Biomineralization: from Paleontology to Materials Science

Proceedings of the 9th International Symposium on Biomineralization,

(Eds.) Arias, J.L., Fernández, M.S.
Editorial Universitaria, Santiago, Chile, 2007, pp. 215-224.

Structural and Biochemical Patterns at the Micro- and

Nanometre Scales in Mollusc prisms and Coral fibres.
Implication for Coral Growth mode and Environment
Recording

Jean-Pierre Cuif1*, Yannicke Dauphin1, Anders Meibom2 and Nury Guzman3

1
*Lab. Géologie-IDES, Bat. 504, Faculté des Sciences, 91405 Orsay, France
2
LEME, Museum National Histoire Naturelle, 57 Rue Cuvier, 75231 Paris, France
3
UR Paléotropique, IRD, 32 Av. Henri Varagnat, 93143 Bondy, France
cuif@geophy.geol.u-psud.ff

ABSTRACT

In contrast to common knowledge, coral fibres exhibit a series of structural and

biochemical patterns that suggest a close similarity with skeletal units produced in
“matrix mediated” biomineralization processes. As coral skeletons are widely used as
environmental archives in climate change studies (chemical and isotopic measurements
are always empirically interpreted on the assumption of purely inorganic precipitation),
this paper summarizes the evidence for biological control over the skeletal formation
process and discusses the practical consequences with respect to environmental
recording.

INTRODUCTION

Twenty-five years ago, two independently published papers (Johnston, 1980,

Cuif et al., 1980) focussed attention on the submicrometre scale patterns within
two different calcareous units of invertebrate calcium carbonate skeletons.
Johnston investigated the organization of the aragonitic fibres in the skeletons
of Scleractinia, whereas the other paper described the calcitic prisms that build
the external shell layer of the Pteriomorphid Bivalve Pinna nobilis. Surprisingly,
rather similar results were obtained. In both cases, an organic network was visible,
embedded within the crystal-like units. In the Pteriomorphid prism, which was
well established as a typical example of “matrix mediated” biomineralization
(Lowenstam, 1981), the presence of intra-crystalline organic components was
not a matter of controversy. But the Johnston’s result was in strong contrast with
the common opinion about coral fibres. Following an interpretation proposed
by Bryan and Hill (1941) (which is still in use: Veis, 2005), the coral fibre is
an example of a “biologically induced” calcareous structure, crystallized with
minimum (if any) of biological regulation. Building on the work of Bryan and
Hill, Barnes (1970) suggested that the three-dimensional arrangement of fibres

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within coral septa is a result of “crystal growth competition”, again promoting

the physio-chemical view of the precipitation process.
Gathering data from the last few years, this paper aims at emphasizing the
similarities that can be found between coral fibres and two matrix mediated
prisms from Mollusc shells when considering the overall growth-mode, the
structural organisation down to the nanometre scale, and the distribution of the
biochemical compounds involved in crystallization process.

MATERIAL AND METHODS

The reference material for prismatic structures is the Pteriomorphid Pelecypod

Pinna nobilis from the Mediterranean sea. Additionally, Concholepas
concholepas (Bruguières 1789), which is a Muricid Gastropod from the Chilean
and Peruvian coasts is also used to illustrate certain points. Both species built
external shell layers made of elongated densely packed crystal-like units.
Coral fibres were studied through a wide diversity of species from very
different geographical sites, ranging from the Guadalupe Islands (Caribbean
Sea), over the Moorea Island in Polynesia, to New-Caledonia (mostly from the
Noumea lagoon).
In all cases specimens were collected alive. Structural, chemical or
biochemical data were obtained by using the most recent parts of the shells or
coral skeletons, in order to avoid changes that may rapidly occur in the deeper
parts of the calcareous structures. The observational techniques and methods
are simply summarized here. Exact details regarding the preparative processes
can be found in the cited references.
Structural information was collected by scanning electron microscope (SEM)
observation of polished and etched surfaces. Action of enzymatic solutions or
acidic+fixative mixtures enables the growth periodicity to be revealed. Because
of the taxonomy linked diversity of mineral composition and organic matrices,
composition of etching solutions concentration and etching duration must be
adapted specifically to each sample in order to obtain useful results. Usually,
submitting polished skeletal surfaces to low concentrations of formic acid
(typically a 1 per mil concentration + 3 to 5% glutaraldehyde) results in well-
prepared sample surfaces.
Distribution of polysaccharides within calcareous units was studied by using
Synchrotron radiation facilities at ESRF Grenoble (France). Due to an extremely
fine tuning of the X-ray energy, the speciation of sulphur can be determined. I.e.
one can clearly distinguish between S bound to amino acids, disulphur bonds in
cystine, organic sulphate, and S in solid solution in the calcium carbonate phase.
Various mapping scales are available that allow is situ characterization or regions
on the order to 300x300 μm2 with the sub-micrometre spatial resolution.

