CRYSTAL

GROWTH
Flowerlike Agglomerates of Calcium Carbonate Crystals Formed on & DESIGN
an Eggshell Membrane
2006
Masakazu Takiguchi, Koichi Igarashi, Masayuki Azuma, and Hiroshi Ooshima* VOL. 6, NO. 12
Graduate School of Engineering, Osaka City UniVersity, 3-3-138 Sugimoto, Sumiyoshi-ku, 2754-2757
Osaka 558-8585, Japan

ReceiVed July 15, 2006; ReVised Manuscript ReceiVed September 27, 2006

ABSTRACT: Calcium carbonate was crystallized on an eggshell membrane using a specially designed crystallizer. The crystallizer
used was a jacketed cylindrical glass vessel with two compartments divided by an eggshell membrane. The calcium chloride and
sodium carbonate solutions were separately placed in the two compartments. Several kinds of peculiar agglomerates of calcium
carbonate crystals formed on both surfaces of the eggshell membrane. Flowerlike agglomerates formed on the inner surface of the
eggshell membrane that is the egg white side, but those were not formed on the outer surface of the membrane. The flowerlike
agglomerate was composed of a hemispherical core and petal-like crystals growing around the core. We named it the CC (calcium
carbonate)-flower. The different types of agglomerates formed on the outer surface of the membrane. For instance, when the outer
surface of the membrane faced the calcium chloride solution, spherical agglomerates composed of long, needlelike crystals were
obtained. We inferred that the surface structure of the eggshell membrane played an important role for the formation of such peculiar
agglomerates.

1. Introduction
There are many examples that inorganic crystals are the
structural materials of the organisms. The eggshell is a typical
example of such biominerals. The eggshell protects an inner
embryo from external physical hazard and biological encroach-
ment. The eggshell is mainly composed of calcium carbonate
crystals (95% in weight).1 The other materials (3.5%) are organic
matrices mainly consisting of glycoprotein and proteoglycans.
The eggshell membrane is a thin film adhering inside the
eggshell. The membrane plays an important role for the
construction of the eggshell. The carbonate ions generated by
respiration of the embryo pass through the eggshell membrane
and react with calcium ions in the womb. The calcium carbonate
crystals deposit on the surface of the eggshell membrane. The
role of the eggshell membrane is to provide a nucleation point
and affect structuring of the crystalline solid.2
In the previous work, we reported that the tubular structure
of agglomerates of calcium carbonate crystals were formed on
a cation-exchange membrane3 and that the cation-exchange
membrane played an important role as a kind of template. In
the present study, we attempted to crystallize calcium carbonate
on the eggshell membrane. The method of the contact of calcium
ions to carbonate ions was the same as that adopted in the
previous work.3 The method was similar to that in the formation
of the eggshell as described above. Namely, calcium chloride
and sodium carbonate solutions were separately placed in the
two vessels divided by an eggshell membrane. Calcium ions
gradually transfer to the opposite side through the membrane
to react with carbonate ions. The aim of the present work was
to examine if the eggshell membrane might act as a template
as functioned in nature. We also expected a novel morphology
of agglomerates of calcium carbonate crystals. As a result, we
found several structures of calcium carbonate agglomerates,
including the flowerlike structure.
2. Materials and Methods
The calcium chloride dihydrate and sodium carbonate used were of
reagent grade (Wako Pure Chemicals Co. Ltd., Japan). The eggshell
* To whom correspondence should be addressed. E-mail: ooshima@
bioa.eng.osaka-cu.ac.jp. Tel: 81-6-6605-2700. Fax: 81-6-6605-2701.
Figure 1. Schematic diagram of the crystallizer.
membrane was prepared as follows. A hen egg was broken in half,
and the contents were removed. The thin membrane adhering to the
inside of the eggshell was carefully peeled off and washed in deionized
water. It is known that the eggshell membrane is composed of three
thin membranes, namely, the outer shell membrane, the inner shell
membrane and the limiting membrane, from outside to inside.4
Therefore, the surface structure is different between both sides of an
eggshell membrane. We used a whole eggshell membrane as a sheet
of membrane. Here, we call the surface that makes contact with a shell
the outer surface, and the opposite surface the inner surface.
Figure 1 presents a schematic diagram of the crystallizer adopted in
this study. The crystallizer was placed horizontally, as shown Figure
1. The crystallizer is composed of two jacketed cylindrical glass tubes,
a membrane, silicon rubber sheets, and a horseshoe-shaped clamp. The
inner diameter of the glass tube is 20 mm. The eggshell membrane
was tightly fixed to the glass vessels by silicon rubber sheets and a
horseshoe-shaped clamp.
Calcium chloride and sodium carbonate were individually dissolved
in deionized water and degassed to avoid the generation of bubbles
during crystallization. The initial concentration of calcium chloride and
sodium carbonate were 1.0 M; this concentration was arbitrarily adopted
only to get a sufficient driving force for the penetration of calcium
ions. The pH of sodium carbonate solution was adjusted to 12.0 with
5 M NaOH to allow the equilibrium between carbonate and bicarbonate
to shift completely to the carbonate-ion side. The calcium chloride and
the sodium carbonate solutions were placed in each compartment.
The crystallization was carried out in two different modes; one in
which the calcium chloride solution faced the inner surface of the
membrane (Experiment A), and the other was the inverse (Experiment
B).
The crystallization was carried out at 15-50 °C for 1-10 days
without stirring. Calcium ions and carbonate ions transferred to the
opposite compartment through the eggshell membrane. As the result,
calcium carbonate crystals grew on both surfaces of the membrane.
After the crystallization for a given time, the membrane was recovered,
gently rinsed with deionized water three times, and dried in air. The

