in table
20, one sample of cells was suspended in the carbonate mixture,
and an equal sample of cells of the same culture in the nutrient
solution, to which no carbon dioxide was added. In the carbonate
mixture the photosynthesis is much higher than in any other
mediimi (Emerson 1936). Both samples of cells were supplied
with an equal amount of KNO3. The cells were high in content
of carbohydrates. By the higher assimilation rate in the carbona-
te mixture, this sample of cells could make still more, but the
other sample being suspended in the nutrient solution could
build up carbohydrates from the glucose.

In the cells suspended in the carbonate mixture the extra
oxygen output at the end of the experiment amounts to 121 — 57
= 64 mm' per half an hour. If this extra oxygen production
would originate from the nitrate reduction, the oxygen pro-
duction of the cells suspended in the nutrient solution should
have raised to 23 64 r= 87 mm' per half an hour. This is not
the case as is seen from the table. So the increase of the oxygen
production must not be ascribed to nitrate reduction but to a
higher rate of carbon dioxide reduction as only the latter
is affected by the lower concentration of carbon dioxide in the
medium.

The form in which nitrogen is supplied to the cells is of
great influence. I tried to affect the photosynthesis with
NH4CI instead of KNO3. The results, obtained by Pirson with
NH4CI are rather questionable. A distinct increase of the photo-
synthetic rate is not ascertained by Pirson and in a later paper
(1938) he tends to speak of an inhibition.

Like in the nitrate addition, I experimented with a higher
concentration (0,025 N) than Pirson did (0,01 N). The effect of
the NH4CI application was such that, after a dark period of

one hour during which NH4CI could exert its influence on the
cells, the photosynthesis was completely inhibited. This is
shown by the data tabulated in table 21.

The respiration rate is still more stimulated by NH4CI than
by KNO3 supply. The chlorophyll formation appears to be able
to go on in spite of the inhibition of the photosynthesis. This
was established by Pirson too (1938). The increase of the chlo-
rophyll content at NH4CI addition in this experiment is not
beyond the experimental error. It is, however, certain that the
final chlorophyll content in the cells supplied with NH4CI was
higher than in the other samples of cells.

On the basis of the mentioned data it is evident that the de-
crease of the photosynthetic rate at aging of the cultures must
be ascribed to a deficiency of available nitrate. By addition of
a new amount of nitrate the assimilatory capacity is recovered
if the Chlorella cells are able to form new proteins. The photo-
synthetic increase is independent of the chlorophyll content.

Growth and assimilatory Activity of Chlorella grown
in darkness and in inorganic media.

In one of the experiments with aged cells (cf. table 22), to
which nitrogen was added in the Warburg vessels, the first of
the samples of cells was kept in the dark for 12 hours, the second
one was exposed to light for 6 hours and the third one was
in darkness for the entire duration of the experiment. The whole
experiment extended over 24 hours.

When at the end of the experiment the chlorophyll content
of the cells was determined, the chlorophyll content of the three
samples of cells supplied with nitrate appeared to have equally
increased, independently of the time of illumination. Only in
the control sample of cells the chlorophyll content had decreased.

It must be concluded that the chlorophyll formation took place
independently of the light and so Chlorella proves to be able
to build up chlorophyll in complete darkness.

The ability to form chlorophyll in darkness belongs, as Schim-
per (1885) reports, to all lower plants up to and including the
Bryophyta. The Pteridophyta still are partly able to form chlo-
rophyll without light, but a number of ferns requires light
for greening. With the Anthophyta this capacity of greening in
darkness is limited to the cotyledons of the Coniferae (Sachs
1865) (except Larix europaea (Wiesner 1876) ) and the seeds
of some Angiosperms (Lopriore 1904).

(1934) the phase in which Hght is required, is always set quite
at the end of the process of the construction of the complicated
chlorophyll molecule, because etiolated leaves, when illuminated,
show a high rate of chlorophyll formation.

ScHARFNAGEL (1931) presumes that chlorophyll is formed out
of protochlorophyll by means of light. Rudolph tends to regard
protochlorophyll as photosensibilisator in the process of chloro-
phyll formation. According to Liro (1908) the chlorophyll for-
mation by light out of its precursor is a process observed also
in vitro; this, however, is denied by Scharfnagel.

