Simultaneous Measurement of the Silicon Content and

Physiological Parameters by FTIR Spectroscopy in Diatoms

with Siliceous Cell Walls
Anne Jungandreas, Heiko Wagner* and Christian Wilhelm
Institute of Biology, University of Leipzig, Johannisallee 23, D-04103 Leipzig, Germany
*Corresponding author: E-mail, hwagner@rz.uni-leipzig.de; Fax, +49-341-9736899.

Techniques
(Received December 22, 2011; Accepted October 15, 2012)

Diatoms are the most successful biomass producers world-
wide. Therefore, physiological and chemical methods to
measure the cell response to a variety of abiotic factors
are the focus of recent research. We used the two model
diatoms Cyclotella meneghiniana and Skeletonema costatum
for the development of Fourier transform infrared (FTIR)
Nevertheless, diatoms are the only known phytoplankton
group depending directly on silicon (Si) for growth. Due to
their siliceous frustule, diatoms can accumulate considerable
amounts of silica, with reported values of up to 30% of the dry
weight (Werner 1967). However, the Si content is dependent on
various factors including time of day (Werner 1967), the salt

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spectroscopy-based methods to measure simultaneously
the elemental composition of the cells and their cell-specific
physiological properties. The cells were grown in chemostat
cultures to study the response of Si limitation. The model
organisms showed different reactions in terms of their cell
properties. Si limitation was accompanied by a drop in the
growth rate, a reduced content in Si per cell and a decreased
Si : C ratio. Furthermore, the C allocation pattern was chan-
ged in both diatoms under Si limitation, as shown by FTIR
spectroscopy. Moreover, we used FTIR spectra to develop
PLS (partial least square) regression methods to predict
the Si content and the Si : C ratio for single as well as mul-
tiple species. All PLS regression models were validated by
standard chemical methods and showed good prediction
accuracy, with the coefficient of determination R2 being
0.93. We could show that it is possible to monitor phyto-
plankton properties such as C allocation, the Si content and
the Si : C ratio at the same time via FTIR spectroscopy.
Keywords: Cyclotella meneghiniana  Elemental composition
 Macromolecular composition  PLS regression  Si limita-
tion  Skeletonema costatum.
Abbreviations: C, carbon; FTIR, Fourier transform infrared;
N, nitrogen; O, oxygen; P, phosphorus; PLS, partial least
square; R2, coefficient of determination; RMSECV, root
mean square error of cross-validation; Si, silicon.
Introduction
Diatoms are widely used for water quality monitoring
(Potapova et al. 2004), since they contribute about 50% to
phytoplankton primary productivity (Nelson et al. 1995).
concentration of the surrounding medium (Conley 1989) or
nutrient limitation. While nitrogen (N) and phosphorus (P)
limitation affects all phytoplankton cells, Si limitation causes
a species shift in phytoplankton communities, favoring
non-siliceous species when Si is scarce (Egge and Aksnes
1992, Roberts et al. 2003, Carrick and Lowe 2007). The physi-
ology of diatoms under Si limitation or starvation has been
studied intensively, including the kinetics of Si uptake
(Conway et al. 1976, Olsen and Paasche 1986, Claquin
Martin-JÕzÕquel 2005), the changes in the Si content and
elemental ratios (Harrison et al. 1977, Brzezinski et al. 1990,
Shafik et al. 1997, Hoffmann et al. 2008) and the dynamics of
the macromolecular composition (Harrison et al. 1990). These
studies showed that Si limitation usually leads to a drop in Si per
cell as well as a rise in the C : Si and C : N ratio (Harrison et al.
1977, Shafik et al. 1997). Furthermore, lipid accumulation is
documented as a common response in Si-limited diatoms
(Roessler 1990, Lombardi and Wagnersky 1991, Lynn et al.
2000). Other physiological effects are species specific and
show no similar adaptation pattern. These include the C : P
ratio, changes in the cell volume, Chl a per cell or the fluores-
cence yield in photosynthesis (Harrison et al. 1977, Shafik et al.
1997, Lynn et al. 2000, Hoffmann et al. 2008).
To analyze the effects of Si on phytoplankton, the available
measurements are often limited since conventional methods
need material in the range of a few mg DW. In particular, the
analysis of elemental ratios or the macromolecular cell com-
position is time consuming and impossible for small sample
sizes, e.g. from outdoor experiments. Therefore, monitoring
the C allocation and the Si deposition from natural water sam-
ples is hard to accomplish, and sampling with sufficient time
resolution is impossible. These limitations can be overcome by
the use of Fourier transform infrared (FTIR) spectroscopy, since

