Limnol.
Oceanogr.,
25(3), 1980, 474480
@ 1980, by the American
Society of Limnology
and Oceanography,
Inc.
Silicon content of five marine plankton
measured with a rapid filter method1
diatom
species
E. Paasche
Department
of Marine Biology and Limnology,
P.O. Box 1069, Blindern, Oslo 3, Norway
University
of Oslo,
Abstract
The silicon content of five species of marine planktonic diatoms grown in laboratory culture
was measured by a method involving
soda hydrolysis
of cells collected on polycarbonate
filters. An increase in the temperature
or light intensity during growth resulted in an increase
in the Si content in some species and a decrease in others. The magnitude of these changes
depended on whether the results were expressed as pg Siacell-r, pg Si*prnd2 cell surface
area, or pg Si per pg C.
Although silicon in the form of polymerized silicic acid (biogenic
silica) is a
major component of the cell walls of all
important plankton diatoms, there is considerable variation between species in Si
content (Parsons et al. 1961; Paasche
1973u; Harrison et al. 1977). Within a given species, the Si content may vary as a
function of cell size (Paasche 197%; Durbin 1977) as well as of external factors.
When plankton diatoms are grown in media with low silicic acid concentrations,
their Si content may be reduced by as
much as 70% (Paasche 1973b; Tilman
and Kilham 1976; Harrison et al. 1977).
The degree of silicification
may decrease
with increasing
temperature
(Durbin
1977) or may show a maximum at some
intermediate
temperature (Furnas 1978).
Diatoms collected in the field in the
warm season sometimes are weakly silicified (Gran 1912; Hart 1942; Hasle and
Smayda 1960). Laboratory results suggest
that this could be either a temperature
effect or a consequence of low silicic acid
concentrations
in the summer in temperate and polar waters. In late spring and
summer 1978, Skeletonema
costatum
produced large and apparently
weakly
silicified
populations
in the Oslofjord
(Paasche and e)stergren 1980). We wanted data especially on the effect of growth
temperature
on silicification
in this
species to facilitate interpretation
of the
r This work was supported by the Norwegian
search Council for Science and the Humanities.
Re-
field observations.
Four other species
were included
for comparison,
and the
effect of light was also studied.
The method used to measure diatom
silicon in this work was a modified version of the procedure used by Conway et
al. (1977) to study silica production in the
plankton of Lake Michigan. The plankton
is collected
on filters which are then
heated in a dilute soda solution to hydrolyze the silica to orthosilicic
acid. The
advantages of this are twofold: soda hydrolysis is fairly specific for diatom silica,
thereby reducing the danger of contamination inherent in the soda fusion or other drastic alkali treatments often used;
and concentrating
the algae by filtration
rather than centrifugation
makes the
analysis rapid and reproducible
even
with dilute diatom suspensions.
I thank T. Rgrtveit for technical assistance, S. Myklestad for the gift of a Chaetoceros affinis culture, and P. Jorgensen
for the gift of a silt sample.
Materials and methods
The diatoms used were S. costatum
(Greville)
Cleve, Thalassiosira
pseudonana Hasle & Heimdal,
Rhixosolenia
fragilissima
Bergon, Cerataulina
pelagica (Cleve) Hendey, and Chaetoceros affinis var. willei (Gran) Hustedt. The first
four species were isolated from the Oslofjord, while C. affinis was obtained
from the Trondheimsfjord
by S. Myklestad. All species were made bacteria-free
by a penicillin-streptomycin
treatment
474
Silica
in plankton
475
diatoms
Table 1. Growth rates of five diatom cultures used for chemical analyses. Light intensity in temperature
in light intensity experiment was 18°C.
experiment
was 8 * 1Oi5 quanta *cmm2*s-~. Temperature
Growth
Temp
Skeletonema costatum
Thalassiosira
pseudonana
Chaetoceros affinis
Rhixosolenia
fragilissima
Cerataulina
pelagica
* 0, No growth;
-,
rate*
(divisions.d-1)
Light intensity
(10’” quanta* cnYs2. s-*)
(“C)
8
13
18
23
1
2
4
8
16
0.8
1.0
1.0
0.7
0
1.5
1.4
1.6
1.1
1.1
1.8
1.6
1.8
1.5
1.4
1.3
2.6
1.3
1.8
1.8
0.2
0
-
0.5
-
0.8
0.5
-
1.8
1.5
-
1.7
-
G
-
no observations.
followed by either plating on agar or single-chain isolation by capillary pipette.
