Scholarly article on topic 'Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis'

Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Julien Alleon, Sylvain Bernard, Corentin Le Guillou, Damien Daval, Feriel Skouri-Panet, et al.

Abstract Most ancient organic microfossils delicately preserved in 3D have been found in cherts. Although entombment within silica has been shown to promote morphological preservation, the impact of early silicification on the molecular evolution of fossilized microorganisms during burial remains poorly constrained. Here, we report results of advanced fossilization experiments performed under pressure (250bars) and temperature (250°C) conditions typical of sub-greenschist facies metamorphism for different durations up to 100days on microorganisms experimentally entombed (or not) within a silica gel. The experimental residues have been characterized using XRD and XANES spectroscopy. The present study demonstrates that entombment within silica limits the degradation of microorganism molecular signatures, likely through specific chemical interactions, despite the progressive conversion of silica into quartz during the experiments. Extrapolation of the present results suggests that such protection may persist during geological timescales. The present experimental study provides molecular evidence that, in addition to morphologies, cherts may support the chemical preservation of remains of ancient life. The present results thus constitute a step forward towards the reconstruction of the original chemistry of putative fossilized microorganisms.

Academic research paper on topic "Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis"

CHEMICAL GEOLOGY

ISOTOPE GEOSCIENCE

Accepted Manuscript

Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis

Julien Alleon, Sylvain Bernard, Corentin Le Guillou, Damien Daval, Feriel Skouri-Panet, Sylvain Pont, Ludovic Delbes, Francois Robert

PII: S0009-2541(16)30284-4

DOI: doi: 10.1016/j.chemgeo.2016.05.034

Reference: CHEMGE 17957

To appear in:

Chemical Geology

Received date: Revised date: Accepted date:

25 March 2016 28 May 2016 30 May 2016

Please cite this article as: Alleon, Julien, Bernard, Sylvain, Le Guillou, Corentin, Daval, Damien, Skouri-Panet, Feriel, Pont, Sylvain, Delbes, Ludovic, Robert, Francois, Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis, Chemical Geology (2016), doi: 10.1016/j.chemgeo.2016.05.034

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Early entombment within silica minimizes the molecular degradation of microorganisms during advanced diagenesis

11 12 Julien Alleon , Sylvain Bernard , Corentin Le Guillou , Damien Daval ,

Feriel Skouri-Panet1, Sylvain Pont1, Ludovic Delbes1, François Robert1

1IMPMC, CNRS UMR 7590, MNHN, UPMC, 61 rue Buffon, 75005 Paris, France. 2LHyGeS, CNRS UMR 7517, Université de Strasbourg / EOST, 1 Rue Blessig, 67084 Strasbourg, France

KEYWORDS: Experimental Silicification %I Experimental Fossilization | Organic Microfossils I Molecular Biosignatures | XANES Spectroscopy

ABSTRACT

Most ancient organic microfossils delicately preserved in 3D have been found in cherts. Although entombment within silica has been shown to promote morphological preservation, the impact of early silicification on the molecular evolution of fossilized microorganisms during burial remains poorly constrained. Here, we report results of advanced fossilization experiments performed under pressure (250 bars) and temperature (250 °C) conditions typical of sub-greenschist facies metamorphism for different durations up to 100 days on microorganisms experimentally entombed (or not) within a silica gel. The experimental residues have been characterized using XRD and XANES spectroscopy. The present study demonstrates that entombment within silica limits the degradation of microorganism molecular signatures, likely through specific chemical interactions, despite the progressive conversion of silica into quartz during the experiments. Extrapolation of the present results suggests that such protection may persist during geological timescales. The present experimental study provides molecular evidence that, in addition to morphologies, cherts may support the chemical preservation of remains of ancient life. The present results

thus constitute a step forward towards the reconstruction of the original chemistry of putative fossilized microorganisms.

1. INTRODUCTION

Ancient organic microfossils provide a great deal of information about the evolution of cellular life on Earth. Yet, in addition to hindering their identification, the inevitable degradation of the organic molecules comprising these microorganisms over the course of geologic time likely muddles the reconstruction of their original chemistry (e.g. Bernard and Papineau, 2014; Briggs and Summons, 2014). As a result, the search for the earliest fossil evidence of life on Earth has been and is still fraught with controversies (e.g. Schopf et al., 2002; Brasier et al., 2002; Schopf and Kudryavstev, 2012; Brasier et al., 2015; Wacey et al., 2016).

Remarkably, most of the oldest (putative) organic microfossils delicately preserved in 3D reported so far have been found in cherts (i.e. silica-rich rocks), which are now recognized as windows of exceptional morphological preservation (i.e. "Bitter Springs-type preservation", sensu Butterfield, 2003; Xiao and Schiffbauer, 2009) of organic microfossils (e.g. Walsh and Lowe, 1985; Ueno et al., 2001, 2006; Westall et al., 2001, 2006, 2011, 2015; Tice and Lowe, 2004, 2006; Glikson et al., 2008; Javaux et al., 2010; Sugitani et al., 2010, 2013, 2015a, 2015b; Wacey et al., 2011a, 2011b, 2012; Lepot et al., 2013). Despite this recognition and recent analytical progress, studies addressing the molecular signature of (putative) organic microfossils morphologically preserved in ancient cherts remain rare (e.g., De Gregorio and Sharp, 2006; Igisu et al., 2009; De Gregorio et al., 2009, 2011; Alleon et al., 2016). Intriguingly, although these (putative) microfossils have experienced diagenetic temperatures similar to overmature clay-rich gas shales, i.e. prehnite-pumpellyite to greenschist facies metamorphism, their molecular signatures are significantly less degraded than those of overmature kerogens (Bernard et al., 2010b, 2012a, 2012b).

