FISHERIES RESEARCH BOARD OF CANADA
TranSlation-Series No. 2718
Changes in the chlorophyll content 'and in the cell volume in
plankton'algae, brought aboüt by different,life Conditions
by Eva-Maria Bursche
Original title:.Anderungen im . ChlorophYllgehalt undiM
Zellvolumenbei'Planktonalgen, hervorgerufen durch':
.
unterschiedliche Lebensbedingungen
From:
Int. Revue ges. Hydrobiol., 46(4) : 610-652,1961
•
Translated by the Translation Bureau( PJW) Multilingual Services Division'
Department Of the Secretary of State of Canada
• Department of the Environment
Fisheries Research Board of Canada
Great Lakes Biolimnology Laboratory
Burlington, Ont.
84 pages typescript
•
FR/3 r.3-27/
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AUTHOR — AUTEUR
Bursche, Eva-Maria
TITLE IN ENGLISH — TITRE ANGLAIS
Changes in the chloroohyll content and in the cell volume in
plankton alrme, brougilt about by different life conditions
Title in foreign language (transliterate foreign characters)
Xnderunrr,en im Chlorophyllgehalt und im Zellvolumen bel Planktonalgen, hervorgerufcn durch unterschiedliche Lebensbedingungen
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Translation of articl a published in "Int. Revue ges.
Hydrobiol.", Vol. 46, No. 4, 1961, pp. 610 - 652.
Hydrobiological Institute of the Max-Planck-Gesellschaft,
Fuldastation Schlitz/Hessen.
CHANGES IN THE CHLOROPHYLL CONTENT AND IN THE CELL
Li
VOLUME 17 PLANKTON ALGAE BROUGHT ABOUT BY' DIEYERENT LIFE
CONDITIUS
anderungen im Chlorophyllgehalt und im Zellvolumen
bel Planktonalgen, hervorgerufen durch unterschiedliche
Lebensbedingungen)
UNEDITED DRAFT TRANSLATION
Only for information
By Eva-Maria Bursche
TRADUCTION NON REVISÉE
lrina
seulemont;
Table of Contents
610
1. Introduction
612
2. Method
•
3. Experiments with diatoms
a) Diatoma elonF.atum
SOS-100-10-31
614
- 614
622
b) Stephanodiscus Hantzschii
o) Diato.:2a elon^atum and :^tephanodiscus Hantzsehii
629
in a mixed culture
4. Experiments with planktonic ;hlorococcales
632
a) Scenedes-.qus guadricauda
632
b) Pediastrum bor^ranum
637
c) Ankistrodesmus falcatus
643
5. Discussion
645
6. Su.mr,la ry
650
7. Bibliography
651
,
1. Int'rbduction
The counting method is still regarded as indispensible for the quantitative determination of the phytoplankton.
This is due to the faât that with,it only the
algae themselves are counted; the other sestonic components
are disregarded.
The counting of the plankters, however,
require.s much time and also a special knowledge of algology.
For this reason the counting method is not a laboratory
serial method such as would be required at least for
production-biological investigations.
Nor does the number
of algae give any information about their amount of substance,
since the individual al-,ae vary greatly as to size, while
-3
only the amâunt of substance would be of any production-
biolordcal value to us.
To ascertain the algal volume it
is thus necessary to carry out various measurements.and
calculations.
Attempts are made to overcome the above difficulties
by means of chemical determinationsof, e.g. dry substances,
ashes, proteins. From these it should be possible to draw
conclusions about the algal quantity. But in carrying out
these experiments detritus and zooplankton are also included
in the experimental result, so that the latter may contain
almost incalculable errors of a considerablyohangeable.ordar
CIle
of magnitude.
For some time the less time-consuming chlorophyllextraction method has been used in limnology and oceanography to determine the algal volume indirectly.
In thiS
method the colour intensity of the green pigment solution
is taken as the measure of the phytoplankton contained in
a given volume of water.
However, as has already been
discussed on severpl occasions, the evidential value of
this method is only limited.
Thus, quantitative conclusions regarding the nuMber
of algae, based on the colour intensity of a plankton
ce
chlorophyll extract are unsatisfactory already because t1-1
'size of the algae and consequently their volume are not
P.611
- 4 taken into consideration here. Moreover, through the
species-specific differences in the pigment the heterogeneous composition of a pl:ytoplankton association makes
the estimation of the amount of chlorophyll more difficult
(Tucker 1949, Berardi e Tonolli 1953). Nor does the plankton
ash or the plankton dry substance represent a constant
reference value for the pigment content of the algae
(Margalef 1954, Harris and Riley 1956), not even within a
species, due to the fact that varying growing conditions
may affect the chlorophyll content of the algae.
Nutrition
(Boresch 1913, 7ande1s 1943, Pirson, Tichy and Wilhelmi
1952, Aach 1953, Dersch 1960) as well as light and temperature (Warburg 1925, Harvey 1952/53, Garnier 1958, Handal
1958) exert an influence on the pigment development. These
investigation results must be considered in coMbination
with the fact that there exists no direct dependence between
amount of chlorophyll and photosynthetic action of the algae
(person 1937, MacMillan Conover 1956, Yentsch and Ryther
1957, Sorokin 1957, Ryther and Yentsch 1958).
To this must
be added the methodical difficulties inherent in the extraction process itself and, in addition, the dead chlorophyll
from the seston (Gillbright 1952, Banse 1956), which is also
included in the investigation results.
In a quite general observation Krey (1958) has given
a gynopsis of the level of knowledge in the use of the
chlorophyll method in limnology and oceanography lje as
well as ''›yther and Yentsch (1957) demand that the inves-tizrations on the applicability of the chlorophyll extraction
method be expanded to increase its evidential value.
Since,
furthernore, in our own studies (Bursche, KUhl and Mann
1958, 1959 a, b) in the region of the Lower Elbe and of the
Lower Weser the plankton, seston, and protein values and
the values of the organic substances did not correspond to
the chlorophyll values, it seemed worthwhile to examine the
question regarding the value of the chlorophyll method price
more.
iiie
p-Lecut,
111Veble1t.I.CLU1V11
UVvca. a.clUv.icipj
ments with monocultures of the plankton algae Diatoma
elongatum, Stephanodiscus Hantzschii, Scenede_sauslE17
cauda, Pediastrum boryanum, and Ankistrodesmus faleatus.
These algae were tested for their c'nlorophyll value during
the growth periodi determined by the duration of tlie eneri=
ment. The nutrition conditions in particular underwent a
variation. Since there is frequently only a minimum amount
of nitrate in the waters and since, as is generally known,
a lack of nitrate leads to chlorosis manifestations, t. he
Ca(NO 3 )2-content of the nutrients was essentially changed.
The work with monocultures makes it possible to
detect not only the differences in the chlorophyll content
of the individual algae, but also, possibly, the fluctuations
6
in the chlorophyll content in the course of the development
of the algae. In contra st to the purely physiolof;ical
mode of operation, no value was placed on absolutely optimum
conditions of life.
lishing
We were rather only interested in estab-
experimentally to what extent we must reckon
with fluctuations of the chlorophyll content when algae
develop under varying living conditions.
As reference magnitude we chose that algal volume
which was based on the number of cells and on the volume
calculated from the measured cell sizes: in the first place,
we have here a biologically given measure and not an experimentally obtained value and, in the second place, it was tu
be expected that the. sizes of the cells changing in the
course of the development would, at the same time, provide
an insight into the manner of reaction of the algae to
various influences of the milieu. In addition, the dry
weight
of the algae was also determined.
2. Method
For the experiment the algae were kept in a photothermostat bath (Fi7. 1) under equal external conditions.
It consi st s of a flat frame-aquarium with a vertically
adjustable lighting system under its glass floor. The five
luminescent tubes mounted parallel to one another on a board
•
,
-
_
.
173-
-
-•
.;
;• •
;
•
r
•
4-
a
1::.
--1----
.
T7------'-ir
.
;
i
!( -
..
:
j
s•
,!'
.•
-
Fig. 1. Photothermostat bath with outer filter
installed in a laboratory.
correspond to the type: Warmton "De Luxe", 40 watts of
"Philips". By means of a "Thermomix II" the temperature
of the water is raised to the desired delreleund then kept
constant.
If necessary, the work can also be cerried out
at low temperatures with a cooline coil rcrtinl ac,LIn,-3t
the walls of the aquarium and connected to a cooling- a. , ! ,regate. Because of a continuously appearing bbcterlu-turbidity
the water had to be filtered with the aid of a coal-outside
filter.
The small experimental flasks with the algae cul-
tures were suspended in a frame up to their neck in the
water bath. The frame is movable in its longitudinal
direction by a motor drive so that a slight movement of the
nutrient solution may be achieved.
The culture flasks of a test series were charged
under sterile conditions with equal amounts of nutrient
solution and equal amounts of inoculation material. Within
one experimental series one part of the flasks frequently
contained a nutrient solution different from that of the
other part. The algae were thus able to develop at equal
temperature and lighting conditions, but in a varying nutrient
milieu.
Lighting daily for 12 hours.
After definite time intervals two experimental flasks
were removed at the same time of day from each culture series
in order to make comparisons'.
Basic nutrient solutions were: nutrient solution
No. 8 put together for the green algae by Rodhe (1948) and
the nutrient solution designated by Lefévre, Jakob and
Nisbet
(1952) by "L
C"
(however without (71.phagnum extract).
For the diatoms we used the nutrient solution No. 10 of
9
Chu (1942) : as source of iron, however, we did not use
iron chloride but an iron complex with ethylene diamine
tetraacetic acid (hDTT! iron) (Table 1). The nutrient
salts were individ'ially sterilized and only then corm-
P. 613
tined to the desired nutrient solution. The water was
twice quartz-distilled.
The culture dishes were cleaned
before the beginning of each ézperimental series according
to the specifications of Lund (1949).
The sterile mode of operation should offer protection against contamination of the cultures, especially
against moulding.
The algae themselves, however, were not
quite free from bacteria.
In order not to make the experimental conditions
too extreme in comparison to the conditions in nature, we
used neither clone nor synchrone cultures. The inoculation
material was added to the culture flasks containing the
experimental nutrien solution by cc's. As a matter of fact,
in this manner parent-culture nutrient solutions entered
the experimental flasks with the inoculation algae. But
since all investigation dishes were allotted the same
amounts the comparison conditions were preserved.
C
- 10 Table 1. Compbsition of the basic nutrient
solutions used (g/1)
------------------------------------------------------------l.ouul:
1 Cuti \r. 10 i
1 L1:rÈ.N•1... -..
r,zoaifica -- :
1.^;0:1
Ca(\0,)
K,1IPO;
\IrSO.,
\:i tii0s
Na.,COa
linti0,
0,0.1
0,01
0,0?5
0,0?6
0,0?0
-
I
0,00ri
0,005
-T
0,1
0,1
0,0-1
0,03
-0,0003
I 1
ISIS(•RC1iraE j- ^^ltl'ORCllS:1tiC(:I(
FCC13
'1DT ;
• 0,060
et -N I8B1,:
- i r on
6oil extract
Spur
0enl)1 -1-0,001
0,005
7,5 cllla
Iron nitrate and citric acid.
The algae were counted out by means of 1 mm-high
plankton chambers,the cover plate of which has al mm-9 net.
