Available online at www.sciencedirect.com
ScienceDirect
Geochimica et Cosmochimica Acta 126 (2014) 78–96
www.elsevier.com/locate/gca
Biomineralization of iron-phosphates in the water column of Lake
Pavin (Massif Central, France)
Julie Cosmidis a,b, Karim Benzerara a,⇑, Guillaume Morin a, Vincent Busigny b,
Oanez Lebeau b, Didier Jézéquel b, Vincent Noël a, Gabrielle Dublet a,
Guillaume Othmane a
a
Institut de Minéralogie et de Physique des Milieux Condensés – Université Pierre et Marie Curie, UMR CNRS 7590, 4 place Jussieu,
75252 Paris cedex 05, France
b
Institut de Physique du Globe de Paris - Sorbonne Paris Cité – Université Paris Diderot, UMR CNRS 7154, 1 rue Jussieu, 75238 Paris cedex
05, France
Received 8 June 2013; accepted in revised form 22 October 2013; Available online 13 November 2013
Abstract
The availabilities of iron and phosphorus have considerably impacted biological productivity in past and present natural
aquatic environments, and therefore have been key regulators of climate changes over geological time scales. Microbial
organisms are known to play important roles in reactions that drive Fe and P cycling at redox interfaces in Earth’s surface
environments. Here we study the depth variations of Fe and P speciation in Lake Pavin (Massif Central, France), a deep
(bottom depth 92 m) and permanently stratified lake with anoxic and ferruginous conditions in the water column below
60 m depth. We particularly focus on the potential roles of microbes on Fe and P transformations and traces left by these
processes in the sedimenting particular matter.
Bulk chemical analyses, powder X-ray diffraction and X-ray absorption spectroscopy (XAS) at the Fe K-edge were
performed to characterize the mineralogy and Fe oxidation state of solid particles at different depths in the water column
and in sediments deposited at the bottom of the lake. Fe is mainly hosted by Fe(III)-(oxyhydr)oxides and phyllosilicates
in the shallower oxygenated water column of the lake (25 m). The amount of Fe in suspended matter increases with depth,
and an additional amorphous Fe(II)–Fe(III)-phosphate phase is detected close to the chemocline (at 56 m depth), while vivianite (an Fe(II)-phosphate with a formula of Fe(II)3(PO4)28(H2O)) becomes dominant in the deeper anoxic water (67 m and
86 m depths). Fe-(oxyhydr)oxides are preserved down to these depths in the water column suggesting that Fe-reduction has
little impact on the particulate Fe budget over the monimolimnion. These Fe-(oxyhydr)oxides undergo reductive dissolution
at the surface of the sediments, where vivianite is the main Fe-bearing phase. These results are confirmed by imaging at the
micrometer and nano-scales using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), as well as high spatial and energy resolution XANES spectra at the Fe L2,3-edges and the C K-edge. Moreover, polyphosphate-accumulating microorganisms were observed by
microscopy and chemical imaging at all depths except in the deeper part of the lake (86 m), and some Fe was associated with
these polyphosphate inclusions below 56 m. Finally, the Fe-phosphates encrust microbial bodies, suggesting that microbial
activities might be involved in the formation of these phases. Based on comparisons with laboratory experiments, we propose
that Fe-oxidation (in contrast to Fe-reduction) may play a role in the precipitation of Fe-phosphates in the water column of
Lake Pavin. Polyphosphate-accumulating microorganisms could also be involved in Fe-phosphate formation in the lake, for
instance by increasing dissolved phosphate concentrations in the monimolimnion.
Ó 2013 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +33 1 44 27 75 42.
E-mail address: [email protected] (K. Benzerara).
0016-7037/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.gca.2013.10.037
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
1. INTRODUCTION
Phosphorus (P) is a limiting nutrient essential to life and
its availability controls biological production and climatic
responses in past and present aquatic environments (e.g.,
Filippelli and Delaney, 1994; Föllmi, 1996; Van Cappellen
and Ingall, 1996; Paytan and McLaughlin, 2007; Papineau,
2010). Microorganisms are known to exert important controls on the P biogeochemical cycle in marine and lacustrine
environments (Froelich et al., 1982; Gächter and Meyer,
1993). In particular, they participate in the mineralization
of organic P compounds, generating local gradients of inorganic phosphate (PO43) (O’Brien et al., 1981; Froelich
et al., 1988; Filippelli and Delaney, 1996). Some microorganisms can also concentrate phosphate in the form of
intracellularly stored polyphosphates, which can be hydrolyzed back into PO43 in anoxic sediments (Gächter and
Meyer, 1993; Sannigrahi and Ingall, 2005; Schulz and
Schulz, 2005; Hupfer et al., 2007; Goldhammer et al.,
2010). In marine environments, both processes are thought
to be involved in the precipitation of authigenic calciumphosphate minerals such as apatite, the main constituent
of marine phosphorite deposits, which represent a major
sink in the global P cycle (Föllmi, 1996). PO43 adsorption
to – or coprecipitation with – iron (Fe) (oxyhydr)oxides is
another important mechanism of P removal in marine
and freshwater environments (Buffle et al., 1989; Leppard
et al., 1989; Sundby et al., 1992; Jensen et al., 1995; Hongve,
1997; Lienemann et al., 1999; Gunnars et al., 2002; Thibault
et al., 2009; Dellwig et al., 2010). In anoxic sediments, the
reductive dissolution of these Fe-(oxyhydr)oxides then generates a large local flux of PO43, which might precipitate to
form phosphate minerals such as apatite (Heggie et al.,
1990; Scopelliti et al., 2010) or vivianite (a Fe(II)-phosphate
with a formula of Fe3(PO4)28(H2O)) (Emerson and
Widmer, 1978; Manning et al., 1991, 1999; März et al.,
2008; Jilbert and Slomp, 2013). This process, known as
Fe-redox pumping, is thought to have played an important
role in the formation of Precambrian phosphorites (Nelson
et al., 2010). Therefore, the P biogeochemical cycle is highly
sensitive to redox conditions. It is also intimately linked
with the cycle of Fe, another element essential to biological
production (e.g., Martin et al., 1991; Boyd et al., 2007;
Sterner, 2008). A broad diversity of microorganisms catalyze redox transformations of Fe (e.g., Kappler and Straub,
2005; Weber et al., 2006a; Konhauser et al., 2011). At
circumneutral pH, Fe solubility depends on its oxidation
state, and therefore bacterially-mediated Fe redox transformations of Fe can induce precipitation or dissolution of
Fe-minerals. Many previous studies have focused on the
coupling of Fe and P cycles at redox interfaces (e.g.,
Hongve, 1997; Gätcher and Müller, 2003; Crowe et al.,
2008a; Dellwig et al., 2010; Jilbert and Slomp, 2013).
Lake Pavin is a recent (7000 yr old) deep crater lake
located in the Massif Central (France). It is meromictic,
i.e., permanently stratified with two water layers that do
not intermix. The upper layer, called the mixolimnion (from
0 to 60 m depth), is affected by seasonal mixing, while the
deeper layer, called the monimolimnion (from 60 m to
92 m depth, the bottom depth of the lake), remains
79
permanently anoxic. Because of the occurrence of a redoxcline in the water column, the oxic–anoxic interface is relatively readily accessible compared to the majority of
lacustrine or marine settings where it is located within the
sediments. The water column of Lake Pavin is thus well suited to the study of the biogeochemistry of P and Fe at redox
interfaces. In contrast with most permanently stratified
water bodies which are euxinic below the chemocline, the
monimolimnion of Lake Pavin is sulphide-poor and
Fe(II)-rich (maximum dissolved concentration 1200 lM;
Michard et al., 1994). Several studies, mostly based on geochemical measurements of the aqueous phase, have focused
on the modeling of the geochemical cycles of major and
trace elements including Fe and P in the water column of
Lake Pavin (Michard et al., 1994; Viollier et al., 1995,
1997). These authors proposed an “iron wheel” model in
which highly concentrated dissolved Fe(II) diffuses upward
from the bottom of the lake to the chemocline, where it is
oxidized to Fe(III). Fe(III) precipitates and subsequently
sinks back in the monimolimnion as particulate Fe-(oxyhydr)oxides, where it is assumed to be reduced back to dissolved Fe(II) by organic matter (Michard et al., 1994). The
monimolimnion is also highly concentrated in PO43 (maximum dissolved concentration 300 lM; Michard et al.,
1994) and supersaturated with respect to vivianite. Moreover, a mineral phase showing a Fe/P molar ratio of 1.8
has been identified in the water column of Lake Pavin by
Viollier et al. (1997), and is referred to as “protovivianite”,
which has not been well characterized yet. Based on mass
balance calculations performed on the chemical composition of the sediments in Lake Pavin, it was suggested that
vivianite represents up to 70 wt% of the particulate Fe
in these sediments (Schettler et al., 2007). In addition to
vivianite formation, adsorption to or co-precipitation with
colloidal Fe-(oxyhydr)oxides may also scavenge PO43.
The trapping of PO43 by Fe minerals may reduce the P
flux from the monimolimnion to the mixolimnion (where
PO43 concentrations are comprised between 0 and 1 lM;
Michard et al., 1994), and therefore significantly limit the
primary production in the upper part of the lake (Schettler
et al., 2007).
