BACTERIALLY INDUCED MINERALIZATION OF CALCIUM CARBONATE IN TERRESTRIAL
ENVIRONMENTS: THE ROLE OF EXOPOLYSACCHARIDES AND AMINO ACIDS
OLIVIER BRAISSANT, GUILLAUME CAILLEAU, CHRISTOPHE DUPRAZ,
AND
ERIC P. VERRECCHIA
Institut de Ge´ologie, Universite´ de Neucha ˆtel, Rue Emile-Argand 11, CH-2007 Neuchaˆtel, Switzerland
e-mail: eric.verrecchia@unine.ch
A
BSTRACT
: This study stresses the role of specific bacterial outer struc-
tures (such as glycocalix and parietal polymers) on calcium carbonate
crystallization in terrestrial environments. The aim is to compare cal-
cium carbonate crystals obtained in bacterial cultures with those ob-
tained during abiotically mediated synthesis to show implications of
exopolysaccharides and amino acids in the mineralogy and morphology
of calcium carbonate crystals produced by living bacteria. This is done
using various amounts of purified exopolysaccharide (xanthan EPS)
and L-amino acids with a range of acidities. Amino acids and increas-
ing xanthan content enhance sphere formation in calcite and vaterite.
Regarding calcite, the morphology of crystals evolves from rhombo-
hedral to needle shape. This evolution is characterized by stretching
along the c axis as the amino acid changes from glutamine to aspartic
acid and as the medium is progressively enriched in EPS. Regarding
vaterite, the spherulitic habit is preserved throughout the morpholog-
ical sequence and starts with spheres formed by the agglomeration of
short needles, which are produced in a xanthan-free medium with glu-
tamine. Monocrystals forming spheres increase in size as xanthan is
added and the acidity of amino acids (glutamic and aspartic acids) is
increased. At high xanthan concentrations, amino acids, and mainly
aspartic and glutamic acids, induce vaterite precipitation. The role of
the carboxyl group is also probably critical because bacterial outer
structures associated with peptidoglycan commonly contain carboxyl
groups. This role, combined with the results presented here, clearly
demonstrate the influence of bacterial outer structure composition on
the morphology and mineralogy of bacterially induced calcium car-
bonate. This point should not be neglected in the interpretation of cal-
cite cements and carbonate accumulations in terrestrial environments.
INTRODUCTION
Microbiologically induced mineralization is defined as processes leading
to inorganic mineral deposits by adventitious precipitation. This precipi-
tation arises from secondary interactions between various metabolic pro-
cesses producing carbonate species and the surrounding environment
(Mann 2001). Mineral precipitation by microbes has been known about for
a long time (see historical aspects in Ehrlich 1996, 1998). Among bacte-
rially precipitated minerals, carbonates, and in particular calcium carbonate
(CaCO
3
) in numerous forms, are probably the most important (Ferris et al.
1989). Bacterially precipitated calcite (in terrestrial environments), and ara-
gonite and high Mg calcite (in marine environments) have been described
many times in the literature (e.g., Boquet et al. 1973; Castanier et al. 2000;
Chafetz 1986; Krumbein 1979). Although other species of calcium carbon-
ate minerals are only rarely associated with bacterial activity, there are a
couple of recently described examples: vaterite precipitation by the soil
bacteria Xanthobacter autotrophicus (Braissant et al. 2002; Braissant and
Verrecchia 2002) and monohydrocalcite (CaCO
3
·H
2
O) by the halophilic
bacteria Halomonas eurihalina (Rivadeneyra et al. 1998). The ecology of
the microbes associated with vaterite and monohydrocalcite is consistent
with the lacustrine environment in which the crystals formed (Giralt et al.
2001; Krumbein 1975) and not due to the activity of exotic strains under
exceptional conditions. To our knowledge, microbially induced ikaite
(CaCO
3
·6H
2
O) has not been reported, probably because of its instability.
F
IG
. 1.—X-ray diffractograms of CaCO
3
crystals associated with A) Xanthobacter
autotrophicus and B) Ralstonia eutropha cultures. These diffractograms correspond
perfectly to vaterite (A) and calcite (B), respectively.
