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Microbial mediation of complex subterranean mineral structures

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OPEN ACCESS ARTICLE AT: http://www.nature.com/articles/srep15525 Helictites—an enigmatic type of mineral structure occurring in some caves—differ from classical speleothems as they develop with orientations that defy gravity. While theories for helictite formation have been forwarded, their genesis remains equivocal. Here, we show that a remarkable suite of helictites occurring in Asperge Cave (France) are formed by biologically-mediated processes, rather than abiotic processes as had hitherto been proposed. Morphological and petro-physical properties are inconsistent with mineral precipitation under purely physico-chemical control. Instead, microanalysis and molecular-biological investigation reveals the presence of a prokaryotic biofilm intimately associated with the mineral structures. We propose that microbially-influenced mineralization proceeds within a gliding biofilm which serves as a nucleation site for CaCO3, and where chemotaxis influences the trajectory of mineral growth, determining the macroscopic morphology of the speleothems. The influence of biofilms may explain the occurrence of similar speleothems in other caves worldwide, and sheds light on novel biomineralization processes.
Detail of a BGS bouquet: Hybrid speleothems (detail 2) formed between acicular speleothems (detail 1) and tubular speleothems (detail 3). (b) Photomicrograph under cross polarized light of the longitudinal section of an acicular speleothem, which is made of crystals growing in optical continuity (CAC). (c) Mineralogy of acicular speleothems was investigated with X-ray diffraction showing that these speleothems are made of aragonite. The black spectrogram is the analysis, which is compared to the aragonite XRD peaks. (d) SEM image of an acicular speleothem whose composition is indicated by the EDX analysis reported in panel (e) upper curve. (f) Photomicrograph under cross polarized light of the transverse section of a hybrid speleothem. The center and the rim have morphologies similar to those of acicular and tubular speleothems, respectively. (g) Photomicrograph under cross polarized light of the transverse section of a tubular speleothem, which is made of crystals growing in non-optical continuity. (h) Mineralogy of tubular speleothems was investigated by means of X-ray diffraction showing that these speleothems are made of calcite. The black spectrogram is the analysis, which is compared to the calcite XRD peaks. i) SEM image of the outer wall of a tubular speleothem which is covered by a biofilm (). i) EDX analysis performed on the outer wall of a tubular speleothem indicating that the biofilm is mainly made of C. However, as the EDX analysis penetrates few micrometers the surface the Ca and O peaks suggest that the first layer covering the wall is made of a carbonate mixed with organic matter (). Photos by: Nicola Tisato, Francesco Sauro and Tomaso R. R. Bontognali.
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SCIENTIFIC RepoRts | 5:15525 | DOI: 10.1038/srep15525
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Microbial mediation of complex
subterranean mineral structures
Nicola Tisato1,†, Stefano F. F. Torriani2, Sylvain Monteux3, Francesco Sauro4, Jo De
Waele4, Maria Luisa Tavagna5, Ilenia M. D’Angeli4, Daniel Chailloux6, Michel Renda6,
Timothy I. Eglinton5 & Tomaso R. R. Bontognali5
Helictites—an enigmatic type of mineral structure occurring in some caves—dier from classical
speleothems as they develop with orientations that defy gravity. While theories for helictite
formation have been forwarded, their genesis remains equivocal. Here, we show that a remarkable
suite of helictites occurring in Asperge Cave (France) are formed by biologically-mediated processes,
rather than abiotic processes as had hitherto been proposed. Morphological and petro-physical
properties are inconsistent with mineral precipitation under purely physico-chemical control.
Instead, microanalysis and molecular-biological investigation reveals the presence of a prokaryotic
biolm intimately associated with the mineral structures. We propose that microbially-inuenced
mineralization proceeds within a gliding biolm which serves as a nucleation site for CaCO3, and
where chemotaxis inuences the trajectory of mineral growth, determining the macroscopic
morphology of the speleothems. The inuence of biolms may explain the occurrence of similar
speleothems in other caves worldwide, and sheds light on novel biomineralization processes.
For centuries, caves were considered as mostly barren and inhospitable environments to life. Only
recently, thanks to the advent of new techniques in molecular biology, it has been possible to demonstrate
that subsurface environments are instead populated by a vast diversity of microbes that use unconven-
tional energy sources to perform their metabolic reactions1,2. It has been proposed that some of these
microorganisms may also be involved in the precipitation of speleothems (i.e., mineral deposits that form
in caves)3. e investigation of such mineralization processes is of interest not only for identifying and
understanding new types of biomineralization pathways – that may have applications in industry – but
also because these biogenic speleothems can potentially be preserved in the geological record for billions
of years, becoming a useful biosignature for the search for early life on Earth and on other planets4–7.
It has oen been hypothesized that life arose in the dark, protected from the intense UV radiation that
characterized the early Earth as well as the surface of other planets8–10. From both a paleontological
and geobiological prospective, caves therefore represent a very interesting environment for the study of
biomineralization processes and primitive microbe-mineral interactions.
Asperge Cave, located in the region of the Montagne Noire-Hérault (France), developed in
Cambrian rock following the contact between schists and carbonates11. A limited portion of Asperge,
the “Blue Gallery”, contains a suite of ornate white and blue speleothems resulting from the complex
intertwining of numerous branches and needles of CaCO3 (Fig. 1, S1). Due to the presence of these
1University of Toronto, 35 St. George street, M5S 1A4 Toronto, (CA) . 2ETH Zurich, Institute of Integrative Biology,
8092 Zurich, (CH), now at: Syngenta Crop Protection, Münchwilen AG, Werk Stein, Schaauserstrasse, 4332
Stein AG, (CH). 3Université Montpellier 2, Master EcoSystèmeS, Place Eugène Bataillon, 34095 Montpellier
cedex5, (FR), now at Umeå Universitet, Climate Impacts Research Centre, 98 107 Abisko (SWE). 4Bologna
University, Department of Biological, Geological and Environmental Sciences, Italian Institute of Speleology,
Via Zamboni 67, 40126 Bologna (IT). 5ETH Zurich, ERDW, Sonneggstrasse 5, 8092 Zurich (CH). 6FFS –Fédération
Française de Spéléologie, 28 rue Delandine, 69002 LYON, (FR). Present Address: University of Texas, Jackson
School of Geosciences, Department of Geological Sciences, 1 University Station C1100, Austin, TX 78712 (US).
