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J. Phycol.
36, 821840 (2000)
REVIEW
SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH 1

Vronique Martin-Jzquel 2
Universit de Bretagne Occidentale, UMR 6539, CNRS, Institut Universitaire Europen de la Mer, Technopole Brest-Iroise,
Place Nicolas Copernic, F-29280 Plouzane, France
Mark Hildebrand 2,3

Marine Biology Research Division, Mail Code 0202, Scripps Institution of Oceanography, University of California,
San Diego, 9500 Gilman Dr., La Jolla, California 92093-0202
and
Mark A. Brzezinski
Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, California 93106
Diatoms are the worlds largest contributors to biosilicification and are one of the predominant contributors to global carbon fixation. Silicon is a major limiting
nutrient for diatom growth and hence is a controlling
factor in primary productivity. Because our understanding of the cellular metabolism of silicon is limited, we
are not fully knowledgeable about intracellular factors
that may affect diatom productivity in the oceans. The
goal of this review is to present an overview of silicon
metabolism in diatoms and to identify areas for future
research.
Numerous studies have characterized parameters of
silicic acid uptake by diatoms, and molecular characterization of transport has begun with the isolation of
genes encoding the transporter proteins. Multiple types
of silicic acid transporter gene have been identified in a
single diatom species, and multiple types appear to be
present in all diatom species. The controlled expression and perhaps localization of the transporters in the
cell may be factors in the overall regulation of silicic
acid uptake. Transport can also be regulated by the rate
of silica incorporation into the cell wall, suggesting that
an intracellular sensing and control mechanism couples
transport with incorporation. Sizable intracellular pools
of soluble silicon have been identified in diatoms, at
levels well above saturation for silica solubility, yet the
mechanism for maintenance of supersaturated levels
has not been determined. The mechanism of intracellular transport of silicon is also unknown, but this must be
an important part of the silicification process because
of the close coupling between silica incorporation and
uptake. Although detailed ultrastructural analyses of silica deposition have been reported, we know little about
the molecular details of this process. However, proteins
occluded within silica that promote silicification in vitro
have recently been characterized, and the application

of molecular techniques holds the promise of great advances in this area. Cellular energy for silicification and
transport comes from aerobic respiration without any
direct involvement of photosynthetic energy. As such,
diatom silicon metabolism differs from that of other
major limiting nutrients such as nitrogen and phosphorous, which are closely linked to photosynthetic metabolism. Cell wall silicification and silicic acid transport
are tightly coupled to the cell cycle, which results in a
dependency in the extent of silicification on growth
rate. Silica dissolution is an important part of diatom
cellular silicon metabolism, because dissolution must
be prevented in the living cell, and because much of the
raw material for mineralization in natural assemblages
is supplied by dissolution of dead cells. Perhaps part of
the reason for the ecological success of diatoms is due
to their use of a silicified cell wall, which has been calculated to impart a substantial energy savings to organisms that have them. However, the growth of diatoms
and other siliceous organisms has depleted the oceans
of silicon, such that silicon availability is now a major
factor in the control of primary productivity. Much new
progress in understanding silicon metabolism in diatoms is expected because of the application of molecular approaches and sophisticated analytical techniques.
Such insight is likely to lead to a greater understanding
of the role of silicon in controlling diatom growth, and
hence primary productivity, and of the mechanisms involved in the formation of the intricate silicified structures of the diatom cell wall.
Key index words: biomineralization; cell cycle; cell
wall synthesis; diatoms; primary production; silicic
acid transport; silicification; silicon metabolism
Abbreviations: DSi, dissolved silicon; Ge, germanium; SDV, silica deposition vesicle; SIT, silicic acid
transporter
1 Received
7 February 2000. Accepted 23 May 2000.

have contributed equally to the manuscript.
3 Author for correspondence: e-mail mhildebrand@ucsd.edu.
2 Authors
821
822
VRONIQUE MARTIN-JZQUEL ET AL.
From unicellular algae to vascular plants, numerous

organisms are found to produce siliceous structures,
such as internal or external skeletons, scales, or secondary walls (Simpson and Volcani 1981). Diatoms
(Fig. 1) are the predominant siliceous organisms in the
marine environment and are a major component of
the phytoplankton community. The number of recognized diatom species is 10,00012,000 (Werner 1977,
Gordon and Drum 1994, Norton et al. 1996), and the
total number may be many times greater than that
(Norton et al. 1996). It is estimated that diatoms account for 40% of the total marine primary production of
carbon (Nelson et al. 1995). The cell walls of diatoms are
silicified (Fig. 1), forming a structure called a frustule
containing hydrated amorphous silica with the general
formula [Sin O2n(nx/2) (OH)nx], with x 4. Amorphous
silica is an essential component of the diatom cell
wall, causing Si availability to be a key factor in the
regulation of diatom growth in nature (e.g. Paasche
1980, Ragueneau et al. in press). In turn, the use of silicon by diatoms dominates the biogeochemical cycling
of Si in the sea, with each atom of Si weathered from
land passing through a diatom an average of 39 times
before burial in the sea bed (Trguer et al. 1995).
In the diatom, the intricate siliceous structures in
the cell wall (Fig. 1) are reproduced with fidelity for
each generation, creating unique morphotypes that

are used as taxonomic keys. Pickett-Heaps et al. (1990),
Pickett-Heaps (1991), and Gordon and Drum (1994)
reviewed numerous studies on silica morphogenesis
and valve formation during cell division. Studies on the
biochemistry and molecular biology of diatom silicon
metabolism are not so extensive, and the biochemical
pathways involved, and their regulation, are not well
understood (Sullivan and Volcani 1981, Volcani 1981,
Sullivan 1986). The overall process of silicification involves transport of silicon across the plasmalemma and
then through the cytoplasm to the site of polymerization within the silica deposition vesicle (SDV). The cell
must transport enough silicon to make the wall yet prevent uncontrolled autopolymerization from occurring
in the cytoplasm. Hence, cellular silicon metabolic processes must be under tight physiological control.
We present an overview of diatom silicon metabolism
and its effect on the growth and ecology of diatoms. We
focus on physiological constraints influencing the ability
of diatoms to acquire dissolved Si over the 1- to 70-M
average concentration range of silicic acid that diatoms
experience in the ocean, on the regulation of intracellular Si pools, and on the polymerization process. The relationship between silicon metabolism and other aspects
of cellular function, such as photosynthesis, respiration,
and the cell cycle, will be explored.
uptake of dissolved si
Lewin (1954, 1955) was the first to demonstrate
that diatoms could take up silicon. Paasche (1973a, b)
and Azam et al. (1974) first demonstrated active silicon transport. Since then, numerous studies have
shown that the specific rate of Si uptake (V ) and the
specific Si-dependent cell-division rate () follow
Michaelis-Menten (eq. 1) or Monod (1942) (eq. 2)
saturation functions:
Fig. 1. Scanning electron micrographs of (top) an intact

cell of Thalassiosira weisflogii and (bottom) the acid-cleaned
valve top of an unidentified Thalassiosira species.
V max [ Si ( OH ) 4 ]
V = ------------------------------------------K s + [ Si ( OH ) 4 ]
(1)
max [ Si ( OH ) 4 ]
= ------------------------------------------K + [ Si ( OH ) 4 ]
(2)
where Vmax and max represent the maximum rates of

uptake and division, respectively, at infinite substrate
concentration; Ks is the Si(OH)4 concentration at 0.5
Vmax; and K is the the Si(OH)4 concentration that
limits to 0.5 max (Goering et al. 1973, Guillard et al.
1973, Harrison et al. 1976, Nelson et al. 1976, Davis et
al. 1978).
The kinetic parameters Ks, K, Vmax, and max for silicic acid uptake and Si-dependent growth have been
measured in many diatom species (Table 1). Typically, the half saturation constant for growth, K, is
less than that for uptake, Ks (Table 1), and diatoms
can maintain division rates very close to max at extracellular Si(OH)4 concentrations that limit Si uptake
(Paasche 1973a, Brzezinski et al. 1990, Nelson and
823
DIATOM SILICON METABOLISM

