Acta Biomaterialia 42 (2016) 258–264
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Full length article
Coccospheres confer mechanical protection: New evidence for an old hypothesis B.N. Jaya a,⇑, R. Hoffmann b, C. Kirchlechner a, G. Dehm a, C. Scheu a, G. Langer c,⇑ a
Max-Planck-Institut für Eisenforschung GmbH, 40237 Düsseldorf, Germany Department of Chemistry, Ludwig-Maximilian-University, 81377 Munich, Germany c The Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth, Devon PL1 2PB, UK b
a r t i c l e
i n f o
Article history: Received 5 May 2016 Received in revised form 27 June 2016 Accepted 19 July 2016 Available online 21 July 2016 Keywords: Coccospheres Calcite In-situ compression Mechanical protection
a b s t r a c t Emiliania huxleyi has evolved an extremely intricate coccosphere architecture. The coccosphere is comprised of interlocking coccoliths embedded in a polysaccharide matrix. In this work, we performed insitu scanning electron microscopy based compression tests and conclude that coccospheres have a mechanical protection function. The coccosphere exhibits exceptional damage tolerance in terms of inelastic deformation, recovery and stable crack growth before catastrophic fracture, a feature, which is not found in monolithic ceramic structures. Some of the mechanical features of the coccospheres are due to their architecture, especially polysaccharide matrix that acts as a kind of bio-adhesive. Our data provide strong evidence for the mechanical protection-hypothesis of coccolithophore calcification, without excluding other functions of calcification such as various biochemical roles discussed in the literature. Statement of Significance Although bio-mechanics of shell structures like nacre have been studied over the past decade, coccospheres present an architecture that is quite distinct and complex. It is a porous cell structure evolved to protect the living algae cell inside it in the oceans, subjected to significant hydrostatic pressure. Despite being made of extremely brittle constituents like calcium carbonate, our study finds that coccospheres possess significant damage tolerance especially due to their interlocking coccolith architecture. This will have consequences in bio-mimetic design, especially relating to high pressure applications. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Organisms produce extraordinary crystals in terms of shape and properties. Echinoids for example are able to regenerate their spines, while maintaining the original pattern of the macrostructure [1]. The greatest number of biologically formed crystals belong to the classes of sulfides, sulfates, phosphates and carbonates [2]. Coccolithophores are unicellular haptophyte algae, which produce calcium carbonate platelets (coccoliths) and assemble them into a hollow sphere, the so called coccosphere. Since the hard inorganic parts of coccolithophores are built inside the cell, they differ from those of most other calcifiers. The most common species of the approx. 200 extant species is Emiliania huxleyi (E. huxleyi) [3]. The coccospheres of this species are ca. 5–10 lm in diameter and consist of several layers of coccol-
⇑ Corresponding authors. E-mail addresses: [email protected] (B.N. Jaya), [email protected] (G. Langer). http://dx.doi.org/10.1016/j.actbio.2016.07.036 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
iths that are interlocked and glued to each other by means of polysaccharides (Fig. 1a, [4,5]). The strain RCC1238 used in this study possesses on average 2–3 coccolith layers and 20 coccoliths per cell [4] (Fig. 1b). The coccoliths are similar to a cable reel, consisting of a central tube that connects the lower proximal shield and the upper distal shield and encloses the central area (Fig. 1c and d) [6]. This morphology enables the coccoliths to interlock closely on the coccosphere and form a robust structure [6]. A single segment of an E. huxleyi coccolith comprises of two crystal units, the radial R-unit that is built up of a single crystal with the c-axis oriented parallel to the coccolith plane and the c-axis of the vertical V-unit is perpendicular to the coccolith plane (Fig. 1d grey arrow) [7,8]. This differs from other biological crystals like the ones of bone, corals, sea urchin and mollusk shells, which show a mosaic like crystal assembly [9–14], i.e. consisting of co-oriented crystallites in the mesoscopic size range (1–1000 nm) [15]. It was suggested that these mesocrystal-composite structures, e.g. mollusk shells, have a high fracture toughness compared to the one of single crystalline structures [14].
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
259
Fig. 1. SEM micrograph of (a) a complete and (b) a FIB cut coccosphere of the investigated E. huxleyi strain RCC1238. An individual coccolith and the interlocking of coccoliths are marked with white lines. In (c) secondary electron images of a coccolith viewed from top and bottom are shown. The model of a single discrete element of a coccolith is displayed in (d) (modified after [34,8]), showing the morphologically complex bi-crystalline calcite units (primary R and interstitial V whose orientations are shown by arrows) [7].
Despite decades of research the function of calcification in coccolithophores is a matter of debate, with the seminal discussion by Young [16] (see also the extended version, Young [17]) still being the central work of reference. Among the many hypotheses put forth, a mechanical/protective role of coccospheres was suggested more than a century ago [18], and remains one of the most plausible ideas [16]. The type of protection provided by coccospheres is not identical to the one provided by diatom frustules. While the latter protect the algae from macro-grazers such as copepods, coccospheres do not seem to have this function [19,20]. This, however, is not due to the mechanical properties (i.e. stability) of the coccosphere, but merely reflects the difference in prey/predator size ratio. While copepods, more often than not, have to destroy the diatom frustule in order to eat the diatom, coccolithophores are swallowed whole and coccoliths are only accidentally crushed by copepod mandibles [21,22]. Coccospheres most likely confer a general mechanical stability to the algae. This hypothesis has traditionally been based on morphological observations, but still awaits corroboration by micro-mechanical measurements [18,23,16]. In other words, how much load is needed to break a coccosphere? The mechanical stability of diatom frustules is regarded as being instrumental in the evolutionary success of diatoms [24,25,20]. The average force needed to break a diatom frustule ranges from 0.2 to 0.8 mN [20]. If the coccospheres also showed the ability to accommodate increasing loads by inelastic dissipation before undergoing fracture, this would be strong evidence in favor of the mechanical protection role of coccospheres. In this study, we performed in-situ compression measurements on coccospheres of E. huxleyi by means of a flat punch indenter acting as a micro-crusher inside the scanning electron microscope (SEM). This allows us to determine the loads corresponding to the initial deviation from elastic deformation and the maximum
load before fracture and collapse of the coccosphere occurs. Simultaneously, the deformation process was closely monitored for signs of failure of coccospheres, individual coccoliths and coccolith segments.