Despite the important implications of Fe and P cycling
in Lake Pavin, we still lack a detailed assessment of the
mineralogy of the settling solid particles and the changes
with depth of the particulate Fe-redox state in the water
column of the lake. Moreover, it is likely that microorganisms play an important role in most reactions involving Fe
and P in Lake Pavin. The diversity of anaerobic communities of Bacteria and Archaea and their distribution with
depth in the lake have been characterized (Lehours et al.,
2005, 2007; Borrel et al., 2010; Biderre-Petit et al., 2010).
In particular, bacteria able to perform dissimilatory Fe
reduction have been isolated from the monimolimnion (Lehours et al., 2009, 2010). However, the potential interactions between microbial metabolisms and Fe and P
cycling in the lake are still poorly documented.
In the present study, we performed detailed bulk X-ray
diffraction (XRD) and X-ray absorption spectroscopy
(XAS) analyses to characterize the mineralogical compositions and the Fe redox state of Fe-bearing particles
80
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
collected in sediment traps at different depths of the water
column, as well as in sediments deposited at the bottom
of the lake. We also used electron and X-ray microscopies
to (1) image Fe-bearing phases, (2) determine their chemical
composition and Fe redox state at the submicrometer-scale
as a function of depth in the water column, and (3) seek evidence for some specific associations between Fe-bearing
phases and microbial cells. The combined results contribute
to a better knowledge of the Fe- and P-bearing phases present in the water column of Lake Pavin and their transformations at the chemocline and sediment–water interface,
and provide valuable insights into the potential bacterial
involvement in these transformations.
2. MATERIAL AND METHODS
2.1. Geochemical parameters in the water column of Lake
Pavin
Classical physicochemical parameters (temperature,
conductivity, dissolved oxygen, pH, and turbidity) were
measured in situ with CTD (Conductivity–Temperature–
Density) probes (nke STPS-O2 and/or YSI 6600) and a
NKE STBD 300 probe (turbidimeter) in September 2011.
Light intensity was measured in October 2010 as photosynthetically active radiation (PAR). The photon flux in
lmol m2 s1 was recorded in the spectral range 400–
700 nm simultaneously at the surface of the lake and in
the water column using a LI-192 SA atmosphere Spherical
Quantum Sensor and a LI-193 underwater Spherical Quantum Sensor, respectively. Light penetration rate as a function of depth was then obtained by dividing PAR in the
water by PAR at the surface.
2.2. Collection of particulate matter in the water column and
sediments at the bottom of the lake
Different types of samples were analyzed: (1) suspended
matter collected in sediment traps placed at different depths
in the water column were analyzed by XRD, XAS, scanning and transmission electron microscopy (SEM and
TEM) and scanning transmission X-ray microscopy
(STXM), (2) particles filtered from water samples were analyzed by TEM, and (3) sediment sampled by a gravity corer
at the bottom of the lake (92 m depth) were analyzed by
XRD and XAS. Samples from the sediment traps and core
(types (1) and (3)) were also used for measurements of bulk
iron and organic carbon concentrations. Overall, several
subsamples were analyzed by different techniques, which
provide consistent data as shown in the result section.
Sediment traps (Uwitec) (e.g., Rosa, 1985; Rosa et al.,
1991) consisted of a pair of 2 L bottles each topped with
a tube measuring 1 m in length and 90 mm in diameter.
They were placed at 25, 56, 67 and 86 m depth in the water
column of Lake Pavin (i.e., just below the primary production zone, just above and below the chemocline, and a few
meters above the sediment respectively) and left for 89 days
(from June 30, 2011 to September 27, 2011) in order to collect settling particles. The tubes were designed to avoid particle losses and mixing during the collection of the traps.
After collection, bottles were immediately closed in a glovebag flushed with N2 and kept under anoxic conditions by
sealing within aluminized foils (Protpack, UK). Bottles
were then transported to the laboratory and placed within
an anoxic glove-box (Coy Laboratory Products) under a
N2/H2 (<5% H2) atmosphere (<50 ppm O2). Seven days
after collection, bottles were opened in the glovebox, and
their contents were pooled and centrifuged 3 min at
8000 rpm in 50 mL Falcon tubes in order to separate solid
particles from solution. Pellets were vacuum dried in a dessicator at ambient temperature for 48 h within the glovebox
and stored under anoxic conditions until XRD and XAS
analyses.
Matter suspended in the water column of Lake Pavin
was also collected on filters for SEM observations. For that
purpose, 1 L of water was collected at the same depths as
the sediment traps (i.e., 25, 56, 67 and 86 m). Each sample
was filtered immediately using 0.2 lm GF/F filters. The
filters were dried in a dessicator within the glovebox at
ambient temperature.
The sediment core from the bottom of the lake was collected in September 2009, using a gravitational Uwitec
corer with a diameter of 90 mm. The total length recovered
from this core was 167 cm. In the present work, we focused
only on the first top 6 cm of this core, corresponding to
the last 20 years according to Schettler et al. (1997). Sediment core was transferred into a glove-bag and placed
under anoxic conditions (N2 atmosphere) immediately after
collection. It was then processed and split into cm-scale
fractions along the core’s vertical axis. The porewater was
separated from the solid phases once extracted from the
core barrel using RhizonÒ samplers connected via tubing
to septum capped vials under vacuum (filtration to
0.2 lm). During all the separation process, oxygen levels
were monitored with an Oxi 340i WTW oxygen meter
and were always below the detection limit of 0.1 mg/L.
The solid phases were then frozen at 20 °C and transferred to the laboratory. Samples were dehydrated by
lyophilization and stored in a glove-box under anoxic conditions before preparation for XRD and XAS.
2.3. Chemical analyses of the solid samples
Fe concentration measurements of particulate samples
from the sediment traps and core from the bottom of the
lake were performed in a clean laboratory. Weighed
samples of about 10 mg were loaded in Teflon beakers
and digested in a sequence of acid mixtures including
concentrated HF, HCl and HNO3. They were evaporated
to dryness and dissolved in 5 ml of HNO3 2%. The
solutions were diluted by 1000 in HNO3 2% for Fe concentration determination on a Neptune ThermoFinnigan
MC-ICP-MS (Multiple Collector Inductively Plasma Mass
Spectrometer). Fe concentrations were measured on mass
56 using high-resolution mode and standard bracketing
technique with a solution of known concentration. The
precision on Fe concentration is better than ±8% (relative
to the sample concentration), based on replicate analyses
of international standards (IFG, BCR2, ACE). The concentration of the blank was 28 ng Fe for 10 mg, thus
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
representing a negligible amount relative to the total Fe
measured on the samples (>20 lg Fe for 10 mg).
Organic carbon concentrations were determined on
powders from sediment traps and core after sample combustion on an elemental analyzer coupled to a Delta Plus
XP mass spectrometer. The method can be briefly described
as follows. Samples were decarbonated with diluted HCl
(0.5 M) at room temperature prior to combustion. The
residual powders were then rinsed with deionized water
and dried. Dry sample powders were weighed (10 mg),
loaded in tin capsules and placed into an autosampler.
The capsules were dropped into a combustion chamber oxidizing organic carbon to CO2. The combustion gases were
transferred in a helium carrier gas and analyzed by mass
spectrometry. Carbon concentrations were calculated based
on the peak area at mass 44 (12C16O16O), compared to standard of known concentrations. The concentration of the
blank was negligible compared to the samples concentrations. The precision on carbon concentration is better than
±5% (relative to the sample concentration).
2.4. Characterization of sample mineralogy
2.4.1. X-ray diffraction
Particles from sediment traps and sediment core samples
were studied by XRD. Samples were ground in an agate
mortar and deposited on a flat aluminum sample holder
in the glovebox. Measurements were performed using Co
Ka radiation on a Panalytical X’Pert Pro MPD diffractometer mounted in Bragg-Brentano configuration within an
anoxic sample chamber. Data were recorded in the continuous-scan mode over a 3–100° 2h range with a step of
0.033° and a counting time of 6 h per sample. Under these
conditions, the detection limit for a mineral phase in a complex natural sample is in the order of 1 wt%.
2.4.2. X-ray absorption spectroscopy
XAS were performed at the Fe K-edge on sediment trap
and core samples in order to determine the bulk speciation
of Fe, i.e., redox state and mineralogy of Fe-bearing phases.
2.4.2.1. XAS data collection. Pellets of 7 mm in diameter
were prepared in the glovebox. The sediment trap sample
collected at 86 m and the sediment core sample were prepared as pellets mixing 25 mg of sample and 15 mg of
cellulose. Cellulose is a weak X-ray absorber and is often
used as a mixing material to dilute samples for transmission
XAS measurements (e.g., Cancès et al., 2005). Pure pellets
(without cellulose) of 50 mg were prepared from sediment
trap samples collected at 25, 56 and 67 m. The pellets were
sealed between two layers of Kapton tape in the glovebox
and stored within aluminized paper sealed bags in order
to maintain anoxic conditions during transfer to the synchrotron. X-ray absorption near-edge structure (XANES)
and extended X-ray absorption fine structure (EXAFS)
spectra were collected at the Fe K-edge in fluorescence
mode on the SAMBA beamline at the SOLEIL synchrotron
(Saint Aubin, France), using a Si-drift detector. The analyses were conducted at a temperature of 10–15 K. For each
sample, between 3 and 6 spectra were collected within the
81
7000–7860 eV energy range, and stacked in order to
improve the signal-to-noise ratio. Energy was calibrated
by setting to 7112 eV the first inflection point of the Fe
K-edge XANES spectrum of a reference Fe(0) foil,
recorded simultaneously with the sample.