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 A. Jungandreas et al.
it has been shown to be a fast and sensitive method for the
analysis of microbial cells. In the last decade, FTIR spectroscopy
has emerged as a new method to study a variety of biochemical
compositions of algal cells. It is based on the distinct absorption
bands of carbohydrates (C-O-C bonds at 1,200 cm 1 to
900 cm 1), lipids (C = O of esters at 1,740 cm 1), proteins
(C = O of amides at 1,650 cm 1; N-H of amides at
1,540 cm 1) and silica (Si-O of silica at 1,075 cm 1) according
to Giordano et al. (2001), which together represent >90% of the
cells dry biomass. Several methods have been developed to use
these absorption bands to analyze relative changes in the ratio
of the macromolecular composition. A number of studies used
the comparison of band ratios to analyze the relative contents
of the macromolecules (Beardall et al. 2001, Giordano et al.
2001, Heraud et al. 2005, Stehfest et al. 2005, Liang et al. 2006,
Dean et al. 2008, Heraud et al. 2008, Dean et al. 2010). Recently
two methods have been developed to quantify the absolute
macromolecular composition of non-siliceous species via FTIR
spectroscopy. Both use FTIR spectra of reference substances to
quantify the abundance of carbohydrates, lipids and proteins
based on either band integration (Pistorius et al. 2009) or FTIR
spectra reconstruction (Wagner et al. 2010). However, as carbo-
hydrates and silica show strongly overlapping absorption
bands, these methods are not applicable as yet for diatoms.
To analyze the carbohydrate content in diatoms, Su et al.
(2012) proposed a combination of the FTIR spectral analysis
according to Wagner et al. (2010) and measured the elemental
composition (C : N ratio) to quantify carbohydrates, proteins
and lipids in diatoms without the use of the Si and carbohy-
drate absorbance. This method is able to analyze all macromol-
ecules in diatoms except the amount of cellular Si.
Since Si shows a strong absorption in FTIR spectra, variations
in the cellular Si concentration cause clear changes in the shape
of FTIR spectra. Based on these changes, it is supposed that the
Si content can be quantified directly from the FTIR spectra.
Stehfest et al. (2004) were able to quantify rosmarinic acid via
FTIR spectroscopy in plant cell cultures applying a partial least
square (PLS) regression method.
In line with this methods, the aim of the recent study was (i)
to follow changes in the macromolecular composition in re-
sponse to different Si limitation; and (ii) to develop a PLS re-
gression method based on FTIR spectra to quantify Si in
diatoms. Here, we present data for the two diatom model spe-
cies Skeletonema costatum and Cyclotella meneghiniana to
show that FTIR spectroscopy can be a useful tool for the physio-
logical characterization of cells in response to changing Si con-
centrations as well as the determination of the Si : C cell quota.
Results
Growth and cell characteristics under Si limitation
The induction of Si limitation in both diatom species S. costa-
tum and C. meneghiniana caused several species-dependent
physiological changes (Fig. 1). Under full nutrient supply, a
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higher maximum growth rate (m = 0.67 d 1) has been observed
