J. Phycol.
35, 1162–1170 (1999)
THE CHEMICAL FORM OF DISSOLVED SI TAKEN UP BY MARINE DIATOMS1
Yolanda Del Amo2 and Mark A. Brzezinski
Department of Ecology, Evolution and Marine Biology and Marine Science Institute, University of California at Santa Barbara,
Santa Barbara, California 93106
Results of past studies of the pH-dependent Si
uptake kinetics of Phaeodactylum tricornutum Bohlin
suggested that the anion SiO(OH)23 is the chemical
form of dissolved Si taken up by marine diatoms.
We determined the chemical form of Si taken up by
three other marine diatom species and P. tricornutum
by examining the kinetics of Si use under two dra-
matically different SiO(OH)23 :Si(OH)4 ratios in sea-
water by varying pH from ø8 to ø9.6. Uptake rates
were determined using a precise and sensitive 32Si
tracer methodology. The pH-dependent uptake ki-
netics obtained for all species except P. tricornutum
suggest that marine diatoms transport Si(OH)4. The
half-saturation constant (Km) varies strongly as a
function of pH for all species when the substrate of
transport is assumed to be SiO(OH)23 . Kinetic
curves for Thalassiosira pseudonana (Hustedt) Hasle
et Heimdal, Thalassiosira weissflogii (Grunow) G.
Fryxell et Hasle, and Cylindrotheca fusiformis Rei-
mann et Lewin have statistically identical values of
Km at each pH when the substrate for transport is
assumed to be Si(OH)4 (T. pseudonana and T. weiss-
flogii) or total dissolved silicon (C. fusiformis). In con-
trast, P. tricornutum exhibits unusual biphasic uptake
kinetics: uptake conforms to Michaelis–Menten ki-
netics up to 15 to 25 mM, above which uptake in-
creases linearly. This enigmatic response may have
biased conclusions drawn from past experiments us-
ing this species. However, based on the consistency
of the results for the three other species, a new
model of Si transport in marine diatoms is proposed
on the basis of the direct formation of a complex
between the Si-transport protein and Si(OH)4.
Key index words: marine diatoms; pH; silicic acid
transport; silicon; substrate; uptake kinetics
Diatoms are the major component of phytoplank-
ton in many aquatic ecosystems. At present, from
12,000 (Werner 1977) to 60,000 (Gordon and Drum
1994) diatom species have been identified around
the world. Diatoms are a major food source for pe-
lagic and benthic organisms and have seldom been
found a source of ecological problems in nature
(Officer and Ryther 1980). As a group, they account
for 40% of total primary production of carbon in
the marine environment (Nelson et al. 1995) and
are thought to contribute significantly to the export
1Received 29 April 1999. Accepted 3 August 1999.
2Author for reprint requests; e-mail delamo@lifesci.lscf.ucsb.
edu.
1162
of materials from the surface ocean to the deep sea
(e.g. Honjo et al. 1995, Karl et al. 1996, Dugdale
and Wilkerson 1998). A unique characteristic of di-
atoms compared to other phytoplankton is that
their cell walls are silicified forming a structure
called a frustule, which is composed of hydrated
amorphous silica. The need to form the frustule is
absolute, causing Si availability to be a key factor in
the regulation of diatom growth in nature (e.g. Paas-
che 1980, Ragueneau et al. 1999). In turn, the bio-
geochemical cycle of Si in the ocean is controlled
mainly by diatom use (Tréguer et al. 1995). Numer-
ous studies have focused on morphogenesis and
frustule formation during cell division (reviewed in
Pickett-Heaps et al. 1990, Pickett-Heaps 1991, Gor-
don and Drum 1994). However, studies on Si bio-
mineralization processes are not as extensive (Sul-
livan and Volcani 1981, Volcani 1981, Sullivan
1986), and the biochemical pathways involved in si-
licification and their regulation are still not well un-
derstood.
The aim of this paper is to address the chemical
form of Si that marine diatoms take up from sea-
water to construct their cell walls. The dependence
of the uptake rate of dissolved silicon (dSi) by dia-
toms on the external dSi concentration has been
described in many studies by hyperbolic saturation
kinetics (e.g. Sullivan 1976, Conway and Harrison
1977). Changes in the uptake rate (V ) with increas-
ing [dSi] can be described by a rectangular hyper-
bola, similar to the Michaelis–Menten equation (1)
for enzyme kinetics:
[dSi]
V 5 Vmax · (1)
K m 1 [dSi]
where Vmax is the maximum uptake rate and Km is
the half-saturation constant for uptake defined as
the substrate concentration where V 5 ½ Vmax. The
kinetics constants Vmax and Km are species specific.
More than one chemical form of dissolved silicon
is available to diatoms in seawater because of the
hydrolysis of silicic acid, resulting in an equilibrium
between the nonionized form Si(OH)4 and anionic
forms SiOx(OH)x422x. The chemical speciation of the
various interchangeable forms of dSi in seawater can
be calculated as a function of pH by using the ap-
propriate dissociation constants (Sjöberg et al.
1981). At the natural pH of seawater (pH ø 8), un-
dissociated silicic acid Si(OH)4 is the dominant form
(97% of total dSi), with the remainder being largely
SiO(OH)23 .