Correspondence and requests for materials should be addressed to N.T. (email: nicola.tisato@utexas.edu) or
T.R.R.B. (email: tomaso.bontognali@erdw.ethz.ch)
Received: 27 March 2015
Accepted: 24 September 2015
Published: 29 October 2015
OPEN
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“Blue-Gallery-Speleothems” (BGS), Asperge cave was proposed as a UNESCO natural world heritage
site12. However, since its discovery in 1992, the genesis of these spectacular formations has remained
unexplained1,2,13,14.
e present contribution sheds light on the genesis of the BGS showing how biological factors con-
tribute to create such spectacular morphologies.
Results
e blue coloration of part of the BGS reects the high Cu concentrations found in these speleothems15.
e Cu derives from a heavy-metal-bearing geological stratum on which the speleothems grow (Fig.
S2). e morphology of the BGS is very unusual and includes the following distinctive features: 1)
gentle curves and “bights”, 2) bridges, 3) splitting and coalescences, 4) welding points, 5) large tubular
cross-sections, and 6) preferential upward growth direction. e “bights” are composed of decimetric
branches of CaCO3 growing downwards and orthogonally to the ceiling, then switching direction and
growing upward (Fig.1b,d). e term “bridges” refers to speleothems that span a shorter distance and are
more gently curved (Fig.1b, S1). Branching structures are evident (Fig.1c) and, in some cases, curved
speleothems, originating several decimeters apart from each other, meet and merge in the center of the
gallery (Fig.1d,e). is “coalescence” seems dicult to explain as simply coincidence. Finally, the “weld-
ing points” are sites where curbed speleothems re-join the walls of the gallery, producing a mineral coat-
ing that radiates from the contact point (Fig.1f, S1b). Such coatings spread over the substrate without
following an evident gradient, which might be imposed, for example, by gravitational or capillary ow.
Shapes similar to those described above that deviate from a vertical growth axis have been previ-
ously observed at a smaller scale in other caves. ey are referred to as helictites and their growth has
been attributed to a combination of capillary pressure, surface energy, and gravity, requiring a central
conduit with sub-millimetric diameter, and an impermeable wall2,13–16 (see Supplementary Information).
However, most branches of the BGS have a permeable thin wall with a large inner conduit (Fig.2) that
results in a tubular morphology. Moreover, while helictite branches grow in random directions, the BGS
tend to develop preferentially upward14. Finally, the petrography and the mineralogy of conventional
helictites also diers from that of the BGS. While helictites are usually comprised of aragonite crystals
grown in optical continuity (CAC) (Fig. 3b,d), the BGS are made of non-continuous calcite crystals
(NCC) (Fig.3e,g). Conventional helictites and concretions do occur in close spatial association with the
BGS, oen forming hybrid speleothems comprised of NCC and CAC, respectively (Fig.3f, S1e). e
Figure 1. (a) A bouquet of Blue Gallery speleothems (BGS)—the unusual speleothems of Asperge Cave.
(b) Astonishing “bight” connecting two points ~50 cm apart from each other. e arrow indicates a “bridge.
(c) BGS splitting point (detail of panel b). (d) Two branches meet and merge in the center of the room
forming a bight (detail of panel a). (e) Detail of the panel d showing the “coalescence”, the arrows indicate
the growing direction of the two branches. (f) Typical “welding point”, the arrow indicates the growth
direction of the coating (i.e. against gravity). Photos by: Michel Renda, Nicola Tisato and Tomaso R. R.
Bontognali.
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Figure 2. Biolm-calcite crystals . (a) BGS sample having tubular morphology. (b) BGS sample presenting
biolms (i.e. white dots) and inner crumbly mass. (c) False color CT-scan image of a section of a BGS
sample. Calcite crystals and biolm are represented by pixels with gray levels ~52000 (i.e. yellow) and
~40000 (i.e. red), respectively. (d) e 3D model of the biolm was obtained from the CT-scan image stack
selecting voxels with gray levels between 38500 and 46500. (e) e 3D model of the calcite crystals was
created from the CT-scan image stack segmenting voxels with gray levels between 48800 and 56800. (f) 3D
model obtained as sum of 3D models in panel d and e. Calcite crystals are represented by the yellow solid,
which is covered by the biolm (i.e. red solid). (g,h) SEM images also suggest that the biolm (detail 1
panel h) covers the calcite crystals (detail 2 panel h) (Fig.3). (i) Photomicrograph of a transverse section of
a BGS sample. (j) Photomicrograph under cross-polarized light of the external wall of BGS. Calcite crystals
are surrounded by a microcrystalline mass (highlighted by the red line), which is comprised of biolm and
microcrystalline calcite (Fig.3). Such a biolm-calcite mixture is also suggested by the EDX analysis (Fig.3e
bottom panel). Photos by: Nicola Tisato, Francesco Sauro and Tomaso R. R. Bontognali.
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coexistence of these dierent types of speleothems under the same physico-chemical conditions under-
lines the distinctive mode of mineralization by which the BGS formed.
Abiotic models that explain “non-random coalescences, “welding points” and “tubular cross sec-
tions” in helictites do not currently exist. Moreover “U-loops”, which are structures similar to “bights”,
and crumbly masses in “pool-ngers” have been previously inferred to be the result of biological factors
Figure 3. (a) Detail of a BGS bouquet: Hybrid speleothems (detail 2) formed between acicular speleothems
(detail 1) and tubular speleothems (detail 3). (b) Photomicrograph under cross polarized light of the
longitudinal section of an acicular speleothem, which is made of crystals growing in optical continuity
(CAC). (c) Mineralogy of acicular speleothems was investigated with X-ray diraction showing that these
speleothems are made of aragonite. e black spectrogram is the analysis, which is compared to the
aragonite XRD peaks. (d) SEM image of an acicular speleothem whose composition is indicated by the EDX
analysis reported in panel (e) upper curve. (f) Photomicrograph under cross polarized light of the transverse
section of a hybrid speleothem. e center and the rim have morphologies similar to those of acicular and
tubular speleothems, respectively. (g) Photomicrograph under cross polarized light of the transverse section
of a tubular speleothem, which is made of crystals growing in non-optical continuity. (h) Mineralogy of
tubular speleothems was investigated by means of X-ray diraction showing that these speleothems are
made of calcite. e black spectrogram is the analysis, which is compared to the calcite XRD peaks. i) SEM
image of the outer wall of a tubular speleothem which is covered by a biolm (Fig.2). i) EDX analysis
performed on the outer wall of a tubular speleothem indicating that the biolm is mainly made of C.
