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The failure to bring about carbon assimilation by means
of chlorophyll isolated from the leaf may be attributed, accord-
ing to the authors, to the absence of the enzyme. Also they ob-
served that carbon assimilation continued in leaves from which
the lower epidermis had been removed whilst a slight pres-
sure applied to the leaves brought the process to a standstill.
This is explained on the assumption that the enzyme is situated
at the surface of contact between the chloroplast and the plasma.

This belief in the existence of a controlling enzyme also is
shared by Osterhout and Haasj who experimented with

* Willstatter and Stoll : " Ber. deut. chem. Gesells.," 1915, 48, 1540.

t/rf., p. 1552.

J Osterhout and Haas : "Proc. Nat. Acad. Sci.," 1918,4, 85.



UNKNOWN FACTORS 35

various plants, both fresh water and marine, grown in a culture
solution containing gaseous carbon dioxide. The removal of
carbon dioxide was followed by the method of determination
of the hydrogen ion concentration in the culture medium. It
was found that photosynthesis began immediately on exposure
to light; at first the removal of carbon dioxide was rapid,
then more slowly until a maximum was reached when the
rate remained constant. These authors consider that under
the influence of light some catalyst is developed which facili-
tates the photosynthetic process. Finally Briggs concludes
that the activity of the photochemical phase of the carbon
assimilation mechanism in a seedling, as compared with that
of more mature leaves, is in some way limited : the photo-
chemical phase depends for its intensity not only on the chloro-
phyll but also upon some other factor which increases with age
during the early stages of leaf development.

There is thus strong evidence of the existence of a control-
ling factor essential in the earlier phases of carbon assimilation :
whether it be enzymatic or protoplasmic, it is impossible to say.

THE PRODUCTS OF CARBON ASSIMILATION. Much
investigation is necessary before the precise significance
of this factor can be determined : of the products of car-
bon assimilation, carbohydrate and oxygen are the most
conspicuous ; others there are, but it is unprofitable, if not
impossible, to consider them on this immediate occasion
since accurate knowledge of their part in carbon assimilation
does not exist.

It is well known that according to the law of mass action,
chemical action diminishes as the products of the activity
accumulate ; the initial rate can only be maintained provided the
initial proportions of the reacting substances are preserved and
the products of the reaction removed. In the leaf a further
complication arises since sugar is an osmotic substance and its
undue accumulation may lead to a physical disturbance in the
synthesizing machine such, for example, as the closure of the
stomates. This may be averted by the conversion of the
sugar into starch, a marked and well known phenomenon in
many plants, which temporary reserve is translocated at
nightfall after hydrolysis. Translocation is most apparent

3*



36 THE SYNTHESIS OF CARBOHYDRATES

in the dark, but it takes place also by day and it is only in
sugar leaves that the undue concentration of -soluble carbo-
hydrates, owing to hindrances to rapid translocation, or to a
high assimilatory efficiency, is likely to prove an important
factor in carbon assimilation. In this respect the observations
of Broock * are of considerable interest and value. He found
that the leaves of the sugar beet on bright sunny days showed
a rapid increase in their dry weight up to mid-day, at which
hour there was a sudden decrease: this loss in weight was
more or less uniform until about midnight. The sudden drop
in the carbon assimilation at noon indicates that the process
was brought to a standstill owing, in all probability, to the
closure of the stomates resulting from the accumulation of
materials which were produced at a greater rate than that at
which they could be translocated.

With regard to the oxygen evolved, since carbon assimila-
tion by green leaves is unaffected by various concentrations of
oxygen in artificial atmospheres, and since the oxygen pressure
of the internal atmosphere of plants is a very variable quantity,
it would not appear that the amount of oxygen is a limiting
factor of any great significance.

THE ORGANIC PRODUCTS OF CARBON ASSIMILATION.

The ultimate products of carbon assimilation are carbo-
hydrates : Brown and Morris,f in their classical work on the
chemistry and physiology of foliage leaves, identified sucrose,
dextrose, levulose, and maltose in the leaves of Tropceolum,
the sucrose being in greatest abundance. These results were
generally accepted, reinvestigations of the subject being of
recent date. Parkin { in his investigation of the sugars of the
leaf of Galanthus nivalis, selected for its convenience as a sugar
leaf, found that a considerable quantity of sugars, 20 to 30 per
cent of the dry weight, occurred in the active leaves ; sucrose,
dextrose, and levulose were recognized but maltose was never
found, a result which was to be expected, since starch is not

* Broock: " Uber tagliche und stundliche Assimilation einiger Kultur-
pflanzen. Halle: 1892. See Thoday : " Proc. Roy. Soc.," Lond. B., 1910, 82,

443-

t Brown and Morris: " Journ. Chem. Soc.," Lond., 1893, 63, 604.
J Parkin; "Biochem. Journ.," 1912, 6, i.