2. Experimental procedure Coccospheres cultured in aged, sterile-filtered (0.2 lm pore-size cellulose-acetate filters) North Sea seawater enriched with 100 lmol L 1 nitrate, 6.25 lmol L 1 phosphate, trace metals and vitamins as in f/2 medium [26] were filtered onto an Omnipore polycarbonate membrane filter by using a vacuum pump. The filter was dried at 60 °C and the material was afterwards removed with a spatula and dissolved in ethanol. The sample was then dropped on a silicon wafer and dried. In the end a thin carbon film was deposited to avoid charging effects in the SEM. For more details on the coccolithophore culturing see Langer et al., [27] and Hoffmann et al., [4]. Loading was carried out in situ using the ASMEC Unat II indenter (ASMEC GmbH, Radeberg, Germany) inside a JEOL-JSM 6490 tungsten filament SEM. Individual coccospheres were loaded in compression under displacement control at a rate of 10 nm/s. This represents a uniaxial testing condition, which is different from the hydrostatic (tri-axial) stress state normally experienced by the coccospheres under water. Uniaxial compression of the sphere in the vertical direction leads to tensile opening stresses in the perpendicular direction, in addition to flexural (bending) loading of individual coccoliths. This is a more potent stress state to cause fracture compared to hydrostatic stress state. Therefore the failure loads obtained from these tests represent the lower limit of what the coccospheres can sustain in its underwater environment. The
260
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
Fig. 2. (a and b) Coccospheres marked using FIB for identification and tracking before and after failure. (c) Load-displacement response of one of the coccospheres tested and (d) different stages of deformation and failure of a coccosphere placed on a Si substrate when compressed using a flat diamond punch, as recorded in-situ in the SEM. The numbers I–IV connect the P-d curve with the corresponding still images.
deformation behavior of the coccosphere was closely followed by the SEM while displacement was increased until fracture occurred (Fig. 2). Loading was monotonic in most cases, but cyclic loading was carried out in some until complete failure. Focused ion beam (FIB) machining was used to carve markers adjacent to 6 of the coccospheres for identifying them (Fig. 2a and b) and subsequently imaged in a high resolution SEM (Zeiss AurigaÒ crossbeam) before and after loading to visualize the damage more clearly. A total of 23 coccospheres were tested. A finite element simulation (FEM) was carried out using ABAQUS to model coccolith elements as single cantilevers under bending (more details in the Appendix).
3. Results and discussion Snapshots from the recorded video and the corresponding signatures in the load (P)-displacement (d) curve of a typical compression test of a coccosphere are shown in Fig. 2. After contact (I), the load typically increases linearly, which is a sign of pure elastic (fully reversible) deformation. A deviation of the elastic behavior (II) indicates either the onset of plastic (i.e. irreversible) deformation, OR crack initiation and stable crack growth (III). Shortly later, a tremendous load drop can be monitored for all samples, documenting the loss of structural integrity due to unstable crack growth. This is defined as the point of coccosphere failure. The coccosphere then takes a low but constant force. The maximum load – the peak load – that the coccospheres sustains before failure varied between 0.07 and 0.37 mN with a mean value of 0.20 mN ± 0.07 mN. The variation in sustained maximum load may be a consequence of the number of coccolith layers comprising the coccosphere and will be discussed later. Thus, the coccospheres do not show linear elastic fracture behavior but accommodate a significant amount of the imposed displacement by plastic deformation/stable crack growth. This behavior is not common for purely ceramic materials (e.g. calcite), and is indicative of the mechanical protection function of this composite shell. How is the stability of the coccosphere achieved? Besides the stability of the individual coccoliths, the architecture of the
coccosphere and the presence of polysaccharides acting as glue seem to be instrumental [5,4]. In an E. huxleyi coccosphere the single coccoliths form an interlocking hollow sphere, which houses the cell. The coccosphere can be single- or multi-layered [4]. In any case the coccoliths have room to move relative to each other if an external force is applied because of the flexible mechanical interlocking. This feature combined with the polysaccharide glue in between the single coccoliths, probably accounts for the recovery upon unloading before the first large load drop (Fig. 3 (Coccosphere 1–2)). The onset of inelastic deformation occurs typically at 70% of the maximum load. The load corresponding to the first deviation from linear elastic behavior (Fig. 2c (Stage II)) represents the shear deformation of the polysaccharide glue, which possibly reaches its adhesion limit at the maximum load prior to the first load drop. Smaller load drops culminate in a large load drop of 0.15 mN or more, corresponding to calcite fracture (Figs. 2 and 3 (Coccosphere 3)). When unloaded before this first large load drop, substantial recovery of more than 50% strain follows (Fig. 3 (Coccosphere 1–2)). Fracture initiates at the contact point between the flat punch and the coccosphere, and propagates in the direction parallel to the compression axis. This is natural given the fact that tensile stress is perpendicular to the loading axis along the equatorial plane and drives the crack through. Once fracture has propagated through a couple of coccoliths, the shell continues to deform and buckle in the form of two separate hemispheres on continued loading without incurring much damage to the rest of the coccoliths (Fig. 2d-IV). Fracture of the individual coccoliths occurs like that of a series of cantilevers under bending, with the crack propagating along the junction of the individual single crystalline calcite shield elements attached to the outer- and inner tube element (Fig. 4). This is not surprising considering the maximum tensile stress occurs close to the fixed end of a cantilever (Fig. 5). During the tests, the coccospheres are randomly aligned along the contact surface with the flat punch. There is no way to determine or control the exact contact point, which could be a point of overlap between two intersecting coccoliths OR the center of a single coccolith. The flat punch being much larger than the coccospheres, can come in contact with two or more coccoliths at different contact points simultaneously (like in Fig. 4b). But we
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
261
Fig. 3. Images of 3 different coccospheres (a) before (b and c) after deformation and (d) corresponding load-displacement response, clearly distinguishing signatures during recovery and failure in well-formed coccospheres. All three coccospheres have been subjected to single loading cycles.
observe that cracking always initiates at the attachment of the individual distal shield elements of the coccolith with the tube element (similar to a single cantilever in Fig. 5) which are subjected to bending loads due to the contact made with the flat punch. We can say that the scatter in the fracture loads suggest that the contact point difference leads to the distribution in fracture load. When the contact point is at the overlap region, the load for fracture is expected to be lower compared to when the contact point is predominantly in the central area of a single coccolith. This is because the bending moment experienced by the distal shield elements is higher when loaded close to their free ends, in comparison to being loaded close to their fixed ends. Failure of the coccosphere does not correspond to complete crushing or flattening out of the coccosphere under controlled loading conditions. It is treated simply as the point at which the sphere does not offer any mechanical stability in terms of resisting increasing load. At the peak load, a major crack propagates and on continued loading breaks the sphere into two hemispheres, which further continue to bend and take much lower load. From a biological perspective, at this point the coccosphere has lost its ability to mechanically protect the cell. Once calcite crystals break in a single coccolith, crushing of the coccosphere is controlled by the amount of imposed displacement (how much we move the punch down) and not on the applied load. But tracking true displacements is challenging in a high compliance indenter system [28] and hence this part is not discussed further. So the force needed to break an E. huxleyi coccosphere equals the one needed to break a Thalassiosira punctigera or Coscinodiscus wailesii frustule [20]. Considering the size difference between these species and the mineral constitution of the hard phase, the stresses (load divided by contact area) accommodated before failure by the coccopsheres could be substantially higher. It is concluded that the
E. huxleyi coccosphere architecture has evolved to withstand mechanical stress, as the diatom frustules have [24,25,20]. This establishes mechanical protection as one of the functions of calcification in coccolithophores, as has been suggested many times based on morphological observations (e.g., [18,16]). While this does not imply that mechanical protection is the only function of calcification, our data rule out the hypothesis that calcification has a purely biochemical function and that the product of calcification, i.e. the coccosphere has no evolutionary significance. The biochemical-function-hypothesis comes in various guises, the most popular one being the CO2-production hypothesis, which has already been called into question on physiological grounds ([29] and references therein). Assuming a purely biochemical role of calcification, it would be impossible to explain the enormous effort coccolithophores make to produce their specific coccolith and coccosphere morphologies. Coccolith morphogenesis includes a fine-tuned cellular shaping machinery with the cytoskeleton at its heart, and the formation of a coccosphere is an equally sophisticated process featuring coccolith exocytosis at a specific spot on the cell surface [30,31]. That these morphogenetic mechanisms should have evolved makes sense when considering that the coccosphere serves as a mechanical protection device. Out of the 23 coccospheres tested, a near Gaussian distribution in maximum load before failure was observed (Fig. 6a). The maximum load before failure does not seem to depend on the number of layers constituting the coccosphere and therefore on its final size (Fig. 6b). This is because the cracks initiate at the outermost layer that is in contact with the indenter punch, at the position of maximum stress during bending of the cantilever shaped calcite crystals i.e. at the region between the distal shield and inner/outer tube element (Fig. 5). Please note that ‘‘failure load” does not imply a total destruction of the whole coccosphere, but can mean
262
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
Fig. 4. Fractured coccospheres (a–c). Cracking initiates and propagates at the region connecting the distal shield element and the outer/inner tube elements (b) like a series of cantilever under bending (top view).
