eschweizerbart_xxx

Calcium carbonate bio-precipitation in counter-diffusion systems using the

soluble organic matrix from nacre and sea-urchin spine

MARIA SANCHO-TOMAS1 SIMONA FERMANI2 JAIME GOMEZ-MORALES1 GIUSEPPE FALINI2

and JUAN MANUEL GARCIA-RUIZ1

1 Laboratorio de Estudios Cristalograficos IACT (CSIC-UGR) Avda Las Palmeras n 4 18100 Armilla Spain2 Dipartimento di Chimica lsquolsquoG Ciamicianrsquorsquo Alma Mater Studiorum Universita di Bologna via Selmi 2

40126 Bologna ItalyCorresponding authors e-mail giuseppefaliniuniboit (Giuseppe Falini) jaimeleccsices (Jaime Gomez Morales)

Abstract The biomineralization process of nacre and sea-urchin spine occurs under the biological control of specific macromole-cules in gelling environments through diverse mechanisms In both cases the formation of the crystalline phases ndash aragonite in nacreand magnesian calcite in sea-urchin spine ndash takes place through amorphous calcium carbonate precursor phases Here thesebiomineralization processes were investigated by means of counter-diffusion crystallization experiments using agarose highlyviscous sols entrapping the soluble organic matrix (SOM) extracted from the two mentioned biominerals The presence of theseSOMs did not increase the supersaturation needed for precipitation but narrowed the permitted supersaturations with respect tocalcium and carbonate ions when compared to those observed in experiments using synthetic polypeptides or SOM from corals Inthe presence of SOMs and diffusing magnesium ions the analyses of the precipitates suggested that crystallization proceeded throughtransient amorphous calcium carbonate phases These favoured the crystallization of aragonite or calcite according to the biomineralfrom which SOM was extracted when a specific concentration was used This study showed a control of SOMs on the mineralizationprocess which was more specific for nacre and sea-urchin spine than that for coral skeletons It also validates the counter-diffusionsystem as a tool to investigate biomineralization processes in vitro

Key-words nacre sea-urchin spine soluble organic matrix counter-diffusion system highly viscous sol calcium carbonatebiomineralization

Introduction

The biomineralization refers to the process by whichorganisms form mineral phases (Weiner amp Lowenstam1989) A family of acidic macromolecules entrapped inthe biomineral referred as organic matrix (OM) was foundto be the main responsible for the organism control overmineral composition structure polymorphism morphol-ogy and shape (Crenshaw 1972) In marine calcifyingorganisms two common strategies to produce minerals incontrolled way are used Some organisms first form anorganic matrix framework that then acts as a site for thenucleation and growth of single nano-crystals Then theassembly of nano-crystals in an ordered array is guided bythe structure of the preformed framework (Nakahara1979) Other organisms form vesicles where the mineralcomponent grow here the nano-crystals self-assemble in atri-dimensional way to finally generate single crystals withcomplex and usually curved surfaces (Towe 1967)

The nacreous layer of the mollusc Nautilus pompilius andthe spines of the echinoderm Paracentrotus lividus (Fig 1) are

representative examples of the two above reported biominer-alization mechanisms and are formed of aragonite and calciterespectively the two most common calcium carbonate(CaCO3) biominerals (Lowenstam amp Weiner 1989) Thenacre of N pompilius is formed by a columnar brick-and-mortar structure (Nakahara 1979 Heinemann et al 2011)Aragonite tablets (bricks) are separated by interlamellarsheets of a protein-b-chitin complex (mortar) (Watabe1965 Weiner et al 1983 Weiss et al 2009) (Fig 1) Eachtablet was formed by the 3D registered assembly of nano-particles that diffract X-ray as a twin single crystal of arago-nite The tablet grew parallel to the organic-b-sheet andperpendicular until achieving the consecutive b-sheet layerBetween the b-sheets crystals grew embedded in a silkfibroin-like gel matrix (Levi-Kalisman et al 2001) Sea-urchin spines are composed by the 3D registered assemblyof elongated nano-crystals of magnesian calcite They show aglassy cleavage (Fig 1) and diffract X-ray as single crystals(Schmidt 1924 Towe 1967 Seto et al 2012) Calcoblastsare associated with the growing surface of the spine and areresponsible for the mineral growth occurring on the top of the

Biomineralization andbiomimetic materials

0935-1221140026-2389 $ 585DOI 1011270935-122120140026-2389 2014 E Schweizerbartrsquosche Verlagsbuchhandlung D-70176 Stuttgart

Eur J Mineral

2014 26 523ndash535

Published online February 2014

eschweizerbart_xxx

spine Spine growth was first studied by regenerating a spine(Dubois amp Ameye 2001) Then it was observed that anamorphous calcium-carbonate (ACC) phase first formed invesicles was then deposited on the tip of the spine andgradually transformed into calcite (Politi et al 2004)

In agreement with the above mineralization processesbiominerals without exception appeared to be built byspheroidal grains in which calcareous material was crystal-lized (Baronnet et al 2008) This observation suggestedan interaction between the OM molecules and the mineralphase which could take place also along specific crystal-line planes (Addadi amp Weiner 1985) However the pre-sence of an organic cortex covering the mineral particlescould witness also the non-specific OM entrapping into thecrystal In supporting this it should be considered thatbiominerals nucleation and growth processes occurred insites having the features of highly viscous sols due to thepresence of a high concentration of acidic macromoleculesmaking the OM This knowledge was experimentallyproved in vivo for the nacre (Heinemann et al 2011 Sunamp Bhushan 2012) the opercolum (Khalifa et al 2011)the sea-urchin spine (Politi et al 2004 Rao et al 2013)and the coral (Gagnon et al 2012 Venn et al 2013)

In vitro experiments on the crystallization in highlyviscous sols or gels were carried out mainly to studynucleation and growth under conditions in which ionictransport was controlled only by diffusion For these stu-dies a widely used experimental set up was the counterdiffusion system (CDS) in which a gel or highly viscoussol separated the two reservoirs from which the ionscounter diffused into the gel The CDS permitted toexplore a continuous variation of ion activities and super-saturation both in space and time (Henisch 1970 1988)

The CDS was recently (re)proposed as a tool to study invitro biomineralization processes in conditions that maysimulate the gelling environment observed in vivo(Silverman amp Boskey 2004 Asenath-Smith et al 2012)Some parameters measurable in the CDS were rationa-lized The supersaturation conditions at early stages ofcrystallization were qualitatively inferred from the

position in the highly viscous sol column of the first pre-cipitate (xo) and the time (tw) it was first observed Similartw and xo in different experiments were interpreted ascrystallization occurring under similar supersaturationconditions in all of them The length of the crystallizationregion (D) was assumed to reflect how restrictive thesupersaturation and the equality of cation-anion concentra-tions were for nucleation to occur (Henisch et al 1986aand b Garcıa-Ruiz 1991) higher D values were indicativeof less restrictive supersaturation and cation-anion concen-tration ratios for nucleation (Sancho-Tomas et al 2013)

In this research we investigated the influence of solubleorganic matrix from N pompilius (NpoSOM) andP lividus (PliSOM) on the precipitation of CaCO3 in anagarose highly viscous sol by CDS The main goal was todetermine their effects on the crystallization of CaCO3

taking advantage of the reported peculiar characteristicsof the CDS which allows the discrimination of additiveeffects on nucleation andor growth and also to estimatethe supersaturation condition at which the nucleationoccurs (Prieto et al 1994 Katsikopoulos et al 2009Fernandez-Gonzalez amp Fernandez-Dıaz 2013)

2 Materials and methods

21 Extraction of SOM

Fragments of the septa of N pompilius and spines of P lividuswere suspended in a NaClO solution (3 vv) for 10 minutesto remove traces of organic material from the body of organ-isms The pieces were air dried for one night and ground in amortar up to obtain a fine and homogeneous powder (grainsize 100 mm) Then 35 g of powdered biominerals weredispersed in 5 mL of milli-Q water (resistivity 182 MV cm at25 C filtered through a 022 mm membrane) and poured intoan osmotic tube for dialysis (MWCO frac14 35 kDa CelluSepMFPI) The sealed tubes were put into 1 L of 01 MCH3COOH solution under stirring The decalcification pro-ceeded for 48 h At the end the tube containing the extracted

Fig 1 Composite images showing the living organism in situ (insets) and scanning-electron microscope (SEM) pictures of the fractured shellof N pompilus (A) and of the spine P lividus (B) In (A) and (B) are observable the typical organization of tablets in the nacre structure andthe glassy cleavage of a single-crystal sea-urchin spine The images of the organisms were modified from wikicommons (online version incolour)

524 M Sancho-Tomas et al

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organic matrix was dialyzed against milli-Q water until thefinal pH was about 6 The obtained aqueous solution contain-ing the whole organic matrix was centrifuged at 30 g for 5minutes to separate the SOM and the insoluble fraction whichwere then freeze-dried

22 Preparation of agarose highly viscous sols

Firstly an agarose stock solution of 02 (wv) was heatedup to 90 C for 20 minutes to dissolve completely theagarose powder (Agarose D-5 Hispanagar) Then thesolution was cooled down to 50 C and thereafter mixedwith the required volume of heated milli-Q water in dif-ferent beakers partially submerged in a water bath at 55 Cto obtain a final 01 (wv) agarose highly viscous solconcentration In each beaker different amounts ofNpoSOM or PliSOM were added to reach a final concen-tration of 50 mgmL (c) or 250 mgmL (5c) The preparedsolution was stirred for 1 minute and transferred to U-tubeswith a 1 mL syringe The final pH of the highly viscous sols(pHhvs) was about 65

The low rigidity of the highly viscous sol posed a series ofproblems in the settling of the experiment Particular care waspaid in the filling process of the reservoir that occurred at thesame time on both sides of the tube The horizontal position ofthe tube was also checked If these main parameters werecarefully controlled the reproducibility was acceptable