for S. costatum in comparison with C. meneghiniana
(m = 0.38 d 1). For both species, a linear decrease in growth
was observed under Si limitation, matching the amount of
the nutrient dilution (Fig. 1A). This could be expected from
the theory of chemostat cultures, where m is linearly correlated
with the limiting nutrient concentration. With increasing Si
limitation, different patterns of dry weight and cellular Chl a
content have been measured. In S. costatum, the dry weight per
cell did not change, whereas in C. meneghiniana this ratio
changed drastically under severe Si-limiting conditions
(Fig. 1B). In contrast, the Chl a content in C. meneghiniana
remained constant at each Si limitation, while the Chl a content
strongly increased in S. costatum with rising Si availability
(Fig. 1C).
The chemical composition also showed species-dependent
differences in response to Si limitation. The Si content with
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respect to the dry weight of S. costatum steadily decreased
with rising limitation, while the Si : C ratio remained constant
up to an Si supply that matched half of the maximum growth
rate. It only dropped at the highest Si limitation studied
(Fig. 1D, F). However, the Si per cell was only slightly decreased
with Si limitation (Fig. 1E). In comparison with S. costatum, the
Si content with respect to the dry weight in C. meneghiniana
increased with slight Si limitation as a result of the lower dry
weight (Fig. 1D), whereas the Si content per cell (pg) was found
to be constant (Fig. 1E). Further Si limitation, however, resulted
in a decreased Si cell content as well as lower Si concentration
with respect to the dry weight (Fig. 1D). The Si : C ratio in
C. meneghiniana showed the same pattern compared with
the Si with respect to the dry weight. After the increase at
slight limitation, more limited cells showed a decreased Si : C
ratio (Fig. 1F). Furthermore, the cellular C : N ratios were af-
fected through Si limitation. In both species, a steady rise in the
C : N ratio could be observed (Fig. 1G).
FTIR spectra of diatoms under Si limitation
The effect of Si limitation on FTIR cell spectra of diatoms in the
range of 1,800–750 cm 1 is shown in Fig. 2. Further examples of
FTIR spectra of diatoms are shown in Fig. 4A. Band assignments
have been realized according to Movasaghi et al. (2008). In
general, the spectra consist of the specific peak frequencies
for ester bonds of lipids (1,740 cm 1), amide I and II bands
of proteins (1,650 cm 1 and 1,540 cm 1) and the overlapping
carbohydrate and Si region (1,200–950 cm 1). As can be ex-
pected from the decreased Si : C ratio (Fig. 1), the absorption
intensities of the Si vibration bands Si-O at 1,078 cm 1
(Giordano et al. 2001) were changed. For S. costatum, a sus-
tained decrease of this IR band in relation to the amide I band
was observed (Fig. 2a), which is in accordance with the chem-
ical reference. In C. meneghiniana, the slight increase and sub-
sequent decrease of this band within the IR spectra (Fig. 2b) are
again in agreement with the Si : C ratio, especially with the dry
weight pattern under Si limitation (Fig. 1). The decrease in Si
 FTIR spectroscopy in silica-containing diatoms