However, as the EDX analysis penetrates few micrometers the surface the Ca and O peaks suggest that the
rst layer covering the wall is made of a carbonate mixed with organic matter (Fig.2). Photos by: Nicola
Tisato, Francesco Sauro and Tomaso R. R. Bontognali.
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and calcied bacterial aggregates1,2,16,17. Our investigations show that a biolm is, indeed, associated
with the BGS. Under an optical microscope such biolms appear as a white or transparent aggregate of
so organic material. Transmitted light microscopy, scanning electron microscopy (SEM) imaging and
X-Ray computed microtomography (μ CT) show how the biolm is closely associated with the crystals
and almost completely cover the inner and outer wall of the BGS (Figs2 and 3). Its presence confers to
the BGS a powdery habit that diers from that of the more translucent conventional helictites present
in the same cave chamber (Fig. S1). e organic composition of the biolm was conrmed by SEM-
Energy-dispersive X-ray spectroscopy (EDX) analyses (e.g. Fig.3e).
A biolm with the same color and habit to that associated to the BGS was found also on the mud
covering the walls of the “Blue Gallery”. ere, thanks to the color contrast, the biolm is visible to the
naked eye as a multitude of white dots. Locally the dots develop and merge forming larger patches that
show a progressive hardening (i.e., mineralization). ese encrusted areas are made of CaCO3 and appear
to constitute the initial substrate upon which the BGS develop (Fig.4). e white dots are more abundant
around BGS bouquets and exclusively occur in the Blue Gallery; only conventional speleothems adorn
the other rooms of Asperge cave. ese observations suggest that the biolm may be involved in the
formation of the BGS.
A diversity survey based on the 16 SrRNA gene revealed that the microbial community of the biolm
is dominated by Proteobacteria, Acidobacteria, and Actinobacteria (Fig. S3). Tests using dierent com-
binations of primers failed to reveal a signicant fungal population.
Mixed cultures, inoculated in situ in Ca-amended agar plates directly from the BGS, induced the for-
mation of rhombohedric dark- or light-colored crystals, recognized as calcite under optical microscopy.
One of these cultures was also observed and analyzed under the SEM and EDX showing the precipitation
of complex Ca-carbonate hemispheres (Fig. S4). Plates inoculated with pure cultures showed dierent
crystals with various habits. e latter induced biomineralization of at coating-like crystals, forming
on the surface of the colonies morphologically distinct from the other previously observed using mixed
cultures. ese cultivation-based methods show that various bacteria that are part of the biolm (mainly
Actinobacteria) can precipitate CaCO3 in vitro (Figs S3, S4). Actinobacteria, which are oen present in
Figure 4. Typical sequence of features associated to the BGS. Some decimeters away from the speleothems
(detail 3) aggregates of biolm are visible as white dots (detail 1, zoom in b and c). e biolm is locally
characterized by an unusual morphology (detail 4) that may be due to the presence of microbes capable of
gliding. Between the BGS (detail 3) and the mud covered by white dots (detail 1), a calcite coating covers
the mud (detail 2). Aggregates of biolm are also present below such a coating. Photos by: Nicola Tisato.
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cave environments, have been previously documented in promoting the formation of Ca-carbonate3,17–19.
In general, the metabolic reactions of these microbes cause a local increase in pH and alkalinity, inducing
supersaturation with respect to Ca-carbonate. Moreover, the extracellular polymeric substances (EPS)
that microbes produce, and that form the biolm, act as preferential nucleation sites, inuencing the
morphology and the mineralogy of the precipitate6,7,20,21. Our results indicate that similar biomineraliza-
tion processes may occur in the Blue Gallery. However, none of the previously described cases produce
morphologically complex speleothems such as those present in Asperge, suggesting that a yet uncharac-
terized microorganism, or biomineralization process, may be responsible for the observed phenomenon.
Discussion
Identifying the specic organism(s) involved may prove challenging as the majority of strains found in
the BGS cannot be currently cultivated in vitro, and we cannot exclude that the mineralization process
may be extremely slow and orchestrated by more than one species. Nevertheless, among the identied
strains one is particularly intriguing as it shows 11% dissimilarity from other known 16 SrRNA sequences
(i.e., potentially representing a new order of microorganism, GenBank accession number: KJ750906),
and it is most closely related to Myxococcales. Some Myxobacteria species are known to eciently induce
the precipitation of calcium carbonate, as well as to tolerate high concentrations of Cu22–24. Moreover,
they possess the unusual ability of gliding, a process whereby the bacterial community can move over
solid substrates, forming morphologically complex swarms of cells and EPS25,26.
e presence of a biolm, including microbes whose gliding behavior may be directed by chemot-
axis, would provide a simple explanation for the distinctive morphology of the speleothems, including
irregular curves, “bridges”, “bights” and upward-growing shapes. e organic material constituting the
biolm provides a nucleation site for calcium carbonate, which precipitates from the same water that
the microorganisms need to survive. e factors that control the direction of biolm growth - and
subsequently the morphology of the speleothem - are dicult to be precisely identied. e biolm
may develop in random directions or in a direction that allows the microbial community to avoid being
completely entombed by the carbonate mineral (e.g., preferentially upward against gravity) or, even,
it may move toward the most ecologically favorable regions of the cave room (e.g., areas with higher
humidity, or where localized air currents transport aerosols containing nutrient for the microorganisms).
