RESPIRATION OF PHYGOMYGES

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RIJKSUNIVÊRSITEIT

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RESPIRATION OF PHYCOMYCES

PROEFSCHRIFT

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. B. J. H. OVINK. HOOGLEERAAR IN DE
FACULTEIT DER LETTEREN EN WIJSBE-
GEERTE. VOLGENS BESLUIT VAN DEN
SENAAT DER UNIVERSITEIT TE VERDE-
DIGEN TEGEN DE BEDENKINGEN VAN DE
FACULTEIT DER WIS- EN NATUURKUNDE.
OP MAANDAG 21 NOVEMBER 1927,
DES NAMIDDAGS TE VIER UUR

DOOR

SIEBE RIEKELE DE BOER.

GEBOREN TE DRACHTEN

AMSTERDAM T MCMXXVII

). n. DU bUobY

BIBLIOTHEEK DER
RIJKSUNIVERSITEIT
UTRECHT.

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AAN MIJNE OUDERS

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Nu ik gekomen ben aan het einde van mijn academische
studie, past mij een woord van dank aan LI allen, hoog-
leeraren der philosophische faculteit, voor Uw onderricht,
vooral U, hooggeleerde Van Romburgh, Nierstrasz,
Jordan, Pulle, Kruyt, We st er dijk en Went.

Drie Uwer hebben in het bijzonder tot mijn vorming
bijgedragen.

Gij, hooggeleerde Kruyt, zijt het geweest die den
doorslag hebt gegeven tot de richting die ik in mijn
studie zou volgen. Voelde ik aanvankelijk meer voor de
morphologische vakken in de biologie, Gij hebt mij geleerd
welke bekoring er uit gaat van het experiment. Uw
colleges waren voor mij een openbaring. Hoewel ik de
laatste jaren minder met de kolloïdchemie in aanraking
kwam, denk ik nog steeds met zeer veel genoegen terug
aan den tijd toen ik op Uw laboratorium werkte.

Hooggeleerde West er dijk. Mogen sommigen de toe-
gepaste botanie niet tot de exacte wetenschappen willen
rekenen, de wijze waarop men in Uw laboratorium de
vraagstukken der phytopathologie leert bestudeeren bewijst
de onjuistheid dezer opvatting en moet bij ieder belang-
stelling voor die wetenschap wekken, ook bij hen. die in
het begin „im Herz abgewandt" zijn.

Ik ben U zeer erkentelijk voor de wijze waarop Gij
mij met de problemen der praktijk in aanraking hebt
gebracht, zoowel door Uw persoonlijke omgang, het
werken in Baarn, als door Uw excursies, waarvan ik
vooral die naar Denemarken wil noemen.

Hooggeleerde Went, hooggeachte promotor. Ik ben
in mijn studietijd wel buitengewoon bevoorrecht geweest,
doordat ik reeds vroeg Uw assistent werd en zoodoende
jaren lang voortdurend met U in aanraking was, niet
alleen in het laboratorium maar ook in Uw huis. De
gastvrijheid van U en Mevrouw Went zal steeds in mijn
herinnering blijven.

Het is moeilijk onder woorden te brengen wat ik U
verschuldigd ben. Ik dank U zeer voor de hulp, die Gij
mij steeds hebt verleend, de wijze waarop Gij mij als
botanicus hebt gevormd door Uw leiding en critiek en de
gelegenheid die Gij steeds hebt geboden om kennis te
maken met geleerden uit binnen- en buitenland.

Bij de bewerking van mijn dissertatie heb ik verder veel
hulp mogen ondervinden in andere laboratoria, waarvoor
ik U hooggeleerde Kolthoff, Schoorl en Ornstein
veel dank zeg.

Van U, waarde Greta Mes, waarde Mulder en
hooggeleerde Baas Becking, heb ik veel bijstand mogen
ondervinden bij de vertaling van dit proefschrift.

Aan U, P. A. de Bout er, zij dank gebracht voor de
hulp bij het toestel en U. A. de Bouter, voor het
vervaardigen der teekeningen, terwijl verder de heer
Lobel mij steeds zeer behulpzaam was bij het maken
der voedingsbodems.

{Extrait du Recueil des Travaux botaniques néerlandais, Vol. XXV, 1928).

RESPIRATION OF PHYCOMYCES

by

S. R. DE BOER.

INTRODUCTION.

METHODS AND APPARATUS.

In the following pages a description is given of experiments
on the respiration of Phycomyces Blakesleeanus Burgeff.
This fungus is wellknown under the name of Phycomyces
nitens Kunze, but two years ago, Burgeff (16) showed
that the latter name really belonged to another Phycomyces,
so he called the former Phycomyces Blakesleeanus.

In all the experiments transfers were used from a one
spore culture of a 4- strain, obtained from the „Centraal-
bureau voor Schimmelcultures" at Baarn, Holland.

Two kinds of respiration apparatus were used, one of
the air current type and the other of the closed space or
Regnault type.

Respiration, consisting in evolving carbon dioxide and
absorbing oxygen, is generally measured.
1® by determining the CO.^ given off
2° „ „ „ O2 taken in

3° „ using both methods simultaneously.
The first method has already been worked out by
Pettenkofer (69); later on Pfeffer (70, 95) introduced
it into plant-physiology. This socalled Pettenkofer-Pfeffer
method consists in forcing or sucking a current of air depriv-
ed of carbondioxide through the respiration vessel. In
leaving it the air has to pass a solution of bariumhydroxide
in long horizontal glass tubes, called Pettenkofer-tubes.
The quantity of COo given off by the plant can be deter-

mined from its absorption by the barytawater and sub-
sequent titration. The air current can be led alternately
into different tubes. Another absorber with barytawater
behind the Pettenkofer-tubes will tell that all the COg
has been absorbed in the tube, if the solution in the absorber
remains clear.

This method is a very simple and handy one and can
always be used when the only thing to be settled is the
amount of the COg, given off by the plant. It was in such
cases that I made use of it, the air being sucked through
the system by an aspirator of the kind described by
Fernandes (31). Attached to it was an open mano-
meter with mercury which enabled me to check the sucking
force of the aspirator.

The respiration chamber was placed in a glass basin
filled with water, kept at a constant temperature by elec-
trical heating. Inside the glass basin there was also a metal
spiral tube through which the air had to pass before entering
the respiration vessel in order to take on the same tem-
perature.

Before entering the vessel the air was purified in different
washing-bottles filled with solutions of sodium hydroxide,
strong sulphuric acid, silver nitrate and potassium per-
manganate. In leaving the respiration vessel the air passed
a small absorbing flask with sulphuric acid. Without this
precaution, the air, rich in watervapour given off by the
respiring plant, would render the bariumhydroxidesolution
less concentrated. The sulphuric acid should not be taken
too strong, for in this case an absolutely dry air enters the
Pettenkofer-tube and a humid one leaves it, also chan-
ging the strength of the barytawater.

Half a century ago Ad. Mayer (61, 96) determined
the Og- absorption by measuring the decrease in a volume
of air, which was shut off from the atmosphere by means

of mercury, a strong alkaline solution absorbing all the
CO2 evolved by the plant.

By measuring the COg absorbed by the alkaline solution,
Godlewsky (35) determined the Og taken in and the
CO2 given off by the plant at the same time.
Bonnier and Mangin (11) examined the O2 absorbed
and the COg given off by the plants by analysing samples
of air. It is obvious that in both methods a lack of oxygen
may effect the respiration. Moreover, in the last method,
a lot of CO2 may be formed in the experimental vessel,
which may injure the plants. Besides this, the more there
is of CO2, the more will be absorbed by the water and the
plants, so the amount of COo found will be too small.

In zoo-physiology an apparatus is used without these
drawbacks, the prototype of which was published by
Regnault and Re is et (76). It has subsequently been
modified and improved by Benedict (7) and by
Fredericia (33). In these apparatus a supply of oxygen
has to compensate the decrease in the oxygen pressure
inside, so as to keep it as nearly as possible to the original
pressure. Various devices are in use. Connected with the
chamber a volume recorder is often arranged, which will
at a certain point close an electric circuit and admit oxygen
from a cylinder and reduction valve.

In 1923 Fernandes (30 and 31) introduced into
plant-physiology an ingenious respiration apparatus on the
Regnault-Reiset principle. The oxygen is obtained by a
new\'method, i.e. by hydrolyzing a sodium hydroxidesol-
ution. As much O2 as is taken up by the plant is formed
and led to the respiration vessel. So the amount of oxygen
in the closed space always remains the same. The hydrogen
formed at the other electrode is collected in an eudiometer.
It is equal to twice the amount of oxygen absorbed by the
plant. A pump circulates the constant volume of air through
the system. By means of tubes filled with barytawater the

COa can be absorbed. As both Og and COg are determined
the respiratory quotient is known.

Fe man des did not pay much attention to the respir-
atory quotients. I made the determination of the respir-
atory quotients an important part of my investigations.
I soon discovered that in order to be able to determine the
quotients accurately, certain alterations had to be made.

For a detailed description of the original apparatus I beg
to refer the reader to Fernandes\' publication. Here
I am only going to give a schematic description of the appa-
ratus and its improvements as shown in fig. 1. The closed
space with the constant volume of air is formed by the
system A, B, etc... H. By means of H the air is pumped
in the direction indicated by the arrows. A is the respiration
chamber, the air entering at the top and leaving the vessel
at the bottom. Then the air has to pass the washing-bottle
B filled with a solution of sulphuric acid of which the same
may be said as on p.ll8. D and E are Pettenkofer tubes, long
35 c.m., diameter 2.1 c.m., each filled with 80 ccm. barium-
hydroxide solution, absorbing the COo given off by the plant.
G is the control barytatube. The level in the right leg of
the manometer L would rise if oxygen was not formed m
the electrolyzing vessel M. The electrolyzing current can
be regulated by means of a resistance. In the circuit I placed
amilliamperemeterwhich was a great convenience as will

be proved afterwards.

It appears from the diagram that the most
improvement made is the compensating vessel K. In star-
ting an experiment the vessels A and K are shut off from
the atmosphere by closing the taps I and J. Changes in
barometric pressure can have no influence then. It stands
to reason that the electrolyzing vessel M must be shut off
too. The glass tube at the left in which the hydrogen is formed
and conducted to the eudiometer N is in connection with
the atmosphere. O is a levelling device consisting of a glass

H

TZJ

to

tube with water in connection with the lower end of the
eudiometer N. During or at the end of each experiment
O has to be raised or lowered till the pressure at the point
* is the same as at the beginning of the experiment, which
can be seen from the level in M being the same in the elec-
trolyzing vessel and both its tubes.

With the suction velocity employed, one Pettenkofer tube
is not sufficient. Two are necessary though the upper tube
has to absorb only a small amount of COo. The use of
2 Pettenkofer tubes instead of 3 vertical absorptiontubes
has many advantages. The difference in pressure is now
only 3 c.m. water, whereas Fernandes had a difference
of 35 c.m. This lessens the chance of leakage. The two
tubes are attached to a frame. There is room enough for
two of such sets of tubes. By means of the three-way-
taps C and F these sets of tubes can be changed at
any time so that any new experiment can go on without
interruption.

As far as possible the connections are of glass, the rest
consisting of heavy vacuum-tube. As rubber will absorb
CO2 the section between A and D is kept as short as possible
Behind E (e.g. in the pump H) CO« cannot be lost by
diffusion, because there is no more CO™ present.

When the apparatus is ventilated, purified air enters
at F, passes the vessel A and leaves the system at C. I ven-
tilated for instance when taking long experiments where
measurements cannot continually be carried out.

Except for O and N the whole apparatus is fixed in a
glass basin with water, which is kept at a constant tempera-
ture (it 0.03° C.) by electrical heating.

The humidity in the respiration vessel during the experim-
ents was always very high and nearly saturated for two
reasons, 1° because the entering air was sucked through
water and 2° because the respiring plant gives off water.
It was checked by means of a hygrometer.