From almost every sample 50 algae were measured off (objective 45-fold, micrometer eyepiece 3- or 10-fold). The
volume of the algae was calculated by means of the formulae
developed from the plasticine models (Bursche 1959 b; the
calculations were based on the cell width),or for Stephanodiscus Hantzschii simply as cylinder. The measurements
on which the volume calculations were based corresponded
to the average values which usually were formed from 100
measurements according to the multiplication method (Weber
1957).
If there were fewer than 100 measurer.lents used
a notation to that effect is made at the respective place.
The paper filters of Schleicher and Schiill No. 1575
•
- 11 -
were compact enough to keep back all experimental algae.
For the dry weight determinations the algae were dried for
one hour at 90 0 •
Since chlorophyll extractions according to the
rapid method of Oorshot (1955) produced in preliminary
experiments lower values than chlorophyll extractions according to the method of Krey (1939), only the latter were
used in the actual studies. Deviating from the instructions of Krey the algae on the filters were not killed off
by water steam; they were rather held for l minutes in a
• small glass tube in boiling water.
After
the addition of
methanol (an addition of MgCO 3 remained without effect)
the samples remained for 4 to 5 hours in the dark at room
temperatures.
This time was sufficient to extract the
chlorophyll quantitatively. Extraction difficulties were
encountered now and then with respect to Pediastrum boryanum.
In_such case neither repeated cold extraction nor hot extrac-
tion (Ungemach 1960) led to our objective. Pirson, Tighy
and Wilhelmi (1952) report that it was remarkably difficult
to extract Ankistrodesmus falcatus where a lack of phosphorus,
magnesium,and potassium prevailed. Similar causes may have
obtained temporatily in the case of Pediastrum.
The absorption maximum at 660 mil_ (interference
band filter) was photometered with the visual "Leifo"
P. 614
- 12 (Leitz).
Following the suggestion of Krey (1958) and the
already generally practiced manipulation, the calibration
curve was set up with crystallized chlorophyll "a" (Firm:
Sandoz, Basle). It must, however, be emphasized that the
chlorophyll quantities determined in this manner do not
represent the chlorophyll "a" present in the algae, but
rather the green pigment content measured at 660 m»...
. The samples were usually worked when fresh. When
this was not possible they were kept in the dark in a
refrigerator.
No changes occurred than in the chlorophyll
value or in the other reference
values.
Air-dried algae material could not be extracted
quantitatively.
Nor did a carefull drying in the exsiccator
and storing there in the refrigerator (Creitz and Richards
1955) make possible a complete extraction in the green algae.
The two diatom species, on the other.hand, could be completely extracted, provided MgCO 3 had been added during
the filtering and they had been dried in the exsiccator.
It was necessary to kill of the fresh algae in the water
bath.
- 13 3. Experiments with Diatoms
a) Diatoma elon,matu~n
The diatoms were cultivated in the modified nutrient
solution No. 10 of Chu (Table 1). In the experimental
series the Ca(DT03)2-content was varied and, in some cases,
CaC12 was added to the nutrient solution instead of Ca (N03)2*
An orientation experiment carried out at 6000 lux,
15° C waterbath temperature, and with the modified culture
solution No. 10 of Chu which contained 60 mg of Ca (r?03) 2,
provided primarily information on the chlorophyll content
of a Diatoma cell amd aon the standard cell volume of 1 mm3.
Within 11 culture days there occurred threshold values
a
between 4.4 x 10-b y and 2.3 x 10-6^1 chlorophyll per cell
and 3.7 y and 2.2/ chlorophyll in 1 mmr5 of cell volume. A
visible chlorophyll decomposition did not take place during
the experimental period.
In order to reproduce also a state of nitrate defioiency without extending the experimental period,onp pax't.of
the experimental flasks has been provided in the following
culture series with a nutrient solution which contained
calcium chloride instead of calcium nitrate. Only with the
inoculation material did these cultures receive nitrAe.
In the following two experimental series one part
- 14of the culture flasks was thus now provided with a nutrient
solution ("I") containing 60 mg Ca(NO 3 ) 2/litre, a second
part with nutrient solution ("II") containing 18 mg 0a(Nq 2/1,
and the other experimental flasks with a calcium nitratefree nutrient solution ("Ill"); instead of clacium nitrate
the latter contained 18 mg Ca01 2 /1.
Six thousand lux were
directed at the algae at the bottom of the culture dishes.
Ap..e and amount of inoculation material were the saine in
both culture series.
The first of these two experiments was carried out
at a waterbath temperature of 15 0 C.
The cell multiplication proceeded fairly unïforMly
1
in the two Ca(M0) -containing culture series (Fig. 2) .
2
With the exception of the two values for nutrient solution
series I and II from the last culture day -- which represent
individual values -- all others are the average of the
investigation results from two parallel cultures.
At the beginning the rate of division was greater
in the Ca01 -containing culture series than in the "nitrate
2
cultures". But toward the end of the experimental period
1 For greater clarity we chose the curve representation. The lines between two measurinp., points represent
only connecting lines and not a development.
- 15 -
:Nur^ber of
cells in
Chtorophyll
d /10cm
Vcjlumep
/U I t cF1
X10
1 ur ^'
xh0
200r
200
(.
150
100
50
1
3
5
7
9
11
5
7
9
11
3
5'
.7
9
11 Days.
Fig. 2. Cell multiplication, algae volumes and
chlorophyll content in the course of the culture experiment
with Diatoma elonLatum at 150 C.
o
o
nutrient solution series I
p ---* nutrient solution series II
x -•- •--Z nutrient solution serie s III.
--------------------------------------------------------------
- 16 -
the number of cells decreased to such an extent that
P. 616
there were fewer diatoms in this nutrient solution series
than in the two other culture series. The total algae
volume in 1 cc of culture solution,corresponding to the
number of cells, manifested the same tendencies as the increase and decrease of the number of cells. In contrast,
the chlorophyll content of the algae showed striking deviations since in spite of the increase in the number of -oells
and Iri the volume in nutrient solution series III, the
amount of pigment decreased already at the same time.
These conditions, however, become clearly apparent
only when the chlorophyll content is referred back to the
standard measure of a cell or, preferably -- since the size'
of a cell is changeable -- to a standard cell volume of
1 mm3 (Table 2). The numerals show that in the cultures
containing calcium nitrate the chlorophyll content in a
cell or in a standard volume changed fbr less than in the
calcium chloride-containing medium.
The deficiency symptoms
which at first found no expression in the cell production soon
showed themselves in a lower chlorophyll content of the
algae material and in the smaller averuge cell volume. The
chlorophyll decomposition in nutrient solution III is
noticed also in the dry weight.
The deficiency cells had a pale aj.pearance; the
chloropla st s were to a large degree degenerated. :11hen the se
- 17diatoms were transferred into fresh parent nutrient solution
growth and chlorophyll formation set in again.
The experiment was not extended any farther, since
the objective had been to establish only the fact that the
amount of chlororhyll can be affected by the nutrient milieu.
This was quite evident from the results of the investigation.
The conditions of the next experimental series
with Diatoma corresponded -- with the exception of the
temperature which was lowered to 8 0 C -- to those of the
preceding culture experiment. According to Huber-Pestalozzi
(1942), Diatoma elongatum appears as spring form at temperatures between 4 ° C and 10 ° C.
According to this, good
growth might have been expected at 8 0C.
Utermahl (1925) still found numerous chains of these
diatoms even in the summer months in the Plane Lakes.
The
isolated cells which led to the Diatoma monoculture used here
originated from summer plankton catches in the Small PlOne
Lake.
At 8 °C the cell partition proceeded slower than at
15 °C (Fig. 2 and 3).
However, at 8 °C it continued in all
three culture series to the last culture dey. This time, too,
the partition frequency was again greater at the beginning
in the nitrate-poor chloride-containing medium than in the
- 18nitrate-rich nutrient solutions.
While the tôtal algae
voluneincreased in the suspension unit in culture series
I and II apuroxinately to the same extent as the number of
individuals, this was soon not the case any more in nutrient
solution series III. The chlorophyll degeneration in the
Diatoma cells from nutrient solution series III became
noticeable already starting with the 2nd experimental portion.
Nor did the chlorophyll content in a cell such as
the one in the standarà volume of 1 mm 3 reach the values
established in the preceding experiment (Table 3). In the
(-11
nitrate-poore chloride-containing milieu Diatoma contained
at the end of the experimental period only one third of the
chlorophyll contained in the algae of culture series I and
In this experimental series the dry substance could
be determined only in a few specimens.
But even from the
limited data obtained we may draw the conclusion that the
chlorophyll poverty of the algae material from nutrient
solution series III stood out also with regard to the dry
weight.
II.
Table 2. Cell volume, chlorophyll, and dry-weight values of Diatoma elonatum
in a culture experiment at 15 00
Culture Volume of one ychlorophyll 3, Chloroeyll % chlorghyll y dry weight
'in 1 mmD of in dry weight of one cell
days
cell (»)
in one cell
(x 10 -4 )
(x10-6 )
oeil volume
•
I9
3
.5
1225
1214
1157
1116
(1135) 2)
7
9
11
II')
1185
1168
1144
1073
(1109) 2)
III 1 )
I
Il
III
I
II
III
1097
959
984
937
987
4,4
4,3
3,5
(3,8)
(4,2)
4,9
3,9
3,9
3,6
(3,9)
3,7
2,9
1,5
1,6
1,2
3,6
3,5
3,0
(3,4)
(3,7)
4,1
3,4
3,3
3,3
(3,5)
3,4 .
3,0
1,5
1,7
1,2
TH- H
1,04
1,01
(1,2)
(1,2)
1,03
1,15
1,3
(1,4).
ni
i
n
ni
0,95
0,48
0,43
0,3
4,0
3,6
3,2
(3,4)
3,9
3,2
2,9
(2,9)
3,1
3,0
3,7
3,9
1) 1, II, and III denote the nutrient solution series
2) () rumerals in brackets are individual values
Table 3. Cell volume, chlorophyll, and dry-weight values of Diatoma elongatum
in a culture experiment at 80C .
Culture Volume of one ychlorophyll chloroehyll % chlorophyll ydry weight
In 1 mm'à of in dry weight of one cell
cell (je)
days
in one cell
)
-6
(x 10 -4 )
cell volume
(x 10
I')
II 1 )
III 1 ) '
I
ll-
1241
1218
1183
1156.
(1193) 2)
1232
1192
1137
1177
(1145)
1103
1003
951
993
913
3,3
3,0
2,9
3,9
(3,5)
3,2
3,2
3,2
3,5
(3,5)
.
3
5
7
9
11
In
2,9
1,8
(1,4)
1,7
(0,95)
•
I
H
III
I
II
III
I
II
III
2,6
2,3
2,4
3,4
(3,1)
2,6
2,6
2,8
3,0
(3,1)
2,6
2,0
2,8
1,6
(1,0)
0,68
1,0
(1,4)
0,78
1,3
(1,5)
(0,82)
(0,36)
4,2
3,8
(2,6)
4,0
2,7
(2,4) .
-
II and III dEnote the nutrient solution series
2 ) () rumerals in brackets are individual values.
(2,3)
(2,6)
-
20 -
------------------ -------------------------------------------
Nunl e r of
çin
x'.c^
Ch► orop hylj
ffl l0cm
Volume
U' / t qm'
A10
10
5
1-
3
5
•7
9
11
3
5
.7
9
11
7
9
11
dr.̀iy.S..