To avoid the complicating
effect of a sizedependent variation in chemical composition (Durbin
1977), we reisolated
R.
fragilissima,
C. pelagica, and C. affinis
into clonal culture shortly before the experiments
started. The cell widths in
these cultures were 12.0 pm (R. fragilissima), 12.2 pm (C. pelagica), and 9.0
pm (C. affinis). The S. costatum and T.
pseudonana cultures consisted of a mixture of cells of slightly different size, with
mean widths of 4.0 and 3.8 pm.
The growth medium was based on scawater diluted to 24%0 salinity with distilled water, to which was added 100 PM
nitrate, 100 PM orthosilicic
acid, 10 PM
phosphate,
and vitamins
and chelated
trace metals (Paasche 1973a). It was stcrilized by filtration through membrane filters (0.2-pm pore). The cultures were
grown in 500-ml polycarbonate
conical
flasks in growth cabinets in which the
temperature was 8”, 13”, 18”, or 23°C and
was constant to within &O.l”C. Light was
provided 12 h * d-l from “cool-white”
fluorescent lamps giving a light intensity of
8 * 10’” quanta. cmp2 *s-r in the temperature experiments.
In the light intensity
experiments, temperature was constant at
18°C; light intensity was varied by adding more lamps or by wrapping the flasks
in one or more layers of black nylon
gauze, the transmittance
of which had
been ascertained with a light meter,
The cultures were started from inocula
of about 1 lg Chl n * liter-l (5-10 ,cLgChl
a *liter-l at the two lowest light intensi-
ties in the light experiments).
Growth
was followed by daily measurements
of
in vivo chlorophyll
fluorescence
by
means of a UME FM3 fluorometer.
Exponential
growth constants
(Table 1)
were calculated from measurements
on
the last 3 days of growth, with no great
claim to precision
due to the limited
number of measurements
and also bccause of a variable in vivo chlorophyll
fluorescence yield in R. frugilissima
and
C. pelagica.
Cerataulina
pelagica
did
not grow at 8°C and R. frugilissima
failed
to grow at the lowest light intensity tried.
The cultures were harvested when they
had reached a density of ca. 50 pg Chl
cI *liter-‘,
and samples were filtered for
chemical analysis or preserved with iodine for later counting and measuring of
cells.
Before counting,
the preserved
samples were shaken vigorously
to break up
chains to short fragments of 24 cells.
The cells were counted under a microscope in hemacytometers
or Palmer-Maloney chambers. At least 1,000 cells were
counted for each sample. Mean cell dimensions were determined
from microscopic measurements of 50 or more cells
from each culture. Surface areas were calculated on the assumption that the cells
were cylinders
with an elliptical
crosssection in C. affinis (h = Iha, where a
and h are the axes) and a circular crosssection in the other four species. Spines
and connecting
processes were not included in the calculations.
Carbon content of cells collected
on
glass-fiber filters was determined
as de-
476
Paasche
scribed by Strickland and Parsons (1972),
using a Carlo Erba elemental analyzer.
To determine the silicon content of the
diatoms, we filtered 25 ml of cell suspension through a 25-mm Uni-Pore (Bio-Rad)
polycarbonate
filter of 0.6-pm pore size,
using an all-plastic
filtering
apparatus.
The filter was rinsed with about 2 ml of
prefiltered
seawater. The unfolded filter
was immediately
placed in a 50-ml polypropylene
centrifuge
tube, which was
then stoppered to prevent any loss of particulate Si during storage. Hydrolysis was
carried out by adding 18 ml of 0.5%
Na,CO, solution to the centrifuge
tube
and heating it to 85°C for 2 h. Agitation
of the tube during heating proved unnecessary. When cool, the contents were
neutralized with 0.5 N HCl to the turning
point of methyl orange (pH 3-4) and
made up to 25 ml in a volumetric
flask.