From the fact that chlorophyll in lower plants can be formed
in darkness, it is evident that these theories have no general
validity, since lower plants contain a factor that makes the action
of light superfluous; consequently the effect of light in this
process is not specific.

The absorption spectrum of chlorophyll developed in darkness
in Nostoc punctiforme was tested by Etard and Bouilhac (1898),
of Chlorella vulgaris by Radais (1900). None of these authors
could state differences between the absorption spectra of chloro-
phyll formed in darkness and of chlorophyll formed in light.

I too compared the methanol extracts of the chlorophyll
formed in darkness and in light and found no discrepancy. As,
according to the absorption spectra, the chlorophyll developed
in the dark was identical with normal chlorophyll, it was in-
vestigated whether there exists some difference between the
photosynthetic activities. This research still has another interest,
because it enables to examine whether the decrease in assimila-
tion number, described in the previous chapters, is caused by
the illumination.

Pantanelli (1903) has found that by intensive illumination
the photosynthesis comes to an end. These quot;fatiguequot; effects
involve a ceasing of the protoplasmic streaming and a chloro-
phyll destruction. It might be possible, that this phenomenon
appeared to a minor extent in my experiments, for the assimi-
lation number is the lower, the greater the amount of light the
cells have been exposed to. This phenomenon can also be
ascribed to other factors, and in the experiments of the foregoing
chapter has been proved that the exhaustion of the nutrient
solution is the principal cause of the decrease of the assimilation
number.

However, there is a possibility that chlorophyll having never
been exposed to light would show an abnormal behaviour when
illuminated, and also it was of interest to investigate whether

the further assimilatory system was affected by the complete
absence of light during the growth of the cells.

As the increase of the chlorophyll in the experiment mentioned
in the beginning of this chapter is rather slight and so the for-
mation of chlorophyll in darkness is not quite convincm^ I
have grown cultures of Chlorella in complete darkness. The
cultures were supplied with the normal nutrition or with the
nitrate plus nutrient medium both containing 1.5 per cent of glu-
cose Immediately after the inoculation the culture flasks were
placed in a light tight cupboard in a dark room. The development
of the culture then was scanty. The cells poorly divided and did
not form much chlorophyll. The highest measured ratio

If, however, during the growth the cultures were continuously
shaken the development improved noticeably and the chloro-
phyll formation did not vary from that in cultures grown m
light To this purpose, the culture flasks, wrapped up in black
paper were put in the shaking machine, which was boarded
up with black paper. To which factor this disparity in growth
of standing and shaken cultures must be ascribed is not quite
conceivable. Of course the shaking does provide an even dis-
tribution of the cells and of the nutrient compounds. That
the shaking would aid the ventilation and so the respiration
does not look probable. The ventilation must have been rather
poor by the double packing in paper (cf. also results with
cultures cultivated in inorganic medium in this chapter). ')

When the cultures were to be used in an experiment, a sample
was taken out of the culture, centrifuged and suspended in the
carbonate mixture at as little light as possible. The exposure to
light during the determination of the photosynthesis consequently
must be considered as the very first illumination of the cells.

As the temperature in the incubating room in darkness was
low, the cells grew slowly in the beginning and the further

in^th higher plants it proved to he impossible to stimulate the chloro-
phyll formation in darkness by shaking. Seedlings of Zea Mays and Pisum
sativum growing well in the dark at the cost of the storage material m the
seed, remained fully etiolated in the shaking machine.

growth too was a little slower than in the preceding experiments.
This appears from the length of time elapsing after the inoculation
and the moment on which the first experiment could be carried
out and from the smoother slope of the curves in figure 17.
This figure and table 23 represent the data of assimilation per
nutrient solution per half an hour and the chlorophyll

It is apparent that there exists no essential difference with
the cultures grown in light.

The readings per half an hour of photosynthetic measuring
are constant from the beginning and show the normal time of
induction (Smith 1937). The chlorophyll, however, is highly
sensitive to the intensive illumination. It appears that the chloro-
phyll concentration decreases if the cells are exposed half an
hour to an illumination of 12000 Lux. The percentage of the
chlorophyll, decomposed during the determination of photo-
synthesis varied between 0 and 15 per cent and is the higher,
the older the cells. Young cells preserve their chlorophyll con-
tent or form even more chlorophyll. In computing the results,
the average of the chlorophyll concentration before and after
the experiinent was taken.