Fig. 5. (a) Schematic of individual element of a coccolith under bending load and (b) a finite element simulated stress distribution of a cantilever under bending load.
destruction of the outermost layer only. We conclude that, although no difference in maximum load was observed between coccospheres with a different number of coccolith layers, the amount of layers is critical for the mechanical protection. Our results indicate that even if the most outer layer gets damaged, the remaining layers still can form an intact albeit smaller coccosphere. The large loads sustained by these structures suggest that the complex motif and geometry of the coccosphere enables a predominantly calcite based system to be used as a structural material in spite of its inherent brittleness and sensitivity to flaws. Conventionally brittle materials like single crystalline oxides, silicon and metallic glasses have shown increased strength and damage tolerance in compression at length scales smaller than 500 nm [32]. Individual calcite crystals in the coccoliths are already in the
domain of this size regime where substantial improvement in strength, fracture toughness and damage tolerance is expected owing to their smaller size. This exceptional damage tolerance displayed by the coccosphere showing accommodation of deformation by inelastic deformation and/or stable cracking before catastrophic fracture would not be possible in monolithic calcite structures without the special architecture of the coccosphere and the constitution of the polysaccharide glue encompassing the calcite. It is important to note that the testing conditions inside the SEM in vacuum are extremely unnatural to the coccosphere, which, in its natural environment, remains continuously hydrated. This implies that the organic components including polysaccharides are possibly dry in the SEM, and have partially lost their adhesive properties. Therefore our results represent the lower limit
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
263
Fig. 6. (a) Histogram showing a near Gaussian distribution of maximum loads, recorded from 23 coccospheres and (b) maximum load as a function of coccosphere size showing no correlation between the two.
Fig. 7. (a) Assembly of modeled single cantilever beam (b) Cantilever post deformation showing stress contour and meshing as applied to the model.
of damage tolerance. In hydrated state, the polysaccharides are expected to accommodate loads with increased degree of inelastic deformation before undergoing unstable fracture. It would be interesting to test to what extent the stability of the coccosphere hinges upon the interlocking architecture of the coccosphere (e.g., [4,22]). There is already a hint that mechanical interlocking and the polysaccharide glue that holds them together contribute to the initial recovery as well as prevent complete collapse of the coccosphere after the inorganic component of the coccolith (the calcite) fractures. Before the one individual coccolith segment reaches the critical bending displacements, these interlocks accommodate compression by shearing/slipping along each other, which is characterized by the deviations from linear elastic deformation. Of course, such accommodation will not be possible under tensile loads, for which they are not intrinsically designed. A comparison of E. huxleyi and Pontosphaera discopora could shed more light on that question, because the latter species does not feature interlocking coccoliths [16]. However, if present, the interlocking of coccoliths seems to be important for the damage tolerance of the coccosphere, because severe coccolith malformations, which preclude an efficient overlap of the single shields, are accompanied by very fragile coccospheres which do not withstand typical sampling and preparation for SEM imaging [33]. This again emphasizes the evolutionary significance of coccolith morphogenesis. In this context the effects of ocean acidification on coccolithophore calcification are interesting. Seawater acidification has been shown to hamper calcification in various ways, and in a species- and even strain-specific fashion ([27] and references
therein). In some cases calcification rate is reduced and in some cases the percentage of coccolith malformations increased. A combination of these two effects is occasionally, but not always, observed. Traditionally, the focus has been on calcification rate, a reduction of which has been interpreted as bad news for the respective species. This is curious, considering that it is unknown whether a maximum calcification rate is important in ecological/ evolutionary terms. Our data, on the other hand, consolidate coccolith morphogenesis as an evolutionary relevant process, and therefore a detrimental effect of ocean acidification on morphogenesis should indeed impact the organism’s fitness. Overall, our study emphasizes the importance of the product of calcification over the process of calcification in coccolithophores. 4. Summary Deformation patterns of calcite based coccospheres of E. huxleyi were studied in-situ in the SEM. Interface failure between the polysaccharide glue and the calcite based coccoliths (as shown by deviation from linear elasticity) precedes calcite fracture. While the deformation is largely recoverable after interface shear, damage is permanent after the distal shield elements undergo brittle fracture. The maximum load that the coccospheres can sustain is independent of their size and therefore independent of the number of layers forming them. Nevertheless, the coccosphere still remains intact even when one coccolith of the outer coccolith layer has fractured. Thus a larger number of coccolith layers provide more efficient protection. The bending based geometry of the naturally
264
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
complex cellular architecture of coccospheres is able to sustain fairly large compressive stresses despite being made of more than 90% ceramic constituents and is able to outperform other technical ceramics in the strength to weight ratio. It is concluded that coccospheres have evolved to withstand mechanical load. This conclusion rules out exclusively biochemical functions of coccolithophore calcification.
[9]
[10]
[11]
Acknowledgement This work was supported by the grant from Natural Environment Research Council (NE/N011708/1).
[12]
Appendix A.