SUBSTRATE FOR SI UPTAKE IN DIATOMS
It has been implicitly assumed by most investiga-
tors that the entire pool of dissolved Si (often noted
as Si(OH)4) is indiscriminately taken up by diatoms.
However, Riedel and Nelson (1985) obtained evi-
dence that SiO(OH)23 is the chemical species trans-
ported across the membrane. They examined
changes in the Si-uptake kinetics of Phaeodactylum
tricornutum Bohlin (the only diatom with no known
absolute requirement for Si) as a function of pH
over a small pH range that alters the chemical spe-
ciation of dissolved Si to a large degree. In their
study, P. tricornutum exhibited hyperbolic saturation
kinetics for Si uptake at both pH 8.6 and 9.5, but
the Km values obtained at the two pHs differed con-
siderably when uptake was expressed as a function
of total dissolved silicon concentration or the con-
centration of Si(OH)4. In contrast, very similar Km
values were obtained at both pHs when SiO(OH)23
was assumed to be the substrate transported by the
cells. The pH-dependent uptake kinetics observed
by Riedel and Nelson (1985) suggest that diatoms
may not indiscriminately take up all forms of dis-
solved silicon as widely presumed but transport the
first conjugate base of silicic acid, SiO(OH)23 .
We reinvestigated this possibility by examining
the pH dependence of Si-uptake kinetics using four
marine diatoms, including three common species
with known absolute Si requirements (Thalassiosira
weissflogii (Grunow) G. Fryxell et Hasle, Thalassiosira
pseudonana (Hustedt) Hasle et Heimdal, and Cylin-
drotheca fusiformis Reimann et Lewin) and also P. tri-
cornutum. Uptake rates were determined using a pre-
cise and sensitive 32Si tracer methodology (Tréguer
et al. 1991, Brzezinski and Phillips 1997).
MATERIALS AND METHODS
Diatom cultures. Diatom species (Thalassiosira pseudonana, clone
3H–CCMP 1335, and Phaeodactylum tricornutum, clone PETPD–
CCMP 630) were obtained from the Provasoli-Guillard National
Center for Culture of Marine Phytoplankton (CCMP) at the Big-
elow Marine Laboratory for Ocean Sciences (Maine, U.S.A.). Cul-
tures were unialgal and axenic. Thalassiosira weissflogii has been
maintained in culture at the University of California at Santa Bar-
bara for several years. Cylindrotheca fusiformis was obtained from
Scripps Institution of Oceanography (San Diego) through the
courtesy of Dr. M. Hildebrand. All clones were maintained in f/
2 enriched seawater medium with added dissolved silicon (Guil-
lard and Ryther 1962, Guillard 1975) at 178 C under a continuous
photon fluence rate of ø100 mE·m22·s21.
Si-free seawater. Filtered seawater from Santa Barbara Channel
(California) was used to produce Si-free seawater by growing T.
weissflogii to remove dissolved Si. Forty liters of Santa Barbara
Channel seawater were filtered through 0.6-mm polycarbonate fil-
ters, enriched to Guillard’s f/10 level of macronutrients (21.2
mM-Si, 7.2 mM–P, 176 mM–N) and inoculated with a 1-L culture
of T. weissflogii in f/2 medium. The 40-L culture was incubated
at 178 C under continuous illumination, and the [dSi] was mon-
itored until Si depletion was reached ([dSi] , 0.5 mM). Seawater
was then filtered through 0.6-mm polycarbonate filters and stored
at 38 to 58 C in the dark. Final dSi concentration was 0.4 mM.
Aliquots of the Si-free seawater were filtered through 0.2-mm poly-
carbonate filters and autoclaved just prior to each kinetic exper-
iment.
Uptake experiments. Cultures (250-mL) of T. weissflogii, T. pseu-
donana, C. fusiformis, and P. tricornutum were grown at a constant
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1163
photon fluence rate of ø100 mE·m22·s21 at 178 C in nutrient-
enriched f/2 seawater media with the [Si(OH)4]:[NO23 ] ratio tai-
lored for each species to cause preferential depletion of Si (see
Brzezinski et al. 1990). The Si:N molar ratios used were 1:10 for
T. weissflogii, 1:18 for T. pseudonana, 1:200 for C. fusiformis, and 1:
880 for P. tricornutum. The N:P ratios were held at 24:1 through-