2.4.2.2. XAS data analysis. EXAFS data were extracted
with the Athena XAS data analysis software (Ravel and
Newville, 2005), using a threshold energy of 7125 eV. The
principal component analysis (PCA-TT) module of the
SIXPack software provided the minimum number of components needed to analyze the EXAFS dataset and the
target transformation procedure was used to identify the
most appropriate reference compounds for the linear combination fit. EXAFS spectra of the field samples were then
fit over a k-range of 2.5–9.5 Å1 using the linear combination least square fitting (LC-LSF) module of SIXPack.
Only the reference spectra improving the reduced v2 value
of the fit by >10% and accounting for more than 5% of
the total Fe absorption were kept in the final fit. According
to O’Day et al. (2004), 5% is approximately the detection
limit of a particular mineral in a mixture of Fe-bearing
compounds, although this value depends on several parameters, including the spectral uniqueness of this component
and the number of components in the mixture. Typical
errors on the fractions of each component in the mixture
provided by the LC-LSF procedure are in the order of
10%.
2.4.2.3. Iron reference compounds. The EXAFS spectra of
both natural field samples and synthetic samples were
recorded and used for the LC-LSF procedure. These reference samples were prepared for EXAFS measurements and
analyzed in fluorescence mode using the same procedures as
the Lake Pavin samples (e.g., reference samples were
diluted with cellulose to avoid overabsorption). Their
mineralogy was checked by powder XRD. Vivianite and
amorphous Fe(III)-phosphate were synthesized using the
procedure described in Eynard et al. (1992). Pyrite and
mackinawite were synthesized following Wei and
Osseo-Asare (1996) and Donald and Southam (1999),
respectively. Siderite was synthesized according to a protocol adapted from Jimenez-Lopez and Romanek (2004) and
Romanek et al. (2009). A field sample of Fe(III)-organic
matter (humates) complexes originating from the Rio
Negro (Amazon basin, Brazil) was also analyzed (Allard
et al., 2011). Reference phyllosilicate samples measured by
XAS include illite from Le Puy en Velais, France (Ildefonse
et al., 1998), Mg-rich chlorite (kindly provided by
D. Beaufort, Université de Poitiers, France) and biotite
(collection of minerals, Institut de Minéralogie et de
Physique des Milieux Condensés, Paris, France). XAS data
of carbonated green rust synthesized from the reduction of
lepidocrocite by Shewanella putrefaciens strain ATCC
12099 according to Ona-Nguema et al. (2002) and
published in Pantke et al. (2012) were also used. We finally
used previously published XAS data of 2-Line and 6-Line
ferrihydrite, hematite and goethite (Maillot et al., 2011).
Determining the exact proportions of Fe-phyllosilicates
present in the Lake Pavin samples based on EXAFS is
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J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
not achievable because the reference spectra for these compounds are not distinct enough from each other in the krange of 2.5–9.5 Å1. This is also the case for the spectra
of reference Fe-(oxyhydr)oxides. We therefore created a
model EXAFS spectrum representing Fe-bearing phyllosilicates using a linear combination of illite, chlorite and biotite spectra in proportions 50/30/20. We also created a
model EXAFS spectrum representing Fe-(oxyhydr)oxides
using a linear combination of 6-Line ferrihydrite and hematite spectra in proportions 80/20. The compositions of these
model spectra provided the best EXAFS fit for the Lake Pavin 25 m depth sample. These model spectra were finally
added to the library of reference spectra used for the EXAFS fitting of the sample spectra.
2.5. Scanning electron microscopy
The morphology of the particles settling in the water column of Lake Pavin was studied by SEM and the elemental
composition of the particles was determined by energy dispersive X-ray spectrometry (EDXS). Pieces of filters
(<0.2 lm) collected at different water depths were mounted
on aluminum sample holders using double-sided carbon
tape, and were carbon coated. SEM analyses were performed using a Zeiss ultra 55 SEM equipped with a field
emission gun. Images in the secondary electron (SE) mode
were acquired with the microscope operating at 3 kV and a
working distance (WD) of 2.5 mm, using the SE2 detector. Some SE images were also acquired at 10 kV and
7.5 mm WD. Backscattered electron (BSE) images were
acquired at 10 kV and a WD of 7.5 mm using the AsB
detector. Additional analyses were performed on sediment
trap samples. These samples were gently crushed in an
agate mortar and deposited on double-sided carbon
mounted on aluminum stubs, then carbon coated. For each
sample, ten to twenty Fe-bearing particles were spotted
using the BSE mode. SEM-EDXS analyses were performed
on these selected particles at 15 kV and a WD of 7.5 mm
after copper calibration. A total of 100,000 counts were
recorded for each measurement. Semi-quantification of
the spectra was achieved using the ESPRIT software
(Bruker) and the phi–rho–z method. SEM analyses were
performed within two weeks after sample preparation. As
no Fe oxidation state measurement can be performed with
SEM, above-described SEM sample preparation procedures were not designed to ensure the preservation of the
redox state of the samples.
microscope chamber flushed with nitrogen. Analyses were
then performed in a helium atmosphere. It has been shown
in previous studies that this protocol allows the preservation of the Fe oxidation state of the samples (e.g., Miot
et al., 2009).
STXM and X-ray absorption near edge structure
(XANES) analyses were performed on beamline 11.0.2.2.
of the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, USA) (Bluhm et al., 2006).
Energy calibration was achieved using the well-resolved
3p Rydberg peak of gaseous CO2 at 294.96 eV (Ma et al.,
1991). A 25 nm zone plate was used. Data included images
and image stacks, from which XANES spectra and maps
were retrieved. The aXis2000 software (Hitchcock, 2012)
was used for data processing.
The relative distributions of Fe(II)- and Fe(III)-phosphates in the samples were mapped using STXM stacks
of images taken at three different energies: below the Fe
L2,3 absorption edge (702 eV), at 708.3 (maximum absorption by Fe(II) phases), and at 710 eV (maximum absorption
by Fe(III) phases). The absorption intensities of a reference
Fe(II)-phosphate (vivianite) and an amorphous Fe(III)phosphate at these three energies were extracted from their
normalized XANES Fe L2,3-edge spectra (Miot et al.,
2009), and used to fit the 3-energy stacks of the samples
using the stack fit tool of aXis2000. The Fe(II) and Fe(III)
components of the fits were then mapped. The Fe(II) over
total Fe ratio (Fe(II)/Fetot) was determined for selected
Fe-phosphate particles by fitting the Fe L2,3-edge XANES
spectra obtained on these particles with linear combinations
of the normalized reference Fe(II)- and Fe(III)-phosphate
compounds using the CGO (Conjugate Gradient Optimization method) curve fit tool of aXis2000, as described in
Miot et al. (2009). Typical errors on Fe(II)/Fetot ratio estimations using this method are typically lower than 10%.
2.7. Transmission electron microscopy
Samples from the sediment traps were analyzed by
TEM. The same TEM grids used for previous STXM analyses were later used for TEM. The analyses were performed
using a JEOL 2100F (FEG) operating at 200 kV and
equipped with a field emission gun, a high resolution
UHR pole piece, and a Gatan energy filter GIF 200. Scanning transmission electron microscopy (STEM) observations were performed in the high angle annular dark field
mode (HAADF) and a probe size of 1 nm. EDXS mapping
was performed using the STEM mode.
2.6. Scanning transmission X-ray microscopy
3. RESULTS
Samples from the sediment traps were analyzed by scanning transmission X-ray microscopy (STXM) at the carbon
K-edge and at the Fe L2,3-edges. Each sample was resuspended in degassed distilled water and deposited on formvar-coated TEM grids within an anoxic glove-box. Grids
were dried at ambient temperature and mounted on a
STXM aluminum sample holder within the glove-box.
Then, they were stored in sealed bags made of aluminized
foil to preserve anoxic conditions during transfer to the synchrotron. The bags were opened at the beamline within the
3.1. Geochemical profiles in Lake Pavin water column
Fig. 1 shows the profiles of temperature, pH, conductivity at 25 °C (C25), turbidity and oxygen concentration in
the water column of Lake Pavin in September 2011. The
oxygen concentration profile shows that in September
2011 the redoxcline lies at a shallower depth (50 m depth)
compared to previous studies (usually around 60 m depth
for Michard et al., 1994; Viollier et al., 1995; Lehours et al.,
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
83
T (°C), O2 (mg/L), pH, Turbidity (NTU)
0
2
4
6
8
10
12
14
16
18
0
T
Turbidity
Vivianite
Depth (m)
20
O2
30
pH
40
Mixolimnion
Redoxcline
Monimolimnion
50
60
70
v
Intensity (a.u.)
10
v
v
v
vv
C25
i
i
v
v
v
v vv v
v
80
i
90
0
100
200
300
400
500
10
C25 (μS/cm)
Fig. 1. Geochemical profiles in the water column of lake Pavin.
Temperature (T), conductivity (C25), pH, oxygen concentration
(O2) and turbidity were measured in September 2011.