Also the “welding points” might reect biological activity: once the microbes, located on the tip of the
speleothems, reach the nutrient-rich mud, the biolm that promotes precipitation of calcite expands
following the nutrient gradient. Finally, the presence of a biolm might also provide an explanation for
the “coalescences”. Attraction between microorganisms—for instance through quorum sensing, which is
typical of Myxococcales27—may provide a more plausible explanation than coincidental coalescence of
two abiotically-formed speleothems within a large three-dimensional space.
A passive, microbially-inuenced mineralization process, whereby biolms serve as a nucleation
sites for Ca-carbonate, and where biological growth—in random directions or controlled by chemot-
axis—inuences the orientation and trajectory of speleothem growth, represents a conservative expla-
nation for the observed morphological features. However, we cannot exclude a more sophisticated role
for the biolm whereby microbes actively control the formation of the speleothems in order to obtain
yet-to-be-identied ecological advantages. While details of the underlying processes are lacking, we con-
clude that the biolm must play a key role in determining the morphology of the BGS. e occurrence
of “normal” abiotic aragonitic speleothems within the same room of the Asperge cave system provides a
natural “negative control”, supporting our conclusion of the biogenicity of BGS. Why the biogenic spe-
leothem are made of calcite and not of aragonite, as well as why they occur exclusively in specic and
limited areas of the cave are two aspects that deserve to be attentively evaluated in future studies. e
fact that we observed hybrid acicular–tubular morphologies (Fig.3, S1), with biolm only associated
to tubular morphologies, suggests that microbes might obtain a yet-to-be-dened benet in converting
aragonite- to calcite-speleothems. Similarly, the occurrence of the BGS exclusively in the areas of the cave
that are enriched in heavy metals (e.g., Cu—commonly toxic for most living organisms) suggests that the
unusual speleothems may be the result a biological detoxication process.
e results of this study may require a revised interpretation of the formation of some other types
of speleothem. e remarkable similarity between the BGS and speleothems in a cave in Colorado,
USA2 demonstrates this mineralization process is not unique. It is only in rare cases that prokaryotic
microorganisms have been shown to mediate the formation of morphologically complex macroscopic
mineral structures4. In this regard, a better understanding of the mechanisms involved may provide a
basis for dening a new type of fossil structure of biogenic origin that can be recognized in the geo-
logic record, while also yielding important insights into biomineralization processes relevant to the elds
of engineering, materials science and environmental remediation6,7. Finally, as the BGS develop in an
extreme environment, further investigations could be signicant for studies regarding extremophiles and
extraterrestrial life5,8,9.
Methods
Asperge cave was visited 4 times between July 2012 and August 2013 to nalize morphological descrip-
tions of the BGS, eld observations and sampling activity. e BGS mineral structure has been extensively
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photographed and described in the eld. Sampling of speleothems was intentionally limited to preserve
the BGS bouquets.
Light microscopy. Polished thin sections (30 μ m thickness), suitable for transmitted light microscopy
and SEM observation, were prepared from the original samples and analyzed with a Zeiss Axioplan Optic
Microscope at the Department of Biological, Geological and Environmental Science of the University of
Bologna. e microscope was equipped with a Deltapix DP200 camera for acquisition of high-resolution
images.
Sample mineralogy (XRD). For X-Ray diraction (XRD) analyses, few milligrams of sample were
ground to an ultrane powder and suspended in ethanol. en a drop of suspension was deposited on
a glass slide. Aer ethanol evaporation XRD analysis was performed using a Bruker AXS D8 Advance
instrument equipped with a Lynx eye super speed detector by means of Cu K radiation, an antiscattering
slit of 20 mm, and rotating sample. e sample patterns were recorded from 5.3° to 84.9° 2θ in steps of
0.033°, 2 s counting time per step.
Scanning Electron Microscopy (SEM). Scanning electron microscopy analyses were performed
with a Zeiss Supra 50 VP at the University of Zurich, equipped with an energy-Dispersive X-ray-Detector
for element analysis (EDX). A 7 nm platinum coating was applied to the samples. e image and EDX
analysis were obtained with a secondary electron detector, an accelerating voltage of 15 kV, and a working
distance of about 10 mm. In order to preserve the organic structures constituting the biolm - which
are usually destroyed during the drying procedure necessary to apply the metal coating—most of the
analyzed samples were shock-frozen in liquid nitrogen and subsequently freeze-dried.
X-Ray microtomography (CT-scan). X-Ray microtomography analysis was performed using a
micro-CT scanner (Phoenix X-ray V-tome-x, General Electric Sensing and Inspection Technologies)
installed at the Department of Civil Engineering at the University of Toronto. e sample, a section of
tubular BGS, was mounted on a 5-axis rotation stage and irradiated with X-rays on its external curved
surface by rotating it 360° in 1080 equally spaced increments. At each angle, 3 projections were acquired
and averaged to obtain a 2D 16-bit grey scale projection. e chosen magnication of the specimens
within the eld of view corresponded to a voxel resolution of ~10 μ m. A 0.5 mm thick copper lter was
used to reduce beam hardening artifacts in the reconstructed 3D volume.
Image reconstruction was performed using the Phoenix X-ray datos—x-reconstruction soware (v.
1.5.0.22), including: a beam hardening correction of 3/10, automatic ring artefact reduction, and a ‘scan
optimization’ which compensates for small translations of the specimen during scanning and correctly
locates the centre of reconstruction.
Reconstruction produced an image stack formed by 1024 16-bit grey scale images with dimensions
of 1018 by 1018 pixels. Segmentation of the image stack and production of the three-dimensional (3D)
models were performed using the free version of the commercial soware MeVisLab28 and includes the
following passages:
(1) e image stack was rescaled to a voxel size of 15 μ m by means of a “lancsoz 3” lter;
(2) We produced a single DICOM le from the rescaled image stack;
(3) Using a “2D marked view editor”, we placed, on the DICOM le, 77 and 81 seeds (original seeds) on
voxels which represented biolm and calcite, respectively;
(4) Two “region growing” procedures searched for voxels adjacent to the seeds and having grey level
equal to the original seed grey level ± 6%. e new identied voxels became new seeds and the
procedure was repeated until no new seeds were found. is generated two distinct volumes, repre-
senting the biolm and the calcite, comprised of voxels with grey value ranging 38500—46500 and
48800—56800, respectively;
(5) e volumes were rendered and captured as images with a “3D view” toolbox.