During my experiments I also investigated the influence
of pure oxygen on respiration. The oxygen entered at tap
F, passed absorber G, the respiration vessel and absorber
B, and left the system at tap C. In this way the liquid in the
system, for instance that in B and G, was saturated with
oxygen. It stands to reason that, when starting an experiment,
the barytawater in the Pettenkofer tubes also had to be
totally saturated with oxygen before connecting them to
the taps C and F. Otherwise oxygen would be absorbed,
because oxygen is more soluble in water than air. In 160 ccm.
of solution at 25° C., 2.8 ccm. air and 4.6 ccm. oxygen are
absorbed at a pressure of 1 atm. It was necessary to lead an
oxygen current of the same temperature as that of the
basin through each new set of tubes for 10 minutes before
using it in a new experiment. Otherwise oxygen would
dissolve and one might get the impression that there was
a higher respiration than was actually the case.

An analogical difficulty was encountered when using
different temperatures. In 160 ccm. of solution, air is dissolved
to an amount of 3.7 ccm. at 10°; 3.05 ccm. at 20°; 2.65 ccm.
at 30° and 2.25 ccm. at 40°. Suppose the temperature in the
room is 15°. Then the temperature of the solution in the
Pettenkofer tubes before putting them in the waterbasin
will be the same. When experiments are carried out at
37°5 about 1 ccm. of air will be given off. A mistake of
1 ccm is a very large one, as may be seen afterwards. So
whenever there was a difference between the temperature
in the room and the temperature of the water an air current
was pumped through each set of tubes for 10 minutes before
starting the new experiment, because in this way the equil-
ibrium was much more quickly attained than when the
liquid remained undisturbed. This air first passed through
a metal spiral in order to assume the temperature in question.

The rectangular respiration vessel in the circulating
apparatus had a width of 3.5 c.m., a length of 8.5 c.m, and

a depth of 10.5 c.m. These were the inside dimensions.
With the washing-bottle B its contents were 325 to 350 ccm.
The lid and the bottom were double. The inner walls were
perforated at different places, except near the openings
of the tubes. This aids the distribution of the air current.
The sides of 10.5 X 8.5 c.m. were of glass. The glass was
provided with horizontal lines at a distance of % c.m. so
that the growth of the sporangiophores could be observed.
Two slides, impenetrable to light, were usually covering
the glass walls, preventing the phototropic curving of the
sporangiophores.

Many blank experiments were carried out in order to
test the apparatus. The decrease in the strength of the
barytawater in the Pettenkofer tubes, probably owing to
a condensation of water vapour, on an average did not exceed
an amount corresponding to 0.1 ccm per hour. I neglected
it, the more readily as the decrease in the constant volume
of air in the apparatus was on an average 0 15 ccm per hour.
These two facts compensated each other. It seems that
the metal of the vessel was oxydized to a slight degree. In
pure oxygen the decrease in volume was much larger,
namely 0.6 ccm per hour, whereas the change in the bary-
tawater had remained the same as in air. With the experi-
ments in pure oxygen the error of 0.6 — 0.15 = 0.45 ccm
per hour was taken into account.

In the air current apparatus I sometimes used another
vessel measuring 13 X 12 X 5 c.m. inside, the contents
being about 800 ccm. By means of different blocks of metal
exactly fitting in the vessel I could reduce the volume to
any required degree.

It takes some time for the COo given off by the plant
to reach the Pettenkofer tubes. This will take longer, as
the experimental chamber is bigger and the suction-velocity
smaller. Therefore the relation between the suction velocity
and the volume of the respiration vessel is of the utmost

importance in studying the influence of some external
factor on respiration. I studied this relation by liberating
in the vessels of 325 and 800 ccm one milligrammolecule
of COg (22.4 ccm). At a certain moment I emptied a tube
with 3 ccm of n. hydrochloric acid in a small glass basin,
containing one milligrammolecule of sodiumcarbonate (106
mgm). Air was sucked through the vessel at different velo-
cities. The tubes were very often changed. The results are
to be found in table 1 and 2.

In my experiments I mostly used the respiration vessel
of 325 ccm at a suction-velocity of 3 or 3^4 per hour.

TABLE 1.

Vessel of 325 ccm. cubic contents.

la. Suction velocity 1.81. per hour.

After 5 min. 7.4 ccm. COj.

„ 10 „ 13.1

,, 20 f, 18.7 ,, ir

„ 30 „ 21.1

ft 60 tt 22.8 »» ft

Half the amount has disappeared
after 8 min.

Ic. Suction velocity 3 1. per hour.

After 5 min. 11.55 ccm. COa.

10 „ 17.3 „

» 20 „ 20.8 „

»r 30 21.7 ff ft

n 70 „ 22.15 „

Half the amount has disappeared
after about 4^/i min.

Ife. Suction velocity 2}4 1. per
hour.

After 5 min. 9.15 ccm. COj.
„ 10 „ 14.9 „
„ 20 „ 19.4 „
30 „ 21.1

60 ft 22.1 It tt

Half the amount has disappeared
after nparly 7 min.

Id. Suction velocity 3\'^ 1. per
hour.

After 5 min. 12.75 ccm. COj.

„ 10 „ 17.95 „

„ 20 „ 20.75 „

„ 30 „ 21.65 „
70 „ 22.1

Half the amount has disappeared
after about 4 min.

2c. Suction velocity 3 1. per hour.

After 10 min. 12.3 ccm. COj.
„ 20 „ 17.4 „
„ 30 „ 19.7 „ »
„ 50 „ 21.7
„ 95 „ 22.35

Half the amount has disappeared
after 8% min.

2d. Suction velocity 3>/2 1. per
hour.

After 10 min. 13.4 ccm. CO,.
„ 20 „ 18.6
„ 30 „ 20.7
„ 50 „ 22.2 „
„ 90 „ 22.9

Half the amount has disappeared
after 7% min.

It follows from table 1 that in this case the CO« on an
average reaches the barytawater after fully 4 minutes.

As regards former investigators .K u y p e r (52) used a
cylindrical vessel, height 16 c.m., diameter 10 c.m., so
measuring about 1250 ccm. The air was sucked through at
the rate of 3 1. per hour. The relation was about the same
as in table 2a. So in Kuyper\'s experiments CO« newly
formed needed on an average 25 minutes to reach the Petten-
kofer tubes.

Usually I employed a solution of about 0.05 n. of barium-
hydroxide, equal to about 7.9 grm. per 1., the molecular

weight of Ba(0H)2 8 aq. being 315.6. As the commercial
bariumhydroxide is impure I had to dissolve about 9 grm.
per 1.1 dissolved the hydroxide in hot water in flasks of about
8 1. and allowed the insoluble carbonates to settle. The
clear solution was then siphoned. A little more than 1 grm.
BaClo per 1. was added in order to repel the solubility-
product of the BaCOg.

The titer of the barytawater before and after an experiment
was determined by means of 0.05 n. HCl. The latter was
made to correspond to a 0.05 n. NaOH solution, in its
turn corresponding to a 0.05 n. solution of oxalic acid.

CHAPTER L

THE RESPIRATION OF PHYCOMYCES ON DIFFERENT MEDIA.

§ 1. Introduction.

At first my purpose was to carry out respiration experi-
ments with cultures of Phycomyces on a culture medium
of a definite well-known composition. It is possible to
cultivate Phycomyces on sugar media. Lindner (56) for
instance found that the strain grew very well on maltose,
raffinose and dextrinesolutions, the — strains moreover also
on glucose, fructose and saccharose. Grete Orban (65)
on the contrary thought that the -f strains were less par-
ticular and consumed more different kinds of sugar than
the — ones. More contradictions are to be found in literature
as to which kind of sugar is the best medium. The reason
may be the existence of a considerable number of races
with different physiological properties, as Satina and
Blakeslee (e.g. 84) have shown, so that the investiga-
tors probably have worked with different races. The race
I investigated for instance grew better on saccharose than
on maltose, but probably this fact does not hold good
for all races.

The more sugar has been dissolved in the liquid, the
better the fungus will grow. Of course there are limits,
because at higher concentrations the osmotic pressure
prevents the growth. The same is to be seen on agar media.
Neither can the growth be continually increased by taking
thicker layers, as the aerophilous mycelium remains in the
uppermost part of the agar. Schmidt (86) has investig-
ated the same subject in detail. The growth was therefore
never strong enough to work with when the fungus was
cultivated on sugars.

On starchagar the growth of the fungus can be increased
by enlarging the amount of starch, as the harmful influence
of higher osmotic pressure does not exist in this case. On
starchagar media however, oxygen cannot enter sufficiently.
With fatty media we see the same thing. F1 i e g (32)
found that Phycomyces did not grow very well on fat;
probably there was also a lack of oxygen in this case. With
the above media sufficient respiration can of course be ob-
tained by taking a great quantity of them in thin layers.
But in doing this a large respiration vessel is necessary,
the drawback of which has been shown on p. 126. Secondly
the larger the amount of culture medium the more CO2
will remain dissolved in it.

In my experiments I therefore used such solid culture
media as always contain a sufficient amount of food and
allow the air to enter freely. For a starch-medium I used
bread, for an oil-medium ground linseed, both soaked in
water. Some of the culture media were analysed in the
pharmaceutical laboratory of Prof. Schoorl, to whom
I am very much indebted for his kind assistance.

In the carbohydrate medium there are proteins and some
fats, in the oil-medium we also find carbohydrates and
proteins. With the apparatus described in the introduction
I was able to measure the respiratory quotients very accurat-
ely. My purpose next was to determine by means of these
quotients what kind of food Phycomyces would take from
such a heterogeneous culture medium. When carbohydrates
arc consumed the respiratory quotient will ordinarily be
about 1.00, when fats or proteins are combusted it will
be smaller as more oxygen has to be absorbed in comparison
with the carbohydrates.

During the growth of the fungus the p« of the culture
medium is changing. As the principal subject of my investi-
gations was respiration I only examined the change in the
acidity of the medium qualitatively by means of Ph measure-

merits. If examining the whole metabolism of Phycomyces,
I should also have had to determine the quality and quantity
of the acids formed, as p« is not a measure for the quantity
of acids in a culture medium. For instance it depends on

its buffer-capacity.

I determined Ph by means of the quinhydroneelectrode.
I am very thankful to Dr. K o 1 t h o f f for helping me
with this part of my investigations. Media with a small
buffer capacity cannot be measured, except when very
pure quinhydrone is used (46). My culture media, however,
had a large buffer capacity so that these precautions could
be omitted.

The fungus was cultivated in two ways. Firstly in small
earthenware troughs, containing about 13 ccm, area 6
sqcm, depth about 2 c.m. The dry weight of the bread in
it was about 6 grm. and of the ground linseed, etc. about
41/2 grm. Secondly the food was given to the fungus in a
thin layer on rough linen, which was stretched over a small
glass frame of 7 X 2Y> c.m., in such fashion that the medium
was in contact with the air both above and below. I always
used two of such frames fitted above each other in the
respiration vessel. For the sake of brevity they will be
spoken of in the tables as "two layers".

All experiments in this chapter were carried out at 25° C.

§ 2. Respiration on Carbohydrate Medium.

The analysis of the bread\') used per dry weight was as
follows: carbohydrates 77 %, proteins about 10 % and
fat nearly 1 %. Of the carbohydrates about 10 % are soluble,

such as maltose.

On bread first a generation of thin sporangiophores appears,
followed by a generation of thick ones. In course of time

In Holland the bakers sometimes mix the flour with water. I
always used this "waterbread". If milk is used the percentage of
fat is higher (see p. 139.)

new generations of sporangiophores follow. Fig. 2 (table 3)
shows the whole grand period of the respiration of Phyco-
myces on a small trough with bread. Both the amount of
COo given off and of Oo taken in increase very rapidly,

the more the mycelium penetrates through the culture
medium. About 31/2 days after transferring, i.e. 2 days
after the surface has been covered by mycelium, the respi-
ration reaches a maximum. The sporangiophores of the
first generation are now very thin and have nearly stopped
growing, the thick ones are about 21/2 c.m. long. From this
moment respiration decreases gradually: to half the amount

after 3 or 4 days.

