Chemical composition of the bleached spicules measured by means of XRF.
Many animals base their skeleton on calcium containing compounds and in particular calcium phosphates are prevalent as the main bone constituents. There are, however, animals that rely on different compounds for their skeleton. An example of such a class of animals is sponges, which have a skeleton constituted of silica (Müller et al, 2007). The fact that silica is used as skeleton provides the sponges with the ability to live in calcium-poor and acidic environments. Despite the characteristic composition of the skeleton, sponges have managed to spread to various living environments such as seawater (Bavestrello et al., 1995; Croce et al., 2003; W. E. G. Müller et al., 2005; Pisera, 2003; Schwab & Shore, 1971; Uriz et al., 2000) and lakes (Kaluzhnaya et al., 2005; Schröder et al., 2003). Furthermore, the prevalence of sponge across the entire planet witnesses that they can grow at different temperatures. Due to the unique features of sponges, they have been intensively studied by scientists through the last decades. Many of the studies focused on the evolutionary and biological aspect of sponges (Barthel 1986; Barthel, 1995; Calcinai et al., 2007). After clarification of the structure of the sponges, it is known that they possess unique features. As a consequence of the unique features of sponges, these creatures have attracted interest from material scientists (Wang et al., 2010) since study of sponges could lead to new materials or facilitate the production of already existing materials through biomimetic approaches.
The composition and structure considerably vary from one type of sponge to another, and therefore it is not possible to address all kinds of sponges in one chapter. In this chapter, we focus on the sponge called
2. Materials and methods
2.1. Extraction of the sponge
The freshwater sponge
To separate the inorganic part from the organic part of the sponge, the organic part of the sponge was removed by means of bleaching. A selected amount of the sponge was added to a Teflon container and mixed with 35 % H2O2, 69 % HNO3, and water in the volume ratio 8:7:1. The Teflon container was placed in a 90 °C water bath for 20 min as the heat accelerates the breakdown of organic material. The solvents were then removed and fresh solvents added. The container was replaced in the water bath for 20 min. The procedure was repeated until a white material without any colorants appeared in the Teflon container. The solvents were then removed and the material rinsed once with water. The material was heated at 160 °C for 2h in order to evaporate any remaining bleaching agent without changing the properties of the bleached material.
2.3. Calorimetric measurements
Calorimetric and gravimetric responses of the sponge to temperature were measured using a simultaneous thermal analyzer (NETZSCH STA 449C Jupiter (Selb, Germany)), which provides functions of both differential scanning calorimeter (DSC) and a thermogravimeter. For the measurements, a platinum crucible containing the sample and an empty reference platinum crucible were placed on the sample carrier at room temperature. Initially both crucibles were held 5 minutes at an initial temperature of 333 K. Thereafter an upscan to 1543 K and a subsequent downscan were performed at 10 K/min. The samples underwent a second up- and downscan with 10 K/min in order to achieve uniform thermal histories (Yue, 2008). The purge gas was air with a flow of 40 mL/min. Before measuring each sample, a baseline was measured by using two empty crucibles according to the above-stated heating procedure, which was used for correcting the DSC signal of the samples. The isobaric heat capacity (
2.4. Heat treatments
To remove the organic material from the inorganic part by another approach than bleaching, a part of the sponge was put in a porcelain crucible and heat-treated in a muffle furnace (Scandiaovnen A/S, Denmark) in air at 823 K for 7 h. This temperature was selected in such manner that all organic substances could be completely removed.
Another sample was prepared by heating untreated sponge in an alumina crucible in air to 1723 K for 17h in an Entech SF6/17 electric furnace (Ängelholm, Sweden) in order for the material to crystallise.
2.5. Scanning electron microscopy (SEM) imaging
SEM measurements were done on a Zeiss 1540 XB scanning electron microscope (Oberkochen, Germany) using uncoated samples. The secondary electrons were recorded using an acceleration voltage of 1 kV. Images of untreated, bleached, and heat treated samples were acquired.
2.6. Transmission electron microscopy (TEM) imaging
Since it was not possible to obtain any images with sufficient contrast on the untreated sponge, heat treated samples were used for transmission electron microscope (TEM) imaging. After annealing the untreated sponge in an electric furnace at 1723 K for 17 h it was finely ground using an agate mortar. The obtained powder was dispersed in isopropanol and after thorough dispersing, a droplet of the dispersion was allowed to dry on a carbon-coated copper grid. The ground, heat-treated sponge fragments kept on the grid were imaged in a high-resolution TEM (JEOL JEM 4010, acceleration voltage 400 keV, point-to-point resolution: 0.155 nm).
