Cyanobacterial calcification in modern microbialites at the submicrometer scale

Abstract

The search for microfossils in the geological record has been a long-term challenge. Part of the prob-lem comes from the difficulty of identifying such micro-fossils unambiguously, since they can be morphologically confused with abiotic biomorphs. One route to improve our ability to correctly identify microfossils involves studying fossilization processes affecting bacteria in modern settings. We studied the initial stages of fossilization of cyanobac-terial cells in modern microbialites from Lake Alchichica (Mexico), a Mg-rich hyperalkaline crater lake (pH 8.9) host-ing currently growing stromatolites composed of aragonite [CaCO 3 ] and hydromagnesite [Mg 5 (CO 3)4(OH) 2 · 4(H 2 O)]. Most of the biomass associated with the microbialites is composed of cyanobacteria. Scanning electron microscopy analyses coupled with confocal laser scanning microscopy observations were conducted to co-localize cyanobacterial cells and associated minerals. These observations showed that cyanobacterial cells affiliated with the order Pleurocap-sales become specifically encrusted within aragonite with an apparent preservation of cell morphology. Encrustation gra-dients from non-encrusted to totally encrusted cells span-ning distances of a few hundred micrometers were observed. Cells exhibiting increased levels of encrustation along this gradient were studied down to the nm scale using a combi-nation of focused ion beam (FIB) milling, transmission elec-tron microscopy (TEM) and scanning transmission x-ray mi-croscopy (STXM) at the C, O and N K-edges. Two differ-ent types of aragonite crystals were observed: one type was composed of needle-shaped nano-crystals growing outward from the cell body with a crystallographic orientation per-pendicular to the cell wall, and another type was composed of larger crystals that progressively filled the cell interior. Ex-opolymeric substances (EPS), initially co-localized with the cells, decreased in concentration and dispersed away from the cells while crystal growth occurred. As encrustation de-veloped, EPS progressively disappeared, but remaining EPS showed the same spectroscopic signature. In the most ad-vanced stages of fossilization, only the textural organization of the two types of aragonite recorded the initial cell mor-phology and spatial distribution.

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Biogeosciences, 10, 5255–5266, 2013
www.biogeosciences.net/10/5255/2013/
doi:10.5194/bg-10-5255-2013
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Cyanobacterial calcification in modern microbialites at the
submicrometer scale
E. Couradeau
1,2,3,*
, K. Benzerara
1
, E. G
´
erard
3
, I. Est
`
eve
1
, D. Moreira
2
, R. Tavera
4
, and P. L
´
opez-Garc
´
ıa
2
1
Institut de Mineralogie et de Physique des Milieux Condenses UMR7590, Paris, France
2
Laboratoire Ecologie Systematique Evolution UMR8079, Orsay, France
3
Institut de Physique du Globe de Paris UMR7154, Paris, France
4
Departamento de Ecolog
´
ıa y Recursos Naturales, Universidad Nacional Aut
´
onoma de M
´
exico, DF Mexico, Mexico
*
now at: Arizona State University, Tempe, Arizona, USA
Correspondence to: E. Couradeau (estelle.couradeau@asu.edu)
Received: 18 December 2012 – Published in Biogeosciences Discuss.: 22 February 2013
Revised: 24 June 2013 – Accepted: 26 June 2013 – Published: 1 August 2013
Abstract. The search for microfossils in the geological
record has been a long-term challenge. Part of the prob-
lem comes from the difficulty of identifying such micro-
fossils unambiguously, since they can be morphologically
confused with abiotic biomorphs. One route to improve our
ability to correctly identify microfossils involves studying
fossilization processes affecting bacteria in modern settings.
We studied the initial stages of fossilization of cyanobac-
terial cells in modern microbialites from Lake Alchichica
(Mexico), a Mg-rich hyperalkaline crater lake (pH 8.9) host-
ing currently growing stromatolites composed of aragonite
[CaCO
3
] and hydromagnesite [Mg
5
(CO
3
)4(OH)
2
· 4(H
2
O)].
Most of the biomass associated with the microbialites is
composed of cyanobacteria. Scanning electron microscopy
analyses coupled with confocal laser scanning microscopy
observations were conducted to co-localize cyanobacterial
cells and associated minerals. These observations showed
that cyanobacterial cells affiliated with the order Pleurocap-
sales become specifically encrusted within aragonite with an
apparent preservation of cell morphology. Encrustation gra-
dients from non-encrusted to totally encrusted cells span-
ning distances of a few hundred micrometers were observed.
Cells exhibiting increased levels of encrustation along this
gradient were studied down to thenm scale using a combi-
nation of focused ion beam (FIB) milling, transmission elec-
tron microscopy (TEM) and scanning transmission x-ray mi-
croscopy (STXM) at the C, O and N K-edges. Two differ-
ent types of aragonite crystals were observed: one type was
composed of needle-shaped nano-crystals growing outward
from the cell body with a crystallographic orientation per-
pendicular to the cell wall, and another type was composed
of larger crystals that progressively filled the cell interior. Ex-
opolymeric substances (EPS), initially co-localized with the
cells, decreased in concentration and dispersed away from
the cells while crystal growth occurred. As encrustation de-
veloped, EPS progressively disappeared, but remaining EPS
showed the same spectroscopic signature. In the most ad-
vanced stages of fossilization, only the textural organization
of the two types of aragonite recorded the initial cell mor-
phology and spatial distribution.
1 Introduction
The search for microbial cell fossils (microfossils) in the ge-
ological record has been a long-term challenge causing mul-
tiple debates (Schopf and Packer, 1987; Brasier et al., 2002,
2005; Schopf et al., 2002, 2010). Part of the problem comes
from the difficulty to identify microfossils unambiguously,
since they are small and their morphology can be confused
with abiotic biomorphs (Garcia-Ruiz et al., 2002). Micro-
bialites are organosedimentary formations resulting from the
microbially mediated precipitation of carbonates (Burne and
Moore, 1987) and are favored targets in the search for mi-
crofossils (Riding, 2000). Stromatolites (i.e., laminated mi-
crobialites) are considered among the oldest records of life
on earth (e.g., Allwood et al., 2006; Altermann and Divi-
sion, 2006; Altermann, 2004; Grotzinger and Knoll, 1999).
