Page:Nanostructural Organization of Naturally Occurring Composites Part II.pdf/3

Hermann Ehrlich et al.

(Germany) membranes with a MWCO of 14 kDa. Dialysis was performed for 5 days at 4°C. The dialyzed material was dried at room temperature and used for structural and analytical investigations.

2.2. Development of the silica nanoparticular fraction using mechanical disruption of cleaned R. fibulata spicules.
The spicules of R. fibulata were treated using enzymes as described above, cleaned, dialysed, and cut in 3 cm long pieces. These fragments of clean spicules were placed in deionized water and disrupted mechanically using glass covered stirring bars (25 × 6 mm) and magnet stirrer during 24 hours at 25◦ C. After centrifugation (1500× g) for 5 minutes the debris of mechanically disrupted spicules was collected and milky colour supernatant was carefully decanted. Obtained debrisfree suspension (Figure 1(b)) was used for SEM and TEM investigations.

2.3. FTIR spectroscopy
IR spectra were recorded with a Perkin Elmer FTIR Spectrometer Spectrum 2000, equipped with an AutoImage Microscope using the FT-IRRAS technique (Fourier transform infrared reflection absorption spectroscopy) as described previously [21].

2.4. Transmission electron microscopy (TEM)
Conventional transmission electron microscopy [1] was performed with a Philips CM200 FEGST Lorentz electron microscope at an acceleration voltage of 200 kV. For electron microscopy, a drop of the water suspension containing the sample was placed on the electron microscopy grid. After one minute, the excess was removed using blotting paper and thereafter dried in air. The electron microscopy grids (Plano, Germany) were covered with a holey carbon film.

2.5. Scanning electron microscopy (SEM) analysis
The samples were fixed in a sample holder and covered with carbon, or with a gold layer for 1 minute using an Edwards S150B sputter coater. The samples were then placed in an ESEM XL 30 Philips or LEO DSM 982 Gemini scanning electron microscope.

2.6. Chitinase digestion and test
Dried 20 mg samples of purified sponge spicules, previously pulverized to a fine powder in an agat mortar, were suspended in 400 μL of 0.2 M phosphate buﬀer at pH 6.5. Positive control was prepared by solubilizing 0.3% colloidal chitin in the same buﬀer. Equal amounts of 1 mg/mL of three chitinases (EC 3.2.1.14 and EC 3.2.1.30): N-acetyl-βglucosaminidase from Trichoderma viride (Sigma-Aldrich) number C-8241, and two poly (1,4-β-[2-acetamido-2-deoxy-D-glucoside]) glycanohydrolases from Serratia marcescens (Sigma-Aldrich) number. C-7809 and Streptomyces griseus (Sigma-Aldrich) number. C-6137, respectively, were suspended in 100 mM sodium phosphate buﬀer at pH 6.0. Digestion was started by mixing 400 μL of the samples and 400 μL of the chitinase-mix. Incubation was performed at 37◦ C and stopped after 114 hours by adding 400 μL of 1% NaOH, followed by boiling for 5 minutes. The eﬀectiveness of the enzymatic degradation was monitored using optical microscopy (Zeis, Axiovert). The Morgan-Elson assay was used to quantify the N-acetylglucosamin released after chitinase treatment as described previously [22]. The sample which contains chitinase solution without substrate was used as a control.

2.7. Preparation of α-chitin
Alpha-chitin was prepared from a commercially available crab shell chitin (Fluka). The material was purified with aqueous 1 M HCl for 2 hours at 25◦ C and then refluxed in 2 M NaOH for 48 hours at 25◦ C. The resulting α-chitin was washed in deionized water by several centrifugations until neutrality was reached. The whole procedure was repeated twice. α-chitin was also used as a standard for FTIR and for Fourier transform (HRTEM) studies.

2.8. Preparation of colloidal chitin
Ten grams of α-chitin (Fluka) was mixed with 500 mL of 85% phosphoric acid and stirred for 24 hours at 4◦ C. The suspension was poured into 5 L of distilled water (DW) and centrifuged (15000× g for 15 minutes). The resulting precipitate was washed with DW until the pH reached 5.0 and then neutralized by addition of 6 N NaOH. The suspension was centrifuged (15000× g for 15 minutes) and washed with 3 L of DW for desalting. The resulting precipitate was suspended in DW and dialyzed. The chitin content in the suspension was determined by drying a sample.

2.9. Silicification of colloidal chitin and NAG
In the first step, 0.93 g of colloidal chitin or NAG (SigmaAldrich, Miss, USA) previously suspended in 34.6 g of methanol was added to 51.8 mL of deionized water. The suspension pH was then raised above 10 with the addition of 100 μL of 1 N NaOH solution. Finally, 169 μL of tetramethylorthosilicate (TMOS, 99 wt%, ABCR GmbH, Germany) were added and the solution was stirred at room temperature. After 1 hour the suspension was filtered and the recovered precipitate rinsed with deionized water, then with methanol, and finally air-dried.

3. RESULTS AND DISCUSSION
Most of the glass sponges inhabit soft muddy substrates. One of the strategies of survival under such conditions is the formation of root structures that prevent the body of the animal from sinking into the ground [23]. Due to their preferred deep-sea habitat, the Hexactinellida have been poorly investigated with respect to their general biology [24, 25] and the nature of organic components which build their skeletal