[13]
Simulation procedure of single cantilever bending [14]
Single cantilever bending was simulated in the commercial software ABAQUS 6.10 to compare it to the bending and fracture of the coccoliths. A three dimensional cantilever beam of dimensions 600*100*100 nm were modeled. The beam was allowed to deform only elastically, with a modulus E of 70 GPa and a Poisson’s ratio m of 0.25, based on the properties of calcite single crystals that were experimentally determined by indentation. The fixed end of the cantilever was prevented from displacement/rotation of any kind. It was loaded up to 3 mN close to the free end using a wedge indenter of 40 nm tip radius. A 20 nm long starter cracks was inserted to model intrinsic flaw 50 nm away from the fixed end. The beams were partitioned in the pre-cracked region to separate out the crack domain, where the enriched mesh was assigned to accommodate the extended finite element formulation. A total of 50,000 elements were used in the modeled structure with 3-D linear hexagonal reduced integration elements. The modeled assembly is shown in Fig. 7. In the cracked domain, a maximum principal stress to failure (MAXPS) of 1 GPa was imposed on the material ahead of the crack tip for crack initiation. A stress based criterion was used since the material is assumed to be nominally brittle and the value itself was obtained from independent experiments on fracture stress of unnotched specimens. The stress contour of the beam was extracted as output at every loading step. References [1] Y. Politi, T. Arad, E. Klein, S. Weiner, L. Addadi, Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase, Science 306 (2004) 1161– 1164. [2] H.C.W. Skinner, Biominerals, Mineral. Mag. 69 (5) (2005) 621–641. [3] M.E. Marsh, Regulation of CaCO3 formation in coccolithophores, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 136 (2003) 743–754. [4] R. Hoffmann, C. Kirchlechner, G. Langer, A.S. Wochnik, E. Griesshaber, W.W. Schmahl, C. Scheu, Insight into Emiliania huxleyi coccospheres by focused ion beam sectioning, Biogeosciences 12 (2015) 825–834. [5] J.R. Young, K. Henriksen, Biomineralization within vesicles: the calcite of coccoliths, Rev. Mineral. Geochem. 54 (2003) 189–215. [6] J.R. Young, M. Geisen, L. Cros, A. Kleijne, C. Sprengel, I. Probert, J. Östergaard, A guide to extant coccolithophore taxonomy, J. Nannoplankton Res. 1 (2003) 1–132 (Special issue). [7] J.R. Young, J.M. Didymus, P.R. Bown, B. Prins, S. Mann, Crystal assembly and phylogenetic evolution in heterococcoliths, Nature 356 (1992) 516–518. [8] R. Hoffmann, A.S. Wochnik, C. Heinzl, S.B. Betzler, S. Matich, E. Griesshaber, H. Schulz, M. Kucˇera, J.R. Young, C. Scheu, W.W. Schmahl, Nanoprobe
[15] [16] [17] [18] [19] [20]
[21]
[22] [23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33] [34]
crystallographic orientation studies of isolated shield elements of the coccolithophore species Emiliania huxleyi, Eur. J. Mineral. 26 (4) (2014) 473– 483. J.M. Neff, Ultrastructure of the outer epithelium of the mantle in the clam Mercenaria mercenaria in relation to calcification of the shell, Tissue Cell 4 (4) (1972) 591–600. M. Rousseau, E. Lopez, P. Stempflle˙, M. Brendle˙, L. Franke, A. Guette, R. Naslain, X. Bourrat, Multiscale structure of sheet nacre, Biomaterials 26 (31) (2005) 6254–6262. W.W. Schmahl, E. Griesshaber, C. Merkel, K. Kelm, J. Deuschle, R.D. Neuser, A.J. Goetz, A. Sehrbrock, W. Mader, Hierarchical fibre composite structure and micromechanical properties of phosphatic and calcitic brachiopod shell biomaterials – an overview, Mineral. Mag. 72 (2008) 541–562. K. Benzerara, N. Menguy, M. Obst, J. Stolarski, M. Mazur, T. Tylisczak, G.E. Brown, A. Meibom, A study of the crystallographic architecture of corals at the nanoscale by scanning transmission X-ray microscopy and transmission electron microscopy, Ultramicroscopy 111 (8) (2011) 1268–1275. J. Seto, Y. Ma, S.A. Davis, F. Meldrum, A. Gourrier, Y.-Y. Kim, U. Schilde, M. Sztucki, M. Brughammer, S. Maltsev, C. Jäger, H. Cölfen, Structure-property relationships of a biological mesocrystal in the adult sea urchin spine, Proc. Natl. Acad. Sci. 109 (18) (2012). 7126–7126.. L. Bergström, E.V. Sturm (née Rosseeva), G. Salazar-Alvarez, H. Cölfen, Mesocrystals in biominerals and colloidal arrays, Acc. Chem. Res. 48 (2015) 1391–1402. R.-Q. Song, H. Cölfen, Mesocrystals – ordered nanoparticle superstructures, Adv. Mater. 21 (2010) 1–30. J.R. Young, Possible functional interpretations of coccolith morphology, Abhandlungen der geologischen Bundesanstalt Wien 39 (1987) 305–313. J.R. Young, Functions of coccoliths, in: A. Winter, W.G. Siesser (Eds.), Coccolithophores, Cambridge University Press, Cambridge, 1994, pp. 63–82. H.H. Dixon, On the structure of coccospheres and the origin of coccoliths, Procs. R. Soc. London, B 64 (1900) 305–315. C.S. Sikes, K.M. Wilbur, Functions of coccolith formation, L&O 27 (1982) 18–26. L. Friedrichs, M. Hörnig, L. Schulze, A. Bertram, S. Jansen, C. Hamm, Size and biomechanic properties of diatom frustules influence food uptake by copepods, MEPS 481 (2013) 41–51. G. Langer, G. Nehrke, S. Jansen, Dissolution of Calcidiscus leptoporus coccoliths in copepod guts? – a morphological study, Mar. Ecol. Prog. Ser. 331 (2007) 139–146. S. Jansen, Copepods grazing on Coscinodiscus wailesii – a question of size?, Helgol Mar. Res. 62 (2008) 251–255. H. Tappan, The Paleobiology of Plant Protists, W.H. Freemann and Company, San Francisco, 1980, p. 1028. C. Hamm, R. Merkel, O. Springer, P. Jurkojc, C. Maier, K. Prechtel, V. Smetacek, Architecture and material properties of diatom shells provide effective mechanical protection, Nature 421 (2003) 841–843. C.E. Hamm, The evolution of advanced mechanical defenses and potential technological applications of diatom shells, J. Nanosci. Nanotechnol. 5 (2005) 108–119. R.R.L. Guillard, J.H. Ryther, Studies of marine planktonic diatoms, I, Cyclotella nana (Hustedt) and Detonula convervacea (Cleve), Can. J. Microbiol. 8 (2) (1962) 229–239. G. Langer, I. Probert, G. Nehrke, P. Ziveri, The morphological response of Emiliania huxleyi to seawater carbonate chemistry changes: an inter-strain comparison, J. Nannoplankton Res. 32 (2011) 27–32. B.N. Jaya, C. Kirchlechner, G. Dehm, Can micro-scale fracture tests provide reliable fracture toughness values? A case study in silicon, J. Mater. Res. 30 (5) (2015) 686–698. S. Trimborn, G. Langer, B. Rost, Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi, Limnol. Oceanogr. 52 (2007) 2285–2293. A.R. Taylor, M.A. Russell, G. Harper, T. Collins, C. Brownlee, Dynamics of the formation and secretion of hetercoccoliths by Coccolithus pelagicus (ssp braarudii), Eur. J. Phycol. 42 (2007) 125–136. G. Langer, L.J. de Nooijer, K. Oetjen, On the role of the cytoskeleton in coccolith morphogenesis: the effect of cytoskeleton inhibitors, J. Phycol 46 (2010) 1252– 1256. B.N. Jaya, V. Jayaram, Fracture testing at small length scales: from plasticity in Si to brittleness, Pt. JOM 68 (1) (2015) 94–108. G. Langer, M. Bode, CO2 mediation of adverse effects of seawater acidification in Calcidiscus leptoporus, Geochem. Geophys. Geosyst. 12 (2011) Q05001. J.M. Didymus, J.R. Young, S. Mann, Construction and morphogenesis of the chiral ultrastructure of coccoliths from marine alga Emiliania huxleyi, Proc R Soc Ser B 258 (1994) 237–245.
Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Full length article
Coccospheres confer mechanical protection: New evidence for an old hypothesis B.N. Jaya a,⇑, R. Hoffmann b, C. Kirchlechner a, G. Dehm a, C. Scheu a, G. Langer c,⇑ a
Max-Planck-Institut für Eisenforschung GmbH, 40237 Düsseldorf, Germany Department of Chemistry, Ludwig-Maximilian-University, 81377 Munich, Germany c The Marine Biological Association of the United Kingdom, The Laboratory, Citadel Hill, Plymouth, Devon PL1 2PB, UK b
a r t i c l e
i n f o
Article history: Received 5 May 2016 Received in revised form 27 June 2016 Accepted 19 July 2016 Available online 21 July 2016 Keywords: Coccospheres Calcite In-situ compression Mechanical protection
a b s t r a c t Emiliania huxleyi has evolved an extremely intricate coccosphere architecture. The coccosphere is comprised of interlocking coccoliths embedded in a polysaccharide matrix. In this work, we performed insitu scanning electron microscopy based compression tests and conclude that coccospheres have a mechanical protection function. The coccosphere exhibits exceptional damage tolerance in terms of inelastic deformation, recovery and stable crack growth before catastrophic fracture, a feature, which is not found in monolithic ceramic structures. Some of the mechanical features of the coccospheres are due to their architecture, especially polysaccharide matrix that acts as a kind of bio-adhesive. Our data provide strong evidence for the mechanical protection-hypothesis of coccolithophore calcification, without excluding other functions of calcification such as various biochemical roles discussed in the literature. Statement of Significance Although bio-mechanics of shell structures like nacre have been studied over the past decade, coccospheres present an architecture that is quite distinct and complex. It is a porous cell structure evolved to protect the living algae cell inside it in the oceans, subjected to significant hydrostatic pressure. Despite being made of extremely brittle constituents like calcium carbonate, our study finds that coccospheres possess significant damage tolerance especially due to their interlocking coccolith architecture. This will have consequences in bio-mimetic design, especially relating to high pressure applications. Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Organisms produce extraordinary crystals in terms of shape and properties. Echinoids for example are able to regenerate their spines, while maintaining the original pattern of the macrostructure [1]. The greatest number of biologically formed crystals belong to the classes of sulfides, sulfates, phosphates and carbonates [2]. Coccolithophores are unicellular haptophyte algae, which produce calcium carbonate platelets (coccoliths) and assemble them into a hollow sphere, the so called coccosphere. Since the hard inorganic parts of coccolithophores are built inside the cell, they differ from those of most other calcifiers. The most common species of the approx. 200 extant species is Emiliania huxleyi (E. huxleyi) [3]. The coccospheres of this species are ca. 5–10 lm in diameter and consist of several layers of coccol-
⇑ Corresponding authors. E-mail addresses: [email protected] (B.N. Jaya), [email protected] (G. Langer). http://dx.doi.org/10.1016/j.actbio.2016.07.036 1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
iths that are interlocked and glued to each other by means of polysaccharides (Fig. 1a, [4,5]). The strain RCC1238 used in this study possesses on average 2–3 coccolith layers and 20 coccoliths per cell [4] (Fig. 1b). The coccoliths are similar to a cable reel, consisting of a central tube that connects the lower proximal shield and the upper distal shield and encloses the central area (Fig. 1c and d) [6]. This morphology enables the coccoliths to interlock closely on the coccosphere and form a robust structure [6]. A single segment of an E. huxleyi coccolith comprises of two crystal units, the radial R-unit that is built up of a single crystal with the c-axis oriented parallel to the coccolith plane and the c-axis of the vertical V-unit is perpendicular to the coccolith plane (Fig. 1d grey arrow) [7,8]. This differs from other biological crystals like the ones of bone, corals, sea urchin and mollusk shells, which show a mosaic like crystal assembly [9–14], i.e. consisting of co-oriented crystallites in the mesoscopic size range (1–1000 nm) [15]. It was suggested that these mesocrystal-composite structures, e.g. mollusk shells, have a high fracture toughness compared to the one of single crystalline structures [14].
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
259
Fig. 1. SEM micrograph of (a) a complete and (b) a FIB cut coccosphere of the investigated E. huxleyi strain RCC1238. An individual coccolith and the interlocking of coccoliths are marked with white lines. In (c) secondary electron images of a coccolith viewed from top and bottom are shown. The model of a single discrete element of a coccolith is displayed in (d) (modified after [34,8]), showing the morphologically complex bi-crystalline calcite units (primary R and interstitial V whose orientations are shown by arrows) [7].
Despite decades of research the function of calcification in coccolithophores is a matter of debate, with the seminal discussion by Young [16] (see also the extended version, Young [17]) still being the central work of reference. Among the many hypotheses put forth, a mechanical/protective role of coccospheres was suggested more than a century ago [18], and remains one of the most plausible ideas [16]. The type of protection provided by coccospheres is not identical to the one provided by diatom frustules. While the latter protect the algae from macro-grazers such as copepods, coccospheres do not seem to have this function [19,20]. This, however, is not due to the mechanical properties (i.e. stability) of the coccosphere, but merely reflects the difference in prey/predator size ratio. While copepods, more often than not, have to destroy the diatom frustule in order to eat the diatom, coccolithophores are swallowed whole and coccoliths are only accidentally crushed by copepod mandibles [21,22]. Coccospheres most likely confer a general mechanical stability to the algae. This hypothesis has traditionally been based on morphological observations, but still awaits corroboration by micro-mechanical measurements [18,23,16]. In other words, how much load is needed to break a coccosphere? The mechanical stability of diatom frustules is regarded as being instrumental in the evolutionary success of diatoms [24,25,20]. The average force needed to break a diatom frustule ranges from 0.2 to 0.8 mN [20]. If the coccospheres also showed the ability to accommodate increasing loads by inelastic dissipation before undergoing fracture, this would be strong evidence in favor of the mechanical protection role of coccospheres. In this study, we performed in-situ compression measurements on coccospheres of E. huxleyi by means of a flat punch indenter acting as a micro-crusher inside the scanning electron microscope (SEM). This allows us to determine the loads corresponding to the initial deviation from elastic deformation and the maximum
load before fracture and collapse of the coccosphere occurs. Simultaneously, the deformation process was closely monitored for signs of failure of coccospheres, individual coccoliths and coccolith segments.