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Fig. 1 Cell characteristics of the two diatom species S. costatum (open circles) and C. meneghiniana (filled circles) with respect to different Si
availabilities per day. (A) Growth rate per day, (B) dry weight, (C) Chl a and (D) Si (%) with respect to the dry weight, (E) Si per cell (pg), (F) Si : C
ratio and (G) C : N ratio. The asterisks show the P-values of the respective data point compared wiht the replete culture (*P < 0.05; **P < 0.01,
***P < 0.001).

peak intensities in relation to the amide I band was, however,
more pronounced in C. meneghiniana than in S. costatum. This
was not expected since the Si : C ratio of both species was
decreased to a similar extent from 0.18–0.16 down to 0.09
for the highest Si limitation. Therefore, overlapping spectral
FTIR bands from cellular compounds other than Si within
this spectral range must be changed. The first indications for
an altered biochemical composition were given by the meas-
urements of the cell characteristics and especially the altered
C : N ratios. Since all relevant biomolecules have distinct vibra-
tional bands, the variance in the biochemical structure of the
biomass is reflected in the shape of FTIR spectra. The most
remarkable changes within the IR spectra can be detected in
the region around 1,750 cm 1 representing the cellular lipid

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 A. Jungandreas et al.

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Fig. 2 FTIR spectra of (A) S. costatum and (B) C. meneghiniana cultures adapted to different Si availabilities per day. The spectra are normalized
to the amide I vibration band (1,545 cm–1) and plotted as r.u. (relative units).

content. In contrast to C. meneghiniana, the FTIR spectra of
Si-limited S. costatum cells showed no changes in the ratio of
the amide I to the lipid spectral band (1,650 cm 1 vs.
1,750 cm 1), indicating that there is no Si-dependent lipid ac-
cumulation in this species. In contrast, the spectra of C. mene-
ghiniana showed an increased absorbance ratio of the lipid to
the amide I band. This led to the conclusion that accumulation
of lipids in Si-limited cells is not uniform in all species. The
results for S. costatum indicated further responses in the bio-
chemical cell composition. The lack of lipid accumulation in S.
costatum and the increase of the C : N ratio with rising Si limi-
tation leads to the conclusion that the small decrease of the
vibrational Si band in comparison with C. meneghiniana arises
from an overlapping absorption increase due to an accumula-
tion of carbohydrates. For C. meneghiniana, the C-O stretching
vibration peaking at 1,155 cm 1, which is related to carbohy-
drates, corresponds to the Si limitation. This peak correlates
with the changed dry weight in the same way as the Si absorp-
tion under intermediate Si limitation. In contrast, for the high-
est applied Si limitation, the peak rises and, therefore, indicates
a slight increase in carbohydrate accumulation. However, the
spectral analysis of the FTIR spectra showed very clearly that
the two diatom species correspond in a different way to Si
limitation with respect to the biochemical cell composition.

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 FTIR spectroscopy in silica-containing diatoms

Carbohydrates, lipids and proteins respect to the dry weight (38 samples) was R2 = 0.952
(RMSECV = 0.413) and for Si : C (19 samples) was R2 = 0.953
To check if biochemical cell homeostasis is altered by Si limita-
(RMSECV = 0.0159). Here the model ranged between 3.5%
tion, FTIR spectra and the results of the CHN analysis were used
and 11.8% Si with respect to the dry weight and a from 0.064
to estimate the amount of carbohydrates, lipids and proteins
to 0.338 for the Si : C ratio (Fig. 5C, D). With this spectral ana-
with respect to the dry weight according to Su et al. (2012).
lysis, it could be shown that FTIR spectroscopy can be used for
Carbohydrates, lipids and proteins make up to about 80% of the
the prediction of the cellular Si content.
dry weight, whereas about 20% of the cell dry weight is formed
Furthermore, we tried to develop PLS regression models that
by the siliceous frustule, and minor cell compounds. Under
are able to predict characteristics not only for one but more
replete nutrient supply, both S. costatum and C. meneghiniana
generally for a number of different diatom species (Fig. 6).
cells contained similar amounts of about 40% carbohydrates
Therefore, additional FTIR spectra of Thalassiosira weisflogii
(Fig. 3A). With increasing Si limitation, the carbohydrates con-
and Phaeodactylum tricornutum were added to develop a uni-
tent was increased up to 60% of the dry weight in S. costatum,
form model for four diatoms together with S. costatum and
whereas the carbohydrate content in C. meneghiniana was
C. meneghiniana. Thalassiosira weisflogii was chosen as a
slightly decreased. The protein content was higher in S. costa-
widely used model organism and P. tricornutum was included
tum and decreased with increasing nutrient limitation (Fig. 3B).
as a diatom which has just marginal amounts of Si with respect
However, the changes in the protein content were marginal in
to the dry weight (Tesson et al. 2009).