Permeability of tubular BGS. e permeability of tubular BGS was qualitatively estimated using
a falling head permeameter29. e samples consisted in two portions of speleothem ~2 cm in length.
Both the samples were like small tubes with outer and inner diameter of ~1 and ~0.4 cm, respectively.
Bi-component epoxy resin was applied at one of the two extremities of each tubular BGS occluding, for
~0.4 cm in length, the internal conduit. e other extremity was glued onto a 0.5 cm inner diameter sil-
icon pipe. As a consequence, the portion of tubular BGS not sealed by epoxy resin was ~1 cm in length.
e silicon pipe was connected to the tip of a glass burette (50 ml, 0.1 ml precision). e burette was
lled with ~30 ml of distilled water exerting a uid pressure, on the inner wall of the tubular helictites, of
~40 cm of water. Aer < 5 min water started percolating from the external wall of the helictites suggesting
that the tubular helictites are rather permeable structures. However, due to the highly irregular thickness
of the wall and the imprecision in dening the area of the non-sealed portion of the tubular BGS it was
impossible to assess a quantitative estimation of the permeability.
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Chemical analysis with X-Ray Fluorescence Spectrometer (WD-XRF). For wavelength disper-
sive X-ray uorescence (WD-XRF) samples were ground to ultrane powder and kept 24 hours at 110 °C.
Ten grams of sample were mixed with 5 ml of solution of Elvacite polymer resin and acetone. e mixture
was stirred to allow the acetone to evaporate and the resultant powder was placed in a penny-shaped
mold and compressed with a vertical pressure of 40 MPa for one minute.
A WD-XRF spectrometer Axios–PANalytical (Institute for Mineralogy and Petrology - IMP of ETH–
Zurich) equipped with ve diraction crystals was used for this study. e SuperQ soware package
provided by PANalytical was employed for calibration and data reduction. Calibration is based on 30
certied international standards. e precision of analysed elemental abundances are better than ± 0.2%
for SiO2, ± 0.1% for the other major elements. For trace elements, relative errors are better than 10% for
concentrations of 10–100 ppm, better than 5% for higher concentrations and can reach as much as 50%
at levels below 10 ppm. erefore the detection limit is considered to be approximately 5–10 ppm.
e measured sample returned total element concentrations of less than 100%. is discrepancy is
related to the possible presence of water and organic matter in the sample. In addition, elements lighter
than Na cannot be recognized with WD-XRF30.
In vitro Ca-carbonate precipitation. Microorganisms associated with the BGS were sampled using
a cultivation-based method. Samples of biolm associated to BGS were scratched out directly into yeast
peptone agar plates (YPA; yeast extract 3 g/L, peptone 5 g/L, agar 18 g/L) using a sterile lab spatula.
Bacterial strains showing distinct phenotypic characteristics were transferred several times on yeast tryp-
tone agar plates (YTA; yeast extract 3 g/L, tryptone 5 g/L, agar 18 g/L) to get single pure bacterial colonies.
Mixed and pure bacterial strains isolated from the BGS were tested for their ability to induce
Ca-carbonate precipitation in vitro on YTA, amended with 3.16 g/L Ca (CH3COO)2•H2O. Negative con-
trols consisting of Ca-amended YTA medium inoculated with sterile H2O allowed us to rule out con-
tamination during inoculation and growth. Aer inoculation, plates were incubated in the dark at 19 °C
for 2–3 weeks.
16SrRNA sequencing analysis and taxonomy of the microbiome. We characterized the micro-
bial diversity of the white biolm associated to the BGS via targeted 16S rRNA gene sequencing. Total
DNA was extracted using the FastDNA SPIN Kit for Soil according to the manufacturer’s instructions
and quantied with a NanoDrop ND-1000 spectrophotomer. Universal primers 27F (5 -AGA GTT TGA
TCM TGG CTC AG-3 ) and 1492R (5 -TAC GGY TAC CTT GTT ACG ACT T-3 ) were used to amplify
by PCR 16S rRNA. Combination of ITS5-4 and ITS1-4 primers was used to characterize the Internal
Transcribed Spacer (ITS) of eukaryotic species (ITS5, 5 -TAC GGY TAC CTT GTT ACG ACT T-3 ;
ITS4, 5 -TCC TCC GCT TAT TGA TAT GC-3 ; ITS1, 5 -CC GTA GGT GAA CCT TGC GG-3 ). PCR
conditions were initial denaturation at 98 °C for 30 s followed by 35 cycles including denaturation at
98 °C for 8 s, hybridization at 65 °C for 30 s and elongation at 72 °C for 25 s. A nal elongation lasted
6 min at 72 °C. Phusion high delity DNA polymerase was used. 16S rRNA PCR and a negative control
PCR sample were cloned using Clone Jet PCR kit (ermo Scientic). DH5α E. coli strains were trans-
formed by thermal shock, at 42 °C for 45 s. Transformed cells were then incubated on ice for 2 min and
950 μ L of SOC medium was added. Samples were incubated at 37 °C on a horizontal shaker at 225 rpm
for 1 h. Samples were inoculated on yeast-tryptone agar plates amended with ampicillin (100 μ g/mL)
and incubated overnight at 37 °C. Single colonies were transferred to 50 μ L ddH2O and heat shocked
for 10 min at 95 °C to burst bacterial cells and release DNA. A total of 4 μ L were used to amplify the
cloned insert using DreamTaq polymerase (ermo Scientic) and primers pJET 1.2_F (5 -CGA CTC
ACT ATA GGG AGA GCG GC-3 ) and pJET 1.2_R (5 -AAG AAC ATC GAT TTT CCA TGG CAG-
3 ). Amplicons were puried through NucleoFast® PCR plates (Macherey-Nagel) and sequenced using
either 27F or 1492R primers. Sequencing reactions were performed using BigDye Terminator v3.0 ready
reaction cycle sequencing kit (Applied Biosystems). e sequencing PCRs were in a total volume of
10 μ L using 20 to 40 ng DNA, 10 pmol of primer and 2 μ L BigDye reaction mix previously 1:4 diluted.
e cycling prole was 10 s denaturation at 95 °C, 5 s annealing at 50 °C and 4 min extension at 60 °C for
100 cycles. Sephadex G-50 DNA Grade F (Amersham Biosciences) was used to clean sequencing reaction
prior loading the samples into a 3100 ABI automated sequencer. Sequences were quality checked and
visualized using Sequencher version 4.2 soware package (Gene Codes Corporation). Sequences were
blasted to the NCBI database using BLASTN algorithm31 for similarities to previously deposited species.