No remarkable difference in the viscosity of the highlyviscous sol estimated by tilting a tube containing thehighly viscous sol with and without SOMs was observedThe SOM was added in very low concentration withrespect to the one of the agarose which was kept constant01 (wv) in all the experiments

23 CaCO3 crystallization experiments

The experiments were carried out by using a U-tube setupThis tube has a column length of 45 mm which is acces-sible to diffusing reagents from two side reservoirs 02 mLof a 05 M solution with MgCa ratio equal to 0 or 3 wasadded in the cation reservoir These solutions were pre-pared by dissolving the required amounts of CaCl22H2Oand MgCl26H2O (Sigma-Aldrich) in milli-Q water 02mL of 05 M NaHCO3 solution (Fluka Biochemika) wasadded in the anion reservoir Cation- and anion-bearingsolutions counter-diffused through the column filled withthe agarose highly viscous sol entrapping the SOM Whenthe first crystals appeared after a waiting time tw thedistance from the cationic reservoir to the first observedcrystals xo was measured The length of the crystallizationregion in the U-tube D was measured 14 days after theonset of the experiment

The values of x0 and D were normalized to the tubelength They are thus dimensionless numbers that assumevalues from zero (x0 at the cationic reservoir) to one (x0 atthe anion reservoir)

The experiment using 250 mgmL PliSOM and diffusingMg2thorn showed a variability in the tw value to which wasassociated a high value of the standard deviation

Precipitates were taken out from the column tube andplaced on top of a 045 mm pore size cellulose filter Theprecipitates were washed several times with hot milli-Qwater in order to remove the agarose residue and then air-dried All the experiments were performed at room tem-perature (23 2 C) Each set of experiments was repli-cated at least three times and in each set an additive-freecontrol experiment was present (Fig 2)

24 Characterization of the precipitates

The optical observations were carried out with anoptical microscope (Nikon AZ100) connected to adigital camera (Nikon DS-Fi1) Some samples wereinspected in a PhenomTM scanning electron micro-scope (SEM) In addition scanning electron micro-graphs of carbon-sputtered samples were recordedusing a GEMINI SMT field-emission SEM (CarlZeiss Germany) The structural properties of the pre-cipitates were analyzed by X-ray diffraction (XRD)Fourier transform infrared spectroscopy (FTIR) andRaman spectroscopy The mass of obtained precipi-tates was not enough to be analyzed by X-ray powderdiffraction Therefore the precipitates were groundand mounted on a Bruker X8 Proteum X-ray diffract-ometer equipped with a Microstar copper rotatinganode generator a goniometer and a SMART 6000CCD detector The calculated X-ray powder diffrac-tion pattern was obtained after integrating the diffrac-tion frames with the XRD2DSCAN software(Rodrıguez-Navarro 2006) The FTIR analyses wereperformed by using a FTIR Nicolet 380 (ThermoElectron Co) The measurements were done in thewavelength range 4000ndash400 cm-1 at a resolution of 4cm-1 Disks were made by mixing a small amount( 1 mg) of sample with 100 mg of KBr and applyinga pressure of 486 psi to the mixture using a hydraulicpress Raman spectra were collected using aLabRAMHR spectrometer with backscattering geome-try (Jobin-Yvon Horiba Japan) The excitation linewas provided by a diode laser emitting at a wave-length of 532 nm and a Peltier cooled charge-coupledevice was used as detector The spectrometer resolu-tion was better than 3 cm-1 The spectra were base-line corrected for clarity Crystals were poured into aPetri dish and observed under the Raman microscope(10x) before collecting the spectrum of several crys-tals from each condition (only conditions where Mg2thorn

were diffusing from cationic reservoir)A Rietveld program (Quanto) for quantitative phase

analysis of polycrystalline mixtures from powder diffrac-tion data was used to quantify calcium carbonate phasesand to evaluate the unit-cell parameters (Altomare et al2001) The isomorphic substitution of magnesium to

Calcium carbonate bio-precipitation in counter-diffusion systems 525

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calcium in the calcite structure was estimated from the unit-cell parameters according to Paquette amp Reeder (1990)

3 Results

31 CaCO3 precipitation into highly viscous sols

A reference experiment of CaCO3 precipitation was carriedout using the CDS in absence of the SOM Calcium chlorideand sodium hydrogen carbonate solutions counter-diffusedthrough the agarose viscous sol column from the cationicand anionic reservoirs respectively The first precipitatesappeared after a tw of 22 8 h at the position equal to 062 005 The precipitation evolved symmetrically with respectto xo and after 14 days from the onset of the experiment D was030 003 (Fig 2 Table 1) Isolated crystals were observedunder an optical microscope (Fig 3A and Fig S1A in supple-mentary material freely available online linked to this articleon the GSW website of the journal httpeurjmingeoscien-ceworldorg) and they were identified as calcite by XRDanalyses (Fig 7) Calcite appeared as single crystals of sizesbetween 75 and 200 mm and displaying rhombohedral 104faces plus less developed hk0 faces (Fig 4AndashC) as alreadyreported (Li et al 2009)

32 CaCO3 precipitation into highly viscous solscontaining SOMs

The NpoSOM was added to the highly viscous sol atconcentrations of 50 mgmL (c) or 250 mgmL (5c)

Using the same experimental set-up of the referenceexperiment tw was of 20 10 h and 24 4 h whilexo was of 069 003 and 065 003 respectivelyThe evolution of the precipitation was slightly asymme-trical with respect to xo and roughly stopped in theposition 064 001 and 063 001 in the cationicreservoir direction and in the position xo equal to 073 001 and 071 003 in the anionic reservoir directionwhen using c and 5c NpoSOM respectively (Fig 2Table 1) In the presence of c NpoSOM optical micro-scope pictures showed that in the precipitation zone (D)crystalline aggregates with sizes between 80 and 400 mmwere formed (Fig S1C) When 5c NpoSOM was usedthe sizes of the observed aggregates oscillate between 40and 350 mm (Fig S1E) The SEM pictures (Fig 5AndashF)showed that in the presence of entrapped NpoSOM onlyaggregates of rhombohedral crystals of calcite formed andthat the aggregation increased with the concentration ofNpoSOM When 5c NpoSOM was present the crystalsappeared formed by the assembly of elongated sub-micro-meter particles (Fig 5F) an effect that was not evidentwhen c NpoSOM was used (Fig 5C)

The dissolution of PliSOM into the highly viscoussol gave a tw of 23 3 h and of 22 4 h and a xo of065 004 and of 066 001 when using a con-centration equal to 50 mgmL (c) or 250 mgmL (5c)respectively The precipitation evolved slightly asym-metrically with respect to xo and stopped in the posi-tion 060 002 and 061 001 in the cationicreservoir direction and in the position 071 001and 069 001 in the anionic reservoir direction

Fig 2 Graphical representation of the measured parameters in the precipitation experiments of calcium carbonate carried out by counter-diffusion system Left-column and right-column refers to the experiments without and with the presence of Mg2thorn in the cation reservoirrespectively The parameters were measured in the absence (A) and in the presence of NpoSOM at concentrations equal to 50 mgmL (B) andto 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) The length of the tubes has beennormalized from cation reservoir (0) to anion reservoir (1) The real length of the tubes was 45 mm Red and blue colours indicate thecrystallization region from the starting point of crystallization (xo bold numbers) to the cation reservoir (left-lower corner) and anionreservoir (right-lower corner) respectively Arrows indicate the waiting time (tw hours left-upper corner) and the number of replica isshown in the right-upper corner Horizontal black lines in the middle of each figure show the variability in the measurements Polymorphswere also indicated as calcite (C) Mg-calcite (MgC) and aragonite (A) (online version in colour)

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calcium in the calcite structure was estimated from the unit-cell parameters according to Paquette amp Reeder (1990)

3 Results

31 CaCO3 precipitation into highly viscous sols

A reference experiment of CaCO3 precipitation was carriedout using the CDS in absence of the SOM Calcium chlorideand sodium hydrogen carbonate solutions counter-diffusedthrough the agarose viscous sol column from the cationicand anionic reservoirs respectively The first precipitatesappeared after a tw of 22 8 h at the position equal to 062 005 The precipitation evolved symmetrically with respectto xo and after 14 days from the onset of the experiment D was030 003 (Fig 2 Table 1) Isolated crystals were observedunder an optical microscope (Fig 3A and Fig S1A in supple-mentary material freely available online linked to this articleon the GSW website of the journal httpeurjmingeoscien-ceworldorg) and they were identified as calcite by XRDanalyses (Fig 7) Calcite appeared as single crystals of sizesbetween 75 and 200 mm and displaying rhombohedral 104faces plus less developed hk0 faces (Fig 4AndashC) as alreadyreported (Li et al 2009)

32 CaCO3 precipitation into highly viscous solscontaining SOMs

The NpoSOM was added to the highly viscous sol atconcentrations of 50 mgmL (c) or 250 mgmL (5c)

Using the same experimental set-up of the referenceexperiment tw was of 20 10 h and 24 4 h whilexo was of 069 003 and 065 003 respectivelyThe evolution of the precipitation was slightly asymme-trical with respect to xo and roughly stopped in theposition 064 001 and 063 001 in the cationicreservoir direction and in the position xo equal to 073 001 and 071 003 in the anionic reservoir directionwhen using c and 5c NpoSOM respectively (Fig 2Table 1) In the presence of c NpoSOM optical micro-scope pictures showed that in the precipitation zone (D)crystalline aggregates with sizes between 80 and 400 mmwere formed (Fig S1C) When 5c NpoSOM was usedthe sizes of the observed aggregates oscillate between 40and 350 mm (Fig S1E) The SEM pictures (Fig 5AndashF)showed that in the presence of entrapped NpoSOM onlyaggregates of rhombohedral crystals of calcite formed andthat the aggregation increased with the concentration ofNpoSOM When 5c NpoSOM was present the crystalsappeared formed by the assembly of elongated sub-micro-meter particles (Fig 5F) an effect that was not evidentwhen c NpoSOM was used (Fig 5C)

