calcite reinforced silica–silica joints in the ...€¦ · skeleton of deep-sea glass sponges the...

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Hermann Ehrlich ,* Eike Brunner , Paul Simon , Vasily V. Bazhenov , Joseph P. Botting , Kontantin R. Tabachnick , Armin Springer , Kurt Kummer , Denis V. Vyalikh , Serguei L. Molodtsov , Denis Kurek , Martin Kammer , René Born , Alexander Kovalev , Stanislav N. Gorb , Petros G. Koutsoukos , and Adam Summers

Calcite Reinforced Silica–Silica Joints in the Biocomposite Skeleton of Deep-Sea Glass Sponges

The hierarchically structured glass sponge Caulophacus species uses the fi rst known example of a silica and calcite biocomposite to join the spicules of its skeleton together. In the stalk and body skeleton of this poorly known deep-sea glass sponge siliceous spicules are modifi ed by the addition of conical calcite seeds, which then form the basis for further silica secretion to form a spinose region. Spinose regions on adjacent spicules are then joined by siliceous crosslinks, leading to unusually strong cross-spicule linkages. In addition to the biomaterials implications it is now clear, from this fi rst record of a biomineral other than silica, that the hexactinellid sponges are capable of synthesizing calcite, the ancestral skeletal material. We propose that, while the low concentrations of calcium in deep sea waters drove the evolution of silica skeletons, the brittleness of silica has led to retention of the more resil-ient calcite in very low concentrations at the skeletal joints.

1. Introduction

By virtue of their embedded, mineralized skeletons, sponges are of great interest to materials scientists, chemists,

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DOI: 10.1002/adfm.201100749

Dr. H. Ehrlich , Prof. Dr. E. Brunner , M. Kammer Institute of Bioanalytical Chemistry Dresden University of Technology Bergstr. 66, D-01069 Dresden, Germany E-mail: [email protected] Dr. P. Simon Max Planck Institute of Chemical Physics of Solids Noetnitzer Str. 40, D-01187 Dresden, Germany V. V. Bazhenov Institute of Chemistry and Applied Ecology Far Eastern National University Sukhanova 8, 690650 Vladivostok, Russia Dr. J. Botting Leeds Museum Discovery Centre Carlisle Road,1, Leeds LS10 1LB, UK Dr. K. Tabachnick P.P. Shirshov Institute of Oceanology RAS Nahimovski Prospect 36, 117312 Moscow, Russia Dr. A. Springer , R. Born MBZ and Institute of Materials ScienceDresden University of Technology Budapester Str. 27, D-01069 Dresden, Germany

Dr. K. Kummer , Dr. D. Institute of Solid StateDresden University of Zellescher Weg 16, D-0 Prof. Dr. S. L. MolodtsEuropean XFEL GmbHNotkestr. 85, D-22607 Dr. D. Kurek Centre “BioengineerinProspekt 60-ya Oktiabr Dr. A. Kovalev , Prof. DrFunctional MorphologZoological Institute Christian-Albrecht-UniAm Botanischen Garte Prof. P. Koutsoukos Department of ChemicUniversity of Patras 1, Stadiou Str., GR-265 Prof. A. Summers Friday Harbor LaboratoUniversity of Washingt620 University Road, F

palaeontologists, geologists, and biologists. As the most basal metazoans, they are the key to understanding the evolution of both calcium- and silicon-based biomineraliza-tion. The manifestation of this minerali-zation, a skeleton of spicules embedded in the body of the sponge, is typically a complex arrangement of calcite or silica. For example, the skeletal spicules of glass sponges (Hexactinellida, Porifera) are valu-able model systems for the investigation of structure-function relationships in bio-materials, with the ultimate goal of iden-tifying design strategies for new synthetic materials. [ 1 ] Natural structural composites often show a variety of desirable properties, including transient properties in the pres-ence of water. [ 2 , 3 ] The hexactinellid sponges have silica skeletal structures that are both

fl exible and tough, [ 1 , 4 , 5 ] because of their hierarchically layered structure and the hydrated state of the silica. [ 6 ] Emblematic of the complexity of sponge skeletons is Euplectella aspergillum , in which the skeleton is an elaborate cylindrical latticelike

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structure with at least six hierarchical levels of organization spanning the length scale from nanometers to centimetres. [ 5 ] The basic elements are laminated spicules that consist of a proteinaceous axial fi lament surrounded by alternating concen-tric domains of consolidated silica nanoparticles and organic interlayers.