out. Cultures were sampled every day for [dSi] analysis. When
[dSi] was ,20 mM (ø4–8 days), a 20-mL aliquot of each culture
was added to each of two larger polycarbonate flasks containing
1100 mL of Si-free f/2 seawater at 178 C. The pH of one of the
larger flasks had been previously adjusted to ø8.0 (7.98–8.45)
and the other to pH ø 9.6 (9.25–9.80) by using 10% HCl or 1-N
NaOH. The ratio of [Si(OH)4]:[SiO(OH)23 ] in each media, cal-
culated using the dissociation constants of Sjöberg et al. (1981),
is (97%:3%) 5 32.3 at pH 8.0 and (43%:57%) 5 0.75 at pH 9.6.
Each 1100-mL culture was then sampled in triplicate for liquid
scintillation counting blanks (54 mL each), for biogenic silica
analysis (50 mL each), for cell counts, and for [NO23 1 NO222]
and [HPO242] analysis. In all experiments, nitrate 1 nitrite and
phosphate concentrations were kept higher than f/2 level (800
mM nitrate and 20 mM phosphate) to guarantee that those nutri-
ents were not limiting during the time course of the experiments.
For each species, the two cultures (pH ø 8 and ø 9.6) were
dispensed into a series of 60-mL polycarbonate incubation bottles.
Increasing volumes of 1-mM Si stock solutions of Na2SiO3-9H2O,
previously adjusted to pH ø 8 or ø 9.6, were then added to pro-
duce a series of dSi concentrations ranging from 0 to 50 mM
above ambient. For P. tricornutum, which has less efficient uptake
kinetics (Riedel and Nelson 1985), pH-adjusted 10.6-mM Si stock
solutions were used to produce the three highest dSi concentra-
tions, ranging from 50 to 300 mM above ambient. The dSi con-
centrations in the experimental flasks were well below the limit
for Si polymerization (ø2 mM; Stumm and Morgan 1996). The
1-mM Na2SiO3 stock solution used for the majority of Si additions
is also below the concentration threshold for the formation of
colloidal or polymeric Si; however, the 10.6-mM Si stock solution
could theoretically polymerize spontaneously. Such a high con-
centration was required to prevent the additions of Si from de-
creasing significantly the salinity of the media in the experimental
flasks. The probability of polymerization was minimized by pre-
paring both the 1-mM and the 10.6-mM stock solutions just prior
to each experiment.
After the addition of Si to a 60-mL flask, a 6-mL volume was
withdrawn and filtered for dSi concentration analysis, and 250 to
446 mL of 32Si (830–1670 Bq as sodium silicate) were added im-
mediately. All flasks were then incubated for 3 to 4 h at 178 C
with illumination provided continuously by cool-white fluorescent
lamps at a photon fluence rate of 133 mE·m22·s21.
At the end of the incubation, the contents of each flask were
filtered in their entirety through a 0.6-mm Nuclepore filter. Each
filter was placed in the bottom of a 20-mL plastic liquid scintil-
lation vial and dried overnight with the cap off in a closed cabinet.
Then each filter was covered with 2.0 mL of 2.5-M HF to dissolve
all biogenic silica, capped, and allowed to sit overnight. Finally,
after addition of 10 mL of HP Ultima Gold XR liquid scintillation
cocktail, the activity of 32Si was assayed using liquid scintillation
counting (Beckman LS 5000TA) following Brzezinski and Phillips
(1997).
The pH of the cultures was measured at the beginning and
end of the incubations. Increases in pH were minor for T. pseu-
donana (0.003 and 0.04 pH units for both pH’s cultures) and for
T. weissflogii (0.02 and 0.03 pH units). The two pH’s cultures of
C. fusiformis showed increases of 0.2 pH units, and changes in P.
tricornutum cultures were 0.3 and 0.3, 0.2 and 0.3, 0.4 and 0.5
during each of three experiments.
Specific Si-uptake rate and kinetic parameter calculations. To calcu-
late the specific uptake rates of dSi (V in h21) during the incu-
bation time t, we used the formulation given in Brzezinski and
Phillips (1997):
1 2
[bSi]0 1 [bSi]N 1
V 5 ln · , (2)
[bSi]0 t
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1164 YOLANDA DEL AMO AND MARK BRZEZINSKI