Table 1.
Saturation (Vmax, max) and half-saturation (Ks, K) constants for silicic acid uptake and Si-limited growth in diatoms.
Species
Ks (m)
K (m)
Vmax (h1)
max (d1)
Asterionnella formosa
Ohrid clone
1.93
1.11
Windermer clone
1.09
0.61
Frains Lake clone
3.94
1.06
35.8b
7.70
Asterionnella ralfsii
0.478.32
Chaetoceros debilis
2.2
Chaetoceros gracilis
Cyclotella meneghiniana
1.1
3.3
0.47
3.21
15.1b
7.6
Cyclotella sp.
Ditylum brightwelli
0.18
1.44
1.33
8.3
9.3
26.6c
2.96
Hantzschia sp.
1.1
2.3
Leptocylindrus danicus
0.330.74
2.55
Licmophora sp.
2.58
2.15c
Nitzschia alba
4.5
3.35d
62
4.1e
Navicula pelliculosa
4.4
334f
Nitzschia angularis
4.2
560f
Phaeodactylum
tricormutum
Skeletonema costatum
97.4
4.25b
11.854.8
1.21.5b
0.8
0.095c
Culture conditionsa
Batch culture
L/D: 14/10, 20 C
1.5610.6 M
Batch culture
L/D: 14/10, 20 C
1.611 M
Batch culture
L/D: 14/10, 20 C
0.422 M
Batch culture
L/D: 14/10, 20 C
0.530 M
Continuous culture
Aluminum
Continuous light, 18 C
020 M
Continuous culture
Perturbation
L/D: 16/8, 18 C
05 M
Batch culture
L/D: 12/12, 25 C
050 M
Batch culture
Continuous light
22 C, 6 M
Batch culture
L/D: 14/10, 20 C
0.530 M
Batch culture
L/D: 14/10, 20 C
0.422 M
Batch culture
L/D: 12/12, 25 C
050 M
Batch culture
L/D: 18/6, 20 C
015 M
Batch culture
L/D: 12/12, 25 C
050 M
Batch culture
Continuous light
22 C, 0.14 M
Batch culture
Continuous light
20 C, 015 M
31Si, 30 C
0.2% glucose
31Si, 30 C
Vesicular uptake
68Ge, batch culture
Synchronized
Continuous light
20 C, 0100 M
68Ge, batch culture
Synchronized
Continuous light
20 C, 0100 M
Batch culture
Continuous light
20 C, 0500 M
Batch culture, pH
Continuous light
20 C, 0200 M
Batch culture
Continuous light
20 C, 015 M
References
Kilham 1975
Kilham 1975
Tilman and
Kilham 1976
Tilman and
Kilham 1976
Gensemer et al.
1993
Conway and
Harrison 1977
Taguchi et al. 1987
Thomas and
Dodson 1975
Tilman and
Kilham 1976
Tilman and
Kilham 1976
Taguchi et al. 1987
Paasche 1973b
Taguchi et al. 1987
Thomas and
Dodson 1975
Paasche 1973b
Azam et al. 1974
Bhattacharyya and
Volcani 1980
Sullivan 1976
Blank and
Sullivan 1979
Nelson et al. 1984
Riedel and
Nelson 1985
Paasche 1973b
(continued)
824
Table 1.

Continued
Species
Ks (m)
K (m)
Vmax (h1)
1.11.8
0.0880.074
0.7
0.12
1.3
0.1
0.481.36
Thalassiosira pseudonana
Clone 3H (estuarine)
2.442.77
0.073c
1.39
0.98
0.82.3
3.6
0.0620.092
0.19
Clone 13
(Sargasso Sea)
1.41.5
Thalassiosira
nordenskioldii
max (d1)
2.1
0.0310.028
0.04
2.75
8.6
2.75
0.022
1.26
0.088
0.89
Thalassiosira decipiens
2.27
4.09c
Thalassiosira gravida
0.2
0.02
Culture conditionsa
Continuous culture
Continuous light
18 C, 010 M
Continuous culture
Perturbation
L/D: 16/8, 18 C
05 M
Continuous culture
Perturbation
L/D: 16/8, 18 C
05 M
Continuous culture
Continuous light
20 C, 014 M
Batch culture
Continuous light
20 C, 015 M
Batch culture
L/D: 14/10, 20 C
0.3216.5 M Si
30Si, Batch culture
L/D: 14/10, 20 C
0.2515 M
Batch culture
L/D: 14/10, 20 C
0.3216.5 M Si
30Si, Batch culture
L/D: 14/10, 20 C
0.2515 M
Batch culture
Continuous light
Marine medium
20 C, 010 M
Batch culture
Continuous light
Freshwater medium
20 C, 0100 M
Batch culture
L/D: 9/15, 10 C
030 M
Batch culture
L/D: 9/15, 3 C
030 M
Batch culture
Continuous light
20 C, 015 M
Continuous culture
Perturbation
L/D: 16/8, 18 C
05 M
References
Davis 1976
Conway et al. 1976
Conway and
Harrison 1977
Paasche 1973a
Paasche 1973b
Guillard et al. 1973
Nelson et al. 1976
Guillard et al. 1973
Nelson et al. 1976
Olsen and
Paasche 1986
Olsen and
Paasche 1986
Paasche 1975
Paasche 1975
Paasche 1973b
Conway and
Harrison 1977
aL/D is light/dark followed by the time in each. Temperature and range of DSi in the culture are also presented. Other variations
are noted; please see reference for complete details.
bmolcell1h1
cpgcell1h1
dmolg wet wt1min1
enmolmg protein1min1
fpmol106 cell1min1
Dortch 1996). Average Ks values tend to be lower than

the dissolved silicon (DSi) concentration in nutrientrich environments in the ocean (e.g. Nelson and
Trguer 1992, Brzezinski et al. 1997) but often exceed
ambient silicic acid concentrations in oligotrophic
gyres (Brzezinski and Nelson 1996, Brzezinski et al.
1998). Comparison of the maximum uptake rates in
individual cells with the amount of cell wall silica
shows that Si transport and silica deposition are

closely coupled in some species. For example, in synchronized cultures of Navicula pelliculosa, Vmax was
measured at 27.8 fmolcell1h1 (Sullivan 1977) and
cell wall silica at 4275 fmolcell1 (M. Hildebrand,
unpublished data), suggesting that under conditions
of maximum uptake the cell could transport enough
silicic acid to make the wall in 1.52.7 h. This is consis-
tent with the observed rate of cell number increase in

synchronized N. pelliculosa (Sullivan 1977), suggesting
that essentially all Si taken up was deposited without
any Si diverted to internal pools for storage.
The identification of a saturable uptake system indicates that Si uptake is carrier mediated (Paasche
1973a, b, Azam et al. 1974). Transport is sodium dependent in marine diatoms (Bhattacharyya and Volcani 1980) and may be sodium and perhaps potassium coupled in freshwater species (Sullivan 1976),
although at much lower ionic strength. Thus, the
transporter in marine species has characteristics of a
sodium/silicic acid symporter (Bhattacharyya and Volcani 1980, Hildebrand et al. 1997). Indirect evidence
suggests that transport is electrogenic and that the
Si(OH)4:Na ratio is 1:1 (Bhattacharyya and Volcani
1980). Metabolic energy is required for uptake (Azam
et al. 1974), and transport is inhibited by sulfhydryl
blocking reagents (Lewin 1954, Sullivan 1976).
The chemical forms of Si available to marine diatoms are undissociated silicic acid, Si(OH)4, which
comprises about 97% of the DSi in seawater at pH 8.0,
and SiO(OH)3, which comprises most of the remaining 3% (Ingri 1978, Stumm and Morgan 1981). Most
diatom species transport undissociated silicic acid
(Del Amo and Brzezinski 1999); however, Riedel and
Nelson (1985) and Del Amo and Brzezinski (1999)
showed that Phaeodactylum tricornutum could also take
up the less abundant anion.
Chemostat studies suggested three different modes
of silicic acid uptake in some diatom species: surge
uptake, internally controlled uptake, and externally
controlled uptake (Conway et al. 1976, Conway and
Harrison 1977) (Fig. 2). Surge uptake occurred upon
replenishment of silicate-starved cells, when intracellular
silicon pools were depleted, and the concentration gradient into the cell was maximum (Fig. 2). Maximum
uptake rates were observed during surge uptake (Conway et al. 1976, Conway and Harrison 1977), and there
is some evidence that the size of the internal soluble
pool after surge uptake was related to the external Si
concentration (Table 2). In internally controlled uptake (Fig. 2), uptake was regulated by the intracellular
utilization of Si, presumably the rate of silica deposition into the cell wall (Conway et al. 1976, Conway and
Harrison 1977). There is a coupling of uptake to deposition (M. Hildebrand, unpublished data); however, it
should be noted that this does not occur in all diatom
species (Chisholm et al. 1978). In externally controlled
uptake (Fig. 2), extracellular concentrations are at
very low levels, and rates become a function of decreasing substrate concentration. Because Vmax changes
under different modes of uptake and the timing of
these changes differs depending on the species and
external conditions, Harrison et al. (1989) introduced
the concept of presenting Vmax values with a superscript
denoting different time periods, such as Vm0-1, Vm1-5,
etc., to compensate for changes in Vmax with time.
Molecular characterization of the silicic acid transport system began with the first cloning and func-
825
Fig. 2. Diagram of three modes of silicic acid uptake by diatoms, after Conway et al. (1976) and Conway and Harrison
(1977). The three largest boxes represent the external environment, and the large rectangular object inside each box represents a diatom cell. The oblong feature at the top inside of the
cell is the silica deposition vesicle (SDV). Arrow thickness denotes the relative extent of transport of silicic acid. Shading inside and outside the cell denotes the relative presence of silicic
acid. (Top) Conditions when surge uptake occurs, when intracellular Si levels are low and maximum uptake from the environment occurs. (Middle) Internally controlled uptake, when
intracellular Si levels are replete and uptake is controlled by the
rate of silica incorporation into the cell wall. Zig-zag lines denote a feedback mechanism coupling uptake to incorporation
in which the cell senses the rate of incorporation and controls
uptake accordingly. (Bottom) Externally controlled uptake,
when extracellular levels are low and uptake is controlled by decreasing extracellular Si levels.
tional identification of cDNAs encoding a silicic acid

transporter from Cylindrotheca fusiformis (Hildebrand
et al. 1997). RNA transcribed in vitro from a fulllength cDNA clone injected into oocytes of the frog,
Xenopus laevis, enabled the uptake of 68Ge (a radiotracer analog of Si) from the medium. Competition experiments using unlabeled silicic acid demonstrated the specificity of the cloned transporter.
Transport by transformed oocytes was sodium dependent and sensitive to the sulfhydryl blocker N-ethyl
maleimide (Hildebrand et al. 1997, and unpublished
data) as observed in diatoms (Sullivan 1976, Bhattacharyya and Volcani 1980). The cDNAs encoded an integral membrane protein originally proposed to have
826
Table 2.

