Supporting Information

Iron Incorporation in Biosilica of the Marine Diatom Stephanopyxis  turris:

Dispersed or Clustered?

Janine Kaden1, Stephan, I. Brückner1, Susanne Machill1, Christoph Krafft2, Andreas Pöppl3, Eike

Brunner1

1 School of Mathematics and Science, TU Dresden, 01062 Dresden, Germany2 Leibniz Institute of Photonic Technologies (IPHT) Jena, 07745 Jena, Germany3 Faculty of Physics and Earth Sciences, University Leipzig, 04103 Leipzig, Germany

E-mail: eike.brunner@chemie.tu-dresden.de

Infrared spectroscopy

IR spectra of high and low wavenumbers of the silicagels and the isolated biosilica from S.  turris are

provided in Figure S1. The silicagel spectra just show bands related to silica vibrations. For biosilica,

bands due to tightly silica-associated organic material occur in addition to the bands caused by SiO 2.

The region between 3000 and 3800 cm-1 and the band at 1640 cm-1 correspond to the stretching and

bending vibrations of O-H groups, respectively. The broad band at approximately 3500 cm-1 belongs

to water molecules bound via H-bonds to silanol groups. The range of 3720 to 3320 cm-1 indicates

strongly interacting vicinal O-H groups (Innocenzi 2003; Khraisheh et al. 2005; Aguiar et al. 2009).

Between 2800 and 3000 cm-1, stretching vibrations of C-H are visible. The band located at around

1735 cm-1 shows the stretching vibration of carbonyl groups (C=O) according to esters. Typical bands

arising from proteins are amide I (mainly stretching of C=O) and amide II (mainly bending mode of N-

H) at 1650 cm-1 and 1540 cm-1, respectively. C-O-C stretching vibrations mainly of polysaccharides

occur at 900 – 1200 cm-1. Overlapping bands caused by tightly silica-associated organic matter are

characteristic for this spectral region. The band at approximately 1227 cm-1 is due to the

antisymmetric stretching vibration of phosphate, for instance found in phosphorylated proteins like

silaffins (Kröger and Poulsen 2008), and in carbohydrates, such as mannose-6-phosphate (Hedrich et

al. 2013). The antisymmetric stretching vibration of siloxane (Si-O-Si) is located between 1000 and

1200 cm-1 and consists of two transverse optical (TO) modes: TO1 known as the antisymmetric

stretching vibration of Si-O-Si is located at about 1080 cm-1 and TO2 at around 1200 cm-1 as a

prominent “shoulder”. For Biosilica 1 to 3, the former band is located at 1085, 1090, and 1091 cm-1,

respectively. For the silicagels, the band occurs in the range from 1085 cm-1 to 1073 cm-1 depending

on the iron content in the silicagels. The higher the amount of incorporated iron in the silica

framework, the larger is the shift (see Figure S2). Further bands caused by silica are located at about

975, 800, and 465 cm-1 and are assigned to the stretching vibration of Si-OH from silanol groups, the

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symmetric stretching vibration of Si-O-Si, and to the rocking vibration of O-Si-O (Doelsch et al. 2001;

Innocenzi 2003). Furthermore, the altered shape of the band at 970 cm-1 indicates an increasing

number of defects within silica framework for the silicagels depending on the incorporated amount

of iron. Cations are able to disrupt a regular silica structure by breaking Si-O-Si bonds thus leading to

the formation of Si-O-NBO (non-bridging oxygen) (Serra et al. 2002). These shifts are also observable

for the samples prepared at pH 5.5. That means, iron incorporation into the silica framework takes

place at both pH values.

Since the present work focuses on iron incorporation into the biosilica of the marine diatom

S.  turris, the bands due to vibrations of silanol groups and especially the antisymmetric stretching

vibration of siloxane groups are of major interest. The wavenumber of the antisymmetric stretching

vibration for Biosilica 1 to 3 is, however, not indicative. Silicon substitution by iron leads to a

significant shift only for higher amounts of iron (see Figure S2). At the low iron contents observed for

biosilica, no significant effect can be expected.