Published by Copernicus Publications on behalf of the European Geosciences Union.

5256 E. Couradeau et al.: Cyanobacterial calcification in modern microbialites
It has been traditionally suggested that the formation of an-
cient stromatolites was mediated by cyanobacterial oxygenic
photosynthesis based on comparison with modern analogues
(Altermann et al., 2006; Riding, 2006a; Buick, 2008). A
few alternative scenarios involving purely abiotic processes
(Lowe, 1994; Grotzinger and Rothman, 1996; McLoughlin
et al., 2008), anoxygenic photosynthesis (Bosak et al., 2007)
or sulfur metabolism (Wacey et al., 2011; Bontognali et al.,
2012) have been tentatively proposed. Putative cyanobacte-
rial microfossils have been reported in the Warrawoona stro-
matolitic formation (∼ 3.45Ga) (Schopf and Packer, 1987)
but both their biogenicity and their cyanobacterial affiliation
have been questioned (Brasier et al., 2002). Microfossils of
undefined taxonomic affiliation have been proposed in stro-
matolites from the Tumbiana formation at 2.7Ga (e.g., Lepot
et al., 2008). Interestingly, while microfossils of calcified
cyanobacteria are commonly found in the geological record
since the base of the Cambrian–Precambrian stromatolites
most often lack microfossils, the earliest undisputed oc-
currence being Girvanella at 700Ma (Riding, 2006a). This
lack of cyanobacterial microfossils in Precambrian rocks de-
spite the fact that stromatolites were well developed and
cyanobacteria were already present since at least 2.3 Ga has
been called the “Precambrianenigma”(Riding and Voronova,
1982; Arp et al., 2001; Riding, 2012). Understanding the pro-
cesses leading to microfossil formation would help to under-
stand why cyanobacteria did not get encrusted and did not
fossilize in the Precambrian era.
It is assumed that the formation of cyanobacterial micro-
fossils results from the local impregnation of their cell wall
or sheath by carbonate precipitation, which is induced by
photosynthetic activity (Riding, 1982, 2006a). Cyanobacte-
ria import carbon in the form of HCO
−
3
in environments
where dissolved CO
2
is limiting. HCO
−
3
is then converted
to CO
2
and CO
2−
3
or, depending on the authors, CO
2
and
OH
−
(Jansson and Northen, 2010). CO
2
is then fixed by
photosynthesis into organic carbon while CO
2−
3
and/or OH
−
are exported to the extracellular medium. This raises the
saturation of the surrounding solution with various carbon-
ate minerals, depending on the cation content of the extra-
cellular solution (e.g., Mg
2+
and/or Ca
2+
). An additional
source of oversaturation is provided by an active export of
Ca
2+
from the cells coupled with import of H
+
(Belkin
et al., 1987). Finally, cyanobacteria produce extracellular
polymeric substances (EPS) that form a diffusion-limited
micro-environment where pH and other chemical gradients
(e.g., [Ca
2+
]) build up (Arp et al., 2001). As a result, CaCO
3
crystals may precipitate around the cells and entomb them
(e.g., Riding, 2006a; Pentecost and Franke, 2010). Alter-
natively, it has been suggested that the production of large
amounts of EPS by cyanobacteria may inhibit carbonate pre-
cipitation by sequestering cations. In that case, prior degra-
dation of EPS by heterotrophic bacteria may be necessary
for carbonate precipitation to occur (Dupraz and Visscher,
2005).
Two types of causes, highly debated, have thus been pro-
posed to explain the lack of Ca- and/or Mg-carbonate im-
pregnation of cyanobacterial cells during the Precambrian.
First, the chemical composition of the Precambrian ocean
may have not provided suitable conditions for calcium car-
bonate precipitation in cyanobacterial sheaths (Arp et al.,
2001). A high concentration of carbonate ions (due to a high
partial pressure of carbon dioxide at that time) and a low con-
centration of calcium ions would promote calcium carbonate
precipitation far from cells which consequently would not get
encrusted, and would be thus not preserved as microfossils.
Other authors argue that biological parameters such as sheath
EPS composition (Dupraz and Visscher, 2005; Obst et al.,
2009) and/or cell activity (Kupriyanova et al., 2011) being
additional critical parameters, ancestral cyanobacteria may
have not been able to provoke extracellular carbonate nu-
cleation and precipitation efficiently (Jansson and Northen,
2010; Couradeau et al., 2012).
The mechanism of cyanobacterial calcification by sheath
impregnation is still debated in several occurrences includ-
ing the oldest calcified microfossils. For instance, the mecha-
nisms leading to the formation of the Girvanella-type micro-
fossils, which are widespread in Paleozoic formations, have
been questioned. Based on petrographic evidence, some au-
thors have proposed that the calcification took place post-
mortem (Pratt, 2001); others suggest it is a result of cell
metabolic activity (Arp et al., 2002). In any case, all authors
agree on the fact that a better understanding of processes in-
volved in cyanobacterial calcification in modern settings is
required.
While there is an extensive record of fossil calcified
cyanobacteria (Arp et al. (2001) mention 864 occurrences of
fossil calcified cyanobacteria reported in the literature), only
a few modern field occurrences have been studied thoroughly
(Table S1).
Modern stromatolites/microbialites form in marine as well
as lacustrine environments. Marine stromatolites such as Ba-
hamas and Shark Bay stromatolites have been studied in de-
tails (e.g., Goh et al., 2009; Planavsky and Ginsburg, 2009;
Reid et al., 2000; Visscher et al., 1998) and have been con-
sidered as good analogues to ancient stromatolites since they
exhibit the same laminated macrofabric (see for instance R
Pamela Reid et al., 2003). However, some Archean stroma-
tolites have been shown to form in lakes e.g., those from
the Tumbiana formation (2.7 Ga) (Awramik and Buchheim,
2009). Therefore, the study of lacustrine microbialites is also
relevant and is needed to complete the available reference
database used to interpret ancient microbialites.