As for the respiratory quotient, at the moment of the
most intensive respiration it.is higher than 1.00. As respir-
ation decreases the quotient drops from about 1.15 to
about 1.00. I wondered whether in the long run the quotient
would continue decreasing or remain at 1.00. I therefore
carried out an experiment with an old culture, which had
remained at room-temperature for about 2 or 3 weeks.
From table 4 it appears that old cultures also show a quotient
of about 1.00 on these starch media.

Why is the quotient higher than 1.00 when respiration
is at its full strength? The thought occurred to me, that
the cause might be that the air could not enter sufficiently
into the culture troughs, which are relatively deep. Thus
COo might have been formed anaerobically. If so, the
quo\'tient would be smaller if the air could enter better.
I now used for the first time the frames with the thin layers
of culture medium, mentioned above. The result of the
experiment with 2 X P/t grm. bread is shown in fig. 3
(table 5). Contrary to .expectation the quotient is raised
and fully 1.20. I therefore carried out many experiments
on this point and the quotient always turned out to lie

between 1.20 and 1.25.

Also from fig. 3 it follows that in the end the quotient
approaches the value 1.00. As here the amount of culture
medium is smaller, the grand period of the respiration is
less extended than in fig. 2, whereas the sporangiophores
are yet smaller during the maximum respiration.

Phycomyces does not behave like the Mucoraceae of the
Mucor group, which are able to ferment sugars, as
Brefeld (15) already showed. Kostytschew (48) grew
Mucoraceae on bread without finding 5hy fermentation;
on sugar being added to the bread fermentation appeared.
In case the high respiratory quotient of Phycomyces
were due to a fermentative process, the quotient should
rise if the bread were soaked in a sugar solution instead
of in water. This is not so, however, no such rise being
found for instance with bread soaked in a 5 % solution

of saccharose. Respiration as such however is more intense
in this case.

As the high respiratory quotient on bread is a normal
characteristic of the fungus, substances are apparently
manufactured containing less oxygen than carbohydrates.
The presence of alcohol etc. cannot be demonstrated, so
I suppose that the value of the respiratory quotient is prob-
ably at first higher than 1.00 because carbohydrates are
changed into fats. In the mycelium fats can be demonstrated.
Also the facts mentioned in chapter III render the suppo-
sition probable.

The more respiration decreases the lower sinks the
respiratory quotient. In the troughs the respiration in the

upper part of the culture medium will be in the descending
part of the grand period, whereas below the respiration
is at its maximum. So the average respiratory quotient
never reaches tlie value measured on the thin layers of
culture media. In examining different external influences
afterwards, I therefore always used the last culture method,
as processes are simpler here.

As for the acidity of the medium, Schmidt (86)
found that Phycomyces turned the medium acid. This
holds good for the above carbohydrate medium. The pn
of bread is about 5.85 and is made smaller by the fungus,
for instance after 2 weeks it is 5.3, after 3 weeks 4.5.

§ 3. Respiration on Media with a varying Amount

of Fat.

As has already been stated I used ground linseed as an
oily medium. Here the fat and also the other food proteins
and carbohydrates are distributed through the medium, so
that the air can enter better than on pure fat. The compos-
ition of linseed per dry weight is as follows: fat 35 % and
proteins 25 %. According to the text-books (e.g. 99)
carbohydrates occur as much as 9 % sometimes, the greater
part however consists of pectin-mucilage, the smaller part
of sugars. Phycomyces thrives exceedingly well on fatty
media. Fat is well-known to have a high combustion energy
(H o e b e r 40). F 1 i e g (32) cultivated Aspergillus niger
on fatty media. On sugar media however respiration was
more intensive. Phycomyces on the contrary is a true fat
consumer as will be shown presently. Respiration is inten-
sive on fatty media. The fungus prefers fat out of a mixed
culture medium. The sporangiophores also grow better.
As a rule a thin generation is not to be observed on ground
linseed. One generation of very thick sporangiophores
appears, growing faster than the ones on bread. The spo-
rangiophores are very numerous and arise as it were all

at the same time, leaving no space for following generations,
for in contradistinction to the starch-media new generations
hardly follow.

In fig. 4 (table 6) the grand period"of respiration is shown

Fig. 4. Grand Period of Respiration on a Small Earthenware Trough with
Ground Linseed (oil 35 per cent.). Sec table 6.

on a small trough with ground linseed. Respiration increases .
very rapidly and about 3)4 days after transferring, i.e.
nearly 2 days after the surface was covered with mycelium,
it reaches its maximum. The sporangiophores then have
a length of from 4 to 5 c.m. Then respiration decreases

gradually as on the starch-media. In consequence of fat-
combustion the respiratory quotient is low. It drops to
about 0.66 and remains there during the maximum respira-
tion. When respiration decreases the respiratory quotient
rises in a few days to about 0.75.

From the composition of linseed-oil (93) it appears that
in consuming only the oil, the quotient should be about
0.72. As will be proved hereafter carbohydrates are also
consumed in some measure. Therefore the quotient should
be found still higher, whereas it is lower. Phycomyces on
oil-media apparently takes up more oxygen than is neces-
sary for the combustion of fats. Oxygen is apparently fixed
by changing fats into carbohydrates.

This fact has been mentioned in earlier literature for
seedlings. Sachs (82) found microchemically that, in
germinating fatty seeds, fat was changed into carbohydrates.
G o d 1 e w s k i (35) determined the respiration of fatty seeds
and found the respiratory quotient falling from 1.00 to
about 0.60. So carbohydrates were manufactured. Later
on the quotient again approached 1.00, so the carbohy-
drates formed were consumed. Fungi, growing on fatty
substances, show this phenomenon very often in a very
strong degree as Flieg (32) records.

I also examined the respiration on linseed-meal, con-
taining 14^4 % oil. Fig. 5 (table 7) shows the respiration
on a small earthenware trough. The course of the grand
period resembles that on ground linseed. Respiration in-
creases less rapidly at first and remains a little lower. The
maximum of respiration again takes place when the spo-
rangiophores are about 4 c.m. long. As there are less fatty
substances, the influence of the carbohydrates is more
obvious, as the respiratory quotient is much higher. It falls
during the most intensive respiration only to 0.76 and
rises during the following days to about 0.85.

UJ

In order to get a medium as the above with still less
oil, I kept linseed-meal for a considerable time in a bottle
with petrol ether. Now and then I renewed the ether. After
it had been removed quantitatively, the medium contained

about % fat. I grew Phycomyces on this medium.
The respiration now was completely changed as may be shown
in fig. 6 (table 8). Fat with the best nutritive value has

nearly disappeared so that the respiration is much lower,
though the remaining 21/0 still have great influence!
as the respiratory quotient is about 0.95 and the respiration
much higher than in the next case without fat.

On this medium appear always more generations with fewer

and thinner sporangiophores, and not one numerous gene-
ration with thick sporangiophores as in the former two cases.

I totally deprived a little of the above named medium
of fat by extracting it for 4 days in a Soxhiet with petrol
ether. The respiratory quotient is now about 1.00. Fig. 7
(table 9). A kind of carbohydrate-respiration takes place.
It seems that the abundance of proteins has no influence.
The respiration is smaller than on the medium with 2i/. %
oil, which proves that Phycomyces uses oil by preference
when it is there.

This also appears from the following experiment. Once
by mistake instead of waterbread, the so-called "milkbread"
was used, which contained about 2^2 % fat. This fact
lowers the respiratory quotient very much as may be seen
from table 10.

Afterwards, whenever I studied the influence of external
foctors on the respiration of Phycomyces on media rich
in oil, I did so on the method of thin layers on a set of frames.
In fig. 8 (table 11) the grand period of respiration is given
for such a culture on 2 X 1 \'/, grm. of ground linseed. The
respiratory quotient is again about 0.65. In consequence
of the smaller quantity of culture medium and the larger
surface of it as compared with fig. 4, the mycelium relat-
ively spreads more quickly and reaches the maximum
respiration sooner, namely about 2 days after transferring.
At this moment the sporangiophores are not yet visible.
When they appear the respiration is already decreasing.
The curve descends more rapidly than the one in fig. 4.
The whole curve is sharper and shorter.

§ 4. Consumption of Proteins.

Butkewitsch (17) showed that fungi only consume pro-
teins when no carbohydrates or fats are present. In this
case Aspergillus niger liberates a lot of ammonia. Pénicillium
and fungi like Mucor especially amino-acids.
Kostytschew (48) carried out respiration experiments

with fungi on proteins as a culture medium and found the
respiratory quotient to be about 0.50. Klotz (44) did not
find an ammonia production with fungi until the carbohy-
drates had entirely been consumed.

As has already been remarked it follows from fig. 7 that
Phycomyces does not consume proteins either if carbohy-

drates are to be had, as the respiratory quotient would
otherwise be much smaller. I investigated this question in
another way by baking bread containing respectively 10 %
and 20 % peptone. Moreover, it was just possible that in

10.0

5.0

the presence of simple proteins the whole respiration would be
raised. Fig. 9 (table 12) shows the respiration on two layers
of bread plus 10 % peptone. The curve looks like fig. 3,

where bread without peptone was used. Neither the res-
pirationvelocity, nor the respiratory quotient has changed.
The same is to be seen in fig. 10, giving the respiration
on a small trough with bread plus 20 % peptone. The
curve corresponds on the whole to fig. 2. At last the res-
piratory quotient seems to be lower than on bread.

So proteins take no essential part in the respiration of
Phycomyces. They are probably taken in a small measure,
as follows also from the change in the acidity of the medium.
Whereas on bread the Ph decreases, as stated above, on
bread plus peptone the p« rather increases, especially on
bread with 20 % peptone. The Ph of linseed meal is about
5.5, after one week of growth the Ph has risen to about
6.0, after 234 weeks to 6.8 and later on to 8.0, so that the
Ph cannot be measured well by means of the quinhydrone-
electrode.

When a culture of Phycomyces on linseed medium is
kept sterile for several weeks, ammonia is at last produced,
as indicated by its strong odor.

§ 5. Discussion.

In the above experiments, by measuring very accurately
the respiratory quotients, I determined the substances
consumed by Phycomyces in its respiration when cultivated
on heterogeneous culture media. Fat has a high nutritive
value, carbohydrates also are good culture media. Secondly
the grand period of the respiration was determined on food
substances of different quantities and qualities. The curves
ascend rapidly to a maximum of respiration, then descend
more or less slowly.

What may be the cause of this dropping of the curve?
Is it the decrease in food or the formation of harmful sub-
stances, products of metabolism? The same phenomenon
was already observed half a century ago with the respiration
of seedlings. R i s c h a w i (78) found that with Vicia Faba

seedlings, which have big cotyledons and therefore a lot of
food, respiration remained constant for a long time. So
want of food might be the cause of the decrease in the
respiration of other objects of investigation. Lately it was
Fernandes (31) who said that, when respiration decreas-
es with seedlings of Pisum sativum, there is no question
about want of food. The fall is probably the result of the
abnormal conditions of the seedlings in the respiration vessel.
Krzemieniewski (51) managed to raise the respiration
by adding minerals to the plants.

In our case abnormal conditions cannot be responsible.
One gets the impression that the decrease in the respiration
results from the food being more difficult to obtain at the
moment. When the mycelium starts to grow over the surface
of the culture medium, respiration increases very strongly
till the maximum has been attained. By that time the myc-
elium has penetrated the whole culture medium, if the
method of the two thin layers is used (fig. 8). A small part
of the food has indeed been consumed, but the hyphae
cannot spread in an untouched medium, so that the hyphae
approach each other and have to grow in every hole
and corner. When a small trough with medium is used
this process is more gradual, because the mycelium can
grow downwards for a long time. The curve in fig. 4 is
therefore less sharp than in fig. 8 and the maximum of
respiration is later.

It seems therefore probable that the shape of the curves
is governed by the available food supply, though special
investigations would be necessary to prove that no harmful
metabolic substances play a part.