FIG. 1. Si-uptake rates (V ) obtained at two different pHs as a function of the concentration of different forms of dissolved Si for
three diatom species: (A) Thalassiosira pseudonana, (B) Thalassiosira weissflogii, (C) Cylindrotheca fusiformis. (V) Low pH, corresponding to
7.98, 8.03, and 8.01 for (A), (B), and (C), respectively. (v) High pH, corresponding to 9.45, 9.47, and 9.25 for (A), (B), and (C),
respectively. Fitted curves are the Michaelis–Menten hyperbolas obtained by the nonlinear regression of Wilkinson (1961).

where [bSi]0 and [bSi]N are the biogenic silica concentration
measured directly at the beginning of the incubation and the
increase in biogenic silica concentration during incubation, re-
spectively; [bSi]N is given by:
[bSi]N 5 [dSi]BqCell/BqTotal (3)
where [dSi] is the concentration of total dissolved silicon in the
flask and BqCell and BqTotal are the disintegrations per second
(dps) of 32Si in cells at the end of the incubation and the total
activity added to the culture, respectively. This equation assumes
that the distribution of 32Si remains in equilibrium among the
various chemical forms of dSi during the incubation. For all ex-
periments, specific Si-uptake rates (V ) were plotted against the
corresponding total dissolved silicon, Si(OH)4, SiO(OH)23 , and
SiO2(OH)222 concentrations for both pH treatments, and the ki-
netic parameters, Vmax and Km, were calculated for each curve
using the nonlinear regression technique of Wilkinson (1961).
Analytical methods. Except for dissolved silicon samples that
were immediately analyzed, nitrate 1 nitrite and phosphate sam-
ples were stored at 48 C until analysis. Total dissolved silicon (dSi)
was analyzed according to Strickland and Parsons (1972) modi-
fied to use the reagent blank of Brzezinski and Nelson (1986).
Concentrations of the various chemical forms of dSi in seawater
were calculated by using the dissociation constants of Sjöberg et
al. (1981) and the mean pH over the incubation period.
Nitrate 1 nitrite (NO23 1 NO222) and phosphate (HPO242) con-
centrations were measured by flow injection analysis according to
Murphy and Riley (1962) using a Lachat Instruments Quikchem
8000 (precision 5%). Filters containing cells for biogenic silica
concentration analysis were dried for 24 h at 608 C and stored in
closed plastic petri dishes until analysis. Filters were then even-
tually measured using the method of Paasche (1973), modified
to use the reagent blank of Brzezinski and Nelson (1986).
The activity of the 32Si stock solutions used were determined
by placing 20 mL of each solution into a scintillation vial contain-
ing 2 mL of 2.5-M HF, followed by the addition of 10 mL of liquid
scintillation cocktail and measured by liquid scintillation as de-
scribed previously. Samples for cell counts were preserved with a
diluted formaldehyde/nitric acid mix solution. Species were
counted by microscopic examination using a Fuchs–Rosenthal he-
macytometer slide (Guillard 1978).
RESULTS
For all species except P. tricornutum, uptake rates
exhibited hyperbolic saturation kinetics at both pHs
for all presumed substrates (Fig. 1). The corre-

SUBSTRATE FOR SI UPTAKE IN DIATOMS
FIG. 2. Si-uptake rates (V ) obtained at two different pHs as a
function of the concentration of different forms of dissolved Si
for Phaeodactylum tricornutum, experiment #3. (V) Low pH (8.12,
8.16, and 8.45 for experiments #1, #2, and #3, respectively). (v)
High pH (9.43, 9.49, and 9.80 for experiments #1, #2, and #3,
respectively). Fitted curves are the Michaelis–Menten hyperbolas
obtained by the nonlinear regression of Wilkinson (1961).
sponding statistical correlation coefficients (r2) ob-