Fig. 3. Cell multiplication, algae volume, and
chlorophyll content in the course of the culture experiment
with Diatoma elongatum at 8oC.
O -o nutrient solution series I
nutrient solution series II
x-.---x nutrient solution series III
---------------------------------------------------------------
- 21-
An insight into the distribution of the cell magnitudes within the different culture series is provided by
the length and height measurements of the diatoms on which
the volume calculations are based.
The volume of Diatoma elongatum has been calculated
as parallelepiped from which one twentieth of the total
volume was deducted (manner of calculation derived from
experiments with plasticine models). From the respective
parallel specimens we were able to form an average value
for the length and height of cell. For technical reasons,
however, we were able to ascertain only an average shellwidth for each nutricnt solution series.
No masurement
were undertaken of dividing cells in which cell-wall
formations were already recognizable.
In the first serial experiment with the three culture series the cell lengths showed not only approximately
the same variation Tange in all three nutrient media, but
the distribution of the cells of equal length also indicated
no significant differences (Fig. 4).
The average length
of the cells amounted in nutrient solution I to 50.68p
t 2.66, in nutrient solution II to 50.49 p. ± 2.66, and in
nutrient solution III to 50.50 p ± 3.15 (average values in
each case from 450 measurements).
P. 619
- 22 -
-------------------------- -------------------------------
•r.
Fig. 4.
Percentage distribution of the apical-axes
class means of Diatoma elon ^a t,ain (experiment at 15°C).
o -o nutrient solution series I
ça------q nutrient solution series II
x- --.-x nutrient solution series III.
----------------------------------------------------------On the other hand, we get a quite different picture
when examining the height of the cells (Fig. 5). Here we
notice somethins that was readily revealed by a look into the
microscope: in the calcium chloride-conttaining, nitrate-poor P. 620
nutrient solution the cells were on an average flatter than
- 23 -
50
•
40
i
•
*xf
30
I/
1
i
20
1
1
11
II
'\ II
\
-1
1
•. 1,
•
I
1
10
,
I.
i i
I
1i
-
•
2,5
3
■
\' >
41,5
5,5
6i5
f7,5
class
(»). means
Fig. 5. Percentage distribution Of pervalvar-axes
class means of Diatoma elongatum (experiment at 15 0C).
o
o nutrient solution series I
0
o nutrient solution series II
x--.-----x nutrient solution series III.
in the nitrate-rich culture series. The average length of
the pervalvar axes (calculated in each case from 450 measurements) amdounted for the cells frOm nutrient solution I to
4.88 fu * 0.83, for those from nutrient solution II:to
- 24 -
4.76 Éu 1 0.80 and for the cells from nutrient solution III
to 4.36;u t 0.75. The ran ;E of vai iation of the lenFths
of the pervalfar axes and the frequency disti-,ibution of the
cells corresponded in the nitrate-rich media far-reachingly
with one another. In nutrient solution III we did not
find the maximum heiphts of 7.0 tu and 8.2 p measured in
nutrient solutions I and II; instead we found there a
remarkably high percentage of cells with c comparatively
small pervalvar-axis length.
Cells with a pervalvar height
of more than 5.5 jtz could be found in the nitrate-rich cultures to the end of the experiment. Among the measured
cells from nutrient solution series III we found, on the
other hand, after the third experimental day only two specimens which were higher than 5.5 p.
During the course of the experiment the cells in
nutrient solution II manifested generally.a somewhat lower
cell height than those in nutriènt solution I. While during
the first culture days the average pervalvar heights of the
eells from nutrient solution III were only slightly less:
than those of the cells in the nitrate-rich nutrient solution
series, later they were considerably less. These differences
in the average height of the Diatorna-cells can be regarded
only as nutrition-dependent.
This conclusion is supported
by the dry weight of the cells (Table 2). The increase in
weight of the Diatoma deficiency cells toward the end of
P. 621
- 25 -
the experimental period may possibly be traced back to
the formation of reserve substances.
The frequency distribution of the oeil lengths in
the experiment which was carried out at 800 was practically
the same as in the experiment discussed above.
More than
40% of the 450 cells measured in each case from the individual nutrient solution series belonged to the same class
of magnitude.
length
According to our calculations, the average
of the cells from nutrient solution series I amounted
to 50.43 1.1.
2.5,
of those from nutrient solution series
II to 50.15 p. 1 2.9, and for the cells from nutrient solution series III the average length amounted to 50.08e 2.7.
The frequency polygons of the cell height (Fig. 6) also
resembled those of the preceding experiment.
This means,
the 1engths of the pervalvar axes of the cells from the
nitrate-rich culture series showed a far-reaching agreement
in the distribution of the magnitudinal classes, while most
of the cells from nutrient solution III showed pervalvar
axes of ric lower magnitudinal classes. The average height
of the cells from nutrient solution
4.98 fi
I was calculated at
0.8, that of cells from nutrient solution II at
4.62 la 0.88, and that from cells of nutrient solution Ill
at 4.35 p ± 0.76. Cells which were hir7,her than 5.5
occurred in culture series III (with the exception of very
few specimens) only up to the fifth culture day, while in
P. 622
- 26 -
;5
6,5
7,5
8,5
c l a s^s me a n s
^
Fig. 6. Percenta;;e distribution of the pervalvar-axes
class means of Diatoma elon^^tum (experiment at 80C).
0
o nutrient solution series I
9-------m nutrient solution series II
x- .---x nutrient solution series II1
------------------------------------------------------------the two other nutrient solution series such high cells still
occur to the vary last culture day.
1, few Diatoma deficiency cells which had been exposed
to the experimental conditions for 9 days were then placed
- 27 in fresh parent nutrient solution.
After 8 days the average
length of the freshly pigmented cells measured 49.2 p as
compared to an original value of 49.7iu.
However, the average
height now amounted to 5.4 du, while the deficiency cells
showed only an average length of the pervalvar axes of 4.2
F.
These correlations between the cell dimensions and
the nutrient milieu hold true for the given experimental con-
Diatoma elongatum need not .tre. form flat cells under
ditions.
unfavourable conditions. This conclusion was reached when
measuring the height (and length) of cells from an old parent
culture the nutrient solution of which was not changed within
10 weeks.
The average height of Liles° cells amounted to
5.3p (their average length, to be sure, amounted to only 37.8 0.
It should also be mentioned that Diatoma -- at 15 °C
as well as at 8 00 -- formed typical chain colonies only in a
nitrate-poor, chloride-containing medium. In the nitraterich culture series there occurred at most short pieces of
a chain, but usually three- or four-limbed stars were formed.
In the described experiments the colony form thus cannot -as reported by Huber-Pestalozzi (1942) -- have been caused by
the water temperature, but rather by the nutrition factor.
The deviating colony forms were observed in both experimental series
only when investigating the 5 day-old cultures, i.e. at a tine
when the chlorophyll decomposition was already noticeable
in culture series III.
- 28 -
h) ntphanodiscus Hantzschii
Like Diatoma, Stflpanodiscus Hantzschii originated
from the PlCne Lakes.
This diatom was also cultivated in
nutrient solution No. 10 of Chu, with ethylene diamine
tetraacetic acid-iron (XDTE-iron) instead of Fe01 3'
The experimental set-up was the same as the one in
the Diatoma culture series.
The temperature of the photo-
thermostat bath was not varied; in the experiments with
Stephanodiscus it amounted to 15 °C.
In the first culture series we studied growth, volume,
chlorophyll content, and dry weight in the nutrient solutions
I and II at 6000 lux.
It was found that the chlorophyll
content of a cell or of a cell-volume unit lay in the same
order of magnitude as for Diatoma (Table 4).
In Staphano-
discus, too, fluctuations in the .chlorophyll content occurred
during the experimental Deriod; they proceeded independently
of one another within the two nutrient solution series.
Pronounced deficiency symptoms were not noticed. The
of the
percentage/chlorophyll portion of the dry weight corresponded also with regard to the order of magnitude to
the values ascertained for Diatoma. The dry weight of one
single Staphanodiscus cell was less than that of a Diatoma
cell. In this res-ect conditions were approximately equal
to the volume ratios.
P. 623
- 29 -
Table 4. Cell-volume, chlorophyll, and dry-weight
values of Stephsnodiscus Hantzschii in the culture experiment
at 6000 lux
Culture
days
Ydrÿ
-ychloro- vchloro- ;o of
Volume of phyll in phyll in chloro- weight
of one
one cell one cell 1 mm3 of phyll
cell
in dry^
ce 11(x 10-6)
( 3)
I"
weight (x 10-4)
volume
11)
/
3
5
7
9
10
11
1101
1070
978
914
939
906
I
II 1)'
1070
983
897
864
898
846
I
3,75
2,95
2,80
2,75
2,85
3,40
i
II
I
4,70
2,45
2,75
3,20
2,65
2,50
3,5
2,7
2,8
3,0
3,0
3,8
I
II
4,4
2,5
3,1
3,6
3,0
2,9
i
I
li I
1
1
1,3
(1,3)2)
1,4
(1,5)2)
1,45 1 (44)
I
2,85
(2,3)
(1,7)
2,35
I
II
2,8
2,5
1,9
(1,8)
1)I and II denote the nutrient solution series.
2)
() Numerals in brackets represent individual values.
On the basis of these experimental results the next
culture series with St^hanodiscus was carried out with
nutrient solutions deviating more strongly from one another:
nutrient solutions II and III and a nutrient solution which
instead of Ca(N03)z or-CaC12 contained 18 mg CaSO4/litre
(IV).
The temperature of the ph`^tothermostat bath amounted
aF.ain to 15°C. The illumination was increased to 8000 lux.
The dry weight could not be determined since.due to
the insi-,nificant chlorophyll development in the deficiency
series, the. entire algae material was needed for the pigment
determination.
- 30 e
it
AccordinF; to Fia. 7 there was no complete correla-
tion between number of cells, total volume, and chlorophyll
content.
The multiplication effect produced in nutrient
solution III in Diatoma did not appear in Stephanodiscus
Hantzschii.
The cell partition was impeded in comparison
to the multiplication performance of the cells in nutrient
solution II.
In nutrient solution IV the nu:aber of cells
was still less. In this culture series the algae material
was so chlorophyll-poor that the pigment content of 70 ce
suspension could not be detected any more with the "Leifo".
That Ste hanodiscus was nevertheless still viable in this
nutrient solution was demonstrated by transferring the cells
into fresh, nitrate-containing culture solution in which
the diatoms developed well and pigmented anew.
In nutrient solution III the chlorophyll content
of
Sta_phanodiscus
also decreased so sharply that after
the eleventh culture day no pi^ment value could be determined any more of the 70 cc of algae culture. In this case
the addition of nutrient substances also led to new growth
and renewed pigment formation.
In comparison to the chlorophyll amounts in Stephanodiscus cells from nutrient solution II in the above-desoribed
experiment,.the cells at 8000 lux contained toward the end
of the Pxperimental period somewhat less chlofophyll (Table 5).
- 31-
liul.;_.er of
oPll
in
lcrq
xi°
Vglumep
,u /1cm
xlO
Cht0r0 P h y 1
2(110 c m
15
150
150
/. 0
-0
10
100
100
o
0
,o
50
50
.•
o/ .
- 4--
-
o
•
•
. • ---
..x, .. x
0
e
•
1
4
6
8
1
M
1
4
.•
1
6
8
1
11
•,•
1
1
13
4
6
--
8
. •t
n
u days
Fig. 7. Cell multiplication, algae volume, and
chlorophyll content in the course of the culture experiment
with Stephanodiscus Hantzschii at 8000 lux.
o------o nutrient solution series II
nutrient solution series III
nutrient solution IV.
