Francès Westall

Barbara Cavalazzi
email: cavalazzib@uj.ac.za

Laurence Lemelle
email: laurence.lemelle@ens-lyon.fr

Yves Marrochi

Jean-Noël Rouzaud
email: rouzaud@mailhost.geologie.ens.fr

Alexandre Simionovici
email: alexandre.simionovici@ujf-grenoble.fr

Murielle Salomé
email: salome@esrf.fr

Smail Mostefaoui

Caroline Andreazza
email: caroline.andreazza@univ-orleans.fr

Frédéric Foucher

Frances Westall
email: frances.westall@cnrs-orleans.fr

Yves Marrocchi
email: marrocchi@mnhn.fr

Jan Toporski
email: jan.toporski@witec.de

Andrea Jauss

Volker Thiel
email: vthiel@gwdg.de

Gordon Southam
email: gsoutham@uwo.ca

Lachlan Maclean

Susan Wirick
email: swirick@bnl.gov

Axel Hofmann
email: ahofmann@uj.ac.za

Anders Meibom

François Robert

Christian Défarge
email: christian.defarge@univ-orleans.fr

Frances Westall

Implications of in situ calcification for photosynthesis in a 3.3 Ga-old microbial biofilm from the Barberton greenstone belt, South Africa

Keywords: Barberton, microbial mat, photosynthesis, calcification, aragonite

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The earliest traces of photosynthesis occur in well-preserved sedimentary rocks of Early-Mid Archaean age (4.0-3.3 Ga) in the Barberton (South Africa) and Pilbara (Australia) greenstone belts (see review in [START_REF] Westall | Early life: Nature, distribution, and evolution[END_REF]. In the commonly accepted understanding of the evolution of life, anoxygenic photosynthetic microorganisms appeared first in the Early-Mid Archaean [START_REF] Tice | Photosynthetic microbial mats in the 3,416-Myr-old ocean[END_REF]Westall et al., 2006) while the more sophisticated and more efficient oxygenic photosynthesisers appeared in the Late Archaean [START_REF] Buick | The antiquity of oxygenic photosynthesis: evidence from stromatolites in sulphate-deficient Archaean lakes[END_REF][START_REF] Altermann | Microfossils from the Neoarchaean Campbell Group, Griqualand West Sequence of the Transvaal Supergroup, and their palaeoenvironmental and evolutionary implications[END_REF][START_REF] Brocks | Archean molecular fossils and the early rise of eukaryotes[END_REF][START_REF] Summons | Molecular fossils for cyanobacteria recording a geological history of oxygenic photosynthesis[END_REF].The most common expression of photosynthesis in the rock record is fossilised photosynthetic microbial mats.

These are finely laminated structures that are formed by photosynthetic microorganisms in generally oligotrophic environments where the primary producing microorganisms have access to sunlight that they can use as an energy source. These mats are highly complex consortia of different kinds of microorganisms living off the organic biomass produced initially by the photosynthetic microorganisms [START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF]. The preservation of photosynthetic microbial mats is serendipitous because they often form in ephemeral, littoral environments that are active and subject to physical destruction (Noffke, 2009). Moreover, early diagenetic processes may also lead to complete degradation of the mats or blurring of their signatures. A concatenation of events is therefore required to preserve them, such as burial by fine detritus to produce recognisable, microbially-influenced sedimentary structures (MISS, Noffke, 2009), or impregnation of the layered photosynthetic structure by minerals, such as carbonate or silica [START_REF] Walter | Stromatolites[END_REF][START_REF] Cady | Fossilization processes in thermal siliceous springs: trends in preservation along the thermal gradient. In: Evolution of hydrothermal ecosystems on Earth (and Mars?)[END_REF][START_REF] Konhauser | Microbialsilica interactions in Icelandic hot spring sinter: possible analogues for Precambrian siliceous stromatolites[END_REF][START_REF] Jones | Taphonomy of silicified filamentous microbes in modern geothermal sintersimplications for identification[END_REF]. The latter process will concomitantly dilute any organic or geochemical proxy signature.

Given the vagaries of the taphonomic process, identification of the remains of photosynthetic microbial mats in ancient rocks needs to be based on a range of complementary data that support its formation within the photic zone, as well as identifying proxies that indicate that the mat was formed by photosynthetic microorganisms. Proxy features that are commonly used to identify fossil photosynthetic microbial mats include (1) a mat-like, laminated morphology (planar or three dimensional, as in domical or columnar stromatolites), (2) the presence of fossil microorganisms of known photosynthetic affinity (e.g., the later-evolved oxygenic photosynthesising cyanobacteria, many species of which have readily identifiable morphologies), (3) a carbon isotopic signature consistent with photosynthesis, (4) evidence of in situ calcification as a by-product of photosynthetic activity (Supplementary figure 1). It should also be demonstrated that these combined characteristics do not occur in microbial mats formed by non-photosynthesising microorganisms. Bailey et al (2009) note that almost all of the physical and chemical characteristics used for identifying fossil photosynthetic mats can also be produced by non-photosynthetic mats, such as those formed by sulphur/sulphate or methane oxidisers in cold seep, hydrothermal or other types of environments. Table 1 compares the characteristics of modern photosynthetic and non photosynthetic microbial mats, underlining the similarities between them in terms of environment of formation, structure, composition and metabolic signatures.