29Si MAS NMR spectroscopy

Figure S3 a) demonstrates the correlation between the second moment M2 for the 29Si NMR signals

of Q3 and Q4 groups and the Fe:Si mass ratio. Data for silicagels and biosilica are provided. A linear

correlation between M2 and the iron content in the silicagels is observed. At the relatively low iron

contents found in biosilica, M2 is, however, not significantly influenced. That means, it is not possible

to use M2 as an indicator for the type of iron incorporation (dispersed or clustered) within the

biosilica. In contrast, the effect observed for T1 is remarkably different for dispersed or clustered iron

in the silica network even at low iron concentrations. As shown in Figure S3 b), the iron-induced

contribution ΔR1 to the relaxation rate T1-1 in silicagels is huge even at low iron contents. That means, T1 is very sensitive to dispersed iron incorporation.

The maximum amount (upper limit) of iron dispersed in the SiO2 of biosilica could therefore

be estimated as follows: Assuming that the iron is fully dispersed in the synthetic silicagels, we have

determined an iron-induced contribution ∆ R1=1T 1

− 1T 10 to the longitudinal relaxation rate. T1

0 is

the relaxation rate of the iron-free sample (Silicagel 1). The dependence of ΔR1 on the Fe:Si mass

ratio (denoted as x) is shown in Figure S3 b) for the Q4 groups and can be described by the function:

∆ R1/ s−1=5015.9 x2+70.81x

Under the assumption that the longitudinal relaxation rate measured for biosilica is solely due to

iron, one can deduce that the maximum amount of iron dispersed in the SiO2 matrix is 3.0 % for

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Biosilica 1 and 1.6 % for Biosilica 2. We therefore conclude that more than 95 % of the iron in the

biosilica is clustered and only less than 5 % is dispersly incorporated into in the SiO2 matrix.

References

Aguiar H, Serra J, González P, León B (2009) Structural study of sol – gel silicate glasses by IR and

Raman spectroscopies. J Non Cryst Solids 355:475–480.

Doelsch E, Stone WEE, Petit S, Masion A (2001) Speciation and Crystal Chemistry of Fe (III) Chloride

Hydrolyzed in the Presence of SiO4 Ligands. 2. Characterization of Si - Fe Aggregates by FTIR and

29 Si Solid-State NMR. Langmuir 17:1399–1405.

Hedrich R, Machill S, Brunner E (2013) Biomineralization in diatoms-phosphorylated saccharides are

part of Stephanopyxis turris biosilica. Carbohydr Res 365:52–60.

Innocenzi P (2003) Infrared spectroscopy of sol-gel derived silica-based films: A spectra-

microstructure overview. J Non Cryst Solids 316:309–319.

Khraisheh MAM, Al-Ghouti MA, Allen SJ, Ahmad MN (2005) Effect of OH and silanol groups in the

removal of dyes from aqueous solution using diatomite. Water Res 39:922–932.

Kröger N, Poulsen N (2008) Diatoms—From Cell Wall Biogenesis to Nanotechnology. Annu Rev Genet

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Serra J, González P, Liste S, et al (2002) Influence of the non-bridging oxygen groups on the bioactivity

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Captions of Figures

Figure S1. IR spectra of the spectral region from 450 cm-1 to 4000 cm-1 of the silicagel series prepared

at pH 7 (a) and biosilica (b).

Figure S2. Band shift of the antisymmetric stretching vibration as(Si-O-Si) as a function of Fe:Si mass

ratio determined by ICP-OES in the silicagel for pH 5.5 (black points) and pH 7 (black triangles). The

range characteristic for biosilica (Fe:Si mass ratios of 0.0014 to 0.0031) is shaded in grey.

Figure S3. a) Correlation between second moments M2 for 29Si NMR signals in silicagels and biosilica

for Q3 (black symbols) and Q4 (grey symbols) as a function of the Fe:Si mass ratio determined by ICP-

OES. b) Complete diagram and fitting curve of the correlation between ΔR1 of Q4 for silicagel (black

rectangles). ΔR1 is the iron-induced contribution to the longitudinal relaxation rate, i.e.,

∆ R1=1T 1

− 1T 10 , where T 1

0 denotes the measured T1 value for the iron-free sample (Silicagel 1).

Figure S4. Correlation between the offered Fe:Si mass ratios in the culture medium and the Fe:Si

mass ratio found in the SDS/EDTA isolated biosilica (Biosilica 1: Fe:Si 0.13:1; Biosilica 2: Fe:Si 0.25:1;

Biosilica 3: Fe:Si 0.5:1)

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Figure S1

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Figure S2

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Figure S3

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Figure S4

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