In that framework, Lake Alchichica is a hyperalkaline lake
(pH∼ 8.9) in Mexico and harbors a high density of mod-
ern microbialites composed mostly of hydromagnesite and
aragonite (Ka
´
zmierczak et al., 2011). Recently, the system-
atic association of colonies of Pleurocapsales with patches of
Biogeosciences, 10, 5255–5266, 2013 www.biogeosciences.net/10/5255/2013/

E. Couradeau et al.: Cyanobacterial calcification in modern microbialites 5257
aragonite has been evidenced in these microbialites (Gerard
et al., in press). Pleurocapsales are an abundant cyanobacte-
rial groups identified in Alchichica microbialites, which oth-
erwise harbor a wide microbial diversity (Couradeau et al.,
2011). Aragonite is usually in smaller abundance than hy-
dromagnesite in these microbialites. Hence the precipitation
of aragonite in Lake Alchichica is not the dominant process
involved in the accretion of the microbialites. However, it is
a particularly interesting process since it allows a delicate
preservation of cells remnants and a model for the first stages
of fossilization. Aragonite has been suggested as the primary
phase in the Tumbiana stromatolites (e.g., Lepot et al., 2008),
stressing the importance of studying modern calcification by
this phase. Here, we aimed at studying at the submicrome-
ter scale this association between Pleurocapsales and arago-
nite, as a modern case of cyanobacterial calcification. This
allowed for getting details on the different steps of the cal-
cification process of Pleurocapsales cells. For that purpose,
we characterized the assemblages of Pleurocapsales cells and
aragonite using a combination of confocal laser scanning mi-
croscopy (CLSM), scanning and transmission electron mi-
croscopies (SEM, TEM), focused ion beam (FIB) milling and
synchrotron-based scanning transmission x-ray microscopy
(STXM). The study of mineral growth around and within
cells and resulting mineral textures as well as the assessment
of the distribution of organic matter in these systems provide
an unprecedented and important modern reference at thenm
scale for future studies of fossil calcified cells.
2 Material and methods
2.1 Sample collection and preparation
The microbialite sample analyzed in this study was collected
at a depth of 4m in Lake Alchichica in 2007 (Ka
´
zmierczak
et al., 2011) and placed in a sterile zip plastic bag. A micro-
bialite fragment was subsequently fixed in the laboratory in a
4% formaldehyde solution (methanol free, ultra pure; Poly-
sciences, Inc.) 4h at 4
◦
C then washed in phosphate-buffered
saline (PBS) solution and finally stored in (1/1) ethanol/PBS
at −20
◦
C. A millimeter-sized fixed fragment was stained
first by calcein at a concentration of 0.1 mg mL
−1
for 48 h
at 4
◦
C, then by DAPI at 1µgmL
−1
for 2h at room temper-
ature. Samples were then dehydrated through a graded se-
ries of ethanol solutions (i.e., ethanol/water volume ratios
at 30 %, 50 %, 70 %, 90 %, and 100 %), and progressively
embedded in hard grade LR-white resin (Polysciences, Inc.).
This was followed by incubation at 4
◦
C for 18h in (1/1)
then (2/1) mixture of LR-white/ethanol and finally in pure
LR-white resin. After 3h at room temperature, samples were
embedded in pure LR-white resin for 1h at 40
◦
C and then
for 24h at 60
◦
C. Transverse cross sections were cut using
a diamond wire before polishing using diamond powder at
1/4µm.
2.2 Bulk x-ray diffraction (XRD)
A non-treated fragment of microbialite was ground in 100%
ethanol. XRD patterns were recorded with a Panalytical
X’Pert Pro MPDH mounted in the Bragg Brentano config-
uration. Data were recorded with a monochromatic CoKα
beam (λ = 0.17889nm) in continuous scan mode within a
(3–100
◦
) 2θ range with steps of 0.017
◦
and a counting time
of 813.98s per step.
2.3 Confocal laser scanning microscopy (CLSM)
Polished sections were observed using an Olympus Flu-
oViewTM FV1000 confocal laser scanning microscope. The
microscope was equipped with a 405nm laser diode, and
multi-line argon (458nm, 488nm, and 515 nm), helium-
neon-green (543 nm) and helium-neon-red (633nm) lasers.
Fluorescence images were obtained by concomitant ex-
citation at wavelengths of 405 nm, 488 nm, and 543 nm
and collection of the emitted fluorescence between 425–
475nm, 500–530 nm, and 560–660nm. Despite the possi-
ble occurrence of crosstalks between DAPI and autoflu-
orescence when using simultaneous excitation, Gerard et
al. (2013) have shownthe efficiency of thisapproach on Lake
Alchichica microbialites to image diverse cyanobacteria, in-
cluding Pleurocapsales.
2.4 Scanning electron microscopy and FIB milling
The section of the microbialite sample collected at a 4 m
depth and analyzed by CLSM was coated with gold-
palladium and observed by scanning electron microscopy
(SEM). Images were collected in backscattered and sec-
ondary electron modes using a Zeiss Ultra 55 FEG-SEM op-
erating at 10 kV with a 30µm aperture and a working dis-
tance of 8 mm. Elemental compositions were determined by
energy dispersive x-ray spectrometry (EDXS) using an EDS
QUANTAX detector and the software ESPRIT. EDXS anal-
yses were operated using a 20keV acceleration voltage, a
60µm aperture and a working distance of 7.5mm. Two ultra-
thin foils transparent to electrons (< 200 nm) were prepared
by FIB milling with a Zeiss dual FIB-NEON 40EsB using
the FIB “lift-out” technique (e.g., Benzerara et al., 2005). A
30kVGa
+
beam operated at ∼ 5nA was used for the ini-
tial steps of the milling. Progressive excavation from both
sides of the section area was performed through repeated
milling of steps. Depth of milling was approximately 6 mi-
crons. An in situ micromanipulator was attached to the foil
by FIB-assisted platinum deposition and the foil was liber-
ated from the substrate by a U-cut milling pattern. The foil
was transferred to an Omniprobe grid and welded to it. Final
thinning of the section was performed with Ga
+
beam oper-
ated at 100pA current. The foil measured 15µm in length,
∼ 6µm in width and ∼ 100–200nm in thickness.
www.biogeosciences.net/10/5255/2013/ Biogeosciences, 10, 5255–5266, 2013

5258 E. Couradeau et al.: Cyanobacterial calcification in modern microbialites
2.5 Transmission electron microscopy (TEM)
TEM observations were carried out on a JEOL2100F mi-
croscope operated at 200kV, equipped with a field emis-
sion gun, a high resolution UHR pole piece, and a US4000
GATAN camera. Selected area electron diffraction (SAED)
was performed using the smallest aperture allowing retrieval
of diffraction patterns from a 100× 100nm
2
area.