In examining in the following chapters the influence of
external factors such as temperature, air of different com-
position, etc. on respiration, it should be borne in mind
that I always used cultures at the maximum of the grand

period. The experiments as a rule take about 12 hours,
during which time the respiration is nearly constant. Some-
times I employed the small earthenware troughs, where
the maximum appears about 3^4 days after inoculation at
25°. For the greater part I proceeded on the method of the
thin layers of culture medium on a set of two frames, and
mostly with 2x1^/4 grm. medium. In this case the maximum
of respiration begins about 21/2 days after the transfer on
the linseed medium. The medium is penetrated by the
mycelium, the thick sporangiophores already mentioned
being however not yet visible. Fully 12 hours later, when
the experiment is generally finished, they are about 1 c.m.
long. On bread, where the respiration is less intensive and
does not rise so rapidly as on oil-media, the cultures have
to be half a day older before an experiment can be begun.
The sporangiophores are then about 1 c.m. in length.

As stated above the length of the sporangiophores is an
index of their stage of development corresponding to a
specific point in the grand period. After some training this
can be seen at a glance.

When fungi are grown on liquid solutions the dry weight
of the fungous mat can be determined and so the respiration
can be compared per dry weight, although this method is
not at all an exact one, as dead cells etc. do not respire and
are included in the dry weight. As Phycomyces was cul-
tivated on solid media, dry weights could not be determined.
Notwithstanding this the different experiments can very
well be compared as, by cultivating on the described lines,
all cultures respired almost with equal strength.

In my cultures two factors affect the rate of respiration,
first the method of transferring and secondly the amount
of water in the culture medium.

The influence of the former is especially visible on the
starch-media. Ordinarily the thick generation of sporangio-

phores is preceded by one with very thin sporangiophores
as has been described above. When the medium is ino-
culated with very few spores, for instance by means of a
strongly diluted spore suspension, on bread only thick
sporangiophores arise. On the other hand when the whole
surface has been inoculated with a great number of spores,
a great number of sporangiophores appears, which are all
very thin. No thick ones appear. In this case the respiration
is also lower. All my cultures were therefore inoculated
in the same way: each frame was just touched in two spots
with the platinum needle, covered with spores.

The second influence is still of more importance. As stated
above the bread and the oil-media are mixed with water,
as no growth takes place on dry media. But when too much
water is used, the respiration is also too low, as may be seen
in table 13, where the respiration does not reach 75 % of
the amount of table 5 (fig. 3). The difference sometimes
is still larger. Therefore always the same amount of water
has to be mixed through the medium, namely about 2 grm.
of water per 11/^ grm. of ground linseed and fully 1 grm.
per gr^i- bread.

After some time it is easy to tell how much the respiration
of a culture will be, from the humidity of the culture medium,
as also the amount of water necessary for an intensive
respiration. On an average a culture on 2 X 1 ^^ grm. of
bread takes in during the maximum of respiration 5.75 ccm.
0« and gives off 7.0 ccm CO«, a culture on ground linseed
absorbs 15 ccm O., and evolves 10 ccm COj. (50 seedlings
of Pisum sativum, dry weight about 8 grm., evolve about
12.5 ccm CO, at 25°).

In former investigations on respiration with seedlings
etc. as a rule no allowance was made for the fact that bacteria
might accompany the objects of investigation; Fernandes
(31) proved that this may very often be the case. As I

worked with a pure culture of a fungus this did not occur
in my experiments. I always took as many precautions as
possible. Before starting an experiment the respiration vessel
was sterilized by means of a 1 "/oo corrosive sublimateso-
lution. Whenever the apparatus was ventilated the air was
sucked through a Pettenkofer tube with strong sulphuric
acid keeping back to some extent spores of the atmos-
phere. After finishing an experiment I always examined the
cultures for the possibility of an infection by fungi or
bacteria. I inoculated on malt media and broth, the latter
being an excellent medium for all kinds of bacteria. Only
once in all my experiments did an infection take place, but
never again when I transferred from below the surface.
When I transferred from the surface only in a few cases
bacteria appeared in the broth after some days, Phycomyces-
mycelium having already been formed. Apparently bacteria
had fallen on the culture but had not developed.

Considerable mistakes may be made by neglecting the
amount of COo dissolved in the culture medium (especially
in certain respiration apparatus, see p. 119). It seems to
me that this was the case with Puriewitsch (74) who
carried out determinations of the respiratory quotients
with Aspergillus, by analysing air-samples.

I tried to make sure whether a great amount of CO«
was absorbed by the solid medium I used.

To begin with, CO« may be bound chemically to sub-
stances in the medium. By adding acid the COo will be
liberated as the compounds are decomposed. This can be
done in a Barcroft-apparatus (5). The increase in volume
indicates the COg evolution. I found that CO., was only
chemically bound in old cultures on linseed medium and
on bread plus 20 % peptone, where the medium finally
becomes alkaline as stated on p. 142. I never used these
old cultures in my experiments however.

Secondly an amount of COo will be dissolved in the
water of the culture medium. I determined it as follows.
The culture was placed in a glass flask with a wide neck.
First I measured respiration for some hours. Then COo-free
water was poured into the flask. The water was boiled,
which took about 5 minutes. The COo dissolved in the
medium was driven out and absorbed in the Pettenkofer
tube. In a trough with bread at the utmost 4^ ccm COg
appeared to be dissolved, with linseed somewhat more.
With the 2Yy grm. media on the set of frames the amount
of CO2 dissolved was probably about half the amount.

At all events the amount of COo absorbed by the culture
media cannot have had any influence on the figures found.

CHAPTER IL

THE INFLUENCE OF LIGHT ON RESPIRATION.

I Started my investigations by studying the influence of
light on respiration. Opinions are still divided on this point,
although the question was already studied half a century
ago. I chose Phycomyces because it possesses strongly
developed sporangiophores, and in consequence of their
rapid growth an energetic metabolism should take place
in them. Also a part of the aerophilous mycelium remains
above the culture medium, so that in contrast with other
fungi a large part of the whole will receive light, namely
sporangiophores and mycelium above the surface of the

culture medium.

I was only acquainted with the publications of
Bonnier and Mangin (11) on the influence of light on
the respiration of Phycomyces. They found a decrease in
respiration in light. If I had known about Shorawski\'s
publication (87) I should certainly have chosen a different
fungus.

As regards the other publications I will not discuss those
by Drude (26), Pauchon (67) and Pringsheim (72)
because they are not accurate enough for the present time.
Wolkoff and Mayer (96) studied the influence of light
on the consumption of oxygen by seedlings. Whereas a small
increase in the respiration was discovered, a decrease was
never determined. Wilson (95) found no influence of
light on the respiration of seedlings or mushrooms.

The experiments of Bonnier and Mangin (11, 12)
were detailed and apparently very accurate. With all their
objects of observation (mushrooms, fungi, rhizomes, roots,
flowers, etc.) they found a strong decrease in respiration

in light. It amounted to 10 %, 20 % or more, and became
stronger as the intensity of the light increased. Puriewitz
(73) confirmed these results for mushrooms. With roots
and rhizomes the decrease was not always noticeable. With
flowers and etiolated plants there was rather an increase
in respiration.

Elfving (29) found no influence on the respiration
of older cultures of fungi such as Pénicillium. The dry
weight of cultures grown- in light was however far less than
the dry weight of cultures grown in the dark. Therefore
the light seemed to have influenced the respiration of the
fungi when they were still growing. Accordingly
Elfving analysed the gas from the closed flasks in which
the spores of the fungus had germinated and grown. It
now appeared that not only the dry weight remained smaller
but also that the respiration was lower in light.
Elfving therefore agreed with Bonnier and Mangin,
whose objects, according to him, all indicated growth.

It is questionable in Elfving\'s experiments whether, if
the respiration had been calculated per dry weight, it would
then also have been smaller in the light.

Aereboe (1, see also Detmer, 24) could not discover
the slightest influence of light on respiration. As for mush-
rooms he did not state his opinion on the influence of light
because his material was unfavourable. He often used the
petals of flowers. There is room for doubt whether in his
experiments the petals were not too closely packed together
so that only part of them received light.

I had a translation made of a paper by Shorawski
(87), the original of which appeared in Russian. For young
heads of Agaricus campestris he found a decrease in respir-
ation of about 20 % in light, with Mucor, cultivated on
bread, there was an increase of about 20 %. With Phy-
comyces the respiration was independent of the light.
Bonnier and Mangin may have found a decrease

through making no allowance for the grand period of the
respiration.

Finally Kolkwitz (45) found an increase in the respi-
ration in light of about 10 % for Pénicillium, Mucor and
others. For Aspergillus and Mucor, Maximow (60)
failed to discover any influence on young cultures where
there was sufficient food, while there was an increase in res-
piration of the older cultures in light, especially at first.

According to Lowschin (57)* the increase of respira-
tion in light, found by Kolkwitz and Maximow, is
due to a rise in temperature.

Summarizing it therefore looks as if light has a retarding
influence on the respiration of mushrooms. For the lower
fungi there may be an accelerating influence on the respir-
ation under special circumstances. It is possible that the
investigators did not allow for the fact that there may be
certain substances in the culture media which develop COg
under the influence of light, as for instance oxalic acid.
This substance is indeed often formed by fungi such as
Aspergillus, with which the experiments are often carried out.

Like Shorawski, I have not been able to find any in-
fluence of light on the respiration of Phycomyces. I tried
the effect of daylight at different times of the day and the
year and also of strong and weak electric light. As the lamps
were placed at a distance of only 20 c.m. from the basin,
a large glass cuvette, 10 c.m. thick, was filled with water
and placed between the lamp and the basin to absorb the
heat. The light intensities used, were measured photo-
metrically and were about 800 and 6000 M. C. The different
intensities of the daylight were not measured as the influence
was negative. All the experiments in this chapter were
carried out at 20° C.

The tables 14, 15 and 16 illustrate some of the many
experiments with Phycomyces. It follows from the tables

that the results are negative, but we also see that Phycomyces
is a poor object for studying the influence of light on respi-
ration, because contrary to expectation the respiration of
the sporangiophores and the mycelium that receive light
forms only a small part of the total respiration.

In table 15 and 16 the sporangiophores were namely
removed at 20.20 and 19.00 respectively. We see that after-
wards respiration has diminished very little.

The supposition might be made that for some time after
the removal of the sporangiophores, respiration is found
too high in consequence of the woundstimulus. But it seems
to me that this is not the case, because if so, respiration would
be sure to decrease in the long run. Separate experiments
showed however that it does not.

Table 15 shows that the removal of the culture for a few
minutes (from 18.00 to 18.03) out of the respiration vessel
as such has no influence.

These experiments are not of great consequence for
answering the question as to the influence of illumination
on respiration. In the experiments described in this chapter
I therefore mainly used the higher fungi namely mushrooms,
where a decrease in respiration has often been stated.
Besides the mushrooms found in nature, I used pure cultures
of mycelium. It has to be remembered however that in
nature the fructifications are exposed to the light, whereas
this is not the case with the ordinary mycelium.

I am giving some tables of the respiration of Polyporus
destructor on fiat pieces of carrot on which it grows well.
These pieces are easily illuminated on both sides.

Of the experiments with mushrooms I only give those
carried out with little ones. The respiration of the larger
ones, such as Boletus species and others is more intensive,
but with the little ones the surface illuminated is larger in
proportion to the weight.

It follows from the tables 17, 18, 19, 20 and 21 that hght
does not influence the respiration of mushrooms, either
of the naturally-occurring fructifications, or of the pure
cultures, in contrast with the findings of Bonnier and
Mangin, Puriewitsch and Shorawski. Our results are
in agreement with those of Wilson.

Moreover some time ago a paper of Rise hards (77)
appeared, who also failed to discover any influence.

It therefore seems to me that there is no direct influence
of light on respiration, except of course under accessory
circumstances. Spoehr (89) for instance found a higher
respiratory activity caused by ionizated air due to the ultra-
violet rays of the sunlight. So in my experiments this factor
was eliminated, the light always being deprived of ultra-
violet rays.