A priori, silicification might be invoked as the explanation for the limited molecular degradation experienced by these microfossils during advanced (thermal) diagenesis. The critical role of authigenic mineralization (e.g.., pyritization, calcification, phosphatization, chloritization, glauconitization) in the morphological survivability of organic fossils through geological times has indeed been recognized and highlighted by many authors (e.g., Orr et al., 1998; Briggs, 2003; Briggs and Wilby, 1996; Bernard et al., 2010a; Anderson et al., 2011; Galvez et al., 2012; Kremer et al., 2012; Broce et al., 2014; Schiffbauer et al., 2014; Wacey et al., 2014; Muscente et al., 2015). Consistently, a number of experimental studies have highlighted the importance of the close association with minerals (such as sulfides, carbonates, phosphates, hydroxides, clays) for the morphological (e.g., Briggs and Kear, 1993; Briggs et al., 1993; Grimes et al., 2001; Martin et al., 2003; Wilson and Butterfield, 2014; Iniesto et al., 2015) and chemical (e.g., Li et al., 2013; 2014; Picard et al., 2015a, 2015b) preservation/degradation of soft tissues during fossilization processes.

Silicification processes have received even more attention: silicified microbial morphologies have been studied in situ by many authors in modern silicifying hydrothermal systems (Walter et al., 1972; Schultze-Lam et al., 1995; Cady and Farmer, 1996; Konhauser and Ferris, 1996; Jones et al., 1998, 2001, 2003, 2004; McKenzie et al., 2001; Konhauser et al., 2001, 2003, 2004; Handley et al., 2005, 2008; Campbell et al., 2015). In parallel, a number of authors have experimentally investigated the processes of silica polymerization and precipitation in the presence of microorganisms (e.g. Fortin and Ferris, 1998; Hinman, 1990; Fein et al., 2002; Yee et al., 2003; Benning et al., 2004a, 2004b). Further, the impact of early silicification on the morphologies of microorganisms has been extensively studied through laboratory experiments conducted under various conditions, from room temperature to pressure and temperature conditions typical of diagenesis (e.g. Oehler and Schopf, 1971; Oehler, 1976a; Walters et al., 1977; Francis et al., 1978a, 1978b;

Birnbaum et al., 1989; Westall et al., 1995; Phoenix et al., 2000; Toporski et al., 2002; Lalonde et al., 2005; Orange et al., 2009, 2011a, 2011b, 2012, 2013a, 2013b, 2014).

Altogether, these studies have shown that the precipitation of amorphous silica is mainly controlled by the dehydroxylation of the active hydroxyl groups present at the surface of silica (silanols) and that the rate of amorphous silica polymerization and precipitation is independent of the presence of biomass, i.e. that the microbial role in silicification is predominantly incidental. During silicification, microorganisms likely act as templates for the binding and nucleation of silica, even though they have only little affinity for monomelic silica. Long exposure to silicifying media eventually leads to the complete impregnation of microorganisms by silica, with cell walls and intracellular granules progressively becoming the only recognizable features. Besides the pioneering study of Oehler (1976a), which investigated the geochemical evolution of algal soluble organics (lipids and pigments) during thermal diagenesis, and the recent study of Orange et al. (2012), which documented the changes in composition of archeal soluble organics (amino acids, polysaccharides and lipids) during silicification, the above-mentioned experimental studies have all only focused on morphologies. Although these studies have highlighted the high potential of silica matrices for the morphological preservation of microfossils, i.e. that early silicification of microorganisms allows their morphological preservation, the impact of advanced (thermal) diagenesis on the molecular signatures of silicified microorganisms remains to be precisely documented. In fact, this appears as the prerequisite to, eventually, clear the way for the reconstruction of the original chemistry of putative fossilized microorganisms.

Here, we report results of advanced fossilization experiments performed on prokaryotic cyanobacteria Gloeobacter violaceus and eukaryotic microalgae Euglena gracilis that we experimentally entombed within a silica gel initially consisting of opal-A, i.e. a hydrated amorphous form of silica. Following the philosophy of Oehler (1976a), who

performed fossilization experiments under pressure and temperature conditions typical of diagenesis, the present advanced fossilization experiments have been performed at 250 °C and 250 bars for different durations (1, 10 and 100 days) to simulate burial-induced diagenetic processes. These temperature conditions typical of sub-greenschist facies metamorphism are similar to those experienced by most Archean cherts (e.g., Tice et al., 2004; Glikson et al., 2008; Sugitani et al., 2010; Javaux et al., 2010; Lepot et al., 2013). Regardless its value, pressure is important as it authorizes microtextural transformation (Beyssac et al., 2003). A sample of microorganism-free silica gel and silica-free microorganisms have also been submitted to the same conditions to serve as control groups. Experimental residues have been characterized using X-ray diffraction (XRD) and synchrotron-based X-ray absorption near edge structure (XANES) spectroscopy to document both mineralogical and organic geochemical evolution of these silicified microorganisms. The present results demonstrate that silica restrains the molecular degradation of microorganisms during experimental fossilization at 250 °C and 250 bars and still offers effective protection even after the conversion of silica into quartz. Extrapolating the present results suggests that the mechanism of impeding chemical degradation via silica emplacement may be relevant in fossilization of organic materials over geological timescales.