10.1021/cg0604576 CCC: $33.50 © 2006 American Chemical Society

Published on Web 11/07/2006
Flowerlike Agglomerates of Calcium Carbonate Crystals Crystal Growth & Design, Vol. 6, No. 12, 2006 2755

Figure 2. SEM images of the eggshell membrane. (A) Cross-section of the eggshell membrane. (B) Outer surface of the eggshell membrane. (C)
Inner surface of the eggshell membrane.

crystals on the membrane were coated with gold and observed by a

scanning electron microscope (SEM; SHIMADZU EPM-810 and JEOL
JSM-6500FE). The polymorphs of calcium carbonate crystals were
identified by a powder X-ray diffraction meter (Rigaku, MiniFlex).

3. Results and Discussion

Figure 2 shows the SEM images of the eggshell membrane
used for this study. As mentioned above, the eggshell membrane
is composed of three membranes, namely, the outer shell
membrane, the inner shell membrane, and the limiting mem-
brane.4 Panel A of Figure 2 shows a cross section of the
membrane. The left upper side corresponds the outer surface
of the eggshell membrane, which adheres to the eggshell. The
inner shell membrane is sandwiched between the outer shell
membrane and the limiting membrane. The total thickness of
the eggshell membrane was about 70 µm. The outer surface of
the eggshell membrane, namely, the surface of the outer shell
membrane, is made of fibers, as shown in the panel B of Figure
2. Panel C of Figure 2 shows the inner surface of the membrane,
namely, the surface of the limiting membrane. It was smoother
than the outer surface.
3.1. Experiment A: The Inner Surface of the Eggshell
Membrane Facing the Calcium Chloride Solution. Peculiar
agglomerates of calcium carbonate crystals were obtained when
the inner surface of the eggshell membrane faced the calcium
chloride solution. Figure 3 shows the SEM images of the Figure 3. (A) SEM images of the calcium carbonate crystals formed
calcium carbonate crystals that grew on the inner surface of on the inner surface of the eggshell membrane by crystallization at 15
the membrane at 15 °C. Those are like flowers. Panel C of °C for 10 days. (B) Enlarged view of the rectangular area in panel A.
Figure 3 displays the flowerlike structure in more detail. We (C) Enlarged image of the flowerlike structure (the CC-flower). (D)
named the flowerlike agglomerate the CC (calcium carbonate)- Another type of the CC-flower differing in the morphology of petal-
flower. Such a flowerlike structure of calcium carbonate crystals like agglomerates. (E) CC-flowers obtained by repeating the same
experiment. (F) Enlarged view of panel E.
has not been reported. The size of the CC-flowers was about
30 µm in diameter. We found a hemispherical core at the center
of the CC-flower, as presented in panel C of Figure 3. The size However, it should also be true that the surface of the limiting
of the core presented in panel C is about 11 µm in diameter. membrane plays an important role for the formation of the CC-
Because the hemispheric core structure had not been observed flowers. The structure like the CC-flower has not been also
on the inner surface of the eggshell membrane before crystal- observed in a natural egg.
lization as shown in panel C of Figure 2, the core must be made Figure 4 shows an X-ray powder diffraction pattern of the
from calcium carbonate; it is composed of scalelike crystals. whole precipitate forming on the inner surface of eggshell
The hemispheric core is surrounded by many petal-like ag- membrane. The datum indicated that the crystals were a mixture
glomerates. The length of the petal-like agglomerate was about of calcite and vaterite. The platelike crystals should be calcite
10 µm. A different type of petal was observed, as presented in and the scalelike crystals constructing the spherical cores should
panel D; the petals are composed of small platelike calcium be vaterite.