The influence of light on the respiration of green plants
is quite a different question. Borodin (14) already
showed that the respiration is the more intensive the more
carbohydrates are present. As carbohydrates are formed
in light, the light will indirectly increase the respiration
of green plants.

CHAPTER in.

THE INFLUENCE OF GASMIXTURES CONTAINING DIFFERENT
PERCENTAGES OF OXYGEN ON THE RESPIRATION
OF PHYCOMYCES.

It may be concluded from the behaviour of Phycomyces
as described in chapter I that the fungus will grow better
the more it is exposed to the air. It was therefore important
to determine how Phycomyces will behave in pure oxygen,
in gas with different percentages of oxygen and in the total
absence of it.

§ 1. Literature.

Many researches about respiration under the above
conditions were carried out with other objects, also with
some fungi. The respiration in the total absence of oxygen,
especially in connection with the intramolecular respiration,
has repeatedly been made a matter of investigation. In the
absence of oxygen the evolution of COo does not as a rule
cease directly, but goes on for some time owing to the split-
ting of carbohydrates in the culture medium. This was
already noticed by Le char tier and Bellamy (54 cf. also
58) with fruits, by Deh^rain and Moissan (23) with
leaves and by Borodin (13) with seedlings.

The quotient, giving the proportion between the amount
of COo given off anaerobically and the amount given off
in air, varies in different plants. It is 1.0 for seedlings of
Vicia Faba (97) but mostly smaller, e.g. i/i ^or Lupinus
(70, 95). Moeller (64) found this quotient to be indep-
endent of the reserve food in the seeds. It may be the same
for fatty seeds as for those containing starch. Chudia-
kow (18) also found that fatty seeds give off much COj
anaerobically. On the other hand Diakonow (25) showed

that the amount of COg given off anaerobically is larger
with seeds containing starch than with fatty ones.
Godlewski and Polszenius (36) explained these con-
tradictions: Chudiakow first soaked the seeds in water,
allowing the air to enter freely, by which fats changed into
carbohydrates.

As far as fungi are concerned, Mucor species give off a
good deal of COo in the absence of oxygen (Brefeld, 15),
Pénicillium and Aspergillus one fourth of the amount
given off in air (Diakonow, 25). This holds for cultures
on sugar. On chinic acid and tartaric acid they die. Saccha-
romyces is also unable to give off intramolecular COg without
sugar (Chudiakow 19).

Tissues, poor in carbohydrates, give off a lot of COo
anaerobically on sugar solutions (etiolated leaves: Palladin
66; seeds rich in proteins: Godlewski 37, 38). The
smaller the number of carbohydrates, the sooner there-
fore, the fungi and the tissues of higher plants will die in
the absence of oxygen.

If CO2 is given off anaerobically on media without carbo-
hydrates, it generally is supposed that these have been
manufactured by the fungus itself. Kostytschew (49)
demonstrated sugars in cultures of Aspergillus niger on
chinic acid and tartaric acid (cultures on peptone appear
to behave otherwise). F lie g (32) cultivated Aspergillus
niger on pure oil-media and found that young cultures
died very soon in the absence of oxygen, older ones could
stand it better, carbohydrates having alreadyb een manuf-
actured from the fat.

As for high oxygentensions, the older investigators like
Bert (8) and Boe h m (10) expected from them an increase
in growth and respiration but neither of the two happened.
On the contrary in the long run they found a decrease.
Dehérain and Moissan (23) discovered no influence of

oxygen on respiration. Proceeding on Pettenkofer\'s method,
at different temperatures. Rise ha wi (78) sent air and
oxygen through his experimental vessel in turns, without
finding the slightest influence on the respiration of seed-
lings. Godlewski (35) found, at least with seeds con-
taining oil, that in pure oxygen the respiration increased a
little in the beginning and afterwards went down.
Johannsen (42) worked with different oxygentensions up
to a few atmospheres. At first the amount of CO« given
off became greater. If the pressure continued the respi-
ration became weaker and weaker until death occurred.
The higher the pressure the sooner death was brought
about. During the experiments rapid changes appeared in
the tension and this may also have affected the plants.

Kolkwitz (45) found that in pure oxygen the respira-
tion of fungi such as Aspergillus is at the least double its
usual amount.

Flieg (32) investigated the influence of pure oxygen
on fungi cultivated on fat. In this case the respiration was
doubled and decreased very gradually in course of time.
On sugar where ordinarily the respiration is much higher,
the increase in oxygen was small. Moreover oxygen is here
more harmful than on fat, as within few days the respiration
was reduced to a minimum.

Th. de Saussure (85) discovered no difference in res-
piration when the oxygentension was reduced to half its
amount. Wolkoff and Mayer (96) afterwards also stated
that in a gasmixture with 10 % oxygen the amount of oxygen
taken in did not vary. Borodin (14) did not agree with
them. Wilson (95) found no change in the CO« evolved
in 4 % oxygen with Helianthus, where the quantity of CO«
given off anaerobically equals only one fourth of that given
off in air. The decrease was considerable in 1 % oxygen.

At low oxygentensions CO. will probably be given off

intramolecularly. Stich (91) found that with different
seedhngs the respiratory quotient sometimes begins to
increase when the amount of oxygen is 3 to 4 %.
Puriewitsch (75) affirmed that in a 4 to 5 % oxygen-
mixture the intramolecular respiration has not yet begun.

The influence of different oxygentensions on the growth
of the sporangiophores of Phycomyces has been the subject
of two examinations. Jentys (41) found that they grow
as well in pure oxygen as they did in air. Wieler (94)
said that the growth ceases at 0.2 % oxygen. This fact does
not say anything about the respiration, because in the
absence of oxygen an intensive intramolecular respiration
can take place, without any noticeable growth.

The influence of different oxygentensions on the respi-
ration of Phycomyces has never been studied. Other Muco-
raceae may give off a large amount of COo in the absence
of oxygen (48) but this does not say anything for Phycomyces,
as the latter also behaves quite differently as regards the
culture conditions (see chapter I). It is not possible to say
beforehand how Phycomyces will behave in different gas-
mixtures. I will therefore proceed to the description of my
own experiments.

§ 2. The Influence of pure Oxygen.

Commercial "oxygen" taken from a bomb, containing
more than 96 % oxygen, was purified by means of washing-
bottles with solutions of permanganic soda, strong sul-
phuric acid and strong alkaline solutions.

A. Starch-medium. Table 22 shows the effect of a
long exposure to the action of pure oxygen of a culture on
bread. After the maximum in the grand period, the respir-
ation continues to decrease in the same way as in the air.
Apparently oxygen neither raises the evolution of COo
nor lowers it, no matter how long the exposure.

Fig. 11 (table 23) gives the in-
fluence on the respiratory quotient.
Compared with the COg given off,
the Og absorbed is a little more
than in the air. The quotient there-
fore has diminished. Table 24 shows
the same for an older culture.

B. Oil-medium. The grand
period of the respiration on ground
linseed also takes about the same
course in pure oxygen as in the
air, as is shown in table 25.

Fig. 12 (table 26) gives the influence on the respiratory
quotient. Table 27 shows another experiment. As on starch,
the quotient is a little lower in pure oxygen, as some more
oxygen is absorbed.

§ 3. The Influence of Gasmixtures containing no
Oxygen or less Oxygen than Air docs.

I used commercial bombs of "nitrogen" containing a
gas consisting of nearly 98 % nitrogen and fully 2 % oxygen.

By mixing different amounts of this gas with air, I obtained
mixtures of different compositions. The percentage of oxygen
was determined by means of the Jordan pipet (43).

The nitrogen from the bomb was purified in washing-
bottles containing strong sulphuric acid and a strong solution
of sodium hydroxide.

Some of the experiments were carried out with the small
earthenware troughs. Others with the medium on a set of
frames. The quantity mostly used in this case was about
grm.

A. Starch-medium. The result of the experiments on
the effect of different percentages of oxygen on bread-
cultures of Phycomyces is summarized in the following
table.

TABLE 28.

The influence of different percentages of oxygen on cultures
of Phycomyces on bread.

12 % oxygen

9Î4% »

8\'/2% „

6^2 % „

3 % ,

o/
/o

2

1%%

no influence.

I influence?

; 94 % of the COj-evolution in air.
R\'i o/

/O tt It It It II II

; 64 % „ „ ,, ,, ,, ,,
so o/

/O II II It It II ■ It

% „ ,, ,, ,, „ ,,

It follows from fig. 13 that in a 2 % oxygenmixture
where the respiration has been reduced to half the normal
amount, it will remain nearly constant. In young cultures
where in the air there is a rapid increase, the respiration
also becomes constant in 2 % oxygen (fig. 14).

5.0

12?*

Fig. 14.

lOO

3.0

qo

12"

Fig. 15.

100

50

QOO

,2?o

Fig. 16.

Influence of Low Oxygen Tensions on the Respiration on Starch-media, see Text and Table 30, 31, 32 and 33.

Fig. 15 shows the respiration of such a young culture in

1 Vz % oxygen.

From the values given in table 28, it appears that the
COg-evolution gradually decreases to about 3 %, where the
fall becomes faster so that probably at a very low oxygen
tension respiration will stop.

By forcing the gas containing 2 % of oxygen through
two Pettenkofer tubes, filled with a strongly alkaline pyro-
gallic solution, I obtained nitrogen with only a trace of oxygen.

Fig. 17 and 18. Influence of an Atmosphere containing 81/2 and
3 per cent, of Oxygen on the Respiratory Quotient on Starch-
media. (Sec table 36 and 37).

It follows from fig. 16 (table 33) that in this case respiration
indeed soon falls to a minimum.

Tables 34 and 35 show the transition from air to a mixture
containing no oxygen at all. I used hydrogen, liberated in
a Kipp-apparatus by means of pure zinc and 5-normal
sulphuric acid. The hydrogen was passed through a washing-
bottle with potassium permanganate and Pettenkofer tubes
with alkaline pyrogallic solutions. In the total absence of
oxygen Phycomyces practically gives off no CO« but dies

in a few hours. It does not recover,
for after remaining in air for 24 hours
no CO2 has yet been given off.

On bread, contrary to expectation,
the respiratory quotient does not
change in smaller percentages of
oxygen. Fig. 17 (table 36) gives the
facts for a mixture containing %
of oxygen, fig. 18 (table 37) for a
mixture containing 3 % of oxygen.

\'o oxygen

•oOo, cOj

100

50

so

18?"

Fig. 19. Influence of an Atmosphere containing 21/3 per cent, of

Oxygen on the Respiration on Oil-medium, sec Table 40.
Fig. 20. Influence of an Atmosphere containing 3 per cent, of
Oxygen on the Respiratory Quotient on Oil-medium, see Table 42,

These experiments will be discussed after the description,
of the behaviour of the cultures on ground linseed.

B. Oil-medium. 8 % of oxygen has not yet the
slightest influence on the respiration of cultures on ground
linseed. At reduction to about 6 % of oxygen, the CO2
evolution goes down to about 92 % (table 39) of the nor-
mal amount. At 2% % of oxygen to about 74 % (fig. 19
table 40). On oil-media, apparently, Phycomyces can stand
low oxygen tensions better than on starch-media, at least
as far as the evolution of CO2 is concerned.

In contradistinction to the starch-medium, the respiratory
quotient changes on oil-media, as has been ascertained
for 41/4 (table 41) and 3% oxygenmixtures (table 42, fig. 20;
table 43). The COo-evolution diminished in these cases
to 85 % and 81 % respectively, the Oo-consumption to
77 % and 71 % respectively. The respiratory quotient
therefore has increased.

At very low oxygentensions respiration stops, as was the
case on bread. In 1 % oxygen the COo-evolution has dropped
to about 30 % (table 44), in hydrogen Phycomyces dies
(table 45).

§ 4. Discussion.

The experiments reveal many peculiarities about the
respiration of Phycomyces. It cannot give off CO« anaero-
bically, neither on oil-media nor on carbohydrates. In respect
to the latter fact Phycomyces differs from what has been
found for fungi and higher plants. The greater part of the
bread indeed consists of starch. But it is probable that
Phycomyces consumes this starch by changing it first into
sugars. Moreover, bread contains already some sugars.

