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Nanoscale environments associated with
bioweathering of a Mg-Fe-pyroxene
Karim Benzerara
†‡
, Tae Hyun Yoon
†
, Nicolas Menguy
§
, Tolek Tyliszczak
¶
, and Gordon E. Brown, Jr.
†储
†Surface and Aqueous Geochemistry Group, Department of Geological and Environmental Sciences, Stanford University, Stanford, CA 94305-2115;
§Laboratoire de Mine´ ralogie-Cristallographie, Unite´ Mixte de Recherche, 7590 Centre National de la Recherche Scientifique, and Institut de Physique du
Globe de Paris, 4 Place Jussieu, 75252 Paris Cedex, France; ¶Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and
储Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center, 2575 Sand Hill Road, MS 99, Menlo Park, CA 94025
Communicated by W. G. Ernst, Stanford University, Stanford, CA, December 3, 2004 (received for review September 13, 2004)
Microorganisms are believed to create microenvironments leading
to reaction products not predictable from equilibrium thermody-
namics and to unique biomineral morphologies. Unambiguous
evidence for such environments is, however, rare in natural sam-
ples. We have used scanning transmission x-ray microscopy and
spectromicroscopy at the sub-40-nm scale, coupled with transmis-
sion electron microscopy, to examine bioweathering products on a
meteoritic Fe-Mg-orthopyroxene colonized by a filamentous mi-
croorganism. Our measurements reveal an amorphous Al-rich layer
beneath the microorganism, calcium carbonates of unique mor-
phology intimately associated with polysaccharides adjacent to the
microorganism, and regions surrounding the microorganism with
different iron oxidation states. Our results confirm the presence of
different microenvironments at this microorganism–mineral inter-
face and provide unique nanometer-scale views of microbially
controlled pyroxene weathering products.
aluminosilicate 兩biomineralization 兩geomicrobiology 兩STXM 兩
Urey reaction
Weathering of silicates at Earth’s surface has major impacts
on the environment, including the formation of soils and
the release of cationic nutrients (e.g., Fe and Mn) necessary for
the growth of microorganisms and plants (1). Other released
cations, such as Ca and Mg, can form Ca- and兾or Mg-carbonates,
resulting in sequestration of atmospheric CO
2
. This reaction,
known as the Urey reaction, has been suggested to control
Earth’s climate on geological time scales (2, 3). Pyroxenes are
among the most abundant silicates contributing to these reac-
tions (4). Abiotic dissolution of pyroxenes has been studied
extensively in the laboratory (e.g., see ref. 5), but significant
differences relative to dissolution rates measured in the field are
still not well understood (4, 6). The dissolution of Fe-containing
pyroxenes is even more complex because of the redox chemistry
of iron.
The role of microorganisms in the dissolution of silicates and
the cycling of Fe has been the focus of many experimental studies
(e.g., see ref. 7), some of which have shown that microorganisms
can radically modify the dissolution rates of silicates. They are
thought to do so by creating microenvironments in which pH and
other solution variables can dramatically differ from bulk con-
ditions (8, 9), by producing diverse metal-chelating organic
ligands (10), or by promoting metabolic redox reactions (11).
The pathways of microbially mediated Fe-silicate dissolution
reactions in nature remain, however, largely unexplored, partic-
ularly at the nanometer scale. Here we report new results on the
effects of a microorganism on the dissolution of an Fe
2⫹
-Mg
orthopyroxene [(Mg
0.75
Fe
0.23
Ca
0.01
)(Si
0.99
Al
0.01
)O
3
] that was ex-
posed at Earth’s surface for 70 years. Although it may not be
broadly representative of weathering environments, this sample
from the Tatahouine meteorite, which fell in the southern
Tunisian desert in 1931, provides an unusual opportunity to
examine biotic reaction pathways in a well known time frame and
under defined conditions in an arid environment (12–15). Pre-
vious SEM and transmission electron microscopy (TEM) anal-
yses of the same sample (15) showed that some of the fragments
were colonized by filamentous microorganisms whose taxonomy
remains unknown. Evidence for a biological origin of this
filament comes from the carbon, nitrogen, and phosphorus
content as discussed in ref. 15. The microorganism is in contact
with the orthopyroxene and is bordered by a cluster of nanom-
eter-sized, rod-shaped calcite single crystals. Removal of the
calcite crystals showed that the underlying pyroxene surface is
pitted, suggesting some causal relationship involving the micro-
organism, calcite precipitation, and enhanced dissolution of the
orthopyroxene. This meteorite fragment was selected for further
analysis by using focused ion beam (FIB) milling to prepare an
ultrathin (80 nm) cross section through the microorganism, the
calcite crystals, and the pyroxene. Scanning transmission x-ray
microscopy (STXM) was used to perform high spatial and
energy resolution near-edge x-ray absorption fine structure
(NEXAFS) spectroscopy at the C K-edge, Al K-edge, and Fe
L
3
-edge at the microorganism–calcite–pyroxene interface.