Both types of mats can be formed in the photic zone but non-photosynthetic mats can also form in caves or deep water. Both can form on sediment surfaces but non-photosynthetic mats also form within the sediment. Both mat types can be laminated (chemical mineral precipitates, such as calcareous travertines can also be laminated, [START_REF] Pentecost | Travertines[END_REF]. Whereas non-photosynthetic microbial mats have also been described as being sediment stabilising and having crenulated, contorted and wrinkled surface (Bailey et al., 2009). However, interlayering with chemically-precipitated evaporite deposits in littoral/sabkha environments seems to be restricted to photosynthetic mats. Certain biomarkers distinguish the microbial composition of photosynthetic and non-photosynthetic microbial mats. For example, [START_REF] Summons | Molecular fossils for cyanobacteria recording a geological history of oxygenic photosynthesis[END_REF] describe 2-methylhopanes from 2.7 Ga-old oil shales from the Pilbara of Australia as being the degradation products of phototrophic bacterial lipids. Lipid biomarkers of sulphate reducing bacteria include mono-O-alkyl glycerol ethers [START_REF] Arning | Lipid biomarker patterns of phosphogenic sediments from upwelling regions[END_REF]. However, biomarkers have a limited life time (albeit very long) and even at 2.7 Ga the syngenicity of the 2-methylhopanes is questioned [START_REF] Rasmussen | Reassessing the first appearance of eukaryotes and cyanobacteria[END_REF]. Elemental sulphur deposits are associated with mats formed by sulphur-oxidising bacteria (Nelson and Castenholz, 1981) but not with photosynthetic microbial mats. Phosphorite deposits are also characteristic of non-photosynthetic microbial mats, such as those formed by the sulphuroxidisers Beggiatoa [START_REF] Reimers | The role of bacterial mats in phosphate mineralization with particular reference to the Monterey Formation[END_REF] and Thiomargarita (Schultz et al., 1999).

Calcification, on the other hand, occurs in non-photosynthetic microbialites, such as carbonate mud mounds formed around cold seeps [START_REF] Barbieri | Microbial fabrics from Neogene cold seep carbonates, Northern Apeninne, Italy[END_REF], as well as in photosynthetic microbial mats [START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF].

Although many species of cyanobacteria are readily recognisable in the fossil record when well preserved (e.g., [START_REF] Hofmann | Precambrian microflora, Belcher Islands, Canada: Significance and systematics[END_REF][START_REF] Knoll | Exceptional preservation of photosynthetic organisms in silicified carbonates and silicified peats[END_REF], not all photosynthetic microorganisms possess distinct morphological attributes and not all microorganisms can be/are fossilised [START_REF] Westall | The influence of cell wall composition on the fossilisation of bacteria and the implications for the search for early life forms[END_REF][START_REF] Orange | Experimental silicification of the extremophilic Archaea Pyroccus abyssi and Methanocaldococcus jannaschii. Applications in the search for evidence of life in early Earth and extraterrestrial rocks[END_REF]. Carbon isotope signatures are not sufficiently distinctive of photosynthesis since photosynthetic microorganisms are characterised by a wide range of δ 13 C values (e.g., -3 to -28‰ for oxygenic photosynthesisers and -9 to -36‰ for anoxygenic photosynthesisers) that overlap with those of other nonphotosynthetic microorganisms [START_REF] Schidlowski | A 3800 million-year isotopic record of life from carbon in sedimentary rocks[END_REF][START_REF] Schidlowski | Carbon isotopes as biogeochemical recorders of life over 3.8 Ga of Earth history: Evolution of a concept[END_REF], as well as those of carbon formed by non-biogenic processes [START_REF] Van Zuilen | Reassessing the evidence for the earliest traces of life[END_REF]. However, bulk carbon isotope signatures from photosynthetic microbial mats will be heavier than those for mats produced by sulphur/sulphate oxidisers or methane oxidisers. Moreover, carbon isotopic measurements are generally made on bulk rock samples that contain traces of the hypothetical photosynthetic microbial mats, as well as traces of any other microorganisms in that particular habitat. The resulting isotopic signature will, thus, be mixed.