Phycomyces evidently behaves altogether differently
from Mucor species, which ferment sugars in a hydrogen
atmosphere, and consequentely the less oxygen the atmos-
phere contains, the larger their respiratory quotient will be.

The respiratory quotient of Phycomyces remains unchanged
on carbohydrates at lower oxygentensions, because Phyco-
myces cannot give off CO2 intramolecularly formed. If
the fact that it is a little higher than 1.00 in air was not the
result of fat manufacture but of sugar fermentation, it
would rise at lower oxygentensions. It is true that in pure
oxygen the quotient is lowered, but probably certain sub-
stances in the medium are oxidized, independently of the
plant, the consumption of oxygen on ground linseed being
also raised.

The quotient on oil-media increases at low oxygentensions.
At first sight it might be supposed that in this case a carbo-
hydrate respiration partly takes the place of the oil respir-
ation, in consequence of the latter requiring more oxygen.
But then respiration on starch-media ought to be higher,
whereas it is higher on oil-media at low oxygentensions.

As facts in literature would rather lead one to expect the
reverse, it is very curious that on oil-media respiration is
the least affected by small quantities of oxygen. There is
nothing similar to be found in literature.

By many investigators, in accordance with Pfeffer\'s
views, respiration is considered as consisting of two pro-
cesses, at first a splitting of sugars into alcohol and CO^,
secondly an oxidation of the alcohol to CO2 and HoO.
The CO2 formed anaerobically is due to the first process,
which is of course less sensitive to a lack of oxygen than
the second one. On fat, anaerobic C02-cvolution only takes
place when the fungus itself has manufactured carbohydrates
from the fat (Flieg).

The phenomena found for Phycomyces, both on starch-
and oil-media do not fit in this scheme.

Apart from the mutual differences both on oil-and starch-
media, a decrease in oxygen will affect Phycomyces much
sooner than is ordinarily the case with other plants, as may

be seen from § 1. In chapter I it has often been remarked
that Phycomyces appeared to be very aerophilous. For all
that the mycelium will grow well so long as the oxygen
percentage does not drop below 8 or 9 %.

I described the small respiration on liquid media on p. 128.
Is it due to the water as such, or to a small diffusion of
oxygen? In the latter case respiration will increase when
the fungus is brought into an oxygen atmosphere. An exper-
iment was carried out (table 46) showing that on media
mixed with much water respiration remains low in pure
oxygen.

The fact, therefore, that Phycomyces grows badly in a
liquid culture medium is for the greater part due to the
liquid as such and is probably only to a slight degree the
result of a small oxygen diffusion.

CHAPTER IV.

THE RESPIRATION VELOCITY AND THE MAGNITUDE OF THE
RESPIRATORY QUOTIENT OF PHYCOMYCES AS A
FUNCTION OF THE TEMPERATURE.

The influence of temperature on respiration has often
been studied. The disciission of the literature on the subject,
however, will show that there are still various problems
which have not been solved. This was the reason why I
decided to investigate the question more in detail, espe-
cially as it gave me an experimental object with which I
was well acquainted.

§ 1. Discussion of the Literature.

Sachs in 1860(83), in investigating the influence of
the temperature on the growth of seedlings, introduced
the concept of the three cardinal points: minimum, optim-
um and maximum; these three points were in later years
-also determined for other processes for instance the photo-
synthesis.

As regards respiration, it struck the older investigators
that the optimum, if there is one, is always much higher
than with other processes (cf. e.g. Ad. Mayer, 62). A
question of much discussion was how respiration reached
this high optimum. According to Wolkoff and Mayer
(96), Ad. Mayer (62) and Bonnier and Mangin (11)
the intensity of respiration was nearly a linear function of
the temperature. Rischawi (79), instead of a straight
line, found a curve convex towards the temperature axis,
the curve given byDehérain and M o i s s a n (23) and also
the one of Pedersen (68) was even convex to a higher
degree. Both ascend rapidly.

The contradictions may be the consequence of a difference

in objects or in methods, but generally the respiration curve
was found more or less convex towards the temperature
axis. The curve was much steeper than those for other
processes, such as photosynthesis (Kreusler 50): the
optimum was found near the lethal temperature.

Contrary to this opinion Clausen (20) and Z i e g e n-
b e i n (98) found that optimum and lethal temperature do not
lie close together. At higher temperatures above the optimum
the respiration may be lower than at the optimum, and
for all that constant.

If in these experiments the temperature was lowered,
the respiration appeared to be smaller than it was before
at the same temperature. Pfeffer (71) remarks that ap-
parently part of the plants had died. Pfeffer therefore
holds the view that an optimum never can be spoken of
with respiration, because in this case the curve would bend
at a temperature not yet noxious to the plant, as happens
according to him with photosynthesis and the growth of
plants.

Another explanation of the optimum is suggested by
Tammann (92) and Duel aux (27) for enzymes. Accor-
ding to them, it is the result of the enzymes being des-
troyed at higher temperatures, and the higher the tempera-
ture, the more of them there are destroyed. The optimum
therefore is the result of the noxious effect of the higher
temperatures. The process itself would not be impeded
by a rise in temperature. Without this harmful effect the
enzyme action would increase with the temperature in a
continuously ascending curve (Duclaux).

It has been the great merit of F. F. Blackman (9)
that he proposed a similar theory, independent of
Tammann and Duclaux for all physiological processes,
in connection with a research by one of his pupils Miss
Matthaei. Miss Matthaei (59) studied the COo assimi-
lation of the leaves of Prunus Laurocerasus. When care is

taken that there is always sufficient COo and light, no
optimumcurve is found, but the rate of assimilation increases
rapidly with the temperature, according to a curve, convex
towards the temperature axis. The reaction velocities at
these high temperatures are however not constant. A leaf
cannot keep up this maximal assimilation, and the higher
the temperature, the more rapidly it decreases. The tempe-
rature curves of assimilation values will therefore vary in
proportion to the time the plant is kept at the high inju-
rious temperatures.

From the data obtained by Miss Matthaei, F. F.
Black man claims that:

lo. Physiological reactions are influenced by temperature
in a similar way as chemical reactions, if only the orga-
nism is not injured. According to Van \'t Hoff\'s law
(or rather according to the interpretation the biologists
of that time gave of Van \'t Hoff\'s law) the reaction
velocity is doubled or trebled for a rise of ten degrees
in temperature (also expressed by the formula: tem-
perature coefficient Qio = 2 or Q,o = 3).
2o. The optimum curve is the result of the time factor.
The shorter the time of observation, the less the injury
and the higher the optimum. If it were possible to
observe after a "time o" a "Van\'t Hoff\'s curve" would
be found.

3o. This "Van\'t Hoff\'s curve" can therefore be constructed
in two ways by-extrapolation. After the first method
the Qio is used, determined at lower temperatures where
no injury takes place. By means of this Q^^ the curve
is constructed for higher temperatures. After the second
method the values of the theoretical curve at higher
temperatures are extrapolated from the values obtained
after 1, 2, 3 etc. hours of observation.

It is now obvious that Ziegenbein (98) would have
found the optimum at another temperature if he had observed

either sooner or later. The experiments of Chudiakow
(19) who found the optimum of fermentation of yeast at
40°, 35°, 30° or 25°, according to the time elapsed, fit in

with the above theory.

Kuyper (52) was the first who tried to apply B 1 a c k-
man\'s theory to respiration. He experimented with
green peas and other seeds on the Pettenkofer-Pfeffer
Lthod. According to him the "Van\'t Hoff\'s" rule holds
good up to 20°. Qio = ± 2.8. By means of this coefficient
it was possible to construct by extrapolation the exponential
curve for higher temperatures. The values of this theore-
tical curve are much higher than those, which would be
found by means of the second extrapolation method of
Blackman. Kuyper however still believes that Black-
man\'s theory in its general lines is correct.

In my opinion the different result with the two methods of
extrapolation may have been caused by the following facts.

When Kuyper moves his plants from a low to a
higher, thought not injurious, temperature, the objects
take on the new temperature after about 10 minutes. He
has, however, to wait an hour before starting his experi-
ments. Otherwise he will get too low an amount of COo
at the beginning. According to Kuyper the respiration
apparently does not adapt itself directly to the new tempe-
rature. The same thing must happen on the objects being
brought to a higher injurious temperature. Here there are
two tendencies, an increase in the COo-evolution owing to
the adaptation to the new higher temperature and a decrease
owing to injury. Kuyper now experimentally determines
the time at which the first measurement must be taken.
Before as well as after this time the amount is lower.

In my opinion the reason for this so-called adaptation to
the new temperature, is that during the first half hour after
the temperature has been raised, gas is collected which was
still formed at the lower temperature (see p. 126).

Further, it has lately become evident that there are many
objections against the materials used in Kuyper\'s expe-
riments. Stalfelt (90) namely has shown that the seed
coat offers an obstacle to the diffusion of gas. From the
experiments of Sierp (88) it also appears that peas are,
physiologically, very complex structures (See also the
recent publication of Frietinger, 34).

Van Amstel and Van Iterson (2, 3) object against
Kuyper that in the course of long periods of observa-
tion, say of several hours, adaptation and growth processes
may appear. The authors used yeast and so always worked
with cells which are small and will therefore quickly assume
the new temperature. They used a definite quantity of the
yeast, of which the velocity of fermentation, respiration etc.
at the lower uninjurious temperatures is known. The velocity
was determined after 5, 10, 15 and 20 min. pre-heating.
After the measurement had been taken the temperature
was at once changed to a harmless one and the fermentation
velocity determined. This fermentation velocity is only a
part of the velocity the yeast would have at the same tempera-
ture if it had not been injured at the high temperature.
This proportion supplies a certain value with which the
velocity at the high temperature has to be multiplied, to
get the velocity at high temperatures if nothing had been
injured.

For all the examined functions the theoretical "zero-
hour" curve does not show Blackman\'s exponential
curve, but an optimum curve, so that according to
Van Amstel and Van Iterson the theory of Duclaux-
B lack man must be rejected for these processes. This
also follows from the behaviour at harmless temperatures.
Up to 45° namely, the reaction velocity is independent
of the preliminary heating, but yet the Qio already decreases.

Rutgers (80, 81) determined the influence of tempe-
rature on the geotropic presentation time. He concludes

from his experiments that the "zero-hour" curve never can
be extrapolated from the values found after 1, 2, 3 hours
etc., because the reaction-velocities only adapt themselves
gradiially to a new temperature. The greatest support for
this theory Rutgers finds in Kuyper\'s publication. I
have shown that in this case the adaptation is probably due
to experimental errors. I therefore do not agree with
Rutgers when he suggests that this gradual adaptation
has influenced the results of Van Amstel and Van
Iter son, who moreover have refuted (2, 4) this and
other criticisms from Rutgers (80, 81) and Kuyper
(53).

It was already mentioned by Rutgers (81) that with
chemical reactions Qjo may diminish at higher temperatures.
Cohen Stuart (21) studied this subject more in detail
and pointed out that Van \'t Hoff\'s law is often wrongly
interpreted by biologists. Especially in physiological proc-
esses when there is a heterogeneous system, Q^ as a rule
will decrease at higher temperatures. The "zero-hour" line
therefore need not be an exponential curve, as
Blackman supposed, and cannot be constructed by
means of the Qio found at low harmless temperatures.

In the following pages the results are stated of investig-
ations as to whether or not respiration will directly adapt
itself to a new temperature. As this proved to be the case
it was tried to construct the "zero-hour" line according to
the second method of Blackman, namely by an exact
extrapolation from the points on the "injury"-curves.

The present writer also investigated the influence of

CO«

temperature on the respiratory quotient q of Phycomyces

growing on fat-and starch-media.

Very little has been done about studying the influence
of temperature on respiratory quotients. The older inves-

tigators were divided in their views. Bonnier and
Mangin (11, 12) found that the respiratory quotient is
independent of the temperature. According to Dehérain
and Moissan (23), Moissan (63) and Dehérain and
Maquenne (22) it increases with the temperature.
Puriewitsch (75) confirmed their experiments. The
younger the objects, the greater is the increase of the quo-
tient at a higher temperature. He found something similar
for fungi. The influence of temperature here is namely
smaller, in proportion as the lack of food is greater.