tained by the linearized Woolf transformation of the
Michaelis–Menten equation (S/V vs. S) are all
.0.98 (ranging from 0.982–0.999; n 5 9–10).
In contrast, P. tricornutum exhibited unusual up-
take kinetics with a biphasic response where uptake
conformed to Michaelis–Menten kinetics up to 15
to 25 mM total dSi, with uptake increasing linearly
at higher concentrations (Fig. 2). To confirm this
biphasic response, the pH-kinetic experiment for P.
tricornutum was repeated three times (experiments
denoted #1, #2, and #3), leading to highly variable
kinetic curves but with all three showing a biphasic
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1165
response. Controls for nonbiological uptake (ad-
sorption and precipitation; see Wollast et al. 1968,
Nelson et al. 1984) were conducted at the highest
dSi concentration, where precipitation or nonbio-
logical adsorption were most likely (Nelson et al.
1984). Two sets of controls consisting of formalde-
hyde-killed cells in the experimental media and cell-
free media were incubated alongside flasks contain-
ing living cells. All controls showed either nonexis-
tent 32Si uptake (#0.02% of total added 32Si) or no
precipitation of inorganic silica (particulate silica
concentration #0.05% of dSi concentration), indi-
cating that the biphasic kinetic response of P. tricor-
nutum was not due to an experimental artifact. In
addition, in all three experiments with P. tricornu-
tum, four to six data points out of 10 form the linear
part of the curve at high concentration. This dis-
cards an artifact due to the possible presence of col-
loidal Si introduced by 10.6-mM Si stock solution
used to create the highest three dSi concentrations
in each experiment (see Materials and Methods).
Estimation of the kinetic parameters for P. tricor-
nutum was performed by considering only the data
points from rate measurement conducted at dSi
concentrations ,30 mM. Linear regressions of S/V
versus S for those data points yielded correlation co-
efficients ranging from 0.757 to 0.999 (n 5 4–6).
Table 1 gives the Si-uptake kinetic parameters
(Vmax and Km) calculated for the four species at both
low (ø8.0) and high (ø9.6) pH. As expected, Km
and Vmax generally varied strongly with pH (Table
1). In the lower pH series, that is, the pH closest to
natural seawater pH, Km values are considerably low-
er for all three common diatom species, and Vmax is
higher, except for one experiment (#2) out of the
three with P. tricornutum (Table 1).
The half-saturation constants obtained for each
species at the two pHs are compared using t-tests
with pooled sample variances (Moore 1996) in Ta-
ble 2. For T. pseudonana and T. weissflogii, the Km
values at each pH are very similar in magnitude with
Si(OH)4 as the presumed substrate (Table 1) and
statistically indistinguishable (Table 2). For C. fusi-
formis, Km values obtained at each pH are nearly
equal when using total dSi or Si(OH)4 as the sub-
strate (Table 1) and statistically indistinguishable
when using total dSi (Table 2). The results for the
three experiments with P. tricornutum are inconsis-
tent (experiments #1, #2, and #3; Tables 1, 2). Sim-
ilar Km values at the two pHs are obtained only in
experiment #1, in which Si(OH)4 is assumed to be
the substrate. All other experiments show large dif-
ferences of Km values at the two pHs for all pre-
sumed substrates, and a high variability exists in the
value of Km obtained among experiments. In exper-
iments #2 and #3, different values of Km were ob-
tained at the two pHs when using any form of Si as
the substrate (Table 1), although the values in Table
2 suggest that we cannot reject the null hypothesis
that the Km are equal when Si(OH)4 is considered