PRODUCTS OF CARBON ASSIMILATION 37

formed in the leaves in any appreciable quantity. The amount
of sugar increases towards the base of a leaf and concurrently
the ratio of the sucrose to the hexoses diminishes. The sucrose
increases rapidly in amount after sunrise, reaches a maximum
and diminishes during the night, the percentage amounts of
the hexoses of the leaf, however, remain fairly constant. As
the season advances, the proportion of sucrose to the hexoses
decreases, the latter being formed at the expense of the former.
Parkin confirms Brown and Morris in the observation that the
levulose is, as a rule, more abundant than the dextrose and
supports the contention that sucrose is the first recognizable
sugar in carbon assimilation, the hexoses- being formed from
it by inversion. The reason for levulose being more abundant
than dextrose is that the latter is more used by the leaf in its
respiratory processes.

Davis, Daish, and Sawyer,* in the instances of the potato
and the mangold, agree that sucrose is a primary sugar of
carbon assimilation and from it the hexoses arise. The cane
sugar and hexose are at their maximum at mid-day and the
amounts increase with the season. Unlike Brown and Morris
they found pentose to be present whilst maltose was consist-
ently absent, both by day and by night, from the leaves men-
tioned. In view of the presence of starch in the potato leaf,
it is surprising that the presence of maltose was not estab-
lished : there is good reason for supposing that maltase is
commonly present in plants in which starch occurs and is
digested : this enzyme is rather indiffusible, is destroyed by
alcohol and chloroform under ordinary conditions, and is
very susceptible to heat, a temperature of 50 C. having a
marked adverse effect upon its activity, whilst at 55 C. it may
be destroyed. It is these properties of maltase which account
for the varying statements regarding the occurrence of mal-
tose in green leaves : if the procedure of experiment is such
as to destroy the maltase, maltose will be found ; if, on the
other hand, the enzyme is not destroyed, the maltose is con-
verted into hexose sugars. f

Gast I examined the carbohydrate content of the leaves of

* Davis, Daish, and Sawyer : " Journ. Agric. Sci.," 1916, 7, 225.
f Davis: " Biochem. Journ.," 1916, 10, 31, 49, 56.
JGast: " Zeitsch. Physiol. Chem.," 1917,99, i.



38 THE SYNTHESIS OF CARBOHYDRATES

Tropceolum, Vitis> Musa and other plants collected in the early
afternoon and early morning : he found that sucrose always
was more abundant than maltose and the other sugars identi-
fied ; he is in accordance with Brown and Morris regarding
the absence of pentose. Kylin * found that in general terms
the amount of glucose varies inversely as the amount of starch.
Sucrose is generally the most abundant sugar, especially in
sugar leaves, but Gentiana brevidens^ a sugar leaf, does not
contain any sucrose, whilst Tilia europ<za> a starch leaf, con-
tains much sucrose.

In conclusion it may be said that leaves generally contain
sucrose, maltose, dextrose, and levulose, whilst the occurrence
of pentose is uncertain.

With regard to starch, its occurrence and relationships to
sugar have already been considered,^ and since agreement
regarding the sequence of events in carbon assimilation which
culminate in the formation of sugar has not been reached, it
is not desirable here to discuss which is the primary sugar of
the synthetic process.

Fats sometimes have been described as direct products of
carbon assimilation, more especially in plants, such as Van-
cheria, which have an abundance of fat-like substance in their
green tissues. The evidence for the contention is anything but
satisfactory, especially in view of the transmutation of carbo-
hydrate into fat in various plants and under the influence of
certain external conditions such as low temperature.* The
problem has been resuscitated by Meyer who describes the
presence of a colourless fluid of unknown composition and
having the physical properties of an oil which occurs in the
chloroplasts of Tropceolum. This substance, which is not a
fat, since it gives on distillation hexylenealdehyde, increases
with assimilation, and is considered by Meyer to be a definite
product of carbon assimilation.

HYPOTHESES CONCERNING THE SYNTHESIS OF CARBO-
HYDRATE BY THE GREEN PLANT.

With the problems associated with the march of events
from the initial carbon dioxide and water to the final carbo-

* Kylin : id., 1918, loi, 77. f Vol. I. J See Vol. I.

Meyer : " Ber. deut. hot. Gesells.," 1917, 35, 586; 1918, 36, 235, 674.