2. METHODS

2.1. Selected Microorganisms

Although most experimental silicification studies have been historically performed on cyanobacteria (Oehler and Schopf, 1971; Oehler, 1976a; Walters et al., 1977; Francis et al., 1978a, 1978b; Phoenix et al., 2000; Yee et al., 2003; Benning et al., 20004a, 2004b; Orange et al., 2013a, 2013b), the response to silicification of a wide range of

microorganisms has also been investigated, including other bacteria, either Gram-positive (Fortin and Ferris, 1998; Fein et al., 2002; Orange et al., 2014) or Gram-negative (Birnbaum et al., 1989; Fortin and Ferris, 1998; Toporski et al., 2002; Lalonde et al.,

2005), archaea and viruses (Orange et al. 2009, 2011a, 2011b, 2012), and eukaryotes (Walters et al., 1977; Francis et al., 1978a, 1978b; Westall et al., 1995).

Here, we selected two strains of unicellular oxygenic photosynthetic microorganisms for advanced fossilization experiments: the prokaryotic cyanobacteria Gloeobacter violaceus (PCC 7421) and the eukaryotic microalgae Euglena gracilis (n°1224-5d - Cambridge). Fresh G. violaceus exhibit purple spherical cells of about 1 |im in diameter, while fresh E. gracilis cells are green and approximately 30 |im in length and 10 |im in width (Figure 1). Obviously, these two strains have not been selected to exemplify all prokaryotic and eukaryotic microorganisms, but rather to serve as two different well-known precursors for the present experiments.

G. violaceus are believed to have diverged phylogenetically prior to the endosymbiotic event responsible for the apparition of the first eukaryotic cells (Rexroth et al. 2011). G. violaceus produce intracellular Mg-rich polyphosphate vesicles which are used by the cells as a storage form of phosphorus (Benzerara et al., 2014 - Figure 1). TEM observations of ultrathin sections revealed an atypical cell wall structure compared to other cyanobacteria, comprising, from interior to exterior, an electron-dense peptidoglycan layer which serves a structural role, an intermediate electron-dense cell wall layer, a double track-structured outer membrane, and an adherent external sheath layer (Rippka et al., 1974; Guglielmi et al., 1981; Schneider and Jürgens, 1991). In addition to a peptidoglycan-polysaccharide complex, Gloeobacter violaceus contain lipopolysaccharides (20-25wt%), proteins (20-25wt%), carbohydrates (15-20wt%), and fatty acids (2-10wt%) as main constituents of their cell walls (Schneider and Jürgens, 1991).

E. gracilis belong to an ancient lineage near the base of the eukaryotic tree of life and are among the most documented photosynthetic euglenoids (e.g., Cramer and Myers, 1952; Guttman, 1971; Gibbs, 1978; Vismara et al., 2000; Miot et al., 2008, 2009; Leloup et al., 2013; Williams, 2016). These flagellated protists contain chlorophyll-rich chloroplasts which control the synthesis of carbohydrates stored intracellularly as starch granules and paramylon which can reach up to 90% dry weight (Guttman, 1971). Structurally, E. gracilis do not have a cell wall, but a pellicle made up of a protein layer supported by a substructure of microtubules arranged in strips spiraling around the cells (Sommer, 1965; Vismara et al., 2000). The complexity of this structure is also reflected in its chemical composition: the pellicle dry weight consists of about 70% of proteins, 15-20% of lipids, and 10-15% of carbohydrates (Barras and Stone, 1965; Nakano et al., 1987).

Both strains have been cultured at IMPMC. G. violaceus have been cultured in a regular BG-11 culture medium as done by Couradeau et al. (2012) following the recommendations of the Pasteur institute while E. gracilis have been cultured at ambient temperature, under permanent light exposure with ethanol as a carbon source, in a culture medium composed of KH2PO4 (0.5 g.L-1), MgSO4,7H2O (0.5 g.L-1), CaCh^O (0.26 g.L-1), (NH4)2HPO4 (0.5 g.L-1), a complement of vitamins, zinc (as ZnSO4), iron (as FeCl3), and manganese (as MnSO4) as done by Miot et al. (2008, 2009). Cells of both strains have then been rinsed 3 times in bi-distilled water dried in an oven at 50 °C under vacuum for 3 days.