It cannot be decided here whether it was the light which
was the cause of the differences in the pigment formation.
While the inoculation material was equally old in the two
culture experiments, the number of initial cells and the
P. 625
- 32
nutrient solutions of the parent cultures were not quite
the same.
The That that the algae form less chlorophyll
at high liP.ht intensities than at a weaker illumination
seens to justify the assumption that our present findings
could possibly be connected with it.
Table 5. Oeil-volume and chlorophyll values of
Stephanodiscus Hantzschii in the culture experiment at
8000 lux.
Culture Volume qf one
cell (FQ)
days
0
0
11 1
4
6
8
11
13
)
1007
1099
983
944
866
III')
IV 1 )
946
896
1036 ... . 847
921 818
(962) 2)
(897) 2 )
ychlorophyll Vchlorophyll
in one cell
ih 1 mm 3 of
cell volume
(x 10 -6 )
II
III
IV
II
III
IV
4,90
2,75
2,25
1,95
2,30
1,85
1,40
0,79
-
-
4,8
2,5
2,3
2,0 •
2,7
1,9
1,4
0,88
--
-
•
1) II, III, and IV denote the nutrient solution series
2) ( ) NuIlerals in brackets are individual values.
Even if the growth of SteDhanodiscus was extremely
poor in nutrient solution III, a multiplication nevertheless
did take place.
The chlorophyll decompositinn was just as
noticeable as in Diatoma.
The lowest still determinable
piment content amounted to 18% with reference to the highest
chlorophyll value in 1 mm 3 cell volume found in thisexperimental series.
- 33-
In the first experiment with Stephanodiscus the
cells of both nutrient solution series (I and II) showed
approximately the same frequency distribution of the cell
diameter during the culture exi:eriment.
As regards the
lengths of the pervalvar axes it was, in contrast, found
that the nitrate-richest culture series contained more
higher and fewer flat cells than the,nutrient solution series
with only 18 mg (Ca(NO3)2/litre.
The average height for
the cells from nutrient solution I was calculated at 10.3)u,
for the cells from nutrient solution II at 9.97)u (average
values from 360 measurements in each case). From the first
portion of the experiment on the average cell volumes were,
however, somewhat larger in culture series 1 than in culture series II.
The frequency distribution for diameter (Fig. 9) and
height (Fig. 10) of the Stephanodiscus cells from the second
experimental series showed basically the saine distribution
tendency as the Diatoma cells.
The cell diameters (which are
to be compared with the cell lengths of Diatoma) hardly distinguished themselves from each other in the three nutrient
media. Approximately 70% (350 measurements per culture
series) belonged to the sanie order of magnitude.
Again the
nutrient solutions without calcium nitrate contained a number of cells with a smaller diamater than the calcium nitraterich culture series, but also fewer cells with a maximum
- 34 r
14,5 class mean s
(N
Fig. S. Percentage distribution of the class means
of pervalvar axes of Stephanodisous Hantzschii. (Experiment
at 6000 lux.)
o -o nutrient solution series I
e)-------o nutrient solution series II
e-1110
-7
7071
GO
50
,
f
40
1.
30
•
•
20
• 1\
• -
.
10
•
• 11
/1
•
915
ic
ue
14
ije
class means
9. Percentage distribution of the class means of
the diameters of Sterhanodisous Hantzschii (expriment at
8000 lux).
0------0 nutrient solution sQ ries II
nutrient solution eries III
nutrient solutiQn geries IV
- 36 --
•
X
40
^^ .i• i
^
30
y'V
•/
•^
'• ^
20
x
•
o
•\
•
/
/
-
^^
10
•^
,x / -/
/•
65
7,5
Fip:. 10.
\
^ •I
^ /x\
^^•
^ \\
'
105
11,5
2^
13,5
14,5
1^5
class means
(,U)
Percentage distribution of the class means
of pervalvar axes of Stephanodiscus Hantzschii (experiment
at 8000 lux).
o---o nutrient solution series II
•---.--•---• nutrient solution series III
x-- ••--- ••---x nutrient solution series IV
diameter.
The average diameter of the cells amounted in
nutrient solution II to 3.1.01 V :t 0.83, in nutrient solution
III to 10.8:3P ± 0.82, and in nutrient solution IV to
10,65 Iù x 0.96.
- 37 -
In Stephanodiscus the differences in the cell
P. 626
volume were also due to the plasticity of the pervalvar
axis. This was shown most clearly by the cells from the
nutrient solution series IV. In this series occurred comparatively many flat cells, so that the class-magnitude
distribution of the cell height deviated from that of the
alpae material from the other two culture series. Nutrient
solution III, which in Diatoma exerted a quite definite
influence on the length of the pervalvar axes in comparison
with the cells of the calcium nitrate-containing cultures,
did not act in the same manner in Stephanodiscus. According
to our calculations, the average height of the cells from
nutrient solution II amounted to 10.64 iu t, 1.5, from nutrient
solution III to 10.49 /u ± 1.48, and 'of the cells from
nutrient solution IV to 9.7
± 1.34.
The distribution of the aells with pervalvar axes
P. 627
of the same class means showed that within the individual
culture series basically no deviations from the mode of
distribution registered for the first days of the culture
experiment occurred during the entire experimental period.
Until the . very
end of the experiment there were still
P. 628
comparatively high cells present in nutrient solutions II
and III.
This fact indicates that an enlargement of the 0011 need
not be causally connected with good nutrition conditions,
- 38 Cell enlargements during cell deficiencies have been known;
they will be pointed out later.
Algae material which after the eleventh day was
transferred from culture series IV into fresh nitratecontaining nutrient solution where it multiplied and pigmented well showed after 5 days an average pervalvar-axis
length (calculated from 50 measurements) of 11.76 p. The
Initial cells had had an average height of 9.85 i. The
average cell diameter of the cells grown in the new nutrient
solution amounted to 10.58
while the initial material
had shown an average diameter of 10.4314.
Similar oeil
enlargmts
in fresh parent nutrient solution could, to
be sure, also be determined in Staeanodiscus from cul-,
ture series III.
The average oeil dimensions of algae material from
an old parent culture have also been determined for Stephanodiscus. Just as in Diatoma, the average diameter (= apical
axis in Diatoma) was considerably shorter, while the cell
height barely deviated on an average from the height of the
cells from the culture experiments.
These serial experinents with Stephanodiscus thus
showed that the cell volume as well as the chlororhyll
content of an algal species may'vary considerably in conformance with the environmental conditions.
P. 629
- 39 r
c) Diato!na elon,atum and Step^ianociiscus T?antzschii
in a mixed culture.
To find out ^•:hether in a:aixed culture the chlorophyll
vGlues in the standard cell volume and the percentage rate
of the dry weight are the same as in a monoculture we carried
out an experimental series with Diatoma and Staphanodiscus
in a mixed culture.
The experimental set-up was again the same as in
the above-described experiments.
The temperature of the
photothermostat bath amounted to 15oC, the light intensity
to 6000 lux.
In nutrient solution I the multiplication led in
general to somewhat higher numbersof cells than in nutrient
solution II (Fig. 11). Once the culture had 150,000 to
200,000 cells per cc the multiplication was virtually discontinued; this had also been observed 'in the course of the
preceding experiment s.
If the development of Stephanodiscus and Of Diatoma
àre studied separately from one another we find that within
the culture ti^ae the development in the mixed culture
differs from that in the experiments with the monocultures.
During the first 7 culture days rtephanodiscus and Diatoma
showed practically the same growth in the two nutrient
- 40 -
solutions.
Thereafter more pronounced growth deviations
were noticed; a a matter of fact, similar deviations had
already been determined in the monoculture for Stephanodiscus, but not for Diatoma.
In contrast to the Stephano-
discus cells which had multiplied more markedly in nutrient
solution I than in nutrient solution II, Diatoma had multiplied in the monocultures almost evenly in the two nutrient
solutions. In the mixed culture, however, more Diatoma
cells were formed during the last experimental days in nutrient
solution II than in nutrient solution I.
In culture series II,
in which the growth conditions were not as favourable any
more for Stephanodiscus after the seventh culture day, Diatoma
was thus able to develop somewhat better than in hutrient
solution series I; in the latter conditions of life favoured
Staphanodiscus. In contrast to Stmhanodiscus, the growth
of Diatoma appears to have been somewhat inhibited as a whole,
for it did not achieve the same cell production as in the
monocultures.
In nutrient solution I the increase in the number of
individuals was not accompanied any more by an increase in
the volune between the last and the second-last portion of
the experiment.
The alree material in the mixed culture contained
more chlorophyll than that in the monocultures (Table 6). So
far we are unable to give an explanation of this phenomenon.
- 41 ----------------------------------------------------- ------------------TJiatoma e
Stephan. H
1VUilluer of cells ivLUnoer of cells
Number
of cells
in 1;,^,,
x 101
Ch; O' CC?-/l!
ï I l l, cni
in 1cm'
inlcm'
vc; ;,men ,
m
yx10.
150 7 r
2
4
7
8
9
;0
2
4'/ C 9 10
r r-1 A^> ti r c,
1l) 3.► uJ ^
i
`1
Fig. 11. Cell multiplication, algae volume, and chlorophyll
content during the culture experiment with Diatoma elongatum { Stephanodiscus Hantzschii.
o nutrient solution series I
•----- - nutrient solution series II
------------------------------------------------------------------------In the mixed cultures, too, no exhaustion of nutrient substances became noticeable in these two nutrient solution
series during the experimental period.
Since the mass of a1Rae of the first culture days
was required for the chloro^hyll extraction, we were able
to determine the dry substance only from the 8th day on.
- 42 -
The dry weie,hts of the mixed-culture material lay in the
sane order of magnitude as those determined for Diatoma
and Starhanodiscus in monocultures. Percentagewise, too,
the chlorophyll content of the algae was higher in the dry
substance.
The results of the investigation of this_series of
mixed cultures make it seem probable that the allelopathy in
the algae not only controls the growth, but that it also
exerts an influence on the pigment formation.
The percentage frequency distribution of the oeil
P. 630
lengths or cell diameters did not manifest any peculiarities
in comparison with the results of the investigations of the
other culture experiments.
Table 6. Cell volume and chlorophyll values of Diatoma
eloneatum and Stepahnodiscus Hantzschii in a mixed-culture
experiment
Volume of a' Volume of ychlorophyll echlorophyll
in dry weight
Culture Sterhano- a Diatoma 'in 1 mm 3
cell volume
days
discus cell cell
(-1.F3)
(in3)
. .
2
4
7
'
8
9
10
1)
.
I 1)
II')
I
II
I
11
929
922
927
962
891
857
868
902
971
931
917
901
1068
1118
1085
1121
1115
1107
1230
1161
1082
1082
1092
1034
8,5
4,8
4,5
4,5
4,5
4,8
4,6
3,6
4,6
4,9
4,0
4,2
II
1,8
1,9
1,6
-
1,7
2,0
1,4
I and II denote the nutrient solution series.
- 43 <
The variation ranp.e of the lengths of the pervalvar
axes of Stepha^odiscus and Diatorsa was virtual_Ly the saine
as in the monoculture series. The frequency distribution of
the cell heights of the same class means (Fig. 12 and 13),
on the other hand, deviated especially in Diatoma -- whose
growth in the mixed culture was significantly affected by
Stephanodiscus -- considerably from conditions existing in
the monoculture.