A 5-ml aliquot was used for the determination of Si(OH), according to Strickland and Parsons (1972). A blank correction was made by taking an unused filter
through the hydrolysis
and subsequent
steps. The relative standard deviation, as
determined
from eight sets of triplicate
analyses of S. costatum cultures in the
concentration
range of 250-400
pg
Sisliter-l,
was 4.2% of the mean.
Conway et al. (1977) used cellulose ester filters in their version of the method.
The use of polycarbonate
filters is a substantial improvement
since these, unlike
cellulose-based
filters, are not visibly attacked by soda and give a very low blank
reading. The blank was improved further
by reducing the soda concentration
from
3%, as suggested by Conway et al. (1977),
to 0.5%. We used Merck “Suprapur”
soda, but ordinary
reagent grade soda
gave an equally low blank (0.008 optical
density units in a l-cm cell) at the 0.5%
concentration
level.
Comparison with the more drastic procedure of boiling the diatoms with sodium
hydroxide
(Paasche
1973a,b)
showed that the soda treatment caused
complete hydrolysis of the diatom silica.
In a test on a concentrated mixed suspension of S. costatum, R. fragilissima,
and
C. pelagica, the two methods yielded the
following
values (pg Sisliter-I,
+SE of
the mean; n = 6): soda hydrolysis of cells
on filters, 1,160 + 5; sodium hydroxide
hydrolysis
of centrifuged
cells, 1,138 +
4. It is essential for the success of the
soda hydrolysis method that the cells be
spread out on a filter. When the method
was tried on centrifuged
cell material,
hydrolysis was incomplete.
Soda hydrolysis
is considered
to be
specific for amorphous silica (Tessenow
1966; Hurd 1973; Conway et al. 1977). To
test the method for interference
of silicate minerals, we subjected a sample of
moraine silt (from P. Jgrgensen) of 6-20pm grain size, and containing ca. 30% Si
mainly in the form of illite and chlorite,
to the same treatment as the diatom samples. Less than 0.5% of the calculated Si
content of the silt was recovered.
All chemical
analyses were done in
triplicate.
The amount of work involved
precluded
the use of duplicate
flasks.
However, the experiments
with S. costatum were repeated with essentially the
same results as those reported here.
Results
It is generally believed that the siliceous cell wall accounts for all but a
small fraction of the silicon in diatom
cells (see Werner 1977). However, Kesseler (1974) and Chisholm
et al. (1978)
have presented evidence that some of the
large and highly vacuolate marine plankton diatoms store appreciable amounts of
partly polymerized
silicic acid in their
cell sap. To see whether the results reported here would include
significant
amounts of loosely bound Si, we did tests
with R. fragilissima
and C. pelagica as
well as with two other large species, Ditylum brightwellii
(West) Grunow and
Guinardia jlaccida (Castracane) Peragallo, all of which have prominent vacuoles.
In all of these species <lo% of the Si
retained by the filters was lost upon rinsing with 0.01 N HCl, a treatment that effectively
disrupted
the cells. It seems
likely therefore that the values of cellular
silicon reported here represent mostly
cell wall silica.
The growth rates on the last 2 days be-
Silica
in plankton
477
diatoms
Table 2. Cellular
silicon content of five diatom species grown at different
temperatures
and light
intensities.
Light intensity in temperature
experiment
was 8. 1Ol5 quanta-cm-2*s-‘.
Temperature
in light
intensity experiment
was 18°C.
Silicon
Tcmp
Skeletonema costatum
Thalassiosira
pseudonana
Chaetoceros affinis
Rhixosolenia fragilissima
Cerataulina
pelagica
* -,
conlcnt*
(pg Si . cell-‘)
Light intensity
( lW5 quanta. crne2. SP)
(“C)
8
13
18
23
1
2.59
1.47
41.7
51.0
-
2.70
1.90
38.8
47.5
89.0
2.67
1.88
35.3
30.6
88.4
2.61
1.41
33.3
27.6
96.6
1.57
-
2
1.60
3G
-
4
8
16
2.26
27.7
-
2.62
29.9
-
29.2
-
No observntions.
fore harvesting
are shown in Table 1.