Purported photosynthetically metabolising microorganisms and their macrostructures (microbial mats, stromatolites) have been identified in the oldest, well-preserved sedimentary rocks known. [START_REF] Dunlop | A new microfossil assemblage from the archeaen of Western Australia[END_REF], [START_REF] Schopf | Archean microfossils: new evidence of ancient microbes[END_REF][START_REF] Lowe | Stromatolites 3,400-Myr old from the Archean of Western Australia[END_REF][START_REF] Lowe | Restricted shallow water sedimentation of early Archean stromatolitic and evaporitic strata of the Strelley Pool Chert, Pilbara Block, Western Australia[END_REF] first described stromatolites and/or the possible remains of cyanobacteria in well-preserved sediments (cherts) dating from the Early Archaean (3.5-3.3 Ga) in the Pilbara region of NW Australia. Similar interpretations were made in Early Archaean cherts from the Barberton greenstone belt in South Africa [START_REF] Byerly | Stromatolites from the 3300-3500 Myr Swaziland Supergroup, Barberton Mountain Land, South Africa[END_REF]. Further studies have been undertaken since the 1990s in both Early Archaean terrains (e.g. [START_REF] Walsh | Microfossils and possible microfossils from the Early Archean Onverwacht Group, Barberton Mountain Land, South Africa[END_REF][START_REF] Schopf | Microfossils of the Early Archean Apex Chert: New evidence of the antiquity of life[END_REF][START_REF] Hofmann | Origin of 3.45 Ga coniform stromatolites in Warrawoona Group, Western Australia[END_REF][START_REF] Tice | Environmental controls on photosynthetic microbial mat distribution and morphogenesis on a 3.42Ga clastic-starved platform[END_REF]Lowe, 2004, 2006;[START_REF] Allwood | Stromatolite reef from the Early Archaean era of Australia[END_REF][START_REF] Allwood | Controls on development and diversity of Early Archean stromatolites[END_REF][START_REF] Tice | Environmental controls on photosynthetic microbial mat distribution and morphogenesis on a 3.42Ga clastic-starved platform[END_REF][START_REF] Westall | Early Archean fossil bacteria and biofilms in hydrothermally-influenced sediments from the Barberton greenstone belt, South Africa[END_REF]Westall et al., , 2006; see reviews by [START_REF] Westall | Early life on Earth : the ancient fossil record[END_REF][START_REF] Westall | Early life: Nature, distribution, and evolution[END_REF] using structure (laminated and/or domical), and fractionated carbon isotopic signatures as proxies for photosynthesis. The remains of the photosynthetic primary producing organisms are rarely preserved (cf. [START_REF] Altermann | Microfossils from the Neoarchaean Campbell Group, Griqualand West Sequence of the Transvaal Supergroup, and their palaeoenvironmental and evolutionary implications[END_REF], Indeed, the carbon isotope signatures of highly metamorphosed sediments from the Isua Greenstone belt in Greenland (3.8 Ga) were also interpreted as indications of photosynthesis [START_REF] Schidlowski | A 3800 million-year isotopic record of life from carbon in sedimentary rocks[END_REF][START_REF] Schidlowski | Carbon isotopes as biogeochemical recorders of life over 3.8 Ga of Earth history: Evolution of a concept[END_REF][START_REF] Mojzsis | Evidence for life on Earth before 3800 million years ago[END_REF][START_REF] Rosing | [END_REF]. Critiques of some of the studies have concentrated on the biogenicity of the laminated structures [START_REF] Lowe | Abiological origin of described stromatolites older than 3.2 Ga[END_REF][START_REF] Brasier | Questioning the evidence for Earth's oldest fossils[END_REF] and the hypothetical photosynthetic isotopic signatures (e.g. van [START_REF] Van Zuilen | Reassessing the evidence for the earliest traces of life[END_REF]. More recent investigations have addressed the importance of environmental habitat and the effects of local variations on the distribution of the ancient photosynthetic structures. For instance, [START_REF] Allwood | Stromatolite reef from the Early Archaean era of Australia[END_REF][START_REF] Allwood | Controls on development and diversity of Early Archean stromatolites[END_REF] demonstrated by detailed field mapping and macro to microscopic analyses that several varieties of domical structures in a 3.5 Ga-old formation in the Pilbara in Australia formed in a variety of localised habitats on a shallow water carbonate platform. They argued that their environment of formation and their physical and chemical characteristics of the domical structures could only have been produced by photosynthetic microorganisms. Similarly, [START_REF] Tice | Environmental controls on photosynthetic microbial mat distribution and morphogenesis on a 3.42Ga clastic-starved platform[END_REF]Lowe (2004, 2006) and Tice (2009) made a detailed microscopic and isotopic study of laminated structures in the 3.4 Ga-old Kromberg Formation in the Barberton Greenstone Belt, interpreted in terms of a diversity of mats related to specific local environments in a littoral environment. Among all proxies, in situ calcification, which is a common signature of modern, lithifying photosynthetic microbial mats (Supplementary figure 1; [START_REF] Défarge | On the appearance of cyanobacterial calcification in modern stromatolites[END_REF][START_REF] Défarge | Texture of microbial sediments revealed by cryo-scanning electron microscopy[END_REF][START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF], is one feature that remains un-documented in these ancient putative microbial ecosystems. The objective of our study is therefore to present evidence of in situ calcification in a microbial biofilm and microbial mats present in a 3.3 Ga-old sediment from the Josefsdal Chert Formation in the Barberton Greenstone Belt, South Africa, and to examine the relationship between calcification and the possible photosynthetic origin of the biofilm and mats. With this in view, we present an in situ structural and compositional analysis of the biofilm and mats on a micrometric microbial scale. Our approach mimics, as closely as possible, that used in the study of modern photosynthetic microbial mats in which a range of observational, geochemical, metabolic, and genetic methods are used at the microbial mat scale.

GEOLOGICAL BACKGROUND, MATERIALS AND ANALYTICAL METHODS

Geological Context

The Early to Mid Archaean Barberton greenstone belt of South Africa contains some of the world"s oldest and best-preserved supracrustal sequences that provide a wealth of information on Earth"s history and early life (e.g., [START_REF] Byerly | Stromatolites from the 3300-3500 Myr Swaziland Supergroup, Barberton Mountain Land, South Africa[END_REF][START_REF] Walsh | Microfossils and possible microfossils from the Early Archean Onverwacht Group, Barberton Mountain Land, South Africa[END_REF][START_REF] Banerjee | Preservation of 3.4-3.5 Ga microbial biomarkers in pillow lavas and hyaloclastites from the Barberton Greenstone Belt, South Africa[END_REF]Westall et al., 2006;[START_REF] Tice | Environmental controls on photosynthetic microbial mat distribution and morphogenesis on a 3.42Ga clastic-starved platform[END_REF]. The belt consists of a NE-SW striking succession of supracrustal rocks, termed the Barberton Supergroup (or Swaziland Supergroup in the older literature), which ranges in age from ~3550 to 3220 Ma. The belt has a strike length of ~130 km, width of 10-35 km, and an approximate depth of 4-5 km, and is surrounded by granitoid domes and intrusive sheets, ranging in age from ~3.5 to 3.1 Ma. The volcano-sedimentary sequence of the Barberton greenstone belt has been subdivided into three stratigraphic units, the Onverwacht, Fig Tree and Moodies Groups (SACS, 1980). The study locality consists of two chert horizons cropping out in the topmost of the Kromberg Formation of the Onverwacht Group in the upper part of the Josefsdal Valley (S25°57"54", E31°04"41", Figure 1). Based on its occurrence at the top of Onverwacht Group between the top of the Kromberg Formation and the Mendon Formation (Figure . 1b), the age of the Josefsdal Chert is estimated at ~3.3 Ga. The Josfsdal Chert underwent regional greenschist metamorphism dated at 3.32 Ga [START_REF] Dziggel | Mettamorphism of the granite-greenstone terrane south of the Barberton greenstone belt, South Africa: an insight into the tectono-thermal evolution of the "lower" portions of the Onverwacht Group[END_REF].