In nutrient solution II, in which Diatoma
multiplies better, the cells were on an average higher than
in nutrient solution I. The cells of both culture series
were on an average somewhat flatter than the Diatoma cells
.P
from the same nutrient solutions in the monoculture series,
but higher than the cells which grew in the nitrate-poor,
chloride-containing nutrient solution.
In Stephanodiscus the various vital conditions again
did not express themselves _appreciably in the length of the
pervalvar axes. Moreover, the multiplication of this alga
was hardly affected in the mixed culture. However, the oe11s
were on an average not as hidh as in the Stephanodisousmonoculture experiment (also at 6000 lux and with inoculation material that had been cultivated in a.sirsilar nutrient
solution ).
In contrast to the almost continuous decrease In
the lennth of the pervalvar axes in the course of the experiment with the monocultures the average height of the oells
- 44 -
8,5
Ç5
15
11,5
1Z;s
15
(Jo
meens
.
Percentage distribution of the class means
of the pervalvar axes of Stephanodiscus Hantzschii (in the
mixed culture experiment with Diatoma elonpatum).
Fig. 12.
0
nutrient solution series I
nutrient solution series 11
- 45 -
al.
50 r
40
30
20
10
2,5
3,5
4:5
—r
6,5
7,5 C
la()as means
Fig. 13. Percentage distribution of the class means
of the pervalvar axes of Diatoms elongatum (in the mixed
culture experiment with Stephanodiscus Hantzschii).
o nutrient solution series I
o
•
...tem.
nutrient solution series II
,
varied irregularly in the mixed culture.
But the average
cell volumes calci.ilated for the individual experimental
portions (each calculation was.based on 60 measurements)
(Table 6) corresponded to those determined for the monocultures.
From the cell volumes we cannot derive any con-
nections with the processes within the nutrient solution
series.
The mutual effect of the algae on one another, which
found its expression in the manner of multiplication of the
al.,gae in culture series I and II, in contrast to the process
of development in the monoculture series may also have
affected the algal volume.
4. Experiments with Plan^tonic Chlorococcales
P. 632
The culture series were carried out with the same
objective and with the same experimental set-up as the
üiatom- experirnent s above.
a) Scenedesmus auadricauda
As nutrient medium we used in the first experiment
the nutrient solution "L 4 C" and the nutrient solution
^,To. 8 of Rodhe (Table 1) alongside each other. The original
nutrient solution according) to Rodhe will be desi;nated by
t'Ra"
The • illumination amounted to 6000 lux, the temperature
of the water bath to 25°C. The objective was to determine
whether the chlorophyll content of the al.;ae differs
- 47 -
quantitatively in the variably composed nutrient solutions.
rumber of cells, chlorophyll content, and dry weight were
determined in this experimental series.
Scenedesmus occurred in the L + C-nutrient solution
predominantly in the form of individual cells, in the Ranutrient solution, on the other hand, it occurred as twocell stage. The chlorophyll content of a coenobium con- P. 6 20
sistinp- of two cells) corresponded as to size approximately
to the above-mentioned diatom cells.
The pigment content
fluctuated durinp; the experiment in the L 4 C-nutrient
solution between 3.5-and 2.8x10 -6 , in the R -nutrient
a
solution between 4.8- and 4.2 x 10 -6 'y in a two-cell coeno-
bium.
The percentage of chloropiv11 in the dry weight of
'
the alpae from the L + C-nutrient solution was also lower
(2.9 to 1.1%) than that of_the algae from the Ra -nutrient
solution (5.6 to 1.3%).
In the following experiments with Scenedesmus we
used only the Rodhe nutrient solution. The first objective
was to determine growth and chlorophyll content of the
algae in graduated calcium nitrate content.
One part of
the test tubes vas therefore given Rodhe nqtrient solution
with 120 mg (Ca(NO3)2/1(R b ), the other part of the test
111,
tubes Rodhe nutrient solution with 30 m,); Ca(U0,) 2 /1(R 0 ).
temperature of the water bath amounted to 25 00, the illumination to 6000 lux.
The
- 48 -
We were unable to determine the dry substance
P. 634
here because an attempt was made with the al,;ae material
to deterïnir_e the dry ehlororYi,yll.
During the five-day experimental period the chlorophyll content fluctuated between 0.0- and 4.3 x 10-6j/ in
the culture series containing the most nitrate, and between
4.7- and 3.9 x 10-6'y in the nutrient solution series conLI
The numerical development
taining only 30 mg Ca (I?U3) 2/1..
proçeeded almost uniformly in the two culture series.
For the third culture experiment we used only one
culture series with original Rodhe nutrient solution. This
was used to test to what extent cell volumes, chlorophyll
content, and dry weight -changPd from day to day.
The water bath had again a temperature of 25°C. The
illumination was increased to 8000 lux in order to change
thereby, if possible, the chlorophyll value of the algae.
Fluctuations in the size of the volume, in chlorophyll
content, and in the dry weight were considerable and during
the first two culture days obvioully determined by the
distribution rhythm (2 parallel series. Table 7). The coenobi a did not contai n as much chlorol.h,yll as in the preceding
experic:ient.
They even contatined less chlorophyll than the
I-' denede :nis from the nutrient solution series with only
- 49 -
SO mg Ca(NO 3 ) 0 /1 in the second culture experiment.
On the
basis of these investigations it cannot be decided whether
the higher intensity of illumination is the cause of the
lower pigment content since ae: e and amount of inoculation
material was not quite equal.
Table 7.
Number of individuals, coenobia volumes,
chlorophyll and dry-weight values of Scenedesnus quadricauda
in the culture experiment with nutrient solution Ra
...»._i______,____ ___,L.,
ydry
Number ychloro- Vo- ychloro- cier-of
chloro- Wseight
Culture of coe- Phyll in lume'phyll
in lmra3 phyll
nobia
one coe- of
of a
days
coenoin.1 cc nobium
one
of
in
dry
bium
(x 10 3 (x 10 -6 ) coe- cell
weight (x 1 0 -5
nobi- vdlime
unt
Al)
B 1)
A
B
67
224
217
387
450
570
620
0
1
2
3
4
5
7
1)
74
217
184
396
421
624
520
3,1
1,9
3,2
2,5
2,5
2,2
2,4 .
3,5
2,0
3,2
2,5
2,5
2,2
2,8
.
'
s
i
293
156
258
197
262
229
237
11,0
13,0
12,0
13,0
9,7
9,6
11,0
3,9
4,1
3,3
3,8
4,0
3,8
4,7
•
8,5
4,5
9,7
6,6
6,5
5,9
6,1
A and B denote the parallel cultures
It was only the values related to the standard cell
volume of'l mr0 which made us realize that Scenedesmus contained approximately 1' ive time as much chlorophyll as Diatoma
and Stephanodiscus.
ye
A decomposition of chlorophyll did not
take place during the experimental period. During the last
few culture days we even noticed a slight increase in chlorophyll.
- 50 -
The share of chlorophyll in the dry substance was also
lar^rer in S)cenFdecrnus than in the two diatoms.
The fluc-
tuations in chlorophyll content, revealed during the consideration of the cell volume, were not reflected in the
seme manner by the dry weight. Nor was this to be expected, P. (535
s,nce the volume must not be identical with the mass.
To see whether an addition of CaCl 2 to the nutrient
solution is able to exert an influence on growth and amôunt'
of chlorophyll the following culture series were prepared
for the next experiment: 1) Rodhe nutrient solution with
120 mg Ca(N03)2/1; 2) Rodhe nutrient solution with 30 mg
Ga(N03)2 + 90 mg CaCl2%1(Rd). Tempe rature and illumination
were left at 25°C and 8000 lux. The 'algae volume was deter-,
mined only in the first and in the two last parts of the
experiment.
In the nutrient solution containing more nitrate the
algae in the end did not tnultiply as well as in the nutrient
solution which contained less nitrate but which, instead,
contained CaCl2 (Fig. 14). In contrast to the increasing
number of coenobia, the chlorophyll content in culture
series Rd decr.eased during the last days. In nutrient
solution Rb practically the opposite conditions prevailed.
The dry weight of the algae did not correspond fully neither to
the number of individuals, nor to the amounts of chlorophyll.
:
eîil
number of
coerobie
Dry substance mg/
Ch1orophyll
r/10cre
x10'
1000 r-
50c
1.■■
•
•
SOO
30
3
20
21-
10
MO
I-
2
4
G
8
10
2
4
8
1
10
2
1
4
6
8
Ito daYd
Fig. 14. Coenobia multiplication, chlorophyll content and dry weight
of Scenedesmus quadricauda in the culture experiment with the two nutrient
solution series R b o
o
and
1
- 2 The coenobla were larr-er and contained somewhat more chlorophyll than in the previous exTeriment (Table 8). A reduction of piment in the standard volume and in the percentar;e of dry weight became noticeable only at the end of the
culture time in nutrient solution series R d.
Table 8. Coenobia volume and amount of chlorophyll
of Scenedesmus quadricauda in the culture experiment with
nutrient solution series Rb and Rd
ychloronhyll Volume
of a
Culture .in a coeicoenobium
nobium
days
(x 10-6 )
ychloro- ' % of chlorophyll in
phyll in
dry weight
1 mm 3
cell volume
(11-3
2
:
8
10
Itb 1)
Rd ! )
Rb
-.,
..,d
Rb
Rd
4,50
4,03
4,23
3,70
4,75
4,40
4,05
4,18
3,80
2,60
400
—
—
343
392
343
—
—
332
311
11,6
—
—
11,0
12,2
13,0
—
—
10,8
8,4
r,
-t.b
-n
,..d
3,4
4,2
5,0
3,6
5,0
3,5
. 3,8
4,9
3,6
3,0
P
'
1 )Rb and Rd denote the nutrient solution series.
In a further experimental series the nutrient solutions this time were lacking sodium silicate. The one nutrient
solution was, moreover, prepared with 120 mg Ca(NO 3 ) 2/1(Re ),
the other one with 20 mg Ca(NO 3 ) 2 4 100 mg Ca01 2 /1(Rf ). In
this experiment we also used 200-cc Erlenmeyer flasks into
which wag introduced air that had been filtered through cotton.
The temperature of the water bath amounted to 25 °C, the
illumination amounted to 8000 lux.
- 53 During the last culture days the Scenedesmus multiplied better in the nutrient solution containing more
nitrate than in the culture series containing less calcium
nitrate (Table 9).
The nutrition conditions which had
been changed several times from thoséof the other experimental series led to results differing from those of the
other investigations. Thus, during the first experimental
days there were practically only individual cells in the
two nutrient solution series. With regard to the number of
individuals the Scenedesmus of nutrient solution series Rf
in chlorophyll than those of culture series Re werpo ;
cf
but the coenobia volume varied to such an extent in the
two nutrient solution series that an exactly opposite rela- ,
was calculated for the pigment content in the stan-tionshp
dard volume.
The smaller volume of the algae from culture
series Rf was paralleled by a lower dry weight.
In view of
the percentage of chlorophyll in the dry substance, however, P. 637
were -f
disregarding the first experimental portion -- poorer in
the Scenedesmus of nutrient solution series R
chlorophyll than the algae material from the culture series
containing more nitrate.