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Observation using atomic force microscopy (AFM) were made by using a

DIMENSION 3100 instrument (Veeco) in the tapping mode. The AFM preparative
process also involves polishing and etching, but adaptation to sample properties
is even more critical than for the SEM: no routinely standard method is available
at this point.

RESULTS

A common stepping growth-mode at the micrometre scale - Indication about the

periodicity of the secretory process
Figures 1 to 4, 7 to 10 and 13 to 15 show the common growth pattern in
these three different skeleton units. In spite of a monocrystalline appearance,
and a distinct longitudinal continuity in both the Pinna prisms (Fig. 3c), the
Concholepas prisms (Fig. 8) and in the coral fibres (Fig. 14a-14b, Porites), these
distinct crystal-like units are constructed by superimposition of growth layers
that are perfectly synchronous between adjacent units. Within these micrometre
thick growth units, crystallization occurs simultaneously while the overall
crystallographic orientation of the mineral phase is preserved.
Taking advantage of the incorporation of calcein into the skeletal structure,
labelling experiments have been carried out on the Concholepas shells. Figure 9
shows the two fluorescent lines and the irregular day-growth of the shell between
22/1 and 29/11. It is important to point out that changes in shell pigmentation
during diurnal cycle provide us with a precise chronological control of the shell
growth. SEM observations of the mineral layers deposited during a single day
clearly show that the prisms constructed in this time span have been constructed
by superimposition of about 45 crystallized layers, each of which is about two
micrometres in thickness.
Equivalent timing information have not been obtained yet for the growth
periodicity in the Pinna prisms and for the coral fibres, but in all three cases the
superimposition of synchronic growth layers appears as the basic mechanism
for the growth of the skeleton.
The submicrometre interplay of organic compounds and mineral phase
revealed by synchrotron radiation XANES mapping
In situ mapping of the sulphated polysaccharides in longitudinal sections
of the skeleton units also reveals the layered organization of the glucidic
intraskeletal compounds. At all observation scales (i.e., medium- (Fig. 5, Pinna
prism), low- (Fig. 11, Concholepas shell) or high resolution mapping (Fig. 16,
Goniastrea fibres)) the distribution of the organic compounds corresponds to the
organization of skeletons and skeletal units.
Correspondence between the layering of the mineral phase and the banding
patterns visible in distribution of S-bearing organic compounds at the micrometre

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 Micro- and nanometre structures in mollusc and coral biominerals
Figures 1-17. 1 to 6: Prisms of Pinna nobilis 1-2 Overall view of the Pinna shell external side (1), and close
view of the prisms and lamellae (2). 3: Morphology of the prisms (3a); transversal and longitudinal sections-
polarized light (3a, 3b). 4: Enzymatic etching of a polished surface: the perfect synchronism of growth
layers between adjacent prisms indicates that mineralizating activity of the mantle is controlled at the
overall scale. 5: XANES map showing the exact correspondence of polysaccharide distribution to spatial
arrangement of mineral phase. 6: Nanograins within a growth layer of the Pinna prisms: note the presence
of the grain cortex. 7 to 13: Prisms in the external shell layer of Concholepas concholepas 7: Radial
section of the Concholepas shell. 8: Monocrystalline appearance of the prisms of the external shell layer in
Concholepas. 9: Two calcein growth-marks separated by a week of shell growth. Note the irregularity of the
daily growth and the color difference between the day and night parts of the shell. 10: Layered structure of
the mineral component within a one-day growth of the Concholepas prisms. During this 24 hours of growth,
producing a thickness increase of about 100: m, more than 45 separated mineral layers have crystallized.
11: Distribution of sulfated polysaccharides within the Concholepas prismatic layer. At this scale, the
daily growth rythm is clearly visible through the overall distribution of the sulfated polysaccharides. 12:
Nanograins within the Concholepas prisms. Very similar appearence compared to the Pepecypod prisms of
the Pinna. 13-17 From morphology to nanostructure in coral skeletons 13: Morphology of a Porites corallite.
14: Fibres that built the skeleton units. SEM picture (14a) and thin section of an equivalent sector observed
in polarized light (14b). 15: Layered organization of the Porites skeleton. The superimposed mineralized
layers surround the scattered points where early mineralization occurs (the “centres of calcification”). 16:
High resolution XANES map of the sulfated polysaccharides within coral fibres (Montastrea). The rich
sulfated layers (about three micron in thickness) correspond to the banding pattern of the mineral layers. 17:
Nanograins within coral fibres. This phase contrast image emphasizes the difference between nanograins
and their cortex, which causes a very high phase lag.