2.6 Scanning transmission x-ray microscopy (STXM)
Scanning transmission x-ray microscopy (STXM) and near-
edge x-ray absorption fine structure (NEXAFS) spectroscopy
measurements were carried out on molecular environmen-
tal science 11.0.2.2 beamline at the Advanced Light Source
(ALS, Berkeley, USA).
The rationale for STXM data acquisition and analysis and
examples of applications can be found in Bluhm et al. (2006)
and Moffet et al. (2010). For STXM imaging, the x-ray beam
is focused on an x-ray transparent sample using a zone plate,
and a 2-D image is collected by scanning the sample at a
fixed photon energy. The achieved spatial resolution is de-
pendent on the zone plate (∼ 25nm in the present study) and
the scanning step (which varies from one image to another).
The image contrast results from differential absorption of x-
rays, which partly depends on the chemical composition of
the sample. In addition to imaging, it is possible to perform
at the same spatial resolution, near-edge x-ray absorption
fine structure (NEXAFS) spectroscopy at the carbon K-edge
(and other absorption edges in the 80–2000eV energy range)
which gives information on the speciation (i.e., type of func-
tional group and bonding) of carbon (and other elements).
Measurements were performed at the C, O and N K-edges
and at the Ca L
2,3
-edges. ALS storage ring was operated at
1.9GeV and 500 mA current. Energy calibration was done
using the well-resolved 3p Rydberg peak at 294.96eV of
gaseous CO
2
and the L
3
most intense peak of calcite at
349.3eV (Benzerara et al., 2004). Methods used for STXM
data acquisition and analysis and examples of STXM appli-
cations can be found, for example, in Benzerara et al. (2010,
2011) and Obst et al. (2009). AXis2000 software was used
to extract NEXAFS spectra from image stack measurements
and STXM map construction.
2.7 Saturation index calculation
The saturation indices of the Lake Alchichica solution with
respect to aragonite and hydromagnesite were calculated for
different pH (between 8 and 12) using the software Visual
Minteq 3.0 and the Minteq thermodynamic database. Con-
centrations of major ion concentrations, total alkalinity and
temperature were measured during collection of the sam-
ples and were reported in Ka
´
zmierczak et al., 2011 (i.e., in
meqL
−1
:Cl
−
87.3/SO
2−
4
16.73/Br
−
0.1/F
−
0.008/Na
+
100.5/Mg
2+
35.61/K
+
5.32/Ca
2+
0.735/Li
+
0.26 and al-
kalinity 30.9. Calculations were performed considering a
temperature of 15
◦
C).
3 Results
3.1 SEM and CLSM analyses of cyanobacteria-mineral
assemblages
Bulk XRD analyses (Fig. S1) showed that Alchichica
microbialites collected at 4m were composed of two
main phases: aragonite (CaCO
3
) and hydromagnesite
(Mg
5
(CO
3
)
4
(OH)
2
· 4H
2
O). These two mineral phases could
be clearly discriminated by SEM in the backscattered elec-
tron mode (BSE): hydromagnesite appeared as light grey ar-
eas and composed the major part of the samples (85% of the
section observed), while aragonite appeared as bright discon-
tinuous patches located preferentially at the surface of the
samples in contact with microbial biofilms. The biofilms ap-
peared as dark grey discontinuous layers lying at the surface
of the microbialite and measuring 10 to 500µm in thickness
(Fig. 1a). Previous CLSM and Raman spectroscopy obser-
vations showed colonies of Pleurocapsales in contact with
aragonite patches based on their typical autofluorescence and
pseudo-filamentous morphology (Gerard et al., 2013). More-
over, the presence of Pleurocapsales has been consistently
shown by molecular analyses based on 16SrRNA gene se-
quencing performed on the same sample (Couradeau et al.,
2011). Here, we confirm the specific association of Pleu-
rocapsales cells encrusted in aragonite by SEM and CLSM
(Fig. 1b and c). Increasing levels of encrustation could be fol-
lowed over a distance of 100 µm starting with non-encrusted
cells in the biofilm at the surface of the microbialites and end-
ing in areas where cells were completely encrusted (Fig. 2a).
Calcification consisted on the formation of a mineral layer
around the walls of cells that were located at the periphery
of the sample. Twenty micrometers deeper in the sample, the
inner part of cells was partly encrusted, still keeping a signif-
icant portion of their organic content (Fig. 2b). At fifty mi-
crometers deep in the sample, the inner part of the cells was
completely calcified and, in some cases, the cell wall was
not visible anymore. Completely encrusted cells still showed
some residual fluorescence by CLSM (Fig. 1c). As shown by
Gerard et al. (2013) based on the acquisition of spectra, this
fluorescence is specific of cyanobacterial cells and cannot be
confused with the blue signal of aragonite. At this most ad-
vanced fossilization stage, encrusted cells formed some sort
of pavement in the aragonite (Fig. 2a).
In order to get further insight in the mineral-cell assem-
blages down to the nm scale, two FIB foils were cut across
encrusted cells (see locations on Figs. 2a and S2): FIB foil
A was cut across partially encrusted cells located close to
the surface of the microbialite; FIB foil B was cut across
completely encrusted cells, which were located 44 µm deeper
within the aragonite patch.