All experiments carried out with Scenedesmus were
discontinued before a complete exhaustion of the nutrient
solution.
However, volu-le and chlorophyll content of r;cene...-
desmus which had not received any new nutrient solution for
- 54 Table 9. 1?umber of individuals, volume and chlorophyll amount of ;cenede:mus auadricauda in a culture experinient w ith nutrient solutions Re and R f
chtôro
ychlotiTolu^é Volume
^u iber
rrophyll
Culture of. coe- of one of one i)hyll
coeno- in one in lmm 3
indinobia
coenocell
in 1 cc vidual bluta
days
bium
(u.3)
volume
cell
(x 103)
(x10-^
(^.t3)
!
(
3
6
9
i
Re 1) I 1111>
249,5
505,0
1115,4
249,0
531,0
654,0
Re I
266
296
225
g1
146
165
225
R1
Rl
Re I
R,
322
2,1
2,6
2,3
197
RB
RI
2,0
2,0
1,9
7,9
8,5
7,6
14,0
11,6
9,6
% of
chlorophyll
in dry
weight
RB
R`
3,4
3,3
3,4
3,9
2,9
2,2
I
1)Re and Rf denote the nutrient solution series.
61 . months and which had lain in a thick layer at the bottom
of the culture dish were still determined. A two-cell
coenobium from this old stock culture contained only
1.3 x 10-6 ;` chloroph,yll and 1 mm3 cell volume even only as
little as 1.5y. The coenobia had an average volume of
885 ^3. ^',hen new stock nutrient solution was added to these
algae they multiplied strongly and formed again chlorophyll.
The chlorophyll contert of ;:cenedesmus thus is not
an unchar.Feable value; it fluctuates within the dimensions
^ef1.ned by s;rovvth and milieu influendes.
In order to be able to detFrm^ne the volume of
- 55 Scenedesmus, the calculations were based either on the
width of a two-cell coenobium or, if necessary, on the
width of the individual cells after it had been found that
it was the cell diameter rather than the cell length which
varied predominantly with the nutrient content.
As an
example of this we may mention the frequency distribution
of the cell measurements in the culture experiment discussed just prior to this one. The lengthsof the cells
barely differed from each other in the two nutrient media
(Fig. 15).
But the individual cells (Fig. 16) as well as
the two-cell coenobia (Fig. 17) were in the majority of
cases clearly wider in the nutrient solution containing
more nitrate(R e e) so that the volumes were on an average
larn;er than those of the algae from the nutrient solution
containing less nitrate but, inetead, more calcium, nitrate.
The frequency distribution of the coenobia widths
of equal class means of Scenedesmus from culture series
which contained only different amounts_of Ca(NO 3 ) 2 and
CaCl 2 did not vary as much as in the experiment in which,
in addition, sodium nitrate was lacking (Fig. 18).
- 56 -
6,5
-T-r7,5
q5
9^5
145
class mear s
(P)
Fig. 15. Percentage distribution of the class means
of the cell lengths of Scenedesmus quadricauda in the culture
experiment with the two nutrient solution series Re o
and R f •------- •
o
- 57 -
50
r-
I\
I \
40
I
30
\
PA\
20
10
7:5
35
.2,p
0-ass means
(
j
'1)
Fig. 16. Percentage distribution of class means
of cell widths of individual cells Of Scenedesmus auadri°suds in the culture experiment with the two nutrient
solution series R e o
o
and R f
C.
- 58 -
oI
GO
50
40
30
10,5
11,5
12,5
0 lef. Set Mfet.8
Percentage distribution of the class means
of the coenobia widths of Scenedesmus quadricauda in the
culture experiment with the two nutrient solution series
Fig. 19.
Re o-----o
and Rf .------.
- 59 -
---------------------------------------------------------
60
50
40
30
I
20
I
I
10
i
7^5.
Fig. 18.
8,5
9,5
t415,5 Q Z E1 ^9^ Itl t? ^ i] 3
Percentage distribution of the olass means
of the coenobia widths of Scenedesinu squadricauda in the
culture experiment with the nutrient solution
ser.ies Rb o'o
and Rd
d
--------------------------------------------------------- -- _-__
- 60 h) Pediastrum boryanum
For the culture experiments with Pediastrum we used
Rohde nutrient solution with gradated amounts of calcium
nitrate.
The main objective here was to determine the
amount of chlorophyll in the alga and, possibly,detect
ae,e-conditioned differences in the chlorophyll content.
The serial experiments were carried out in the photo-
P. 638
thermostat bath with the same experimental set-up as in
all experiences discussed so far.
In_ the first Pediastrum experiment we prepared two
ki
nutrient solution series (with 120 mg and 30 mg Ca(N0) 0 /1).
The illumination was adjusted to 8000 lux; the temperature ,
of the water bath amounted to 25 °C.
The development of Pediastrum took place almost
uniformly in the two culture series.
In nutrient solution
R b the algae contained somewhat more pigment than in nutrient
solution R 0. The amounts of chlorophyll contained in the
standard volume as well as the percentage of chlorophyll in
the dry substance corresponded as to order of magnitude to
the values established for Scenedesmus. Only the chlorophyll content of a colony was almost by one power of ten
larl;er than in the alr-ae investigated so far.
With the following culture series it was intended
P. 639
to investigate whether and to what extent volumes and amounts
- 61 -
of chlorophyll vary in algae of different ages under equal
conditions of life.
For this reason the culture flasks of this experiment
have been provided only with original Rodhe nutrient solution.
The illumination amounted to 8000 lux, the temperature
of the water bath to 25 °C.
One part of the specimen flasks
has been inoculated with Pediastrum which had been transferred
three days previously into a fresh stock nutrient solution
(series A), the other part with Pediastrum which for approximately 5 weeks had not received any new nutrient solution
(series B).
In the course of the experiment the old Pediastrum
at first multiplied more vigorously than the young inoculation
material (Fig. 19).
But between the second last and the last
portions of the experiment the descendants of the young
inoculation algae then developed better than the ones of
the old inoculation material. Following this mode of observation the total volume of algae in 1 cc nutrient solution
already indicates the differences in the size of the colonies.
While the increase or decrease of the chlorophyll content
of 10 cc culture suspension of series A did not correspond
exactly to the increEse or decrease in the number of colonies and the algLe volume, it nevertheless correspondEd
approxinately.
This did not hold true for series B.
P. 640
- 62 -
rumber of
iionâble in 1 cc
cht0r0phytj
vsturne
» /1Fre
x10
- x10
..V10cde
.
50
50
•
o
o
•
I\
1
o
40
\o
40
1
■
o
I
/ •
o
,
o
//
•
/0
/
1
I o
o
a
30
•0
I
I
o
of
1
30
o
10.-
s
o
/1
1/
I/
20
/
20
o
•
•
•
•
/
/
10
10
0
3
5
7
0
3
5
7
3
5
7
.
days
•■■•
Fig. 19. Coenobia multiplication, algae volume, and
chlorohyll content of rediastrum borvanum in the culture experiment
with different old inoculation material.
o----o----o culture series A
•••••••■•
■•••••
■••••
• culture series B
,
-
63 -
The averarrF volume of Pediastrurl from the fresh
stock culture was not even half as large as that of the
algae from the nutrie.nt medium which was several weeks old
(Table 10).
The chlorophyll content - of each colony was
almost the same with both inoculation substances. Taking,
into corsideration the volume, we noticed however a significant difference in the pigment content of the varying
inoculation. material.
Table 10. Coenobia volume hnd amount of chlorophyll
of Pediastrum boryanum in the culture experiment with vâz'iably
old inoculation matérial
Culture
days
chTorôvOT-1 )F0
Volume
phyll in a ihyl l in
of*a
1 mm3
coenobium coenobium.
cell
(x 10-F)
(p3)
70 0.
chlorophyll
in dry
weight
.
volume
0
3
b
7
I V A1)
B1)
A
B
A
B
1564
1369
1348
1227
3946
813
1054
1237
2,3
2,2
2,6
2,5
2,0
0,7
1,8
2,1
14
15
19
20
5
9
16
17
A I
3,3
3,9
4,2
B
1,9
2,0
-
14 and B denote the tw.o culture series with variably
old inoculation material.
The chlorophyll content of a colony in a standard
volume and calculated as f.ercentane of dry weifTht did not
show in a co-:I,ar i.son of the two culture series during the
P. 641
I
- 64 -
experiment a complete approximation of the values to one
another.
"Pediastrum descer_dinc- from the old inoculation
culture always contained less chlorophyll than PediaGtrum
which had developed f rom the fresh inoculation culture..
The extreme difference in age of the inoculation substance
and the fact that Pediastrum grew only ver .y slowly in the
Rodhe nutrient solution helped to make the differences in P. 643
chlorophyll content of variably old algae material stand
out especially clearly.
The results achieved so far in the investigations
were corroborated in two other culture experiments which
CP
are not cze scribed in detail here.
No pronounced deficiency phenomena have been evoked
in the Pediastrum serial experiments. To demonstrate
liowever that in this alga, too, the chlorophyll content
decreases with an exhaustion of the nutrient medium without
resulting, in a permanent loss to its vitality, we determined the volume and chlorophyll content of Pediastrum which
for 6z months had not received any ^iew nutrient solution.
These a1x,,ae had a barely greenish effect; the carotenoids
gave it a redish-yellow appearance. The average colonial
volume amounted to 3163 1a3, the chlorophyll content in the
conobium to 1.3 x 10-5
and in 1 mM3 cell volume to 4.2 ^^ .
The values thus still corresponded to those which had been
ascertained for the old inoculation material of the one
- 65culture experiment described above. After transferring
into a fresh nutrient solution a multiplication set in
again and soon the culture had a freshgreen appearance.
The determination of the volume of Pediastrum
created difficulties since the height of the colonies could
not be established. Vie thus measured only the diameter.
The height of the colony has been determined in conformance with the length of the cell from a typical multi-cell
conobium.
As a result of this fact it can only be demonstrated
that in the experiment with the variably old inoculation
algae most of the descendants of the two inoculation substance s showed colony-diameters of the same order of magnitude (Fig. 20).
But the percentage of large conobia
was noticeably smaller in the culture series which had been
inoculated with the old stock culture.
- 66 -
--------------------------------------------------------------60
Ir.
50
40
30
20
o
\
o
1,p
;^
p
^•\
a
^ D
IT
7r5
T7-
125
Fi^. 20.
1 7r5
22,5
275
325
375 •
-T
42,5
47,5
I . 1
I
Ta^5
: 575
G2,5 c lt3 sR!?] e Ei n s
1-'
Percentage distribution a.f -the .class means cf
coenobia diameters of Pediastrum boryanum 3n-the culture
experiment tivith vsriGbly old inoculation .mate--Liâl.
0
0
o culture series A
culture series B
- 67 c) Ankistrodesmus falcatus
Two culture exreriments were carried out with
Ankistrodes-lus falcatus. In both experiments the illumination amounted to 8000 lux and the temperature of the water
bath to 25 °C.
In the first experiment the culture flasks
contained Rodhe nutrient solution with 120 mg or 30 mg
P. 644
C8(NO 3 ) 2 /1. In this case the algae in the nutrient solution containing less calcium nitrate grew better than in
the culture series with more nitrate. In the course of the
experiment nutrient substances were presumably set free in
culture series R 0 by dying algae so that during the last
culture days new inereasuS became possible. In the nutrient
solution series containing more nitrate, on the other hand, ,
a steady sliplit increase in the number of cells had been
noticed which almost came to a standstill only between the
9th and the last (12th) culture day. There was no constant
relationship between the amounts of chlorophyll of the two
nutrient solution series and the number of individuals that
must be allotted to them (the volume was not determined in
this experimental series. Things were not much different
as far as the dry weights were concerned.