The chlorophyll in aging cells became ever more photo-
unstable. For this reason it was of no sense to extend the ex-
periments over a longer period. Moreover, a curious phenomenon
was met with: the chlorophyll in aged cultures came out of the
cells and formed a colloidal chlorophyll solution in the nutrient
medium. When the cells had been centrifuged out of the nutrient
solution, the supernatant liquid was brightly green of colour.
This chlorophyll solution did not give fluorescence and was
decolourized within a few days when standing in diffuse day-
light in an open vessel. The phenomenon that the chlorophyll

diffuses out of the cells gives the explanation of the decrease
of the chlorophyll content of the cultures grown in light (see
Chapter III). In the latter cultures the chlorophyll when dif-
fused out IS photo-oxidized in the light with the oxygen origin-
ating irom the photosynthesis in the culture. In the cultures
grown in the dark the chlorophyll is preserved in colloidal
solution as no oxydation can occur in the medium that must
be very low in oxygen tension by the respiration of the cells
As the powth of the cultures in the dark does not show any
essential deviation from that of the cultures grown in light it
seemed rather probable that the latter were growing heterotro-
phically too. For this reason cells were grown in the normal
nutrient solution, however, not containing glucose. The growth

On the 97th day the chlorophyll concentration amounts only
to one fourth of the maximal chlorophyll concentration of the
cultures provided with a nutrient solution containing glucose
u Jnbsp;be remarked that it was investigated whether

the addition of a growth promoting substance had any influence
on the growth of the cells. To Dr. L. Pons I am indepted for
supplying a biotme preparation of known activity. An influence
of biotine could not be stated. (For the preparation of biotine cf
Pons 1938).

In spite of the slight development, the ratio Photosynthesis
• 1 . 1 ,, ,nbsp;chlorophyll

IS low m aged cells, which is not due to a deficiency of inorganic
nutrient compounds The poor development of the cells must
be ascribed to a deficiency of organic substances. The conditions
m the culture are apparently not of a nature to make possible
a sufficient assimilation to supply the want of carbohydrates of
tlie cells. As the photosynthetic activity of the cells in the
carbonate mixture in the Warburg vessels is sufficiently high
to form a considerable amount of carbohydrates, the assimilation

in the culture flasks must be inhibited by deficiency of carbon
dioxide. The cottonwool stoppers do not allow a sufficient aerati-
on. Therefore I made a strong air current pass through
cultures provided with inorganic nutrient solution. Growth is
then only a little slower than in cultures provided with a
nutrient solution containing per cent of glucose. The cells
grown under these circumstances give an assimilation curve
and a chlorophyll curve which do not deviate from the curve
found in cells supplied with an organic carbon source. The maxima
of chlorophyll per cm'' nutrient solution and of photosynthesis
per cm' nutrient solution too lie approximately on the same level.

A divergence is only found in the rate of respiration which
was in the beginning of the growth of inorganically cultivated
cells so small that no measurable change in pressure per half
an hour was stated. This low respiration rate of the cells must
be ascribed to the deficiency of carbohydrates. When the cells
are still in their state of rapid division no carbohydrates are
accumulated. When the cell-division in the older cultures de-
creases, more carbohydrates are accumulated and the rate of
respiration increases.

Cultures of Chlorella covered by cottonwool stoppers have
an insufficient aeration. Supplied with glucose the growth is
for the greater part heterotrophic and goes on equally well in
complete darkness, if care is taken for a good distribution of the
nutrient compounds by shaking of the culture. It is of no im-
portance for the assimilatory capacity of the cells whether the
cultures have build up their carbohydrates from carbon dioxide
or from glucose; brought under equal external conditions the
assimilatory activity is the same.

In the preceding chapters the results are described of in-
vestigations on the growth and on the photosynthetic activity of
Chlorella pyrenoidosa.