Intracellular soluble silicon pools in diatoms, measured by 31Si, 68Ge, or molybdate.
Species
Analysis
Cylindrotheca fusiformis
Chaetoceros gracilis
Coscinodiscus granii
Ditylum brightwellii
Nitzschia alba
Molyb.
68Ge
Molyb.
68Ge
31Si
Molyb.
68Ge
Navicula pelliculosa
Stephanopyxis turris
Thalassiosira weissflogii
Thalassiosira weissflogii
aRecalculated
68Ge
Molyb.
Molyb.
Molyb.
Molyb.
Molyb.
Molyb.
Molyb.
Culture conditions
Exponential to Si limited
Exponential
Exponential
Exponential
Si limited
Synchrony
Si starvation
Cell wall synthesis
Exponential
Exponential limited
Exponential enriched
Surge uptake
Surge uptake (20 M)
Surge uptake (40 M)
Surge uptake (80 M)
Continuous culture NO3
limited
Soluble pool
(fmolcell1)
% total
Reference
2.17.9
1.6
10006000
12.3 (pmol/cell)
1530a
5.615.2
3.612.4
4.5
18
50
5.2
1.02.1
Hildebrand, unpublished data

Chisholm et al. 1978
Taylor 1985
Chisholm et al. 1978
Azam et al. 1974
Sullivan 1979
11.1
28
6.8
1.63.4
1.67.0
10
30
520
100
16
40
13
2.54.8
5.027
1
5
35
20
150
500
0.94.6
27
56
0.53.5

Binder and Chisholm 1980
Martin-Jzquel, unpublished
data
Claquin 1999
using direct measurements of intracellular water volume (M. Hildebrand, unpublished data).
12 membrane-spanning segments, but recent analysis

suggested that 10 were present (Hildebrand 2000).
The transporter protein was also predicted to have a
long hydrophilic carboxy-terminal domain.
Five different types of silicic acid transporter (SIT)
genes have now been isolated and characterized in C.
fusiformis, which likely represent all of them (Hildebrand et al. 1998). The SITs have no known homologs
and thus represent a new class of transporter, although a portion of a signature sequence for sodium
symporters (Deguchi et al. 1990, Reizer et al. 1994)
was present (Hildebrand 2000). The SITs were highly
conserved in their transmembrane domains and less
conserved at the carboxy-terminus. The carboxy-terminus had a high probability to form a coiled-coil structure, suggesting that the transporters interacted with
other proteins or other copies of themselves. The overall pattern of SIT gene mRNA accumulation during
cell wall synthesis in C. fusiformis correlated with silicic
acid transport activity as determined in N. pelliculosa
(Sullivan 1977), reaching high levels quickly just before
maximum silica deposition and then decreasing rapidly. The pattern of mRNA accumulation of four genes
(SIT25) was nearly identical, being induced at 2 h,
reaching maximum levels by 2:20, and then decreasing to postinduction levels by 3:20 (Hildebrand et al.
1998). For SIT1 the pattern differed; although induction occurred at 2 h, mRNA abundance did not peak
but leveled off until 3 h, and then decreased less than
the other SITs by 3:20. Levels of mRNA accumulation
of the five SITs varied substantially. SIT4 was the most
highly expressed, with levels 24-fold higher than SIT3
and 5, which were the least abundant. These data indicated that the different forms of transporter were
present in different amounts during cell wall synthesis, suggesting specific roles in the overall process of
transport. Hybridization experiments showed that

multiple SIT gene copies were present in all diatom
species tested, which included marine, fresh water,
pennate, and centric species (Hildebrand et al. 1998).
A working hypothesis is that in a given diatom species,
the different SIT genes encode transporters with different transport characteristics or intracellular locations (Fig. 3). Thus, the regulated synthesis and localization of the SITs may be important in the overall
control of silicic acid transport in diatoms.
The discovery of multiple transport proteins and
their different levels of expression in a single diatom
cell is consistent with earlier work by Sullivan (1977),
who showed that Ks and Vmax values for uptake varied
during the course of cell wall synthesis. In N. pelliculosa, uptake kinetics indicated a low affinity (high Ks)
and low capacity (low Vmax) for Si uptake before frustule deposition and a high affinity (low Ks) high capacity (high Vmax) during deposition (Sullivan 1977).
The decrease in Ks could be due to the synthesis of
higher affinity transporters and the increase in Vmax
from an increase in the number or efficiency of transporters in the plasma membrane (Fig. 3) (Sullivan
and Volcani 1981, Sullivan 1986). Consistent with this
latter point, levels of overall SIT mRNA expression in
C. fusiformis were induced 4-fold just before maximum
cell wall synthesis (Hildebrand et al. 1998).
The relationship of Si transport with metal ions has
been investigated but is not well understood. An interaction between zinc and copper affects diatom silicic
acid uptake (Rueter and Morel 1981), but it is unclear
whether this directly affects the transporter. Changes
in the kinetics of silicic acid uptake due to Fe2/3 and
Zn2 stress have been observed in culture (De La Rocha
et al. 2000) and in natural diatom assemblages (V. Franck
et al., University of California, unpublished data). Re-
Fig. 3. Model for changes in silicic acid transporter (SIT)

location and composition before and during diatom cell wall
synthesis. Small dots represent silicic acid. (Top) Diatom cell
before extensive silicification; only a low affinity, low capacity
type of SIT is localized at the plasma membrane. (Bottom) Cell
during silicification; higher affinity and capacity forms of SITs
and greater numbers are present in the plasma membrane.
Two possible localization sites of intracellular forms of SITs are
shown, in a putative silicon transport vesicle (middle left) and
in the silicon deposition vesicle (upper).
sults from laboratory and field studies show consistent

effects. Both Fe2/3 and Zn2 stressed cells have maximum uptake rates 2- to 3-fold lower than nutrient replete cells (De La Rocha et al. 2000). Whether this response reflects a direct role of these metal ions in Si
metabolism or an indirect response to the effect of
these metal ions on other aspects of cellular function
is unclear. Interestingly, low Fe2/3 does not change
the half saturation constant for Si uptake significantly,
whereas Ks increases significantly under low Zn2, suggesting a more direct role for Zn2 than Fe2/3 in diatom Si uptake (De La Rocha et al. 2000).
intracellular pools of silicon
Werner (1966) obtained the first evidence of soluble intracellular pools of silicon in diatoms. Since
then, studies have shown that intracellular pools can
account for a sizable fraction of the total cellular silicon in some species under certain conditions (Table
2). The actual concentration of internal Si depends
on measurement of intracellular water volume, which
has resulted in some discrepancies. Therefore, these
values are not presented in Table 2. Sullivan (1979)
reported pools (assuming monosilicic acid) between
438 and 680 mM in N. pelliculosa. However, cell water
(by weight) was estimated as 20%, and recent measurements suggested a value of 85% for this species
(M. Hildebrand, unpublished data). By adjusting Sullivans data accordingly and also by new direct measure-
827
ments of cell water volume (M. Hildebrand, unpublished

data), intracellular concentrations ranged between 58
and 162 mM. In other species where cell water volume
was directly measured, pool concentrations (assuming
monosilicic acid) ranged from 19 mM in C. fusiformis
to 340 mM in Stephanopyxis turris (M. Hildebrand, unpublished data). These values are well above saturation for silica solubility, which is around 2 mM at pHs
below 9 (Iler 1979). All results (Sullivan 1977, Chisholm et al. 1978, Binder and Chisholm 1980) indicate
that diatoms maintain supersaturated concentrations
of intracellular silicon in a soluble form.
Azam et al. (1974) and Sullivan (1979) questioned
how such high concentrations could be maintained
and suggested that this was due to partially polymerized or colloidal silica, or organosilicon compounds. A
very high concentration gradient from inside to outside the cell has been measured (Sullivan 1979), which
is consistent with intracellular Si having a different
chemical form than extracellular. Partially polymerized forms of intracellular silicon would still require
some type of stabilization at the high concentrations
measured, and colloidal silica would disrupt cellular
membranes (Iler 1979). Binder and Chisholms (1980)
measurement of molybdate-reactive silicon from sonicated Thalassiosira weissflogii cells suggested that the
predominant form was monosilicic acid. The results of
Blank et al. (1986) indicated that most intracellular Si
was molybdate reactive in the first hour after silicate addition to starved Navicula saprophilia, but from 1 to 6 h
the fraction of molybdate-reactive Si decreased by an
unspecified amount. If most intracellular Si was monomeric silicic acid, then cellular components must be
responsible for maintaining it at supersaturated concentrations. Werner (1966) obtained data suggesting
an association of silicon with organic material or proteins. This was confirmed by Azam et al. (1974), who
found that 80% of the intracellular pool of dissolved Si
in Nitzschia alba was precipitable with trichloroacetic
acid. Thus, it is possible that pool silicon is maintained
in a soluble form by an interaction with organic silicon-binding components. This would be consistent
with the intra- and extracellular chemical forms being
different. Another proposed mechanism was that silicic acid was sequestered in specialized vesicles with
intralumenal conditions able to maintain solubility
(Schmid and Schulz 1979, Sullivan 1986). However,
there was no direct evidence that these vesicles contained silicon, either in soluble or polymerized form.
In summary, it has not been unambiguously determined what form of Si is present in the pools, but it is
probably safe to say that it consists of mono- or perhaps lower molecular weight poly-silicic acid complexed with organic material (Werner 1966, Azam et
al. 1974, Sullivan 1977, Bhattacharyya and Volcani
1983), with an unsubstantiated possibility that it is sequestered in membrane-bound vesicles (Schmid and
Schulz 1979).
All diatom species maintain a range of internal silicon concentrations (Sullivan 1977, Binder and Chis-
828
Table 3a.
Species