scale indicates that in order to understand the functional interplay between

organic substrates and mineral phases, investigation must be conducted at a
submicrometre scale.

The common nanogranular organization at the AFM scale

Atomic force microscopy allows a granular nanostructure to be observed in both
calcitic prisms and coral fibres. Growth layers are built by irregularly shaped
nanograins, the dimensions of which being in the range of 10-100 nanometres.
Additionally, the phase-image mode shows that the each granular nanograin
is surrounded by a layer of material of which the physical properties (such
as elasticity, viscosity, etc.) cause a strong interaction with the AFM tip. The
clear correlation between topographic image and the high-contrast phase-mode
image, which is produced by the difference in material properties between the
nanograins and the material enveloping the nanograins, suggests that the latter
is the organic component of the skeletal units.

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DISCUSSION
The growth mode of coral fibres compared to typical matrix mediated units
The observations presented here suggest that, in spite of important biological
differences and phylogenetic distance, these different organisms show strong
similarities in the way they form their calcium carbonate skeleton. For both
corals and shells, the mm-sized crystal-like units are built by superimposing
micrometre-thick growth-layers. This demonstrates a permanent control
exerted by the mineralizing epithelium, i.e. the external cell layer of the
mantle in Molluscs or the basal ectoderm of the coral polyp. In the prismatic
layers of Molluscs, synchronization of the secretory rhythm over the entire
epithelium surface leads to formation of parallel growth units. In corals, a
very similar stepping mechanism controls the development of the fibres. This
controlled stepping growth-mode explains how coral polyps can built their
species specific septal morphologies by modulating thickness, and perhaps
also the rhythm of growth layer deposition in the different domains of a given
septum. In contrast, no mechanism has ever been proposed to explain how
the hypothesized “crystal growth competition”, which produces “randomly
oriented” crystals, may result in these species-specific morphologies and
microstructures. The permanent control exerted on the stepping growth mode
of fibres by coral polyps provides us with a possible explanation for this long
recognized species specific three-dimensional arrangement of coral fibres
(Ogilvie, 1896) in coral skeletons.

Similarity in distribution and overall composition of the organic components

associated to mineral crystallization
In both Mollusc prisms and in coral fibres, the structural banding pattern revealed
within the crystal-like units has a direct correspondence with the layered
distribution of the acidic sulphated polysaccharides. In the development of
coral biomineralization studies, glucids were first characterized and considered
to be the mineralizing organic phase acting as a crystallization template (Goreau,
1956, 1959). Then attention was focused on proteins, after Mitterer (1978)
demonstrated the very distinct spectrum of aminoacids of the protein-like
compounds in the skeletal matrices. Unusually high concentrations of acidic
amino-acids (aspartic and glutamic) are a common feature that has lead to the
suggestion that these organic molecules create nucleation sites. In this respect,
the coral fibers do not differ from more well-studied matrix mediated skeleton
units. The Pinna prisms, for instance, also exhibit very high concentrations in
acidic amino acids (Cuif et al., 1987).
But recent data acquired by synchrotron radiation capabilities have drawn
new attention to glucids, and more precisely sulphated polysaccharides. These

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compounds can produce very large molecules with complex architectures and
carrying numerous acidic groups (Dauphin, 2001). Also in this respect, the coral
skeleton share similarities with other “matrix mediated” skeletal constructs.
Additionally, water has been detected in structural association with the matrix
components in both coral and mollusc skeletons (Cuif et al, 2004). Large molecule
polysaccharides, such as aggrecan or versican, are well known for their ability to
be associated with water.
Taking into account obvious taxonomy-linked differences, this biochemically
common composition of skeletal matrices represents an additional evidence of
similarity between coral fibres and other materials known to be the result of
typical matrix mediated mineralization.