2.2. Encapsulation within the silica gel and fossilization experiments

Dried micro-organisms have been experimentally entombed within a silica gel following the procedure developed by Nassif et al. (2002). Silica sources were sodium silicate solutions SiNa (27 wt% SiO2, 10 wt% NaOH from Aldrich) and colloidal silica (LUDOX HS-40 from Aldrich). SiNa (0.4 M, 1 mL) has been mixed with LUDOX (8.5 M, 1

mL) and hydrochloric acid (4 M) has been added until neutralization. The mixture has been homogenized under mild stirring before the addition of 1 mL of cyanobacterial or algal suspension (which leads to a final total organic content of the gels of about 5wt%). Gelation of these mixtures usually occurs within a few minutes at room temperature.

After a 3 day-long drying in an oven at 50 °C under vacuum, 1 mg of microorganism-rich silica gel has been placed into individual gold capsules which have then been sealed under argon atmosphere using an electrical arc and placed in Parr © autoclaves that have been pressurized using argon (> 99.99% purity). Pressure has been maintained for all experiments at 250 ± 2.5 bars while temperature has been fixed at 250 ± 2.5 °C using a cylindrical electrical oven. Experiments have been performed over 1, 10 and 100 days, thereby allowing kinetic investigations.

Microorganism-free silica gels and silica-free microorganisms have also been submitted to the same conditions to serve as control groups. All experiments have been repeated three times. At the end of these 1, 10 and 100 day-long experiments, solid residues have been recovered from the capsules and stored at 4°C before being prepared for XRD and XANES measurements.

2.3. Characterization techniques 2.3.1. X-ray diffraction (XRD)

The bulk mineralogical composition of the experimental products has been determined using a Panalytical X'pert Pro X-ray diffractometer (IMPMC - Paris, France) operating at 40 kV and 40 mA Co Ka radiations. Sample analyses have been carried out on finely ground powders deposited on a silicium sample holder, in the 20°-110° 20 angle range, with a step size of 0.016° (20) for a total counting time per sample of about 6 h. XRD

patterns have been analyzed using the Eva software (Bruker) for background subtraction and peak finding.

2.3.2. Scanning Electron Microscopy (SEM)

SEM observations have been performed on pieces of experimental products affixed to aluminum stubs and coated with 15 nm of gold using a SEM-FEG ultra 55 Zeiss (IMPMC -Paris, France) microscope. The operating conditions were as follows: 2 kV accelerating voltage and a working distance of 2 mm for secondary electron imaging (SE2 detector), and 15 kV accelerating voltage and a working distance of 7.5 mm for examination using backscattered electrons (AsB detector).

2.3.3. Synchrotron-based XANES spectroscopy

The 'bulk' carbon speciation of the experimental residues has been investigated by X-ray Absorption Near Edge Structure (XANES) spectroscopy at the carbon K-edge (270 - 330 eV) using the 11-ID-1 Spherical Grating Monochromator (SGM) beamline located at the Canadian Light Source (CLS - Canada) (Regier et al., 2007).

The CLS storage ring is operated at 2.9 GeV and between 250 and 150 mA current. Two horizontal deflecting mirrors and one vertical focusing mirror direct the light from the 45 mm planar undulator through the entrance slit and onto a 1700 lines/mm grating (low energy grating) which diffracts the light through a movable exit slit that has been set at 25 |im for the present experiments. A pair of toroidal refocusing mirrors are used to focus the beam

onto the samples in the endstation chamber maintained at a pressure of about 10- Torr. A 100 nm thick titanium filter is used to remove the contribution of second order light.

The beam spot size is approximately 1 mm x 100 p,m with a flux (normalized to 100

mA) greater than 1012 photons at 250 eV. This beamline measures the fluorescence yield of

samples exposed to the incident beam as a function of its energy with a spectral resolution of about 0.1 eV. When normalized to the scattering intensity from a clean gold foil used as a blank, fluorescence yields are directly comparable to absorption data, especially as the SGM uses an Amptek silicon drift detector placed at exactly 90 degrees to the incident beam, a configuration which allows exclusion of the scattered light.

Experimental residues have been pressed onto gold foils (99.99 % purity - Sigma Aldrich) then mounted on a copper sample holder. Calibration in energy has been done using a set of pure reference materials (calcium carbonate, benzoic acid and histidine (> 99 % purity - Sigma Aldrich)). Each spectrum reported in the present study corresponds to the average of about 50 measurements collected at different locations over the sample surfaces. Of note, only the first 100 nanometers of the sample surface are probed using the SGM setup. Spectra have been averaged, background subtracted and normalized using the Igor Pro software.

2.4. XANES data deconvolution procedure

C-XANES spectroscopy has been successfully used to document the carbon speciation of organic microfossils (Bernard et al., 2007, 2009; Lepot et al., 2008, 2009; De Gregorio et al., 2009, 2011; Cosmidis et al., 2013a, 2013b; Robin et al., 2015). Absorption peaks of a C-XANES spectrum constitute sensitive indicators of the local chemical bonding environment surrounding the carbon atoms and correspond to transitions from core shell (1s) electrons to both unoccupied n* (antibonding) and low lying o* orbitals.