Length of the stages in the cell cycle in diatoms under exponential growth conditions.
Gen.
time (h)
Growth conditions
P. tricormutum Exponential
19 C, 100 molm2s1
C. fusiformis
Exponential
19 C, 100 m2s1
T. weissflogii
Exponential
19 C, 100 m2s1
T. pseudonana Exponential
19 C, 100 m2s1
C. muellerii
Exponential
19 C, 100 molm2s1
C. simplex
Exponential
19 C, 100 molm2s1
T. weissfloggi Exponential
20 C, 100 molm2s1
Cell-cycle stage
duration (h)
G2M
Reference
9.4 1.4 7.6 0.3
7.9
50.3
7.5 40.6
1.6
42.2
Brzezinski et al. 1990
16.2
9.4 1.1 4.0 1.7
5.7
58
6.8 24.7 10.5
35.2
13.3
4.5 3.6 2.1 3.1
5.2
33.8 27.1 15.9 23.3
39.1
9.8
6.0 0.3 3.1 0.4
3.5
61.2
3.1 31.6
4.1
35.7
16.6
12.9 0.9 1.9 0.5
2.4
79.6
5.6 11.7
3.1
14.8
9.6
7.8 0.8 0.3 0.7
1.0
81.3
8.3
7.3
10.4
8.1
2.1 2.7
3.4
26
41
Vaulot et al. 1987
G2
holm 1980), indicating that pools can expand or contract. In C. fusiformis, pools increased in a regular way
during the course of cell wall synthesis (M. Hildebrand, unpublished data). Pool sizes differ in different species, which could be due to the relative timing
of silicic acid uptake and silica incorporation into the
frustule (Chisholm et al. 1978). In Chaetoceros gracilis
and other species, uptake and deposition were nearly
simultaneous, resulting in a small soluble pool of Si,
but in Ditylum brightwellii, uptake and deposition were
temporally uncoupled, allowing the accumulation of
soluble pools between 70 mM and 2 M (Chisholm et al.
1978). After giving Si-starved T. weissflogii a pulse of silicic acid, the amount of pool silicon was sufficient for
the later synthesis of an entire cell wall (Binder and
Chisholm 1980, Brzezinski and Conley 1994). Thus,
some diatom species can accumulate large amounts of
soluble silicon, whereas in other diatoms (and perhaps most; see Chisholm et al. 1978) pools are smaller
because uptake occurs only during cell wall synthesis.
The size of internal soluble pools can be also influenced by environmental variables. For example, low
light increased soluble pool size, and silicification, in
Coscinodiscus granii (Taylor 1985) and Thalassiosira pseudonana (P. Claquin et al., Institut Universitaire Europen de la Mer, personal communication). Pool sizes
were also maximum at intermediate external Si con-
Table 3b.
Percent of total cell cycle
18.7
G1
G2M
G1
G2
3.1
33
centrations (15 to 50 M) for a given light intensity

and less at lower or higher Si concentrations (Taylor
1985). Nitrogen or phosphorous limitation resulted in
a smaller pool size in T. pseudonana (P. Claquin et al.,
personal communication). Temperature effects on pool
sizes have been noted, which result from changes in
relative rates of incorporation and uptake (Blank et al.
1986). Growth rate can also affect pool size. Pool sizes
may be expected to vary depending on the cell cycle
stage, because more silicon would be required during
cell wall synthesis in G2 and M. The relative amount of
time a given diatom species spends in different parts of
the cell cycle can vary depending on the growth rate
(Brzezinski et al. 1990, Claquin 1999, P. Claquin et al.,
personal communication) (Tables 3a and 3b). C. fusiformis spends a greater portion of time in G1 during rapid
growth (Brzezinski et al. 1990) (Tables 3a and 3b),
and faster growing populations of cells had smaller average pools (M. Hildebrand, unpublished data), reflecting relatively less time spent in cell wall synthesis.
In diatoms where the timing of uptake and incorporation are coupled, internally-controlled uptake occurs (Fig. 2), and transport is regulated by the rate of
silicification (Conway et al. 1976, Conway and Harrison 1977). It was proposed that this control occurred
via a mechanism involving intracellular soluble pools
(Conway et al. 1976, Conway and Harrison 1977). Af-
Length of the stages in the cell cycle in diatoms under limiting growth conditions.
Cell-cycle stage duration (h)
Species
Growth conditions
T. weissflogii Light-limited 10/70

molm2s1
T. weissfloggi T -limited
13 23 C
T. weissfloggi NH4-limited
T. weissfloggi Si-limited
T. weissfloggi Si-limited
C. fusiformis Si-limited
C. simplex
Si-limited
Gen. time (h)
G1
G2
G2M
Percent of total cell cycle

G1
G2
G2M
Reference
40
18
20
45
50
Olson et al. 1986
50
15
15
20
30
30
40
Olson et al. 1986
70
69
27
70
60
50
10
2.3 7.6
1.5 7
25
2
35
1
14
90
61
61
40
Olson et al. 1986

Brzezinski 1992
Brzezinski 1992
56.7 2.8
17.1 1.8
40
3
12
12
10
72
14
59.5 4.5 14.3
19
7.3 31.4
43
36
3
24
58
1.5
78.4 2.8
56.8 4.6
57
4
20
20
ter surge uptake in silicate-limited cells, pools were assumed to increase to maximum levels and then feedback to control transport (Conway et al. 1976, Conway
and Harrison 1977) (Fig. 2). However, in C. fusiformis
(M. Hildebrand, unpublished data), soluble pools did
not have to increase to maximum levels before uptake
was controlled. In fact, soluble pool levels were rigorously maintained, changing only gradually over long
time periods, and they did not transiently expand to
accommodate uptake. This suggested that other cellular factors were involved in the transport control
mechanism. It was proposed that soluble pool levels
were determined by the capacity of intracellular silicon-binding components (M. Hildebrand, unpublished
data). More or less of these components could be present, explaining the observed range of pool levels, but
at a given time, pool sizes would be rigorously fixed by
the amount of binding component. Control of transport was proposed to occur according to the relative
amounts of bound and unbound Si (Hildebrand 2000);
when unbound silicon-binding component was in excess, uptake was favored, and with excess unbound Si,
uptake was inhibited or efflux induced.
Once taken up, silicon can efflux from diatom cells,
as has been measured in radiotracer assays (Azam et
al. 1974, Sullivan 1976). Interestingly, efflux did not
occur in the absence of external silicate (Sullivan
1976), which is consistent with intracellular Si being
bound or sequestered. Increasing amounts of external
silicate up to a saturating level actually increased efflux
(Sullivan 1976). This trend is consistent with the transport regulation mechanism of Hildebrand (2000) described above. In this hypothesis, efflux is a consequence of transient imbalances between uptake and
deposition during surge uptake caused by a level of
transport that exceeds the capacity of intracellular silicon-binding components. With increasing amounts of
external Si, surge uptake would increase, and the resultant higher intracellular excess would be more
readily effluxed. Because the intracellular excess would
consist of unbound silicic acid, efflux could be driven
by an outward concentration gradient.
Ultimately, mechanisms that control uptake must
act upon the silicic acid transporters. Because their
carboxy-terminal portions are very likely to interact
with other proteins (Hildebrand et al. 1998), it may be
that the control mechanism operates through proteins
that bind to and affect the activity of the transporters.
It is not clear whether internal pools are directly involved in cellular processes other than frustule formation. Because silicon may be bound to intracellular components or possibly sequestered in vesicles, the effect of
Si on processes such as DNA synthesis (Darley and Volcani 1969) and cell cycle progression (Brzezinski et al.
1990) could be due to external sensing mechanisms.
intracellular transport of si
The intracellular transport of silicon is not well understood, but a recent review discusses possible mechanisms (Hildebrand 2000). Silicon has been found in
829
all major cellular organelles (Mehard et al. 1974),