The dissolution of PliSOM into the highly viscoussol gave a tw of 23 3 h and of 22 4 h and a xo of065 004 and of 066 001 when using a con-centration equal to 50 mgmL (c) or 250 mgmL (5c)respectively The precipitation evolved slightly asym-metrically with respect to xo and stopped in the posi-tion 060 002 and 061 001 in the cationicreservoir direction and in the position 071 001and 069 001 in the anionic reservoir direction

Fig 2 Graphical representation of the measured parameters in the precipitation experiments of calcium carbonate carried out by counter-diffusion system Left-column and right-column refers to the experiments without and with the presence of Mg2thorn in the cation reservoirrespectively The parameters were measured in the absence (A) and in the presence of NpoSOM at concentrations equal to 50 mgmL (B) andto 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) The length of the tubes has beennormalized from cation reservoir (0) to anion reservoir (1) The real length of the tubes was 45 mm Red and blue colours indicate thecrystallization region from the starting point of crystallization (xo bold numbers) to the cation reservoir (left-lower corner) and anionreservoir (right-lower corner) respectively Arrows indicate the waiting time (tw hours left-upper corner) and the number of replica isshown in the right-upper corner Horizontal black lines in the middle of each figure show the variability in the measurements Polymorphswere also indicated as calcite (C) Mg-calcite (MgC) and aragonite (A) (online version in colour)

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Table 1 Summary of data obtained from precipitation experiments of calcium carbonate by CDS in the absence and presence of SOM fromN pompilius or P lividus entrapped in agarose viscous sol in the absence and presence of Mg2thorn in the cationic reservoir The precipitationparameters refer to measures of the mineral precipitated in the U-tube starting point of precipitation (xo) length of the region around xo (D)waiting time (tw) The precipitate features refer to the minerals after removal from the agarose matrix

Mg2thornCa2thorn frac14 0 Mg2thornCa2thorn frac14 3

ref Npo c Npo 5c Pli c Pli 5c ref Npo c Npo 5c Pli c Pli 5c

xo1 062 (005) 069 (003) 065 (003) 065 (004) 066 (001) 063 (003) 059 (005) 069 (009) 065 (013) 066 (008)

tw2 22 (8) 20 (10) 24 (4) 23 (3) 22 (4) 34 (11) 30 (18) 36 (24) 36 (15) 50 (17)

D 030 (003) 009 (001) 008 (003) 011 (002) 008 (001) 035 (004) 010 (003) 009 (001) 011 (002) 009 (003)phase3 C C C C C MgC A A MgC A A MgC A MgCshape rhom r ag r ag r ag r ag ac sp

sp agac sp sm

sp pesm sp ac sp sm sp

sp agsize4 75ndash200 80ndash400 40ndash350 100ndash550 75ndash500 80ndash150 100ndash200 50ndash200 80ndash300 150ndash500

1 These values are normalized with respect to the length of the U-tube from the cation (0) to the anion reservoir (1) The variability in themeasurements is reported in parentheses 2 The tw is measured in hours (variability in the measurements) 3 Precipitated mineral phase CMgC and A indicate calcite Mg-calcite and aragonite respectively Shape of crystals observed by SEM rhom indicates modifiedrhombohedra r ag indicates aggregates of modified rhombohedra sp indicates spherulites ac sp indicates acicular spherulites sm spindicates spherulites with smooth surface sp ag indicates aggragates of spherulites pe indicates peanut-shape 4 Size distribution ofprecipitates measured along the main axis (mm)

Fig 3 Optical microscope pictures of crystal growing spaces (D) after 14 days in the absence (A B) and in the presence of NpoSOM atconcentration 50 mgmL (C D) and 250 mgmL (E F) and from PliSOM at concentration 50 mgmL (G H) and 250 mgmL (I J) (A) (C)(E) (G) and (I) and (B) (D) (F) (H) and (J) refer to the experiments carried out without and with the presence of Mg2thorn diffusing from thecationic reservoir respectively (see also Fig S1) (online version in colour)

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using PliSOM concentrations equal to c and 5crespectively (Fig 2 Table 1) Under the opticalmicroscope (Figs 3 and S1G) the precipitatesappeared formed by aggregated particles The increaseof PliSOM concentration from c to 5c provoked anarrowing of D and a small decrease of the aggregatessize from 100ndash550 mm to 75ndash500 mm respectively

In Fig 6AndashF are reported SEM pictures showing cal-cium carbonate crystals formed in the presence ofentrapped PliSOM using c PliSOM (Fig 6AndashC) and 5cPliSOM (Fig 6DndashF) In these conditions only modifiedrhombohedral crystals formed Their aggregation stateincreased with the concentration of PliSOM and theshape and size of the building nano-blocks (Fig 6C andF) making the crystalline units were not affected by thePliSOM concentration Calcite was the only phasedetected by XRD analysis (Fig 7)

33 CaCO3 precipitation into the viscous solcontaining SOMs and diffusing Mg2thorn

The addition of Mg2thorn to the cation reservoir (MgCa molarratio equal to 3) always provoked an increase of tw Whenthere was no added SOM xo was very similar to that of theprevious reference experiment (063 003) and to thatfound in the presence of PliSOMs (065 013 when usingc and 066 008 when the SOM concentration was 5c)Notably xo value in the presence of c NpoSOM appearedcloser to the cationic reservoir (059 005) while atconcentration 5c it shifted toward the anionic reservoir(069 009) as compared to the xo value of the referenceexperiment In the presence of Mg2thorn D was asymmetricbeing longer from xo toward the cationic reservoir thantoward the anionic one All the D values were similar tothose observed in Mg2thorn-free experiments (Fig 2 Table 1)

Fig 4 Scanning electron microscope (SEM) images showing the morphology of calcium carbonate crystals precipitated in the agarose highlyviscous sol in the absence of SOMs (A-C) show different magnifications of a calcite crystal grown in absence of Mg2thorn In them therhombohedral 104faces of calcite were indicated together with those hk0 due to the interaction with agarose molecules (D-I) images atdifferent magnification of spherulites formed in the presence of diffusing Mg2thornfrom the cation reservoir (D-F) show a spherulite ofmagnesian calcite In the high-magnification image (F) the crystallographic faces typical of magnesian calcite are indicated (G-I) show aspherulite of aragonite In (I) the hexagonal needle-like 001 capped crystal of aragonite are shown These pictures are representative of theentire populations of crystals

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Fig 5 SEM pictures showing calcium carbonate crystals precipitated in the presence of entrapped NpoSOM (A-F) images of crystals obtained inthe absence of diffusing Mg2thorn from the cationic reservoir using c NpoSOM (A-C) and 5c NpoSOM (D-F) In these conditions only calcite crystalsformed Their aggregation state increased with the concentration of NpoSOM When 5c NpoSOM was present the crystals appeared formed by theassembly of spheroidal nano-grains (F) this effect was less evident when c NpoSOM was used (C) (G-O) Images of crystals obtained in the presenceof diffusing Mg2thorn from the cationic reservoir using NpoSOM In the presence of c NpoSOM (G-I) only aragonite precipitated The needle-likecrystals lost the crystalline morphology observed in the absence of SOM and appeared entrapped in the NpoSOMagarose matrix When 5cNpoSOM was present magnesian calcite co-precipitated with aragonite and two types of spherulites were observed In one case (J-L) the crystallineunits making the spherulite were formed by the assembly of nano-particles and were rhombohedral capped (L) In the other case (M-O) the spherulitewas made by the assembly of irregular needle-like shapes that resembled a poor (N) or completely lost (O) crystalline morphology in both cases theneedle-like shapes were formed by nano-spheroidal particles The particles shown are representative of the whole sample populations

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Fig 6 SEM pictures showing calcium carbonate crystals formed in the presence of entrapped PliSOM (A-F) Images of crystals obtained in theabsence of diffusing Mg2thorn from the cationic reservoir using c PliSOM (A-C) and 5c PliSOM (D-F) In these conditions only calcite crystals formedTheir aggregation state increased with the concentration of PliSOM and shape and size of the building nano-blocks (C and F) making the crystallineunits were not affected by the PliSOM concentration (G-R) Images of particles obtained in the presence of diffusing Mg2thorn from the cationicreservoir using PliSOM In the presence of c PliSOM (G-L) magnesian calcite co-precipitated with aragonite Two families (G-I and J-L) ofspherulites were observed Between them differences were clearly observable only at high magnifications In one case (I) plate-like crystals wereobserved where in the other one (L) needle-like crystals appeared Their crystalline morphologies were poor and irregular When 5c PliSOM waspresent magnesium calcite and aragonite precipitated At low magnification all the spherulites showed a similar morphology and were randomlyaggregated (M and P) At high magnification at least two different scenarios were observed In one case the surface (O) or a fractured region of thespherulite (N) showed the presence of nano-spheroidal particles preferential aligned in one direction making needle-like structure In the other casethe presence of nano-particles rhombohedral capped was observed (Q and R) The particles shown are representative of the whole samplepopulations

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The diffusion of Mg2thorn through the agarose column ledto the precipitation of big rounded and small peanuts-shaped particles which appeared close to each other andpartially overlapped (Fig S1B) Images at different mag-nification of spherulites are shown in Fig 4DndashI Fig 4DndashFshow a spherulite of magnesian calcite In the image athigh magnification (Fig 4F) the crystallographic facestypical of magnesian calcite crystals are indicatedFig 4GndashI show a spherulite of aragonite in Fig 4I thehexagonal needle-like crystals of aragonite 001 cappedare shown

When SOMs were added we observed spherical and iso-lated particles and not well defined D borders Only whenusing 5c NpoSOM the particles appeared more integrated andsharp D borders were observed In this last condition sphe-rical aggregates with small peanut-shaped particles precipi-tated Morphological changes were observed when usingNpoSOM or PliSOM with respect to the reference