1
Published in Journal of Sedimentary Research 73, issue 3, 485-490, 2003,
which should be used for any reference to this work
→
F
IG
. 2.—Morphology of crystals obtained during bacterial growth. A–H, Xanthobacter autotrophicus ; I–P, Ralstonia eutropha. A) General view of vaterite spherulites
in cross-polarized light (XPL). B) Close-up of part A showing two different crystal arrangements. Spheres showing a black cross are constituted by needles, whereas other
spheres are formed by clusters of small monocrystals. C, D) Scanning electron microscope (SEM) view of individual (C) and coalescent (D) vaterite spherulites. The
surface of the sphere is not smooth and shows a mammillated rough topography. E, F) ‘‘Fried egg’’ morphology of accessory crystals. These clusters are constituted by
two parts, a calcitic bottom part having a lens- to plate-shape and a central hemispherical upper part constituted by vaterite crystals of various sizes. G) Detail of the
hemispherical part showing small hexagons of vaterite. H) Imbricated rhombohedra forming imperfect spherical calcite clusters. I) Subhedral to euhedral rhombohedra.
XPL view. J) ESEM view of a calcite flower formed by flat sparitic crystals. K) SEM view of the heart of a calcite flower constituted by crystals forming a spiral-like
structure. L) SEM view of a calcite flower with petals formed by thin palissadic crystals. M) XPL view of a calcite cluster constituted by four triangular-shaped crystals
joined at their tip to form a thickened Maltese cross. N) SEM view of a similar crystal shown in part M. O, P) XPL and SEM view of funnel-like crystals.
Dolomite (CaMg(CO
3
)
2
) has been reported by Warthmann et al. (2000) to
be precipitated by Desulfonatovibrio in anoxic marine environments.
Despite numerous reports of calcium carbonate precipitation by microbes
and the important biomass they represent in soils, accumulation of terres-
trial carbonates is still generally attributed to physicochemical processes
(Lal et al. 2000). In this paper, it is demonstrated that relationships between
morphologies and mineralogies of CaCO
3
encountered in soils and surficial
sediments reveal a bacterial influence related to microbial biofilms. This
study stresses the role of a specific exopolysaccharide (xanthan EPS) and
amino acids on calcium carbonate crystallization in terrestrial environ-
ments.
In marine environments, outer structures such as the cyanobacterial S-
layer have already been recognized as the main crystalline biostructure able
to act as a nucleus for calcium carbonate growth (see review in Schultze-
Lam et al. 1992, 1996 and Smarda et al. 2002). Most microbial cells in
natural environments form communities inside microbial biofilms (Decho
1990, 2000; Sutherland 2001 a, 2001 b). Therefore, exopolysaccharides
(EPS) and amino acids most likely play an essential role in calcium car-
bonate morphology and mineralogy. This study investigates the potential
relationships between EPS, amino acids, oxidative soil bacteria, and cal-
cium carbonate crystals using conventional bacterial cultures and abioti-
cally mediated calcium carbonate synthesis. The aim is to compare calcium
carbonate crystals obtained in bacterial cultures with those obtained during
abiotically mediated synthesis. This is done using various amounts of pu-
rified EPS and L-amino acids with a range of acidities and frequently as-
sociated with mucilages.
MATERIALS AND METHODS
Bacterially mediated calcium carbonate crystals were obtained after 20
days culturing of Xanthobacter autotrophicus (syn: Corynebacterium au-
totrophicum, DSM 432, ATCC 35674) and Ralstonia eutropha H16 (syn:
Alcaligenes eutrophus, DSM: 428, ATCC 17699) on a B4 medium kept at
26
8
C (Merck yeast extract 4.0 g/L; Merck calcium acetate 2.5 g/L; Merck
agar-agar 15 g/L: Boquet et al. 1973). These two strains are oxidative
terrestrial bacteria commonly found in soils and sediments. In the cultures,
the main difference between the two strains is the amount of polysaccharide
produced: X. autotrophicus is surrounded by a large amount of slime,
whereas R. eutropha produces only an extremely low amount of glycocalix
(Holt et al. 1993). The pH was not monitored during culturing, but final
pH was
.
9 as revealed by a droplet of pH indicator. Uninoculated Petri
dishes were kept as a sterility control.
Abiotically mediated crystals have been obtained by precipitation from
a CaCl
2
/ (NH
4
)
2
CO
3
reaction inside a polysaccharidic medium (EPS) with
various types of L-amino acids. The aim of this experiment is to simulate
precipitation of calcium carbonate in an EPS-rich environment without in-
volving living bacteria. Ten runs of this experiment have been conducted.