As any electric-dipole transition spectroscopies, XANES spectroscopy adheres to Beer's law. Variations in peak intensities are thus directly proportional to variations in concentration of the absorbing functional groups. Assuming that the oscillator strength of a given functional group is essentially the same in organic compounds of similar chemistry

(Ishii and Hitchcock, 1988; Francis and Hitchcock, 1992), the relative concentrations of the different functional groups for the residues investigated can be discussed qualitatively.

To obtain a more quantitative insight on the molecular signatures of the experimental samples investigated, the C-XANES spectra normalized to the total carbon amount (measured above 320 eV) have been deconvolved using the Athena software package (Ravel and Newville, 2005) following the procedure described by Bernard et al. (2015), based on the recommendations of Braun et al. (2006).

The absorption edge itself has been modeled using an arctangent function which center position, amplitude and width at half-height have been fixed to 291.5 eV, 1.0, and 0.4 eV, respectively. The remaining absorption signal has been fitted using Gaussian functions, each of them having a fixed energy position and a constant width (0.4 eV below 295 eV and 2 eV above).

Although the natural lineshape of a XANES peak is a Lorentzian function, Gaussian functions have been used to take into account the contribution to the linewidth produced by instrumental broadening effects. It should be kept in mind that most of the Gaussian functions used for these deconvolutions do not account for the presence of functional groups but for broad spectral features corresponding to highly delocalized excited states, sometimes referred to as 1s—virtual state transitions, or for the overlapping contribution of Feshbach resonances.

The normalized 'aromaticity index' is defined here as the sum of the areas of the Gaussian functions accounting for the absorption related to the presence of multiple carboncarbon bond species and used to deconvolve the broad peak at 285 eV according to Bernard et al. (2010c, 2015) and Le Guillou et al. (2013, 2014), normalized, for each strain, to the aromaticity index of the starting material.

3. RESULTS

The C-XANES spectrum of fresh G. violaceus is typical of bacteria (Figure 2), with a main peak at 288.4 eV, attributed to the joint contributions of electronic transitions in

amide groups (occurring at 288.2 eV) and of electronic transitions in carboxylic

(COOH), ester (COOR) or acetal (C(OR)2) groups (occurring at 288.5-288.7 eV), a peak at 285.1 eV, attributed to electronic transitions in aromatic or olefinic groups (C=C),

and a shoulder centered at about 287 eV, attributed to ls^-rc* electronic transitions in carbonyl groups (C=O) (Benzerara et al., 2006, Solomon et al., 2009; Li et al., 2014). This spectrum is consistent with the initial composition of G. violaceus cell walls (Schneider and Jürgens, 1991).

The C-XANES spectrum of fresh E. gracilis appears slightly different (Figure 2). In fact, it also exhibits an absorption feature at 289.4 eV corresponding to 1s^-3p/o* transitions in hydroxyl functional groups (Cody et al., 2011; Bernard et al., 2012a, 2012b). In addition, instead of being centered at 288.4 eV, its intense peak is centered at 288.7 eV and can be attributed to ls^-rc* transitions in carboxylic groups (COOH), ester (COOR) or acetal groups (C(OR)2) (Johansson and Campbell, 2004; Bernard et al., 2012a, 2012b; Rouchon and Bernard, 2015). These contributions are likely related to a high content of carbohydrates (Lawrence et al., 2003), which, in the case of E. gracilis, are stored intracellularly as starch granules and paramylon (Guttman, 1971).

In the absence of silica, G. violaceus and E. gracilis have evolved towards dark oily residues with no specific morphologies during experimental fossilization at 250 °C and 250 bars, even after only one day. The C-XANES spectra of the experimental residues reveal that G. violaceus and E. gracilis have encountered a significant chemical evolution (Figure 2). For both strains, the relative concentrations of aromatic/olefinic carbons and carbonyl/phenolic

carbons have significantly increased (absorption features at 285.1 and around 287 eV have become more intense) while the relative concentrations of amide/acetal/carboxylic and hydroxylated carbons have significantly decreased (intensities of absorption peaks at 288.4/288.7 and 289.4 eV have decreased) after only one day. While the molecular degradation of G. violaceus seems to be complete after 1 day, the molecular signature of E. gracilis has evolved more progressively with increasing experimental duration (Figure 2).

The molecular signatures of G. violaceus and E. gracilis entombed within silica and submitted to experimental fossilization under the same conditions have evolved following similar trends, i.e. E. gracilis have evolved more progressively than G. violaceus. Yet, little or no oil has formed from microorganisms entombed within silica and the transformations encountered by these microorganisms appear significantly less drastic than the ones they have encountered in the absence of silica (Figures 2 and 3). In particular, G. violaceus entombed within silica still contain amide groups after 100 days. Interestingly, the C-XANES spectra of the residues of G. violaceus entombed within silica exhibit an additional absorption feature at 290.3 eV which can be attributed to 1s—n* transitions in carbonate groups (Benzerara et al., 2006, Brandes et al., 2010), which increases in intensity with increasing experimental duration from 1 to 100 days. These carbonates may result from the destabilization of intracellular polyphosphates during the experiments.