Images of particles obtained in the presence of diffusingMg2thorn from the cationic reservoir using NpoSOM areshown in Fig 5GndashO In the presence of c NpoSOM(Fig 5GndashI) the needle-like crystals partially lost the crys-talline morphology observed in the absence of SOM andappeared entrapped in the NpoSOMagarose matrix When5c NpoSOM was present two typology of spherulites wereobserved In one case (Fig 5JndashL) the crystalline unitsmaking the spherulite were formed by the assembly ofnano-particles and were rhombohedral capped (Fig 5L)In the other case (Fig 5MndashO) the spherulite was made bythe assembly of irregular shapes preferentially alignedalong a preferential direction that resembled a poor(Fig 5N) or completely lost (Fig 5O) crystalline morphol-ogy in both cases the assembled shapes were formed bynano-spheroidal particles

In Fig 6GndashR images of particles obtained in the pre-sence of diffusing Mg2thorn from the cationic reservoir usingPliSOM are shown Two families (Fig 6GndashI and 6JndashL) ofspherulites were observed in the presence of c PliSOMBetween them the differences were clearly observable onlyat high magnifications In one case (Fig 6I) plate-likecrystals were observed whereas in the other one (Fig 6L)needle-like crystals appeared Their crystalline morpholo-gies were poor and irregular When 5c PliSOM was pre-sent all the spherulites showed at low magnification asimilar morphology and were randomly aggregated(Fig 6M and P) At high magnification at least two differ-ent scenarios were observed In one case the surface (Fig6O) or a fractured region of the spherulite (Fig 6N)showed the presence of nano-spheroidal particles alignedpreferentially in one direction thus making needle-likestructures In the other case the presence of rhombohedralcapped nanoparticles was observed (Fig 6Q and R)

Aragonite and magnesian calcite were identified byX-ray diffraction and FTIR spectroscopy (Figs 7 andS2) except for NpoSOM at concentration c condition atwhich only aragonite was detected The quantification ofthe crystalline mineral phases was carried out by Rietveldrefinement of the X-ray powder diffraction patterns Thedata (Table S1) show that in the absence of SOMs thecontent of aragonite was 95 2 ww It increaseadonly in the presence of c NpoSOM to 100 ww whiledecreased at 63 2 ww 85 2 ww and 89 2 ww in the presence of 5c NpoSOM c PliSOM and 5cPliSOM respectively

Raman spectra showed aragonite and calcite crystals inall conditions (Fig 8) All precipitates were inspected byoptical microscopy before collecting the Raman spectrumbut pictures did not reveal morphological differences

Fig 7 X-ray diffraction patterns of calcium carbonate precipitates The left-figure corresponds to the experiments in absence of Mg2thorn andthe right-figure in the presence of Mg2thorn The precipitates were obtained in the absence (A) and in the presence of NpoSOM at concentrationsequal to 50 mgmL (B) and to 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) (onlineversion in colour)

Calcium carbonate bio-precipitation in counter-diffusion systems 531

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

using PliSOM concentrations equal to c and 5crespectively (Fig 2 Table 1) Under the opticalmicroscope (Figs 3 and S1G) the precipitatesappeared formed by aggregated particles The increaseof PliSOM concentration from c to 5c provoked anarrowing of D and a small decrease of the aggregatessize from 100ndash550 mm to 75ndash500 mm respectively

In Fig 6AndashF are reported SEM pictures showing cal-cium carbonate crystals formed in the presence ofentrapped PliSOM using c PliSOM (Fig 6AndashC) and 5cPliSOM (Fig 6DndashF) In these conditions only modifiedrhombohedral crystals formed Their aggregation stateincreased with the concentration of PliSOM and theshape and size of the building nano-blocks (Fig 6C andF) making the crystalline units were not affected by thePliSOM concentration Calcite was the only phasedetected by XRD analysis (Fig 7)

33 CaCO3 precipitation into the viscous solcontaining SOMs and diffusing Mg2thorn

The addition of Mg2thorn to the cation reservoir (MgCa molarratio equal to 3) always provoked an increase of tw Whenthere was no added SOM xo was very similar to that of theprevious reference experiment (063 003) and to thatfound in the presence of PliSOMs (065 013 when usingc and 066 008 when the SOM concentration was 5c)Notably xo value in the presence of c NpoSOM appearedcloser to the cationic reservoir (059 005) while atconcentration 5c it shifted toward the anionic reservoir(069 009) as compared to the xo value of the referenceexperiment In the presence of Mg2thorn D was asymmetricbeing longer from xo toward the cationic reservoir thantoward the anionic one All the D values were similar tothose observed in Mg2thorn-free experiments (Fig 2 Table 1)

Fig 4 Scanning electron microscope (SEM) images showing the morphology of calcium carbonate crystals precipitated in the agarose highlyviscous sol in the absence of SOMs (A-C) show different magnifications of a calcite crystal grown in absence of Mg2thorn In them therhombohedral 104faces of calcite were indicated together with those hk0 due to the interaction with agarose molecules (D-I) images atdifferent magnification of spherulites formed in the presence of diffusing Mg2thornfrom the cation reservoir (D-F) show a spherulite ofmagnesian calcite In the high-magnification image (F) the crystallographic faces typical of magnesian calcite are indicated (G-I) show aspherulite of aragonite In (I) the hexagonal needle-like 001 capped crystal of aragonite are shown These pictures are representative of theentire populations of crystals

528 M Sancho-Tomas et al

eschweizerbart_xxx

Fig 5 SEM pictures showing calcium carbonate crystals precipitated in the presence of entrapped NpoSOM (A-F) images of crystals obtained inthe absence of diffusing Mg2thorn from the cationic reservoir using c NpoSOM (A-C) and 5c NpoSOM (D-F) In these conditions only calcite crystalsformed Their aggregation state increased with the concentration of NpoSOM When 5c NpoSOM was present the crystals appeared formed by theassembly of spheroidal nano-grains (F) this effect was less evident when c NpoSOM was used (C) (G-O) Images of crystals obtained in the presenceof diffusing Mg2thorn from the cationic reservoir using NpoSOM In the presence of c NpoSOM (G-I) only aragonite precipitated The needle-likecrystals lost the crystalline morphology observed in the absence of SOM and appeared entrapped in the NpoSOMagarose matrix When 5cNpoSOM was present magnesian calcite co-precipitated with aragonite and two types of spherulites were observed In one case (J-L) the crystallineunits making the spherulite were formed by the assembly of nano-particles and were rhombohedral capped (L) In the other case (M-O) the spherulitewas made by the assembly of irregular needle-like shapes that resembled a poor (N) or completely lost (O) crystalline morphology in both cases theneedle-like shapes were formed by nano-spheroidal particles The particles shown are representative of the whole sample populations

Calcium carbonate bio-precipitation in counter-diffusion systems 529

eschweizerbart_xxx

Fig 6 SEM pictures showing calcium carbonate crystals formed in the presence of entrapped PliSOM (A-F) Images of crystals obtained in theabsence of diffusing Mg2thorn from the cationic reservoir using c PliSOM (A-C) and 5c PliSOM (D-F) In these conditions only calcite crystals formedTheir aggregation state increased with the concentration of PliSOM and shape and size of the building nano-blocks (C and F) making the crystallineunits were not affected by the PliSOM concentration (G-R) Images of particles obtained in the presence of diffusing Mg2thorn from the cationicreservoir using PliSOM In the presence of c PliSOM (G-L) magnesian calcite co-precipitated with aragonite Two families (G-I and J-L) ofspherulites were observed Between them differences were clearly observable only at high magnifications In one case (I) plate-like crystals wereobserved where in the other one (L) needle-like crystals appeared Their crystalline morphologies were poor and irregular When 5c PliSOM waspresent magnesium calcite and aragonite precipitated At low magnification all the spherulites showed a similar morphology and were randomlyaggregated (M and P) At high magnification at least two different scenarios were observed In one case the surface (O) or a fractured region of thespherulite (N) showed the presence of nano-spheroidal particles preferential aligned in one direction making needle-like structure In the other casethe presence of nano-particles rhombohedral capped was observed (Q and R) The particles shown are representative of the whole samplepopulations

530 M Sancho-Tomas et al

eschweizerbart_xxx

The diffusion of Mg2thorn through the agarose column ledto the precipitation of big rounded and small peanuts-shaped particles which appeared close to each other andpartially overlapped (Fig S1B) Images at different mag-nification of spherulites are shown in Fig 4DndashI Fig 4DndashFshow a spherulite of magnesian calcite In the image athigh magnification (Fig 4F) the crystallographic facestypical of magnesian calcite crystals are indicatedFig 4GndashI show a spherulite of aragonite in Fig 4I thehexagonal needle-like crystals of aragonite 001 cappedare shown

When SOMs were added we observed spherical and iso-lated particles and not well defined D borders Only whenusing 5c NpoSOM the particles appeared more integrated andsharp D borders were observed In this last condition sphe-rical aggregates with small peanut-shaped particles precipi-tated Morphological changes were observed when usingNpoSOM or PliSOM with respect to the reference

Images of particles obtained in the presence of diffusingMg2thorn from the cationic reservoir using NpoSOM areshown in Fig 5GndashO In the presence of c NpoSOM(Fig 5GndashI) the needle-like crystals partially lost the crys-talline morphology observed in the absence of SOM andappeared entrapped in the NpoSOMagarose matrix When5c NpoSOM was present two typology of spherulites wereobserved In one case (Fig 5JndashL) the crystalline unitsmaking the spherulite were formed by the assembly ofnano-particles and were rhombohedral capped (Fig 5L)In the other case (Fig 5MndashO) the spherulite was made bythe assembly of irregular shapes preferentially alignedalong a preferential direction that resembled a poor(Fig 5N) or completely lost (Fig 5O) crystalline morphol-ogy in both cases the assembled shapes were formed bynano-spheroidal particles