1166 YOLANDA DEL AMO AND MARK BRZEZINSKI
TABLE 1. Maximum uptake rates (Vmax) and half-saturation constants (Km) for Si uptake using the different chemical forms of dissolved
Si as the substrate (S) for each of the four diatom species studied. Values in parentheses represent 695% confidence limits determined
by the nonlinear regression method of Wilkinson (1961). Bold characters highlight overlapping confidence limits.
Vmax (h21)
Diatom species Low pH High pH
T. pseudonana 0.091 (60.002) 0.042 (60.002)
T. weissflogii 0.089 (60.004) 0.066 (60.004)
C. fusiformis 0.086 (60.002) 0.057 (60.001)
P. tricornutum,
exp. #1 0.015 (60.002) 0.0086 (60.0001)
P. tricornutum,
exp. #2 0.0346 (60.0005) 0.05 (60.02)
P. tricornutum,
exp. #3 0.238 (60.002) 0.075 (60.004)
the substrate transported in experiment #2. This re-
sult is due to the large confidence limit for Km ob-
tained at the higher pH. For all four species, when
the substrate of transport is assumed to be
SiO(OH)23 or SiO2(OH)222 , Km is always statistically
different at both pHs.
Thus, P. tricornutum exhibited biphasic kinetics,
and the kinetic parameters for the low-concentra-
tion hyperbolic response region were highly variable
among the three experiments conducted (Tables 1,
2). However, the kinetic curves have statistically
identical values of Km independent of pH when the
substrate for transport is assumed to be Si(OH)4 for
T. pseudonana and T. weissflogii and total dissolved Si
for C. fusiformis.
DISCUSSION
Variable kinetics for P. tricornutum. None of the
three experiments performed with P. tricornutum
shows the same pattern as in the previous study by
Riedel and Nelson (1985). Both our study and that
of Riedel and Nelson used clone PETPD, but the
incubation temperature differed by 38 C (178 C here
instead of 208 C in their study). It is known that for
most enzymes, kinetic parameters change with tem-
perature, although they remain constant within a
certain temperature range (e.g. Turner and Pollock
1993). P. tricornutum growth is sensitive to changes
in temperature from 158 to 208 C (Hayward 1968),
which may cause our results to differ from those of
Riedel and Nelson; however, incubation conditions
did not differ among the three experiments per-
formed in our study, yet different kinetic responses
were observed in all three experiments. Thus, in-
cubation conditions are not likely to be the cause of
the differences between our results and those of Rie-
del and Nelson (1985).
To better compare our data with those of Riedel
and Nelson (1985), cellular uptake rates were cal-
culated in fmol·cell21·h21 (Table 3) to match the
units that they reported. Because Vmax values are not
specified in their paper, an approximation of Vmax
was derived from their graphical representations at
60 and 160 mM, that is, their maximum total dSi
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S 5 total dSi S 5 Si(OH)4 S 5 SiO(OH)23
Km (mM) Km (mM) Km 5 (mM)
Low pH High pH Low pH High pH Low pH
1.4 (60.1) 2.2 (60.4) 1.4 (60.1) 1.1 (60.2) 0.044 (60.003)
4.7 (60.5) 8.0 (61.1) 4.5 (60.5) 4.0 (60.5) 0.16 (60.02)
0.88 (60.09) 0.9 (60.1) 0.85 (60.09) 0.59 (60.06) 0.030 (60.003)
4.2 (61.3) 6.19 (60.02) 4.0 (61.3) 3.24 (60.01) 0.2 (60.1)
9.7 (60.3) 57 (623) 9.2 (60.3) 28 (611) 0.45 (60.02)
6.9 (60.1) 3.9 (60.5) 6.3 (60.1) 1.2 (60.1) 0.60 (60.01)
concentrations in their pH series. Differences in
Vmax between our experiments and those of Riedel
and Nelson (1985) are noteworthy (Table 3).
It is interesting to note that the lowest total dSi
concentration used by Riedel and Nelson (1985)
was ø15 mM, similar to our experiment #2, in which
the lowest concentration was ø10 mM of total dSi.
Within our three experiments, experiment #2 gives
the most pronounced differences between the ki-
netic parameters from the two pH series (Table 1).
Given the biphasic kinetics observed for P. tricornu-
tum, the lack of data in the low-concentration range,
where the kinetic curve conforms to the Michaelis–
Menten equation (Fig. 2), may bias the values of the
kinetic parameters from these experiments.
The natural habitat of P. tricornutum may explain
its variable response in our experiments. P. tricor-
nutum has been found in coastal brackish waters and
commonly in small bodies of water (fishery tanks
and rock pools; Rushforth et al. 1988). Those can
be high-pH environments where SiO(OH)23 would
be more abundant and where diatoms may be spe-
cifically adapted to extreme conditions. Further-
more, this diatom has been described as having
three possible morphotypes (with a possible nonsili-
ceous morphology of the cells; Lewin 1958, Lewin
et al. 1958, Gutenbrunner et al. 1994). Silicified
ovate cells represented .99.9% of all cells in each
of our three experiments, but the unusual plastic
morphology of P. tricornutum may be part of the ex-
planation for its unusual uptake kinetics.
Lomas and Glibert (1999) recently reported sim-
ilar biphasic uptake kinetics for nitrate use by nat-
ural diatom populations. However, they interpret
that phenomenon as an adaptation to modulate the
flow of photosynthetic electron energy via nitrate re-
duction during periods of imbalance between light
energy harvesting and utilization. Here the unusual
uptake kinetics for P. tricornutum could be related
to the possible presence of two different transport
systems with each induced at different external Si
concentrations. This hypothesis is consistent with re-
cent observations that up to six different Si-transport
molecules may coexist within individual diatom cells
 15298817, 1999, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1529-8817.1999.3561162.x by CONICET Consejo Nacional de Investigaciones, Wiley Online Library on [07/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
SUBSTRATE FOR SI UPTAKE IN DIATOMS 1167