BAEYER'S HYPOTHESIS 39

hydrate, the realms of theory are entered. The hypotheses
are many and have this in common none entirely satisfy the
plant physiologist ; for which reason it is proposed to consider
but two in any detail on the present occasion, not that those
discarded are barren of ideas indeed, some contain valuable
suggestions but that their examination, more especially when
unsupported by experimental evidence, would tend rather to
obscure than to clarify the problem.

In 1870 Baeyer put forward the hypothesis that the carbon
dioxide is split up by the plant into carbon monoxide and
oxygen, and that the water is concurrently resolved into its
constituent elements. The carbon monoxide and hydrogen
thus produced then combine to produce formaldehyde, which
undergoes polymerization, and so forms a hexose.

These changes may be represented in the following equa-
tions :

/ 1. co 2 = CO + o

\ 2. H 2 O = H 3 + O

3. CO + H 2 = CH O

4. 6(CH 2 0) ' = C 6 H 12 8

Thus, according to the theory, there are two distinct actions,
the first leading to the formation of formaldehyde, and the
second to the production of sugar.

Considering the first part of Baeyer's theory, it is seen that
both carbon monoxide and hydrogen are supposed to be pro-
duced, but carbon monoxide has been found but once in a free
state in the living plant (p. 73), and it is a substance which
does not lend itself at all readily to constructive metabolism,
the evidence as to whether plants are able to make use of it
for synthetic purposes being contradictory. Bottomley and
Jackson * state that if the carbon dioxide normally present
in the atmosphere be replaced by about twenty times as much
carbon monoxide the increase in the amount of the latter
being necessary on account of its lesser solubility in water as
compared with carbon dioxide plants of Tropaolum formed
starch and flourished. Preliminary analyses also showed that,
in the case of seeds germinated in an atmosphere in which the
carbon dioxide had been replaced by carbon monoxide, there
was in the seedlings an increase in organic carbon. Further,

* Bottomley and Jackson : " Proc. Roy. Soc.," Lond. B., 1903, 72, 130.



40 THE SYNTHESIS OF CARBOHYDRATES

they found that a negative pressure obtained in the vessels
containing the plants assimilating carbon monoxide. This
was to be expected if the hypothesis be accepted, for if the
carbon monoxide be used up in photosynthesis, then the
amount of oxygen set free would be half that evolved in
normal photolysis. On the other hand, Kraschennikoff* has
come to the conclusion, based on a number of experiments,
that green plants cannot make use of carbon monoxide ; he
points out, however, that his evidence does not prove that
carbon monoxide is not formed in the early stages of photo-
synthesis. It may also be remarked that according to the
investigations of Sulander,f carbon monoxide acts as an
anaesthetic, but is much weaker in its action than chloro-
form. He found that 0*5 per cent of this gas was sufficient to
inhibit the growth of seedlings of the lupin and the germina-
tion of the spores of many Fungi. It does not therefore
follow that carbon monoxide is not formed in plants. As is
well known, carbon dioxide is itself an anaesthetic if present
in a sufficient amount, and possibly it is more potent in this
respect than is carbon monoxide, for Sulander found that in
many cases the streaming movements of protoplasm were not
affected even after several hours' exposure to the last-named
gas.

A modification of Baeyer's theory thus appears to be
necessary. Erlenmeyer, long before the experimental work
on carbon monoxide just referred to was done, suggested that
the carbonic acid in the cells undergoes a reduction which
leads to the formation of formic acid and oxygen, and that
the formic acid is further reduced to formaldehyde and
oxygen :

T. H 2 C0 3 = CH,(X, + O
2. CH 2 O 2 = CH 2 0" + O

or else that the action is continuous and that the carbon
dioxide and water may directly give rise to formaldehyde
and oxygen :

co 2 + H 2 o = CH 2 o + O 2

According to these views either formaldehyde or formic acid
must be produced,

* Kraschennikoff : "Rev. gdn. Bot.," 1909, 21, 177.
f SulancLr : " Beih. hot. Centrbl.," 1909, 24, I., 357.



ASSIMILATION OF FORMALDEHYDE 41

These substances, as is well known, are poisonous, so that,
if formed, they must be polymerized before they have time to
injure the protoplasm, and experiments have shown that,
under certain conditions, formaldehyde may be made use of
by the plant. Thus Bokorny * showed that Spirogyra in the
absence of carbon dioxide can make starch when supplied with
a compound of formaldehyde and sodium hydrogen sulphite ;
also Treboux f and Bouilhac have stated that Elodea, Sinapis,
and certain Algae can form starch in the dark when supplied
with dilute (-0005 per cent) solutions of formaldehyde.