§ 2. The Respiration of Phycomyces on Oil-media
at different Temperatures.

A. Experiments.

The experiments described in the preceding chapters
were mostly carried out at 25° C. In this chapter the
experiments are given at different temperatures.

In the figures of § 2 A and § 3 A the ordinate-axis repre-
sents the rate of respiration in ccm. per hour, the abscissa-
axis has been taken as the time-axis, graduated into hours.
The solid lines are again the "Oo-lines", the broken ones
the "COa-lines". In the same figures the respiratory quotients
are indicated by a solid line. Here the ordinales represent
the magnitude of the respiratory quotients.

The hatched part represents the time required ,by the
water in the basin to take on the new temperature. The
vertical line in front of it indicates the time at which the
experiments at the initial temperature were finished, the
vertical line behind indicates the time at which the experi-
ments started at the new temperature. The "Oo-and
COo-curves" are extended as far as the hatched part (cf.
page 197).

At higher injurious temperatures, where respiration
decreases, no delay is allowed in starting the new experiment

in order to get as much as possible of the first part of the
"injury-curve".

It does not do to start the new experiment before every
thing has assumed the new temperature. Blank experiments

were carried out in order to
test this. Nearly the whole
apparatus very rapidly takes
on the temperature of the water
in the basin. Only in the culture
medium (see § 2 B) and in the
alkaline solution in the glass-
vessel M (fig. 1) the lag of the
temperature is greater. The
experiments were therefore usu-
ally started 22^ minutes after

50

Oz

CO,

Fig. 21. The Transition from 25° to 10° C. on Oil-media (see

table 47).

the water in the basin had taken on the new temperature.
In the meantime some manipulations have to be carried
out, such as the sucking of air of the required temperature
through the Pettenkofer tubes (see p. 123).

At the average temperatures the measurements are
taken every hour, at the lower temperatures where respiration
is smaller even after longer periods. At higher temperatures,
however, the initial measurements are taken every half hour,
because respiration decreases rapidly and its exact progress
has to be found; the oxygen consumption is even deter-
mined every quarter of an hour. In the last case the
values obtained are indicated in the figures by means of a
small circle instead of a dark dot. Shorter periods of obser-
vation are not desirable as errors will then become relatively
too large.

All experiments were carried out with two thin layers of
culture medium of 1 Yi grm. each on a set of frames. The
respiration was always measured in the "constant" part of
the grand period.

In fig. 21 (table 47) the respiration is given at 25° and
at 10° C. The respiration is less at the lower temperature.
As regards the evolution of COo nothing is seen of a gradual
adaptation to the new temperature. It immediately becomes
about 28.5 % of what it was at 25° C. and remains so during
the following hours.

The amount of 0« taken in, however, becomes constant
only after some hours. At first it only decreases to 38.5 %
and then gradually drops to 28.5 %, which with the COo-
evolution is immediately the case.

As mentioned above, blank experiments showed that
the air in the respiration vessel in every case assumed the
new temperature within 20 min. A contraction of the air
can therefore not be the cause of the higher initial oxygen
consumption.

At a transition from a higher to a lower temperature the
amount of oxygen absorbed is at first apparently too high
as compared with the CO« given off. Consequently the
respiratory quotient is lower at first and reaches its original
value only when the respiration becomes constant.

The same thing happens when the transition is to 15°
and 20° C. The values of the respiration at new temperatures
will not be given separately for each temperature but are
summarized in table 59 (p. 185). Fig. 22 (table 48) shows
the effect of the transition from 25° to 15°, fig. 23 (table 49)
shows it from 25° to 20°. The deviation of the initial oxygen
respiration becomes smaller the less the difference is in
temperature; the same may be said about the initial respi-
ratory quotients.

I also determined the respiration velocities at 10°, 15°
20° and 25° C. by starting the experiments at 15°. Fig. 24
(table 50) gives the situation at the transition from 15° to
10°. The oxygenconsumption is again a little higher at
first and the respiratory quotient smaller.

In the following experiments we for the first time see the
transitions from a lower to a higher temperature. At the
change from 15° to 20°, as is shown by fig. 25 (table 51),

both the COg-evolution and the Og-absorption are constant
almost immediately. The respiratory quotient is not smaller
at first but perhaps even larger. This is more obvious in the
next fig. 26 (table 52), giving the situation at the transition
from 15° to 25°. The respiratory quotient again only reas-
sumes its original value after some time, but this time
because it is higher at first. In contrast with the former
experiments, the initial Og-consumption is namely too low
when the culture is brought to a higher temperature.

From the fact that the COg-production is constant at
once it follows that a temperature of 25° is not yet injurious
to the fungus. As soon however as the higher temperature
has a harmful effect, there will be a decrease in the intensity
of respiration. But if also in this case the amount of Oo
absorbed at first becomes smaller as compared with the

COa given off, the "Oo-curve" will start too low and des-
cend less rapidly than the "COo-curve".

This is indeed the case at higher temperatures. At 27°5
the injurious effect has begun. It is obvious from the COo-
evolution; for the first time we see an initial decrease: fig. 27
(table 53). The Oo-consumption however is constant. The
increase of it, which might be expected in view of its beha-
viour at a transition to a higher temperature from fig. 26,

is apparently compensated by the decrease which would
appear as in the case of the COo-evolution.

At 30° (fig. 28, table 54) the decrease is stronger, as the
respiration starts at a higher level. The "Og-curve" also
descends, but in a slighter degree than the "COg-curve",

y coj

Fig. 27 and 28. The Transitions from 25° to 27°5 and 30° C. on Oil-media (see table

53 and 54).

as the Og-consumption again does not attain its value at
once. In consequence of these facts, in fig. 27 and 28 the
respiratory\'quotients are too high at first jtist as was the
case in fig. 26. When the downward movement ceases and

the respiration again becomes constant, the respiratory
quotient reassumes its original value.

At 32°5 the respiration becomes constant no more: fig.
29 (table 55). The noxious influence of the temperature

continues. The "Oo-curve" and the "COo-curve" start
closer together and also remain so more or less. The respi-
ratory quotient at first already approaches the value of
1.00 and never again reassumes its original value of about
0.67.

The fact that the values of the Og taken in and the CO2
evolved tend to approach each other, becomes more apparent
at higher temperatures.

At 35°, fig. 30 (table 56), the respiration decreases rapidly
and the fungus dies in the long run. The COg-production
and the Og-consumption run about parallel and close
together. The respiratory quotient starts at 1.00 and remains
above or about 0.90.

The transition from 25° to 37°5 is represented in fig. 31
(table 57). The decrease in respiration is still more rapid.
The "Oa-curve" and the "COg-curve" nearly coincide, the
respiratory quotient is about 1,00 (The smaller the values,
the less exactly the quotients can of course be determined).

At 40° the fungus is dead within about two hours (table
58). As the decrease in respiration is too rapid to determine
exactly the "zero-hour" point, the curve has not been
plotted.

B. Respiration on Oil-media as a Function of
Temperature.

It follows from § 2 A that the more injurious the tem-
perature becomes, the more the respiratory quotient of
Phycomyces on a linseed medium approaches the value 1.00.
This suggests that at higher temperatures the fungus
consumes carbohydrates instead of fats. (It should be
remembered that in a linseed medium carbohydrates occur).
Perhaps the fungus is able to stand higher temperatures
better on starch-media than on oil ones. In § 3 therefore
the influence of the temperature on starch-media cultures
will be investigated.

The change in the respiratory quotient gives a peculiar
shape to the "zero-hour"-line. In extrapolating the points
of the "zero-hour"-line from the values found it is neces-
sary to trace back the different "injury"-curves to the
zero time i.e. the time at which the culture medium reached

03

the new temperature. This extrapolation is difficult at high
injurious temperatures, as a slight deviation in the extra-
polated line will cause a great mistake in the position of the
"zero-hour" point. As many experiments as possible were
therefore carried out.

The time passing between the zero time and the beginning
of the first experiment must be known exactly.

The experiments were all carried out with a suction
velocity of 3 to 1. per hour and in the respiration vessel
of 325 to 350 ccm cubic contents. From table 1 it follows
that in this case the COg given off at a certain moment
by the plant needs minutes on an average to arrive at
the Pettenkofer tube. In the curves the values measured
are therefore indicated 4% minutes before the average
time of observation.

• I was unable to measure the temperature of the mycelium,
I think however that the fungus and the culture medium
may be supposed to assume temperatures at the same time.
How much time will elapse between the moment that the
water of the basin in which the apparatus is fixed attains
the new temperature, and the moment at which the culture
medium assumes it? As mentioned on page 172 the tempera-
ture lag in the culture medium is rather large. In the Physical
Laboratory I determined it thermoelectrically When the
water in the basin is brought to a higher temperature the
temperature of the culture medium increases very rapidly
at first and then very gradually till the desired temperature
is reached.

Now it would be wrong to choose as the zero-time the
time at which the culture medium finally reached the new
temperature, as at higher temperatures the noxious action
has already begun. I therefore took one half of the time
necessary for the medium to assume the new temperature.

I have to thank Prof. Ornstein for his kindness in assisting me.

In this case the zero time at 37°5 is to 10 min., at 35°
about 7^ min., at 32°5 5 to 7% min. and at 30° about
5 min. after the basin reached the new temperature.
In table 59 the respiration-velocities are given for the

the COa-production at 25° is assumed to be 10 ccm. per
hour and the Og-consumption 15 ccm. per hour.

(See table 59).

In table 60 the respiration velocities are given for tem-
peratures above 25° C. Fig. 32 represents the "zero-hour"-
line. (See table 60).

The temperaturequotients apparently decrease rapidly.
As there is a small temperature interval I give the Q5.

ISO 495 20® 6.58 to K-V.

= 15» = 43 =

250 9.0

= — = 1.41 (fig. 23).
^20° 6.35 ^ ^

The "Og-curve" is even an optimum curve because at
higher temperatures the Oo values approach the COo values
and therefore decrease.

Fig. 33 gives the respiration-velocities at different tem-
peratures after different periods. Besides, the thin lines at
the top give the "zero-hour"-line when the time at which
the basin assumed the new temperature is taken as the
zero time.

§ 3. The Respiration of Phycomyces on
Starch-media at different Temperatures.

A. Experiments.

If on linseed-media the change in the respiratory quot-
ients, as found in § 2, is really the result of a more or less
intensive consumption of carbohydrates, on bread-media,
where the consumption of carbohydrates will always be the
chief feature, these changes in the respiratory quotients
will not occur at the transitions to other temperatures.

Fig. 34 (table 61) renders the transition from 25® to 10®.
The respiration-velocity is much smaller on bread than on
linseed, the values, moreover, are small in consequence of
the low temperature. A relatively larger error is therefore

TABLE 59.

r\'

èhour

hour

\',1 hour

\'2 hour

\\ 1
\\ •

V

V
\\

3 hour

40"

Fig. 33. Velocities of Respiration on Oil-media (Linseed) at Different Temperatures after Different
Periods. A. The Consumption of Oxygen. B. The Production of Carbondioxide.

187
TABLE 60.

made in determining the respiratory quotient. But at first
the quotient apparently is a little higher. The Oo-consump-
tion immediately becomes constant at the new temperature.
It seems that the COo-production starts a little too high.
The same is to be seen from the transitions from 25° to 15°
or 20°, represented in fig. 35 (table 62) and 36 (table 63).

From the transition from a lower to a higher harmless
temperature e.g. from 15° to 25° (fig. 37, table 64) it is
again evident that in contradistinction to the respiration
on oil-media, the Oo absorbed directly becomes constant
and that the respiratory quotient remains constant.

At 27°5 and 30° the respiration also immediately becomes
constant, fig. 38 (table 65) and fig. 39 (table 66). The COo-
evolution is at best a bit higher at first. The respiratory
quotient remains nearly the same.