Spicules assigned to hexactinellids are known from the ear-liest part of the animal fossil record (Ediacaran, 630 to 542 My). [ 7 ] Their skeletons appear to have been optimized by natural selec-tion to provide structural support, and as an inert scaffold for various tissues encompassing a variety of functions. Organic molecules including silicateins, [ 8 ] or more recently discovered collagen [ 9 ] and chitin [ 10–12 ] are responsible for both the biosil-icifi cation and for the specifi c mechanical properties of their skeletons. These molecules and their functions are currently the subject of detailed discussions in modern chemical biology. The skeletal structures of glass sponges are also remarkable due to their size, durability, fl exibility, and optical properties, [ 13 ] however, numerous questions regarding the chemistry/biology interface of these mostly unique constructions remain open.

Deep-ocean currents are generally perceived as sluggish, but at intermediate depths (1000 to 5000 m) a is steady fl ow is present, in which the current speeds up and slows down as it encounters variations in terrain. [ 14 ] Sponges are sessile organisms that fi lter great volumes of seawater to collect food particles and resolved organic matter, and they rely partly on ambient water currents; many are also raised above the sea fl oor on spicular stalks in order to better exploit them. Photographs of deep-sea hexactinellids, in natural conditions, demonstrate remarkable fl exibility and resilience of the stalks. [ 14 ] Because the spicules are made from a brittle material this fl exibility is unexpected. The primary goal of the current study was to inves-tigate the chemistry and the peculiarities of structural organiza-tion in the spicular stalk and body spicules of the hexactinellid sponge Caulophacus species . ( Figure 1 ). [ 15–17 ]

2. Results

2.1. Clublike Structures Within the Sponge’s Skeletal Framework

Caulophacus , including its subgenus C. (Caulophacus) , are mushroom-shaped sponges that are attached to hard substrates by a long stalk. The atrial cavity is turned out, and represented by the upper surface of the cap (Figure 1 A,B). The tubular stalk is primarily constructed from long diactine (two rayed) spicules that are connected to their neighbors by numerous articulations (Figure 1 C–E) or by bridges (Figure 1 F–G) through secondary silica deposition. The stalk is fi xed to the substratum by a basy-dictional plate, which contains mostly hexactines (six-rayed spi-cules) fused to each other at points of contact. The subgenus is cosmopolitan, being distributed in all oceans and in some seas with “normal” salinity at a depth of 130–6770 m. Currently about 15 species are known.

Individual spicules were isolated from the Caulophacus sp . stalk using pincers. We observed clublike constructions ( Figure 2 A,B) at the ends of separated spicules using scanning electron microscopy (SEM). These spinose, clublike structures

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were also observed in light microscopy images (Figure 2 C). To obtain more detailed information about localization of these specifi c structures within the spicular articulations, we employed gentle desilicifi cation using a 2.5 M NaOH solution at 37 ° C, as described previously. [ 9 , 10 ] After 14 days of immersion, the clublike constructions could be observed as being located within articulations using both light microscopy (Figure 2 D) and SEM (Figure 2 E,F). Detailed analysis of the SEM images [ 18 ] revealed that the spiny surfaces on these spicules could be coupled with each other, preventing slippage against the cor-responding silica-based articulation ( Figure 3 ). This structure is unique and evolved as a natural engineering solution to struc-tural integrity problems in the construction of the hierarchical skeletons of glass sponges.

Siliceous monaxon spicules with various spination pat-terns are well described in the sponge literature. [ 19 ] It has been hypothesized that this phenomenon results from hypersilicifi ca-tion related to a high silica concentration in water. [ 20 ] However, during alkali-based desilicifi cation experiments we observed that the spines of the clublike structures were highly resistant to alkali treatment, in contrast to the surface silica layers, which are deeply corroded under these conditions (Figure 3 ).