The study outcrop consists of banded black and white/greenish white silicified volcaniclastic sediments deposited on top of pillow basalt (Supplementary Figure 2).

Sedimentary structures in the chert indicate deposition in water depths ranging from sub wave base to shallow water. The black horizons contain organic matter and pseudomorphed pyrite crystals (now iron oxide), the organic matter consisting of organic detritus as well as occasional laminated microbial mats (Supplementary Figure 3). The samples used in this study come from a horizon stratigraphically located at the base of the silicified sediments.

We have based our study on microbial mat-like structures observed in thin section and in cut rock surfaces of Josefsdal Chert, and, in particular, on an exceptionally well-preserved microbial biofilm exposed in three dimensions directly on top of a bedding plane surface. A preliminary description of the exposed microbial biofilm, henceforth termed the Josefsdal Chert Microbial Biofilm (JCMB) in this work, was made by [START_REF] Westall | Early Archean fossil bacteria and biofilms in hydrothermally-influenced sediments from the Barberton greenstone belt, South Africa[END_REF]Westall et al. ( , 2006)).

The JCMB occurs on a fresh fracture surface that partially follows a bedding plane between the top of an underlying black (carbon-rich) layer and an overlying greenish white layer. The rock surface on which the biofilm is exposed is uneven and, in a small area, other biofilm fragments alternate with sediment layers deposited above the JCMB (Supplementary figure 4). It consists of portions of a thin carbonaceous film, <10 µm thick, that collectively cover an area of about 6 mm 2 (Figure 2a; Supplementary Figure 2b). The biofilm is only one of a packet of mat-like structures whose combined thickness is <1 mm (the upper portions were almost entirely removed by fracturing). It is similar to the mat shown in a thin section of the Josefsdal Chert in Supplementary Figure 3. The surface of the film consists of fine filaments, 0.25-0.3 µm in diameter, that are thickly coated with a smooth material interpreted as EPS (Figures 6, 8 in Westall et al., 2006). Parallel orientation of the filaments and overturning of portions of the mat in the same direction (Figures 5 and 6a in Westall et al., 2006) suggest formation under flowing water, possibly close to a hydrothermal spring since the biofilm was silicified in vivo by hydrothermal fluids. A subaerial beach setting is suggested by the presence of intercalated layers of pseudomorphed (silicified) evaporite crystals in the upper layers of the film and by cracks in its surface that indicate periodic exposure to the atmosphere (Figure 6d in Westall et al., 2006). Detrital particles including volcanic shards and quartz grains from 10"s of µm up to ~300 µm in size are trapped in the mat (Figure 7b in Westall et al., 2006).

Carbon isotope signatures for the bulk sediment were -22.7 ‰ [START_REF] Westall | Early Archean fossil bacteria and biofilms in hydrothermally-influenced sediments from the Barberton greenstone belt, South Africa[END_REF] and a value of -26.8 ‰ was obtained for a bulk sample of separated carbon-rich black layers (Westall et al., 2006). The turgid filaments in the surface of the biofilm, rather than degraded remnants, (Figures 6f, 8b in Westall et al., 2006) suggest rapid silicification probably by silica-rich hydrothermal fluids as the biofilm was living (cf. Hofmann and Bohlar, 2007). This is supported by the fact that the anions of the evaporite crystals have been replaced by fluorine, most likely present in the same hydrothermal fluids that induced silicification of the mat.

Analytical techniques

FIB preparations

beam (FIB) sections vertically cut using a Ga beam into the 5-10 µm thick mat (Figure 2b) [START_REF] Lekstrom | Using the in situ lift-out technique to prepare TEM specimens on a single-beam FIB instrument[END_REF]. (N.B. the mat had previously been coated with Au for SEM observation, Westall et al., 2006). The FIB sections were prepared at the Centre Pluridisciplinaire de Microscopie Electronique et de Microanalyse at the University Aix-Marseille using a Philips FIB 200, and at the Institute d"Électronique, Microélectronique et de Nanotechnologie (IEMN), Lille, using an FEI Strata DB 235. On some sections, a protective 1-2 μm thick platinum strip was deposited on the sample before milling in order to protect the FIB slice. The primary ion currents used for in situ milling and polishing of the FIB sections were in the range of 0.17 to 7 nA and beam energies ranged from 2 to 30 keV. Sections 90 nm thick were mounted on Cu-TEM grids coated with holey carbon film were prepared for transmission electron microscope (TEM) analyses, whereas thicker FIB sections of 3µm were prepared for scanning electron microscope (SEM), scanning transmission electron microscopy (STEM), NanoSIMS (a high spatial resolution secondary ion mass spectrometry) and synchrotron radiation analysis.

SEM and TEM

Three TEM instruments used were a Philips CM20 TEM fitted with EDX detector available at Centre de Microscopie Electronique, Université d"Orléans, France; a TEM JEOL 2011 microscope at the University of Paris VI, France; and a FEI TECNAI G2 at the Centre Pluridisciplinaire de Microscopie électronique et de Microanalyse (CP2M), Marseille, France.

In all cases, the beam energy used was 200 kV and the microscopes were equipped with a energy-dispersive X-ray spectroscope (EDX) capable of measuring light elements (except for the JEOL 2011 TEM). The SEM instruments used included an Hitachi S4500 FEG-SEM, equipped with a Polaron LT7400 cryopreparation system that can function as a cryoSEM, at the Centre de Microscopie Electronique, Orléans and a Philips XL30 SFEG STEM at the Centre Pluridisciplinaire de Microscopie Electronique et de Microanalyse at the University Aix-Marseille. SEM imaging and elemental mapping were made between 5-10 kV.