The rate of photosynthesis was determined while the cells
were suspended in Warburg's caibonate-bicarbonate mixture
number 9, using the manometric method of Warburg.

ent cultures must be compared, it is a problem per which entity
the measured photosynthesis must be computed. As the chlo-
rophyll IS the only measurable substance in the green plants
which can be said to be involved in the assimilatory prLess

of 2S0 rT ''' (f^t^^^ed per half hour at a temperature
ot ^5 5 C.) per unity of chlorophyll was considered as an item
tor the assimilatory activity. This ratio is proportional to the
assimilation number (quot;Assimilations-Zahlquot;) according to Will-
sxatter and Stoll (1918); only the values in my work cannot
be compared with the values of these authors as in my work
merely relative chlorophyll concentrations were determined.

experiments done with the same culture after different periods
of time It appeared that this ratio is the highest in very young
ce Is and dechnes from the very beginning of the growth in
culture. This means that during the growth more chlorophyll
is tormed than corresponds with the increase of the photo-
synthetic capacity; so the photosynthesis per unity of chlorophyll
decreases. As there was no reason to accept that the pLto-
chemical activity of the chlorophyll was decreased, it m^st be
assumed that the factor of the Blackman reaction, which mav he
an enzyme, increases at a slower rate than the chlorophyll. It
follows from this that the amount of chlorophyll and the Black-
man factor increase independently of each other.

l^iting the rate of photosynthesis
when the cells are satured with carbon dioxide at a surplus of
light intensity, has a high temperature sensitivity. If the decrease
ot photosynthetic activity was caused by the diffusion of carbon
dioxide through the cell walls that become thicker in older cells
or by inactivation of the chlorophyll, the temperature sensitivity
ot the assimilation process should decline in older cells as
diffusion- and photochemical processes have a lower temperature
^efficient than the Blackman reaction (van den Honert 1930)
However it appeared that the temperature sensitivity did not
vary with the age and the cultivating methods of the cultures
so It IS allowed to assume that the Blackman reaction always is
the limiting process.

Different experiments were carried out to investigate which
facto, „i^t a«ec. .He rationbsp;. A„

The experimental results of Fleischer (1934), who found in
young cells cultivated with magnesium deficiency considerable

It appeared that the chlorophyll and the Blackman factor were
differently affected by the external circumstances during the
growth of the cultures. By constant light and high temperature
the chlorophyll formation was stimulated but the Blackman
factor was unfavourably affected and so the assimilation numbers
were considerably lower than in cultures of the same age and
density cultivated in diffuse daylight.

As according to the theory of Warburg and Uyesugi (1924)
the Blackman reaction represents the splitting of a peroxide
and the catalase activity of the Chlorella cells was supposed
to be responsible for this reaction, it was investigated whether
the power of splitting hydrogen peroxide showed the same
variations in activity as had been found for the Blackman reac-
tion. This appeared not to be so. The power of splitting hydrogen
peroxide is much less sensitive to unfavourable conditions in
the culture during the period of growth of the cells. The decrease,
found for the Blackman reaction in all cultures, was never stated
for the catalase activity. Therefore it seems improbable that the
mechanism of the Blackman reaction can be identified with the
catalase reaction. If, indeed, the oxygen liberated by green plants
in photosynthesis originates from the splitting of some peroxide,
it must be assumed that in the dark chemical reaction another
process is involved that limits the rate of photosynthesis and, as
a consequence, represents the Blackman reaction, or it must be
assumed that this peroxide is splitted by another agent than that
which is responsible for the splitting of added hydrogen
peroxide.

By varying the concentration of the nutrient solution the rate
of growth was not affected, but the length of the period of
growth was; consequently the reached maximum of density
of the cell suspension, of the chlorophyll content and of the
photosynthetic capacity of the culture were proportional to
the concentration of the nutrient compounds in the medium.

As the result of the determination of the growth and photo-
synthesis in media in which the nutrient compounds were given
in different mutual proportion, it could be stated that the amount
of available nitrate limits the photosynthetic capacity of the
culture. Even in the cultures with a high amount of nitrate
the photosynthesis decreases by nitrate deficiency in course of

time. It could be demonstrated that, when the concentration
of iron limits the growth of the cells, the photosynthesis does
not decrease by iron deficiency but remains constant until it
is decreased by nitrate deficiency.

In cells of which the assimilatory activity was decreased by
nHrate deficiency, the addition of nitrate appeared to have a
highly stimulating effect on the rate of photosynthesis, while
the chlorophyll content was only slightly increased. The rate of
photosynthesis could, however, only be increased when the
cells contained a sufficient amount of reserve carbohydrates. So
it seemed probable that the added nitrate was used for the
formation of new proteins, by which the photosynthesic rate
was increased. This, however, does not directly require the
assumption that in photosynthesis an enzyme of proteid-like
nature is involved. The proteins are so closely related to the
protoplasm and the actions of the protoplasm are so numerous
and unapproachable that any further conclusion seems premature.