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C. meneghiniana. The lipid content was slightly higher under
replete conditions for C. meneghiniana and did not change
significantly in S. costatum with higher nutrient limitation
(Fig. 3C). In contrast to S. costatum, the lipid content of slightly
Si-limited C. meneghiniana cells decreased, then increased to up
to 40% with higher Si limitation.
The presented data were further validated by a comparison
of the carbohydrate content obtained by the FTIR spectroscopy
method described by Su et al. (2012) with colorimetric meas-
urements. The staining of a carbohydrate extract was per-
formed twice for C. meneghiniana and S. costatum. The two
samples chosen for each species differed in their overall macro-
molecular cell composition as shown clearly by their FTIR spec-
tra (Fig. 4A).
The two methods predicting the carbohydrate content of
the samples showed no significant differences for any of the
four samples (Fig. 4B).
In conclusion, the results showed that the two diatom spe-
cies studied here had a very different C allocation pattern in
response to Si limitation.
FTIR spectra-based prediction models
Since FTIR spectra reflect the cellular response to the applied Si
limitation, the spectra were used to develop PLS regression
models for the prediction of the cellular Si content and the
Si : C ratio in diatoms. For the calibration of these models,
the chemical reference data (Fig. 1) were used and assigned
to the respective FTIR spectra.
For the species S. costatum, cells within the range of 3.7–
10.3% Si with respect to the dry weight (17 samples) and an
Si : C ratio ranging from 0.089 to 0.273 (10 samples) were used
for the calibration of the PLS regression models (Fig. 5A, B). The
results of these PLS regression models showed good prediction
of both Si with respect to the dry weight and Si : C, with a high
correlation coefficient of R2 = 0.93 [root mean square error of
cross-validation (RMSECV) = 0.502] and 0.944 (RMSECV =
0.0134), respectively. The same holds true for C. meneghiniana,
where the correlation coefficient of the prediction of Si with
The combined PLS regression model for the prediction of
the Si concentration with respect to the dry weight in the cells
(53 samples) had a range from 0.2% (P. tricornutum) up to
11.8%, and reached a prediction accuracy of R2 = 0.95
(RMSECV = 0.514, Fig. 6A). This model was further validated
with five independent, randomly chosen data sets of three dif-
ferent species which are not included in the calibration model.
The value of Si with respect to the dry weight was then calcu-
lated from the FTIR spectra with a prediction error of <10%.
Thus, the results showed a successful prediction of the Si con-
tent in the used data sets.
The same procedure has been performed for the Si : C ratio
(29 samples). Here the model showed a prediction accuracy of
R2 = 0.944 (RMSECV = 0.0203). The models were also validated
with the same five independent data sets as the method for Si
content prediction and proved itself workable for all the sam-
ples (Fig. 6B).
Summing up these results, the PLS methods using FTIR spec-
tra were able to predict the Si content with respect to the dry
weight and the Si : C ratio with the same accuracy as chemical
methods.
Discussion
Diatoms are the major group of phytoplankton species depend-
ing on Si for growth and cell division. It was shown in several
studies that Si exhaustion can alter the phytoplankton com-
position and favors non-siliceous species (Egge et al. 1992,
Roberts et al. 2003, Carrick and Lowe 2007). However, the
underlying physiological mechanisms to deal with Si limitation
in diatoms are still not understood.
This study shows that Si limitation does not induce a general
pattern of metabolic changes in diatom physiology, but rather
species-specific responses. The cellular Si content (and there-
fore the thickness) of the frustule is reduced in both examined
species. However, the Si content is also affected by changes in
the cellular dry weight and the cell size, respectively, thus the Si
with respect to the dry weight is a more accurate value. At the