Mineral-Organic interplay at the nanometre scale

The precise correspondence between the mineral banding patterns and
the layering of organic phases at the micrometre scale implies that both
components of the skeletons are associated at a submicrometre level. The
nanogranular sub-organization of the growth layers in coral fibres and Mollusc
prisms fully explain this coincidence. At this observational level, a perfect
parallel exists between Coral fibres and Mollusc prisms. We draw attention
to the fact that, in the organic network observed by Johnston (1980) in the
upper parts of the coral fibres, the size of the cells corresponds, from a purely
dimensional stand-point, to the nanograin cortex that so strongly interacts
with the AFM tip. Factors that influence the substrate-tip interaction suggest
that the embedding material is the hydro-organic phase that also previously
was identified as sulphated polysaccharides and proteins by electrophoresis
and XANES mapping. Due to some possible alteration of the network during
TEM preparative process, Johnston was able to show the presence of this
organic network at the upper parts of the fibres only, and he suggested that
this organic component “disappears” in the deeper part of fibres. We have
now ample evidence that organic materials exist all along the fibres of living
corals. Conversely, there is no doubt that biochemical changes occur during
the early diagenesis of coral colonies, even on relatively small time scales,
such as one decade (Cuif et al., 1997).
With respect to the comparison between coral fibres and well known “matrix
mediated” structures, this suggest that no difference exists in the crystallization
process itself. Crystallization of a growth-unit in coral skeleton does not take
place in a “fluid close to sea water”, as commonly admitted (from McConnaughey,
1989 to Sinclair and McCulloch 2004), but within a specifically secreted and
chemically active hydrated medium.

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Coral fibre crystallization and environmental record

An in-depth understanding of the biomineralization growth layer is of
major importance in paleoclimate research. Firstly, it is worth noting that
each of these well-coordinated growth layers (in both Molluscs and Corals)
represent the fundamental Environment Recording Unit. Labelling of the
corallite or shell growth by fluorescent dies allows very precise correlations
to be made between environmental conditions and the minor element or
isotopic signals recorded in the synchronously crystallized Coral or shell
elemental growth layers. Experiments have shown a very high periodicity
for the biomineralization cycles. In Concholepas concholepas for instance,
up to 50 two-micron thick growth layers can be produced within a single day
(Fig. 10) (Guzman, 2004). This high periodicity of the layered crystallization
mechanism provides climate change investigators with interesting possibility
to calibrate a great diversity of shell producing organisms within a short
period of time.
Due to the availability of high resolution analytical techniques (e.g.
NanoSIMS) the possibility now exists), as recently shown (Meibom, 2004),
to separately analyse the superimposed growth layers, allowing for the first
time a precise correlation to be made between environmental conditions at a
given time and signals recorded in the corresponding growth layer. From both
theoretical objectives (improved analyses of the crystallization process), and
practical applications (proxy-bearer calibrations), coral skeletons and mollusc
shells can be gathered into systematic research programs because of the new
understanding that these organisms share a very similar biomineralization
process.
From a more fundamental viewpoint, explaining the taxonomy-linked
response of organisms submitted to variation of their common environment,
the long standing question of the “vital effect”, is now a target within reach.
As in both corals and molluscs crystallization does not occur in a fluid “close
to sea water” but within a chemically active organic framework, hypothesis
can be reasonably made that the species-specific compositions of the skeletal
matrices may influence the crystallization process, leading to taxonomy-
linked responses concerning chemical or isotopic signals recorded within
Ca-carbonate skeletons. Therefore instruments allowing measurements to be
made at the relevant scale, i.e., the few micron thick crystallization growth
layer, have a key-role to improve the use of calcareous biogenic structures as
climate archives: for the first time in climate change studies, precise correlation
can be made between a given variation and the records simultaneously made
by different organisms within the corresponding growth layers respectively.