The chlorophyll
content of the Ankistrodesmus cells was at least one power
of ten lower than in Pediastrum and was even somewhat lower
thon in Scenedes - ms. Besides, thé algae of nutrient
solution series Re contained less chlorophyll than those
of culture series R b'
The percentage of chlorophyll in
P. 645
the dry substance was also lower in the algae from-nutrient::
solution series R c than in the algae from nutrient:solutivt_:
-- serie.- R b ' The percentages of both culture se
ponded to the values ascertained for Scenedesmusend Pedii- estrum. At any rate, an average cell volume was determinedfor the entire culture series
and as average valuefor:-
1 mm 3 cell volume we ascertained 20 ,lchlorophyll_of_algae of nutrient solution series R b and 18 y chlorophYlL:of:
Ankistrodesmus of nutrient solution series no .
The objective of the second culture experiment - with.
Ankistrodèsmus was - toAmvestigate once more thè'zchlôrophyll:
content, but under somewhat changed nutricnt cOndition:Rndr
by taking into consideration the cell volume . ,
Here we used the original Rodhe nutrient --solution
and such with 19 mg 0a(NO 3 )2 4 38 mg CaCl 2
Tèmpetature
and illumination were set, as in the preceding - experiments,
at 25 ° 0 and 8000 lux respectively.
In both nutrient solution series the algae multiplied fairly evenly (Fig. 21).
The volume Of the tuLai algae
In the suspension unit did not increase to the game extent
as the number of individuals, for in the few CuLture series
containing nitrate the small average cell volume of the algae
became noticeable on the 7th culture day (Table 11).
After
3 days the chlorophyll content in the cultute series with
- (3 9 -
rumber of
Cells in
icig%
x 10
Chlorophyll
VQi me„
,te/ 1 cm'
K10`
rlOce
1000 r
100 r-
500
50
la
10
7
0
20 r
0
3
7
Ô
10
à
7
10
d-ays
Fig. 21. Cell multiplication, algue volume, and chlorophyll content of Ankistrodesmus falcatus in a culture experiment with the two nutrient solution series Ra o-----o and
•
...mu.
Table 11. Cell volume and chlorophyll values of Ankistrodesmus falcatus in a culture experiment with nutrient
solutions R a and R
Volume
of one
cell
(j1 3 )
Culture
daya
•
0
3
7
10
Ra i) 1
vchlorophyll 'ychlorophyll
in 1 mm 3
in one
cell
cell ,
volume
(x 10- °)
Rg i)
Ra
74
38
50
1,0
1,1
1,4
Rg
Ra
1,0
0,9
1,1
14
16
26
1) R and R
a
1
Rq
19
2,6
135
70
70
56
I
13
23
22 '
denote the nutrient solution series.
- 70 -
more nitrate was greuter than in nutrient solution
series Rg.
Per individual cell this alga contained less chlorophyll
than aScenFdesmus coenobium consisting of two cells; but
related to the cell volume of 1 mm^, tinkistrodesmus contained
more pipmFnt. This experiment also made evident that the
chlorophyll content was affected*by growth and environmental factors.
The calculation of the oeil volume was based on the
diartieter of the cells which ehan,r^ed conspicuously with the
growth conditions (Fig. 22). In neither nutrient solution
series did the average length of Ankistrodesmus show any noteworthy differences.
5. Discussion
The culture experiments made possible a more detailed
observation of the reactions of algae to different conditions of life than would have been possible in'fiel:d:.ë±periments6'..They have again corroborated the fact that algae
P. 647
react very noticeably to environmental influences of all
kind
and that the pigment formation may be determined not
only by light and temperature but also by the nutrient
content of the water. A deterioration of the nutrient
substance (brouFht about by a deficiency of calcium nitrate
or by a nutrient consumption in the course of time) generally
caused a distinct decrease in the chlorphyll content of
- 71 -
•
class mEans
(p)
Fig. 22. Percentage distribution of the class means
of cell widths of Ankistrodesmus falcatus in a culture experiment with the two nutrient solution series R a o-----0 and
Rg .------.
Diatoma elomeatum, Stephanodiscus Hantzschii, Scenedesmus .
quadricauda, Pediastrum boryanum, and Ankistrodesmus falcatus.
These chlorosis phenomena appeared in relation to the
individual cell or colony, as in the standard volume of
1 mm 3 , and also as a percentage of the dry substance. The
different temperature and licht(?) conditions also led to
differences in the pigment content.
- 72 The Lmounts of chlorophyll per cell or per conobium
were the same for Diatoma, Stephanodiscus, Scenedesmus, and
Ankistrodesmus; only the Pediastrum colonies had on an
average approximately 10 times as much chlorophyll as the
other algae mentioned above.The algae cell or the colony -the latter is considered only as a number of individuals -shows up, to be sure, in the ca1cUlation completely independent of the frequently substantial differences in the sizes
of the algae. Taking into consideration their volume and
then calculating the amount of chlorophyll for 1 mm 3 cell
volume it was found that of the investigated algae the phyto- P.648
plankter which belonged to the same systematic group also
contained approximately the same amount of pigment.
The
Chlorococcales then contained in comparison with the two
diatoms on an average five times more chlorophyll in the
standard volume of 1 mm 3 .
But under varying milieu condi-
tions the chlorophyll content of the algae varied greatly.
This means, in other words: greatly varying cell sizes
(volumes) may contain one and the same amount of pigment.
In the course of the investigations the following algae
volumes contained 1 mg of chlorophyll:
Dia toma elongatum
Stephanodiscus lianizschii
Diatoma e. ± Stephanodiscus H.
Scencdcsmus quadricauda
Pediastrunt boryanum
Ankistrodesmus falcatus
244-1000 mm'
208-1136 mm3
1) mm'18—27
71— 667 mm'
33— 233 mm'
39— 771 ) mm'
1) These
figures do not include any deficiency cells.
- 73 The 1;. sted figures repre se nt the threshold values
ascertained in the experiments; under other environmental
conditions they May also be different.
Atkins and Parks (1951) determined 1 rng chlorophyll
in 53 mm3 of cell volume of Chiorella from a monoculture;
this value corresponds to that of the Ghlorococcales discussed in this treatise. For the diatom-plankton of the
Kiel Bay Gillbricht (1952) calculated an algal volume of
136 mm3 which contained 1 mg chlorophyll. This value is
similat to the values of the,described diatom mixed culture.
7
He mentione further that,among other things, he has obtained
for one and the same plankton volume two different chlorophyll values one of which was nine times greater than the
ôther.
These variations in the chlorophyll content of the
algae or of the plankton are ascribed to the qualitative
change of the plankton association.
Gillbricht points out
that these facts must be taken into consideration if chlorophyll measurements are to provide a guide for the size of
the production.
The fluctuations in the chlorophyll content brought
about by the most varied enviror_hental influences were
noticed also with respect to the dry weir*ht. In the case
of a deficiency of nutrient substances the percentage of
chlorophyll i-: the dry substance genera].ly.decrearFd.
- 74roreover, in the green algae the percentaF, e of chlorophyll
in the dry weirrht was greater than in the siliclous algae.
According to the experimental results the averge value
for green al7ee amounted to 3.4%, that for diatoms to 1.1%,
of the dry weirrIt (the average value in the mixed culture
amounted to 1.7%).
For a nitrogen-deficiency experiment with Chlorella
Aach (1953) mentions decreaFing percentages from 4.62 to
0.04 chlorophyll in the dry weight. Riley (1941) reports
for the plankton investip.ated by him an average value of
2.9% of chlorophyll in the dry substance.
Neither the volume of the algae nor their dry weight
are reference values for the chlorophyll content of the
alp7ae which are mutually dependent on one another.
As a
•
matter of fact, there is no proportionality even between
photosynthesis and chlorophyll content.
The alc.ae volume of the individual species varied
species-specifically with the environmental conditions and
of course also with the ap,e of the algae.
Soeder (1960) showed that at overoptimum salt Con-
centrations in the nutrient solution Chlorella Dyrenoidosa
assumes an appearance which resembles that of storaP;e ce115
wllich are formed at a N- or P-deficiency.
In other wordo,
no immediate conclusion can be drawn from the size of the
- 75 oeil or of the colony with regard to the phy-s.iolo7ical
P. 649
state -- e.,^. whether nourished well or poorly. From the
previously described experiinents such determinatians may
be derived.
Even if the size of the volume of a species of algae
is subjected to considerable fluctioations, it is,nevertheless, only by taking the size of the volume into considerati on that an a^pproxi:-ation of the pigment contents within
a systematic unit and a differentiation between the systematically non-related groups is possible. As a matter of
fact, in the green algae the change in volume was essentially
based on the widening and narrowing of the cells, in the
diatoms on the shorténin^;_ or. lengthening of the pervalvar
axis.
The composition of the nutrient solution affected
also the development of the colonÿ, as was already reported
in the description of the course of the individual experimental series.
Thus, at variable temperatures Diatoma
elongatum formed zig-zag bands only in the nitrate-deficient
culture series with calcium-chloride addition.
This observation may, pFrhaps, be related to the
investigation re sult s of Schr8der (1954); in experiments
with Stichococcus bacillaris she deterYained that the addition of an earth-alkaline chloride to the nutrient solution
- 76 prevents the decomposition into individual cells. However,
in the experiments with Diatoma the long chain colonies were
observed only after deficiency phenomena had already affected
size of volume end pigment content.
Schroeder further found
that in Scenedesmus species an increase in the salt concentration brought about a disintegration into individual cells.
This determination corresponds to our own findings which
have shown that in the more concentrated nutrient solution
L 4 C Sceredesmus occurred predominantly in the individual
stage, while in the solution with less nutrient salt according to Rodhe two-cell coenobia usually occurred.
It may be regardéd as definite that the pigment
content of algae differs not only intersuecifically, but
also that the chlorophyll content of the algae may vary to
a considerable extent infraspecifically.
This applies not
only to the experiment, but exactly the same to-occurrences
in the open.
It may be argued that a plankton association
in natural waters is exposed also to other influences;
however, the basic presumptions are the same. Conditions
of illumination, temperature, and nutrient content change
in the course of the year. If conditions are favourable
for algae plankton populations are formed; under unfavourable
conditions for the resuective species the latter disintegrate.
Certain combinations of nutrient'substances may brin e?; about
a pronounced multiplication; however, this does not necessarily
- 77 mean that e.g. the algae will contain -- as one would be
led to expect -- correspondim7ly high amounts of pigment
(see culture experiment with Diatoma). Especially in water
blooms the chlorophyll content of the algae will not be the
at
same at the beginning and/the fading away of the high production.
If the amount of chlorophyll is to be used as
measure for the amount of algae in a body of water we must
take into consideration not only the qualitative composition
of the plankton association but, if possible, also the state
of development. Because of the multiplicity of the acting
factors and our hitherto still comparably limited knowledge
of the environmental demands of the individual algae this
1/4!
may, however, not be realizable.