From the differences in behaviour of the rate of photosynthesis
and of the chlorophyll content at nitrate addition, it was stated
in several experiments that the increase of photosynthesis was
never accompanied by an adequate increase but sometimes
even by a decrease in chlorophyll content, while in other cases
it was found that an increase of chlorophyll content did not
involve a higher photosynthetic rate. This proves that the de-
crease of the assimilation numbers can not be explained by
assuming a time effect on the photosynthetic activity of the
chlorophyll and that the Blackman reaction, being the rate
determining process in photosynthesis, is not directly dependent
on the concentration of the chlorophyll.

However, it appeared impossible to raise the assimilation num-
bers above a certain value. The abnormally high assimilation
numbers found by willstatter and Stoll with leaves of
etiolated higher plants could not be reproduced with Chlorella,
for not only the synthesis of proteins took place in darkness
equally well as in light, but also the chlorophyll formation and
growth are possible in complete darkness if only carbohydrates
are present.

The assimilation numbers of etiolated leaves, which are
only surpassed by the ratios found in some aitrea-varieties
of different plants, are so much higher than the assimilation
numbers found in Chlorella (Emerson 1936) and Hormidium (Van
den Honert 1930) that in these lower plants just as in normal
higher plants the chlorophyll must be in excess in proportion to

the rate determining factor of photosynthesis. As this cannot be
changed by cultivation in complete darkness and as also chlorotic
Chlorella cells appeared to have constant assimilation numbers
(Emerson 1929, Fleischer 1934), there must exist — in spite of
the independence of chlorophyll content and Blackman reaction
— an internal factor in the green plants which prevents the
assimilation numbers to diverge widely, a fact which is not
yet understood.

1.nbsp;The photosynthetic activity of Chlorella pyrenoidosa, ex-
pressed by the ratio Pj^o^^nt^es^nbsp;highest in young

2.nbsp;By cultivation under different external conditions of light
and temperature, cells of different photosynthetic activity can
be formed.

3.nbsp;The decline of the photosynthetic activity cannot be ex-
plained by accumulation of photosynthetic products.

4.nbsp;A specific effect of magnesium deficiency on the photo-
synthetic activity of young cells, as has been found by Fleischer,
could not be reproduced.

5.nbsp;The decline of the photosynthetic activity cannot have been
caused by inactivation of the chlorophyll, but must be ascribed
to the activity of the Blackman reaction.

6.nbsp;The changes in the rate of photosynthesis and of catalase
activity do not correspond, so it seems unlikely that the Black-
man reaction and the splitting of added hydrogen peroxide should
proceed by means of the same mechanism.

7.nbsp;If the decline of the total photosynthetic capacity of an
aging culture is caused by nitrate deficiency, it can be raised
by nitrate addition. This can be achieved equally well in light
as in darkness if only the cells contain a sufficient amount of
carbohydrates. If no carbohydrates are present the rate of
photosynthesis can only be increased in light when new car-
bohydrates are photosynthesized.

8.nbsp;The increase of the photosynthetic activity by nitrate
addition is established independently of the chlorophyll.

9.nbsp;The growth and chlorophyll formation of Chlorella in
glucose containing media are equally good in darkness as in
light. The absence of light during the growth does not have an
effect on the photosynthetic power of the cells.

10. The fact whether the cells have built up their carbo-
hydrates of carbon dioxide or of glucose does not affect the
photosynthetic activity.

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Time	pH	Photosynthesis	Chlorophyll	Cell volijine	Photosynthesis
Time	pH	cm' nutr. sol.	cm' nutr. sol.	cm' nutr. sol.	Chlorophyll
6	7	18,4	0,066	1A7	2,80
10	8,2	94,0	0,508	6	1,85
12	8,2	79,1	0,468	7	1,69
16	7,4	44,6	0,386	8,5	1^5
19	7,6	29,5	0,374	10	0,82
25	7,8	16,3	0,370	13,1	0,44
34	7,4	21,3	0,360	11,5	0,59