Following previous studies (Bernard et al., 2010c, 2015; Le Guillou et al., 2013, 2014), the 'aromaticity index' of G. violaceus and E. gracilis has been extracted from C-XANES data. An increase of the aromaticity index indicates an increase of maturity, i.e. a degradation of the initial molecular composition. For each strain, the aromaticity index of the residues of experiments performed within silica is significantly lower than that of experiments performed in the absence of silica (Figure 4). Intriguingly, independent of the presence or absence of silica, the aromaticity index of the residues of G. violaceus is always

lower than that of E. gracilis for a given experimental duration. In addition, for G. violaceus, this index increases during the first day and does not significantly vary for longer exposure. In contrast, the aromaticity index of E. gracilis exhibits a log-linear relationship with experimental duration (Figure 4).

The silica gels in which the microorganisms investigated have been entombed consist of opal-A, a hydrated amorphous form of silica, as indicated by XRD (Figure 5). In the absence of microorganisms, the silica gel is converted into quartz in less than a day when submitted to 250°C and 250 bars (Figure 5). The silica gel originally containing G. violaceus has also rapidly crystallized into quartz (in less than a day) while the silica gel originally containing E. gracilis has remained amorphous during the first day and evolved towards opal-CT in the 10 day-long experiments to become an assemblage of opal-C and quartz with increasing experimental duration (Figure 5).

SEM observations of residues of experiments performed on microorganisms entombed within silica reveal that organics have been redistributed within the mineral matrix, even after the shortest (1 day-long) experiments (Figure 6). Within the samples originally containing G. violaceus, empty silica lepispheres have rapidly formed (in less than a day). The formation of similar silica lepispheres appears more progressive within the samples originally containing E. gracilis (Figure 6).

4. DISCUSSION

4.1. Degradation of microorganisms

The present study provides experimental insights on burial-induced microorganism degradation processes from a molecular point of view. In the absence of silica, the evolution

towards dark oily residues of G. violaceus and E. gracilis likely indicates oil and bitumen generation through thermal cracking reactions (Dieckmann et al., 1998; Behar et al., 1997; Lewan, 1997; Behar et al., 2003; Lewan and Roy, 2011; Bernard and Horsfield, 2014) and a concomitant increase of aromaticity of the solid residue (Harkopf-Fröder et al., 2015). C-XANES data reveal that the evolution of G. violaceus and E. gracilis is typical of organic solids having experienced thermal maturation (Schiffbauer et al., 2012; Li et al., 2014; Bernard et al., 2015), i.e. a loss of oxygen-containing functional groups concomitant to an increase of the relative abundance of aromatic structures (e.g., Bernard and Horsfield, 2014). For both strains, the absence of a sharp 1s^-g* exciton at 291.7 eV, related to the presence of extensive planar domains of highly conjugated aromatic layers (Cody et al, 2008; Bernard et al., 2010c), suggests that significant pericondensation of aromatic rings has not occurred even after 100 days.

Intriguingly, the aromaticity index of E. gracilis exhibits a log-linear relationship with experimental duration which does not correspond to any reaction mechanism commonly operating in solid/solid organic reactions (Rothenberg et al., 2001). Similar log-linear behaviors have been reported for parameters describing the maturation/degradation of carbon materials as a function of experiment durations (Beyssac et al., 2003; Cody et al., 2008; Zhao et al., 2010; Schiffbauer et al., 2012; Bernard et al., 2015). This behavior suggests that the degradation of E. gracilis has mainly occurred through a series of kinetically controlled reactions such as random chain scissions and polymerization/condensation reactions for instance (Rothenberg et al., 2001; Vandenbroucke and Largeau, 2007; Sánchez-Jiménez et al., 2010; Pérez-Maqueda et al., 2014). Of note, this apparent loglinear kinetic behavior likely turns into a plateau when a thermodynamic metastable equilibrium is reached as it is likely the case in natural settings (Helgeson et al., 1993, 2009; Richard and Helgeson, 1998).

In contrast to E. gracilis, the aromaticity index of G. violaceus residues does not continue to evolve after the first day of experiment. This implies that either the aromaticity increase of G. violaceus residues has not followed a log-linear kinetic behavior or that a loglinear relationship has turned into a plateau in less than a day. Such different behavior between G. violaceus and E. gracilis might be a result of the high carbohydrate concentration of E. gracilis. In fact, acetal functional groups, abundant in carbohydrates, are known to be more resistant to thermal degradation processes than amide functional groups (Vandenbroucke and Largeau, 2007). In any case, the present results evidence that slightly different organics may follow significantly different degradation pathways during advanced (thermal) diagenesis.

4.2. Influence of the silica matrix

The present experimental results demonstrate that entombment within silica limits the molecular degradation of microorganisms. The opposite could have been expected as the silica matrix used for the present experiments mainly consists of hydrated opal-A. In fact, the presence of water has been shown to enhance organic maturation processes and oil and bitumen generation at low-temperature (Lewan, 1997; Behar et al., 2003; Lewan and Roy, 2011). Here, despite the high water content of the silica matrix, the C-XANES spectrum of E. gracilis entombed within silica and submitted to 250 °C and 250 bars for 100 days appears very similar to the C-XANES spectrum of E. gracilis submitted to the same conditions for only 1 day in the absence of silica (Figure 3). The preservation of G. violaceus entombed within silica is even more remarkable (Figure 3).