2.2. Identifi cation of Calcite-Silica Composite

In order to proceed with more detailed studies, we mechanically disrupted several spicules showing the clublike constructions, as shown in Figure 4 A. SEM images of these broken elements show that the spines possess central structures, covered with a layer of silica (Figure 4 B). Elemental mapping (Figure 4 C) and energy dis-persive X-ray spectroscopy (EDX) (Figure 4 D) unambiguously show that calcium and not silicon is the main component of this central structure (see the Supporting Information, Figure S1). Atomic absorption spectroscopy [ 18 ] also confi rmed the presence of calcium in this spicular material , at an average concentration of 37.8 ± 9.0 ng mg − 1 (mean ± standard deviation, s.d.), corresponding to 3.78 ± 0.90 mass percent of calcium in the whole spicule.

The chemical nature of the mineral phases within the club-like structures was then investigated by means of photoemission spectroscopy (PES) and X-ray absorption spec-troscopy (XAS) (Figure 4 E,F), performed on untreated spi-cules of Caulophacus sp. The appearance of Si 2p photoemis-sion demonstrates the presence of silicon in all spicules, and the exact position of the peak on the binding energy scale grants an insight into its chemical bonding. Pure silicon is observed at 100 eV bond energy, whereas SiO 2 is found at 105 eV. SiO x compounds are observed between 100 and 105 eV according to their stoichiometry (Figure 4 E). The observed bond energy of about 103 eV suggests that the silicon com-pound in the spicules has a silicium-to-oxygen ratio of 1:1,2. Therefore we hypothesize that silicon oxide is present in these skeletal structures in a bound form. This stoichiom-etry rules out foreign particles as a source of the silica signal. Also, XAS spectra obtained for each of the sponge samples (Figure 4 F) show a strong signal at the Ca 2p absorption edge. The fi ne structure of the near-edge X-ray absorption pat-tern closely resembles that of calcite. Transmission electron microscopy (TEM) and electron diffraction analysis (EDA) of

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Figure 1 . Complex organization of Caulophacus sp . spicular stalks. a) Caulophacus sp . on the rocky surface, 15 cm tall. b) The stalks of this sponge possess a complex network of glassy spicules (c). d,e) Scanning electron microscopy (SEM) images show that spicules within stalks are connected to each other with scaffoldlike articulations. f) SEM image that shows parallel spicules connected to each other by small crosslinks. g) Computer reconstruction of these articulations.

the untreated clublike structures ( Figure 5 A,B) further confi rm these results. TEM diffraction analysis of the spicule shows the presence of two phases, one amorphous (Figure 5 C) and one crystalline (Figure 5 D). EDA for the spines shown as a TEM image in Figure 5 B correspond to the (0001) zone axis of calcite with d spacings: 100 (1010) 4.32 Å; 010 (0110) 4.32 Å and 210 (2110) 2.49 Å. These data correlate well with those reported previously for calcite identifi ed in other invertebrates using the electron diffraction technique. [ 21 ] The microstructure of the central calcite crystals isolated by alkali treatment from the clublike structures (Figure 5 D) is also very similar to the morphology of calcite crystal nuclei described previously [ 18 , 22 ] (see Supporting Information, Figure S2). Further analysis of the crystals (Figure 5 E) isolated from clublike structures

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after alkali-based desilicifi cation was achieved by infrared (IR) (see the Supporting Information, Figure S3) and Raman (Figure 5 F) spectroscopy. The obtained FTIR spectra as well as Raman spectra unambiguously confi rm the calcite nature of these crystals.

3. Discussion

Mechanical testing of the spicules suggests an important strengthening role for the observed structures. Micro-indentation tests of the sponge architecture demonstrated only slight plastic deformation (one micrometer or less), compared with tens of micrometers of elastic deformation. Moreover,

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Figure 2 . Clublike structures within the Caulophacus sp . skeleton. a,b) Isolated and broken stalk spicules, separated using pincers, showing clublike structures. c) Clublike termination of a spicule observed using light microscopy. d) After immersion of the stalks in 2.5 M NaOH for two weeks, partial dissolution of silica-based articulations revealed that club-like construction (arrows) are located within articulation joints. e,f) SEM images of the partially dissolved articulations confi rm the observations made using light microscopy.

buckling events were clearly seen on the indentation curves (see the Supporting Information, Figure S4A). The sponge has rigid outer layer (thickness 150 μ m) where the spicules are inter-connected more densely. This area has a reduced E-modulus (REM) of 14.9 ± 4.5 MPa with the minimal measured value of 1.45 MPa. The bulk material of the sponge is softer and is rela-tively isotropic. The REM of the sponge in the axial direction