Synchrotron analysis: The beamline ID21 SXM set-up for fluorescence imaging and µ-

XANES of S at the K-edge

FIB sections prepared for synchrotron irradiation were mounted on C-coated TEM Cugrids. With its 2-7 keV energy range, the ID21 scanning X-ray microscope (SXM) of the European Synchrotron Radiation Facility (ESRF) in Grenoble [START_REF] Susini | The scanning X-ray microprobe at the ESRF by X-ray microscopy beamline[END_REF] is a unique tool for microspectroscopy at the sulfur K-edge, attaining high spatial (0.3 µm) and spectral (0.25 eV) resolution. The SXM was operated at energies around 2472 eV, close to the sulfur K-edge. Micro fluorescence maps were also performed at the Fe K-edge around 7112 eV with a comparable set up. The FIB cuts mounted on TEM grids were held between 4 m thick Ultralene foils. The sample is positioned at an angle of 60º (90º between beam and fluorescence detector axis) with respect to the incoming beam. Further details of the instrument setup are provided in the Supplementary Methods.

STXM and Near-Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy at the C K-edge

Scanning transmission X-ray microscopy (STXM) and Near-Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy at the C K-edge analyses were made on the X1A beamline at the National Synchrotron Light Source (NSLS, Brookhaven). Data for the Josefsdal Chert were collected using a STACK data collection technique where an image is taken, the monochromator energy is changed by a small increment; another image is collected and so on until a series of images over the energy range of interest is collected. The images are aligned in X and Y using a cross correlation algorithm. Spectra can be obtained from individual pixels in the image. Carbon maps are acquired by taking the difference of the sum of images acquired below 283 eV and the sum of images acquired between 290-293 eV. The STACK energy spacing for this data set was 280-283.6 eV, with 0.3 eV step size, 283.6-292.0 eV with 0.1 eV step size, 292-305 eV with 0.3 eV step size [START_REF] Jacobsen | Soft x-ray spectroscopy from image sequences with sub-100 nm spatial resolution[END_REF]. The image size was 320 pixels in both X and Y with a pixel size of 30nm. Further details of the instrument setup are provided in the Supplementary Methods.

NanoSIMS

Chemical maps of a 10 x 3 x 15 µm FIB section were produced with the Cameca NanoSIMS 50 at the Muséum National d"Histoire Naturelle in Paris, France (N.B. the same FIB section was analysed by µ-XANES at the ESRF, Grenoble). The instrument is characterized by high spatial resolution (down to 50 nm), high sensitivity, and the simultaneous detection of up to five isotopes. In this study, negative secondary ions of 12 C, 16 O, 12 C 14 N, 28 Si and 32 S were mapped simultaneously (multicollection-mode) using electronmultiplier detectors. Using primary current conditions (about 0.5 pA on the sample surface), the primary ion beam of Cs + was focused onto the sample in spots <100 nm in size. Analyses were performed at a mass-resolving power > 5000, sufficient to resolve significant isobaric interferences. A normal incidence electron gun was tuned during analysis in order to compensate for positive primary beam charge deposition on the sample surface. Two ion images consisting of 256 x 256 pixels were acquired by scanning areas of 17 x 17 m and with counting times of 1 s/pixel. Within the imaged regions, two oxygen-rich features were observed and subsequently imaged in frames of 8 x 8 m (256 x 256 pixels) and with a spatial resolution of about 50 nm. Note that, because nitrogen can only be detected as CN -in NanoSIMS, it can only be measured in the presence of carbon. Also, the sample was attached to a metallic mount using carbon tape, which greatly reduces the risk of surface contamination compared to the use of epoxy.

ToF-SIMS

The Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) analyses were made on a cleaned cut rock surface using an ION-TOF IV GmbH instrument (Münster, Germany) equipped with a Bismuth liquid metal primary cluster ion source (see details below) at the Technical Research Institute of Sweden (Borås). Spectra from the sections were recorded using 25 keV Bi 3 + primary ions, with the instrument optimized for maximum mass resolution (bunched mode, m/Δm ~3000-6000). The cleaning protocol for the samples is as follows. The rock surface was sawn with saw blades that had been thoroughly cleaned and rinsed with acetone. MilliQ water was used during sawing. The outer portions of the rock sample were sawn off and discarded to eliminate contamination. A slice of rock from the inside of the hand specimen was then sawn, wrapped in acetone-cleaned aluminium foil and stored in a cooled Ar-filled, Schott glass container until analysed. The sample was removed from the container in a laminar flow hood, placed on the ToF-SIMS sample holder, and then placed directly in the instrument for analysis.

Raman spectrometry

Raman spot analyses and mineralogical maps of a polished thin section of the Josefsdal Chert were carried out using a WITec Alpha500 RA system. The light source used was a frequency-doubled Nd:YAG green laser (wavelength =532 nm). Low energy power of ~6 mW was used in order to prevent deterioration of the samples.

RESULTS

Microbial mats in thin section

Microbial mat-like structures were observed in a number of thin sections from the Josefsdal Chert outcrop. They occur as packets of fine anastomosing laminae tens of micrometers in thickness (Supplementary Figure 3). The laminae are conformable with the underlying surface but have a wispy, undulating structure. They trap detrital particles such as quartz and volcanic grains. The laminae form packets that can be up to several hundred micrometers in thickness suggesting some periodicity to growth. Raman spectral mapping documented their kerogenous composition (Supplementary Figure 5) (D:G peak ratio of about 2.5). Analysis and mapping using an ion probe demonstrated that they are enriched in Ca, a result which triggered further investigation of the Ca-containing phase. Small crystals of pseudomorphed pyrite (1-2 µm) are frequently associated with the black layers (Supplementary Figure 5b). Although now oxidised, EDX and µ-XANES analyses document remnant sulphur in the crystals (data not shown).