These methods, in combination with TEM, provide remarkably
clear chemical-state-specific images of the same sample areas of
the microorganism–mineral interface at sub-40-nm spatial res-
olution and 0.1–0.3 eV (1 eV ⫽1.602 ⫻10
⫺19
J) energy
resolution that could not be obtained by a single method.
Materials and Methods
FIB Milling. FIB milling was performed with an FEI Model 200
TEM FIB system at the Universite´ Aix-Marseille III. The FIB
lift-out method was used to prepare the sample (see Fig. 4) as
described by Heaney et al. (16). The area studied by Benzerara
et al. (12) by SEM (figure 1a in ref. 12) could be easily located
by using the imaging capabilities of the FIB. A thin layer of
platinum was then deposited on the specimen across the filament
and the calcite cluster to protect them during the milling process.
The FIB system uses a Ga liquid metal ion source for milling. A
30 kV Ga
⫹
beam operating at ⬇20 nA excavated pyroxene from
both sides of the Pt layer to a depth of 5
m. Before removal of
the thin slide, the sample was further thinned to ⬇80 nm with a
glancing angle beam at much lower beam currents of ⬇100 pA.
Finally, a line pattern was drawn with the ion beam along the side
and bottom edges of the thin section allowing its removal. The
⬇15-
m⫻5-
m⫻80-nm slide was transferred at room pressure
with a micromanipulator onto the membrane of a C-coated 200
mesh copper grid.
TEM. TEM observations were carried out on a JEOL 2010F
microscope operating at 200 kV and equipped with a field
emission gun, a high-resolution Ultra High-Resolution pole
piece, and a Gatan GIF 200 energy filter.
Abbreviations: FIB, focused ion beam; NEXAFS, near-edge x-ray absorption fine structure;
STXM, scanning transmission x-ray microscopy; TEM, transmission electron microscopy.
‡To whom correspondence should be addressed. E-mail: benzerar@stanford.edu.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0409029102 PNAS
兩
January 25, 2005
兩
vol. 102
兩
no. 4
兩
979–982
GEOLOGY
STXM. STXM studies were performed at A LS branch line 11.0.2.2
with the synchrotron storage ring operating at 1.9 GeV and
200–400 mA stored current. Technical details about this new
branch line and its operation are provided in ref. 17, and recent
applications to colloids are presented in ref. 18. Energy calibra-
tion was made using the well resolved 3p Rydberg peak at 294.96
eV of gaseous CO
2
for C, the Fe L
3
maximum peak of the
Tatahouine pyroxene at 707.8 eV for Fe, and the first major
NEXAFS peak of montmorillonite at 1,567.7 eV. The detector
used in all measurements reported in this study was a photo-
multiplier tube with a phosphor scintillator. Stacks of images
were obtained by scanning in the x–ydirection at each energy
increment over the energy range of interest (280–320 eV for C,
1,555–1,580 eV for Al, and 705–730 eV for Fe). The contrast
results from differential absorption of x-rays, which is dependent
on the chemical composition of the sample. Normalization and
background correction (in particular the carbon signal from the
carbon film of the TEM grid) of the NEXAFS spectra were
performed by div iding each spectrum by a second spectrum from
a sample-free location on the grid. Spectral and spatial resolu-
tions were ⬍0.1 eV and 40 nm, respectively, at the C K-edge and
Fe L
3
-edge, and 0.3 eV and 50 nm, respectively, at the Al K-edge.
AXIS2000 software (Version 2.1n, A. P. Hitchcock, McMaster Uni-
versity, Hamilton, ON, Canada) was used to align image stacks and
extract NEXAFS spectra from image stack measurements.
Results and Discussion
Calcium Immobilization in a Microbial Microenvironment. The C
K-edge NEXAFS spectrum of the filament is a complex mixture
of peaks (Fig. 1). Comparison with spectra from various biolog-
ical polymers (19) suggests the presence of polysaccharides (peak
at 288.6 eV) and proteins (peak at 288.2 eV) in the filament.