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was 614 ± 173 kPa, whereas in areas where single spicules pro-trude, it was just 3.82 ± 1.34 kPa (see the Supporting Informa-tion, Figure S4). The inner surface of the sponge has a reduced number of cross-linking spicules and spicules with orientation parallel to the surface, which explains the small REM values of the innermost part of the sponge (REM 4.3 ± 0.5 kPa). How-ever, after 100 μ m of compression of the soft layer, the sponge


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Figure 3 . Clublike spicules determining the structural integrity of Caulophacus sp . sponge stalks. a) SEM images show that clublike structures are responsible for mechanical coupling between two thin spicules within one large spicule. b) Corresponding computer reconstruction of this coupling. c,d) Alkali treatment led to dissolution of silica, allowing observation of the spines of clublike constructions using SEM, on the tenth day of immersion in alkali. e,f) The spines seem to be resistant to alkali treatment in contrast to the weakly corroded silica-based material that covers the spicules and forms the articulations.

material has REM of 618 ± 200 kPa (see the Supporting Infor-mation, Figure S4B), equaling that of the bulk sponge material.

The challenge now is to understand the mechanisms by which this unique biocomposite is initially formed, at high pres-sure (420 ATM) and at low temperature (between 2 and 4 ° C). In most cases, calcifi cation in sponges is confi ned to an extracel-lular space delineated by tightly adhering sclerocyte cells. [ 20 ] The sclerocytes secrete a macromolecular matrix to the extracellular space, which comprises a genetically programmed, three-dimensional framework that guides spicule formation. It is diffi -cult to differentiate between organically mediated and inorganic precipitation, and a combination of different mechanisms in the same species cannot be ruled out. A decisive factor for the control of nucleation of calcium carbonate is the development of local

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supersaturation with respect to calcite or to any other calcium carbonate polymorph. This in turn depends on the levels of cal-cium and carbonate ion activities. The latter depend strongly on local pH as the dissolution of CO 2 is favored at higher pH values. Seawater may become supersaturated with respect to calcium carbonate especially in spatially confi ned structures. The inner space of the Caulophacus sp . spicule represents a microchannel of approximately 1.0 μ m in diameter fl ooded with ionic solu-tion, while one extremity of the spicule is sealed by the silica. At these conditions, contact with silicic surfaces is expected to favor heterogeneous nucleation of calcite, as in this case lower super-saturation is needed in comparison with the value needed for homogeneous. For example, calculation of the saturation of cal-cite at 2,000 meters, 5 ° C and pH between 7.62 and 8.22 yielded

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Figure 4 . Identifi cation of mineral components within clublike constructions. a) The SEM image of a mechanically disrupted clublike spicule shows that the spines each possess a nucleus that is covered by silica layer. b) Magnifi cation of (a). Elemental mapping (c) and EDX analysis (d) show the presence of calcium as the main component of this nucleus. e) Photoemission spectra of club-formed spicule showing the presence of two kinds of silicon oxides. f) X-ray absorption spectra showing that calcium carbonate in the form of calcite is the second mineral component present within the spicules.

values between 49 and 184, respectively. [ 23 ] Calcite is expected to precipitate at a saturation exceeding 100, a value which corre-sponds for the ion activity of seawater to pH 8.00.

The observed morphology of this silica–calcite biocomposite can be understood as the result of the crystallization of cal-cite crystals in the presence of polymeric silica. [ 24 ] Garcia-Ruiz et al. [ 25 ] obtained numerous peculiar forms of alkaline-earth metal carbonates in high-pH silica gel, which indicated that silicate anions interact strongly with carbonates. Furthermore, layers of silicates have been deposited on the surface of cal-cite, [ 26 , 27 ] a fact supporting crystallographic affi nity for the two inorganic solids. It has also been shown in vitro that a complex crystalline architecture can be induced through the miniaturi-zation of the growth subunits by covering the surface of car-bonate crystals with silicate anions and by the self-organized assembly of the miniaturized subunits. [ 28 ] The silica–calcite-

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based clublike structures made by Caulophacus sp . are the fi rst natural example of this principle, which confi rms that biomin-eralization is a process in which organisms produce mineral solutions for their own functional requirements, [ 29 ] starting on nano- and microlevels of structural hierarchy.