suggesting a general transport process. If intracellular
transport involved the silicic acid transporters (Fig.
3), then a specific form of SIT would have to be targeted to each major organelle. Unless each organelle
has a use for silicon, this may not be likely. Silicate ionophoretic activities that enabled the transport of silicon
across lipid bilayers and bulk organic phases, were isolated from Nitzschia alba (Bhattacharyya and Volcani
1983). Thus, intracellular transport could occur by ionophore-mediated diffusion, consistent with silicons presence in all organelles. It was suggested that these ionophoretic activities could also be involved in maintaining
high intracellular levels of unpolymerized silicic acid
(Bhattacharyya and Volcani 1983), in essence being the
silicon-binding components previously discussed. Another proposed mechanism was that Si was transported by specialized vesicles with intralumenal conditions that would maintain silicic acid solubility
(Schmid and Schulz 1979, Lee and Li 1992). However, as mentioned previously, there is no direct evidence of sequestration of silicon in these vesicles. Of
course, there may be other as yet unidentified cellular
components involved in these processes.
silica deposition
Elegant ultrastructural studies have followed the
course of silicification in the diatom cell wall (e.g.
Schmid et al. 1981, Schmid and Volcani 1983, Li and
Volcani 1985a, b, Pickett-Heaps et al. 1988). In spite
of this, we still know little about the molecular and
biochemical details of the process, although general
models have been presented (Sullivan 1986, PickettHeaps et al. 1990), and the most recent work (Krger
et al. 1999, Vrieling et al. 1999) suggests that a substantial increase in knowledge is to come.
Silica polymerization occurs within a specialized intracellular compartment known as the SDV, bound by
a membrane called the silicalemma (Drum and
Pankratz 1964, Reimann et al. 1966, Schmid et al. 1981,
Crawford and Schmid 1986). The intralumenal pH of
the SDV is acidic (Vrieling et al. 1999), which would favor the gelling of silicic acid (Iler 1979). Diatom silica is
completely enveloped in an organic casing (Reimann
et al. 1965, Volcani 1981), which presumably consists of
SDV proteins or glycoproteins. Silica in forming valves
is always enclosed within the silicalemma, which may
suggest that the silicalemma contributes to silicification, either directly through membrane-associated
components or indirectly by enclosing a specialized microenvironment for polymerization. The silicalemma is
not likely the only controlling factor, however, because
proteins occluded within diatom silica have been identified (Krger et al. 1994, 1996, 1999), and evidence indicates that the cytoskeleton and other cellular components are involved (Schmid 1979, 1980, Blank and
Sullivan 1983, Pickett-Heaps et al. 1990). Some models
have suggested (for review, see Pickett-Heaps et al.
1990) that after completion of the valve a process of
exocytosis occurs and the SDV becomes all or part of
830
the mature diatom cell wall. This concept has driven investigations aimed at isolating diatom cell wall proteins
as a means to characterize proteins of the SDV (Hecky
et al. 1973, Krger et al. 1994, 1996). Hecky et al.
(1973) completely hydrolyzed a cell wall fraction and
determined the amino acid composition in several diatom species and showed that it was enriched in hydroxylated amino acids (serine and threonine) and glycine
and was depleted in the negatively charged glutamic
and aspartic acids. Other novel hydroxylated amino
acids have been isolated from diatom cell walls (Nakajima and Volcani 1969, 1970). Based on these findings,
models have been postulated regarding the involvement of hydroxylated amino acids in silica polymerization (Hecky et al. 1973, Lobel et al. 1996), proposing in
general that coordination of silicic acid by adjacent hydroxyls would lower the activation energy for initiation
of polymerization. More recently, a conserved class of
diatom cell wall proteins (and their genes) called the
frustulins have been isolated and characterized (Krger
et al. 1994, 1996). These were calcium-binding glycoproteins having repeated domains (Krger et al. 1994,
1996). The frustulins were not specifically associated
with cell wall silica and so may not be directly involved
in the silicification process (Krger et al. 1997, Krger
and Sumper 1998). Immunolocalization data suggested
that the frustulins were likely to be outer coat proteins
of the cell wall (Krger et al. 1997, van de Poll et al.
1999). Results from recent immunocytological experiments (van de Poll et al. 1999) suggested that the diatom cell wall and silica casing were distinct entities.
Thus, there is not necessarily correspondence between
proteins of the cell wall and the SDV.
Swift and Wheeler (1992) isolated organic material
occluded within the silicified structures of the cell wall,
which appeared to include glycoproteins enriched in
serine and glycine, that may have been phosphorylated.
A class of proteins (and their genes) isolated from hydrofluoric acid-treated cell wall silica has been characterized, which were greatly enriched in proline, serine, cysteine, and aspartate (Krger et al. 1994, 1997). One of
these proteins was found associated with specific girdle
bands in C. fusiformis, but its function is not yet defined
(Krger et al. 1997, Krger and Sumper 1998). The discovery of a class of proteins occluded within the silicified
spicule of a sponge that had templating and polymerization-enhancing properties (Shimizu et al. 1998, Cha et
al. 1999) suggested possible roles for occluded proteins
in diatom silica. A most significant recent finding has
been the isolation of a class of polycationic peptides occluded within diatom silica that catalyze the formation
of silica nanospheres at low pH (Krger et al. 1999).
These polypeptides, called silaffins, contained a repeated amino acid domain rich in lysine and arginine
clusters, regularly spaced, and were also enriched in
serine. The lysines were modified to -N,N-dimethyllysine and lysine with N-methyl propylamine attached to
the -amino group (Krger et al. 1999). A synthetic peptide without the modified lysines could not promote silica polymerization at low pH, indicating that the post-
translationally modified residues were essential for

activity. Data from characterization of the silaffins were
consistent with two observations on diatom silicification:
polymerization occurs in an acidic environment (Vrieling et al. 1999), conditions under which the silaffins
were most active, and spheres of silica were formed during polymerization, as had been observed in several ultrastructural analyses (e.g. Chiappino and Volcani 1977,
Borowitzka and Volcani 1978, Schmid and Schulz 1979).
However, the identification of amines involved in polymerization (Krger et al. 1999) indicate that previous
models based solely on the involvement of hydroxyls
(Hecky et al. 1973, Lobel et al. 1996) need modification.
energetics of silicification
Indications are that energy for silicification is provided by aerobic respiration (Lewin 1955) and not by
photosynthesis (Sullivan 1976). Transport and biomineralization processes are energized by oxidative phosphorylation (Sullivan 1976, 1980, Blank and Sullivan
1979, Blank et al. 1986). Increases in respiration rate coincide with the transport period (Coombs et al. 1967b,
Coombs and Volcani 1968), and after cytokinesis, cells
have sufficient energy to sustain biomineralization until the valves form (Sullivan 1986).
Photorespiration also provides energy to make
amino acids for proteins associated with the diatom cell
wall. Serine and glycine, enriched in proteins of the wall
(Hecky et al. 1973, Volcani 1981, Swift and Wheeler
1992), are synthesized via a pathway related to the classic glycolate pathway of photorespiration (Burris 1977,
Winkler and Stabenau 1995). Their concentrations are
regulated during the cell cycle, reaching maxima at the
time of cell division and new valve deposition in T. weissflogii (Martin-Jzquel 1992). These same amino acids
were part of the theoretical model of the energetics of diatom silicification by Lobel et al. (1996). According to
their calculations, mediation of silicification by a specific arrangement of serine residues permits a low-energy
polymerization pathway for Si, with an activation barrier
of 15.4 kcalmol1 and a net stabilization level of 28.0
kcalmol1, although as mentioned the involvement of
amines (Krger et al. 1999) must now be incorporated
into models of polymerization.
Because the energy for silicon metabolism is more
closely linked to respiration than photosynthesis, the inhibition of cell wall silicification by Ge had no effect on
the regulation of carbohydrate synthesis and oxygen production by photosynthesis (Werner 1967). Instead, it led
to a decreased utilization of carbohydrates for synthesis
of cell wall organic material during biomineralization.
The uncoupling between the energetics of silicification and photosynthesis may help explain why silicic acid uptake and deposition can proceed in the
dark. Azam and Chisholm (1976) measured uptake
rates in darkness that were 44% of those obtained in
the light for natural populations. Brzezinski and Nelson (1989) and Nelson and Brzezinski (1997) also observed significant Si uptake in the dark by natural
populations that occasionally exceeded rates obtained
in the light. Coombs et al. (1967a) measured Si uptake and cell division during both the light and dark
phases of a photocycle in N. pelliculosa, with carbon
provided by reserves built up in the light. The coupling of silicon metabolism to a photocycle is species
specific. In T. weissflogii, pulsed nutrient supplies can
override the timing of division during a photocycle
(Wheeler et al. 1983, Putt and Przelin 1988). Ditylum
brightwellii can take up Si during the light or dark period of a diel photocycle, but Si deposition occurs
only in the light; in Skeletonema costatum, uptake, incorporation, and division are nearly simultaneous in the
dark period (Chisholm et al. 1978). Also, the length
of the light period alters the extent to which cell division proceeds during the dark in Thalassiosira fluviatilis ( T. weissflogii) (Chisholm and Costello 1980).
relationship with cell division and the
cell cycle
The deposition of new siliceous valves between mitosis and daughter cell separation creates a close coupling between silicon metabolism and the cell cycle
(Crawford 1981, Brzezinski 1992, Schmid 1994). The
observation of Si uptake immediately preceding cell
division to accommodate the deposition of new valves
has been noted for several species (Chisholm et al.
1978, Sullivan and Volcani 1981, and references
therein). However, as mentioned previously, some
species can take up and store silicic acid well before
cell division (Chisholm et al. 1978, Brzezinski and
Conley 1994).
The tight coupling of the cell cycle and silicic acid
uptake has important implications for assessing kinetic parameters for uptake. In asynchronous populations, the values of Ks and Vmax obtained are biased by
the fact that not all cells actively take up Si during an
experiment. This problem was examined quantitatively by Brzezinski (1992) using asynchronous populations of T. weissflogii as a model system. Both Ks and
Vmax are underestimated by up to 8-fold when uptake
by the subpopulation of cells that are actively taking
up Si is averaged over the entire population. The degree of underestimation was found to vary with incubation time and the degree of Si limitation of the population examined. Those results argue that the Si
uptake systems of diatoms could have lower affinity,
and greater maximum capacity, than some current kinetic parameters indicate.
The coupling of Si uptake and its deposition during formation of the cingulum is less well known. The
girdle bands of the cingulum are formed sequentially
after deposition of the new valves and may contain a
large fraction of the Si in many species (Round 1972).
In N. pelliculosa (Brb.) Hilse., sufficient data exist to
deduce the coupling of Si deposition and transport
for both the valves and cingulum. New valves are deposited after cytokinesis followed by the deposition of
the first and often the second girdle band while the
daughter cells are still attached (Chiappino and Vol-
831
cani 1977). Deposition of the third and final girdle