The synthesis was performed using a CaCl
2
0.05 M solution to which was
added: (1) 1.0% (w/v) amino acids (L-glutamine, L-glutamic acid and L-
aspartic acid. These amino acids were chosen because they are commonly
found in the parietal structures of many bacteria, e.g. polyglutamate in
Bacillus anthracis, Mycobacterium tuberculosis, Sporosarcina halophila,
polyglutamine in the genus Xanthobacter and Flexithrix, and finally po-
lyaspartate in Synechococcus sp.); and (2) 0%, 0.1%, 0.5% and 1.0% (w/
v) concentrations of xanthan from Sigma
TM
(a bacterial EPS mainly com-
posed of mannose and glucose). The pH of this solution was adjusted to
8.4 (the pH of calcite stability at 1 atm and 25
8
C) before adding xanthan.
The solution was sterilized by autoclaving at 121
8
C for one hour. After
sterilization, the solutions were placed in Petri dishes that remained for 20
days in ethanol-washed desiccators filled with (NH
4
)
2
CO
3
. Petri dishes
were examined with a binocular microscope to control possible presence
of contaminants.
Crystals obtained in bacterial cultures, as well as in the abiotic experi-
ment, were both isolated by either sedimentation or directly from the cul-
ture using precision tweezers. The crystals obtained were gold coated and
observed with a Philips XL20 SEM. Crystals sampled from bacterial col-
onies were previously washed in saturated calcium hypochlorite solution
in order to remove organic matter from the surfaces of the crystals. Samples
of bacterial colonies bearing crystals were fixed with glutaraldehyde (5%),
immediately dehydrated in ethanol, and air-dried after ethanol replacement
by tetramethylsilane (TMS; Dey et al. 1989). These samples were gold
sputter coated for 60 seconds to give a 23 nm gold coating, and observed
with a Philips XL20 SEM. Crystals were analyzed by X-ray diffraction
(XRD) using a Scintag diffractometer and by an energy-dispersive spec-
trometry (EDS) microprobe coupled with a Philips XL30 environmental
scanning electron microscope (ESEM). Calcite and vaterite mineralogies
were determined by cross checking XRD analyses, crystal habits, and rel-
ative proportions of each phase plus comparison of observed crystal shapes
with shapes documented in the literature.
RESULTS
Calcium Carbonate Crystals Obtained in Bacterial Cultures
Bacterially mediated crystals were found mostly on the surfaces of col-
onies. They have various mineralogies and morphologies. In terms of min-
eralogy, X. autotrophicus produced mainly brown vaterite spherulites rang-
ing from 50
m
mto200
m
m in diameter, with a small fraction of calcite
crystals (Fig. 1A), whereas R. eutropha produced colorless calcite crystals
ranging from 100
m
mto600
m
m in size, with traces of vaterite (Fig. 1B).
Spheres produced by X. autotrophicus show three different monocrystal
(small individual crystal) arrangements (Fig. 2A–D). Although the overall
sphere shape remains, the morphology of individual crystals forming the
sphere varies from a fan shape to a needle (Fig. 3). The crystals are or-
ganized around a central point and grow radially to form the sphere. Ac-
cessory shapes are also associated with the spheres. They are made of either
a polyhedral assemblage of calcite crystals or a mixture of calcite and
vaterite. Two main morphologies have been observed. ‘‘Fried egg’’ shapes
are probably formed at the surface of the colony (Fig. 2E–G). The sub-
spherical part is constituted by vaterite and grows on a lens- to plate-shape
calcite surface. The flat part is in contact with the atmosphere, the sphere
being inside the colony. Therefore, the general shape is a flipped ‘‘fried
egg.’’ Calcite also forms imperfect spherical clusters made of imbricated
rhombohedra (Fig. 2H).
2
F
IG
. 3.—Morphology of individual crystals forming calcium carbonate spheres
associated with Xanthobacter autotrophicus. From left to right: fan-shaped, petal-
like, and needle monocrystals. The two first spheres are vaterite, and the third is
either calcite or vaterite.
←
F
IG
. 4.—Scanning electron microscope photographs of crystal morphologies obtained during abiotically mediated synthesis of calcium carbonate in the presence of
exopolysaccharides and amino acids. Abscissa: Blank (no amino acids present), Gln, L-glutamine, Glu, L-glutamic acid, Asp, L-aspartic acid. Coordinates: xanthan content
(a glucose and mannose polymer), from 0.0% (absence of xanthan) to 0.1, 0.5 and 1.0% w/v. A) Euhedral calcite crystals. B) Subhedral and dendritic calcite crystals.