Grafe \ found that green seedlings grown in the light, in an
atmosphere free from carbon dioxide and containing not more
than I -3 per cent of formaldehyde vapour showed a greater
increase in growth and in dry weight as compared with the
controls. These results were corroborated by Baker who
concluded from most carefully controlled experiments that
formaldehyde could be assimilated to a certain extent in the
light, but not in the dark, in an atmosphere void of carbon
dioxide.

With regard to the presence of formaldehyde in green
leaves there is much positive evidence || and mention has
already been made of the formation of formaldehyde and of
formic acid from carbon dioxide and water under the influence
of different forms of energy.

Willstatter and Stoll 1F argue that the " assimilatory ratio,"
CO 2 /O 2 , should provide evidence regarding the possible inter-
mediate products; they point out that oxalic, formic and
glyoxylic acids and formaldehyde are possible intermediate
products between the carbon dioxide and carbohydrate. The
assimilatory ratios of these substances are respectively, 4, 2,
I -3 3 and I. Under the conditions of their experiments, they
found the assimilatory ratio to be I and it remained constant,
even in succulents, under varying conditions in the amounts of
carbon dioxide and oxygen and with the temperature ranging

* Bokorny: " Biol. Zentrbl.," 1897, 17, i, 1916, 36, 385 ; " Ber. deut. chem.
Gesells.," 1891, 24, 103.

f Treboux: "Flora," 1903,92, 49.

% Grafe: "Ber. deut. hot. Gesells.," 1911, 29, 19.

Baker : "Ann. Bot.," 1913^27, 411.

|| See Vol. I., Section on Aldehydes.

IF Willstatter and Stoll: " Ber. deut. chem. Gesells.," 1917, 50, 1777.



42 THE SYNTHESIS OF CARBOHYDRATES

from I o C. to 3 5 C. Wherefore, it is concluded that the carbon
dioxide is reduced to formaldehyde, this being the only hydrate
having a single carbon atom.

Other authors consider that formic acid is the more likely
intermediate product of early origin. Erlenmeyer was the first
to make the suggestion, but it is only of recent years that
renewed attention has been given to the possibility. The work
of Spoehr has already been mentioned : he demonstrated that
carbon dioxide and water are easily reduced to formic acid
by means of radiant energy ; from formic acid a sugar-like
substance is formed, under conditions such as obtain in an
active leaf, which the plant can utilize as a food stuff. He
points out that in Grafe's and Baker's work the plants were
in reality supplied with formic acid, since formaldehyde is
oxidized in the light, and he himself found that plants in-
creased in dry weight in an atmosphere of formic acid. Wis-
licenus * also supports the formic acid hypothesis.

Laboratory experiments on the polymerization of formalde-
hyde to sugars not infrequently are quoted in support of the
formaldehyde hypothesis.

The first successful attempt to bring about such a polymeriz-
ation was made by Butlerow in 1861, who, by the catalytic
action of lime water, at the ordinary temperatures, on trioxy-
methylene (itself a polymer of formaldehyde), obtained a syrup
with a somewhat bitter taste, which he called methylenitan.
Subsequently Loew undertook an investigation of the action
of milk of lime on formaldehyde, and devised the following
experiment. A 4 per cent solution of formaldehyde is mixed
with an excess of milk of lime and repeatedly shaken for about
half an hour ; after filtering, the mixture is set aside for some
days until the pungent smell of formaldehyde has disappeared.
The solution, which will now reduce Fehling's solution, yields a
colourless syrup with a very sweet taste.f This substance,
which is known as crude formose, was examined by Emil
Fischer, who found it to consist of a mixture of various

* Wislicenus : " Ber. deut. chem. Gesells.," 1918, 51, 942.

fMore recently Ewart (" Proc. Roy. Soc.," Victoria, 1919, 31, 328) has
worked out the best conditions for the polymerization of formaldehyde to sugar,
and finds that the most rapid reaction is produced by sodium hydroxide in the
presence of a neutral calcium salt.



FORMATION OF HIGHER CARBOHYDRATES 43

hexoses and succeeded in isolating from it a small quantity
of a sugar acrose. Acrose can also be obtained by the
action of dilute caustic soda on glycerose, a substance obtained
by the oxidation of glycerol. From the acrose thus formed
Fischer was able by an elaborate series of reactions to prepare
ordinary fructose or levulose. In respect to the action of
weak alkalis, Spoehr found that sunlight had no action on a 3
per cent solution of formaldhyde in the presence of such salts
as calcium carbonate, potassium carbonate and potassium
nitrate in decinormal concentration, no trace of sugar being
found after an isolation period of four months.

