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Figure 2: TEM images of silica-organic matrix-based suspension obtained after mechanical disruption of spicules in deionized water (a). Crystallites of organic nature are embedded in amorphous silica (b).

structures. It was generally accepted that their skeletons are composed of concentric layers of amorphous hydrated silica, containing varying amounts of organic material [26, 27] deposited around a proteinaceous axial filament [28, 29]. The finding of collagen within basal spicules of H. sieboldi [12, 16] and chitin in skeletons of F. occa [17] and in spicules of E. aspergillum [3], stimulated our attempts to find materials of organic nature in other species of glass sponges. In the case of R. fibulata, we have not observed any visible signs of demineralization of these materials using optical microscopy and SEM after 14 days and at the similar experimental conditions as in the study on H. sieboldi and Monorhaphis sp. On the contrary, spicules of R. fibulata show high resistance to alkali treatment even after 3 months of demineralization. This was similar the resistance observed for E. aspergillum [3]. This phenomenon led us to the assumption that siliceous skeletons of investigated sponges possess a material which protects amorphous silica from dissolution in alkali, and is highly resistant to alkali digestion. It is well known that chitin in alkali is stable with respect to degradation [30]. Correspondingly, in our experiments, chitin was the first candidate for a biomaterial with this property. Initially, we performed experiments on mechanical disruption of cleaned R. fibulata spicules (Figure 1(a)) in deionized water as described above. This method of desintegration of spicules was very eﬀective. Visual observations show that water solution became of a milky color typical for silica colloidal suspensions (Figure 1(b)) even after 6 hours. Debrisfree suspensions obtained in this way were stable during 3-4 days. SEM of the suspensions confirmed their nanoparticular structure (Figure 1(c)). Silica nanoparticles of diameter between 20 and 35 nm are associated around oriented organic matrix of nanofibrillar nature as shown in Figure 1(c). To verify whether this kind of silica-organic matrix obtained from the colloidal suspension mimic the biosilicification, we carried out in vitro experiments in which we exposed it to silicic acid solution derived from TMOS. We developed hard and transparent glassy spheres (Figure 1(d)) which were stable in water and in air during several months. TEM investigation of these colloidal suspensions (Figure 2(a)) used for the development of spherical glassy

Figure 3: FTIR spectra of organic matrix isolated after desilicification of R. fibulata spicules show strong evidence for β-1, 4glycosodic linkage at 890–896 cm−1 and for ether bond in pyranose ring at 1153–1157 cm−1 (arrows).There is no evidence for the presence of Si–O–Si bonds.

Figure 4: High-resolution transmission electron microscopy image of the fragment of isolated chitin nanofibril (a); the arrows indicate the presence of crystallite-like structures with a diameter which corresponds to that of chitin crystallites (2 nm). AFM micrograph of chitin nanofibrillar matrix (b).

materials clearly revealed that organic crystallites of approximately 3 nm in diameter are embedded in amorphous silica matrix. Observed HRTEM image (Figure 2(b)) is highly similar to previously reported HRTEM images of chitin nanocrystallites of the same diameter [31]. Therefore, in following experiments it was decided to isolate organic matrix from silica-containing spicules of R. fibulata using a desilicification procedure based on alkali treatment [16, 17]. To test our hypothesis that alkali-insoluble residues of R. fibulata spicules are of chitinous nature, we carried out different highly sensitive structural and biochemical analysis as described below. FTIR observation of purified, dialysed, and dried samples of the alkali-insoluble organic matrix isolated after demineralization of R. fibulata spicules (Figure 3) confirmed occurrence of pyranose rings and β-1, 4 linkages, with peaks at 1156 cm−1 and at 894 cm−1 very similar to those of α-chitin. It was reported previously that the spectral feature between 1153 and 1157 cm−1 is mainly associated with an ether bond in a pyranose ring [32, 33]. The β-linkage was also indicated