2005). Some temporal variations of the redoxcline’s depth
have indeed been evidenced, and are interpreted as
variations in the intensity and duration of the mixing of
the mixolimnion from one year to another (mainly correlated with meteorological conditions at the lake). Conductivity significantly increases below the redoxcline,
reflecting high concentrations of dissolved species in the
monimolimnion (Michard et al., 1994; Viollier et al.,
1995). A turbidity peak occurs at 52 m depth, indicating
either the precipitation of one or several solid phases or
the presence of high cell densities close to the redoxcline.
15
20
25
v
v v v
v v v
i
v v v
v
v v vv v vv
iv
Mn
a vv vv v v
Mn
i
i a
30
35
40
Sediment
Mn
86 m
Mn 67 m
56 m
25 m
45
50
Position [°2θ] (Co)
Fig. 2. XRD spectra of the samples from the water column (25, 56,
67 and 86 m depths) and the sediment from the bottom of Lake
Pavin. The spectrum of a reference vivianite is also plotted. Besides
vivianite (v), identified crystalline phases include Mn-oxide (Mn) as
well as detritic phases such as illite (i) and albite (a). The broad
peak between 20° and 32° (2h) in the sediment spectrum is due to
diatom frustules.
Table 1
Bulk contents of iron and organic carbon (Corg) of the samples
from the water column (25, 56, 67 and 86 m depths) and the
sediments at the bottom of Lake Pavin (92 m).
Depth (m)
Fe (wt%)
Corg (wt%)
3.2. Mineralogy and chemical composition of the particulate
matter in the water column and the sediments
25
56
67
86
Sediment
0.21
0.43
0.74
2.24
1.85
34.2
31.6
29.9
26.1
8.8
SEM observations show that particulate samples from
the water column and sediment core mostly contain diatom
frustules (silica), as previously described by Michard et al.
(1994) and Viollier et al. (1997) (diatom frustules are moreover detected in the sediment by XRD; Fig. 2). The mean
sedimentation flux in the water column, calculated using
masses of material collected in the sediment traps, is
0.12 ± 0.05 g m2 day1. Bulk sample concentrations of
Fe and organic carbon (Corg) were measured (Table 1).
The Corg content of the samples of water column particles
is relatively high, with concentrations from 34.2% at 25 m
depth slightly decreasing to 26.1% at 86 m depth. In contrast, the Corg of the sediment sample is only 8.8%. Bulk
Fe concentrations range between 0.21 and 2.24 wt% and
increase with depth in the water column, as previously
observed by Viollier et al. (1997).
Principal component analyses (PCA) performed on the
EXAFS spectra of the samples showed that the four first
principal components of the PCA explained more than
82% of the total variance of the system. Among the model
compounds spectra analyzed in this study, target
transformation analysis (e.g., Dublet et al., 2012) allowed
identifying four spectra as the most appropriate ones for
the linear-combination fitting of the samples EXAFS
spectra. The best linear combination fits of all samples EXAFS spectra were thus obtained by using vivianite, amorphous Fe(III)-phosphate, and model Fe-bearing
phyllosilicates and Fe-(oxyhydr)oxides EXAFS spectra.
Linear-combination fitting of the EXAFS spectra collected for sediments retrieved from 25 m depth show that
the Fe speciation is dominated by Fe-bearing phyllosilicates
and Fe-(oxyhydr)oxides (Figs. 3 and 4; Supporting
Information Table A). EDXS analyses of the Fe-bearing
particles at 25 m depth scatter on a line spreading between
pure Fe and pure Si endmembers (Fig. 5). This confirms
that these particles, apart from the Si-pure diatom frustules
fragments, correspond to Fe-(oxyhydr)oxides and
phyllosilicates.
The fits of the Fe EXAFS spectra for the sample collected at 56 m depth indicate that Fe-bearing phyllosilicates
and Fe-(oxyhydr)oxides are still present. In addition to
these phases, the best fit is obtained with the addition of
two new components, vivianite and amorphous Fe(III)phosphate (Fig. 4). From bulk EXAFS measurements, it
cannot be determined whether there is a single mixedvalence Fe(II)- and Fe(III)-phosphate phase or a
mixture of two phases, i.e., vivianite and amorphous
84
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
25 m
TF
k3 χ(k)
56 m
67 m
86 m
sediment
2.5
3.5
4.5
5.5
6.5
7.5
8.5
9.5 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
k (Å-1)
R(Å) + Δ R
Fig. 3. Fe K-edge experimental (solid line) and LC-LSF fitted (dotted line) EXAFS spectra (left) and real parts of Fourier transform (right) of
the samples from the water column of Lake Pavin (25, 56, 67 and 86 m depths) and the sediment at the bottom of the lake.
% Fe
0
20
40
wt %
60
80
25 m
100 0
0,5
1
1,5
2
2,5
Phyllosilicates
Fe-(oxyhydr)oxides
56 m
Am. ferric phosphate
Vivianite
67 m
86 m
Sediment
Fig. 4. Bar diagrams showing the results obtained from the least square - linear combination fitting of the Fe K-edge EXAFS spectra of the
samples from the water column (at 25, 56, 67 and 86 m depths) and the sediment at the bottom of Lake Pavin. Left: proportions of standard
Fe compounds used for linear combination fitting of the Fe K-edge EXAS spectra of the samples, normalized to the total Fe concentration of
the samples. Right: mass concentration of each Fe-bearing species to the total mass of the samples (in wt%) deduced from these fits.
Fe
100
25 m
0
56 m
80
20
60
67 m
40
60
80
20
P
0
100
86 m
40
100
80
60
40
20
Si
0
Fig. 5. Ternary Fe, P, Si diagram of settling Fe-bearing particles
collected in the water column of Lake Pavin (at 25, 56, 67 and 86 m
depths). Fe, P and Si relative abundances were obtained from
SEM- and TEM-EDXS measurements. The solid line corresponds
to the Fe/P molar ratios of vivianite mixed with Si (Fe/P = 1.5).
Fe(III)-phosphate. Crystalline vivianite is however not detected by XRD at this depth, being likely below the detection limit for this method (Fig. 2). EDXS analyses confirm
the presence of phyllosilicates and Fe-(oxyhydr)oxides
(Fig. 5). Particles enriched in P are also observed. Their
mean Fe/P ratio is relatively high (Fe/P 2.88), and they
could consist of Fe-(oxyhydr)oxides on which PO43 is
sorbed, and/or Fe-phosphates (e.g., Voegelin et al., 2013).
At 67 m depth, the best EXAFS fit is obtained for a mixture of vivianite and Fe(III)-phosphate, along with phyllosilicates and Fe-(oxyhydr)oxides (Fig. 4), with a higher
proportion of vivianite (37%) as compared to the 56 m
depth sample (12%). The presence of vivianite at this
depth is confirmed by XRD (Fig. 2). EDXS analyses
(Fig. 5) indicate that the mean Fe/P molar ratio for the
Fe-phosphates is 1.92.
Vivianite is the dominating Fe-bearing phase detected at
86 m by EXAFS spectroscopy (Fig. 4). This is supported by
XRD (Fig. 2) and EDXS (Fig. 5) analyses. The average
Fe/P ratio of the Fe-phosphates analyzed by EDXS for this
sample is 1.68, consistent with a higher proportion of
vivianite present at this depth compared to 67 m depth.
Fe-(oxyhydr)oxides and phyllosilicates are detected by
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
EXAFS at 86 m depth as well, but not by the few EDXS
analyses that were performed.
It should be noted that Fe sulfide colloidal species (FeS)
previously described in the monimolimnion of the lake
(Bura-Nakić et al., 2009) are observed neither by EXAFS
nor by EDXS analyses. However, the addition of pyrite
EXAFS spectra significantly improve the quality of
EXAFS fit for the 67 m depth sample (v2 was reduced by
more than 50%), but this compound is discarded since it
contributes to less than 5% to the total Fe absorption.
When corrected from the variations of the total Fe
content of the samples with depth (Fig. 4), the absolute concentrations (relative to the sample mass) of phyllosilicates
and Fe-(oxyhydr)oxides measured by EXAFS in the particulate fraction of the water column remain approximately
unchanged from 25 m down to 86 m. In contrast, the abundance of vivianite clearly increases with depth.
In the sediment core sample (from 92 m depth), the only
Fe-bearing phase detected by EXAFS is vivianite (Fig. 4).
XRD confirmed that vivianite is the main crystalline phase
composing these sediments (Fig. 2). Pyrite, suggested by
previous studies to be an important Fe-bearing phase in
the sediments (Viollier et al., 1997; Schettler et al., 2007),
is not detected by these techniques in the top 6 cm of
the core.
3.3. Evolution of the bulk iron oxidation state of the particles
from the water column and sediments
Fig. 6 shows the normalized XANES Fe K-edge spectra
acquired on the sediment trap samples and the sediment
core (the Fe K pre-edges are also presented in SI Fig. A).