TABLE 1. Extended. TABLE 3. Approximated maximum values of Si uptake rates (V,

in fmol·cell21·h21) for P. tricornutum at 60 and 160 mM of total
dSi, the highest concentrations used by Riedel and Nelson (1985)
in the high and low pH series, respectively. Values are deduced
S 5 SiO(OH)23 S 5 SiO2(OH)222 from figure 1A in Riedel and Nelson (1985). Values in parenthe-
Km (mM) Km (nM) ses are Vmax values.
High pH Low pH High pH
Riedel
1.1 (60.2) 0.0011 (60.0001) 0.8 (60.1) and
4.0 (60.5) 0.004 (60.001) 3.0 (60.4) Nelson
0.35 (60.04) 0.0008 (60.0001) 0.16 (60.02) pH Experiment 1 Experiment 2 Experiment 3 (1985)

Low, V160 0.35 (0.38) 0.45 (0.50) 7.5 (15.84) 1.5

2.95 (60.01) 0.006 (60.002) 1.995 (60.005) High, V60 0.65 (1.29) 0.45 (1.07) 6 (31.81) 1.1
29 (612) 0.0164 (60.0006) 23 (69)

2.7 (60.3) 0.0425 (60.0009)
(Hildebrand et al. 1997, 1998) and observation of
Si-starved cells of C. fusiformis, which showed similar
nonsaturated Si-uptake rates at high substrate con-
centrations (Del Amo et al., in prep.).
Those results strongly suggest that the absence of
low-concentration data points, the high variability in
the response of this species, and the peculiarity of
the kinetic curves obtained for P. tricornutum may
have biased conclusions drawn from past experi-
ments using this species.
Substrate for Si uptake. Riedel and Nelson (1985)
suggested that SiO(OH)23 rather than Si(OH)4 is the
chemical form transported across the membrane
during Si uptake by marine diatoms. This suggestion
was drawn from the near equality of Km values ob-
tained for P. tricornutum in kinetic experiments at
pH 8.6 and 9.5 when SiO(OH)23 was used as the
presumed substrate for Si uptake. We found that
values of Km for the four diatoms that we examined
were extremely different when Si(OH)23 was used as
the substrate. In contrast, the results for the three
diatom species forming true siliceous frustules sug-
gest that Si(OH)4 is the substrate transported across
the cell membrane, with the caveat that for C. fusi-
formis the data also support the indiscriminant trans-
port of all chemical species of dSi. Transport of the
more abundant, undissociated silicic acid (97% of
all dSi at pH 8.0) is consistent with the idea that
marine diatoms would be adapted to transport the
most readily attainable form of Si in their environ-
ment.
As mentioned previously, it is known that the ki-
netic parameters of enzymes are sensitive to tem-
4.2 (60.5) perature, but they are also sensitive to pH (Vega-
Catalan 1990). The enzyme and the substrate exist
in many ionizable states, depending on the pH and
the Michaelis parameters (Km and Vmax), and thus
uptake rates may vary with hydrogen ion concentra-
tion. The protonation or deprotonation of the active
sites is thought to be responsible for the modifica-
tion in uptake rate with pH (Vega-Catalan 1990).
Indeed, Sullivan (1976) showed that Si-uptake rates
for Navicula pelliculosa are influenced by pH, with
the optimum at pH 8.5. However, in nature, en-
zymes are generally confined to a narrow pH range.
Thus, their activity is usually interpreted in terms of
simple kinetic models, where the Michaelis param-
eters are by definition constant and thus indepen-
dent of pH. The entire pH range for all the exper-
iments was from 7.98 to 9.80, but in a single exper-
iment the pH difference was always ,1.5 units pH
and always around the natural pH of seawater.
Whether the Si-transport system of the diatoms was
modified with that pH change is not known. How-
ever, because of the small pH difference imposed in
these experiments, we assume that the Km value was
constant.
On the basis of our results, we constructed a new
formulation of the Michaelis–Menten function. As-
suming that Si(OH)4 is the substrate that binds to
the Si-transport protein (E) and is moved across the
cell wall, we can write the following:
k1 k3
E 1 Si(OH)4 o E 2 Si(OH)4 → E 1 Si 2 P (4)
k2
where Si-P is the intracellular form of Si and k1, k2,
and k3 are the rate constants for each reaction. Such
a model follows a Michaelis–Menten law where the
observed Km is a dimensionless quantity analogous

TABLE 2. t-values from two-sample t-tests for the equality of half-saturation constants obtained at the two pH values for each of the four
diatom species studied. df 5 degrees of freedom.

Diatom species S 5 total dSi S 5 Si(OH)4 S 5 SiO(OH)23 S 5 SiO2(OH)222

T. pseudonana (df 5 17) 2.19 1.18a 6.07 6.32

T. weissflogii (df 5 18) 2.95 0.66a 7.45 7.76
C. fusiformis (df 5 18) 0.45a 2.55 9.04 9.86
P. tricornutum, experiment 1 (df 5 8) 1.63a 0.68a 53.00 432.62
P. tricornutum, experiment 2 (df 5 9) 2.36 1.91a 2.80 2.84
P. tricornutum, experiment 3 (df 5 9) 6.89 28.01 7.35 9.41
a Cases in which the null hypothesis of equal Km values at the two pH values cannot be rejected at the 95% confidence level.