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 A. Jungandreas et al.
Fig. 3 The content of (A) carbohydrates, (B) proteins and (C) lipids of
S. costatum (open circles) and C. meneghiniana (filled circles) cultures
adapted to different Si availabilities per day. The asterisks show the
P-values of the respective data point compared with the replete cul-
ture (*P < 0.05; **P < 0.01, ***P < 0.001).
lowest Si concentration used in this study, the Si ratio with
respect to the dry weight was reduced by 50% in comparison
with optimal growth conditions in both S. costatum and
C. meneghiniana. This is in accordance with earlier studies
(Paasche 1973) which reported a comparable decrease in Si
per cell in Thalassiosira pseudonana following a hyperbolic
function. This hyperbolic function holds true for S. costatum,
where cell dry weight does not change remarkably. Our data,
however, showed that the hyperbolic decrease is not a general
pattern, since cell size measured as dry weight can vary up to
2-fold in C. meneghiniana due to lipid accumulation, resulting
in no general decrease of Si per cell. Furthermore, the Chl a
content of S. costatum is reduced under increasing Si limitation.
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Since the growth rate is decreased, a reduced pigmentation is a
possible adaptation, as it lowers the amount of absorbed light
energy which is necessary to sustain biomass synthesis. In con-
trast, C. meneghiniana does not show a change in the Chl a to
dry weight ratio. Therefore, the two species show very dissimilar
reactions to the limitation, with S. costatum reducing its energy
uptake and accumulating carbohydrates. Cyclotella meneghini-
ana, on the other hand, invests in lipids which have a higher
energy density than carbohydrates or proteins.
For a better understanding of the Si limitation effects on
diatom cells, we used FTIR techniques simultaneously (i) to
analyze changes in the macromolecular content and (ii) to de-
termine the cellular Si content as well as the Si : C ratio based on
PLS regression models. Since Si limitation depresses the growth
of diatoms (Brzezinski et al. 1990), an accumulation of storage
compounds is observed in both species. While in S. costatum
the carbohydrate content rises, C. meneghiniana shows an in-
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crease in lipids, forming highly reduced biomass to store large
amounts of energy. This is in accordance with recent literature,
where nutrient limitation was shown to cause the accumula-
tion of certain macromolecules, mostly resulting in an increase
in lipids and/or carbohydrates, depending on the species
(Beardall et al. 2001, Giordano et al. 2001, Heraud et al. 2005,
Stehfest et al. 2005, Liang et al. 2006, Dean et al. 2008, Heraud
et al. 2008, Dean et al. 2010). In addition, the accumulation of
carbohydrates and lipids results in an increase in the C : N ratio
under Si limitation (Fig. 1), which is in accordance with previ-
ous studies published by Shafik et al. (1997) and Harrison et al.
(1977). The determination of all major cell compounds in dia-
toms, except for the siliceous frustule, has been performed pre-
viously by Su et al. (2012). Within the present work, FTIR
spectra were used to predict both the macromolecular cell
composition and the amount of Si with respect to the dry
weight, as well as the respective Si : C ratio. Models of single
species (for C. meneghiniana and S. costatum) and mixed
models for four diatom species have been designed. It could
be shown that despite species-dependent differences in the
shape of the FTIR spectra and the different adaptations of
C. meneghiniana and S. costatum to low Si availabilities, it was
possible to create PLS regression models that can predict the Si
with respect to the dry weight and the Si : C ratio with high
accuracy, similar to the reliability of the respective reference
method. Jebsen et al. (2012) analyzed the distribution of bio-
mass into the macromolecular pools at different growth rates
caused by nutrient limitation and different light intensities.
Although the accumulation of macromolecules differed, a pre-
diction of the growth rate by FTIR-based PLS regression was
possible. This was due to minor changes in the shape of the
absorption bands. The results indicate that PLS regressions can
be used for a wide range of differently adapted/limited cells,
which would be the basis for prediction methods that can be
used for environmental samples.
The use of such PLS regression methods to analyze FTIR
spectra is not limited to Si-related cell characteristics but can
also be used to quantify other substances with a distinct
 FTIR spectroscopy in silica-containing diatoms

Fig. 4 (A) FTIR spectra and (B) the carbohydrate content of two cultures of C. meneghiniana [C.m. 1 (black) and C.m. 2 (red)] and S. costatum
[S.c. 1 (blue) and S.c. 2 (green)] obtained by FTIR spectroscopy (white) and chemical staining (gray). The FTIR spectra are normalized to the
amide I vibration band (1,545 cm–1) and shown in r.u. (relative units).

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Fig. 5 Validation plots of PLS regression models for the prediction of Si (% DW–1) and Si : C cell ratios. Shown are the predicted ratios from FTIR
spectra as a function of reference data from chemical analysis for the Si content (with respect to the dry weight) in (A) S. costatum and
(C) C. meneghiniana as well as the Si : C ratio in (B) S. costatum and (D) C. meneghiniana.

absorption in the infrared region. Furthermore, the presented
methods are not restricted to algal cells but can be applied to
multicellular organisms. This was shown, for example, by deter-
mination of rosmarinic acid in higher plants (Stehfest 2004) and
by Allison et al. (2009), who predicted several cell parameters in
grasses with ATR-FTIR spectra-based PLS regression methods.
However, it should be considered that these methods require
sufficient reference data sets that include samples which are
similar to the samples used for analysis. As the composition of
the cellular biomass, and therefore the shape of the FTIR