No amplications were obtained using dierent ITS primer combinations (ITS5-4 and ITS1-4), sug-
gesting fungal ecology to be absent or a limited fraction of the microbiome.
16SrRNA from pure cultures cultivated in vitro was amplied using a high-delity polymerase KAPA
3G Plant PCR Kit (Kapabiosystems) and primers 27F and 1492R. PCR cleaning and sequencing was
described above. Pure culture strains isolated from the BGS belonged to Sphingopyxis and Rhodococcus
species (Fig. S3).
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Acknowledgements
We thank Giovanni Grasselli and Bryan Tatone for the CT-scan analysis, Marcello Zala, Patrick Brunner,
Elena Maria Comelli and Giulio Di Toro for critical discussions, Scurion GmbH for technical support.
e present study has been partially supported by: CFI-LOF grant 18285 and Swiss National Scientic
Foundation.
Author Contributions
N.T. has contributed with: concepts and ideas, eld work (including photographic activity), W.D.-,
X.R.F.-, X.R.D.-, S.E.M.-, E.D.X.- analyses, permeability measurements, C.T. scanning, preparing some
microbiological experiments, gure preparation and writing. S.T. has contributed with: concepts and
ideas, eld work (including photographic activity), microbiological experiments and writing. S.M. has
contributed with: concepts and ideas, eld work (including photographic activity), microbiological
experiments, gure preparation and writing. F.S. has contributed with: concepts and ideas, eld work,
thin section preparation and description, text revision, concepts and morphological interpretation.
M.L.T. has contributed with: concepts and ideas, eld work, W.D.-X.R.F., gure preparation, text revision.
I.D.A. has contributed with: concepts and ideas, eld work, thin section description and morphological
interpretation. J.D.W. has contributed with: eld work, concepts and ideas, mostly compiling the abiotic
theories on helictite formation. M.R. has contributed with: eld work including organization and
photographic activity, concepts and morphology interpretation. D.C. has contributed with: eld work
including organization and photographic activity, concepts and morphologies interpretation. T.I.E. has
contributed with concepts, ideas, nancing and text revision. T.R.R.B. has contributed with: concepts and
ideas, eld work (including photographic activity), S.E.M.-, E.D.X.-analyses, microbiological experiments,
and writing.
www.nature.com/scientificreports/
10
SCIENTIFIC RepoRts | 5:15525 | DOI: 10.1038/srep15525
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Tisato, N. et al. Microbial mediation of complex subterranean mineral
structures. Sci. Rep. 5, 15525; doi: 10.1038/srep15525 (2015).
is work is licensed under a Creative Commons Attribution 4.0 International License. e
images or other third party material in this article are included in the article’s Creative Com-
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the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
... The released metals could be trapped locally, directly above the sulfide protolith, or they may be transported and precipitated far away from the sulfide protore [3,[16][17][18][19]. More importantly, recent investigations demonstrated the ability of bacteria and archaea as well to influence metal solubilization, mobilization, and subsequent formation of carbonate-hosted Pb-Zn ± Cu ± Au deposits and their supergene counterparts (e.g., [20][21][22][23][24][25]). ...
... 21 The core of the carbonate platform consists of a succession of greenschist-facies Cambrian- 22 Ordovician to Silurian metasedimentary and volcaniclastic rocks made up dominantly of 23 quartzite and schist. Unconformably overlying the metasedimentary Paleozoic package is 24 a sequence of about 400-500 m thickness of Triassic sedimentary rocks consisting of red-25 bed siltstone and gypsiferous to salt-bearing argillite locally interbedded with CAMP 26 tholeiitic basalts [52]. The reef deposits consist of unmetamorphosed, flat-lying, massive, 27 thickly bedded coral-, algal-, and bivalve-bearing Pliensbachian platform carbonates. ...
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The work is based on the assumption that an electrochemical cell, under the influence of electromagnetic fields emitted by terrestrial crust in conditions of tectonic stress, will function as an electric pile using the electrode potential differences. The signal obtained can be correlated on long term with energy sources that derive from the solar activity, from the relative position of the Sun, Earth and Moon, or from the nearby and very distance tectonic processes, the identification of electric/electromagnetic precursors of significant earthquakes, becoming possible.
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In this chapter, the authors deal with the physical, organic, and chemical deposits found in the dark or semi‐dark areas of caves, excluding those found in cave entrances or rock shelters. Clastic sediments in caves, excluding those found at entrances, have been the subject of numerous studies, mainly during the last 60 years. Phosphorite is a chemical deposit that can be deposited in caves. Speleothems are secondary mineral deposits that form in caves by flowing, dripping, ponded, or seeping water and take on a typical shape. They are mostly composed of minerals such as calcite, aragonite, or gypsum, but other minerals can also form entirely or partially speleothems. Speleothem texture and fabrics are increasingly used to support the interpretation of the geochemical signals (stable isotopes and trace elements) in the paleo‐environmental and paleoclimatic reconstructions based on speleothem archives.