Together with the microbial-like morphology of this filament,
the presence of nitrogen and phosphorus (15) as well as bio-
chemical compounds, such as proteins, strongly support its
biological origin. The peak at 290.2 eV indicates the presence of
carbonates on or in the microorganism, suggesting fossilization.
C K-edge NEXAFS spectra of reference biological compounds
are scarce in the literature and are likely not representative of all
C-containing molecules present in this microorganism, which
helps explain why several spectral features could not be assigned.
Peaks at 284.3 eV, 286.4 eV, and 287.3 eV are thought to be
related to C functional groups present in either pristine microbial
molecules or molecules resulting from very early diagenesis. The
C K-edge NEXAFS spectrum of the calcite crystals is very
different from that of the microorganism, showing a major peak
at 290.2 eV (Fig. 2), which is characteristic of the
* resonance
of the CAO bond of carbonate groups. An additional peak at
288.6 eV was observed inside the calcite crystal cluster (Fig. 2)
and is indicative of carboxyl groups (
*CAO) likely associated
with polysaccharides (19, 20). Many authors have proposed the
involvement of organic molecules, either polysaccharides or
proteins, in the precipitation of calcium carbonates (e.g., see ref.
21). The nanometer-scale mixture of polysaccharides and car-
bonates observed on this sample suggests that calcite precipita-
tion occurred in a polysaccharide matrix likely released by the
filamentous microorganism. This suggestion is consistent with
several previous studies that presented models to explain how
polysaccharides could template carbonate precipitation (e.g., see
ref. 22). Moreover, the polysaccharides observed in this study
help explain the nanometer-sized, rod-shaped calcite crystals
found on the Tatahouine meteorite, which is an unusual mor-
phology for calcite crystals (12). Similar unusual morphologies
can be achieved when calcite growth occurs in the presence of
organic molecules, and polysaccharides in particular (23). More-
over, these calcite crystals are surrounded by an amorphous
calcium carbonate layer as shown by high-resolution TEM (12),
which is normally highly unstable under natural conditions but
can be stabilized in the presence of polysaccharides (24). Both
features thus provide potential biosignatures that could be useful
in the search for past life in earth and planetary materials.
Although some studies have suggested that abiotic dissolution of
Mg-silicates could be inhibited in the presence of carbonates
(e.g., see ref. 25), SEM and TEM observations on the Tata-
houine meteorite have shown that the filamentous microorgan-
ism enhanced the dissolution of the pyroxene below the calcite
Fig. 1. Spectromicroscopy analysis of filamentous microorganism. (A) TEM
image of the microorganism (arrows). Cc, calcite cluster; OPx, orthopyroxene.
The thin electron-dense layer covering the top of the cross-section is the
platinum layer deposited before FIB milling. (B) STXM image of the same area
at 270 eV. (C) C K-edge NEXAFS spectra from the microorganism, and refer-
ence calcite and organic molecules (from ref. 19); sodium alginate serves as a
model compound for polysaccharide, and albumin serves as a model com-
pound for proteins. Dashed lines at 288.2, 288.6, and 290.2 eV highlight the
most prevalent peaks of the model components.
Fig. 2. Spectromicroscopy analysis of the calcite nanocrystals. (A) TEM image
of the calcite nanocrystal cluster (see ref. 12). (B) Equivalent STXM image at
290.3 eV. (C) C K-edge NEXAFS spectra from the calcite cluster outlined on B,
and reference calcite and polysaccharide (sodium alginate) spectra (from ref.
19). Dashed lines at 288.6 and 290.2 eV highlight the mixture of calcite and
polysaccharides in the Tatahouine calcite cluster.
980
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0409029102 Benzerara et al.
cluster (12, 15). We suggest that this microorganism uniquely
coupled calcite precipitation and pyroxene dissolution by releas-
ing molecules that mediated Ca-carbonate precipitation. In
addition, we suggest that the same or other organic molecules
enhanced pyroxene dissolution. A similar Urey-like reaction
occurring in the presence of and promoted by microbes has been
indicated by several studies (26, 27). Such microbially mediated
reactions could be responsible for trapping some of the calcium
and magnesium released by pyroxene dissolution as well as
atmospheric CO
2
(2). The Urey reaction is classically considered
to be a two-step reaction on a time scale of hundreds of
thousands of years, with silicate weathering occurring on the
continents and carbonate precipitation occurring in the oceans
(2). Our results show that this reaction can take place on a very
small scale over a much shorter time scale.