Im^-- Light during growth	Temperature during growth	Age of culture	Photosynthesis	Qs
Im^-- Light during growth	Temperature during growth	Age of culture	Chlorophyll	Qs
diffuse daylight	± 21°	35 days	1,14	1,5
» ),	± 21°	17 days	3,82	1,4
it 11	± 21°	20 days	2,13	1,6
continuous artificial light	± 26°	20 days	1,13	1,5

Amount of magnesium in cm' of the nutrient solution	Time	Photosjmthesis	ChlorophyU
Amount of magnesium in cm' of the nutrient solution	Time	Chlorophyll	CeU volume
0,004 0,006	6	2,52	37
0,004 0,006	6	2,87	27
0,01	7	2,63	43
0,03	6	2,46	36,7
0,05	6	2,34	26
0,07	7	2,76	38
0,1	7	2,68	45
1	6	3,00	39
2	7	3,37	35
5	6	2,64	35

Illumination	Intervals of observation	Photosjmthesis per 1/2 hour	Respiration per V2 hour
4V2 hours Light	30 min. 30 min. 30 min. 30 min. 30 min. 2 hours	90 113 108 96 104 108
18 hoiors Dark	30 min. 30 min. 5V2 hours llVs hours		25 20 12 12
25 hours Light	30 min. 2 hours 1nbsp;hour 2nbsp;hours 3nbsp;hours 2 hours 13 hours 30 min. 30 min. 30 min.	25 85 116 119 115 153 9 155 156 141
19 hours Dark	2 hours 2V2 hours 14 hours 30 min.		36 46 30 31
1 hour Light	30 min. 30 min.	143 153

Cultivation	Photosynthesis Chlorophyll	Chlorophyll cm' nutr. sol.	Cell volume cm' nutr. sol.	Photosjmthesis cm' nutr. sol.
in constant light	1,62	366	8,1	545 little
in constant light	1,62			starch
in diffuse daylight	2,45	386	8,4	947 much
in diffuse daylight	2,45			starch

Time	Photosynthesis per hour	H2O2 decomposition per 10 min.
Time	cm» nutrient solution	cm» nutrient solution
6 8 11 18 26 31 Normal nutri	11,2 35,0 78,4 32.8 25,7 20.9 TABLE 15. ent solution 2 X concentratii	12,1 15,4 54 92 89 58 an. Cultivated in constant light.
Time	Photosynthesis per 1/2 hour cm» nutrient solution	H2O2 decomposition per 10 min, cm» nutrient solution

	Time	control cells suspended in carbonate mixture O2 liberated per V2 hour	cells in carbonate mixture -f- 0,025 n KNO3 O2 liberated per V2 hour	cells in carbonate mixture 0,025 n KNO3 O2 liberated per V2 hour	cells in carbonate mixture -1- 0,025 n KNO, O2 liberated per 1/2 hour
Light	12.05—12.35	86,5	86,0	Dark	Dark
i9	12.35—13.55	102,5	98,0	JJ
99	13.55—14.25	115	102	»
	14.25—14.55	113	120		99
	15.05—15.35	130	105	107	ft
	15.35—16.05	124	106	162	91
	16.05—17.45	123,5	124	150	99
	17.50—19.30	128	124	155	if
99	19.30—21.30	125	133	158	99
99	21.40—2210	115	124	146	134
97	22.10—22.40	119	142	158	212
99	22.40—23.10	120	136	166	212
99	23.10—23.40	112	137	164	208
Dark	23.43—10.43	-53	-63	-63	-65
light	10.55—11.25	130	190	202	259
99	11.25—11.55	136	187	185	300

Time	O2 liberated per hour	O2 liberated per 1/2 hour	O2 liberated per 1/2 hour	O2 liberated per 1/2 hoiir
Light 16.00—16.30	47,4	45,6	Dark	Dark
		KNO3 added	n	KNO3 added
16.40—17.10 19.30—20.00 21.45—22.15	48,6 48,3 43,1	49.4 55.5 56,1	» J) J,	»1 »
Dark 22.20—10.45	-3,0	-5,5	»
Light 10.55—11.05 11.25—11.55 15.40—16.10 „ 16.10—16.40	48,9 51,4 46,4 46,0	107,5 112,0 113,3 108,5	II II 52,0 56,3	99 99 53,0 54,7
Chlorophyll content ^fore the experiment	0,187	0,187	0,187	0,187
Chlorophyll content after die experiment	0,140	0,343	0,185	0,214
mm» cellvolume before the experiment	12	12	12	12
mm» cellvolume after the experiment	9,5	14	12,6	llA