Although poriferan skeletons may be composed of a large variety of minerals, calcium carbonate and siliceous structures very rarely co-occur in the same sponge. The only previously known examples are in the form of solid calcareous skeletons in addition to siliceous spicules, or as small calcareous spher-ules [ 30 ] or granules [ 31 ] in siliceous spiculate sponges. None of these constitute biocomposites, and no sponge with calcareous spicules has yet been found to secrete silica also.

It is less clear how the skeletons of early sponges were con-structed, and this is of great signifi cance in palaeontology and early animal phylogeny. It has been argued that the different


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Figure 5 . Calcite crystals within club-like structures. a) TEM image of the native Caulophacus sp . spicule with a fragment of club-like structure (arrow) (B). Spicule chosen for selected area electron diffraction (SAED). c,d) TEM diffraction analysis of this spicule shows the presence of two phases: amorphous with a diffuse amorphous halo at 2.05 Å (c) and crystalline (d). Electron diffraction pattern corresponding to the spines shown as a TEM image in (b) corresponds to the calcite lattice with d spacings of 4.32 Å (101) and 2.49 Å (210). Crystals isolated after alkali-based desilicifi cation [ 10 ] of club-like structures are well visible using SEM (e). Their Raman spectra (f, above) are absolutely similar with those of calcite standard (f, below).

secretion mechanisms of spicules in Silicispongia ( Demospongiae and Hexactinellida ) and Calcarea preclude homologous biomin-eralization between the sponge classes. [ 32 ] However, the heterac-tinid sponge Eiffelia globosa shows features characteristic of both groups, and spicules have a bilayered structure suggestive of a

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silica–carbonate biocomposite. [ 33 ] The presence of a silica–calcite composite in extant hexactinellids suggests that the mecha-nism of secretion is fl exible in the precise structures formed,and that extant and early sponges share a common biochemical basis for secreting both materials. Although the structures of the

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skeletal elements themselves may have changed, as well as the cytological architecture, the mineralogical secretion processes are likely to be homologous. If similar structures to those in Caulo-phacus are confi rmed in other taxa, the widespread presence of sil-ica–calcite composites in the silicisponge lineage but not the calcar-eans, would tend to support an ancestral carbonate spicule miner-alogy for Porifera . More data are needed from other sponge groups before the full implications of such fi ndings will be known.

4. Conclusions

Structural design of biological materials has evolved under extreme evolutionary pressures to ensure the survival of a spe-cies, often in adverse environments. [ 34 ] We can now outline the factors that are important for the highly deformable and resil-ient structure of the glass sponge stalk. Typically, slender rods of small dimension are stiffer than expected. [ 17 ] Stress concen-tration near holes and notches is dissipated by materials with microstructure such as the glass sponge stalk. In a foamlike construction, a stiff and brittle material of single cells contrib-utes to the volume, but not to the stiffness of the construction. In foamlike sponge construction, the largest structural fi bre-like elements appear to govern the fracture toughness and the localization of microdamage. Twisting or bending of the fi ber-based construction is an additional degree of freedom that dis-sipates stress. Additionally, clavate-shaped, spine-covered ends of spicule, which are made of crystalline calcite, are responsible for a mechanical interlocking of single spicule with each other. A combination of two different materials at the level of single spicule (see the Supporting Information, Figure S5), the inter-locking system between spicules, and the fi brous composite construction of the sponge stalk, make the entire construction mechanically stable and simultaneously very fl exible under mechanically challenging environmental conditions. Intrigu-ingly, the concept of turning weakness into strength based on the university-diversity-principle (UDP), [ 34 ] in the case of Caulophacus sp . sponge, can be discussed as very specifi c because of the presence of two different inorganic materials. In contrast to this siliceous sponge, diatoms, for example, can transform a brittle biosilica into a ductile, strong, and tough material, solely through alterations of its structural arrangement at the nanoscale. [ 35 , 36 ] Thus, mechanical response of diatoms cell walls can be greatly altered by simple alteration of their structural geometry without the need to introduce new con-stituents. [ 35 ] Clearly, the future development of the comparative computational models [ 37 ] of glass sponges and diatoms, as two different biosilica-based and nanostructurally organized hierar-chical systems, represents a scientifi c task of great interest.