Biogeosciences, 10, 5255–5266, 2013 www.biogeosciences.net/10/5255/2013/

E. Couradeau et al.: Cyanobacterial calcification in modern microbialites 5259
25
1
Figure 1. Images of a section prepared from an Alchichica microbialite collected at a 4m 2
depth. (A) SEM (secondary electron mode) picture of the section showing the relative 3
distribution of mineral phases. Aragonite (white) lies mostly at the surface of the microbialite 4
while hydromagnesite (light grey) composes the inner part. LR-white resin appears in dark-5
grey around the sample. Pockets of microorganisms are visible in aragonite, for instance in 6
area A and area B. (B-C) Close-ups of area A obtained by SEM (B) and CLSM (C). Some 7
residual autofluorescence underline cell ghosts in the aragonitic part. 8
9
Fig.1.Images of a section prepared from an Alchichica microbialite
collected at a 4m depth. (A) SEM (backscattered electron mode)
picture of the section showing the relative distribution of mineral
phases. Aragonite (white) lies mostly at the surface of the micro-
bialite while hydromagnesite (light grey) composes the inner part.
LR-white resin appears in dark-grey around the sample. Pockets
of microorganisms are visible in aragonite, for instance in area A
and area B. (B–C) Close-ups of area A obtained by SEM (B) and
CLSM (C). Some residual autofluorescence underline cell ghosts in
the aragonitic part.
3.2 STXM study of organic matter in the FIB foils
The distribution of carbon and calcium within FIB foils was
assessed by EDX spectroscopy (Fig. S3) and the specia-
tion of carbon (carbonates vs. organic carbon) was studied
by scanning transmission x-ray microscopy (STXM). NEX-
AFS spectra were measured at the C K-edge on both FIB
foils A and B (Fig. 3). For each foil, two types of NEX-
AFS spectra were observed. One type of NEXAFS spec-
trum was typical of calcium carbonates and showed a ma-
jor peak at 290.2 eV that was attributed to 1s-π ∗ electronic
transitions in carbonate groups (Benzerara et al., 2006). The
other type of NEXAFS spectrum was characteristic of or-
ganic carbon and showed peaks at 284.8, 286.5 and 288.5eV
that could be attributed to aromatic, ketone and carboxylic
functional groups, respectively (Benzerara et al., 2004). Or-
ganic carbon had the same spectroscopic signature in both
FIB foils. Carbonate groups (i.e., aragonite as determined by
XRD and TEM) vs. organic carbon were mapped in the two
FIB foils based on their spectroscopic differences. In FIB foil
A, two different textures were observed for organic carbon
(Fig. 4): (i) some organic carbon appeared as homogeneous
26
1
Figure 2. Fossilization gradient of Pleurocapsales in aragonite observed by SEM in 2
backscattered electron mode. (A) area B (as outlined in Figure 1) showing progressive 3
encrustation of cells in aragonite. Locations of FIB foils are shown. FIB foil A corresponds to 4
the beginning of the fossilization gradient while FIB foil B was cut in totally encrusted cells. 5
(B) Close-up of area B showing the textural relation between cells and aragonite. In some 6
Fig. 2. Fossilization gradient of Pleurocapsales in aragonite ob-
served by SEM in backscattered electron mode. (A) area B (as out-
lined in Fig. 1) showing progressive encrustation of cells in arago-
nite. Locations of FIB foils are shown. FIB foil A corresponds to
the beginning of the fossilization gradient while FIB foil B was cut
in totally encrusted cells. (B) Close-up of area B showing the tex-
tural relation between cells and aragonite. In some cells, the inner
part remains totally organic while in others only the wall is visible,
while the inner part being filled by aragonite.
3 micrometer wide patches filling the partly encrusted cells;
(ii) the rest of the organic carbon was diffuse and showed
an intimate association with aragonite crystals. Local con-
centrations of this organic carbon drew contour line delim-
iting aragonite clusters, which likely corresponded to the
previous boundaries of encrusted cells (Fig. 3b). Three en-
crusted cells were observed in foil A (Fig. 3b). FIB foil B
was cut across several cells in a more advanced encrusta-
tion stage (Figs. 2a and S2). However, only one cell could
be distinguished in foil B based on a local concentration of
organic carbon (Fig. 3c). Otherwise, most of the organic car-
bon was diffuse and intimately associated with carbonates.
NEXAFS spectra were measured at the O and N K-edges
(Fig. S4). Similarly to the observations performed at the C
K-edge, only one kind of spectrum was retrieved from or-
ganic matter in both FIB foils, indicating that the functional
groups composing organic matter as detected by NEXAFS
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5260 E. Couradeau et al.: Cyanobacterial calcification in modern microbialites
28
1
Figure 3. Scanning transmission x-ray microscopy analyses at the C-Kedge of FIB foils A and 2
B (A) NEXAFS spectra at the C K-edge of organic matter composing the cells (green) and 3
carbonates (red). (B) STXM map with 40 nm spatial resolution showing the distribution of 4
organic matter (green) and carbonate (red) in FIB foil A. Cell morphology is preserved in the 5
first steps of fossilization. (C) STXM map with 40 nm spatial resolution showing the 6
distribution of organic matter (green) and carbonate (red) in FIB foil B. The remnant of one 7
cell can still be seen, whereas in its surroundings the organic matter is diffuse within 8
aragonite. 9
10
Fig. 3. Scanning transmission x-ray microscopy analyses at the C-
Kedge of FIB foils A and B (A) NEXAFS spectra at the C K-edge
of organic matter composing the cells (green) and carbonates (red).
(B) STXM map with 40 nm spatial resolution showing the distri-
bution of organic matter (green) and carbonate (red) in FIB foil A.
Cell morphology is preserved in the first steps of fossilization. (C)
STXM map with 40nm spatial resolution showing the distribution
of organic matter (green) and carbonate (red) in FIB foil B. The
remnant of one cell can still be seen, whereas in its surroundings,
the organic matter is diffuse within aragonite.
spectroscopy did not change qualitatively with increasing en-
crustation (Fig. S4).
3.3 Textural arrangement of aragonite crystals
Two kinds of aragonite crystals that we term in the follow-
ing “type 1” and “type 2” aragonite were observed (Fig. 4).