ToF-SIMS analysis on the carbonaceous layers (Supplementary Figure 6) in the hand

The Josefsdal Chert fossil Microbial Biofilm

There are two distinct types of carbonate phases associated with the JCMB. Silicified pseudomorphs after calcite are part of the evaporite mineral suite interbedded and coating the topmost layers of the filamentous biofilm (Figure 3; Figures 6c,d in Westall et al., 2006). The silicified pseudomorphs of calcite in the top part of the biofilm are blocky crystals, 5-10 µm in size, possessing no other associated elements (Figure 4). There is also a nanocrystalline phase containing traces of Mg, Fe, Cr and sulphate precipitated in association with the kerogen in the body of the biofilm (Figures 456).

Numerous FIB-sections cut vertically through the JCMB show that the 10 µm thick structure is divided into a lower part having a nanometer scale granular texture and an upper part consisting of alveolar-textured kerogen (Figures 3,5a). The thickness of the kerogen layers varies laterally within the biofilm from < 1 µm to a few micrometers, whereas the granular layer is typically thicker and can be up to about 5 µm (e.g., Figure 3b). High resolution images of the FIB sections show that the 5-10 nm nanocrystals in the lower part of the biofilm have nucleated within an alveolar-textured organic matrix that is structurally similar to that in the overlying kerogen layer (Figures 5,6; Supplementary Figure 7). Micro-XRF, EDX and µ-XANES mapping of the FIB sections document the association of Ca, Mg, Fe, Cr and sulphate with the nanocrystalline phase, with Ca the predominant element (Figure 4). HRTEM images of the carbonate crystallites (Figure 6) show interplanar distances of 0.335 nm, corresponding to the d 111 interplane of the carbonate phase aragonite. Fast Fourier Transform imaging of the nanocrystals (inset, Figure 6b) shows a cloudy-spotty ring (corresponding to d = 0.335 nm), typical of the diffraction pattern generated by multiple crystallites with variable rotational orientation.

Elemental mapping by EDX and NanoSIMS show that carbon is ubiquitous throughout the biofilm, and that it occurs especially in the pure kerogen zone in the upper layer of the biofilm, as well as in certain nanocrystal-free pockets within the granular lower part (Figure 7). Traces of N and S are associated with the kerogen. Using µ-XANES we were able to qualitatively estimate up to 1% S in the upper, non-granular portion of the biofilm where both µ-XANES and NEXAFS documented the presence of the S-containing heterocyclic molecule thiophene (Figure 8; Supplementary Figure 8). Note that although the kerogen has an amorphous appearance, HR-TEM shows that, in terms of its nanostructure, it corresponds to a disordered poly-aromatic carbon containing small stacks of fringes ~1 nm long (Figure 5b), indicating that the kerogen is relatively mature. The molecular moieties identified by ToF-SIMS analyses (Supplementary Figure 8) could correspond to these polyaromatic structures.

DISCUSSION

Timing of the formation of the JCMB

The nanostructure and composition of the organic carbon is consistent with degraded ancient organic carbon that has undergone lower greenschist facies metamorphism, thus indicating that the carbon predates the 3.2 Ga-old metamorphic event that affected the region [START_REF] Dziggel | Mettamorphism of the granite-greenstone terrane south of the Barberton greenstone belt, South Africa: an insight into the tectono-thermal evolution of the "lower" portions of the Onverwacht Group[END_REF]. While Wacy (2010) suggested that the JCMB might be a later endolithic biofilm supported by a hydrothermal intrusion into the Early Archaean chert, morphological and environmental evidence (Westall et al., 2006) suggest syngenetic formation of the biofilm on a sediment surface in an evaporitic littoral environment.

Moreover, the JCMB is part of a packet of biofilms (Supplementary Figure 4) and shares many of the same characteristics as other delicate sediment stabilising microbial mats observed in thin sections of other samples of the Josefsdal Chert (Supplementary Figure 3, see Westall et al., 2006). The syngenicity of its development and calcification is further corroborated by the geochemical data presented here which indicate that the calcification of the JCMB is related to the activities of a photosynthetic consortium that includes sulphur reducing bacteria. The rationale for this interpretation is explained below.

Calcification of the JCMB

Two, clearly distinct carbonate phases are associated with the JCMB: (1) nanophase aragonite crystals that nucleated onto the alveolar kerogen within the body of the biofilm and

(2) blocky calcite crystals (now pseudomorphed by silica) in the uppermost layer of the JCMB. In terms of crystallography, elemental composition and occurrence, the two phases exhibit different characteristics presumably reflecting different origins. The blocky calcite crystals are large and sub-euhedral relative to the nanophase aragonite. They contain no associated elements apart from Ca and are strictly associated with the suite of pseudomorphed evaporite minerals in the uppermost layers of the JCMB. In contrast, the aragonite crystals are small (5-10 nm), anhedral (spherical to oval in shape), contain traces of Mg, Fe and Cr and sulphate, and occur only in association with the alveolar kerogen within the body of the mat. This phase is always separated from the biofilm surface (and the exterior environment) by a layer of more or less pure kerogen. Calcification of the degraded organic matter in the body of the mat must have occurred as the mat was living because the biofilm was silicified while it was active (the silicified filaments on top of the mat are non-calcified have a turgid morphology, cf. Westall et al., 2006). Nucleation and preservation of 5-10 nm-sized aragonite crystallites within the "degraded" alveolar kerogen below suggests mediation by the organic matrix in the calcification process, that is, "organomineralisation" sensu [START_REF] Défarge | Texture of microbial sediments revealed by cryo-scanning electron microscopy[END_REF] and [START_REF] Défarge | Organomineralization[END_REF]. These observations corroborate the syngenicity of the development of the mat and its calcification.