Dendritic crystals show two different morphologies: diffusion-limited aggregation clusters and ordered dendrites. C) Crystal morphologies become chunkier, leading to
imbricated twins. Some euhedral rhombohedra are preserved. D) Agglomerated twins tending to form spheres. E) Euhedral calcite rhombohedra associated with vaterite
spherulites composed of either short needle monocrystals or an agglomerate of small euhedral crystals. F) Imperfect calcite rhombohedra are present with vaterite spheres.
General structure of vaterite spherulites is similar to part E. G) Rare spherulitic vaterite associated with calcite rhombohedra. Edges of calcite crystals are smoother than
in part A, and they tend to form imbricated twin clusters. H) Vaterite and calcite spherulites. Calcite appears as imbricated twin clusters. In addition, both vaterite and
calcite can be characterized by fibro-radial spheres. I) Upper part: vaterite spiky agglomerates and cauliflower-shaped calcite. Bottom part: left, calcite rhombohedra; right,
epitactic growth of a hexagonal vaterite crystal on a calcite substrate forming cauliflower clusters. J) Spiky agglomerate of vaterite starting to form a sphere, and a rough
calcite spherulite. K) Calcite spheres. The arrow shows remains of a rhombohedron emphasizing the transition between imbricated twin clusters shown in parts G and H
and the structure of spheres formed by styloidic crystals. Double sphere of calcite showing a structure close to fibro-radial spheres and composed by styloidic monocrystals.
L) Fibro-radial calcite and vaterite spheres with a smooth surface associated with spiky vaterite spherulites (arrow). M) Fibro-radial calcite spherulites (right). Spiky vaterite
agglomerate (left). N) General view of spiky vaterite spherulites associated with calcite spheres. Flat shapes are due to contact of the surface of the medium with the
atmosphere. O) Calcite spheres. Some spheres are constituted by stacked flat monocrystals, which can be compared with spheres formed by styloidic crystals shown in
part K. P) General view of vaterite and calcite spheres. Vaterite spherulites are characterized by the agglomeration of large monocrystals, whereas calcite spheres are
smooth and fibro-radial.
Crystals produced by R. eutropha have three main shapes. The two most
common morphologies encountered are euhedral to subhedral rhombohedra
(Fig. 2I) and calcite flowers, composed of crystals with euhedral termina-
tions (Fig. 2J–L). The center of the flower is formed by a circular spiral-
like crystal (Fig. 2K). Two other shapes have been observed: thickened
‘‘Maltese crosses’’ (Fig. 2M, N) and, rarely funnels (Fig. 2O, P). In blank
uninoculated Petri dishes, crystals were not observed, emphasizing the cru-
cial role of bacteria.
Calcium Carbonate Crystals Obtained in Abiotically Mediated
Experiments
The reaction between CaCl
2
and (NH
4
)
2
CO
3
, with neither xanthan nor
amino acids present, produces calcite rhombohedra (Fig. 4A), which is not
surprising. However, synthesis of calcium carbonate crystals in mucilagi-
nous solutions results in numerous crystal morphologies, emphasizing the
potential role of EPS in their shape diversity. Synthesis using increasing
concentrations of xanthan without the addition of amino acids leads mainly
to individual and branching crystals. At a low concentration of xanthan
(0.1%), dendritic crystals form. Dendrites can be disordered, and similar to
structures obtained by diffusion-limited aggregation (Fig. 4B), or ordered
along the main three directions of calcite crystal growth (Fig. 4B). Nev-
ertheless, subhedral rhombohedra remain the most abundant shape (Fig.
4B). At a higher concentration of xanthan (0.5%), the most common shape
(constituting about 90%) is a dendrite-like crystal, which is in fact lacunar
and made of small stacked rhombohedra (Fig. 4C). Around 5% of the
crystals are euhedral rhombohedra (Fig. 4C). The remaining 5% are im-
bricated rhombohedron twins that form irregular spheroidal clusters (Fig.
4C). When the xanthan concentration reaches 1%, more and more imbri-
cated rhombohedron twins agglomerate until they form subspherical clus-
ters. Whatever the concentration in xanthan, all the crystals obtained in the
amino acid free media are calcitic.