In Fig 6GndashR images of particles obtained in the pre-sence of diffusing Mg2thorn from the cationic reservoir usingPliSOM are shown Two families (Fig 6GndashI and 6JndashL) ofspherulites were observed in the presence of c PliSOMBetween them the differences were clearly observable onlyat high magnifications In one case (Fig 6I) plate-likecrystals were observed whereas in the other one (Fig 6L)needle-like crystals appeared Their crystalline morpholo-gies were poor and irregular When 5c PliSOM was pre-sent all the spherulites showed at low magnification asimilar morphology and were randomly aggregated(Fig 6M and P) At high magnification at least two differ-ent scenarios were observed In one case the surface (Fig6O) or a fractured region of the spherulite (Fig 6N)showed the presence of nano-spheroidal particles alignedpreferentially in one direction thus making needle-likestructures In the other case the presence of rhombohedralcapped nanoparticles was observed (Fig 6Q and R)

Aragonite and magnesian calcite were identified byX-ray diffraction and FTIR spectroscopy (Figs 7 andS2) except for NpoSOM at concentration c condition atwhich only aragonite was detected The quantification ofthe crystalline mineral phases was carried out by Rietveldrefinement of the X-ray powder diffraction patterns Thedata (Table S1) show that in the absence of SOMs thecontent of aragonite was 95 2 ww It increaseadonly in the presence of c NpoSOM to 100 ww whiledecreased at 63 2 ww 85 2 ww and 89 2 ww in the presence of 5c NpoSOM c PliSOM and 5cPliSOM respectively

Raman spectra showed aragonite and calcite crystals inall conditions (Fig 8) All precipitates were inspected byoptical microscopy before collecting the Raman spectrumbut pictures did not reveal morphological differences

Fig 7 X-ray diffraction patterns of calcium carbonate precipitates The left-figure corresponds to the experiments in absence of Mg2thorn andthe right-figure in the presence of Mg2thorn The precipitates were obtained in the absence (A) and in the presence of NpoSOM at concentrationsequal to 50 mgmL (B) and to 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) (onlineversion in colour)

Calcium carbonate bio-precipitation in counter-diffusion systems 531

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

Fig 5 SEM pictures showing calcium carbonate crystals precipitated in the presence of entrapped NpoSOM (A-F) images of crystals obtained inthe absence of diffusing Mg2thorn from the cationic reservoir using c NpoSOM (A-C) and 5c NpoSOM (D-F) In these conditions only calcite crystalsformed Their aggregation state increased with the concentration of NpoSOM When 5c NpoSOM was present the crystals appeared formed by theassembly of spheroidal nano-grains (F) this effect was less evident when c NpoSOM was used (C) (G-O) Images of crystals obtained in the presenceof diffusing Mg2thorn from the cationic reservoir using NpoSOM In the presence of c NpoSOM (G-I) only aragonite precipitated The needle-likecrystals lost the crystalline morphology observed in the absence of SOM and appeared entrapped in the NpoSOMagarose matrix When 5cNpoSOM was present magnesian calcite co-precipitated with aragonite and two types of spherulites were observed In one case (J-L) the crystallineunits making the spherulite were formed by the assembly of nano-particles and were rhombohedral capped (L) In the other case (M-O) the spherulitewas made by the assembly of irregular needle-like shapes that resembled a poor (N) or completely lost (O) crystalline morphology in both cases theneedle-like shapes were formed by nano-spheroidal particles The particles shown are representative of the whole sample populations

Calcium carbonate bio-precipitation in counter-diffusion systems 529

eschweizerbart_xxx

Fig 6 SEM pictures showing calcium carbonate crystals formed in the presence of entrapped PliSOM (A-F) Images of crystals obtained in theabsence of diffusing Mg2thorn from the cationic reservoir using c PliSOM (A-C) and 5c PliSOM (D-F) In these conditions only calcite crystals formedTheir aggregation state increased with the concentration of PliSOM and shape and size of the building nano-blocks (C and F) making the crystallineunits were not affected by the PliSOM concentration (G-R) Images of particles obtained in the presence of diffusing Mg2thorn from the cationicreservoir using PliSOM In the presence of c PliSOM (G-L) magnesian calcite co-precipitated with aragonite Two families (G-I and J-L) ofspherulites were observed Between them differences were clearly observable only at high magnifications In one case (I) plate-like crystals wereobserved where in the other one (L) needle-like crystals appeared Their crystalline morphologies were poor and irregular When 5c PliSOM waspresent magnesium calcite and aragonite precipitated At low magnification all the spherulites showed a similar morphology and were randomlyaggregated (M and P) At high magnification at least two different scenarios were observed In one case the surface (O) or a fractured region of thespherulite (N) showed the presence of nano-spheroidal particles preferential aligned in one direction making needle-like structure In the other casethe presence of nano-particles rhombohedral capped was observed (Q and R) The particles shown are representative of the whole samplepopulations

530 M Sancho-Tomas et al

eschweizerbart_xxx

The diffusion of Mg2thorn through the agarose column ledto the precipitation of big rounded and small peanuts-shaped particles which appeared close to each other andpartially overlapped (Fig S1B) Images at different mag-nification of spherulites are shown in Fig 4DndashI Fig 4DndashFshow a spherulite of magnesian calcite In the image athigh magnification (Fig 4F) the crystallographic facestypical of magnesian calcite crystals are indicatedFig 4GndashI show a spherulite of aragonite in Fig 4I thehexagonal needle-like crystals of aragonite 001 cappedare shown

When SOMs were added we observed spherical and iso-lated particles and not well defined D borders Only whenusing 5c NpoSOM the particles appeared more integrated andsharp D borders were observed In this last condition sphe-rical aggregates with small peanut-shaped particles precipi-tated Morphological changes were observed when usingNpoSOM or PliSOM with respect to the reference

Images of particles obtained in the presence of diffusingMg2thorn from the cationic reservoir using NpoSOM areshown in Fig 5GndashO In the presence of c NpoSOM(Fig 5GndashI) the needle-like crystals partially lost the crys-talline morphology observed in the absence of SOM andappeared entrapped in the NpoSOMagarose matrix When5c NpoSOM was present two typology of spherulites wereobserved In one case (Fig 5JndashL) the crystalline unitsmaking the spherulite were formed by the assembly ofnano-particles and were rhombohedral capped (Fig 5L)In the other case (Fig 5MndashO) the spherulite was made bythe assembly of irregular shapes preferentially alignedalong a preferential direction that resembled a poor(Fig 5N) or completely lost (Fig 5O) crystalline morphol-ogy in both cases the assembled shapes were formed bynano-spheroidal particles

In Fig 6GndashR images of particles obtained in the pre-sence of diffusing Mg2thorn from the cationic reservoir usingPliSOM are shown Two families (Fig 6GndashI and 6JndashL) ofspherulites were observed in the presence of c PliSOMBetween them the differences were clearly observable onlyat high magnifications In one case (Fig 6I) plate-likecrystals were observed whereas in the other one (Fig 6L)needle-like crystals appeared Their crystalline morpholo-gies were poor and irregular When 5c PliSOM was pre-sent all the spherulites showed at low magnification asimilar morphology and were randomly aggregated(Fig 6M and P) At high magnification at least two differ-ent scenarios were observed In one case the surface (Fig6O) or a fractured region of the spherulite (Fig 6N)showed the presence of nano-spheroidal particles alignedpreferentially in one direction thus making needle-likestructures In the other case the presence of rhombohedralcapped nanoparticles was observed (Fig 6Q and R)

Aragonite and magnesian calcite were identified byX-ray diffraction and FTIR spectroscopy (Figs 7 andS2) except for NpoSOM at concentration c condition atwhich only aragonite was detected The quantification ofthe crystalline mineral phases was carried out by Rietveldrefinement of the X-ray powder diffraction patterns Thedata (Table S1) show that in the absence of SOMs thecontent of aragonite was 95 2 ww It increaseadonly in the presence of c NpoSOM to 100 ww whiledecreased at 63 2 ww 85 2 ww and 89 2 ww in the presence of 5c NpoSOM c PliSOM and 5cPliSOM respectively

Raman spectra showed aragonite and calcite crystals inall conditions (Fig 8) All precipitates were inspected byoptical microscopy before collecting the Raman spectrumbut pictures did not reveal morphological differences

Fig 7 X-ray diffraction patterns of calcium carbonate precipitates The left-figure corresponds to the experiments in absence of Mg2thorn andthe right-figure in the presence of Mg2thorn The precipitates were obtained in the absence (A) and in the presence of NpoSOM at concentrationsequal to 50 mgmL (B) and to 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) (onlineversion in colour)

Calcium carbonate bio-precipitation in counter-diffusion systems 531

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

Fig 6 SEM pictures showing calcium carbonate crystals formed in the presence of entrapped PliSOM (A-F) Images of crystals obtained in theabsence of diffusing Mg2thorn from the cationic reservoir using c PliSOM (A-C) and 5c PliSOM (D-F) In these conditions only calcite crystals formedTheir aggregation state increased with the concentration of PliSOM and shape and size of the building nano-blocks (C and F) making the crystallineunits were not affected by the PliSOM concentration (G-R) Images of particles obtained in the presence of diffusing Mg2thorn from the cationicreservoir using PliSOM In the presence of c PliSOM (G-L) magnesian calcite co-precipitated with aragonite Two families (G-I and J-L) ofspherulites were observed Between them differences were clearly observable only at high magnifications In one case (I) plate-like crystals wereobserved where in the other one (L) needle-like crystals appeared Their crystalline morphologies were poor and irregular When 5c PliSOM waspresent magnesium calcite and aragonite precipitated At low magnification all the spherulites showed a similar morphology and were randomlyaggregated (M and P) At high magnification at least two different scenarios were observed In one case the surface (O) or a fractured region of thespherulite (N) showed the presence of nano-spheroidal particles preferential aligned in one direction making needle-like structure In the other casethe presence of nano-particles rhombohedral capped was observed (Q and R) The particles shown are representative of the whole samplepopulations