XANES spectra of vivianite, amorphous ferric phosphate
and ferrihydrite reference compounds are also shown. The
positions of the Fe K-edge (e.g., Waychunas et al., 1983)
and the pre-edge feature (Wilke et al., 2001) are used to
estimate the Fe redox state of Fe-bearing minerals. The
measured positions of the crest of the Fe K-edge differ by
more than 4 eV between vivianite and amorphous ferric
Normalized µ(E)
Vivianite
Sediment
86 m
67 m
56 m
Am. ferric
phosphate
25 m
7100
Ferrihydrite
7120
7140
7160
7180
Energy (eV)
Fig. 6. Normalized XANES Fe K-edge spectra of the samples
from 25, 56, 67 and 86 m depths in the water column and from the
sediment at the bottom of the lake. Spectra of reference ferrous
phosphate (vivianite), amorphous ferric phosphate and ferrihydrite
are given for comparison. Dashed lines correspond to the fits of the
samples spectra with linear combinations of vivianite and amorphous ferric phosphate spectra, used for Fe(II)/Fetot ratio calculations (see Table 2).
85
phosphate and the positions of their pre-edge features are
separated by 1.2 eV. Lake Pavin samples all fall between
these two end-member values. A rough estimate of the
Fe redox state of the samples is obtained by fitting the Fe
K-edge XANES spectra with linear combinations of Fe
K-edge XANES spectra of vivianite and amorphous
Fe(III)-phosphate (Table 2). These estimates were only performed on samples containing Fe-phosphates (i.e., samples
from 56, 67, 86 m depths and the sediment) and are more
accurate for the deeper samples which contained higher
concentrations of these phases. The calculated bulk
Fe(II)/Fetot ratio is 20% for the sample at 56 m depth
and progressively increases up to 100 % in the sediment.
For comparison, ranges of Fe(II)/Fetot ratios calculated
with the mineralogical compositions determined by EXAFS
fits (using estimated Fe(II)/Fetot ratios of the phyllosilicates
of 60–90% for chlorite, 80–100% for biotite and 0–50% for
illite) are also given in Table 2. The ratios calculated based
on XANES spectra fall within these ranges. These results
are in good agreement with an increase of the vivianite
content with depth in the water column. Moreover,
XANES spectra show that about 20 % of Fe(III) is still
present at a 86 m depth, consistently with the presence of
Fe-(oxyhydr)oxides detected by EXAFS at this depth. In
contrast, all the Fe is ferrous in the sediment, in agreement
with EXAFS data, which indicate that Fe is mostly present
as vivianite.
3.4. Microscopy analyses of the particles from the water
column and sediments
Fe-bearing particles were located by STXM at the Fe
L2,3-edges using maps of Fe obtained by subtracting an image acquired below the edge (702 eV) and converted into
optical density (OD) from an image acquired above the
edge (708.3 or 710 eV) and converted into OD. The same
particles were then systematically re-observed by TEM
and their elemental composition was determined by
TEM-EDXS. Particles consisting mostly of Fe and O with
some Si are observed at 25 m depth and are interpreted as
Fe-(oxyhydr)oxides. The low Si signal originates from associated diatom frustules and/or Si precipitated on the
particles. TEM-EDXS analyses performed on the Fe-(oxyhydr)oxides sometimes show small amounts of Ca, P, Cl, S,
Table 2
Calculated Fe(II)/Fetot ratios of the samples collected at 56, 67 and
86 m depths in the water column and from the sediment at the
bottom of the lake. Ratios were obtained by fitting Fe K-edge
XANES spectra with a linear combination of the reference
vivianite and amorphous ferric phosphate spectra on the one hand
(second column-XANES), and estimations of the Fe(II)/Fetot of
the reference compounds used for the EXAFS fits on the other
hand (fourth column–EXAFS). The chi squared (v2) values,
measuring the quality of the XANES fits, are provided.
XANES
Sample
Sediment
86 m
67 m
56 m
Fe(II)/Fetot
1.0
0.8
0.5
0.2
EXAFS
v2
0.0154
0.0093
0.0211
0.0239
Fe(II)/Fetot
1.00
0.7–0.8
0.5–0.6
0.2–0.3
86
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
K and Mg. Fe-bearing phyllosilicates, composed mostly of
Si and O with various amounts of Al, K, Mg, Ti and Ca are
also found. At 56, 67 and 86 m depths, besides Fe-(oxyhydr)oxides and Fe-bearing phyllosilicates, particles rich
in Fe, O and P with varying smaller amounts of K, Cl,
Ca, Mg and S are observed and interpreted as Fe-phosphates. Distribution of Fe(III) and Fe(II) in these particles,
as well as their calculated Fe/P ratios, are shown in Fig. 7.
At 56 m depth, all the analyzed Fe-phosphates are
dominated by Fe(III) (Fig. 7a). At 67 and 86 m depths,
a
Fe-phosphates contain both ferric and ferrous Fe (Fig. 7b
and c). Fe(II)/Fetot ratios are calculated for selected
Fe-phosphate particles (Fig. 7e). The limited number of
particles analyzed does not allow us to provide a statistical
analysis of the average Fe(II)/Fetot ratio at different depths
(which is provided by bulk XAS), but it shows that single
Fe-phosphate particles have diverse Fe(II)/Fetot at a given
depth, ranging for example between 20% and 90% at
67 m depth or 40% and 95% at 86 m depth. Mixedvalence Fe-phosphates are therefore present in these
56 m
Fe(II)
Fe(III)
2.8
Fe(II)
Fe(I
Fe
(III)
(I
Fe(III)
Fe(II)
Fe
F
(II)
Fe(III)
Fe(III)
2.9
2.4
2.2
5 µm
b
67 m
2.3
5 µm
c
86 m
1.4
1.7
5 µm
1.9
e
d
1
2
3
Optical density (a.u.)
Fe P Sii
700
vivianite
1
2
3
Fe/P
1.6
1.7
1.9
Fe(II)/Fetot
0.9
0.8
0.4
Am. Fe(III)-PO4
710
720
730
Energy (eV)
Fig. 7. Fe L2,3-edge STXM and TEM analyses of the samples collected at 56, 67 and 86 m in the water column. (a–c) Left: images at 702 eV.
Middle: distribution of Fe(II). Brighter areas are enriched in Fe(II). Right: distribution of Fe(III). Brighter areas are enriched in Fe(III). The
circles correspond to particles identified as Fe-phosphates based on the TEM-EDXS compositions. Calculated Fe/P ratios of these particles
are indicated below the circles. Several such images and distribution maps were acquired for each sample. (d) TEM-EDXS map of Fe, P and Si
performed on the square area in (c), showing three Fe- and P-rich particles labelled 1, 2 and 3. (e) Fe L2,3-edge XANES spectra obtained on
particles 1, 2 and 3. The spectra of vivianite and amorphous Fe(III)-phosphate used to calculate the Fe/Fetot ratios are shown. The Fe/P ratios
of these three particles are also given.
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
samples. The presence of Fe-phosphate particles with
Fe(II)/Fetot ratios close to 1 is moreover consistent with
XRD results showing the presence of vivianite at these
depths.
Particles collected at different depths were imaged by
SEM in the SE mode (Fig. 8, SI Fig. B). At 67 and 86 m
depths, a great number of Fe-phosphate particles 1–1.5 lm
in diameter in size appear as filaments and/or round-shaped
objects with a granulated surface. In the BSE mode, these
particles appear as chains or clusters of cocci-shaped compartments reminiscent of microbial shapes.
STXM and TEM analyses were performed to further
examine associations between microbes and Fe-phosphates.
At 86 m depth, some Fe-bearing particles resembling morphologically encrusted microbial cells were analyzed by
STXM at the Fe L2,3 edges and the C-Kedge (Fig. 9).
The XANES spectra of these particles at the C-K edge
show that they are composed of organic carbon, with peaks
at 285.2, 286.8 and 288.5 eV. These peaks have been
observed previously for biomineralized bacteria and were
assigned to 1s ! p* electronic transitions in aromatic
groups, ketonic or phenolic groups, and carboxylic groups
respectively (Benzerara et al., 2004). Moreover, a peak at
290.4 eV is detected in the C-K edge XANES spectra of
the organic polymers outside the cells. Such a peak has been
previously observed for bacteria and associated polymers
obtained from Fe-rich aqueous environments by Benzerara
a
et al. (2008) and Chan et al. (2009); this peak has been
interpreted to represent Fe complexation by carboxyl
groups of polysaccharides. It is also worth noting that Fe
is consistently distributed at the border of the putative bacterial cells as shown by the Fe L2,3-edge map (Fig. 9b).
Finally, at 25, 56 and 67 m depths, many bacteria containing intracellular round-shaped granules were observed
by STXM and TEM (Fig. 10). These inclusions are
mainly composed of P and O, with smaller amounts of
Ca at 25 m depth, as well as Fe and sometimes Cl and
S at 56 m and 67 m (Fig. 10d and e). The Fe/P molar ratios of these inclusions obtained from TEM-EDXS, using
10 bacteria for each sample, are 0.11 (±0.02) and
0.10 (±0.02) at 56 and 67 m depths, respectively. No
bacterium containing P-inclusions were found at 86 m
depth. Based on their morphology and chemical composition, these inclusions are interpreted as polyphosphate
bodies. Metal ions such as Ca and Fe are known to be
frequently associated with microbial polyphosphates
(e.g., Kornberg, 1995; Irani and Morgenthaler, 1963). Cl
and S probably originate from the microorganisms themselves or are present as salts having precipitated during
sample preparation for TEM.