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 A. Jungandreas et al.
Fig. 6 Validation plots of PLS regression models for the prediction of (A) the Si content (% DW–1) and (B) the Si : C ratios. The FTIR spectroscopic
PLS regression models include data of the four diatom species C. meneghiniana (open circles), S. costatum (open triangles), T. weisflogii (open
diamonds) and P. tricornutum (open stars). The models have been tested by independent data sets (respective filled symbols).
spectra, is affected by various environmental factors, PLS regres-
sion methods should include samples of a wide variety of con-
ditions to minimize false predictions through outliers. The
collection of the required reference data can be very extensive,
as is the case for the carbohydrate or lipid content. Despite
these limitations, once the respective methods are developed,
FTIR spectroscopy can be used to predict various cell charac-
teristics from small sample amounts on the basis of single spec-
tra with very good reliability.
The results biased on the present spectroscopic method for
the determination of the C allocation pattern and the uptake/
concentration of elements in the biomass include important
information for a wide range of physiological studies and
adaptation experiments (Jakob et al. 2007). Furthermore, FTIR
spectroscopy allows not only a chemical analysis of the cellular
biomass composition but also a simultaneous analysis of
physiological key features such as C allocation pattern and
growth performance in one step. Further studies to test if
these methods can be transferred to cells taken from real
nature will show if the use of FTIR spectroscopy can signifi-
cantly improve the analysis of functional traits in phytoplank-
ton communities.
Materials and Methods
Culture conditions and cell properties
To characterize the physiological adaptation of diatoms under
Si limitation, cultures of C. meneghiniana and S. costatum were
grown in Si-limited semi-continuous chemostat cultures at
20 C (Langner et al. 2009). The irradiation was set at
200 mmol photons m 2 s 1 in a light/dark cycle of 13/11 h. For
each species, four different cultures were grown in Si reduced
f/2 medium with daily dilution rates resulting in an Si supply of
0.17–0.67 mg l 1 for S. costatum and of 0.15–0.59 mg l 1 for
C. meneghiniana. Thereby, the highest dilution rate chosen
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for each of the species matched the maximum growth rate
under the present light conditions. The lower dilution rates
corresponded to 75, 50 and 25% of the respective maximum
growth rate.
To develop PLS regression models based on the chemical
composition of diatoms, two additional species were grown in
batch cultures and illuminated with 120 m mol photons m2 s 1
(fluorescent tubes, 36 W/25-1, Osram) with a light/dark cycle of
14/10 h. Thalassiosira weisflogii was grown in f/2 medium with
an initial Si concentration of 7.9 mg l 1, while for P. tricornutum,
Si free ASP2 medium was used.
The salt concentrations of the f/2 media were set to
33.3 g l 1 for S. costatum and 16.6 g l 1 for C. meneghiniana
and T. weisflogii. The cell numbers of C. meneghiniana,
P. tricornutum and T. weisflogii were determined using a particle
counter (Z2 Beckman Coulter GmbH), while a Bürker counting
chamber was used for S. costatum. The Chl a content was
determined spectroscopically after extraction in 90% acetone
and calculated according to the formula of Jeffrey and
Humphrey (1975). For the dry weight measurement, cells
were harvested by centrifugation (3,000  g, 5 min), washed
with distilled water and lyophilized (FreeZone 2.5, Ilmcac)
before weighting.
Silicon, carbon and nitrogen measurement
The Si content was determined using the molybdenum blue
method with 2–4 biological repeats for each culture condition.
A total of 7  107–10  107 cells were centrifuged at 3,000  g
for 5 min, washed with distilled water and centrifuged again at
3,000  g for 2 min. After removing the supernatant, the cells
were re-suspended in 1 ml of distilled water. Then 5  106–
20  106 cells were taken for Si analysis, while the remaining
cells were centrifuged again for dry weight determination.
For the dissolution of the frustule, we used a modified
method according to Kamatani et al. (2000). After adding
4 ml of NaOH (0.4 M), each sample was heated to 95 C for