Such molecular preservation likely results from specific chemical interactions between the investigated microorganisms and the silica matrix, as historically proposed to

explain the fine-scale morphological preservation of silicified wood (Leo and Barghoorn, 1976). Amino acids and peptides can interact with silica-rich solutions through electrostatic interactions and hydrogen bonding (Coradin and Livage, 2001; Benning et al., 2004a, 2004b; Konhauser et al., 2004). Hydroxyl-bearing functional groups of organic molecules can be adsorbed through hydrogen bonding with silanols (Hsu et al., 2000; Coradin and Livage, 2001, Jal et al., 2004; Konhauser et al., 2004; Zou et al., 2008; Ciriminna et al., 2013). The concentration of silanols at the surface of silica is directly related to its degree of hydration (Zhuravlev, 2000; Konecny, 2001). Si-O-C covalent bonds may form during water release, resulting in the stabilization of some organic functional groups despite increasing temperature conditions (Hsu et al., 2000; Jal et al., 2004; Zou et al., 2008). Such stabilization has likely limited oil and bitumen generation through thermal cracking reactions (little or no oil has formed during the experiments with silica) and thereby having restricted the aromatization of organic residues.

Although it remains significantly lower, the aromaticity indices of E. gracilis and G. violaceus entombed within silica exhibit similar relationship with experimental fossilization duration to those of E. gracilis and G. violaceus experimentally fossilized in the absence of silica, respectively. In other words, G. violaceus and E. gracilis entombed within silica exhibit degradation patterns similar to those observed for experiments performed in the absence of silica. The present study thus pinpoints that, even within silica, the molecular signatures of different organic precursors may follow different degradation pathways.

Of note, despite previous observations of silicification preserving microbial morphologies (Oehler and Schopf, 1971; Oehler, 1976a; Walters et al., 1977; Francis et al., 1978a, 1978b; Birnbaum et al., 1989; Westall et al., 1995; Phoenix et al., 2000; Toporski et al., 2002; Lalonde et al., 2005; Orange et al., 2009, 2011a, 2011b, 2012, 2013a, 2013b, 2014), entombment within silica has not promoted the morphological

preservation of G. violaceus and E. gracilis cells during the present experiments. The present closed-system experimental design may have precluded the escape of volatile products, thus promoting the circulation of fluids and the scatter of organics within the silica matrix. The empty molds resulting from the migration of organics likely served as templates for the formation of silica lepispheres similar to those formed in natural silica sinters (Florke et al., 1976; Rimstidt and Cole, 1983; Herdianita et al., 2000; Rodgers et al., 2004; Lynne et al., 2005).

4.3. Evolution of the silica matrix

The silica gel originally consisting of hydrated opal-A has evolved towards opal-CT, opal-C and quartz during the fossilization experiments with E. gracilis. Similar mineralogical changes accompany ageing of silica sinters in natural settings (Florke et al., 1976; Rimstidt and Cole, 1983; Herdianita et al., 2000; Rodgers et al., 2004; Lynne et al., 2005). Interestingly, the evolution of the silica matrix of samples containing microorganisms seems closely related to (if not controlled by) the evolution/degradation of the entombed microorganisms. Silica crystallization has indeed been more progressive in samples containing E. gracilis compared to samples containing G. violaceus, i.e. silica crystallization patterns are similar to the molecular degradation patterns of E. gracilis and G. violaceus.

In the absence of microorganisms, the silica gel is converted into quartz in less than a day when submitted to 250 °C and 250 bars, in good agreement with previous experiments performed under similar conditions (Corwin et al., 1953; Betterman and Liebeau, 1975; Oehler, 1976b; Kastner et al., 1977; Hinman, 1990; Okamoto et al., 2010). As is also the case in the presence of G. violaceus, it is difficult to infer if/how the presence of cyanobacteria has impacted the crystallization of the silica gel. The transition from opal to

quartz has been shown to be faster in the presence of ions such as Mg (Kastner et al., 1977; Kyle and Schroeder, 2007) or minerals such as carbonates (Kastner et al., 1977; Hinman, 1990; Herdianita et al., 2000; Rodgers et al., 2004; Lynne et al., 2005; Lakshtanov et al., 2010). As the intracellular Mg-rich polyphosphate vesicles of G. violaceus have decomposed into carbonates during the experiments, the presence of G. violaceus may have slightly accelerated the crystallization of the silica gel.

In contrast, the XRD data reported here evidence that the presence of E. gracilis has retarded the crystallization of the silica gel. Although organics have only little affinity for monomeric silica, their presence may affect the crystallization rate of amorphous silica and, depending on their chemical composition, impart an influence on the fabric of silica sinters (Hinman, 1990; Fein et al., 2002; Yee et al., 2003; Konhauser et al., 2004; Rodgers et al., 2004; Handley et al., 2008). Previous observations on natural samples have similarly suggested that chemical interactions between organics and silica may hinder crystallization processes (Moreau and Sharp, 2004; Foucher and Westall, 2013). As hydrogen bonds between amorphous silica and organics mainly occur with hydroxyl-bearing functional groups (Hsu et al., 2000; Coradin and Livage, 2001; Jal et al., 2004; Konhauser et al., 2004; Zou et al., 2008; Ciriminna et al., 2013), silica crystallization has likely been retarded in the present experiments because of the high quantity of hydroxyl-rich carbohydrates formed intracellularly by E. gracilis.