band is delayed until just before mitosis (Chiappino
and Volcani 1977). Thus, deposition of the entire
frustule occurs during one continuous segment beginning just before division and ending just before
daughter cell separation. Silicic acid uptake in this
species is confined to this same segment of the cell cycle (Sullivan 1977). The tight coupling between Si uptake and cell division in N. pelliculosa (Sullivan 1977)
is thus reinforced by the fact that all parts of the frustule are deposited near the time when division occurs.
The coupling of uptake and division may be significantly weaker for species that construct a significant
fraction of their frustules at other points in the cell cycle (Brzezinski and Conley 1994).
A series of Si dependencies have been identified in
the diatom cell cycle. Two arrest points appear universal among diatoms (Fig. 4), one at the G1/S boundary
and another during G2/M associated with construction of new valves (e.g. Darley and Volcani 1969, Vaulot et al. 1987, Brzezinski et al. 1990). Additional Si
dependencies have been identified associated with
the deposition of other components of the frustule.
A Si dependency early in G1 (Fig. 4) associated with
the formation of siliceous setae (spines) has been
demonstrated in Chaetoceros sp. (Brzezinski et al.
1990). Not all Si-dependent arrest points are directly
coupled to silica deposition. The arrest point at the
G1/S boundary (Fig. 4) has been argued to be indicative of an Si dependency for DNA synthesis (Darley
and Volcani 1969), although a direct Si requirement
by diatom DNA polymerase (Okita and Volcani 1978)
or other proteins has not been demonstrated. The
data of Vaulot (1985) suggests that this arrest point
serves a regulatory role by testing the environment to
see whether sufficient Si exists to allow completion of
cell division. His data on the release of Si-starved cultures of T. weissflogii by the addition of Si suggest that
cells arresting in G2/M due to a lack of Si do not recover as well compared with those arrested at the
Fig. 4. Arrest points due to silicon starvation in the diatom

cell cycle. Most diatoms arrest predominantly at the G1/S phase
boundary. Another arrest point is during G2 and M phase, the
precise location within these phases has not been defined. Diatom
species that have setae can also arrest in early G1 (Brzezinski et
al. 1990).
832
G1/S boundary. A detrimental effect of prolonged interruption of the division process is implied.
other factors affecting silicification
The amount of silica in a diatom species can vary
substantially, by up to 4-fold (Lewin 1957, Paasche
1980, Taylor 1985, Brzezinski et al. 1990). A consideration is whether changes in the extent of silicification
are due to different frustule thickness or different cell
size. Depending on cell size, diatom growth rates vary,
with larger cells growing more slowly even within the
same species (Paasche 1973c, Parsons and Takahashi
1973, Durbin 1977, Taylor 1985). Larger cells of a species naturally have more silica than smaller cells because of the larger physical size of each component of
the frustule. On the other hand, Durbin (1977)
showed that clones of a given size grown at a lower
temperature had increased silicification relative to
those grown at a higher temperature, implying that
frustules were thicker. Therefore, both cell size and
frustule thickness can contribute to variations in the
extent of silicification. Marine diatoms have one order of magnitude less silica per unit cell volume than
freshwater diatoms, and it was suggested that this
could be due to an adaption to lower DSi availability
in the marine environment, salinity effects, or differences in sinking strategies (Conley and Kilham 1989).
Cell division and growth seem to have the most direct effect on silicification. But factors such as external silicon concentrations and other external growth
conditions including light, temperature, and nutrient
concentrations (N and P) or trace metal ions (Fe2/3,
Zn2, etc.) are involved in regulating growth rate.
They are therefore indirectly involved in controlling
silicification. Under nonlimiting Si conditions, because of the weak direct dependence of silicon metabolism on other aspects of cellular metabolism, the incorporation of Si is mainly linked to the duration of
the cell wall synthesis phase. An increase in the length
of this period under low growth rates allows maximum incorporation of silicon into the frustule, and
the converse occurs under high growth rates (Fig.
5A). Thus, under nonlimiting Si conditions, mineralization is generally inversely correlated to growth rate
(Fig. 5A, Table 4) (Flynn and Martin-Jzquel 2000).
This has been shown for growth rate controlled by
light intensity by Taylor (1985) in Coscinodiscus granii
and by Davis (1976) in Skeletonema costatum; controlled
by nitrogen by Harrison et al. (1976, 1977) in Skeletonema costatum, Thalassiosira gravida, and Chaetoceros
debilis and by P. Claquin et al. (personal communication) in T. pseudonana; and controlled by temperature
by Durbin (1977) in Thalassiosira nordenskioldii, by Paasche (1980) in Chaetoceros affinis and Rhizosolenia fragilissim, and by Furnas (1978) in Chaetoceros cruvisetum;
and controlled by phosphorous by P. Claquin et al.
(personal communication) in T. pseudonana. Limiting
levels of Fe2/3 and Zn2 can also increase silicification in some but not all diatom species examined
(Hutchins and Bruland 1998, Takeda 1998, De La
Fig. 5. Relative cellular silicon levels as a function of

growth rate under limiting conditions. (A) Continuous cultures
under non-silicon-limited conditions. Plots are of () Coscinodiscus granii limited by light (scale 102), () Chaetoceros affinis limited by temperature, () Rhizoselinia fragilissima limited
by temperature, and (*) Skeletonema costatum limited by ammonium (scale 101). (B) Continuous cultures under silicon-limited conditions. Plots are of () Thalassiosira weissflogii and ()
Cylindrotheca fusiformis. (See Table 4 for references.)
Rocha et al. 2000). For all these parameters, their

other effects on cellular metabolism could affect this
relationship. For example, Blank et al. (1986) showed
reduced growth and mineralization at lower temperature (10 C) in Navicula saprophilia due to the decreased
rate of polymerization at lower temperature for that
species. An exception to the inverse correlation between silicification and growth rate under nonlimiting Si conditions is the observation that diatoms growing in higher extracellular Si have increased total
cellular or cell wall Si (Paasche 1973a, Tilman and Kilham 1976, Harrison et al. 1977). Because these should
be optimum conditions for growth, increased silicification may be due to a mass action effect that drives
more silica incorporation per unit time.
Limiting DSi concentrations in the external environment results in reduced levels of silicification (Paasche 1975, Davis 1976, Harrison et al. 1977, Brzezinski et al. 1990). Under Si limitation, the level of
mineralization varies directly with growth rate (Fig.
5B, Table 4). The mechanism responsible is likely
833
Table 4. Total intracellular silicon per cell, per volume, or per surface area as a function of growth rate in cultures under different
limitations.
Species
Skeletonema costatum
Limitation
Nonlimited
Si-starved
Si-limited
NH4-starved
NH4-limited
Silicate
Ammonium
Light
Chaetoceros debilis
Thalassiosira gravida
Coscinodiscus granii
Chaetoceros affinis
Thalassiosira nordenskioldii
Rhizosolenia fragilissima
Nonlimited
Si-starved
Si-limited
NH4-starved
NH4-limited
Nonlimited
Si-starved
Si-limited
NH4-starved
NH4-limited
Light
Temperature
Temperate
Temperature
Growth conditions
d1
pmol Sicell1
7.9
6.5
3.9
6.7
7.7
Dilution rate (h1)
0.022
0.032
0.046
0.054
0.068
0.086
Dilution rate (h1)
0.042
0.054
0.080
0.098
100% I 0.14 lymin1
100%
30%
15%
1%
I molm2s1
14
33
60
100
150
( C)
8
13
18
23
( C)
0
10
( C)
8
13
18
23
References
Harrison et al. 1977

4
3.5
3
4.1
5.8
4.7
7.5
7.7
5.6
5.7
Davis 1976
2.9
3.2
4
3.6
30.2
22.9
11.1
30.1
27.2
200
108
127.8
163
133.6
0.14
0.26
0.29
0.53
0.34
225
157
102
88
24
0.79a
0.76
0.48
1.06
1
0.33a
0.20
0.26
0.44
0.24
11.2a
7.7
4.8
4.4
1.1
Taylor 1985
Paasche 1980c
1
1.6
1.8
1.3
1.39
1.29
1.17
1.11
1.662.66b
0.831.83
0.56
1.21.4
Durbin 1977d
Paasche 1980c
0.7
1.1
1.5
1.8
1.7
1.58
1.02
0.92
1
1.7
2
2.5
0.0200.026
0.0230.036
0.0300.043
0.0430.056
Paasche 1973ac
Silicate
Nitrate
fmol Si
0.41a
0.43
0.29
0.51
0.43
Dilution Rate (d1)

0.31
0.24
0.20
0.20
0.45
0.60
Martin-Jzquel et al.
(unpublished data)
0.50
0.58
0.58
0.94
0.27
0.20
(continued)
834
Table 4.