Altogether, these microscopy observations suggest that
at least some of the Fe-phosphates precipitated in the water
column of Lake Pavin may be formed in association with
bacteria.
b
c
e
87
d
f
Fig. 8. SEM images of Fe-bearing particles from 67 m depth collected on a filter. (a) Large scale view in SE mode. The majority of the
particles are diatoms frustules, but some granulated iron-containing particles are also present. (b) Close-up of a single iron-bearing particle in
SE mode. A fracture on the upper right shows that the particle is empty. (c–f) Several particles observed in the SE mode (left) and in the BSE
mode. In the latter mode, the particles appear to be formed by chains or clusters of single 1–1.5 lm void cocci-shaped compartments. Similar
particles were also observed in the 86 m depth sample. Scale bar is 1 lm for all images.
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
a
b
c
1 µm
1 µm
f
Optical Density (a.u.)
88
280 282 284 286 288 290 292 294
Energy (eV)
c
d
e
500 nm
500 nm
500 nm
Fig. 9. STXM analyses of bacteria encrusted by an iron phase at 86 m depth in the water column of Lake Pavin. (a) Image at 700 eV (below
the Fe L3-edge). (b) Iron map obtained by subtracting an OD-converted image at 700 eV from an OD-converted image taken at 710 eV. (c)
Image at 280 eV (below the C K-edge). (d) Organic carbon map obtain by substracting an OD-converted image at 280 eV from an ODconverted image taken at 288.5 eV (e) Two colors composite map obtained by using the Stack fit procedure in aXis2000. Spectra
representative of the inside (red) and border (blue) of the cells were used as model spectra. The two component maps obtained were then
overlaid (in blue and red). (f) C K-edge XANES spectra resulting from the Stack fit procedure, corresponding to the areas mapped in (e). The
colors (blue and red) correspond to the same color areas in (e). The vertical dotted lines correspond to 285.2, 286.8, 288.5 and 290.4 eV. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. DISCUSSION
The points discussed in the following sections about Fe
and P cycling in the water column of Lake Pavin are
summarized in Fig. 11.
4.1. Origin and fate of Fe-(oxyhydr)oxides in the water
column and sediment of Lake Pavin
Fe-(oxyhydr)oxides are detected both in the mixolimnion and monimolimnion of Lake Pavin (Figs. 4 and 5).
All previous studies proposed that Fe-(oxyhydr)oxides
form authigenically in the mixolimnion and/or at the transition zone between the mixo- and monimolimnion through
O2-driven oxidation of soluble Fe2+ diffusing upward from
the monimolimnion (Michard et al., 1994; Viollier et al.,
1997; Schettler et al., 2007). The formation of Fe-(oxyhydr)
oxides (along with Fe-phosphates) at the redoxcline of the
lake might cause the turbidity peak observed at 52 m
depth. Facultative Fe(III)-reducers were identified in the
monimolimnion of Lake Pavin (Lehours et al., 2007, 2009),
and a fermentative bacterial strain called BS2 was isolated
from the water column/sediments interface at 92 m depth,
and was shown to perform significant and rapid (less than
48 h) reduction of hydrous ferric oxide when cultivated in
the presence of glucose (Lehours et al., 2010). Similarly,
anaerobic methane oxidation coupled with Fe reduction
may also occur in the upper part of the monimolimnion
of Lake Pavin (Lopes et al., 2011). However, bulk
mineralogical analyses suggest that the amount of Fe-(oxy-
hydr)oxides is roughly constant with depth throughout the
mixolimnion and monimolimnion, and decreases drastically
only within the sediments at the bottom of the lake (Fig. 4).
This implies that, at least at the time of sample collection,
dissimilatory Fe reduction occurred actively within or at
the surface of the sediments at 92 m depth and had limited
impact on the Fe(II)–Fe(III) balance in the water column.
This interpretation is consistent with the fact that the Corg
content of the samples only slightly decreases in the water
column, whereas Corg is heavily depleted in the sediment
(Table 1). This indeed indicates that organic matter is
strongly affected by mineralization processes within the
sediments or at their surface, at least in part through
dissimilatory Fe reduction (archaeal methanogenesis is also
a potentially important metabolic pathway at the bottom of
the lake; Lehours et al., 2005; Assayag et al., 2008).
The preservation of Fe-(oxyhydr)oxides in the anoxic
water column of the lake might seem surprising, since the
residence time of the these particles in the monimolimnion
is estimated to be 40–100 days according to sedimentation
rates calculated by applying Stokes’ Law for 1–5 lm diameter Fe-(oxyhydr)oxides particles (Posth et al., 2010). This
lapse of time is most probably a lower estimate since the relatively large Fe-(oxyhydr)oxides particles observed by SEM
are most likely aggregates of smaller colloidal particles
measuring up to a few tens of nm (e.g., Buffle et al.,
1989), and therefore probably have much lower settling
velocities. However, several hypotheses can be proposed
to explain the preservation of Fe-(oxyhydr)oxides in the
monimolimnion and higher reductive dissolution in the
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
1 µm
P
e
Fe
Fe Kα
67 m
67 m
Ca Kα
d
c
1 µm
Counts (a.u.)
1 µm
56 m
P Kα
b
Cl Kα
25 m
C Kα
O Kα
Cu Lα
a
89
1 µm
Ca
25 m
67 m
0
1
2
3
4
5
6
7
Energy (keV)
Fig. 10. TEM images of polyphosphate-accumulating bacteria present in the samples collected at 25, 56 and 67 m depths. (a and b) TEM
images of the samples collected at 25 and 56 m depths, respectively. (c) STEM-HAADF image of the sample from 67 m depth. The
polyphosphates appear as electron-dense granules inside the cells. (d) STEM-DF image and TEM-EDXS maps of P, Ca and Fe obtained on
polyphosphate-accumulating bacteria from 67 m depth. (e) TEM-EDXS spectra obtained on intracellular polyphosphates at 25 and 67 m
depths.
sediments. Electron donors needed for bacterial Fe reduction are likely more concentrated in the bottom sediments
than in the water column. Fe-reducing bacteria might also
be more abundant in the sediments than in the water column of the lake. Such hypotheses will need further testing
in the future.
It is worth noting that Mn-oxide particles are detected
by XRD in the samples from 67 and 86 m depth (Fig. 2).
The formation of Mn-oxides below the redoxcline of Lake
Pavin had previously been predicted by Viollier et al.
(1995). Mn-oxides are not present in the sample of sediment
from the bottom of the lake, suggesting that, similarly as
Fe-(oxyhydr)oxides, they might be dissolved at the sediment–water interface.
4.2. Bacterial origin of the Fe-phosphates formed in the water
column of Lake Pavin
Bulk EXAFS, XRD, STXM and TEM data show that
amorphous mixed-valence Fe(II)–Fe(III)-phosphate phases
form close to the mixolimnion–monimolimnion interface
(56 m). Deeper in the monimolimnion, mixed-valence
Fe(II)–Fe(III)-phosphate particles are still observed, along
with vivianite. The very frequent observation of Fe-phosphates particles having morphologies reminiscent of
encrusted microbial shapes with SEM (Fig. 8) suggest that
a significant fraction of the Fe-phosphates (which will have
to be quantified in the future) is formed at the surface of
microorganisms. Consistently, STXM analyses at the C
K- and Fe L2,3-edges show that Fe is distributed at the rims
of some putative bacterial cells, suggesting that some Febearing phases nucleate at the surface of microorganisms
(Fig. 9). The formation of mixed valence Fe(II)–Fe(III)phosphates in a freshwater environment has previously
been described by Hyacinthe and Van Cappellen (2004).
However, microbial activity was not suggested as a catalyst
in that case. Direct evidence of bacterial Fe-phosphate biomineralization in natural environments is very sparse
(Konhauser, 1998). To our knowledge, the only existing
field example in the literature includes the observation of
cyanobacteria and bacteria found in an epilithic microbial
biofilm from Arctic Canada precipitating amorphous
Fe-phosphate grains with a composition similar to strengite
(FePO42H2O) in their periplasmic space and capsule
90
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
(1)
(2)
MIXOLIMNION
(3)
PO4
PO4
FeOOH
PAO
REDOXCLINE (50-60m depth)*
ACCUMULATION
FeOOH
FeOx
-PO4
MONIMOLIMNION
Fe2+
Fe3+
FeOx
Fe(II)Fe(III)PO4
PRECIPITATION
HYDROLYSIS
PAO
REDUCTIVE
DISSOLUTION
FeOOH
PO4
Fe2+
REDUCTIVE
DISSOLUTION
PO4
Fe2+
PO4
FeOx
Fe(II)Fe(III)PO4
Fe2+ PO4
Fe2+
PO4
vivianite
vivianite
SEDIMENT
Fig. 11. Model of Fe and P cycling in the water column of Lake Pavin. Three mechanisms concentrating dissolved inorganic phosphate (PO4)
in the monimolimnion of the lake are presented: (1) scavenging of PO4 by adsorption to or coprecipitation with Fe-(oxyhydr)oxides (FeOOH)
close to the redoxcline, followed by FeOOH reductive dissolution within or at the surface of the sediments at the bottom of the lake. Dissolved
Fe(II) (Fe2+) and PO4 then diffuse upward in the monimolimnion. (2) Polyphosphate accumulating organisms (PAO) accumulate
polyphosphates intracellularly in the oxic mixolimnion and hydrolyze them in the anoxic monimolimnion, generating PO4 in this layer. (3)
Mixed valence Fe(II)–Fe(III)-phosphates (Fe(II)Fe(III)PO4) precipitate close to the redoxcline in association with bacteria, possibly in
connection with Fe-oxidation (FeOx) in this PO4-rich environment. The Fe(II)Fe(III)PO4 are reductively dissolved within or at the surface of
the sediments, generating a Fe2+ and PO4 flux from the sediments to the monimolimnion. High local Fe2+ and PO4 concentrations created by
these three mechanisms allow the precipitation of vivianite in the sediments and in the bottom of the monimolimnion. *The depth of the
redoxcline can vary from one year to another.