1168 YOLANDA DEL AMO AND MARK BRZEZINSKI
to the Michaelis–Menten constant and is by defini-
tion independent of pH:
[Si(OH)4 ]
V 5 Vmax · (5)
K m 1 [Si(OH)4 ]
By using the equilibrium relation between the Si-
species concentrations and the dissociation con-
stants of Sjöberg et al. (1981), we rearranged this
equation in terms of [SiO(OH)23 ]. This gives a new
expression that has an apparent half-saturation con-
centration that equals ‘‘1029.47·Km/[H1]’’ and de-
creases with decreasing pH:
[SiO(OH)23 ]
V 5 Vmax · 29.47
(6)
10 · Km
1
1 [SiO(OH)23 ]
[H ]
The observed Km values in this study follow this pre-
dicted trend, contrary to those of Riedel and Nelson
(1985).
An alternative model was proposed by Riedel and
Nelson (1985) on the basis of the formation of a
complex between the Si-transport protein and
SiO(OH)23 :
k1
E 1 Si(OH)4 o E 2 SiO(OH)23 1 [H]1
k2
k3
→ E 1 Si 2 P (7)
This model follows a rate law by which the experi-
mental Km obtained when Si(OH)4 is considered as
the substrate equals the product ‘‘Km·[H1],’’ which
must decrease with increasing pH:
[Si(OH)4 ]
V 5 Vmax · (8)
K m · [H1 ] 1 [Si(OH)4 ]
The rearrangement of this equation in terms of
[SiO(OH)23 ] leads to a half-saturation constant for
SiO(OH)23 that does not vary with pH, a trend that
is contrary to our findings:
[SiO(OH)23 ]
V 5 Vmax · (9)
1029.47 · K m 1 [SiO(OH)23 ]
Neither our model nor that of Riedel and Nelson
(1985) accounts for the possible different ionizable
states of the enzymes with pH. Both models also pre-
dict unchanging values for Vmax, although we clearly
observe a noteworthy change in Vmax in our experi-
ments. At present the pKs for Si transporters in dia-
toms are unknown, precluding a more detailed anal-
ysis. In all but one experiment, Vmax is 25% to 69%
lower at pH ø 9.5 than at pH ø 8. Hayward (1968)
showed that the growth rate (cell number increase)
of P. tricornutum is insensitive to pH changes between
8 and 9 but decreases at pHs $ 9.5. Given that the
habitat of most marine diatoms is very near pH 8.2,
it is not surprising that the uptake systems of these
cells exhibited their highest affinity (higher Vmax and
lower Km) at the lower pH tested (pH ø 8).
15298817, 1999, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1046/j.1529-8817.1999.3561162.x by CONICET Consejo Nacional de Investigaciones, Wiley Online Library on [07/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Biological transport of noncharged molecules
through membranes is possible by diffusion when
small polar or hydrophobic molecules are involved.
For large uncharged molecules, such as silicic acid,
the cells have developed specific transporters. For
example, glucose is taken up by active transport (en-
ergy-dependent pump) processes in some bacteria
(e.g. Pseudomonas aeruginosa) and by phosphotrans-
ferase system in others (e.g. Escherichia coli). Trans-
port of other uncharged molecules, such as sugars
or amino acids, can also be linked to electrical
charge differences: the symporter transports both
the substrate and one or more protons. It is known
that diatoms possess an inducible active transport
system for Si transport (Azam and Volcani 1974,
Azam et al. 1974) that requires Na1 in the diatom
Nitzschia alba (Bhattacharyya and Volcani 1980).
These authors also showed that symport is electro-
genic, that is, that a net charge is transported across
the membrane. Because Na1 is already positively
charged, these findings support the idea that undis-
sociated silicic acid is the chemical form of Si trans-
ported.
Recently, Hildebrand et al. (1997, 1998) isolated
and sequenced a family of silicon transporter (SIT)
genes in C. fusiformis. Multiple SIT genes are present
in all diatoms examined to date, including T. pseu-
donana and P. tricornutum, and the transporters re-
quire Na1 to function (Hildebrand et al. 1997), sug-
gesting that diatoms have developed silicon trans-
porters that cotransport Na1 and Si(OH)4 (Bhatta-
charyya and Volcani 1983, Hildebrand et al. 1997,
1998).
Ecological consequences. The use of one of the less
abundant forms of dSi in seawater by diatoms, such
as SiO(OH)23 , which comprises only 3% of total dSi
at natural seawater pH of ø8.0, would have strong
implications for diatom physiology and phytoplank-
ton ecology. To sustain a given growth rate, cells
utilizing a more dilute substrate would have to be
more kinetically efficient to obtain Si at the same
rate as those using a more abundant chemical form
of the element. Furthermore, the hypothesis of the
sole utilization of SiO(OH)23 as the substrate for Si
uptake in diatoms implies serious biases in previous
uptake kinetics found in literature that typically as-
sume that diatoms indiscriminately take up all forms
of silicic acid as well as in their related conclusions
on Si limitation in their natural environment.
Indeed, Km values are often used as indicators of
nutrient limitation by comparing them to in situ nu-
trient concentrations (e.g. Fisher et al. 1988, Dortch
and Whitledge 1992, Del Amo et al. 1997). In situ
nutrient concentrations below Km correspond to nu-
tritional conditions that greatly reduce nutrient up-
take rates and potentially limit algal growth rates.
Thus, erroneous Km values and substrate concentra-
tions obviously lead to inappropriate conclusions on
Si limitation.
However, our results suggest that marine diatoms

SUBSTRATE FOR SI UPTAKE IN DIATOMS
are utilizing Si(OH)4, which is the most abundant
form of Si in seawater of natural pH. In the global
ocean, the pH of seawater is buffered between 7.5
and 8.5 by the CO2–carbonate system (Millero
1996), causing the percentage of total dSi present
as Si(OH)4 to vary from 98.9% to 90.3%. However,
in freshwater lakes when pH values reach up to 10,
available Si(OH)4 is only ø23% of total dissolved
silicon, the majority (77%) being present as
SiO(OH)23 . If freshwater diatoms also selectively
transport Si(OH)4, then changes in the availability
of this chemical form with pH may need to be con-
sidered when assessing substrate limitation of Si up-
take in freshwaters.
The authors thank R. Shipe and V. Franck for their assistance in
the laboratory, Robert Petty for nitrate 1 nitrite and phosphate
analysis, and M. Hildebrand for valuable discussions during man-
uscript preparation. We also thank two anonymous reviewers for
their helpful comments. This work was supported by the U.S.
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