2.7. Optical microscopy
The fibres were investigated on a Zeiss Axioskop microscope. The length of the sponge spicules was measured employing a 10x objective. The width was measured as the broadest point of the spicule with a 100x objective in oil immersion. Both width and length measurements were done on 50 spicules.
2.8. X-ray fluorescence
Bleached sponge powder of about 2 grams was mixed with 12 g Li2B4O7 per gram powder. The mixture was melted at 1573 K and made into a tablet. The tablet was placed in a sample holder, and after calibration, the measurement was initiated. The measurement was conducted on a S4-Pioneer X-ray spectrometer (Bruker-AXS, Karlsruhe, Germany).
2.9. Vacuum hot extraction
The water content of the bleached fibres was measured by mass spectrometry coupled with vacuum hot extraction. Prior to the measurement, the sample was kept at 473 K to remove adsorbed water. The sample was cooled to room temperature in the device. Subsequently, the degassing rate was recorded upon heating of the sample with a rate of 10 or 20 K/min to 1773 K.
2.10. Small angle X-ray scattering
The small angle X-ray scattering (SAXS) measurements were done on a pinhole instrument. The instrument was a modified version of a commercially available small-angle X-ray camera (NanoStar, Bruker AXS). It employs a rotating anode X-ray source (CuKα) (0.1 x 1.0 mm2 source size with a power of 1 kW) and a set of cross-coupled Göbel multilayer mirrors for monochromatising the radiation and for converting the divergent beam from the source to an essentially parallel beam. To reduce the background, the instrument has an integrated vacuum. The instrument covers a range of scattering vectors
For the experiments, a bunch of the thin spicules were collected using a Scotch® tape in such a way that they were randomly oriented. The scattering data of a clean tape was used as background for the data treatment.
2.11. X-ray diffraction
The sample heat-treated at 1723 K was measured with a Seifert FPM HZG4 diffractometer (Freiburg, Germany) with Fe Kα radiation. The scan was conducted in the range 5° < 2θ < 65°.
3. Results and discussion
3.1. Macroscopic structure of the sponge
The sponge itself has a hard, but somewhat brittle and spherical structure with a brownish colour (Fig. 1). The brown colour arises from the organic part of the organic and from humic acids from the river that have precipitated on the skeleton of the sponge (Keding et al., 2010). The initial key findings of the structure of the sponge are reported in Jensen et al., (2009).
To examine the macroscopic structure of the sponge, scanning electron microscopy (SEM) images are acquired. The skeleton consists of needle shaped spicules that are bundled together (Fig. 2). There are branches on the bundles of spicules which give the sponge its porous structures. Due to the porous structure, the density of the sponge is only about 15 g/L.
The presence of an axial channel in the spicules is a common feature for sponges (Aizenberg et al., 2005; Cha et al., 1999; W. E. G. Müller et al., 2005; Schwab & Shore, 1971, Uriz et al., 2000). The axial channel has been found to harbour an axial filament containing the catalytic protein silicatein (Cha et al., 1999; Shimizu et al., 1998). Furthermore, the cross section of the spicules in Fig. 4B shows a solid structure around the channel, whereas other sponges have a layered structure (Aizenberg et al., 2004; Levi et al., 1989; Shore, 1972; Uriz et al., 2000).
To investigate the chemical nature of the junctions, spicules subjected to bleaching and heat-treatment at 823 K are considered. After the two treatments, the sponge loses its macroscopic structure and appears as a powder. The loss of the macroscopic structure is confirmed by SEM, since only isolated spicules are found (Fig. 4).
From Fig. 4 it can be observed that the junctions that cement the spicules have been removed. As a consequence of the removal of the junctions, the macroscopic structure of the sponge collapses and isolated fibres are left. The length and width of the spicules have by optical microscopy been measured to 305 and 15.6 µm, respectively. Since the macroscopic structure collapses after both bleaching and heat treatment at 823 K, it can be concluded that the junction consists of organic material. This is in contrast to spicules in other sponges that are cemented by a silica deposition (Aizenberg et al., 2005). Due to the brittle nature of the spicules, sponges that apply silica cemented spicules, obtain mechanical stability by a hierarchical arrangement (Aizenberg et al., 2005). As
3.2. Chemical composition of the spicules
The composition of the bleached sponge has been examined using the vacuum hot extraction (VHE) method up to 1780 K for volatile components such as water and by X-ray fluorescence (XRF) for the remaining inorganic components. During the hot extraction experiments, volatile species liberated from the sample upon heating are detected by means of a mass spectrometer. Prior to the VHE experiments, the sample is kept at 473 K to remove water absorbed to the surface of the spicules since the surface of silica is known to be hygroscopic. The release of water vapour from bleached spicules at two different heating rates is shown in Fig. 5.