Taking into consideration
that the cultures received only 12 h of light per day,
the growth rates in the temperature
experiments were close to those expected
for cultures growing at light saturation
(Eppley
1972). Light intensities
below
8~10~~ quanta. crnb2 *s-l limited the growth
rate at 18°C in S. costatum and R. fragilissima, the only two species tested.
Values of silicon content per cell are
presented in Table 2. The Si content of
T. pseudonana at 18°C agrees with that
previously
observed in the same species
under similar growth conditions (Paasche
1973b). In S. costatum, the Siecell-’ value at 18°C (Table 2) was some 30% lower
than previously
reported for the same
clone (Paasche 1973a), possibly reflecting a partial loss of silicifying
capacity
during the seven intervening
years of
cultivation.
With another clone grown
under corresponding
conditions,
Harrison et al. (1977) obtained a still lower value of 2.1 pg Si *cell-l. These comparisons
are permissible since the cell dimensions
were about the same in all cases (cell diameter ca. 4 pm in both species). The Si
content of the remaining three species is
reported here for the first time.
There was no common pattern in the
responses of the individual
species to the
environmental
variables. The Si content
of S. costatum cells seemed to be independent of temperature
over the entire
15°C interval. Rhixosolenia
fragilissima
and, to a much lesser extent, C. af$nis
showed declining Si values with increas-
ing temperatures, while there was no definite trend in T. pseudonana and C. peZagica. Reduced light caused a decrease
in Siecell-1 in S. costatum though not in
R. fragilissima.
Values of cell silicon content in diatoms are frequently
recalculated
as
weight units of Si per square unit of cell
surface arca. The Si:surface area ratio is
an approximate measure of the degree of
silicification
of the cell wall. My results,
expressed in this way, are shown in Figs.
1 and 2. The Si : surface area ratios were
in the range of 0.015-0.089 pg Si *PM-~,
which
is close to the 0.01-0.08
pg
Si. prnm2 range found in other marine
plankton
diatoms grown in silicon-rich
media (Durbin
1977; Harrison
et al.
1977; Furnas 1978) and somewhat less
than the 0.04-0.17 pg Si . prnB2 reported
for freshwater plankton diatoms (BaileyWatts 1976). A comparison between Table 2 and Fig. 1 suggests
that the
Si : surface area ratio in plankton diatoms
is not primarily
determined
by cell size.
The high values for C. affinis probably
reflect the silicon
content
of the extremely long siliceous setae carried by
this species. Cerataulina
pelagica
appcared to be twice as heavily silicified as
R. fragilissima
despite the nearly identical shape (long cylinders) and size (12 x
40 pm) of these two species.
An increase in temperature
from 8” to
18°C caused a 50% increase
in the
Si : surface area ratio in S. costatum and
a 50% decrease in R. fragilissima,
with
intermediate
responses in the other three
478
Paasche
0.12
lCHAET.
+-----\
CER.
*-*-
*
/- - ~//-+--+
CI-
I
8
I
13
TEMPERATURE,
I
18
1
23
‘C
Fig. 1. Amount of silicon per unit of cell surface
area in five diatom species as a function of temperature (e--Skeletonema
costatum; O-Thalassiosira
pseudonana;
A4haetoceros
affinis; A-RhizosoX-Cerataulina
pelagica). Verlenia fragilissima;
tical bars indicate range of single observations.
species. The effect of temperature in R.
fragilissima
was of the same direction
and magnitude as in Thalassiosira
nordenskioeldii
Cleve in the O”-10°C range
(Durbin 1977). Th ere was a strong light
effect in S. costatum but none in R. fragilissima (Fig. 2).
Silicon : carbon weight ratios are shown
in Figs. 3 and 4; they are in the range
expected on the basis of similar analyses
by others (Durbin
1977; Harrison et al.