When amino acids are added to the medium, various crystal morpholo-
gies are obtained (Fig. 4E–P). At first glance, it is difficult to clearly see
a trend in morphologies, although the habit becomes more spherical as
xanthan increases. In addition, the two calcium carbonate polymorphs—
vaterite and calcite—are always precipitated during abiotic synthesis in the
presence of amino acids. Nevertheless, the ratio between the two miner-
alogical species seems to be essentially related to the abundance of EPS
and the nature of amino acids. Aspartic acid, glutamic acid, and glutamine
always induced vaterite formation at a high concentration of xanthan
(1.0%), although calcite is still present. Consequently, it is critical to dis-
criminate between the two mineralogies in order to understand the differ-
ence in shapes in Figure 4. Two morphological sequences (Fig. 5) can be
described in relation to increasing EPS content (which acts as a weak acid)
and the acidity of the added amino acid. Glutamine is considered as a basic
amino acid, whereas glutamic and aspartic acids are acidic, aspartic acid
being more acidic than glutamic acid.
Regarding calcite (Fig. 5), the morphological sequence starts with rhom-
bohedra produced in a xanthan-free medium with glutamine (Fig. 4E). The
conditions in this case are the most basic (highest pH) of the total exper-
iment involving amino acids. By adding xanthan and by increasing the
acidity of amino acids (glutamic and aspartic acids), rhombohedron edges
become smoother (Fig. 4G) and crystals form numerous twins, agglomer-
ated around a center (Fig. 4G, H), and finally evolve to fibro-radial spheres
(Fig. 4K–P). This sequence can be summarized by the decrease of the
monocrystal size forming the shape, from calcite rhombohedra to calcite
styloids followed by needles (Fig. 5).
Regarding vaterite (Fig. 5), the spherulitic habit is preserved throughout
the morphological sequence. It starts with spheres formed by the agglom-
eration of short needles, which are produced in a xanthan-free glutamine
medium (Fig. 4E). Nevertheless, some spheres are composed of larger (but
still small) monocrystals (Fig. 4E). These monocrystals increase in size as
xanthan is added and the acidity of amino acids (glutamic and aspartic
acids) is increased (Fig. 4I, J, L–P). Contrary to the sequence described for
calcite, the vaterite sequence can be illustrated by the increase in the size
of the monocrystals forming the spheres. Nevertheless, in the presence of
glumanine and glutamic acid and a high amount of EPS, vaterite can form
fibro-radial spherulites, which show a black cross in polarized light, as
shown by Dedek (1966). The only way to differentiate calcite from this
vaterite is by using XRD.
3
F
IG
. 5.—Sequences of calcite and vaterite morphologies obtained during the abiotic experiment. This sketch shows the morphologies related to increasi ng amino acid
acidity and xanthan content. Scales are relative. The various domains of influence of amino acids are shown. Six main steps can be described for both vaterite and calcite.
The calcite sequence starts with rhombohedra. Size of monocrystals forming the clusters decreases from Step 1 to Step 6 until they form needles. The general shape of
calcite evolves from rhombohedra to fibro-radial spherulites. Vaterite sequence: vaterite is always precipitated as spheres. The monocrystals constituting the spheres increase
in size from Step 1 (short needles) to Step 6 (large hexagons). Between Step 2 and Step 3, vaterite can occur as fibro-radial spherulites. Sketches were traced from
photographs.
In summary, amino acids and increasing xanthan content enhance sphere
formation in both calcite and vaterite. In addition, regarding calcite, the
morphology of rhombohedra evolves from a euhedral shape to a needle.
This evolution is characterized by stretching along the c axis as the amino
acid changes from glutamine to aspartic acid and as the medium is pro-
gressively enriched in EPS.
DISCUSSION
Many spherulitic features are observed in carbonate soils and surficial
sediments. The explanation of their origin is often unclear and usually
attributed to possible organic influence without further elucidation. Spher-
ulitic or oolitic habit have been observed in many other cases involving
different microorganisms and different mineral species (e.g., Folk 1993).
Calcium carbonate spherulites remain the most commonly described fea-
tures. It has been shown that formation of aragonitic spherulites by Deleya
halophila may be the result of cell calcification and aggregation (Rivade-
neyra et al. 1996). In addition, formation of magnesian calcite spherulites
and dumbbells has been related to the slime-producing bacteria, Myxococ-
cus xanthus (Gonza´lez-Mun˜oz et al. 2000; Holt et al. 1993). Various types
of apatite [Ca
10
(PO
4
)
6
2
x
(CO
3
)
x
(F,OH)
2
1
x
] have been shown to precipitate
with a spherulitic habit when under the influence of organic matter or
during (nano)bacterial activity. Kajander and C¸iftc¸ioglu (1998) have shown
that nanobacteria can be responsible for the formation of small spherulites
(about 2
m
m in diameter) alone or inside human cells (the spherulitic habit
is not surprising considering the high density of macromolecules). Apatite
spherulites including proteins (referred to as nanoforms) have also been
shown to precipitate in a medium containing sterile fetal bovine serum
(Vali et al. 2001). Salt ooids have been attributed to halophilic bacterial
activity, which may guide the growth of biominerals (Castanier et al. 1999).