530 M Sancho-Tomas et al

eschweizerbart_xxx

The diffusion of Mg2thorn through the agarose column ledto the precipitation of big rounded and small peanuts-shaped particles which appeared close to each other andpartially overlapped (Fig S1B) Images at different mag-nification of spherulites are shown in Fig 4DndashI Fig 4DndashFshow a spherulite of magnesian calcite In the image athigh magnification (Fig 4F) the crystallographic facestypical of magnesian calcite crystals are indicatedFig 4GndashI show a spherulite of aragonite in Fig 4I thehexagonal needle-like crystals of aragonite 001 cappedare shown

When SOMs were added we observed spherical and iso-lated particles and not well defined D borders Only whenusing 5c NpoSOM the particles appeared more integrated andsharp D borders were observed In this last condition sphe-rical aggregates with small peanut-shaped particles precipi-tated Morphological changes were observed when usingNpoSOM or PliSOM with respect to the reference

Images of particles obtained in the presence of diffusingMg2thorn from the cationic reservoir using NpoSOM areshown in Fig 5GndashO In the presence of c NpoSOM(Fig 5GndashI) the needle-like crystals partially lost the crys-talline morphology observed in the absence of SOM andappeared entrapped in the NpoSOMagarose matrix When5c NpoSOM was present two typology of spherulites wereobserved In one case (Fig 5JndashL) the crystalline unitsmaking the spherulite were formed by the assembly ofnano-particles and were rhombohedral capped (Fig 5L)In the other case (Fig 5MndashO) the spherulite was made bythe assembly of irregular shapes preferentially alignedalong a preferential direction that resembled a poor(Fig 5N) or completely lost (Fig 5O) crystalline morphol-ogy in both cases the assembled shapes were formed bynano-spheroidal particles

In Fig 6GndashR images of particles obtained in the pre-sence of diffusing Mg2thorn from the cationic reservoir usingPliSOM are shown Two families (Fig 6GndashI and 6JndashL) ofspherulites were observed in the presence of c PliSOMBetween them the differences were clearly observable onlyat high magnifications In one case (Fig 6I) plate-likecrystals were observed whereas in the other one (Fig 6L)needle-like crystals appeared Their crystalline morpholo-gies were poor and irregular When 5c PliSOM was pre-sent all the spherulites showed at low magnification asimilar morphology and were randomly aggregated(Fig 6M and P) At high magnification at least two differ-ent scenarios were observed In one case the surface (Fig6O) or a fractured region of the spherulite (Fig 6N)showed the presence of nano-spheroidal particles alignedpreferentially in one direction thus making needle-likestructures In the other case the presence of rhombohedralcapped nanoparticles was observed (Fig 6Q and R)

Aragonite and magnesian calcite were identified byX-ray diffraction and FTIR spectroscopy (Figs 7 andS2) except for NpoSOM at concentration c condition atwhich only aragonite was detected The quantification ofthe crystalline mineral phases was carried out by Rietveldrefinement of the X-ray powder diffraction patterns Thedata (Table S1) show that in the absence of SOMs thecontent of aragonite was 95 2 ww It increaseadonly in the presence of c NpoSOM to 100 ww whiledecreased at 63 2 ww 85 2 ww and 89 2 ww in the presence of 5c NpoSOM c PliSOM and 5cPliSOM respectively

Raman spectra showed aragonite and calcite crystals inall conditions (Fig 8) All precipitates were inspected byoptical microscopy before collecting the Raman spectrumbut pictures did not reveal morphological differences

Fig 7 X-ray diffraction patterns of calcium carbonate precipitates The left-figure corresponds to the experiments in absence of Mg2thorn andthe right-figure in the presence of Mg2thorn The precipitates were obtained in the absence (A) and in the presence of NpoSOM at concentrationsequal to 50 mgmL (B) and to 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) (onlineversion in colour)

Calcium carbonate bio-precipitation in counter-diffusion systems 531

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

The diffusion of Mg2thorn through the agarose column ledto the precipitation of big rounded and small peanuts-shaped particles which appeared close to each other andpartially overlapped (Fig S1B) Images at different mag-nification of spherulites are shown in Fig 4DndashI Fig 4DndashFshow a spherulite of magnesian calcite In the image athigh magnification (Fig 4F) the crystallographic facestypical of magnesian calcite crystals are indicatedFig 4GndashI show a spherulite of aragonite in Fig 4I thehexagonal needle-like crystals of aragonite 001 cappedare shown

When SOMs were added we observed spherical and iso-lated particles and not well defined D borders Only whenusing 5c NpoSOM the particles appeared more integrated andsharp D borders were observed In this last condition sphe-rical aggregates with small peanut-shaped particles precipi-tated Morphological changes were observed when usingNpoSOM or PliSOM with respect to the reference

Images of particles obtained in the presence of diffusingMg2thorn from the cationic reservoir using NpoSOM areshown in Fig 5GndashO In the presence of c NpoSOM(Fig 5GndashI) the needle-like crystals partially lost the crys-talline morphology observed in the absence of SOM andappeared entrapped in the NpoSOMagarose matrix When5c NpoSOM was present two typology of spherulites wereobserved In one case (Fig 5JndashL) the crystalline unitsmaking the spherulite were formed by the assembly ofnano-particles and were rhombohedral capped (Fig 5L)In the other case (Fig 5MndashO) the spherulite was made bythe assembly of irregular shapes preferentially alignedalong a preferential direction that resembled a poor(Fig 5N) or completely lost (Fig 5O) crystalline morphol-ogy in both cases the assembled shapes were formed bynano-spheroidal particles

In Fig 6GndashR images of particles obtained in the pre-sence of diffusing Mg2thorn from the cationic reservoir usingPliSOM are shown Two families (Fig 6GndashI and 6JndashL) ofspherulites were observed in the presence of c PliSOMBetween them the differences were clearly observable onlyat high magnifications In one case (Fig 6I) plate-likecrystals were observed whereas in the other one (Fig 6L)needle-like crystals appeared Their crystalline morpholo-gies were poor and irregular When 5c PliSOM was pre-sent all the spherulites showed at low magnification asimilar morphology and were randomly aggregated(Fig 6M and P) At high magnification at least two differ-ent scenarios were observed In one case the surface (Fig6O) or a fractured region of the spherulite (Fig 6N)showed the presence of nano-spheroidal particles alignedpreferentially in one direction thus making needle-likestructures In the other case the presence of rhombohedralcapped nanoparticles was observed (Fig 6Q and R)

Aragonite and magnesian calcite were identified byX-ray diffraction and FTIR spectroscopy (Figs 7 andS2) except for NpoSOM at concentration c condition atwhich only aragonite was detected The quantification ofthe crystalline mineral phases was carried out by Rietveldrefinement of the X-ray powder diffraction patterns Thedata (Table S1) show that in the absence of SOMs thecontent of aragonite was 95 2 ww It increaseadonly in the presence of c NpoSOM to 100 ww whiledecreased at 63 2 ww 85 2 ww and 89 2 ww in the presence of 5c NpoSOM c PliSOM and 5cPliSOM respectively

Raman spectra showed aragonite and calcite crystals inall conditions (Fig 8) All precipitates were inspected byoptical microscopy before collecting the Raman spectrumbut pictures did not reveal morphological differences

Fig 7 X-ray diffraction patterns of calcium carbonate precipitates The left-figure corresponds to the experiments in absence of Mg2thorn andthe right-figure in the presence of Mg2thorn The precipitates were obtained in the absence (A) and in the presence of NpoSOM at concentrationsequal to 50 mgmL (B) and to 250 mgmL (C) and from PliSOM at concentrations equal to 50 mgmL (D) and to 250 mgmL (E) (onlineversion in colour)

Calcium carbonate bio-precipitation in counter-diffusion systems 531

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

between phases Interestingly few calcite crystals weredetected when adding c NpoSOM in spite that only arago-nite was identified by means of XRD and FTIR analyses

The isomorphic substitution of Mg2thorn into the calcitestructure was evaluated by the reduction of the calciteunit-cell parameters (Table S1) The presence of SOMfavoured the substitution of Ca by Mg as compared to thecontrol experiment in which the substitution was of 6 2mol Values of 13 2 10 2 and 8 2 mol wereobtained when using 5c NpoSOM c PliSOM and 5cPliSOM respectively

4 Discussion

Several studies reported the chemical features of NpoSOMand PliSOM (Weiner amp Hood 1975 Weiner 1984Albeck et al 1996 Marin amp Luquet 2008) They weremainly composed of acidic glycoproteins andor proteo-glycans and had as main feature a high content of acidicresidues in their protein regions about 28 and 30 mol ofAsx and Glx in PliSOM and NpoSOM respectively (FigS3 Weiner 1984) The influence of NpoSOM and PliSOMon the in vitro precipitation of CaCO3 was widely exploredand many aspects of their role in vivo were clarified TheSOMs can control the CaCO3 polymorphism (Falini et al1996) the mechanical properties (Berman et al 1988) oras reported for other biominerals also the morphologysize and orientation of the crystallites (Hernandez-Hernandez et al 2008a b and c) In addition they canalso sculpture calcium carbonate crystals in diverse wayaccording to their composition (eg Albeck et al 1996)More recently it was shown that SOM stabilized the for-mation of ACC which was a precursor of the crystallinephases (Weiner amp Addadi 2011 Cartwright et al 2012)Moreover it was also shown that they guide the formationof biominerals according to synthetic paths that did notfollow the classical theories of nucleation and growth(Gebauer amp Colfen 2011)

However despite this qualified and excellent amount ofinformation some further aspects can be clarified by usingthe information obtainable from the CDS in particularthose related to the supersaturation conditions for precipi-tation and the critical concentrations of Ca2thorn and CO3