carbonate crystals. The reproducibility of the CC-flowers was Calcium carbonate crystals also formed on the outer surface
examined by repeating the same experiments. Panels E and F of the eggshell membrane that faced the sodium carbonate
present the result. We could reproduce the CC-flowers, although solution. Figure 5 presents the SEM image of crystals formed
the morphology of the CC-flowers was a little different from on the fibers constructing the outer surface of eggshell mem-
that presented in panel C. Those petals were short. The CC- brane. Cubic crystals that are calcite were observed all over
flowers with short petals were widely found on the inner surface the surface. We did not find any peculiar morphology.
of the membrane, as shown in panel E. It should be noted that 3.2. Experiment B: The Outer Surface of the Eggshell
in a natural egg, no crystal grows on the surface of the limiting Membrane is Facing to the Calcium Chloride Solution.
membrane that faces egg white. This fact means that the Another experiment was carried out at 30 and 50 °C. Namely,
formation of the CC-flowers is not natural, but artificial. the inner and outer surfaces were faced to the sodium carbonate
2756 Crystal Growth & Design, Vol. 6, No. 12, 2006
Figure 4. XRD data of calcium carbonate crystals shown in Figure
3E; those were precipitated on the inner surface of the eggshell
membrane. Closed squares and open circles indicate characteristic peaks
of calcite and vaterite, respectively.
Figure 5. (A) SEM images of calcium carbonate crystals on the outer
surface of the membrane. (B) Enlarged view of the rectangular area in
panel A.
Figure 6. (A) SEM images of the early state of flowerlike structure.
(B) Enlarged view of the rectangular area in panel A. (C) Enlarged
view of the rectangular area in panel B.
and the calcium chloride solutions, respectively. This situation
is same as that in the formation of natural eggshell, although
the concentration of calcium ions and carbonate ions adopted
here was much higher than that in nature. Calcium carbonate
crystals were formed in both sides of the eggshell membrane
in Experiment B as well as in Experiment A. Figures 6 and 7
show the agglomerates formed on the inner surface of the
eggshell membrane. It is notable that the CC-flowers also formed
on the inner surface even in Experiment B, as shown in panels
A and B in Figure 6. The CC-flowers never formed on the outer
surface in Experiment A either. This result makes us reconfirm
that the structure of the inner surface of eggshell membrane is
responsible for the growth of the CC-flower. The CC-flowers
Ooshima et al.
Figure 7. SEM images of calcium carbonate crystals formed on the
inner surface of the eggshell membrane.
Figure 8. SEM images of calcium carbonate crystals formed on the
outer surface of the eggshell membrane. (A) Spherical agglomerates.
(B) Crack of the spherical agglomerate.
shown in panel B may be in an early stage of the formation,
because the petals are thin compared with those shown in Figure
3. We speculate the mechanism of the formation of the CC-
flower as follows. There might be spots on the surface of the
limiting membrane, where calcium and carbonate ions gush.
The hemispheric precipitate, which is composed of scalelike
vaterite as shown in panel C of Figure 6, grows at the spot.
Then crystals that become petals begin to grow around the
hemispheric core. Even when one layer of petals are formed
around the hemispherical core, calcium or carbonate ions would
continue to spring through the gap between petals and the
hemispheric core. Thus, petals would be formed layer by layer
along the surface of the hemispheric core. If this speculation
might be correct, the surface of the hemispheric core would be
completely covered with petals at the end. Several products
shown at the lower left in panel B of Figure 3 and also the
CC-flower shown in panel D of Figure 3 may imply such an
end. We infer from this speculation that the CC-flower is an