In conclusion, recent research illustrates the numerous possible interactions
between crystals and organic matter. Our results emphasize the importance
of bacterial biofilms containing exopolysaccharides and amino acids in the
precipitation of CaCO
3
, and resemble features encountered in bacterial cul-
tures, soils, and paleosols.
The morphologies of crystals obtained in X. autotrophicus and R. eutro-
pha cultures (Fig. 2) can now be understood given the results depicted in
Figure 4. Xanthobacter autotrophicus is known to produce substantial
amounts of exopolysaccharides (Wiegel 1991). This property leads to the
precipitation of vaterite spherulites (Fig. 2A–D), with some spheres show-
ing a black cross of extinction in cross-polarized light. These morphologies
are equivalent to those observed in Step 3 of Figure 5. Vaterite spherulites
showing a black cross have also been observed, although rarely in the
presence of glutamine with 0.5% xanthan. Regarding calcite, the accessory
shape constituted by imbricated rhombohedra (Fig. 2H) is also equivalent
to Step 3 of Figure 5. These observations indicate that crystals observed
in the X. autotrophicus culture should be produced in a medium enriched
4
F
IG
. 6.—Spherulites associated with a paleosol
in Bahamian eolianites (sample obtained by
courtesy of C. Nawratil, University of Geneva).
A) General view in plane-polarized light (PPL)
of a planar pore infilled with a spherulitic calcite
cement. B) Same view in cross-polarized light
(XPL) showing the black cross of calcite
spherulites. C, D) Detail in PPL and XPL of
calcite spherulites showing some growth
increments emphasized by impurities (organic
matter) layers. These spherulites are virtually
identical to those obtained in the presence of
bacterial exopolysaccharides and amino acids.
F
IG
. 7.—Scanning electron micrographs. A)
Example of a fibro-radial calcite spherulite found
associated with polysaccharides in a tropical soil.
B) Vaterite cluster from a tropical soil showing a
structure identical to those obtained in Figure 4J,
M, and N. This vaterite cluster grew on a silica
substratum in a biofilm rich in
exopolysaccharides and amino acids.
in EPS as well as glutamine. Indeed, X. autotrophicus makes not only large
amounts of exopolysaccharides but also a polyglutamine parietal polymer
(Kandler et al. 1983; Wiegel 1991).
In contrast, the fact that R. eutropha only produces calcite rhombohedra
and flower-shaped agglomerates is linked to an absence or only 0.1% EPS
in the medium. The calcite crystals can be compared with Step 1 of calcite
in Figure 5 or with crystals shown in Figures 4E and F. This is consistent
with the fact that R. eutropha produces only low amounts of EPS.
The results obtained in Figure 4 can also be compared with features
observed in natural environments. Spherulites showing an extinction cross
in cross-polarized light are commonly found in soils and paleosols (Fig.
6). These spherulites infill pores and are associated with micritic and/or
organo-micritic layers (Fig. 6A, B). Their origin has never been clearly
identified. Nevertheless, their morphology (Fig. 6C, D) is identical to crys-
tals produced in the EPS-rich medium (Fig. 4H, K, L, P). This point in-
dicates the influence of biofilms in the precipitation of such spherulites in
soils. A similar calcium carbonate spherulite has also been observed in a
polysaccharide-rich tropical soil (Fig. 7A). Calcite and vaterite spherulites
have also been reported in the mucilagenous sheath of cyanobacteria, in-
dicating the fundamental role exopolysaccharides can play in their precip-
itation (Verrecchia et al. 1995; Giralt et al. 2001). In a different tropical
soil, a feature identical to those shown in Figures 4J, M, and N has also
been observed: it is a vaterite cluster growing on a quartz grain in a biofilm
rich in EPS and amino acids (Fig. 7B). In conclusion, the abiotic experi-
ments conducted in this study shed new light on the potential contribution
of bacterial EPS and amino acids on calcium carbonate precipitation in
terrestrial environments.
In natural environments, the variation of CaCO
3
crystal morphologies
and mineralogies associated with EPS may result from its heterogeneity
due to the diversity of microorganisms living within a single biofilm. Het-
erogeneity in slimes leads to differences in diffusion coefficients (local
supersaturation), surface tensions, and viscosity. The viscosity of the 1.0%
5