2- fornucleation and growth To achieve this goal by compara-tive studies the ions diffusion processes in the high viscoussol must not be affected by the presence of additives(Sancho-Tomas et al 2013) In the present experimentthis assumption was justified by the observation that thefinal concentration of Ca2thorn in the reservoir was notaffected by the presence of SOM dissolved in the highlyviscous sol Another important parameter was the waterdiffusion from the reservoir to the highly viscous solwhich could reduce the sol viscosity upon long periods oftime However we observed post mortem that features ofthe highly viscous sol did not change

In the presence of either NpoSOM or PliSOM andtaking into account the low reproducibility of these CDSexperiments (Table 1) with respect to correspondingSOMs-free experiments the crystallization parameters(Table 1) showed a xo-shift toward the anionic reservoir

Fig 8 Raman spectra of calcium carbonate particles formed in thepresence of diffusing Mg2thorn from the cationic reservoir (ref) indi-cates particles formed in the absence of soluble organic matrix(c NpoSOM) and (5c NpoSOM) indicate particles formed in thepresence of NpoSOM at concentrations 50 mgmL and to 250 mgmL respectively (c PliSOM) and (5c PliSOM) indicate particlesformed in the presence of PliSOM at concentrations 50 mgmL and to250 mgmL respectively The upper-figure reports spectra fromparticles in which the main bands were associated to calcite Thereference spectrum of calcite was characterized by bands at 155 280713 and 1087 cm-1 All peaks are broader than the correspondingpeaks in reference calcite implying a disorder structure (Addadiet al 2003) The lower-figure reports spectra from particles inwhich the main bands were associated to aragonite The referencespectrum of aragonite was characterized by bands at 150 205 701and 1085 cm-1 The two families of particles were undistinguishableunder optical microscope and each particle was identified as calciticor aragonitic only after acquisition of the Raman spectrum (onlineversion in colour)

532 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

a narrowing of D and almost no variation of tw The SOMswere rich in carboxylate and sulphate groups and had astrong capability to chelate calcium ions They could affectthe diffusion of calcium ions and could also change thespeciation of inorganic carbon in favour of HCO3

- Onlythe latter effect agreed with the observed xo-shift accord-ing to previous observations in which the presence ofentrapped polypeptides did not roughgly affect the ionicdiffusion (Sancho-Tomas et al 2013 2014) This hypoth-esis was supported also by the observation that the Ca2thornSOM molar ratio is at least 200 and if we consider thechelating behaviour of SOMs similar to that of poly-aspar-tate thus the amount of Ca2thorn chelated by SOMs moleculeswas very low Importantly and in addition to this argu-ment the tw values were not affected by the SOM concen-tration Although an increase of SOM concentration couldreduce both calcium diffusion rate and supersaturationthreshold for nucleation it was improbable that these para-meters were equally affected by the SOM concentration

Being confident on the data in the presence ofNpoSOM or PliSOM unlike what it was observedusing synthetic charged polypeptides (Sancho-Tomaset al 2013) or intra-skeletal coral macromolecules(Sancho-Tomas et al 2014) the narrowing of D wasnot associated to longer tw In the CDS the xo is thepoint where the system is supersaturated for nucleationand the equality-rule condition is fulfilled (Pucar et al1974) The latter condition is not respected in the regionaround xo where a higher concentration of one of thediffusing ions is present Thus NpoSOM or PliSOMallowed the precipitation of CaCO3 only in specificand confined conditions of supersaturation with respectto both calcium and carbonate ions The NpoSOM andPliSOM adsorbed on growing particles and were poten-tially capable to induce the nucleation (Addadi et al1987 Falini amp Fermani 2013 and references therein)This capability and the fact that the precipitation pro-cess occurred under the control of SOMs was confirmedby the change of morphology in the primary crystals thatformed the precipitates (Figs 5 and 6) In them mainlywhen we used the higher concentration of SOM thepresence of nano-sized spheroidal grains was observedThe shape of these primary grains changed with the typeof SOM indicating interactions along specific crystal-lographic planes (Addadi amp Weiner 1985)

The presence of diffusing Mg2thorn had an inhibition effecton the precipitation of CaCO3 (Lippmann 1973Fernandez Diaz et al 1996 Falini et al 2009) Theaddition of Mg2thorn increased the tw but this value was notaffected by the presence of SOMs (except when using 5cPliSOM for which a low reproducibility of the data wasobserved) The diffusion of both Mg2thorn and Ca2thorn from thecationic reservoir towards the reaction zone must occursimultaneously However although both cations diffusedsimultaneously the observations indicate that they did notwith the same rate The high hydration energy of Mg2thorn

could hinder its diffusion in comparison to that of Ca2thornTherefore the Mg2thornCa2thorn ratio in the crystallizationregion could be smaller than that in the reservoir

Magnesian calcite and aragonite were detected in thefinal mineral particles (Fig 7) when Mg2thorn diffused in thehighly viscous sol The effect of SOMs concentration onthe polymorphic selection showed its capability to promoteor inhibit the appearance of one phase a low concentrationof NpoSOM led to the precipitation of aragonite whilemuch more Mg-calcite precipitated at high NpoSOM con-centration (368 wt) A similar effect was reported usingSOMs extracted from coral skeletons (Sancho-Tomaset al 2014) The PliSOM favoured the precipitation ofmagnesian calcite with respect to the control almost inde-pendently of its concentration The isomorphic substitutionof Ca2thorn by Mg2thorn in the calcite lattice was favoured by thepresence of SOMs as already reported for other miner-alized tissues (Politi et al 2010)

An interesting observation was that the particles preci-pitated in the presence of Mg2thornshowed always almostsimilar shapes irrespective of the associated polymorphas confirmed by the Raman analyses (Fig 8) Moreoverthe Raman bands widened when compared to the referencematerial (Fig 8) and the micronano-structure of the spher-ules revealed that they were formed by the assembly ofmicronanoparticles (Figs 5 and 6) It is known that acooperative effect subsisted between SOM and Mg2thorn instabilizing ACC (Politi et al 2004) Moreover it wasshown that the formation of sea-urchin spine and nacreproceeds through CaCO3 polyamorphs (eg Cartwrightet al 2012) Thus the above observations could fit withthe formation of ACC particles stabilized by magnesiumions and SOM that then evolved in a given crystallinephase depending on the associated SOM (Gebauer et al2010) Indeed in the presence of NpoSOM which wasextracted from the aragonitic nacre mainly the formationof aragonite was observed while in the presence ofPliSOM which was extracted from the calcitic sea-urchinspine the formation of magnesium calcite was favoured

5 Conclusions

We have explored the effects of SOMs extracted fromtwo biominerals ndash nacre from N pompilius and spinesfrom P lividus ndash on the precipitation of CaCO3 by usinga counter-diffusion system where the U-tube was filledwith an agarose highly viscous sol The results show thatthe presence of these SOMs does not increase the super-saturation needed for precipitation in contrast to whatsynthetic polypeptides and SOMs extracted from coralsdo but limit the supersaturation conditions to yield pre-cipitation The experiments carried out in the presenceof diffusing Mg2thorn suggest the formation of a transientamorphous calcium-carbonate phase only when SOMsare present We also found that crystallization of arago-nite or calcite depends on the biomineral species fromwhich the SOM was extracted and also on its concentra-tion In conclusion this study suggests that the controlexerted by SOMs in the mineralization of the sea-urchinspine and nacre is under more restrictive conditions of

Calcium carbonate bio-precipitation in counter-diffusion systems 533

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

supersaturation than those supposed for the formation ofcoral skeletons (Sancho-Tomas et al 2014) This lastobservation is supported by palaeontology data miner-alogical changes in coral fibres have been reported incorals through time whereas no equivalent shift has beencited concerning echinoderm skeletons and nacreoustablets (Cuif 1980 Stanley 2003)

Additionally this study shows the high potentiality ofthe CDS for the study of biomineralization processes invitro

Acknowledgements This research received funding fromJunta de Andalucıa (Excellence project RNM5384)MINECO and FEDER (Factorıa de CristalizacionCSD2006-00015 Consolıder-Ingenio 2010) MSTthanks to CSIC for her JAE-Pre research contract withinthe lsquolsquoJunta para la Ampliacion de Estudiosrsquorsquo co-funded bythe European Social Found (ESF) GF and SF thank theConsorzio Interuniversitario di Ricerca della Chimica deiMetalli nei Sistemi Biologici (CIRC MSB) for the support

References

Addadi L amp Weiner S (1985) Interaction between acidic macro-

molecules and crystals Stereochemical requirements for biomi-

neralization Proc Natl Acad Sci USA 82 4110ndash4114

Addadi L Moradian J Shay E Maroudas NG Weiner S

(1987) A chemical model for cooperation of sulphated and

carboxylated in calcite crystal nucleation Proc Natl Acad

Sci USA 84 2732ndash2736

Addadi L Raz S Weiner S (2003) Taking advantage of dis-

order Amorphous calcium carbonate and its role in biominer-

alization Adv Mat 15 959ndash970

Albeck S Weiner S Addadi L (1996) Polysaccharides of intrea-

crystalline glycoproteins modulate calcite crystal growth in

vitro Chem Eur J 3 278ndash284

Altomare A Burla MC Giacovazzo C Guagliardi A

Molitemi AGG Polidori P Rizzi R (2001) Quanto a

Rietveld program for quantitative phase analysis of polycrystal-

line mixtures J Appl Crystallogr 34 392ndash399

Asenath-Smith E Li H Keene EC Seh ZW Estroff LA

(2012) Crystal growth of calcium carbonate in hydrogels as a

model of biomineralization Adv Funct Mater 22 2891ndash2914

Baronnet A Cuif J-P Dauphin Y Farre B Nouet J (2008)