(Konhauser et al., 1994). Some of these bacterial cells were
completely encrusted by Fe-phosphates. Similar mechanisms of Fe-phosphate biomineralization may occur in
Lake Pavin.
Bacteria can induce the precipitation of Fe-phosphates
by several non-exclusive mechanisms. Firstly, negatively
charged extracellular polysaccharides and/or surface proteins and/or lipids can act as templates for the adsorption
of soluble cations (Beveridge and Murray, 1980; Ferris
and Beveridge, 1985; Geesey and Jang, 1989; Konhauser
et al., 1994; Konhauser, 1998), favoring the nucleation of
minerals at the surface of bacteria. Secondly, bacteria
may locally increase PO43 and/or Fe activities through
metabolic activity, so that the required critical saturation
state is exceeded and precipitation occurs. Hereafter, we
discuss which types of microorganisms may increase the
saturation of Fe-phosphates in Lake Pavin and how biomineralization may proceed.
Bacterial reduction of Fe-(oxyhydr)oxides in a PO43rich solution has been shown to trigger the formation of
Fe(II)-bearing secondary phases such as vivianite (Zachara
et al., 1998; Fredrickson et al., 1998; Jorand et al., 2000;
Glasauer et al., 2003) or other types of Fe(II)-phosphate
phases (Peretyazhko et al., 2010) in cultures. Vivianite formation was also observed in response to the reduction of
dissolved Fe(III) (as Fe(III)-citrate) by Geobacter metallireducens (Lovley and Phillips, 1988). In some cases, the bacteria become encrusted by precipitating Fe(II)-phosphates,
which likely nucleate on extracellular polymeric substances
(Peretyazhko et al., 2010). As mentioned above, Fe-reducing bacteria have been isolated from the anoxic water
column of Lake Pavin (Lehours et al., 2009, 2010). These
bacteria are thus potential candidates for explaining
Fe-phosphate biomineralization in Lake Pavin.
However, several observations suggest that dissimilatory
Fe reduction may not be a major process in Fe-phosphate
formation in Lake Pavin. First, as discussed above, no
significant reductive dissolution of Fe-(oxyhydr)oxides
was observed in the monimolimnion of Lake Pavin.
Second, the Fe-phosphate phases formed at the top of the
monimolimnion contain a substantial amount of Fe(III)
(up to 80%), as shown by EXAFS and STXM analyses
(Figs. 4 and 7). Since dissolved Fe(III) concentrations
should be extremely low at the pH prevailing in the
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
monimolimnion of Lake Pavin (pH 6.2), it is unlikely that
Fe(II)–Fe(III)-phosphates can form by direct precipitation
from the solution following dissimilatory Fe reduction.
As a result, the major redox process leading to Fe(II)–
Fe(III)-phosphates precipitation at the top of the
monimolimnion is more likely Fe oxidation of dissolved
Fe(II) rather than Fe reduction. The formation of colloidal
Fe(II)–Fe(III)-phosphates at the redoxcline of a stratified
lake has previously been described by Buffle et al. (1997)
and similarly interpreted as resulting from the oxidation
of Fe(II) into Fe(III). Moreover, it has been shown
experimentally that oxidation of Fe(II) into Fe(III) in a
solution rich in PO43 (with initial dissolved P/Fe ratio
greater than 0.55, as is the case at the redoxcline of Lake
Pavin; Michard et al., 1994) at neutral pH leads to the
precipitation of amorphous Fe-phosphates (Voegelin
et al., 2010, 2013; Kaegi et al., 2010). The Fe/P molar ratio
of the Fe-phosphates formed during these experiments
(1.82) is close to the mean ratio of the Fe-phosphates
present just below the redoxcline of the lake (1.92).
Interestingly, there is a wide diversity of Fe-oxidizing
bacteria encompassing diverse metabolic processes
(Kappler and Straub, 2005; Weber et al., 2006a). Fe(II)oxidizers are widely scattered phylogenetically (e.g.,
Hedrich et al., 2011) and thus identifying Fe-oxidizing
bacteria based on 16S rRNA analyses only can be difficult.
The isolation of Fe-oxidizers has not been attempted yet on
Lake Pavin samples, and therefore we cannot identify putative Fe-oxidizing bacteria from prior 16S rRNA studies.
Yet, we suggest that microbially mediated Fe oxidation
might be an alternative candidate process involved in Fephosphate biomineralization in Lake Pavin. Indeed the
neutrophilic Fe-oxidizing Acidovorax sp. strain BoFeN1
has been shown to induce biomineralization of amorphous
mixed valence Fe(II)- and Fe(III)-phosphates in a medium
with high Fe2+ and PO43 concentrations similar the concentrations prevailing in Lake Pavin (Miot et al., 2009).
Part of the Fe-phosphate precipitation with BoFeN1 occurs
within the periplasm of the cells, forming a continuous
40-nm thick layer that encrusts the cells and produce fossil
cells similar to the ones observed in Lake Pavin (Miot et al.,
2011).
Bacterial oxidation of Fe(II) at circumneutral pH can
occur in microaerophilic and/or anoxic environments. In
the first case, bacteria use Fe(II) as an electron donor for
lithotrophic growth, and O2 as the electron acceptor (e.g.,
Emerson et al., 2010). Oxygen-dependent Fe-oxidizing
bacteria grow at O2 concentrations comprised between
<5 lM and 50 lM, the upper limit corresponding to the
oxygen level at which abiotic rates of Fe oxidation becomes
significantly higher than biotic rate (Druschel et al., 2008).
In Lake Pavin, such microaerophilic conditions are reached
between 45 and 56 m depths, which is where most mixed
valence Fe-phosphates likely form. Interestingly, the microaerophilic Fe-oxidizing bacterium Gallionella ferruginea has
been identified by 16S rRNA sequencing close to the
redoxcline of Lake Pavin (Lehours et al., 2007) but no
biomineralized stalk was detected in the present study.
In anoxic environments, bacterial oxidation of Fe(II)
results either from anoxygenic photosynthesis (Widdel
91
et al., 1993), or nitrate reduction (Straub et al., 1996).
Anoxygenic phototrophic green sulfur bacteria have been
identified at the >100 m deep chemocline of lake Matano,
the world’s largest ferruginous basin (Indonesia) (Crowe
et al., 2008b). Walter et al. (2009) have demonstrated the
existence of photoferrotrophic activity in the ferruginous
meromictic lake La Cruz (Spain). The maximum rate of
light-dependent Fe(II) oxidation was observed close to the
chemocline, at a depth of 11 m, where light intensities
as low as 0.02–0.002% of surface photosynthetically active
radiation (PAR) were measured. In Lake Pavin, the value
of 0.002% of surface PAR is reached deeper (56 m, SI
Fig. C), i.e., close to where mixed valence Fe-phosphates
likely form. Fe(II) oxidation through anoxygenic photosynthesis conducting to Fe-phosphates precipitation at this
depth can therefore not be ruled out.
Alternatively, Fe-oxidizing nitrate-reducing bacteria
have been observed in a variety of sedimentary environments (e.g., Straub and Buchholz-Cleven, 1998; Ratering
and Schnell, 2001; Hauck et al., 2001; Shelobolina et al.,
2003; Weber et al., 2006b) but they have never been
described in a water column yet. The nitrate concentration
profile in Lake Pavin is consistent with nitrate reduction
just below the redoxcline (Lopes et al., 2011). The activity
of nitrate-dependent or other types of Fe-oxidizing bacteria
in the Lake Pavin water column will have to be investigated
by future studies.