The silica–calcite composite structures described here rep-resent an additional example of multiphase biomineralization-phenomenon recently discussed by us. [ 38 ] Up to know, is is not known why some organisms utilize, for example, silica rather than calcium carbonate as a structural material. However, in the case of extreme deep-sea environmental conditions, Mother Nature decides to use both mineral phases within one very complex designed skeletal network, which is defi nitively essen-tial for biological survival of the fi ttest organism.

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5. Experimental Section Sponge Samples : The investigated Caulophacus sp . specimens were

captured during the 22 nd cruise of the R.V. “Academic Mstislav Keldysh” in the N-E Pacifi c, off Bering Island, stn. 2316, 55 o 36,08-35’N 167 o 23,04-24,46’ E, 4200-4294 m. Stalks of Caulophacus sp . were treated according to the following procedure. Sponge material was stored for several days in fresh sea-water. The sponge was dried afterwards for 4 days at 45 ° C. Finally the sponge skeletal material was cleaned in 10% H 2 O 2 and dried again at 45 ° C. Tissue-free dried sponge material was washed three times in distilled water, cut into 3 cm long pieces and placed in a solution containing purifi ed Clostridium histolyticum collagenase (Sigma) to digest any possible collagen contamination of exogenous nature. After incubation for 24 h at 15 ° C, the pieces of glass sponge skeleton were again washed three times in distilled water, dried and placed in a 15 mL vessel containing chitinase solution (as described by us previously) [ 10 ] to digest any possible exogenous chitin contaminations. After incubation for 48 h at 25 ° C, fragments of skeleton were again washed, dried, and placed in 10 mL plastic vessels for storage and analysis.

Chemical Etching of Glass Sponge Skeleton : 8 mL of 2.5 M NaOH (Fluka) solution was added to the 10 mL plastic vessel, which contains fragments of the Caulophacus sp . stalks, purifi ed as described above. The vessel was covered and placed under thermostatic conditions at 37 ° C without shaking. The same procedure was carrying out using 0.01% solution of HF (puriss. p.a., Fluka). The effectiveness of the alkali desilicifi cation procedures was also monitored using optical microscopy and SEM at different locations along the length of spicular material and within the cross-sectional area.

Atom Absorption Spectroscopy (AAS) : A Varian SpectrAA-10 spectrometer with Varian GTA-96 graphite furnace atomizer was used to determine the concentration of calcium within the purifi ed samples of Caulophacus. sp . spicules. Three samples were weighed into Eppendorf microvials, dissolved by adding 100 μ L HF to each sample and homogenised for 30 s by shaking. Each sample was diluted with 900 μ L purifi ed water and again homogenised. A wavelength of 239.9 nm was used to measure absorption by calcium.

Photoemission Spectroscopy (PES) and X-ray Absorption Spectroscopy (XAS) : Measurements were performed at MAX-lab (Lund University, Sweden) using radiation from the beamline D1011 bending magnet dipole beamline located at the MAX II storage ring. X-ray absorption spectra in the vicinity of the Ca 2p absorption threshold were recorded in total electron yield (TEY) mode with a MCP detector. Photoemission of the Si 2p core-level was obtained at 200 eV photon energy using a SCIENTA SES200 electron energy analyzer.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements We thank Prof. H. Lichte for use of the facilities at the Special Electron Microscopy Laboratory for high-resolution and holography at Triebenberg, TU Dresden, Germany. The authors thank R. Lakes, G. Wörheide, G. Bavestrello for helpful discussions. The authors are deeply grateful to R. Schulze, H. Meissner, G. Richter, O. Trommer for excellent technical assistance. This work was partially supported by the DFG (Grant Nos. EH 394/1-1; MO 1049/5-1, ME 1256/7-1 ME 1256/13-1, and WO 494/17-1) and by the BMBF (Grant No. 03WKBH2G), by a joint program “Mikhail Lomonosov - II” of DAAD (Ref-325; A/08/72558) and RMES (AVCP Grant Nr. 8066), and by Erasmus Mundus Co-operation Window Programme 2009. Part of this study was supported, as part of the European Science Foundation EUROCORES Programme FANAS, by the German Science

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Foundation DFG (contract No GO 995/4-1) and the EC Sixt Framework Programme (contract No ERAS-CT-2003-980409) to SNG.

Received: April 4, 2011 Published online: July 29, 2011

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calcite reinforced silica–silica joints in the ...€¦ · skeleton of deep-sea glass sponges the...

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