At 32°5 the noxious action of the temperature has begun,
fig. 40 (table 67). The respiration still becomes constant
in the long run.

At 32°5 the respiratory quotient, at last approaches the
value of LOO, which is still better seen at the other high
injurious temperatures.

10.0

50-

CPa

illlO»

Fig. 35. The Transition from 25° to 15° C. on Starch-
media (see table 62).

10.0

25\'

coj
Oi .

CO, .......

...... p.. -

co,
0,

tsoo

Fig. 37. The Transition from 15° to 25° C. on Starch-
media (sec table 64).

Fig.<39. The Transition from 25° to 30° C.
on Starch-media (see table 66).

At 35° the curve does not become horizontal, fig. 41
(table 68); at 37°5 it descends rapidly, fig. 42 (table 69).
At 40° the decrease is very rapid (table 70).

B. The Respiration on Starch-media as a
Function of Temperature.

If the "zero-hour"-line is constructed by extrapolation
in the same way as was done in § 2 the values given in table
71 are obtained. The respiration velocity is expressed in
percentages of the value at 25° C. and also in ccm, per
hour if the evolution of COg at 25° is 7.0 ccm. per hour
and the absorption of O2 is 5.75 ccm. per hour.

(See table 71).

193
TABLE 71.

Fig. 43 shows the "zero-hour"-line. The curve reveals
the remarkable fact that the intensity of respiration, as
measured by gas-exchange, is an almost linear function of
the temperature. As the COo-production in nearly all
figures was at first a little too high, the "COo-line" is
not quite straight.

Fig. 43 seems to suggest that the "zero-hour"-line deviates
from this straight line at higher temperatures.

The temperature-quotients decrease even more than on
the oil-media.

^ 15° 5.2 20° 7.7

1,22. (fig. 43).

\'25° 100 ^ & ^

As in fig. 33 for the oil-media, the respiration-velocities
on starch-media are given in fig. 44 for different tempera-
tures after different periods.

§ 4. Discussion. .

It follows from the experiments in this chapter that the
range in temperature between which Phycomyces can
live is smaller than is usually the case with other plants.
At 40° Phycomyces already dies in a few hours.
Bartetzko (6) and Lindner (55) moreover found that
it will be frozen to death at a relatively high temperature.

All the experiments were repeated several times, especi-
ally as the greater part of the results were contrary to expec-

tation. The results were exactly the same, only sometimes
the O2-consumption at 30° on a linseed medium remained
higher for a longer period, so that the respiratory quotients
did not reassume their original value until after 4 or 5 hours,
instead of 2 or 3 hours.

Though the differences are not large, the respiration-
velocity in different experiments on the same culture
medium is not always the same. This is chiefly due to the
amount of water in the cuhure medium (p. 145). It may
be asked whether this fact has no influence on the ratio
of the respiration-velocities at different temperatures.
This is not the case. Several experiments were carried out
with very wet bread or linseed e.g. at the transition from
15° to 25° and the temperaturequotients were always found
to be the same as with those cultures, where the quantity
of water was normal.

In § 2 it was suggested that on linseed media the con-
sumption of oil changed into a consumption of carbohy-
drates at high temperatures. An examination of the figures
in § 2 might lead one to think that the initial values of the
Oa-absorption were caused by some obstacle to the diffusion
of oxygen. From chapter III, however, it may be seen that
the oxygentension can be reduced to less than half the
amount without affecting the respiration. It is therefore
very improbable that the decrease in the oxygenconsump-
tion on linseed at higher temperatures should be the result
of a lack of oxygen.

In § 3 A it was moreover found that the Oa-absorption
had directly adapted itself to the new temperature. If in
§ 2 the diffusion of oxygen had been limiting the respiration,
this would also have been the case in § 3.

The supposition that Phycomyces is able to withstand
high temperatures better on starch-media than on oil is
also supported by the facts found in § 3 A. The respiration
at equal temperatures does indeed decrease more rapidly

on oil-media than on starch-media, (compare fig. 31 and
42 for 37°5; fig. 30 and 41 for 35°). Further, in the long
run respiration at 32°5 again becomes constant on starch-
media, whereas on oil-media the decrease continues. On

10.0

Ohour
•yi hour

I:;

\\1hour

\\ \\\'X hour
\\ •

\\

3hour

bread the respiration is almost immediately constant at 30°,
and hence, in contrast with the linseed media, a harmful
influence cannot yet be spoken of.

It might be argued that as the respiration on linseed
passed into a consumption of carbohydrates at higher
temperatures, there would be no further reason for the
respiration to decrease more rapidly than on the other
carbohydrate medium bread. It must be borne in mind
however, that at the initial temperature 25°, the respiration
on linseed originally was more intensive than it can ever
be on the same quantity of bread. Besides, in linseed there
is a very small amount of carbohydrates.

In fig. 40, 41 and 42 the respiratory quotient approaches
the value 1.00. At high temperatures where the fungus
cannot consume fats, it is probably also unable to manu-
facture fat from carbohydrates.

In § 3 it has been mentioned that, in the case of the carbo-
hydrate consumption, the "zero-hour"-line found by means
of extrapolation seems to deviate at high temperatures
from the straight line found for lower temperatures. The
question might be asked whether this is not due to the
method of extrapolation. I do not think it is. If the respi-
ration proceeded along a straight line also at higher tempe-
ratures, the "zero-hour" values of COo-production and
Oo- consumption would be higher. In fig. 41 and fig. 42
these calculated values are represented on the line indicating
the zero-time i.e. the time at which the culture medium on
an average assumed the new temperature (p. 183). For the
Oo-absorption this value in fig. 41 at 35° is at the point Q.
Of course it is possible that in the 10 minutes between the
zero time and the beginning of the new experiments the curve
may descend very rapidly. As far as the COo-production
is concerned this cannot be denied. For the "Oo-curve"
however, it seems to me impossible because in this case

twice the number of observations were carried out. From the
very beginning of the experiment, moreover, the consumption
of oxygen may be gathered from the rate of electrolyzation
(galvanometer, fig. 1). So in fig. 41 the "Oa-curve" really
runs to point P as has been plotted. It seems improbable
that the curve should continue along the liiie P Q, as in
this case there would be a sharp bend in the curve.

Apart from the behaviour at harmful temperatures, the
unexpected fact is revealed that the intensity of respiration
on starch-media (as measured by gas-exchange) at harmless
temperatures is an almost linear function of the temperature.

Hille Ris Lambers (39) found the same function
for the influence of temperature on protoplasmic streaming.
Here the explanation is obvious as this process is chiefly
a matter of viscosity of the protoplasm, a physical process,
the velocity of which is a linear function of the temperature.

In the case of respiration various complicated processes
take place, ending in a process of combustion. As the inten-
sities of chemical processes are ordinarily exponential
functions of the temperature, a curve convex to the tempe-
rature-axis was to be expected, at least for the lower tem-
peratures, where probably diffusion processes not yet can
be limiting factors. An explanation of the straight line is

therefore difficult.

On the other hand it has been proved that the respiration
adapts itself at once to new temperatures, and in my case
not gradually as Kuyper and Rutgers supposed. When-
ever the adaptation seems to be gradual (the absorption
of O2 on linseed media) there is a definite reason for this
behaviour.

And finally oscillations in the respiration velocity, such as
Kuyper (52) and Fernandes (31) described for seeds,
did not occur in my experiments. It is difficult to say
whether this is the result of the simpler objects used, or
the consequence of more minute measurements.

No direct influence of light on respiration could be
detected. Experiments were further carried out on the
influence of other external factors on the respiration of
Phycomyces Blakesleeanus.

By means of an exact determination of the respiratory
quotients it was possible to find what kind of food the fungus
used from a heterogeneous culture medium.

The grand period of respiration was determined at 25°
C. on different quantities and different kinds of culture
medium. The length of the sporangiophores is an index
of their stage of development, corresponding to a specific
point in the grand period.

As a medium rich in oil I used ground linseed, as a starch-
medium bread. The respiratory quotient varies in different
parts of the grand period of respiration. On linseed it rises
from about 0.65 to about 0.75. On bread it becomes 1.00 in
the long run but at the maximum of respiration it is about
1.20. It was made probable that this is due to the manufac-
ture of fat from carbohydrates.

Phycomyces by preference takes fat when this is to be
had; on fatty media the respiration is more intense than
on starch-media. Proteins do not essentially participate in
the respiration.

The fungus cannot live anaerobically, neither on oil-media
nor on carbohydrates. A decrease in oxygen tension affects
Phycomyces very soon. On starch-media some effect becomes
noticeable in about 9 per cent, of oxygen, in 2 per cent, of
oxygen the respiration is reduced to half the normal amount.
Phycomyces can stand low oxygen tensions on oil-media
better than on carbohydrate media, at least in so far as the
COa-evoIution is concerned. On oil-media there is a visible

influence in about 7 per cent, of oxygen, in 2 per cent, of
oxygen the COg-evolution is reduced to about 70 per cent,
of the normal amount.

On bread the respiratory quotient does not change in
smaller percentages of oxygen, it increases on linseed media.

Pure oxygen has no influence on the COg-respiration,
the.Og-consumption only increases by a small amount.

It was proved that respiration adapts itself at once to
new temperatures and that a gradual adaptation to new
temperatures as found by former investigators may be
caused by experimental errors.

At all temperatures the respiration can be represented
by flowing lines, oscillations in the respiration did not occur.

At harmful temperatures the consumption of fat changes
into a consumption of carbohydrates. On linseed the
"zero-hour"-line has therefore a peculiar course. Both the
"Og-curve" and the "COg-curve" are slightly convex towards
the temperature axis, at high harmful temperatures, the "Oo-
curve" becomes an optimum curve because the Og values
approach the COg values and therefore decrease.

The respiration on carbohydrate media is an almost
linear function of the temperature. The "zero-hour" line
deviates from the straight line at high temperatures.

The foregoing investigations were carried out in the
Botanical Laboratory of the University of Utrecht.

This is the place to express my appreciation to Prof.
Dr. F. A. F. C. Went for his kindly help, interest and
criticism.

TABLES.

TABLE 3. (Fig. 2).

Small earthenware trough with bread.
Date: 29-4-\'26; Time 23.00; Temp, in the vessel 25° C.

9.00 thin sp. ph. 1J cm.

21.00 thin sp. ph. 3 to 3i cm.

thick sp. ph. 1 cm.
9.00 few thin sp. ph. 4 cm.
many thick sp. ph. 1 i to 2 cm.

22.00 some new thick sp. ph.

11.00 thick sp. ph. of different
length.

Old culture on bread.
Placed in the respiration vessel in the evening of 11-5-\'26.

TABLE 5 (Fig. 3).

2% grm. bread in two thin layers.
Placed in the respiration vessel in the evening of 17-5-\'26.

Small earthenware trough with ground linseed.
Placed in the respiration vessel in the morning of 24-6-\'26.

Small earthenware trough with linseed meal.
Placed in the respiration vessel at 14.00, 9-6-\'26.

TABLE 8 (Fig. 6).
Small earthenware trough with linseed-meal containing about 2^ % fat.
Placed in the respiration vessel at 11.00, 5-7-\'26.

TABLE 9 (Fig. 7).
Small earthenware trough with linseed-meal totally deprived of fat.
Placed in the respiration vessel at 9.00, 12-7-\'26.

12-7-\'26 13.30-16.00
16.00-18.30

18.30-21.00

21.00- 9.45
9.45-11.45
11.45-14.45
14.45-16.45
16.45-10.45
10.45-14.45
14.45-18.45
18.45-22.45
22.45- 9.45
9.45-13-45

10.00 sp. ph. I cm.

14.00 sp. ph. about 1 cm. thinner
ones longer.

21.00 thick sp. ph. nearly 2 cm.,
thin sp. ph. 3 cm.

9.00 thick sp. ph. about 3 cm.,
thin sp. ph. 5 cm.

TABLE 10.

Small earthenware trough with "milkbread".
Placed in the respiration vessel at 11.00, 5-8-\'26.