2. Experimental procedure Coccospheres cultured in aged, sterile-filtered (0.2 lm pore-size cellulose-acetate filters) North Sea seawater enriched with 100 lmol L 1 nitrate, 6.25 lmol L 1 phosphate, trace metals and vitamins as in f/2 medium [26] were filtered onto an Omnipore polycarbonate membrane filter by using a vacuum pump. The filter was dried at 60 °C and the material was afterwards removed with a spatula and dissolved in ethanol. The sample was then dropped on a silicon wafer and dried. In the end a thin carbon film was deposited to avoid charging effects in the SEM. For more details on the coccolithophore culturing see Langer et al., [27] and Hoffmann et al., [4]. Loading was carried out in situ using the ASMEC Unat II indenter (ASMEC GmbH, Radeberg, Germany) inside a JEOL-JSM 6490 tungsten filament SEM. Individual coccospheres were loaded in compression under displacement control at a rate of 10 nm/s. This represents a uniaxial testing condition, which is different from the hydrostatic (tri-axial) stress state normally experienced by the coccospheres under water. Uniaxial compression of the sphere in the vertical direction leads to tensile opening stresses in the perpendicular direction, in addition to flexural (bending) loading of individual coccoliths. This is a more potent stress state to cause fracture compared to hydrostatic stress state. Therefore the failure loads obtained from these tests represent the lower limit of what the coccospheres can sustain in its underwater environment. The
260
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
Fig. 2. (a and b) Coccospheres marked using FIB for identification and tracking before and after failure. (c) Load-displacement response of one of the coccospheres tested and (d) different stages of deformation and failure of a coccosphere placed on a Si substrate when compressed using a flat diamond punch, as recorded in-situ in the SEM. The numbers I–IV connect the P-d curve with the corresponding still images.
deformation behavior of the coccosphere was closely followed by the SEM while displacement was increased until fracture occurred (Fig. 2). Loading was monotonic in most cases, but cyclic loading was carried out in some until complete failure. Focused ion beam (FIB) machining was used to carve markers adjacent to 6 of the coccospheres for identifying them (Fig. 2a and b) and subsequently imaged in a high resolution SEM (Zeiss AurigaÒ crossbeam) before and after loading to visualize the damage more clearly. A total of 23 coccospheres were tested. A finite element simulation (FEM) was carried out using ABAQUS to model coccolith elements as single cantilevers under bending (more details in the Appendix).
3. Results and discussion Snapshots from the recorded video and the corresponding signatures in the load (P)-displacement (d) curve of a typical compression test of a coccosphere are shown in Fig. 2. After contact (I), the load typically increases linearly, which is a sign of pure elastic (fully reversible) deformation. A deviation of the elastic behavior (II) indicates either the onset of plastic (i.e. irreversible) deformation, OR crack initiation and stable crack growth (III). Shortly later, a tremendous load drop can be monitored for all samples, documenting the loss of structural integrity due to unstable crack growth. This is defined as the point of coccosphere failure. The coccosphere then takes a low but constant force. The maximum load – the peak load – that the coccospheres sustains before failure varied between 0.07 and 0.37 mN with a mean value of 0.20 mN ± 0.07 mN. The variation in sustained maximum load may be a consequence of the number of coccolith layers comprising the coccosphere and will be discussed later. Thus, the coccospheres do not show linear elastic fracture behavior but accommodate a significant amount of the imposed displacement by plastic deformation/stable crack growth. This behavior is not common for purely ceramic materials (e.g. calcite), and is indicative of the mechanical protection function of this composite shell. How is the stability of the coccosphere achieved? Besides the stability of the individual coccoliths, the architecture of the
coccosphere and the presence of polysaccharides acting as glue seem to be instrumental [5,4]. In an E. huxleyi coccosphere the single coccoliths form an interlocking hollow sphere, which houses the cell. The coccosphere can be single- or multi-layered [4]. In any case the coccoliths have room to move relative to each other if an external force is applied because of the flexible mechanical interlocking. This feature combined with the polysaccharide glue in between the single coccoliths, probably accounts for the recovery upon unloading before the first large load drop (Fig. 3 (Coccosphere 1–2)). The onset of inelastic deformation occurs typically at 70% of the maximum load. The load corresponding to the first deviation from linear elastic behavior (Fig. 2c (Stage II)) represents the shear deformation of the polysaccharide glue, which possibly reaches its adhesion limit at the maximum load prior to the first load drop. Smaller load drops culminate in a large load drop of 0.15 mN or more, corresponding to calcite fracture (Figs. 2 and 3 (Coccosphere 3)). When unloaded before this first large load drop, substantial recovery of more than 50% strain follows (Fig. 3 (Coccosphere 1–2)). Fracture initiates at the contact point between the flat punch and the coccosphere, and propagates in the direction parallel to the compression axis. This is natural given the fact that tensile stress is perpendicular to the loading axis along the equatorial plane and drives the crack through. Once fracture has propagated through a couple of coccoliths, the shell continues to deform and buckle in the form of two separate hemispheres on continued loading without incurring much damage to the rest of the coccoliths (Fig. 2d-IV). Fracture of the individual coccoliths occurs like that of a series of cantilevers under bending, with the crack propagating along the junction of the individual single crystalline calcite shield elements attached to the outer- and inner tube element (Fig. 4). This is not surprising considering the maximum tensile stress occurs close to the fixed end of a cantilever (Fig. 5). During the tests, the coccospheres are randomly aligned along the contact surface with the flat punch. There is no way to determine or control the exact contact point, which could be a point of overlap between two intersecting coccoliths OR the center of a single coccolith. The flat punch being much larger than the coccospheres, can come in contact with two or more coccoliths at different contact points simultaneously (like in Fig. 4b). But we
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
261
Fig. 3. Images of 3 different coccospheres (a) before (b and c) after deformation and (d) corresponding load-displacement response, clearly distinguishing signatures during recovery and failure in well-formed coccospheres. All three coccospheres have been subjected to single loading cycles.
observe that cracking always initiates at the attachment of the individual distal shield elements of the coccolith with the tube element (similar to a single cantilever in Fig. 5) which are subjected to bending loads due to the contact made with the flat punch. We can say that the scatter in the fracture loads suggest that the contact point difference leads to the distribution in fracture load. When the contact point is at the overlap region, the load for fracture is expected to be lower compared to when the contact point is predominantly in the central area of a single coccolith. This is because the bending moment experienced by the distal shield elements is higher when loaded close to their free ends, in comparison to being loaded close to their fixed ends. Failure of the coccosphere does not correspond to complete crushing or flattening out of the coccosphere under controlled loading conditions. It is treated simply as the point at which the sphere does not offer any mechanical stability in terms of resisting increasing load. At the peak load, a major crack propagates and on continued loading breaks the sphere into two hemispheres, which further continue to bend and take much lower load. From a biological perspective, at this point the coccosphere has lost its ability to mechanically protect the cell. Once calcite crystals break in a single coccolith, crushing of the coccosphere is controlled by the amount of imposed displacement (how much we move the punch down) and not on the applied load. But tracking true displacements is challenging in a high compliance indenter system [28] and hence this part is not discussed further. So the force needed to break an E. huxleyi coccosphere equals the one needed to break a Thalassiosira punctigera or Coscinodiscus wailesii frustule [20]. Considering the size difference between these species and the mineral constitution of the hard phase, the stresses (load divided by contact area) accommodated before failure by the coccopsheres could be substantially higher. It is concluded that the
E. huxleyi coccosphere architecture has evolved to withstand mechanical stress, as the diatom frustules have [24,25,20]. This establishes mechanical protection as one of the functions of calcification in coccolithophores, as has been suggested many times based on morphological observations (e.g., [18,16]). While this does not imply that mechanical protection is the only function of calcification, our data rule out the hypothesis that calcification has a purely biochemical function and that the product of calcification, i.e. the coccosphere has no evolutionary significance. The biochemical-function-hypothesis comes in various guises, the most popular one being the CO2-production hypothesis, which has already been called into question on physiological grounds ([29] and references therein). Assuming a purely biochemical role of calcification, it would be impossible to explain the enormous effort coccolithophores make to produce their specific coccolith and coccosphere morphologies. Coccolith morphogenesis includes a fine-tuned cellular shaping machinery with the cytoskeleton at its heart, and the formation of a coccosphere is an equally sophisticated process featuring coccolith exocytosis at a specific spot on the cell surface [30,31]. That these morphogenetic mechanisms should have evolved makes sense when considering that the coccosphere serves as a mechanical protection device. Out of the 23 coccospheres tested, a near Gaussian distribution in maximum load before failure was observed (Fig. 6a). The maximum load before failure does not seem to depend on the number of layers constituting the coccosphere and therefore on its final size (Fig. 6b). This is because the cracks initiate at the outermost layer that is in contact with the indenter punch, at the position of maximum stress during bending of the cantilever shaped calcite crystals i.e. at the region between the distal shield and inner/outer tube element (Fig. 5). Please note that ‘‘failure load” does not imply a total destruction of the whole coccosphere, but can mean
262
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
Fig. 4. Fractured coccospheres (a–c). Cracking initiates and propagates at the region connecting the distal shield element and the outer/inner tube elements (b) like a series of cantilever under bending (top view).