Type 1 aragonite is composed of needle-shaped nano-crystals
measuring 195 ± 55 nm in length and 20± 5nm in width
(based on 25 measurements each on Fig. S5, see also
Fig. S6). They formed clusters in which aragonite crystals
shared a similar crystallographic orientation, as confirmed by
selected area electronic diffraction showing arcs of restricted
angular stretch (Fig. S6). Type 2 aragonite was composed of
larger prismatic crystals less homogenous in size (width be-
tween 100–500 nm, see Fig. S5). In the most advanced stages
of encrustation, each cell was either filled with organicmatter
or type 2 aragonite. For all the different stages of encrusta-
tion of Pleurocapsales, type 1 aragonite needles were perpen-
dicular to the cell surface and formed a radial crown around
the cells (Fig. 4c). The addition of radial layers of type 1
aragonite around the cells likely led to the concentric growth
pattern observed in Figs. 4 and S5. This pattern results from
the succession of ∼ 200nm wide layers, each of those layers
29
1
Figure 4. TEM pictures of FIB foils A and B. (A,D) Picture of the whole FIB foils A and B 2
respectively. Organic matter appears smooth (green) while other areas are filled with fibrous 3
aragonite (aragonite type 1) except for areas surrounded by orange lines where crystals of 4
aragonite are bigger (aragonite type 2). Lines of less resistance in the type 1 aragonite pattern 5
are highlighted by green dotted lines. They delineate the limits between cells. (B) Close-up of 6
the area outlined in (A) showing an encrusted cell. (C) Close-up of the area outlined in (B) 7
showing the radial distribution of aragonite needles at the surface of the cell. (E) Close-up of 8
the area outlined in (D) showing the textural organization of successive aragonite type 1 9
layers. (F) Close-up of the area outlined in (D) showing the limit between type 1 aragonite 10
Fig. 4. TEM pictures of FIB foils A and B. (A, D) Picture of
the whole FIB foils A and B, respectively. Organic matter appears
smooth (green) while other areas are filled with fibrous aragonite
(aragonite type 1) except for areas surrounded by orange lines
where crystals of aragonite are bigger (aragonite type 2). Lines
of less resistance in the type 1 aragonite pattern are highlighted
by green dotted lines. They delineate the limits between cells. (B)
Close-up of the area outlined in (A) showing an encrusted cell. (C)
Close-up of the area outlined in (B) showing the radial distribution
of aragonite needles at the surface of the cell. (E) Close-up of the
area outlined in (D) showing the textural organization of successive
aragonite type 1 layers. (F) Close-up of the area outlined in (D)
showing the limit between type 1 aragonite and type 2 aragonite; no
organic matter remains associated with the inner part of the cell at
this stage of encrustation.
composed of aragonite needles sharing a common crystallo-
graphic orientation that is intermediate between those of the
layers located beneath and above. This progressive change in
crystallographic orientation from one layer to the next one
accommodates the transition between two encrusted cells.
Interestingly, a higher concentration of organic matter was
observed (Fig. 3b) in these transition areas where aragonite
crystals formed around two neighboring Pleurocapsales cells
converge (see dotted lines in Fig. 4a and d). The relative dis-
tribution of type 1 aragonite (outside the cells) and type 2
aragonite (within the cells) correlated with the location of en-
crusted cells. Owing to the mineral texture, it was therefore
possible to infer the presence of former cells even when en-
crustation was much advanced and only little organic carbon
remained (Figs. 4d–f and S5).
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E. Couradeau et al.: Cyanobacterial calcification in modern microbialites 5261
4 Discussion
4.1 Preferential fossilization of Pleurocapsales
A previous analysis of 16S rRNA genes showed that
at least 34 phylotypes of Cyanobacteria were present in
Alchichica microbialites, including 5 phylotypes of Pleuro-
capsales (Couradeau et al., 2011). One phylotype of Pleu-
rocapsales (Alchichica AL52 2 1B 148 CyanoOTU35) was
particularly abundant in Alchichica samples (up to 69% of
all cyanobacteria) (Couradeau et al., 2011). The closest rel-
ative of this phylotype was detected in microbialites from
Lake Van (Lopez-Garcia et al., 2005) suggesting an adapta-
tion of this particular lineage to mineralizing environments
such as alkaline lakes. Permineralization of cyanobacterial
cells, including members of the Pleurocapsales, by aragonite
has been proposed as an important mechanism contributing
to Lake Van microbialite growth (Kempe et al., 1991; Lopez-
Garcia et al., 2005). Moreover, close associations between
Pleurocapsales and aragonite have been reported by other
studies, e.g., in the Bahamian thrombolitic black mats (Mob-
berley et al., 2011) or in Satonda microbialites where the
Pleurocapsa-Dermocarpella zone was associated with arag-
onite aggregates (Arp et al., 2003). In Laguna Mormona stro-
matolites, the permineralization of Entophysalis-like Pleuro-
capsales by aragonite was proposed as the most important
mechanism of stromatolite accretion (Horodyski and Von-
der Haar, 1975). Our observations further suggest that Pleu-
rocapsales are essential players in aragonite formation and
specifically contribute to the formation of Alchichica micro-
bialites.
The Pleurocapsales are often closely associated with car-
bonate minerals (Table S1), suggesting that this group is es-
pecially prone to being encrusted. Assessing this ability to
get encrusted among the microbial diversity associated with
microbialites will be crucial to better determine the fraction
of the microbial diversity that can be expected to be fos-
silized.
4.2 Biomineralization pattern of Pleurocapsales and
fate of organic matter
In Alchichica microbialites, crystals of type 1 aragonite first
appear within clusters of Pleurocapsales cells which ex-
hibit autofluorescence and show texturally preserved traces
of the cell walls at least at the SEM scale. This argues in
favor of in vivo calcification. Chemical processes inducing
cyanobacterial cell encrustation by calcium carbonates have
been proposed by previous studies (Dupraz et al., 2009). It
is classically proposed that oxygenic photosynthesis locally
increases the pH in the cell vicinity leading to carbonate
oversaturation and precipitation (Riding, 2006b; Jansson and
Northen, 2010). Alternatively, induction of precipitation at
the surface of the cells may be due to the presence of nu-
cleating molecules such as those composing cyanobacterial
sheaths (Merz-Preiss and Riding, 1999) or S layer proteins
(Thompson et al., 1997).