With regard to the formation of the higher carbohydrates
from fructose, practically nothing is known. Whilst the syn-
thesis of levulose from formaldehyde in vitro, and the con-
version of levulose into glucose are established facts, no
success has as yet attended attempts to synthesize sucrose
from its two hexose sugars, dextrose and levulose. In this
connexion, however, it may be mentioned that the synthesis
of disaccharides from monosaccharides has been achieved only
in a very few cases. Fisher and Armstrong * were able to
synthesize a disaccharide isolactose by the action of an
enzyme, Kefir lactase, on a mixture of glucose and galactose ;
the same authors also synthesized melibiose. Similarly iso-
maltose has been obtained by Croft-Hill f by the action of
maltase on glucose.

It is obvious from this survey that the hypothesis of Baeyer
has influenced to a remarkable degree investigations on the
synthetic aspects of carbon assimilation ; this to a large extent
is psychological, for the hypothesis is attractive in that it is
plausible and lends itself readily to test tube investigation.
The evidence readily centres around distinct nuclei the identi-
fication in the plant of the critical initial intermediate products ;
the effect of presenting the plant with some of these initial sub-
stances in place of natural raw material ; and the elaboration
of the final from the supposed initial products in the laboratory
and may be briefly considered under these heads :

Carbon Monoxide. This gas, if produced, must be fixed

* Fisher and Armstrong: " Ber. deut. chem. Gesells.," 1902, 35, 3144.
t Croft-Hill: ' Journ. Chem. Soc.," Lond., 1898, 73, 634 ; see also Emmer-
ling : " Ber. deut. chem. Gesells," 1901, 34, 600, 2206.



44 THE SYNTHESIS OF CARBOHYDRATES

with extreme rapidity, for there is no evidence of its occurrence
as such in assimilating tissues, remarkable for a compound so
inert. The balance of evidence is against its use by the plant
when substituted for carbon dioxide.

Formaldehyde. There is no doubt that formaldehyde can
be obtained from green leaves, there being many compounds
which yield it more or less readily on decomposition. The
production of formaldehyde in artificial chlorophyll systems
was once considered strongly to support Baeyer's hypothesis,*
but the work of Jorgensen and Kidd, who were the first to use
pure chlorophyll in this connexion, seriously discounts the
significance of the reactions since the formaldehyde has its
origin in the oxidation of the chlorophyll ; it is not produced in
such systems if oxygen be absent. Much of the evidence re-
lating to the production of formaldehyde from carbon dioxide
and water under the influence of radiant energy is contradictory.
The work of Baly and Heilbron, however, has placed this as-
pect of the subject on a much firmer basis.

The facts relating to the production of formaldehyde in the
presence of catalysts, although of interest in themselves and
important in other connexions, provide no proof that such re-
actions obtain in the normal plant.

The contention of Willstatter and Stoll based on the as-
similatory ratio is ingenious, but, since the formaldehyde was
not actually demonstrated, the evidence is not convincing.

Formic Acid. As compared with formaldehyde the formic
acid hypothesis has received much less attention : Spoehr's
judgment appears to be just ; there is more in its favour than
for formaldehyde.

Feeding Experiments. That plants under abnormal con-
ditions, deprived of their natural raw food materials, may
make use of other substances in the elaboration of their food
shows that the plant is a transcendental chemist and in virtue
of its powers can to a certain degree make use of the sub-
stitutes. The facts do not prove that these substitutes, for-
maldehyde and formic acid, are normal intermediate products
in the natural process. If, on the other hand, it is held that

* See, for example, Usher and Priestley : " Proc. Roy. Soc.," Lond., B., 1906,
77, 369; 1906, 78, 318; 1911,84, 101.



THE VIEWS OF WILLSTATTER AND STOLL 45

this kind of evidence is of direct value then the weight of evi-
dence is on the side of formic acid.

The Production of Sugar from Formaldehyde. The evi-
dence for the condensation of formaldehyde to hexose sugars
in the laboratory under the influence of various catalysts is
convincing : but valuable though such evidence be, especially
in providing points d'appui, it does not follow that such a
sequence obtains in the plant. The formaldehyde hypothesis
requires a hexose as the initial sugar : the general opinion
of those who have concerned themselves with the final products
of carbon assimilation is that sucrose, not a hexose, is the
first recognizable sugar.

For these reasons, Baeyer's hypothesis, a theory which has
lived for fifty years, cannot be considered to be substantiated.


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