intermediate product of an echinus-like agglomerate.
Figure 7 presents the other precipitates formed on the inner
surface of the eggshell membrane at 30 °C. Spherical agglomer-
ates of calcite were observed in panel A. In a different view of
the inner surface, candylike agglomerates were observed, as
presented in panel B.
Figure 8 shows SEM images of calcium carbonate crystals
formed on the outer surface of the membrane. As can be seen
in panel A, spherical agglomerates with rough surface were
formed. The rough surface implies that the spherical agglomer-
ates are composed of plenty of small particles with a uniform
size. However, a crack site of the spherical agglomerate
presented in panel B shows that the primary products construct-
ing the agglomerates are not particles, but needlelike crystals.
It is not clear if the needlelike crystals are single crystals.
Figure 9 shows a different site of the outer surface of the
eggshell membrane. Panel A presents the precipitates formed
on the fiberlike structured surface of the outer shell membrane.
Panel B presents those precipitates in large magnification. Panel
C is an enlarged view of the rectangular area of panel B. The
spherical agglomerates should be vaterite in appearance like the
hemispheric cores of the CC-flowers. However, the crystals
Flowerlike Agglomerates of Calcium Carbonate Crystals
Figure 9. (A) SEM image of the spherical core structure formed on
the outer surface of the eggshell membrane. (B) SEM image of the
spherical core in large magnification. (C) Magnified image of the
surface of the spherical core.
formed around the spherical agglomerate were different from
the petal-like agglomerates in the CC-flower. Many short rodlike
crystals grew on the spherical agglomerates. And, as can be
seen in Panel C, the short rodlike crystals were composed of
thin platelike crystals that should be calcite. That short rodlike
agglomerate may grow to the long needlelike crystal shown in
panel B of Figure 8.
Conclusions
Calcium carbonate was crystallized on the eggshell membrane
using a specially designed crystallizer. The eggshell membrane
was placed between the calcium chloride and the sodium
Crystal Growth & Design, Vol. 6, No. 12, 2006 2757
carbonate solutions and precipitates of calcium carbonate on
the inner and outer surfaces of the membrane were observed.
When the inner surface of the membrane faced the calcium
chloride solution, the flowerlike agglomerate formed on the inner
surface of the eggshell membrane. The flowerlike agglomerate
was composed of a hemispheric core and the petal-like crystals
growing around the core. We named the agglometare the CC-
flower. On the other hand, normal cubic crystals of calcite
formed on the outer surface of the membrane. Any other
structuralized precipitates were not observed.
When the outer surface of the membrane faced the calcium
chloride solution, namely, in Experiment B, the spherical
agglomerates formed on the outer surface of the membrane, as
shown in Figures 8 and 9. Those spherical agglomerates were
composed of the long, needlelike crystals. We also obtained
the CC-flowers on the inner surface of the membrane at 50 °C
that were the same as those when the inner surface was facing
the calcium chloride solution.
We conclude that the surface structure of the eggshell
membrane played an important role for the formation of the
peculiar agglomerates of calcium carbonate crystals as the CC-
flowers.
References
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M. D.; Nys, Y. Matrix Biol. 2000, 19, 443-453.
(2) Ajikumar, P. K.; Lakshminarayanan, R.; Valiyaveettil, S. Cryst.
Growth Des. 2004, 4, 331-335.
(3) Takiguchi, M.; Igarashi, K.; Azuma, M.; Ooshima, H. Cryst. Growth
Des. 2006, 6, 1611-1614.
(4) Bellairs, R.; Boyde, A. Z. Zellforsch. Mikros. Anat. 1969, 96,
237-249.
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