In the sediments at the bottom of the lake (92 m
depth), the main Fe-bearing phase is pure-Fe(II), wellcrystallized vivianite. Vivianite is also present in the water
column at 67 and 86 m depth (Figs. 2 and 4). The formation
of vivianite in the monimolimnion of the lake had been predicted by previous studies of the aqueous geochemistry of
the lake (Michard et al., 1994; Viollier et al., 1997). Supersaturation indices with respect to vivianite calculated using
geochemical data from Michard et al. (1994) are indeed 1.6
just below the redoxcline and progressively increase with
depth to reach 5.7 at the bottom of the lake (88 m depth)
(SI Fig. D). Authigenic vivianite formation in lacustrine anoxic sediments has been observed previously (Emerson and
Widmer, 1978; Manning et al., 1999; Fagel et al., 2005;
Sapota et al., 2006; Owen et al., 2010), but to our knowledge Lake Pavin is the only example where vivianite forms
within the water column. The precipitation of vivianite
likely results from the high Fe2+ concentrations generated
in the lower part of the monimolimnion and the sediment
porosity by the reductive dissolution of Fe-(oxyhydr)oxides
and mixed valence Fe-phosphates. The high PO43 concentrations required to reach vivianite supersaturation at the
bottom of the lake are sustained by the combined effects
of (1) bacterial degradation of organic matter within the
sediments of the lake (Michard et al., 1994; Viollier et al.,
1997), (2) liberation of PO43 adsorbed on the Fe-(oxyhydr)oxides, (3) dissolution of Fe(II)–Fe(III)-phosphates
within or at the surface of the sediments, (4) input of dissolved PO43 in the lake by groundwater inflows in the
mixolimnion (Aeschbach-Hertig et al., 2002; Schettler
et al., 2007; Assayag et al., 2008; Bonhomme et al., 2011)
and (5) limited PO43 escape to the mixolimnion due to
scavenging by Fe-(oxyhydr)oxides and Fe-phosphates
92
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
precipitation close to the redoxcline. An additional PO43concentrating mechanism involving polyphosphate-accumulating bacteria is discussed below. Alternatively, the formation of vivianite could be due to the topotactic
conversion of the mixed valence Fe-phosphates, e.g.,
through the progressive reduction of Fe(III) in these
phases. The possibility that such a process occurs under
conditions prevailing in the monimolimnion of Lake Pavin
will have to be further investigated.
4.3. Role of polyphosphate-accumulating microorganisms in
Fe-phosphate precipitation
Another potential process that may trigger Fe-phosphate precipitation in association with microbial cells
involves the potential activity of polyphosphate-accumulating bacteria. A link between polyphosphate accumulation
and Fe-phosphate biomineralization was suggested by
Konhauser et al. (1994), who proposed that the PO43
source for the precipitation of Fe-phosphates in epilithic
microbial biofilms in the Canadian Arctic might be the
decomposition of polyphosphate granules observed within
these cells. A wide diversity of microorganisms including
Bacteria and Eukarya can form intracellular polyphosphates (e.g., Harold, 1966; Seviour et al., 2003). Interestingly, it has been proposed that the precipitation of
calcium phosphates in marine environments leading to
phosphorite formation may result from the activity of polyphosphate-accumulating microorganisms (Schulz and
Schulz, 2005; Diaz et al., 2008; Goldhammer et al., 2010).
For example, the large sulfur-oxidizing bacterium Thiomargarita namibiensis accumulates intracellular polyphosphate
granules under oxic conditions. During anoxic episodes,
the same bacterium derives energy from the hydrolysis of
these polyphosphates, thus liberating PO43. This generates
high local inorganic phosphate concentrations, triggering
calcium phosphate precipitation (Schulz and Schulz, 2005;
Goldhammer et al., 2010). Additionally, sedimentary apatite has sometimes been found to form in close association
with polyphosphate grains in sediments, suggesting that
polyphosphates may also directly nucleate apatite growth
by acting as a mineral template (Diaz et al., 2008).
In Lake Pavin, we observe polyphosphate accumulating microbial cells at 25, 56 and 67 m depths (Fig. 10).
The bacteria likely accumulate intracellular polyphosphates under oxygenated conditions in the mixolimnion.
Then, the cells may hydrolyze polyphosphates during settling in the anoxic monimolimnion. Consistently with this
hypothesis, no polyphosphate inclusion was observed in
the bacteria imaged at 86 m depth, whereas they were
abundant in the mixolimnion (25 and 56 m) and close to
the redoxcline of the lake (67 m). The hydrolysis of
polyphosphates in the anoxic water column of Lake Pavin
might represent a substantial contribution to the monimolimnion PO43 pool. Considering that diatoms form an
important fraction of the settling material in Lake Pavin
and that some diatoms are known to accumulate
intracellular polyphosphates (Diaz et al., 2008; Nuester
et al., 2012), their degradation within the sediments
may also play a role in the PO43 enrichment of the
monimolimnion.
The relative importance of polyphosphates versus PO43
linked to Fe-(oxyhydr)oxides in P cycling has been quantified within the hypoxic water column of Effingham Inlet
(British Columbia), and this study revealed that polyphosphate remineralization could account for 4–9% of soluble
reactive P flux at the hypoxic/anoxic boundary, vs. 65%
for Fe-linked PO43 (Diaz et al., 2012). Applying a similar
study to Lake Pavin would enable an assessment of the role
of polyphosphate-accumulating organisms in the PO43
concentration and subsequent Fe-phosphate formation in
the monimolimnion of the lake.
Interestingly, some Fe is associated with the polyphosphate bodies observed in bacterial cells at 56 m and 67 m.
Polyphosphates are known to be strong chelators of metal
ions such as Ca (e.g., Kornberg, 1995) and Fe (Irani and
Morgenthaler, 1963). Several microorganisms have been
shown to accumulate Fe in their polyphosphate bodies
when cultivated in the presence of this element (Goldberg
et al., 2001; Nagasaka and Yoshimura, 2008; Diaz et al.,
2009; Nuester et al., 2012). Bacteria with intracellular Fecontaining polyphosphate bodies have moreover been isolated from an Fe-rich hydrothermal environment (Lechaire
et al., 2002), and the polyphosphate granules formed by the
Fe-phosphate encrusted bacteria described by Konhauser
et al. (1994) also contained Fe. The measured Fe/P molar
ratios in the polyphosphate bodies of Lake Pavin bacteria
(Fe/P ratio of 0.10) can be compared with those measured
in the diatoms Thalassiosira weissflogii (Fe/P ratio of 0.18)
and Thalassiosira pseudonana (Fe/P ratio of 0.6) grown at
high Fe concentrations (Nuester et al., 2012), or in bacteria
isolated from Fe-rich hydrothermal vents (Fe/P ratio of
0.75; Lechaire et al., 2002). Whether this might be the trace
of a transient precursor within the cells that gives rise to
Fe-phosphate precipitation on/outside the cells will need
to be carefully examined in future studies.
5. CONCLUSIONS
The formation of Fe(II)–Fe(III)-phosphates close to
oxic/anoxic interfaces, possibly through microbial processes (e.g., Fe-oxidation), might play an overlooked role
in P cycling in aquatic environments, in addition to the classical models of Fe-pumping involving Fe-(oxyhydr)oxides
and polyphosphate accumulation and hydrolysis. It is likely
that the two latter processes contribute to the transfer and
accumulation of PO43 at the sediment–water interface in
Lake Pavin, and thus indirectly induce Fe(II)-phosphate
scavenging in the sediment, as summarized in Fig. 11.
Moreover, the bacterial formation of Fe-phosphates
through Fe-oxidation would represent a new and poorly
documented mode of phosphate biomineralization.
Whether this process is specific to Lake Pavin or might
occur in other Fe(II)- and PO43-rich stratified environments (e.g., Buffle et al., 1989) will have to be investigated
in the future. Its role in P cycling in past environments, in
particular during periods of oceanic anoxia should also be
assessed. Another interesting finding of the present study
is the preservation of Fe-(oxyhydr)oxides throughout the
monimolimnion of the lake, which suggest that
dissimilatory Fe-reduction has a limited impact on the
J. Cosmidis et al. / Geochimica et Cosmochimica Acta 126 (2014) 78–96
Fe(II)–Fe(III) balance in the water column. The relative
importance of Fe-oxidation vs. Fe-reduction on the particulate Fe budget in the water column of Lake Pavin will
have to be quantified in future studies.
ACKNOWLEDGEMENTS
This project was partly funded by the Institut National des Sciences de l’Univers (INSU) program “InteractionsTerre/Vie”
(InterrVie) and the Fondation Simone et Cino del Duca, Institut
de France. The SEM and FIB facilities at IMPMC were supported
by Région Ile de France grant SESAME 2006 I-07-593/R, INSUCNRS, INP-CNRS, University Pierre et Marie Curie, Paris. The
TEM facility at IMPMC was supported by Region Ile-de-France
grant SESAME 2000 E 1435, INSU-CNRS, INP-CNRS and University Pierre et Marie Curie (UPMC) – Paris 6. Advanced Light
Source (ALS) Molecular Environmental Science beamline 11.0.2
is supported by the Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences
and Materials Sciences Division, U.S. Department of Energy, at
the Lawrence Berkeley National Laboratory. G. E. Brown Jr.
(Stanford University) is thanked for having provided some beamtime on the 11.0.2 STXM beamline. Tolek Tylisczak (LBNL) is
thanked for providing the best conditions and welcome possible
on STXM beamline 11.0.2 at the ALS. We thank Stephanie Belin
and Valérie Briois at SAMBA (SOLEIL) for their help with the
synchrotron experiment. Jean-Claude Boulliard (Collection de
Minéraux, IMPMC) is thanked for providing reference minerals.
Technical support from Ludovic Delbes and Frédéric Gélebart
(XRD measurements under anoxic conditions) and Jessica Brest
(anoxic chemical synthesis) at IMPMC was greatly appreciated.
We thank Sylvain Huon (UPMC) for providing the PAR measurements and Franck Bourdelle (Université de Lorraine) for his assistance with the estimation of the reference compounds Fe(II)/Fetot
ratios. The authors are indebted to Alexis Templeton (University
of Colorado) for proofreading this article, as well as for helpful discussions and critical comments on this work.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.gca.2013.10.037.
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