Aluminum Fate in a Microorganism Microenvironment. The same
sample area was investigated by STXM imaging and NEXAFS
microspectroscopy at the Al K-edge (1,560 eV; Fig. 3). By taking
images below and above the Al K-edge, it was possible to obtain
an Al distribution map (Fig. 3). This map shows that Al is
preferentially concentrated beneath the microorganism. The Al
coordination number in this 100-nm-thick layer was determined
by acquiring an Al K-edge NEXAFS spectrum, which revealed
an absorption edge position at 1,564.5 eV and a broad peak
centered at ⬇1,568 eV that are indicative of four- and sixfold-
coordinated Al (28). This result is consistent with a disordered
aluminosilicate, such as allophane (Fig. 3), and with TEM
observations showing that the Al-rich layer is amorphous at the
nanometer scale (15). Precipitation of clays at the surface of
pyroxenes has been reported by several workers (e.g., see ref.
29). Our results indicate that the local concentration of Al is
associated with the microorganism. One possible explanation for
this association is that the microorganism reduces the water兾
solid ratio, which has been used to explain local reprecipitation
of secondary products (30). This observation is also consistent
with the finding that microorganisms can mediate the precipi-
tation of aluminosilicates at their surface (31). Similar biologi-
cally induced microenvironments leading to localized etching
and aluminosilicate precipitation have been found in altered
oceanic volcanic glass by using optical microscopy (e.g., see ref.
32). In the present study, we observed the formation of an
Al-rich layer only beneath the microorganism, which may impact
the subsequent dissolution of the pyroxene. Indeed, such resid-
ual layers are potentially responsible for passivation of silicate
surfaces and inhibition of further silicate dissolution (4, 33). In
a Panglossian perspective, it is noted that the formation of this
layer beneath rather than surrounding the microorganism pro-
vides a stable anchor substrate without preventing the microor-
ganism from dissolving the neighboring substrate to take ad-
vantage of the release of essential nutrients from the pyroxene.
It has also been suggested (P. C. Bennett, personal communi-
cation) that the formation of this Al-rich layer may be beneficial
to the microorganism as it sequesters Al, which is a toxic metal
and interferes with iron-chelating siderophores. Characteriza-
tion of the trace element fluxes between this microorganism and
the pyroxene substrate would be of great help in evaluating these
suggestions.
Fe Oxidation at a Microorganism–Mineral Interface. Previous studies
of the abiotic dissolution of Fe(II)-bearing pyroxene have re-
vealed that Fe behavior is mostly controlled by redox conditions
(33). Under oxic conditions at near neutral pH, which is likely the
case for the Tatahouine pyroxene, Fe is oxidized, which may
passivate the surface of the pyroxene (34). In contrast, under
anoxic conditions, dissolution rates of Fe-pyroxenes are similar
to those of Fe-free pyroxenes (33). To verify these earlier
findings for dissolution of Fe-bearing pyroxenes under oxic
conditions, we studied the Tatahouine sample at the Fe L
3
-edge,
which allowed us to determine the iron oxidation state (e.g., see
ref. 35) in the microorganism–calcite–pyroxene microcosm at a
Fig. 4. Fe redox state analysis at the microbe–mineral interface. (A) TEM
image of the cross section showing the microorganism (arrow), the calcite
crystal cluster (Cc), and the orthopyroxene (Opx). (B) Equivalent STXM image
at 707.8 eV. (C) Iron L3-edge NEXAFS spectra from the pyroxene (area 1; see Fig.
2B), the calcite cluster (area 2), the microorganism (area 3), and reference
hematite, representing the Fe3⫹endmember. Dashed lines represent the
positions of Fe L3maxima for Fe2⫹and Fe3⫹at 707.8 and 709.5 eV, respectively.
Fig. 3. Spectromicroscopy study of the microbe–mineral interface at the Al
K-edge. (A) STXM image of the microorganism (same area as the one observed
at the C edge; see Fig. 1) at 1,572 eV. (B) Corresponding TEM image. (C)
Aluminum elemental map obtained from the subtraction of the images of the
microorganism taken above and below the Al K-edge (at 1,572 and 1,560 eV,
respectively), showing high enrichment in Al only beneath the microorganism.
(D) Al K-edge NEXAFS spectrum from the Al-rich layer outlined in Fig. 2 A, and
reference spectrum of an allophane containing both four- and sixfold-
coordinated Al (from ref. 28).