The preservation of aragonite also argues in favour of syngenicity of calcification and JCMB formation. Aragonite is an unstable phase that rapidly converts to calcite and is therefore rare in the geological record [START_REF] Lepot | Microbially influenced formation of 2,724-million-year-old stromatrolites[END_REF][START_REF] Chafetz | Geochemistry of preserved Permian aragonitic cements in the tepees of the Guadalupe Mountains, West Texas and New Mexico[END_REF]. Its preservation in the JCMB is probably due to a combination of nucleation within a kerogen matrix (cf. [START_REF] Lepot | Microbially influenced formation of 2,724-million-year-old stromatrolites[END_REF] and rapid permeation of the biofilm by silica (Figure 9) (cf. Westall et al., 2006[START_REF] Westall | Volcaniclastic habitats for early life on Earth and Mars: A case study from 3.5 Ga-old rocks from the Pilbara, Australia[END_REF]. The formation of aragonite in the EPS of the JCMB rather than another carbonate phase was probably related to its physico-chemical environment of formation. A number of carbonate species may be precipitated during calcification in hydroxyls and subsequent, heterotrophy-generated alkalinity (bicarbonate) contribute to carbonate precipitation.

There are a number of indications that calcification of the JCMB could be related to sulphur reducing bacteria. Sulphur-containing molecules, such as thiophene, occur in the kerogen. Natural bacteriological organic molecules contain sulphur in amounts ranging from 0.4 % (Heldal et al., 2003) to 1% [START_REF] Fagerbakke | Content of carbon, nitrogen, oxygen, sulfur and phosphorus in native aquatic and cultured bacteria[END_REF]. The 1% S in the JCMB kerogen could represent an original composition but may also indicate diagenetic incorporation of sulphur into the organic matrix during anaerobic microbial breakdown of organic matter, such as by sulphur/sulphate-reducing bacteria (sulphurisation; [START_REF] Summons | A review of fundamental aspects of organic matter formation, preservation and composition[END_REF][START_REF] Lemelle | In situ imaging of organic sulfur in 700-800 My-old Neoproterozoic microfossils by X-ray spectromicroscopy at the S K-edge[END_REF]. Moreover, µm-scale pseudomorphed pyrite crystallites (1-2 µm), a diagnostic byproduct of sulphur reducing bacteria activity, are associated with the JCMB and the carbonrich black layers in the Josefsdal Chert (microbial mats and layers of detrital carbon). As in modern microbial mats [START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF], calcification in the JCMB could have occurred in the following manner. The metabolic activity of sulphur reducing bacteria could have caused an increase in dissolved CO 2 within the biofilm, thus increasing the alkalinity. This in turn would have led to desorption of Ca 2+ ions initially adsorbed to the EPS, which combined with the bicarbonate ions in the water-rich organic matrix. Calcium carbonate would have precipitated from the saturated solutions within the EPS framework and would have fixed to the functional groups on the available organic surfaces (EPS).

Implications for photosynthesis

The JCMB formed in an anaerobic beach environment. [START_REF] Kasting | Earth"s early atmosphere[END_REF], modelled 0.2% of present atmospheric levels of O 2 for the Early Archaean period, corroborated by [START_REF] Canfield | The Archean sulfur cycle and the early history of atmospheric oxygen[END_REF] who concluded that the early Archaean seawaters were characterised by low sulphate and oxygen concentrations on the basis of the S isotope record. This would preclude mat formation by aerobic sulphur oxidising microorganisms inhabiting the oxic/anoxic interface (cf. Bailey et al., 2009). The moderately light carbon isotope composition (-26.7‰) indicates that the biofilm was not formed by methane oxidisers.

Furthermore, exposure and interlayering with encrusting evaporite minerals in an evaporitic setting is characteristic of photosynthesising mats (although non-photosynthetic mats can coexist in shallow waters (< 10 cm depth) with photosynthetic mats; [START_REF] Garcia-Pichel | Diel migrations of microorganisms within a benthic, hypersaline mat community[END_REF].

The JCMB is therefore a photosynthetic microbial mat. In the nutrient-limited beach setting in which the Josfsdal sediment formed, the only way to produce the carbon and sulfoxyanions needed by the heterotrophic sulphur reducing bacteria would have been by photoautotrophy.

As in modern photosynthetic microbial mats, it is the layered distribution of the carbonate that suggests that this paleo-record of calcification is not the result of a "pure" culture" but represents a minimum of two complementary metabolic processes, phototrophy and heterotrophy.

Table 1 compares the characteristics of the JCMB with those of modern photosynthesising microbial biofilms. The JCMB formed in the photic zone on an intertidal/supratidal beach surface (cf. Westall et al., 2006) under anaerobic conditions;

exhibits direct interaction with it environment in the form of sediment stabilization, formation under flowing water, overturned filaments/mat portions, desiccation (cf. Noffke, 2009; Westall et al., 2006); despite the modest 10 µm thickness, its finely laminated structure with a vertical architecture (degraded lower calcified part, upper non-calcified active layer) is an indication of a steeply changing physico-chemical gradient that can provided microenvironments for a variety of microorganisms having different nutritional requirements and metabolic strategies [START_REF] Dupraz | Processes of carbonate precipitation in modern microbial mats[END_REF];; it was formed by EPS-secreting microbial filaments; associated pyrite crystallites and possible sulphurisation most likely indicate sulphur reduction of the primary biomass by heterotrophic bacteria; in situ calcification of the lower part of the mat was probably a by-product of the microbial sulphur-reduction; the moderately light carbon isotope signature of -26.7 ‰ suggests a dominantly phototrophic metabolism.