Fig. 5. (a) Schematic of individual element of a coccolith under bending load and (b) a finite element simulated stress distribution of a cantilever under bending load.
destruction of the outermost layer only. We conclude that, although no difference in maximum load was observed between coccospheres with a different number of coccolith layers, the amount of layers is critical for the mechanical protection. Our results indicate that even if the most outer layer gets damaged, the remaining layers still can form an intact albeit smaller coccosphere. The large loads sustained by these structures suggest that the complex motif and geometry of the coccosphere enables a predominantly calcite based system to be used as a structural material in spite of its inherent brittleness and sensitivity to flaws. Conventionally brittle materials like single crystalline oxides, silicon and metallic glasses have shown increased strength and damage tolerance in compression at length scales smaller than 500 nm [32]. Individual calcite crystals in the coccoliths are already in the
domain of this size regime where substantial improvement in strength, fracture toughness and damage tolerance is expected owing to their smaller size. This exceptional damage tolerance displayed by the coccosphere showing accommodation of deformation by inelastic deformation and/or stable cracking before catastrophic fracture would not be possible in monolithic calcite structures without the special architecture of the coccosphere and the constitution of the polysaccharide glue encompassing the calcite. It is important to note that the testing conditions inside the SEM in vacuum are extremely unnatural to the coccosphere, which, in its natural environment, remains continuously hydrated. This implies that the organic components including polysaccharides are possibly dry in the SEM, and have partially lost their adhesive properties. Therefore our results represent the lower limit
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
263
Fig. 6. (a) Histogram showing a near Gaussian distribution of maximum loads, recorded from 23 coccospheres and (b) maximum load as a function of coccosphere size showing no correlation between the two.
Fig. 7. (a) Assembly of modeled single cantilever beam (b) Cantilever post deformation showing stress contour and meshing as applied to the model.
of damage tolerance. In hydrated state, the polysaccharides are expected to accommodate loads with increased degree of inelastic deformation before undergoing unstable fracture. It would be interesting to test to what extent the stability of the coccosphere hinges upon the interlocking architecture of the coccosphere (e.g., [4,22]). There is already a hint that mechanical interlocking and the polysaccharide glue that holds them together contribute to the initial recovery as well as prevent complete collapse of the coccosphere after the inorganic component of the coccolith (the calcite) fractures. Before the one individual coccolith segment reaches the critical bending displacements, these interlocks accommodate compression by shearing/slipping along each other, which is characterized by the deviations from linear elastic deformation. Of course, such accommodation will not be possible under tensile loads, for which they are not intrinsically designed. A comparison of E. huxleyi and Pontosphaera discopora could shed more light on that question, because the latter species does not feature interlocking coccoliths [16]. However, if present, the interlocking of coccoliths seems to be important for the damage tolerance of the coccosphere, because severe coccolith malformations, which preclude an efficient overlap of the single shields, are accompanied by very fragile coccospheres which do not withstand typical sampling and preparation for SEM imaging [33]. This again emphasizes the evolutionary significance of coccolith morphogenesis. In this context the effects of ocean acidification on coccolithophore calcification are interesting. Seawater acidification has been shown to hamper calcification in various ways, and in a species- and even strain-specific fashion ([27] and references
therein). In some cases calcification rate is reduced and in some cases the percentage of coccolith malformations increased. A combination of these two effects is occasionally, but not always, observed. Traditionally, the focus has been on calcification rate, a reduction of which has been interpreted as bad news for the respective species. This is curious, considering that it is unknown whether a maximum calcification rate is important in ecological/ evolutionary terms. Our data, on the other hand, consolidate coccolith morphogenesis as an evolutionary relevant process, and therefore a detrimental effect of ocean acidification on morphogenesis should indeed impact the organism’s fitness. Overall, our study emphasizes the importance of the product of calcification over the process of calcification in coccolithophores. 4. Summary Deformation patterns of calcite based coccospheres of E. huxleyi were studied in-situ in the SEM. Interface failure between the polysaccharide glue and the calcite based coccoliths (as shown by deviation from linear elasticity) precedes calcite fracture. While the deformation is largely recoverable after interface shear, damage is permanent after the distal shield elements undergo brittle fracture. The maximum load that the coccospheres can sustain is independent of their size and therefore independent of the number of layers forming them. Nevertheless, the coccosphere still remains intact even when one coccolith of the outer coccolith layer has fractured. Thus a larger number of coccolith layers provide more efficient protection. The bending based geometry of the naturally
264
B.N. Jaya et al. / Acta Biomaterialia 42 (2016) 258–264
complex cellular architecture of coccospheres is able to sustain fairly large compressive stresses despite being made of more than 90% ceramic constituents and is able to outperform other technical ceramics in the strength to weight ratio. It is concluded that coccospheres have evolved to withstand mechanical load. This conclusion rules out exclusively biochemical functions of coccolithophore calcification.
[9]
[10]
[11]
Acknowledgement This work was supported by the grant from Natural Environment Research Council (NE/N011708/1).
[12]
Appendix A.