Continued
Species
Limitation
Growth conditions
d1
pmol Sicell1
fmol Si
References
Phosphate
Thalassosira weissflogii
Cylindrotheca fusiformis
Asterionella ralfsii
Silicate
Silicate
Aluminium
0.075
0.12
0.24
0.31
0.50
0.65
Doubling time (h)
12
18
28
33
Doubling time (h)
12
18
22
38
52
6.22 molL1
0.96
0.95
0.62
0.48
0.39
0.26
Brzezinski et al. 1990f
1.4
0.9
0.6
0.5
1.4
0.9
0.7
0.4
0.3
0.11
0.22
0.51
0.69
1.6
0.7
0.5
0.4
Brzezinski et al. 1990g
110
65
40
30
25
2.2b
2.8
2.9
3.2
Gensemer 1990h
am3
bm2
values in pg Sicell1.
values in fgm2 from Figure 6 in the reference.
eRecalculated values in pg Sicell1 from Figure 3 in the reference.
fRecalculated values from Figure 6 in the reference.
gRecalculated values from Figure 8 in the reference.
hRecalculated values from Figure 5 in the reference.
cRecalculated
dRecalculated
related to the differential regulation of uptake and

growth. Because diatoms can maintain division rates
near maximum under limiting Si conditions (Paasche
1973a, Brzezinski et al. 1990, Nelson and Dortch 1996),
for a given limiting concentration of silicic acid, uptake
is diminished to a greater extent than growth, leading
to less silica being deposited during the cell cycle (Paasche 1973a, Olsen and Paasche 1986) (Table 4). This
effect is clearly observable morphologically; Si-limited
cells have frustules that are thinner (Paasche 1975,
Davis 1976, Harrison et al.1977, Brzezinski et al. 1990),
and siliceous spines in those species that form them
are diminished or absent (e.g. Harrison et al. 1976,
Brzezinski et al. 1990). The structures of sponge spicules are also affected under limiting Si conditions
(Maldonado et al. 1999).
One question to consider is whether the extent of
silicification affects diatom sinking rates. Culver and
Smith (1989) showed in Chaetoceros gracilis and C. flexuosum that increased sinking rates occurred in cultures with faster growing cells. If the relationship between cell wall silicification and growth rate holds
true in these species, then cells with less highly silicified walls would sink more rapidly. Waite et al. (1997)
showed that sinking rate increased with increased irradiance of T. pseudonana but decreased under the
same conditions in T. weissflogii and Ditylum brightwellii. If growth rate was affected by the different levels
of irradiance, then in these experiments there would
have been no direct correlation between sinking rates
and the extent of silicification. One component of

sinking rate is cell volume, and even though larger diatom cells will sink more rapidly according to Stokes
Law (Waite et al. 1992, 1997) and should have more
total silica (but perhaps less per unit surface area),
these results (Culver and Smith 1989, Waite et al.
1997) indicate that the extent of silicification does not
directly contribute to sinking rate. The results of Bienfang et al. (1982) suggest that even though frustule silicification does not directly affect sinking rates, there
is interaction at another level. The depletion of silicic
acid in batch cultures of four marine diatoms increased cell-sinking rates by 2- to 10-fold despite morphological evidence of reduced silicification. These
results indicate that regulation of cell buoyancy is in
some way affected by Si deprivation but not by the extent of silicification.
dissolution of biogenic silica
Seawater is everywhere undersaturated in silicic
acid (Lewin 1961, Kamatani and Riley 1979, Van Bennekom et al. 1991, Trguer et al. 1995), and the
slightly basic pH of seawater is corrosive to the amorphous silica found in diatoms. Diatoms thus require a
means to protect the frustule from direct exposure to
seawater and the intracellular environment. This is especially important because once it is produced, a frustule is passed from mother cell to daughter cell with
each generation. No one has determined whether dia-
toms can add silicon to the frustule once it has been

incorporated into the mature cell wall.
Cooper (1952) postulated that dissolution of cell
wall silica was prevented by organic material around
the frustule; this was first directly shown by Lewin
(1961). Diatom cell wall silica was more rapidly dissolved when cell wall organic material was removed by
nitric acid treatment, and several other treatments
that killed cells also promoted dissolution (Lewin
1961). In living cells kept in darkness, silica dissolution was minimal (Lewin 1961). Kamatani (1982)
showed that in natural diatom assemblages dominated by Eucampia zodiacus or Coscinodiscus gigas, rate
coefficients of raw diatom cell dissolution were four to
five times less than those of acid-treated frustules.
Some evidence of significant amounts of silica dissolution from frustules of living cells has been obtained
(Nelson et al. 1976). However, a recent report clearly
showed that Si dissolution rates were extremely low,
0.2%0.3% d1, unless diatoms were colonized by bacteria or treated with protease to remove the organic
covering (Bidle and Azam 1999). Nelson et al. (1976)
did not determine whether cultures were axenic, and
cell viability was not monitored, which may explain
their results. Most data (Lewin 1961, Kamatani and
Riley 1979, Kamatani 1982, Bidle and Azam 1999) indicate that silica dissolution occurs appreciably only
in cells in which the organic casing is disrupted.
Kamatani (1982) pointed out that rates of dissolution
differ from one species to another and indicated rate
coefficients ranging over one order of magnitude between the fast dissolving Skeletonema costatum and the
slower C. gigas. He interpreted this as being related to
physicochemical characteristics of the frustule and to
the organic or inorganic matter protecting it, which was
previously suggested by Lewin (1961). Species-specific
differences in dissolution rates can be expected with
regards to the large range of individual specific surface
areas (e.g. Lewin 1961, Lawson et al. 1978, Kamatani
and Riley 1979). Lewin (1961) made the observation
that silica dissolution rates from living diatoms were
much higher than from fossil diatoms and measured a
much higher specific surface area in living species. She
attributed this result to a silica aging process that reduced the specific surface area in the fossils. Kamatani
and Riley (1979) explained the slowing of dissolution
rates with time from acid-washed frustules of Thalassiosira decipiens and Rhizosolenia hebetata by a decrease in
specific surface areas due to selective dissolution of fine
structures such as delicate spines. They also suggested
that the dissolution rate coefficients must be a function of the morphology and the structure of the frustules, not only of the specific surface areas. The dissolution of biogenic silica in seawater is also strongly
temperature dependent (Lewin 1961), with the specific
rate of dissolution increasing approximately 2-fold for
each 10 C increase in temperature (Lawson et al.
1978, Kamatani and Riley 1979, Kamatani 1982).
Impurities in the silica matrix of the frustule appear to affect dissolution rates. According to Van Ben-
835
nekom et al. (1991), variations in Al:Si ratios in diatom frustules have a marked influence on biogenic
silica dissolution rates. Culturing diatoms from the
low Al-high orthosilicic acid waters of the Southern
Ocean with additional Al slowed dissolution rates
(Van Bennekom et al. 1991).
In the ocean, dissolution rates ultimately control
the remineralization of silicic acid in both surface and
deep waters. Thus, the involvement of bacteria in hastening silica dissolution may be a key factor in the
control of diatom growth (Bidle and Azam 1999) by
creating a supply of silicic acid in surface waters independent of that introduced by vertical mixing of highnutrient water from beneath the nutricline. The characterization of bacterial species that may be adapted
to degrade diatom frustule organic material and the
specific enzymes involved should provide insight into
factors affecting this process.
silicification and diatom ecology
Diatoms first appeared in the fossil record about
185 million years ago and in abundance 115110 million years ago (Rothpletz 1896, Gersonde and Harwood 1990). They have since become, on a par with
the grasses and the Pinacea, the most important contributors to global carbon fixation (Werner 1977). Diatoms have radiated to inhabit both marine and freshwater environments, occurring in a variety of habitats
including ice and soils (Werner 1977). Their success
may be due in part to their silicified cell wall. Silicon is
the second most abundant element in the earths
crust; thus, the material for the wall is ubiquitous and
the polymerization of monosilicic acid yielding silica
is a thermodynamically favorable process (Iler 1979).
Raven (1983) estimated that in vascular plants, the use
of a silicified cell wall required, by weight, only 3.7%
of the energy for incorporation of lignin into the wall
and 6.7% for polysaccharide, and by volume, 5% for
lignin and 10% for polysaccharide. Energy saved in
cell wall synthesis may be channeled into other cellular processes. Perhaps by having to use less energy for
cell wall synthesis and by taking advantage of the
abundance of silicon, diatoms and other siliceous plankton not only fit into a previously unexploited ecological niche but can outgrow other species. Before the
evolution of siliceous plankton, DSi was relatively
plentiful in the oceans with concentrations near saturation in the low millimolar range in surface waters
(Holland 1984). Since then, these plankton, which are
now dominated by diatoms, have severely depleted
the oceans of Si. Concentrations are generally 10 M
at the surface, 3 M in the vast mid-ocean gyres (Goering et al. 1973, Azam and Chisholm 1976, Nelson et al.
1981, Brzezinski and Nelson 1989), and no higher than
ca. 160 M in deep water (Bainbridge 1980, Craig and
Broecker 1981). The result is competition for silicon
as a cell wall material in the modern ocean. Limitation of diatom silicification rates has been observed at
least at times in every ocean environment examined,
including both fertile coastal waters (Goering et al.
836
1973, Azam and Chisholm 1976, Brzezinski et al. 1997)