Crystallization of biogenic Ca-carbonate within organo-mineral

micro-domains Structure of the calcitic prisms of the Pelecypod

Pinctada Margaritifera (Mollusca) at the sub-micrometre to

nanometre ranges Mineral Mag 72 539ndash548

Berman A Addadi L Weiner S (1988) Interactions of sea-

urchin skeleton macromolecules with growing calcite crystals

a study of intracrystalline proteins Nature 331 546ndash548

Cartwright JHE Checa AG Gale JD Gebauer D Sainz-

Dıaz CI (2012) Calcium carbonate polyamorphism and its

role in biomineralization how many amorphous calcium carbo-

nates are there Angew Chemie Int Ed 51 11960ndash11970

Crenshaw MA (1972) The soluble matrix from Mercenaria mer-

cenaria shell Biomineralization 6 6ndash11

Cuif J-P (1980) Microstructure versus morphology in the skeleton

of Triassic scleractinian corals Acta Palaeontologica Polonica

25 361ndash374

Dubois P amp Ameye L (2001) Regeneration of spines and pedi-

cellariae in echinoderms A review Microsc Res Tech 55

427ndash437

Falini G amp Fermani S (2013) The strategic role of adsorption

phenomena in biomineralization Cryst Res Tech 48

864ndash876

Falini G Albeck S Weiner S Addadi L (1996) Control of

aragonite or calcite polymorphism by mollusk shell macromo-

lecules Science 271 67ndash69

Falini G Fermani S Tosi G Dinelli E (2009) Calcium carbo-

nate morphology and structure in the presence of seawater ions

and humic acids Cryst Growth Des 9 2065ndash2072

Fernandez Diaz L Putnis A Prieto M Putnis CV (1996) The

role of magnesium in the crystallization of calcite and aragonite

in a porous medium J Sedimen Res 66 482ndash491

Fernandez-Gonzalez A amp Fernandez-Diaz L (2013) Growth of

calcium carbonate in the presence of Se(VI) in silica hydrogel

Am Mineral 98 1824ndash1833

Gagnon AC Adkins JF Erez J (2012) Seawater transport

during coral biomineralization Earth Planet Sci Lett

329ndash330 150ndash161

Garcıa-Ruiz JM (1991) Uses of crystal growth in gels and other

diffusingndashreacting systems Key Eng Mater 58 87ndash106

Gebauer D amp Colfen H (2011) Prenucleation clusters and non-

classical nucleation Nano Today 6 564ndash584

Gebauer D Gunawidjaj PN Ko JYP Bacsi Z Aziz B Liu

L Hu Y Bergstrom L Tai C-W Sham T-K Eden M

Hedin N (2010) Proto-calcite and proto-vaterite in amorphous

calcium carbonates Angew Chem 122 9073ndash9075

Heinemann F Launspach M Gries K Fritz M (2011)

Gastropod nacre structure properties and growth mdashbiological

chemical and physical basics Biophysic Chem 153 126ndash153

Henisch HK (1970) Crystal growth in gels Pennsylvania State

University Press University Park PA

mdash (1988) Crystals in gels and Liesegang rings Cambridge

University Press Cambridge UK

Henisch HK amp Garcıa-Ruiz JM (1986a) Crystal growth in gels

and Liesegang ring formation I Diffusion relationships J

Cryst Growth 75 195ndash202

mdash mdash (1986b) Crystal growth in gels and Liesegang ring formation

II Crystallization criteria and successive precipitation J Cryst

Growth 75 203ndash211

Hernandez-Hernandez A Gomez-Morales J Rodrıguez Navarro

AB Gautron J Nys Y Garcıa Ruiz JM (2008a)

Identification of some active proteins on the process of hen

eggshell formation Cryst Growth Des 8 4330ndash4339

Hernandez-Hernandez A Rodrıguez Navarro AB Gomez-

Morales J Jimenez Lopez C Nys Y Garcıa Ruiz JM

(2008b) Influence of model globular proteins with different

isoelectric points on the precipitation of calcium carbonate

Cryst Growth Des 8 1495ndash1502

Hernandez-Hernandez A Vidal ML Gomez-Morales J

Rodrıguez Navarro AB Labas V Gautron J Nys Y

Garcıa Ruiz JM (2008c) Influence of eggshell matrix proteins

on the precipitation of calcium carbonate (CaCO3) J Cryst

Growth 310 1754ndash1759

Katsikopoulos D Fernandez-Gonzalez A Prieto M (2009)

Crystallization behaviour of the (MnCa)CO3 solid solution in

534 M Sancho-Tomas et al

eschweizerbart_xxx

silica gel nucleation growth and zoning phenomena Mineral

Mag 73 269ndash284

Khalifa GM Weiner S Addadi L (2011) Mineral and matrix

components of the operculum and shell of the barnacle balanus

amphitrite calcite crystal growth in a hydrogel Cryst Growth

Des 11 5122ndash5130

Levi-Kalisman Y Falini G Addadi L Weiner S (2001)

Structure of the nacreous organic matrix of a bivalve mollusk

shell examined in the hydrated state using cryo-TEM J Struct

Biol 135 8ndash17

Li H Xin HL Muller DA Estroff LA (2009) Visualizing the

3D internal structure of calcite single crystals grown in agarose

hydrogels Science 326 1244ndash1247

Lippmann F (1973) Sedimentary carbonate minerals minerals

rocks and inorganic materials Springer-Verlag Berlin

Lowenstam HA amp Weiner S (1989) On Biomineralization

Oxford Univ Press New York

Marin F amp Luquet G (2008) Unusually acidic proteins in biomi-

neralization in lsquolsquoHandbook of biomineralization biological

aspects and structure formationrsquorsquo E Bauerlein Ed Wiley-CH

Verlag GmbH Weinheim Germany

Nakahara H (1979) An electron microscopy study of the growing

surface of nacre in two gastropods species Turbo cornutus and

Tegula pfeifferi Venus 38 205ndash211

Paquette J amp Reeder RJ (1990) Single-crystal X-ray structure

refinements of two biogenic magnesian calcite crystals Am

Mineral 75 1151ndash1158

Politi Y Arad T Klein E Weiner S Addadi L (2004) Sea

urchin spine calcite forms via a transient amorphous calcium

carbonate phase Science 306 1161ndash1164

Politi Y Batchelor DR Zaslansky P Chmelka BF Weaver

JC Sagi I Weiner S Addadi L (2010) Role of magnesium

ion in the stabilization of biogenic amorphous calcium carbonate

a structure-function investigation Chem Mater 22 161ndash166

Prieto M Putnis A Fernandez-Dıaz L Lopez-Andres S (1994)

Metastability in diffusing-reacting systems J Cryst Growth

142 225ndash235

Pucar Z Pokric B Graovac A (1974) Precipitation in gels under

conditions of double diffusion Critical concentrations of the

precipitating components Analytical Chem 46 403ndash409

Rao A Seto J Berg JK Kreft SG Scheffner M Colfen H

(2013) Roles of larval sea urchin spicule SM50 domains in

organic matrix self-assembly and calcium carbonate mineraliza-

tion J Struct Biol 183 205ndash215

Rodriguez-Navarro A (2006) XRD2DScan new software for poly-

crystalline materials characterization using two-dimensional X-

ray diffraction J Appl Crystallogr 39 905ndash909

Sancho-Tomas M Fermani S Duran-Olivencia MA Otalora

F Gomez-Morales J Falini G Garcıa-Ruiz JM (2013)

Influence of charged polypeptides on nucleation and growth of

CaCO3 evaluated by counterdiffusion experiments Cryst

Growth Des 13 3884ndash3891

Sancho-Tomas M Fermani S Goffredo S Dubinsky Z Garcıa-

Ruiz JM Gomez-Morales J Falini G (2014) Exploring

coral biomineralization in gelling environments by means of a

counter diffusion system Cryst Eng Comm doi101039

C3CE41894D

Schmidt WJ (1924) Die Bausteine des Tierkorpers in polarisier-

tem Lichte Cohen Verlag Bonn 528 p

Seto J Ma Y Davis SA Meldrum F Gourrier A Kim Y-Y

Schilde U Sztucki M Burghammerg M Maltsevi S Jageri

C Colfen H (2012) Structure-property relationships of a

biological mesocrystal in the adult sea urchin spine ProcNat

Acad Sci USA 109 3699ndash3704

Silverman L amp Boskey AL (2004) Diffusion systems for evalua-

tion of biomineralization Calcified Tissue Int 75 494ndash501

Stanley GD (2003) The evolution of modern corals and their early

history Earth-Science Reviews 60 195ndash225

Sun J amp Bhushan B (2012) Hierarchical structure and mechanical

properties of nacre a review RSC Advances 2 7617ndash7632

Towe KM (1967) Echinoderm calcite single crystal or polycrys-

talline aggregate Science 157 1048ndash1050

Venn AA Tambutte E Holcomb M Laurent J Allemand D

Tambutte S (2013) Impact of seawater acidification on pH at

the tissuendashskeleton interface and calcification in reef corals

Proc Nat Acad Sci USA 110 1634ndash1639

Watabe N (1965) Studies on shell formation IX Crystal-matrix

relationships in the inner layer of the mollusk shells J Ultrastr

Res 12 351ndash370

Weiner S (1984) Organization of organic matrix components in

mineralizaed tissues Am Zool 24 945ndash951

Weiner S amp Addadi L (2011) Crystallization pathways in biomi-

neralization Annu Rev Mat Res 41 21ndash40

Weiner S amp Hood L (1975) Soluble protein of the organic matrix

of mollusk shells a potential template for shell formation

Science 190 987ndash989

Weiner S Talmon Y Traub W (1983) Electron diffraction of

mollusc shell organic matrices and their relationship to the

mineral phase Int J Biol Macrom 5 325ndash328

Weiss IM Kaufmann S Heiland B Tanaka M (2009)

Covalent modification of chitin with silk-derivatives acts as an

amphiphilic self-organizing template in nacre biomineralisation

J Struct Biol 167 68ndash75

Received 28 December 2013

Modified version received 5 March 2014

Accepted 8 April 2014

Calcium carbonate bio-precipitation in counter-diffusion systems 535