Concerning the localization of mineral nucleation, two
kinds of biomineralization pathways are observed in mod-
ern cases of cyanobacterial calcification as described in the
literature (Table S1): (1) calcification occurs extracellularly
in the biofilm by replacing EPS or (2) directly at the cell sur-
face (on the cell wall or within the sheath), the second case
being more prone to form microfossils. Arp et al. (2001) pro-
posed a model explaining how the prominence of one calci-
fication pathway over the other may depend on the chemical
conditions of the environment. It has been suggested by Arp
et al. (2001) that at low Ca
2+
/high dissolved inorganic car-
bon (DIC), carbonate nucleation may occur randomly in the
biofilm and not specifically in association with cyanobacte-
rial cells. In this particular setting, the pH increase resulting
from photosynthetic activity may not produce a significant
local pH gradient due to the high capacity of pH buffering of
the system (Arp et al., 2001). In contrast, Shiraishi (2012) ob-
served that photosynthesis-induced carbonate precipitation
can occur even at high DIC, arguing that the pH shift due to
photosynthesis is not the main driver of calcification in this
case. In Lake Alchichica, where low Ca
2+
/high DIC con-
ditions prevail, type 1 aragonite needles are organized per-
pendicularly to the cell wall, arguing in favor of nucleation
occurring at the cyanobacterial cell surface supporting the
viewby Shiraishi (2012). This ability might be related to spe-
cial physiological features of cyanobacteria belonging to the
Pleurocapasales order and/or a particular chemical compo-
sition of the surface of the cells. For example, it has been
proposed that the organization of the sheath provides a tem-
plate for mineral nucleation and promotes mineral nucleation
(Reitner, 1993; Braissant et al., 2003; Dupraz and Visscher,
2005). It is possible that the particular sheath of Pleurocap-
sales, also referred to as the fibrous layer (Waterbury and
Stanier, 1978; Pinevich et al., 2008), might provide a suitable
template for mineral nucleation; moreover, it is known that
this kind of sheath, unlike the more common tubular sheath,
is intimately attached to the cell outer membrane (Waterbury
and Stanier, 1978). This particular feature might favor the
formation of microfossils, since the encrusted sheath may re-
main connected to the cell body.
This mechanism may explain the first stage of sheath im-
pregnation by “type 1” aragonite and its associated layered
texture. The later growth of type 2 aragonite may occur post-
mortem since type 2 aragonite sometimes entirely fills the
cell cytoplasm. In some cyanobacterial species, calcification
can occur in vivo intracellularly (Couradeau et al., 2012)
but in that case, precipitates are spherical, amorphous and
keep a small size below 200 nm. Post-mortem calcification
processes have been suggested by other authors. For exam-
ple, in modern microbialites from the Tikehau atoll, carbon-
ate precipitation may start in vivo before pervasive precip-
itation due to organic matter decay (Sprachta et al., 2001).
In vivo calcification allows for the preservation of the size
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5262 E. Couradeau et al.: Cyanobacterial calcification in modern microbialites
and the morphology of the cells (Merz-Preiss and Riding,
1999) while post-mortem calcification (Bartley, 1996) forms
crystals with a morphology controlled by the geometry of
the available space (Chafetz and Buczynski, 1992; Riding,
2006b). Formation of type 2 aragonite within cells may pre-
vent a subsequent collapse of the cell overall structure dur-
ing further compaction as suggested previously (Golubic and
Hofmann, 1976).
Whatever the mechanism of encrustation, the most ad-
vanced stages show some traces of the cells under two forms:
(1) some residual pigments are preserved as indicated by the
detection of autofluorescence by CLSM. Pigments are espe-
cially recalcitrant molecules that can be preserved in sedi-
ments (Leavitt et al., 1997) and can be used as molecular
fossil diagnostic for photosynthetic organisms (Brocks and
Pearson, 2005); and (2) the approximate shape of the cells
is preserved by the specific textural arrangement of the two
types of aragonite crystals. The short-term preservationof the
aragonite crystals is controlled by a delicate balance between
dissolution induced by metabolisms such as fermentation and
aerobic respiration and precipitation induced by metabolisms
such as oxygenic or anoxygenic photosynthesis or sulfate re-
duction (Dupraz et al., 2009; Stal, 2012). The stability upon
aging of this kind of textural biosignature remains to be as-
sessed. It has been shown previously that very fine ultrastruc-
tural features such as cellnucleus in eukaryotes (Huldtgren et
al., 2011) or cell periplasm in bacteria (Cosmidis et al., 2013;
Miot et al., 2011) can occasionally be preserved by fossiliza-
tion, even when rocks have been affected by metamorphism
(Bernard et al., 2007; Galvez et al., 2012).
4.3 Aragonite vs. hydromagnesite precipitation
We have stressed on the point that Pleurocapsales induce
carbonate precipitation at their surface possibly due to their
photosynthetic activity and/or the activity of other microbes
(e.g., sufate-reducing bacteria) and/or the presence of partic-
ular templating polymers at their surface. However, an ad-
ditional issue is raised by our observations: why do Pleuro-
capsales appear specifically associated with aragonite while
the bulk of Alchichica microbialites is formed by hydro-
magnesite? Previous studies have shown that subtle varia-
tions in the chemical composition of the solution can im-
pact significantly the nature of precipitated mineral phases
and that this can be predicted by chemical equilibrium mod-
eling (Gallagher et al., 2013). As discussed above, several
parameters are key in the induction of carbonate precipita-
tion, including the pH. Here, we are more specifically inter-
ested in the parameters that orient the precipitation towards
aragonite instead of hydromagnesite. This orientation may be
driven by several factors including the concentration ratio of
[Ca
2+
]/[Mg
2+
] and pH.
A local increase in the [Ca
2+
]/[Mg
2+
] ratio around Pleu-
rocapsales cells could be one way of explaining why arago-
nite precipitation is favored overhydromagnesite. It is known
that cyanobacteria contain Ca
2+
-ATPases responsible for the
transport of Ca
2+
outside the cytoplasm (McConnaughey
and Whelan, 1997). Their activity allows the maintenance of
a low cytoplasmic Ca
2+
concentration (Dominguez, 2004).
This could result in the increase of the [Ca
2+
]/[Mg
2+
] in
the cyanobacterial cell vicinity. It could thus be speculated
that Pleurocapsales pump out Ca
2+
at a much higher rate
than other cyanobacteria in the microbialites, which do not
calcify or do not induce aragonite precipitation.