1977). In S. costatum grown in a nutrient-rich
medium at 18”C, Harrison et
al. (1977) observed a Si:C ratio of 0.25
(recalculated
from data in their table 2),
which agrees exactly with the 0.24-0.26
in my experiments
at 18°C (Figs. 3 and
4). There was a general tendency for the
Si:C ratio to increase with increasing
temperature
(Fig. 3). This was because
the cells were larger (longer) and contained more carbon at low temperatures.
Rhixosolenia
fragilissima
again was an
o0
I
I
I
5
10
15
LIGHT INTENSITY,
1015quanta
/ cm*.s
Fig. 2. Amount of silicon per unit of cell surface
area in two diatom species as a function of light
intensity. Symbols as in Fig. 1.
exception, with a close to 50% reduction
in the Si:C ratio in the 8”-23°C temperature interval. Light intensity had no pronounced effect on the Si:C ratio in either
of the two species tested (Fig. 4).
Microscopic
observations
indicated
that the variations in silicification
reported here manifested themselves mainly in
the form of changes in cell wall thickness
rather than in the shape and ornamentation of the diatom valves.
Discussion
The consistency between these results
and those of other investigators using different analytical methods adds proof that
the soda hydrolysis method, as used here,
is well suited to the analysis of silicon in
diatom cultures. It is a great advantage
that the same method can be applied to
fieldwork. We used it in our investigation
of the Oslofjord plankton (Paasche and
Q)stergren 1980) to obtain data on natural
S. costatum populations
that we could
compare with the laboratory results presented above.
Silica
in plankton
479
diatoms
+
0.4
SKEL.
0
?
iz
(3, 0.3
.
z
al
2
v
\\
'\ \
----0.2
RHIZ.
--em
t
+
2
z
i
z
0.1
0
GHAL.
I
8
13
18
23
TEMPERATURE,‘C
Fig. 3. Silicon : carbon weight ratio in five diatom species as n function of temperature.
Symbols
as in Fig. 1..
Gran (1912) illustrated
strongly and
weakly silicified
specimens of Chaetoceros decipiens Cleve collected in cold
and warm water and mentioned that similar modifications
occur in other species
of the genus Chaetoceros. Of the diatoms
used in my work, S. costatum, C. pelagica, and R. frugilissima
are known to
form thin-walled
“summer forms” in the
plankton
in the Oslofiord,
at temperatures of 15”-20°C (Braarud 1945; Hasle
and Smayda 1960). The present results
show that even if the Si content of some
species, such as R. fragilissima,
decreases with increasing temperature, the
occurrence of lightly silicified cells in the
plankton cannot always be due to tcmperaturc effects. Low ambient Si concentrations would seem to offer a much more
likely universal explanation.
In the case
of S. costatum, the observations made by
Braarud (1945) 1cave no doubt that the
populations
present in the Oslofjord in
the summer months may at times bc very
Si-deficient.
5
I
10
15
LIGHT INTENSITY, 1OJ5quanta /cm2 es
Fig. 4. Silicon : carbon weight ratio in two diatom species as rz fknction of light intensity. Symbols
as in Fig. 1.
IIarrison et al. (1977) demonstrated the
chemical changes in S. costatum when
grown in Si-limited
chemostat cultures.
Their data show that Si limitation
may
cause a 50% rediiction
in the Si : surface
area ratio and a 75% reduction in the Si:C
ratio. These effects should be contrasted
with the increase of about 50% in the
Si : surface arca and Si:C ratios in the
species when temperatine
is increased
from 8” to 18°C (Figs. 1 and 3). However,
silicification
in S. costatum
in nature
may be affected by other factors besides
silicic acid concentrations,
temperature,
and light intensity. In the Oslofiord, low
Si : surface area and Si:C ratios in S. costatum in late spring and early summer
1978 seemed to bc associated
with
changes in ccl1 morphology that were not
observed in the present temperature and
light intensity
experiments
and which,
apparently, were not primarily the result
of a shortage of Si in the environment
(Paasche and Qstergren 1980).
480
Paasche
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1979