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 Micro- and nanometre structures in mollusc and coral biominerals

CONCLUSIONS

1. With respect to growth mode, organic matrix distribution and organization

at the nanostructural level, coral fibres do not differ from typical matrix
mediated calcareous skeleton units.
2. In climate change studies, data-bases can include corals among other proxy-
bearers to compare taxonomy-linked responses to environmental changes.
3. Simultaneity in occurrence of modern analytical instruments working at the
sub-micrometre range and new concept in Environment Recording Process,
provides investigators with a precise calibration method and new perspectives
in the use of biological archives.

REFERENCES

BARNES, D.J. (1970). Coral skeletons: An explanation of their growth and structure. Science
170: 1305-1308.
BRYAN, W.H., HILL, D. (1941). Spherulitic crystallization as a mechanism of skeletal growth in
the hexacorals. PROC. ROY. SOC. Queensland 52: 78-91.
CUIF J.P., DAUPHIN Y., DENIS A., GASPARD D., KELLER J.P. (1980). Continuité et périodicité du
réseau organique dans les prismes de Pinna nobilis. C.R. Acad. Sc. Paris, sér. D, 290: 759-
762.
CUIF, J.P., DAUPHIN, Y., BERTHET, P., JEGOUDEZ, J. (2004). Associated water and organic compounds
in coral skeletons: quantitative thermogravimetry coupled to infrared absorption
spectrometry.Geochemistry, Geophysics, Geosystems 5: 11, doi:10.2004GC000783.
CUIF, J.P., DAUPHIN, Y., DENIS, A., GAUTRET, P., KIYASHKO, S., MASSAULT, M. (1997). Facteurs
de la diagenèse précoce des biominéraux: exemple d’un polypier de Porites de Nouvelle
Calédonie. Geobios MS 20: 171-179.
CUIF, J.P., FLAMAND, D., FRÉROTTE, B., CHABIN, A., RAGUIDEAU, A. (1987). Fractionnement de la
matrice proteique intraprismatique chez Pinna nobilis L. et composition en acides aminés
des différentes phases. C. R. Acad. Sc. Paris 304, sér. II, 9: 475-478.
DAUPHIN, Y. (2001). Comparative studies of skeletal soluble matrices from some Scleractinian
corals and Molluscs. Int. J. Biol. Macromol. 28: 293-304.
GOREAU, T. (1956). Histochemistry of mucopolysaccharide-like substances and alkaline
phosphatase in Madreporaria. Nature 177: 1029-1030.
GOREAU, T.F. (1959). The physiology of skeleton formation in corals. I. A method for measuring
the rate of calcium deposition by corals under different conditions. Biol. Bull. 116: 59-75.
GUZMAN, N. (2004). Validation d’une approche scléroclimatologique sur le crôte du Chili et
du Pérou par l’analyse microstructurale et biogéochimique des coquilles du gastéropode
Concholepas concholepas Bruguières 1789. Thèse Université Paris XI-IRD, 203 p., Juillet 2004.
JOHNSTON, I.S. (1980). The ultrastructure of skeletogenesis in hermatypic corals. Internat. Rev.
Cytol. 67: 171-213.

223 EDITORIAL U NIVERSITARIA S.A.

Cuif et al.

LOWENSTAM, H.A. (1981). Minerals formed by organisms. Science 211: 1126-1131.

MCCONNAUGHEY, T. (1989).13C and 18O isotopic desequilibrium in biological carbonates; I.
Patterns. Geochim. Cosmoch. Acta 53: 163-171.
MEIBOM, A., CUIF, J.P., HILLION, F., CONSTANTZ, B., JUILLET-LECLERC, A., DAUPHIN, Y., WATANABE,
T., DUNBAR, R.B. (2004). Distribution of magnesium in coral skeletons. Geophys. Res. Lett.
31, L23306, doi: 10.1029/2004GL021313.
MITTERER, R.M. (1978). Amino acid composition and metal binding capability of the skeletal
protein of corals. Bull. Mar. Sci. 28: 173-180.
OGILVIE, M. (1896). Microscopic and systematic study of madreporarian types of corals. Royal
Soc. London Phil. Trans. 187B: 83-345.
SINCLAIR, D.J., MCCULLOCH, M.T. (2004). Corals record low mobile barium concentrations
in the Burdekin River during the 1974 flood: evidence for limited Ba supply to rivers?
Palaeog. Palaeocl. Palaeoecol. 214: 155-174.
VEIS, A. (2005). A window on Biomineralization. Science 307: 1419.

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