5. Concluding Remarks: significance for natural settings

Extrapolating laboratory results to natural settings remains difficult, notably because geological timescales cannot be replicated in the laboratory (Li et al., 2014; Bernard et al., 2015; Picard et al., 2015a, 2015b). Nevertheless, similar to what occurs in natural silica

sinters (Florke et al., 1976; Rimstidt and Cole, 1983; Herdianita et al., 2000; Rodgers et al., 2004; Lynne et al., 2005), quartz has become the main constituent of all residues of 100 day long experiments. Although quartz surfaces are less hydroxylated than other polymorphs of silica (Zhuravlev, 2000; Konecny, 2001), the microorganisms used for the present fossilization experiments seem to have encountered only limited molecular degradation between 10 and 100 days, suggesting that organics and silica are mostly associated through covalent bonds after the first 10 days of experiment. The present degradation patterns of G. violaceus and E. gracilis thus suggest that their molecular signatures could still be (at least partially) preserved even after much longer exposures to such diagenetic conditions.

Although the present experimental results hold great promise for the detection of (partially) preserved molecular signatures of ancient organic microfossils, additional experimental studies appear necessary to thoroughly constrain the molecular evolution of biogenic organic molecules during burial-induced processes and eventually reconstruct the original chemical composition of fossilized microorganisms. In fact, neither biodegradation nor early taphonomic processes that usually occur in natural settings have been simulated here. In addition, quartz may crystallize at lower temperatures, thereby modifying silica-organics interactions. A possible way to overcome this obstacle would be to submit well-characterized, already indurated sedimentary rocks to experimental fossilization. This would allow more accurately simulating natural processes and hence obtaining a more realistic view of the chemical evolution that may have experienced ancient microorganisms during burial processes.

ACKNOWLEDGMENTS

We gratefully acknowledge support from the ERC (project PaleoNanoLife - PI: F. Robert). Special thanks go to Mélinée Deretz for administrative simplification. The SEM facility of the IMPMC is supported by Région Ile de France grant SESAME 2006 N°I-07-593/R, INSU-CNRS, INP-CNRS, UPMC-Paris 6, and by the Agence Nationale de la Recherche (ANR) grant No. ANR-07-BLAN-0124-01. Special thanks go to Imene Esteve for her expert support of the SEM at IMPMC. Bulk XANES data were acquired at beamline 11ID-1 at the CLS, which is supported by the NSERC, the CIHR, the NRC, and the University of Saskatchewan. Special thanks go to Tom Regier and Jay Dynes for their expert support of the SGM beamline.

FIGURE CAPTIONS

Figure 1: Scanning electron microscopy observations of the selected microorganisms (G.

violaceus on the left, E. gracilis on the right).

Figure 2: X-ray absorption near edge structure spectra of experimental residues. C-

XANES spectra collected on the starting materials and on the residues of G. violaceus (top) and E. gracilis (bottom) experimentally fossilized under 250°C and 250 bars for 1, 10 and 100 days in the absence of silica (left) and entombed in silica (right). Peak attribution: 285.1 eV: aromatic; 287.3 eV: carbonyl/phenol; 288.4 eV: amide + carboxyl/ester/acetal; 288.7 eV: carboxyl/ester/acetal; 289.5 eV: hydroxyl; 290.3 eV: carbonate.

Figure 3: X-ray absorption near edge structure spectra of experimental residues. C-

XANES spectra of G. violaceus and E. gracilis experimentally fossilized under 250°C and 250 bars for 100 days entombed in silica and for 1 day in the absence of silica. C-XANES spectra of starting materials are shown for comparison.

Figure 4: Evolution of the aromaticity index of experimental residues. Aromaticity indexes of G. violaceus (circles) and E. gracilis (squares) experimentally fossilized under 250°C and 250 bars for 1, 10 and 100 days in the absence of silica (open symbols) and entombed in silica (filled symbols). Logarithmic curves are used to fit the data for E. gracilis only (see text for details).

Figure 5: X-ray diffraction patterns of experimental residues. XRD patterns of the silica matrix of the starting materials and of the residues of G. violaceus (middle) and E. gracilis (bottom) experimentally entombed in silica and submitted to 250°C and 250 bars for 1, 10

and 100 days. Opal-A (A), opal-CT (CT), opal-C (C), quartz (Q) and halite (H) are indexed. Also shown are the XRD patterns of the microorganism-free silica gel before and after experimental fossilization 250°C and 250 bars for 1 day.

Figure 6: Scanning electron microscopy observations of experimental residues. SEM

images (secondary electrons) of the starting materials and of the residues of G. violaceus (top) and E. gracilis (bottom) experimentally entombed in silica and submitted to 250°C and 250 bars for 1, 10 and 100 days. Black arrows show the microorganisms freshly entombed in the silica gel. White arrows show the silica spheres (lepispheres) formed during the fossilization experiments.

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Experimental Silicification

Experimental Advanced Diagenesis

(250°C - 250 bars)