[13]
Simulation procedure of single cantilever bending [14]
Single cantilever bending was simulated in the commercial software ABAQUS 6.10 to compare it to the bending and fracture of the coccoliths. A three dimensional cantilever beam of dimensions 600*100*100 nm were modeled. The beam was allowed to deform only elastically, with a modulus E of 70 GPa and a Poisson’s ratio m of 0.25, based on the properties of calcite single crystals that were experimentally determined by indentation. The fixed end of the cantilever was prevented from displacement/rotation of any kind. It was loaded up to 3 mN close to the free end using a wedge indenter of 40 nm tip radius. A 20 nm long starter cracks was inserted to model intrinsic flaw 50 nm away from the fixed end. The beams were partitioned in the pre-cracked region to separate out the crack domain, where the enriched mesh was assigned to accommodate the extended finite element formulation. A total of 50,000 elements were used in the modeled structure with 3-D linear hexagonal reduced integration elements. The modeled assembly is shown in Fig. 7. In the cracked domain, a maximum principal stress to failure (MAXPS) of 1 GPa was imposed on the material ahead of the crack tip for crack initiation. A stress based criterion was used since the material is assumed to be nominally brittle and the value itself was obtained from independent experiments on fracture stress of unnotched specimens. The stress contour of the beam was extracted as output at every loading step. References [1] Y. Politi, T. Arad, E. Klein, S. Weiner, L. Addadi, Sea urchin spine calcite forms via a transient amorphous calcium carbonate phase, Science 306 (2004) 1161– 1164. [2] H.C.W. Skinner, Biominerals, Mineral. Mag. 69 (5) (2005) 621–641. [3] M.E. Marsh, Regulation of CaCO3 formation in coccolithophores, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 136 (2003) 743–754. [4] R. Hoffmann, C. Kirchlechner, G. Langer, A.S. Wochnik, E. Griesshaber, W.W. Schmahl, C. Scheu, Insight into Emiliania huxleyi coccospheres by focused ion beam sectioning, Biogeosciences 12 (2015) 825–834. [5] J.R. Young, K. Henriksen, Biomineralization within vesicles: the calcite of coccoliths, Rev. Mineral. Geochem. 54 (2003) 189–215. [6] J.R. Young, M. Geisen, L. Cros, A. Kleijne, C. Sprengel, I. Probert, J. Östergaard, A guide to extant coccolithophore taxonomy, J. Nannoplankton Res. 1 (2003) 1–132 (Special issue). [7] J.R. Young, J.M. Didymus, P.R. Bown, B. Prins, S. Mann, Crystal assembly and phylogenetic evolution in heterococcoliths, Nature 356 (1992) 516–518. [8] R. Hoffmann, A.S. Wochnik, C. Heinzl, S.B. Betzler, S. Matich, E. Griesshaber, H. Schulz, M. Kucˇera, J.R. Young, C. Scheu, W.W. Schmahl, Nanoprobe
[15] [16] [17] [18] [19] [20]
[21]
[22] [23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32] [33] [34]
crystallographic orientation studies of isolated shield elements of the coccolithophore species Emiliania huxleyi, Eur. J. Mineral. 26 (4) (2014) 473– 483. J.M. Neff, Ultrastructure of the outer epithelium of the mantle in the clam Mercenaria mercenaria in relation to calcification of the shell, Tissue Cell 4 (4) (1972) 591–600. M. Rousseau, E. Lopez, P. Stempflle˙, M. Brendle˙, L. Franke, A. Guette, R. Naslain, X. Bourrat, Multiscale structure of sheet nacre, Biomaterials 26 (31) (2005) 6254–6262. W.W. Schmahl, E. Griesshaber, C. Merkel, K. Kelm, J. Deuschle, R.D. Neuser, A.J. Goetz, A. Sehrbrock, W. Mader, Hierarchical fibre composite structure and micromechanical properties of phosphatic and calcitic brachiopod shell biomaterials – an overview, Mineral. Mag. 72 (2008) 541–562. K. Benzerara, N. Menguy, M. Obst, J. Stolarski, M. Mazur, T. Tylisczak, G.E. Brown, A. Meibom, A study of the crystallographic architecture of corals at the nanoscale by scanning transmission X-ray microscopy and transmission electron microscopy, Ultramicroscopy 111 (8) (2011) 1268–1275. J. Seto, Y. Ma, S.A. Davis, F. Meldrum, A. Gourrier, Y.-Y. Kim, U. Schilde, M. Sztucki, M. Brughammer, S. Maltsev, C. Jäger, H. Cölfen, Structure-property relationships of a biological mesocrystal in the adult sea urchin spine, Proc. Natl. Acad. Sci. 109 (18) (2012). 7126–7126.. L. Bergström, E.V. Sturm (née Rosseeva), G. Salazar-Alvarez, H. Cölfen, Mesocrystals in biominerals and colloidal arrays, Acc. Chem. Res. 48 (2015) 1391–1402. R.-Q. Song, H. Cölfen, Mesocrystals – ordered nanoparticle superstructures, Adv. Mater. 21 (2010) 1–30. J.R. Young, Possible functional interpretations of coccolith morphology, Abhandlungen der geologischen Bundesanstalt Wien 39 (1987) 305–313. J.R. Young, Functions of coccoliths, in: A. Winter, W.G. Siesser (Eds.), Coccolithophores, Cambridge University Press, Cambridge, 1994, pp. 63–82. H.H. Dixon, On the structure of coccospheres and the origin of coccoliths, Procs. R. Soc. London, B 64 (1900) 305–315. C.S. Sikes, K.M. Wilbur, Functions of coccolith formation, L&O 27 (1982) 18–26. L. Friedrichs, M. Hörnig, L. Schulze, A. Bertram, S. Jansen, C. Hamm, Size and biomechanic properties of diatom frustules influence food uptake by copepods, MEPS 481 (2013) 41–51. G. Langer, G. Nehrke, S. Jansen, Dissolution of Calcidiscus leptoporus coccoliths in copepod guts? – a morphological study, Mar. Ecol. Prog. Ser. 331 (2007) 139–146. S. Jansen, Copepods grazing on Coscinodiscus wailesii – a question of size?, Helgol Mar. Res. 62 (2008) 251–255. H. Tappan, The Paleobiology of Plant Protists, W.H. Freemann and Company, San Francisco, 1980, p. 1028. C. Hamm, R. Merkel, O. Springer, P. Jurkojc, C. Maier, K. Prechtel, V. Smetacek, Architecture and material properties of diatom shells provide effective mechanical protection, Nature 421 (2003) 841–843. C.E. Hamm, The evolution of advanced mechanical defenses and potential technological applications of diatom shells, J. Nanosci. Nanotechnol. 5 (2005) 108–119. R.R.L. Guillard, J.H. Ryther, Studies of marine planktonic diatoms, I, Cyclotella nana (Hustedt) and Detonula convervacea (Cleve), Can. J. Microbiol. 8 (2) (1962) 229–239. G. Langer, I. Probert, G. Nehrke, P. Ziveri, The morphological response of Emiliania huxleyi to seawater carbonate chemistry changes: an inter-strain comparison, J. Nannoplankton Res. 32 (2011) 27–32. B.N. Jaya, C. Kirchlechner, G. Dehm, Can micro-scale fracture tests provide reliable fracture toughness values? A case study in silicon, J. Mater. Res. 30 (5) (2015) 686–698. S. Trimborn, G. Langer, B. Rost, Effect of varying calcium concentrations and light intensities on calcification and photosynthesis in Emiliania huxleyi, Limnol. Oceanogr. 52 (2007) 2285–2293. A.R. Taylor, M.A. Russell, G. Harper, T. Collins, C. Brownlee, Dynamics of the formation and secretion of hetercoccoliths by Coccolithus pelagicus (ssp braarudii), Eur. J. Phycol. 42 (2007) 125–136. G. Langer, L.J. de Nooijer, K. Oetjen, On the role of the cytoskeleton in coccolith morphogenesis: the effect of cytoskeleton inhibitors, J. Phycol 46 (2010) 1252– 1256. B.N. Jaya, V. Jayaram, Fracture testing at small length scales: from plasticity in Si to brittleness, Pt. JOM 68 (1) (2015) 94–108. G. Langer, M. Bode, CO2 mediation of adverse effects of seawater acidification in Calcidiscus leptoporus, Geochem. Geophys. Geosyst. 12 (2011) Q05001. J.M. Didymus, J.R. Young, S. Mann, Construction and morphogenesis of the chiral ultrastructure of coccoliths from marine alga Emiliania huxleyi, Proc R Soc Ser B 258 (1994) 237–245.