and oligotrophic open ocean gyres (Brzezinski and Nelson 1989, Brzezinski and Nelson 1996). Thus, the potential benefits of producing a silicified cell wall now
come at a price. Si availability in the modern ocean
has a strong effect on the distribution and abundance
of diatoms because of their absolute requirement for
the element (Richter 1906, Lewin 1962). It is now recognized that the rate of supply of silicic acid to diatoms may affect critical aspects of the ocean carbon cycle
by regulating phytoplankton carbon fixation and the
export of phytoplankton-derived organic matter to the
deep sea over large regions of the oceans (Dugdale et
al. 1995, Dugdale and Wilkerson 1998, Ku et al. 1995).
summary
Most investigations into mechanisms regulating cell
growth in microalgae have focused on carbon and nitrogen metabolism and photosynthesis (Turpin et al.
1988, Falkowski and La Roche 1991, Turpin 1991). In
diatoms, one must also take into account the need to
produce a siliceous frustule. Deposition of silica in the
frustule is an obligatory step before the division of vegetative cells, creating a close coupling between the cell
cycle and silicon metabolism, but there is only a weak
direct dependence of Si metabolism on other aspects
of cellular metabolism. The effects of different external conditions (light, temperature, nutrients, trace
metals) lead to different ratios of Si:N and Si:P and
also affect carbon metabolism (Si:C, N:C, P:C) differentially (Brzezinski 1985, Harrison et al. 1990, Hutchins and Bruland 1998, Takeda 1998, De La Rocha et al.
2000, Flynn and Martin-Jzquel 2000).
Silicon mineralization in diatoms results in a structural cell wall material. Apparently, no storage reserve
of silicon is present other than the soluble pools. In
contrast, both nitrogen and phosphorus (Morris 1974,
Kirchman et al. 1986, Brussaard et al. 1997) can be accessed from storage pools or from the breakdown of
intracellular compounds containing these elements
under limiting conditions for growth. A major consequence is that under Si limitation, the internal pool in
most species is not sufficient to complete the wall and
hence cell division and thus cannot sustain a long period of growth. This may explain why diatom growth
is more rapidly inhibited under Si starvation than under nitrogen or phosphorus starvation (Parslow et al.
1984, Harrison et al. 1990). Because most of the silicon
inside the cell is not likely to be free in solution but
rather in the wall or complexed with organic compounds or perhaps sequestered in vesicles, growth models cannot be directly linked to the total cellular Si
quota (Paasche 1980).
This review identifies several areas of diatom cellular Si metabolism to be more clearly elucidated. Understanding the mechanisms that control silicic acid
transport is of much interest, because this is the step
by which the cell takes up the element, and all indications are that transport is an integrated part of the
overall process of silicification (Conway et al. 1976,
Conway and Harrison 1977, Chisholm et al. 1978, Hildebrand 2000). With the cloning of the SIT genes that
encode silicic acid transporters (Hildebrand et al.
1997, 1998) and the ability to reintroduce genes into
diatoms by transformation (Dunahay et al. 1995, Apt
et al. 1996, Fischer et al. 1999, Zaslavskaia et al. 2000),
investigations of the molecular details of silicic acid
uptake, including determination of individual kinetic
parameters and intracellular localization, are now feasible. Characterization of proteins that interact with
the SIT carboxy-termini may provide a means of investigating the mechanism of coupling between transport and incorporation. The way by which the cell
maintains supersaturated levels of soluble silicon has
been a long-standing question, and answering this is
likely to be important in understanding the intermediate steps between uptake and deposition, which include intracellular transport and release into the
SDV. It also may help identify what constitutes physiologically active silicon in the cell, if any, which may
be involved in controlling cellular responses to silicon. Another question is in regard to signaling mechanisms: How does the cell sense extra- or intracellular
silicon levels and transduce this into changes in metabolism and gene expression? Characterization of
factors affecting the expression of silicon responsive
genes (Hildebrand et al. 1993) may be helpful here,
and may help in understanding the cell cycle control
of uptake and silicification, which could involve several sensing and transduction mechanisms. We know
little about the process of silicification and cell wall
synthesis at the biochemical and molecular level, but
recent results (Swift and Wheeler 1992, Krger et al.
1994, 1996, 1997, 1999, Krger and Sumper 1998, Vrieling et al. 1999) promise major advances in this area.
Identification of the silaffin proteins (Krger et al.
1999) is a very significant advance. However, much
still remains to be elucidated about this complex cellular process, including unraveling the details of what
controls silica micro- and macroscale structure formation in the SDV and also in terms of cytoskeletal elements that may be involved. Understanding the molecular details will help to more accurately estimate
the cellular energetic requirements for silicification and
to determine what specific aspects of cellular metabolism are involved in and affected by this process. Although silica dissolution is not often thought of as being part of the cells metabolism of silicon, it really is,
because the cell must protect the frustule from dissolving into its seawater environment and intracellularly, and dissolved Si is the raw material for new frustule formation. Insights from studying dissolution
may identify species- or condition-specific alterations
in the silicification process designed to protect the
silica from the environment. The investigation of diatom cellular silicon metabolism may also have biotechnological applications in devising biomimetic
approaches to materials synthesis.
Diatoms have been objects of aesthetic admiration
and scientific interest for over 200 years and are ex-
tremely important in primary production and the biogeochemical cycling of silicon in the global ocean and
fresh water. Further elucidation of the biochemical and
molecular pathways involved in silicification should lead
to greater understanding of the role of Si in controlling
diatom growth and productivity and the formation of
the ornate silicified structures of the cell wall.
Supported by the CNRS (to V.M.-J.), by the U.S. Army Research
Office under MURI Grant Number DAAHO4-96-1-0443 (to
M.H., M.A.B.), and by gifts from the Dow Corning Corporation
(to M.H.).
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J. Phycol.

36, 821840 (2000)

SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH 1

Mark Hildebrand 2,3

have recently been characterized, and the application

7 February 2000. Accepted 23 May 2000.

VRONIQUE MARTIN-JZQUEL ET AL.

From unicellular algae to vascular plants, numerous

each generation, creating unique morphotypes that

Fig. 1. Scanning electron micrographs of (top) an intact

where Vmax and max represent the maximum rates of

DIATOM SILICON METABOLISM

Frains Lake clone

VRONIQUE MARTIN-JZQUEL ET AL.

Dortch 1996). Average Ks values tend to be lower than

shows that Si transport and silica deposition are

DIATOM SILICON METABOLISM

tent with the observed rate of cell number increase in

tional identification of cDNAs encoding a silicic acid

VRONIQUE MARTIN-JZQUEL ET AL.

Hildebrand, unpublished data

Hildebrand, unpublished data

12 membrane-spanning segments, but recent analysis

transport. Hybridization experiments showed that

DIATOM SILICON METABOLISM

Fig. 3. Model for changes in silicic acid transporter (SIT)

sults from laboratory and field studies show consistent

ments of cell water volume (M. Hildebrand, unpublished

VRONIQUE MARTIN-JZQUEL ET AL.

9.4 1.4 7.6 0.3

Brzezinski et al. 1990

9.4 1.1 4.0 1.7

6.8 24.7 10.5

Brzezinski et al. 1990

4.5 3.6 2.1 3.1

33.8 27.1 15.9 23.3

Brzezinski et al. 1990

6.0 0.3 3.1 0.4

Brzezinski et al. 1990

12.9 0.9 1.9 0.5

Brzezinski et al. 1990

7.8 0.8 0.3 0.7

Brzezinski et al. 1990

Vaulot et al. 1987

Percent of total cell cycle

centrations (15 to 50 M) for a given light intensity

T. weissflogii Light-limited 10/70

Gen. time (h)

Percent of total cell cycle

Olson et al. 1986

Olson et al. 1986

Olson et al. 1986

DIATOM SILICON METABOLISM

all major cellular organelles (Mehard et al. 1974),

VRONIQUE MARTIN-JZQUEL ET AL.

translationally modified residues were essential for

DIATOM SILICON METABOLISM

cani 1977). Deposition of the third and final girdle

Fig. 4. Arrest points due to silicon starvation in the diatom

VRONIQUE MARTIN-JZQUEL ET AL.

Fig. 5. Relative cellular silicon levels as a function of

Rocha et al. 2000). For all these parameters, their