The water release occurs at similar temperatures at both 10 and 20 K/min. with a maximum release rate around 900 K. The spectrometer measures roughly an ion current that at 20 K/min is twice of that measured at 10 K/min. The increased ion current is caused by the higher heating rate, i.e., the experiment at 20 K/min only has half of the time to release the water compared to a heating rate of 10 K/min and consequently, the water release is twice as intense. Since the water is released at the same temperatures at different heating rates, the water release is thermodynamically and not kinetically controlled. Through integration of the degassing curve in Fig. 5 and the measurement of a standard, it is possible to determine the water content (R. Müller et al., 2005). From this approach a water content of 5.5 wt % is determined.
The XRF measurement shows that the chemical composition of the bleached spicules is dominated by SiO2 (Table 1).In addition to the 99.7 wt% SiO2, the spicules contain impurities. The impurities are mainly CaO and Al2O3 and could arise from sample handling and preparation. After the clarification that the spicules mainly consist of SiO2, it is investigated
whether the 5.5 wt% water determined by means of VHE, reside in the glass structure or are present in pores. To do so the glass transition temperature (
3.3. Glass transition temperature
The glass transition temperature (
To determine the water content in the network structure of the
To reveal the presence of possible mesopores, transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS) are employed. Due to pronounced decomposition, TEM images of untreated spicules could not be obtained. Although the bleached spicules underwent massive radiation damage upon TEM inspection, the amorphous nature of the spicules was doubtlessly disclosed. Therefore focus is turned to the sample heat-treated at 1723 K.
The TEM image in Fig. 7 reveal mesoscale crystalline phases (dark areas) embedded in the amorphous phase. The crystalline phases possess a width of ~ 15 nm and a length of up to 200 nm and seem to be randomly orientated in the amorphous phase. To study the crystalline phase a high resolution TEM (HRTEM) image is recorded (Fig. 8).
The HRTEM image in Fig. 8, shows that the crystalline phase indeed is embedded in the amorphous phase. Hence, upon heat treatment at 1723 K, initially causes a removal from some type of mesopores (Fig. 5 and Fig. 6 TG curve) where after a crystallisation of the
surface layer of the mesopores occurs. The presence of mesopores explains that the water is released from the spicules at very high temperatures (Fig. 5). Thus, we can infer that the
On the surface of the spicules, round crystalline structures are found. A comparison with the TEM images in Figs. 7 and 8 shows that only the surface of the fibres and the mesoporous channels crystallise during the heat treatment. X-ray diffraction (XRD) measurements of the heat-treated spicules reveal the presence of cristobalite (Fig. 9B). The presence of cristobalite is expected as it is the stable crystalline form of SiO2 at high temperatures.
From the fitting, cylindrical channels with a length and width of 110 and 23 nm, respectively are found. These dimensions correspond well the size of the channels observed on the TEM images in Figs. 7 and 8. Thus the channel like structures detected by SAXS are the mesoporous channels. In addition to the channels, polymer structures with a radius of gyration of 3.45 nm are determined. This polymer structure is expected to be the protein catalysing the polymerisation of silica, i.e, Silicatein. Silicatein has been reported to be similar to Cathepsin S (Shimizu et al., 1998), which has a diameter of 3.0 nm. Since Silicatein contains 50 % more amino acids than Cathepsin, it is likely that Silicatein or an analogue catalytic protein has a radius of gyration of 3.45 nm. Since
3.5. Formation of mesoporous amorphous silica
The growth mechanism of
The technological and scientific importance of glassy silica is demonstrated by its wide applications such as membranes (de Vos & Verweij, 1998), columns (Dai et al., 2003), heat-proof materials (Saito et al., 2000), optical communication fibres (Tong et al., 2003) and catalysts in organic synthesis (Minakata et al., 2004). At present, silica of high purity is often produced by means of fusing (Brückner, 1970; Tohmon et al., 1989) or argon plasma methods (Tohmon et al., 1989). Due to the costly and advanced methods, a biomimetic approach based on the catalytic proteins in
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