Nor is the "chlorophyll-containing debritus" to be
disregarded in evaluating the amount of chlorophyll obtained
from the seston. The chlorophyll-like dyestuffs extracted
from same may, according to Gillbricht (1952) / represent a
significant portion of the seston chlorophyll. Whether and
to what extent colouring matter extracted from the detritus P. 650
is to be attributed to the chlorophyll may be left an open
question. Suffice it to know in this case that the detritus,
too, prodqces absorption values at 660 mil which are included
in the chlorophyll value and which thus make the determination of the size of the primary production from the
colour intensity of the green pigment extract still more
- 78 uncertGin. (Peut soa:^ed and washed for days, which represents
nothinn else but decer-,posed plant substances and is thus
co.nparb.ble to detritus, was extracted; it absorbed light
of wavelFni7th 660 m)i.).
The detritus content of a body of
water would thus also have to be tested before conclusions
could be drawn from the chloroLhyll extract with re;;urd to
the amount of algae.
Finally, attention would have to be paid also to
tlie varying extractability of the algae, since incomplete
extractions would cause further errors.
(V
In view of the inconstancy of the relationship of
chlorophyll to the hitherto used reference magnitudes the
chlorophyll method may possibly be used only in special
oases and not as a generally applicable method for the quantitàtivè determination of the phytoplankton. The relationship of chlorophyll to the variou's cell reference magnitudes
may, however, provide essential information on the environmental demands of the alFae and on their metabblism.
-
Summar,y
1411onoculture serial experiments (one mixed-culture
experiment) were carried out under different conditions of
development with Diato!7a elors-att.im, Sterlivnodi.^!cus IIantzschii,
Scer..edes-nus guadricauda, PediastrLUn bor%r1inuca, And ;,nhiatrodesnus falcatus.
The number of cella or colonies, the algue
- 79 volume, the chlorophyll content, and occasionally also
the dry substance were determined at certain time intervals.
The algae volume is stated as "average oeil volume"; it is
based on the arithmetical mean from a series of measurements
of the cell axis verying most conspicuously because of the
environmental conditions.
The size of the cell volume depended on the age of
the alf7ae, on their state of nutrition, and on the ion
composition of the nutrient medium.
The colony formation
may also be affected by the composition of the nutrient
solution.
The chlorophyll content per cell or coenobium was
approximately of equal size in the investigated algae. Only
one Pediastrum colony contained approximately ten times as
much chlorophyll as the other experimental algae.
Only when taking into consideration the algae volume
and calculating the chlorophyll content of one standard
cell volume of 1 mm3 it was found that the green algae
contained approximately five times as much chlorophyll as
the two diatoms and that Pediastrum has approximately the
sanie chlorophyll content as the other two ehlorococ.pales.
The percentage of chlorophyll in the dry substance
CP
was lower in the diatoms than in the green algae.
- 80 In states of deficiency produced in the experiments
by calcium-nitrate gradations or which were determined on
old cultures showing a nutrient-substance exhaustion
the
chlorophyll content of the aleae definitely decreased,
dependiru; on the duration and intensity of the state of
deficiency.
A worsening of the growing conditiàns through
low temreratures affEcted also the pigment formation.
A quantitative chlorophyll extraction frcràdry algae
material was possible in the two diatoms by following a
special drying procedure.
Dry green algae could, however,
not be completely extracted.
Since the relatiorship of chlorophyll to the various
cell reference magnitudes is not constant but rather depends
on the environmental conditions and on the species-specific
composition of the pigment, one and the
same amount of
chlorophyll may represent greatly varying amounts of substances.
- 81 -
Biblio^:raphy_
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2 . ATKINS, W. R. G., and M. PARr.E. 1651: Seasonal changes in the phytoplankton as indicated
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i
6. BURSCIIL, E.-ll., H. KüIIL U. H. MAN-.%, 1953: Hyclroehemisehe Faktoren und Phytoplankton
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,^i., subbr..„via Sch..,
d'(Jscillato..,.
.,.idlc (Cyar.ophyceae). C. r. Acad. Sui. 246, 630-632.
13. GILLRRICIIT, ,lI., 1952: Untersuchung,,on zur Procluktionsbiologie des Planktons in der Kieler
1
Bucht. I. Kieler Mecresforsch. 8, 173-191.
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of light intensity and temperature. Physiol. Plantarum 11, 401-420.
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Chemical composition of the phytoplankton. Bull. Bingh. Oceanoor. Coll. 15, 315-323.
16. HARVEY, W., 1952-53: Synthesis of organic nitrogen and chlorophyll by Nitzschia closterium.
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367-549. E. Schweizerbart, Stuttgart.
18. KREY, J., 1939: Die Bestimnttmg des Chlorophylls in lleer«•asser- und Schüpfproben. Jour,
du Conseil. Int. Expl. Mer. 14, 201-209.
19. - 1958: Chemical methods of estimating standing crop of phytoplankton. Rapp. et Proc.
Verb. Conseil Intern. Explor. .lIcr. 144, 20-27.
20. LEFPI'RE, M., H. JAKOB et M. NISBET, 1952: Auto- et Hétèroantaâonismus chez les algues
d'eau douce. Ann. Stat. Centr. cl'Hydrobiol. Appl. 4, 5-19S.
21. Lvlsn, J. W. G., 1949: Studies on Asterionella. I. The origin and nature of the cells producing
seasonal masinla. J. of Ecolo;y 37, 389-419.
22. DIAC.IIILLAN CoNoVEr., S11. A., 1956: Oceanography of Long Island Sound 1952-54 IV. Phytoplankton. Bull. Bingh. Oceanogr. Coll. 1^5, 63-112.
23. bIAxDEr.S, G. R., 19-13: A quantitative study of chlorosis in chlorella under conditions of
sulphur deficiency. Plant Physiol. 1ti, 419-462,
24. 11IARCALF:F, R., 1954: Concicleraciones sobre la determination cuantitativa de)
Aor la valoracion
de pigmentos solubles y los factores (lite afectan a la relacion entre cantidad do pigmento
y peso seco. Publ. Inst. Biol. Apl. 16, 71-û-1. (Referat in: Biol. Abstr. 29, \r. 5208).
25. oORSCI[OT, J. L. P. VAN, 19,55: Conversion of li-lit ener at• in alral culture. Med. Landbouwhogeschool to tilaaelllll^l`Il^^e(ll`C1,uId 5•i, 222-2j6.
2^7. PIRSox,:1., 1937: Ernühruucs- und stoffocchsclphysiolo^ischc Untersuchungen an Fontinalis
und Chlorella. Z. Botanik 711, 193-267.
- 82 -
27
28.
Twin: u. G. .\\rir..nEr.mr, 1952: StoffwechseI und Mineralsalzerniihrung einzelliger
Griinalgen. I. Mittlg. 1. Vergleichende Untersuchungen an Mangelkulturen von Ankistrodesmus. Planta 10, 199-253.
RILEY, G. A., 1941: Plankton studies III. Long Island Sound. Bull. Bingh. Ocean. Coll. 7,
PIRSON', .A., C.
1-89.
1948: Environmental requirements of fresh water plankton algae. Experimental
studios in the ecology of phytoplankton. Symbol. Bot. Upsala 10, 1-149.
30• RYTIIER, J. H., and Ch. S. YENTSCH, 1957: The estimation of phytoplankton production in
the Ocean f Tom chlorophyll and light data. Limnol. a. Oceanogr. 2, 281•286.
31. — 1958: Pritnary production of continental shelf waters off New York. Ibid. 3, 327-335.
32.
SCHROEDER, B., geb. TORNAU; 1954: Untersuchungen über den Einflue versehiedener Neutralsalze aus Wachstum, Zellform und Fadenbildung von StichococCus bacillaris und einigen
•
anderen Grünalgen. Arch. Mikrobiol. 20, 63-88.
33• SOEDER, C. J., 1960: Studien zur Entwicklungsphysiologie von Chlorella pyrenoidosa Chick
unter besonderer Beriicksichtigung der Salzkonzentration im Medium. Flora 14S, 489-516.
314 • SOROKIN, C., 1957: Changles in photosynthetic activity in the course of cell development in
Chlorella. Physiologiti Plantarum 10, 659-666.
35 • TUCKER, A., 1949: Pigment extraction as a tnethod of quantitative analysis of phytoplankton.
Trans. Amer. Mier. Soc. OS, 21-33.
36. UNGEmAcn, H., 1960: Sedimentchemismus und seine Beziehungen zum Stoffhaushalt in
40 europiiischen Seen. Dissertation Kiel.
37 • UTERMI511L, H., 1925: Limnologische Phytoplanktonstudien. Arch. Hydrobiol. Suppl. 5,
29.
RODRE, •
38.
WARBURG, 0.,
.,
1-527.
1925: Versuche über die Assimilation der Kohlensiture. Biochem. Ztschr. 166,
386, 287-307.
9. WEBER, E., 1957; GrundriB der biologisch .en Statistik. YEB Gustav Fischer, Jena 1-466.
0. YENTSCII, C. S., and J. 11. RYTHER, 1957: Short terni variations in phytoplankton chlorophyll
and their significanCe. Limnol. a. Oceaneg,r; e„140-142.
o
Dr. EVA-MARIA BURSCHE
,•
'•
Hydrobiologische Anstalt der
Max-Planck-Geselischaft,
Plün/Holstein
Translation of non-English titles
1. On the disintegration and regeneration of the chloroplast
dyestuffs in Ohlorella.
3. Production,-biological serial determinations in the
southern part of the North Sea in March 1955.
4. Chlorophyll, phytoplankton, and meteorological changes
(Lao Man.giore).
5. The coloration of CyeuPx, hyceae and Chlorophvoehe \ in their
dependence on the nitrogen content of the substratum.
Y
+
f
.40
- 83 6." Hydrochemical factors and phytoplankton during a tide
in the mouth of the Plbe near Cuxhaven.
7. Hydroche:iistry and phytoplankton in the Lower Elbe.
8. Lcire7ntions betwe en the chemi sm and the phytoplankton
development On the Lower ^Ye ser.
11. Deficiency of mineral salts and sec'ondary carotinoids
in green algae.
12. Effects of temperature and of light intensity on the
pigment content of Oscillatoria subbrevis Schmidle
(Cyanophyceae).
13. Investigations on the production biolagy of Plankton...in
the Kiel Bay.
17. The phytoplankton of fresh water.
18. The determination of chloroph;Tll in sea-water and ladle
sample s.
20. Auto- and heteroantagonism among freshwater algae.
24. Considerations regarding the quantitative determination
of ............ for the estimation of soluble pigments,
and factors affecting the correlation between the amount
of. pigment and dry weight.
Translator's note: A word has been omitted in this
sentence which might be 'chlorosis', 'chlorophyll' 2
or some other noun.
26. Nutrition- and r:letabolism-physiolorrical studies on
Fontinalis and Chlorella.
27. Pietabolism and mineral-salt nutrition of one-cell green
algae.
First communication.
Comparative studies on
,
- 84 =
' deficiency cultures of ,'aildstrodesmus.
32. Investigations on the influence of various neutral
its
on growth, cell form, and thread formation of StichocoCcus
bacillaria and several other green algae.
•
33. Studies on the physiolop:y of development of Chlorella
Ezrfn2i4223a
Chick, giving special consideration tO the
salt concentration in the medium.
36. Sediment chemism and its relations to the material economy
in 40 European lakes.
37. Limnological phytoplankton studies.
33. Experiments on the assmilation of carbon dioXide.
(11,
39. Outline of biological statistics.