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We analyzed the microbial community of multicolored speleosol deposits found in Grand Canyon Caverns, a dry sulfuric karst cave in northwest Arizona, USA. Underground cave and karst systems harbor a great range of microbial diversity; however, the inhabitants of dry sulfuric karst caves, including extremophiles, remain poorly understood. Understanding the microbial communities inhabiting cave and karst systems is essential to provide information on the multidirectional feedback between biology and geology, to elucidate the role of microbial biogeochemical processes on cave formation, and potentially aid in the development of biotechnology and pharmaceuticals. Based on the V4 region of the 16S rRNA gene, the microbial community was determined to consist of 2207 operational taxonomic units (OTUs) using species-level annotations, representing 55 phyla. The five most abundant Bacteria were Actinobacteria 51.3 ± 35.4 %, Proteobacteria 12.6 ± 9.5 %, Firmicutes 9.8 ± 7.3 %, Bacteroidetes 8.3 ± 5.9 %, and Cyanobacteria 7.1 ± 7.3 %. The relative abundance of Archaea represented 1.1 ± 0.9 % of all samples and 0.2 ± 0.04 % of samples were unassigned. Elemental analysis found that the composition of the rock varied by sample and that calcium (6200 ± 3494 ppm), iron (1141 ± 1066 ppm), magnesium (25 ± 17 ppm), and phosphorous (37 ± 33 ppm) were the most prevalent elements detected across all samples. Furthermore, carbon, hydrogen, and nitrogen were found to compose 4.7 ± 4.9 %, 0.3 ± 0.4 %, and 0.1 ± 0.1 % of samples, respectively. Finally, Raman spectra compared to the RRUFF Project database using CrystalSleuth found that the mineral composition of the speleosol consisted of calcite, hematite, paraspurrite, quartz, and trattnerite. These data suggest that dry sulfuric karst caves can harbor robust microbial communities under oligotrophic, endolithic, and troglophilic conditions.
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The Karst Waters Institute Breakthroughs in Karst Geomicrobiology and Redox Geochemistry conference in 1994 was a watershed event in the history of cave geomicrobiology studies within the US. Since that time, studies of cave geomicrobiology have accelerated in number, complexity of techniques used, and depth of the results obtained. The field has moved from being sparse and largely descriptive in nature, to rich in experimental studies yielding fresh insights into the nature of microbe-mineral interactions in caves. To provide insight into the changing nature of cave geomicrobiology we have divided our review into research occurring before and after the Breakthroughs conference, and concentrated on secondary cave deposits: sulfur (sulfidic systems), iron and manganese (ferromanganese, a.k.a. corrosion residue deposits), nitrate (a.k.a. saltpeter), and carbonate compounds (speleothems and moonmilk deposits). The debate concerning the origin of saltpeter remains unresolved; progress has been made on identifying the roles of bacteria in sulfur cave ecosystems, including cavern enlargement through biogenic sulfuric acid; new evidence provides a model for the action of bacteria in forming some moonmilk deposits; combined geochemical and molecular phylogenetic studies suggest that some ferromanganese deposits are biogenic, the result of redox reactions; and evidence is accumulating that points to an active role for microorganisms in carbonate precipitation in speleothems.
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French Karst covers the third of the territory. Because calcareous deposits date back to the Cambrian and are now located in tabular region as well as in tectonic areas, French karst is famous for its diversity. Large karst landscapes are well known, such as Les Grands Causses, the Gorges du Tarn and the Gorges du Verdon. Resurgences such as the Vaucluse Fountain and the spring of the Lez are very famous too. Karstic units can show very large dimensions: 400 km for the six hydrological networks of the Pierre Saint-Martin, 104 km for the Arbas Mountain. Maximum depths can reach high records, at Pierre Saint Martin, Berger Cave, Jean Bernard Cave and recently Mirolda Cave was acknowledged to reach 1733-meter depth. Studies proceeded during the last century have revealed an important cave fauna. Considering the heritage value of French karst, the key point is not only the presence of more than 160 decorated caves from Palaeolithic period but also the number of caves showing concretions. Moreover, a rare phenomenon has to be underlined: in Ariege, the Intermittent Fontestorbes Spring is the most regularly and completely studied resurgence in the world, This amount of exceptional features for these caves commands to organise studies and programmes to establish conditions of a good conservation of this heritage for the next generations.
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Although speleothems are usually considered inorganic precipitates, recent work has demonstrated hitherto unsuspected biogenic influence in some twilight areas. We have expanded this notion to the dark zone, examining pool fingers from Hidden Cave, New Mexico, to test for possible bacterial involvement. The pool fingers in Hidden Cave are pendant speleothems that formed subaqueously in paleo-pools. They are 1 to 4 cm in diameter and 5 to 50 cm long. A knobby, irregular external shape is underlaid by a layered interior on two scales, a 0.5 to 1.0 cm alternation between dense and porous layers and a mm-scale alternation between dark micritic calcite and clear dogtooth spar. The micrite is similar to microbialites identified in modern and ancient carbonates. Fossil bacteria were found in all layers. These include (1) calcified filaments 1m in diameter and 5–50m long and (2) micro-rods 0.1 m by 1–2 m. Most filaments are curved rods with a smooth surface but rare examples display a diamond crosshatch surface. The micro-rods occur as isolated crystals to dense meshes. We interpret the micro-rods as calcified bacilliform bacteria and the filaments as calcified filamentous bacteria. Carbon isotopic data are slightly more negative (by - 0.5 to - 1.0% in micritic layers than in dogtooth spar layers, suggesting a greater microbial influence in the micritic layers. Based on these similarities to known microbialites (e.g., petrographic fabrics, the presence of fossil bacteria, and the suggestive carbon isotopic data), we conclude that microbial activity was an intimate part of pool finger formation in Hidden Cave. The significance of such involvement goes beyond speleological contexts to wider questions of identification of biosignatures in rocks on earth and beyond.
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A falling head permeameter is described in which pairs of infrared emitters and detectors on a sight tube are used to measure the flow rate associated with the passage of water through a granular solid under the action of a diminishing pressure head. An equation relating pressure head to elapsed time is derived from which permeability may be calculated. In order to verify the accuracy and sensitivity of the instrument, permeability measurements carried out on a graded quartz sand are compared to those obtained by the more conventional constant head measurement. Excellent agreement is obtained between the permeability values obtained using both measurement methods. Experimental results are also reported for the measurement of the permeability of a range of sieved sand fractions. The falling head permeameter described here is particularly suitable for the measurement of the hydraulic conductivity of granular solids such as sands and soils through which a high flow rate may be expected.