	O2 liberated per ^/s hour	O2 liberated per	1/2 hour
	cells in Ccirbonate mixt.	cells in nutrient	medium
	KNO3 0,025 N.	containing KNO3	0,025 N.
Beginning of the experiment	57	23
9 hours later	121	33

	O2 liberated per Va hour Control	O2 liberated per V2 hoiir KNO3 addition	O2 liberated per 1/2 hour NH4CI addition
Dark 11.00—12.00	-34	-43	-67
Light 12.10—12.40	180	181	-22
Dark 12.45—14.15	-42	-63	-86
Light 14.20—15.20 15.20—17.00	192 186	214 232	-53 -73
Dark 17.15—10.45	-40,5	-71,5	-56
Light 10.50—11.20	182	489	-42,4
Chlorophyll content before the experiment	0,225	0,225	0,225
Chlorophyll content after the experiment	0,189	0,209	0,226

Rate of photosynthesis in the first half hour	31,8	Darkness	1 Darkness	33,0
Addition of nitrate	KNO3 1	KNO3	KNO3 1	—
Period, in hours, of exposure to light	12	6	0	12
Rate of photosynthesis, 24 hours after the beginning of the experiment per half hour	93,9	91,2	__	26,4
Chlorophyll content before the experiment	0,230	0,230	0,230	0,230
Chlorophyll content at the end of the experiment	0,290	0,292	1 0,290	0,172

Time	Photosynthesis Chlorophyll	Photosynthesis cm' nutr. sol.	Chlorophyll cm' nutr. sol.
13 34 97	5,40 2,06 1,04	2,16 3,16 12,9	0,004 0,015 0,123

Time	pH
6	6,9
10	7.1
12	7
18	6,8
26	6,7
31	6,8

Time	pH	Photosynthesis	Chlorophyll	Cell volume	Photosynthesi«
Time	pH	cm® nutr. soî.	cm' nutr. sol.	cm® nutr. sol.	Chlorophyll
9 12 18 25 31 34 38	7,1 6,9 7 7 7 6,4	22,3 68,7 208,0 191.5 234,0 236.6 245,8	0,036 0,146 0,374 0,547 0,700 0,847 0,845	0,77 4,8 9 13.5 8,7 14.6 17.7	7,05 4,38 4,92 3,50 3,35 2,72 2,91
		TABI^ 3.
		1/2 X nutrient solution.
Time	pH	Photosynthesis	Chlorophyll	Cell volume	Photosynthesis
Time	pH	cmquot; nutr. sol.	cm' nutr. sol.	cm® nutr. sol.	Chlorophyll
6 8 11 18 26 31 34	7 7,3 7,1 6,6 6,9 6,9 6,9	11,2 35,0 78,4 32.8 25,7 20.9 20,3	0,015 0,067 0,155 0,133 0^30 0,149 0,115	0,43 1.4 3.5 6,0 9,4 14,3	7,70 5,35 5,08 2,46 1,98 1,40 1,77

Time	pH	Photosynthesis	Chlorophyll	Cell volume	Photosynthesis
Time	pH	cm' nutr. sol.	cm' nutr. sol.	cm' nutr. sol.	Chlorophyll
8 10 12 16 19 25 31	7,6 7,6 6 7 5,4 7,8 Norm	26.5 161,0 157,6 134,6 39,2 39.6 42,8 TAB] al nutrient solu	0,075 0,753 0,850 0,798 0,756 0,574 0,706 LE 6. tion Vs conce	3,9 17.6 14.7 14,9 19,5 25,7 34,3 !ntration.	3,530 2,130 1,850 1,825 0,518 0,691 0,607
Time	pH	Photosynthesis	Chlorophyll	Cell volimie	Photosynthesis
Time	pH	cm' nutr. sol.	cm' nutr. sol.	cm' nutr. sol.	Chlorophyll
5 9 11 16 19 31	6.7 8,2 8,2 8 8,2 6.8	4,35 24,6 17,5 15,5 7,6 3,5	0,016 0,173 0,218 0,247 0,179 0,055	0,27 3.03 3.4 3,48 4,7 5,2	2.664 1,423 1,208 0,435 0,492 0,637

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1 .....	1 , . 1 . 1	1 1	1