Alternatively, the control of local pH might be a key
parameter in carbonate precipitation around cyanobacterial
cells. The calculation of aragonite and hydromagnesite sat-
uration index (Fig. S7) shows that both phases are oversat-
urated at the pH of the lake, with slightly higher saturation
index for aragonite than hydromagnesite. Our calculations
show that if pH increases, e.g., due to photosynthetic activity,
then the saturation index of hydromagnesite would increase
more than that of aragonite suggesting that higher pH would
favor hydromagnesite over aragonite precipitation. This is
not what is observed for Pleurocapsales in Lake Alchichica.
Consequently, an increase of the [Ca
2+
]/[Mg
2+
] ratio seems
the best explanation for the orientation of the precipitation
reaction towards aragonite instead of hydromagnesite. Cal-
cium ions are usually chelated efficiently by the cyanobacte-
rial sheaths (Braissant et al., 2009; Dupraz et al., 2009) and
then released from EPS by the activity of heterotrophic bac-
teria, increasing their concentration and enhancing calcium
carbonate precipitation (Dupraz and Visscher, 2005). Such a
mechanism may apply as well to the precipitation of arago-
nite by Alchichica Pleurocapsales. However, the specificity
of this mechanism on calcium over magnesium has never
been tested experimentally, and will require further investi-
gation with appropriate cyanobacterial strains. In the present
study, we do not have data supporting or invalidating the
biogenic origin of hydromagnesite. Other studies have previ-
ously discussed this point specifically (Gerard et al., 2013).
4.4 Stepwise model of fossilization in Pleurocapsales
As a summary, the fossilization pattern of Pleurocapsales
within aragonite appears to proceed in four main steps
(Fig. 5). In the first step, the cell is photosynthetically ac-
tive and modifies the local chemical environment including
the Ca
2+
/Mg
2+
ratio (step 1). Nucleation starts close on
the cell wall, and needles of type 1 aragonite initially grow
perpendicularly to cell surface, radiating towards the exte-
rior (step 2). The morphology and growth pattern of type 1
aragonite might be controlled by surrounding organic mat-
ter (Braissant et al., 2003). Clusters of type 1 aragonite fill
the space surrounding the cell and their orientation accom-
modates the transition from one encrusted cell to another
(step 3). At this step organic matter is still detectable in the
inner part of the cell. The organic matter around the cell
that initially corresponded to EPS is then pushed towards
the borders of cell clusters. In a final step (step 4) type 2
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E. Couradeau et al.: Cyanobacterial calcification in modern microbialites 5263
31
1
Figure 5. Summarizing sketch of Pleurocapsales encrustation within aragonite. Each step is 2
illustrated by a SEM picture. The mineralization gradient increases from left to right. (1) 3
Living colony of Pleurocapsales. (2) Needles of Type 1 aragonite nucleate and grow from the 4
surface of Pleurocapsales cells. (3) Growth of Type 1 aragonite fills the space surrounding the 5
cell and accommodates the transition from one encrusted cell to another. The inner part is still 6
organic and starts to be replaced by Type 2 aragonite. (4) In the end, the cell is totally filled 7
by aragonite; an organic wall separating Type 1 from Type 2 aragonite is preserved 8
temporarily. 9
10
Fig. 5. Summarizing sketch of Pleurocapsales encrustation within
aragonite. Each step isillustrated by a SEM picture. The mineraliza-
tion gradient increases from left to right. (1) Living colony of Pleu-
rocapsales. (2) Needles of Type 1 aragonite nucleate and grow from
the surface of Pleurocapsales cells. (3) Growth of Type 1 aragonite
fills the space surrounding the cell and accommodates the transition
from one encrusted cell to another. The inner part is still organic and
starts to be replaced by Type 2 aragonite. (4) In the end, the cell is
totally filled by aragonite; an organic wall separating Type 1 from
Type 2 aragonite is preserved temporarily.
aragonite precipitates within cells. Type 2 aragonite shows a
prismatic texture and bigger crystals than type 1 aragonite,
which exhibits the typical needle-shaped crystal. Fluores-
cence of some residual organic matter is detected at this step
in the cell wall. However, most of the organic matter is dif-
fuse at this stage and does not indicate the initial organization
of Pleurocapsales cells anymore. In turn, the relative textural
arrangement of type 1 vs. type 2 aragonite records the ini-
tial shape and distribution of cells. It has been suggested that
aragonite may be replaced diagenetically by hydromagnesite
in Lake Alchichica microbialites (Kazmierczak et al., 2011).
Yet, the oldest microbialites on Lake Alchichica shores are
mostly composed of aragonite with no hydromagnesite, sup-
porting the idea that at least part of the aragonite and possibly
calcified Pleurocapsales cells might be preserved from early
diagenesis. It would be interesting to assess the stability of
this kind of textural biosignature upon aging and to look for
it in increasingly old fossil stromatolites.
Supplementary material related to this article is
available online at: http://www.biogeosciences.net/10/
5255/2013/bg-10-5255-2013-supplement.pdf.
Acknowledgements. We wish to thank especially J. Kazmierczak
and B. Kramer for organizing the sampling expedition to the
Alchichica Lake in 2007 and providing invaluable help during
sampling to P. L
´
opez-Garc
´
ıa and D. Moreira. This project was
financed by the French Interdisciplinary program “Environnements
plan
´
etaires et origines de la vie” (PID OPV-EPOV). The SEM/FIB
facility of the Institut de Min
´
eralogie et de Physique des Milieux
Condens
´
es is supported by R
´
egion Ile de France grant SESAME
2006 I-07-593/R, INSU-CNRS, INP-CNRS, University Pierre
et Marie Curie, Paris. The JEOL JEM-2100F at IMPMC was
supported by Region Ile-de-France grant SESAME 2000 E 1435,
INSU-CNRS, INP-CNRS and University Pierre et Marie Curie–
Paris 6. ALS-MES beamline 11.0.2 is supported by the Director,
Office of Science, Office of Basic Energy Sciences, Division of
Chemical Sciences, Geosciences, and Biosciences and Materials
Sciences Division of the US Department of Energy at the Lawrence
Berkeley National Laboratory.
Edited by: H. Kitazato
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