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The Viking mission has profoundly influenced scientific thinking about the question of life on Mars. Based on the restfits obtained from a number of Viking investigations, it is clear that the surface of the planet is too hostile to support a carbon-based biota. While the existence of biological "oases" elsewhere on the planet have been proposed, none have yet been found. Other information based on Viking data has focused on the possibility that life may have been present on Mars in the distant past, and Viking images have revealed sites on Mars where further exploratory studies for extinct biology are warranted. From the point of view of the search for life on Mars, there is little doubt that the Viking mission, while it did not completely settle the question of life on Mars, put to rest a number of predictions and speculations about this issue. At the same time, new insights derived as a consequence of this mission have resulted in radical revisions in scientific thinking about the possibility of life on Mars. For one thing, up until the Viking mission (and, indeed, also assumed in that undertaking) there was the implicit belief that, if there was life on Mars, it would be confined to the surface and near-surface regions of the planet. By analogy with biology on the Earth, it was reasonable to assume that photosynthetic orgardsms would play a prominent role in any Mars ecological system, and thus require access to solar energy. The data obtained by Viking are best interpreted as indicative of the fact that the surface on Mars is hostile to life and devoid of life.
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Nucleation of calcium carbonate on microbial cell material may have been the dominant mode of microbial carbonate formation during most of Earth's history. Current knowledge predicts that nucleation takes place on the cell surface or on extracellular polymeric substances. However, the initial nucleation steps have not been described in detail and the process remains elusive. Here we describe the bacterial nucleation of calcium carbonate at the nanometer scale. In our precipitation experiment with sulfate reducing bacteria (SRB), the bulk of calcium carbonate precipitates on hundreds of individual globules 60 200 nm in diameter. Globules originate from the SRB cell surface but calcify significantly only when released to the culture medium. Similar globules have been observed, albeit at a much larger scale, in other bacterial precipitation experiments and in many natural microbial carbonates, suggesting that the process we describe could be an important step in microbial calcification.
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Impacts of asteroids and comets posed a major hazard to the continuous existence of early life on Mars, as on Earth. The chief danger was presented by globally distributed ejecta, including transient thick rock vapor atmospheres. On Earth, much of the thermal radiation is absorbed by boiling the oceans. Any surviving life is either in deep water or well below the surface. Global thermal excursions are buffered by the heat capacity of the oceans. But when impacts are large enough to vaporize the oceans (>1028J), thermal buffering serves only to prolong the disaster for thousands of years, while the oceans rain out. Without oceans, thermal buffering does not occur on Mars. Relatively small impacts (1026J) frequently heat the surface everywhere to the melting point. However, owing to the low Martian escape velocity, the most energetic ejecta (including the rock vapor) more easily escape to space, while massive quantities of less energetic ejecta are globally distributed. Survival in deep subsurface environments is more likely on Mars because (1) Mars' lower background heat flow and lower gravity allow deeper colonies, and (2) the thermal heat pulse from a major impact is briefer. Only thermophile organisms could have survived impacts of asteroids large enough to leave heat or boil the entire terrestrial ocean. Studies of terrestrial microorganisms indicate that the last common ancestor may have been thermophile and the survivor of such a catastrophe. This organism should be distinguished from the first common ancestor. An additional refugium is ejection of rock to space by impacts and their return to a habitable planet.
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Crystallization is an important process in a wide range of scientific disciplines including chemistry, physics, biology, geology, and materials science. Recent investigations of biomineralization indicate that specific molecular interactions at inorganic-organic interfaces can result in the controlled nucleation and growth of inorganic crystals. Synthetic systems have highlighted the importance of electrostatic binding or association, geometric matching (epitaxis), and stereochemical correspondence in these recognition processes. Similarly, organic molecules in solution can influence the morphology of inorganic crystals if there is molecular complementarity at the crystal-additive interface. A biomimetic approach based on these principles could lead to the development of new strategies in the controlled synthesis of inorganic nanophases, the crystal engineering of bulk solids, and the assembly of organized composite and ceramic materials.
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Speleothems are secondary mineral deposits whose growth in caves can be studied by mineralogic techniques. One of these techniques is the ontogeny of minerals, which is the study of individual crystals and their aggregates as physical bodies rather than as mineral species. Ontogeny of cave minerals as a scientific subject has been developed in Russia but is poorly understood in the West. This paper introduces the basic principles of this subject and explains a hierarchy scheme whereby mineral bodies can be studied as crystal individuals, aggregates of individuals, associations of aggregates (termed koras), and as sequences of koras (ensembles).
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Microbial mediation is the only demonstrated mechanism to precipitate dolomite under Earth surface conditions. A link between microbial activity and dolomite formation in the sabkha of Abu Dhabi has, until now, not been evaluated, even though this environment is cited frequently as the type analogue for many ancient evaporitic sequences. Such an evaluation is the purpose of this study, which is based on a geochemical and petrographic investigation of three sites located on the coastal sabkha of Abu Dhabi, along a transect from the intertidal to the supratidal zone. This investigation revealed a close association between microbial mats and dolomite, suggesting that microbes are involved in the mineralization process. Observations using scanning electron microscopy equipped with a cryotransfer system indicate that authigenic dolomite precipitates within the exopolymeric substances constituting the microbial mats. In current models, microbial dolomite precipitation is linked to an active microbial activity that sustains high pH and alkalinity and decreased sulphate concentrations in pore waters. Such models can be applied to the sabkha environment to explain dolomite formation within microbial mats present at the surface of the intertidal zone. By contrast, these models cannot be applied to the supratidal zone, where abundant dolomite is present within buried mats that no longer show signs of intensive microbial activity. As no abiotic mechanism is known to form dolomite at Earth surface conditions, two different hypotheses can reconcile this result. In a first scenario, all of the dolomite present in the supratidal zone formed in the past, when the mats were active at the surface. In a second scenario, dolomite formation continues within the buried and inactive mats. In order to explain dolomite formation in the absence of active microbial metabolisms, a revised microbial model is proposed in which the mineral-template properties of exopolymeric substances play a crucial role.