60 min and then filtered through an acetate filter (0.2 mm,
Sartorius). Each filtrate was stained by the molybdenum blue
method using ammonium molybdate, oxalic acid and ascorbic
acid (two technical repeats). Si determination was carried out
spectroscopically by measuring the extinction at 810 nm
(Specord 250, Analytik Jena GmbH).
The C and N contents were measured using a CHN elem-
ental analyzer (vario EL, Analytik Jena GmbH). For this purpose,
a cell suspension (50–70 ml, corresponding to 3–12 mg of or-
ganic dry matter) was harvested by centrifugation (3,000  g,
5 min), washed three times with distilled water and freeze-dried
before measurement.
FTIR spectroscopy
A 5 ml aliquot of the cell culture was filtered through a cellulose
acetate filter (0.2 mm, Sartorius) and re-suspended in 1 ml of
distilled water for washing. The suspension was centrifuged
(1,090 g, 4 min) and the pellet was re-suspended in distilled
water to a final concentration of 50–100  106 cells ml 1.
FTIR spectra were measured using a HTS-XT microtiterplate
module (Bruker) with a DTGS detector as described in
Wagner et al. (2010). A 1 ml aliquot per sample was applied
on a microtiterplate with 384 spots and dried for 10 min at
40 C. FTIR transmission spectra were recorded in the range
between 4,000 and 700 cm 1 with 32 scans per sample and a
resolution of 4 cm 1. FTIR spectra were analyzed using OPUS
Lab Software (Version 5.0, Bruker). Measured cell spectra were
corrected to the background spectra and baseline corrected
with the help of the rubber band method. The CO2 peaks
were excluded.
Carbohydrate, lipid and protein content
FTIR spectra follow Lambert–Beer’s law in a defined absorption
range and can therefore be used to analyze the amount of
contributing molecules (Wagner et al. 2010). The carbohydrate,
lipid and protein contents were analyzed according to Su et al.
(2012). For a verification of the carbohydrate content deter-
mined by the method of Su et al. (2012), the carbohydrates of
four samples (two for each species) were extracted and quanti-
fied according to the method of Granum and Myklestad (2002)
using staining with dihydroxynaphthalene-H2SO4 solution.
PLS regression models to determine Si with
respect to the dry weight and Si : C ratios
FTIR spectra contain a large number of partly overlapping vi-
brational bands, each corresponding to a chemical bond.
Therefore, it is possible to identify the main molecules in the
recorded spectra and compare them semi-quantitatively with
each other. However, since the FTIR cell spectra are very com-
plex, the examination of viable information needs to include
the full spectral range of the cells between 1,800 and 750 cm 1
where most of the vibration bands originating from the cellular
macromolecules are represented.
Plant Cell Physiol. 53(12): 2153–2162 (2012) doi:10.1093/pcp/pcs144 ! The Author 2012.
FTIR spectroscopy in silica-containing diatoms
The differences of the FTIR spectra within the spectral range
from 1,800 to 750 cm 1 were calibrated using reference data
obtained by the chemical analysis. PLS regression models in the
spectral range from 1,780 to 760 cm 1 have been calculated
using the Quant 2 software (OPUS 5.0, Bruker). The FTIR spec-
tra or their first derivative were pre-processed by min–max
normalization or vector normalization to achieve optimal re-
sults. PLS regressions use a number of PLS principal compo-
nents (PLS PCs, which are vectors) to predict substance
concentrations and ratios. All PLS regression models were
checked by cross-validation to determine the coefficient of de-
termination (R2) of the respective model. The R2 represents the
difference between true and recomputed substance concentra-
tions and ratios, with an R2 of 1 indicating a perfect fit.
Therefore, an R2 of 0.9 indicates that 90% of the variance can
be explained by the respective PLS regression. The RMSECV
serves as the standard value for the quality of the fit and
Downloaded from http://pcp.oxfordjournals.org/ by Sumi Sumi on March 31, 2016
gives the approximate standard error between the predicted
and reference values for all data points.
Statistics
The data were analyzed by Welch two sample t-tests with the
help of RStudio (Ver. 0.94.110). If not otherwise specified, all
P-values shown are determined against the values of the
unlimited culture. The asterisks in Figs 1 and 5 correspond to
the P-values of the respective data point compared with the
replete culture (*P < 0.05; **P < 0.01; ***P < 0.001).
Funding
This work was supported by the Bundesministerium für Bildung
und Forschung (BMBF) [02WU0777]; the Deutsche
Forschungsgemeinschaft (DFG) [INST 268/149-1].
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