Benzerara et al. PNAS
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January 25, 2005
兩
vol. 102
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no. 4
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981
GEOLOGY
spatial resolution of ⬍40 nm (Fig. 4). Iron spectra taken on the
pyroxene (area 1) showed a major peak at 707.8 eV, indicative
of Fe
2⫹
(Fig. 4). Fe-rich particles in the calcite cluster (area 2)
displayed a major peak at 709.5 eV, indicating that iron was
oxidized after its release by dissolution of the pyroxene (Fig. 4).
In the microorganism (area 3), however, the Fe L
3
-edge shows
a mixed iron valence (Fe
2⫹
and Fe
3⫹
) (Fig. 4). Spectra were
recorded several times over a several hour period, and all were
identical, indicating that beam damage was not responsible for
this observation in area 3. One possible explanation for variable
iron valence in and around the Tatahouine microorganism is that
the iron oxidation state was modified by this microorganism,
which creates a special microenvironment with a particular pO
2
and兾or modifies the pH, resulting in slower oxidation of iron.
Determining the taxonomy of the microorganism would help in
verifying this hypothesis, although such identification of a single,
partially fossilized microorganism, as in this case, would be
extremely difficult. Another possibility is that organic molecules
produced by the microorganism strongly bind dissolved Fe
2⫹
and
thus inhibit its oxidation as suggested by several previous studies
(e.g., see ref. 36). Fe
3⫹
is likely the stable form of iron at the
surface of the Tatahouine sand. Whatever mechanisms are
involved in iron redox behavior, the microorganism heavily
impacts iron oxidation dynamics in a microenvironment, result-
ing in a major modification of pyroxene reactivity, compared
with a purely abiotic environment. One consequence of the
stabilization of Fe
2⫹
is the delay of entrapment of the microor-
ganism by Fe-oxides. Such stabilization could also provide a
low-energy-cost iron source.
Summary and Conclusions
A filamentous microorganism appears to have had a major
impact on the weathering of a meteoritic Fe
2⫹
-Mg-orthopyrox-
ene during some period of its 70 years of exposure in the
southern Tunisian desert. Modifications of iron oxidation dy-
namics, calcium carbonate precipitation, and Al mobility by this
microorganism may have resulted in different dissolution rates
in regions surrounding the pyroxene separated by tens of nano-
meters. Our observations indicate that this microorganism cou-
pled carbonate precipitation and pyroxene weathering, provid-
ing a potential sequestration mechanism for CO
2
and producing
unique biosignatures in the form of rod-shaped nanocrystals of
calcite. The ability to characterize this microorganism– calcium
carbonate–pyroxene system at the sub-40-nanometer spatial
scale by using STXM and TEM methods provides a unique view
of how one type of microorganism controlled biomineral for-
mation (e.g., see ref. 37) as well as orthopyroxene weathering in
an arid environment. Although more systematic work on similar
samples from various environments will be needed to under-
stand the broader significance of the observations made here,
the methodology presented in this paper should be helpful in
assessing the importance of microorganisms in the evolution of
Earth’s surface chemistry and in identifying them in early Earth
and planetary materials.
We thank the CP2M members in Marseille who granted access to the
JEOL 2010F microscope and to the FEI Model 200 TEM FIB system,
A. P. Hitchcock for providing reference C K-edge spectra for albumin
and sodium alginate, Scott Fendorf (Stanford University) for a helpful
review of this manuscript, and Phil Bennett and two anonymous review-
ers for help in improving this article. The STXM studies were conducted
on branch line 11.0.2.2 at the Advanced Light Source, which is supported
by the Office of Science, Office of Basic Energy Sciences, Division of
Materials Sciences, and Division of Chemical Sciences, Geosciences, and
Biosciences of the U.S. Department of Energy at Lawrence Berkeley
National Laboratory under Contract DE-AC03-76SF00098. This work
was supported by National Science Foundation Grants CHE-0089215
(Stanford University Collaborative Research Activity in Environmental
Molecular Science on Chemical and Microbial Interactions at Environ-
mental Interfaces), CHE-0431425 (Stanford University Environmental
Molecular Science Institute), and EAR-9905755. K.B. thanks the French
Foreign Ministry for a Lavoisier Fellowship.
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982
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www.pnas.org兾cgi兾doi兾10.1073兾pnas.0409029102 Benzerara et al.