Thus, despite the ephemeral nature of many of the characteristics of photosynthetic microbial mats, the JCMB still retains fossil signatures that allow it to be interpreted as a photosynthesising microbial mat formed by a consortium of microorganisms with anaerobic photosynthesisers as the primary producers and sulphur reducing bacteria as the heterotrophs.

Conclusions

The excellent state of preservation of the 3.3 Ga-old Josefsdal Chert Microbial Biofilm by contemporaneous silicification has permitted three-dimensional, nanometre-scale profiling and imaging that documented, for the first time, in situ calcification within a microbial biofilm of this age. Sulphur reducing bacterial activity most likely contributed to the formation and nucleation of nanocrystallites of aragonite onto the degraded organic matter within the body of the biofilm. Preservation of the aragonite (the oldest known occurrence)

was probably due to the combined influence of an organic (kerogenous) matrix and contemporaneous impregnation by hydothermal silica. The JCMB presents all the preservable characteristics of modern photosynthesising microbial mats. It was constructed in the photic zone; it has a laminated, vertically structured architecture including trapped detrital particles; it stabilised the underlying sediment surface; and it was calcified in situ by primary aragonite, probably in association with sulphur reducing bacteria degradation of organic material produced by an anoxygenic photosynthesising primary producer. The -26.7‰ carbon isotope signature is consistent with a photosynthetic metabolism and subsequent heterotrophic degradation of photosynthetic carbon. Their presence indicates that the kerogen is relatively biofilmure. (B) µXANES spot spectral analyses at the S-K-edge in the kerogen and carbonate layers. The kerogen layer is characterised by a high peak at 2474 eV representing a heterocyclic organic compound, identified as thiophene (cf. [START_REF] Lemelle | In situ imaging of organic sulfur in 700-800 My-old Neoproterozoic microfossils by X-ray spectromicroscopy at the S K-edge[END_REF]. It also contains a small amount of inorganic sulphur in the form of sulphate at 2482.5 eV. The calcified part of the biofilm has a higher sulphate peak but still contains a small amount of thiophene.

SUPPLEMENTARY FIGURE CAPTIONS

 In situ calcification (aragonite) of a 3.3 Ga microbial biofilm  Calcification probably due to the activities of a photosynthetic consortium of microorganisms within the biofilm including heterotrophic sulphur reducing bacteria  Preservation of the metastable carbonate phase by the organic matrix and secondary silicification Oldest known occurrence of aragonite.

FIGURE CAPTIONS Figure 1 .

 CAPTIONS1 FIGURE CAPTIONS

Figure 2 .

 2 Figure 2. Location of FIB sections in the Josefsdal Chert Microbial Biofilm (JCMB).

Figure 3 .

 3 Figure 3. Backscatter SEM micrographs of FIB slices cut vertically through the Josefsdal

Figure 4 .

 4 Figure 4. Elemental composition of the carbonate phases in the JCMB.

Figure 5 .

 5 Figure 5. Nanostructure of the JCMB.

Figure 6

 6 Figure 6 Nanostructure of the calcified part of the JCMB. (A) TEM micrograph of a part of

Figure 7 .

 7 Figure 7. NanoSIMS mapping of carbon, nitrogen, and sulphur in the FIB slice shown in

Figure 8 .

 8 Figure 8. µXANES mapping and analysis of sulphur in the FIB slice shown in Figure 4b.

Figure 9 .

 9 Figure 9. NanoSIMS mapping of silica (Si 28 ) in the FIB slice shown in Figures 4b and 7a. (A) Backscatter image of the FIB section analysed. (B) Silica (Si 28 ) map in the FIB section. The vertical dashed white line shows the location of the profile of Si (C).

ACKNOWLEDGEMENTS

Funding was provided by the CNRS, the GDR-Exobiologie, the ANR-Blanc-ARCHAEMAT, and the NSERC. F.W. and C.D. acknowledge A. Richard for help with HR-SEM/cryoSEM and and D. Jalabert HR-TEM at the University of Orléans.

microbial mats, including calcite, aragonite, and dolomite although aragonite appears to be the most common form (Bernd and Seyfried, 1999). The presence of primary aragonite in the JCMB is thermodynamically more plausible than calcite because aragonite precipitates instead of calcite at temperatures > 70°C [START_REF] Kawano | Precipitation diagram of calcium carbonate polymorphs: its construction and significance[END_REF]. This is consistent with the low temperature hydrothermal setting that gave rise to silicification of the Josefsdal Chert (< 150°C, [START_REF] Hofmann | Stratiform alteration zones in the Barberton greenstone belt: a window into subseafloor processes 3.5-3.3 Ga ago[END_REF]. Aragonite is also the favoured Ca-carbonate phase when calcite nucleation and growth are inhibited by the presence of chemical species such as Mg 2+ , Fe 2+ , SO 4 2- [START_REF] Berner | Principles of Chemical Sedimentology[END_REF][START_REF] Bischoff | Catalysis, inhibition and calcite-aragonite problem. I. The calcite aragonite transformation[END_REF], species which, together with Cr, have been detected in the carbonate of the JCMM. The direct source of these metal ions is probably from early diagenetic alteration of the ultramafic-derived volcaniclastic sediments on top of which the mat formed.

The sequence of events that occurred during the lifetime of the JCMB is summarised in Supplementary Figure 8: (1) biofilm formation on a sediment surface, (2) in situ calcification in the lower, degraded part of the film, (3) exposure, desiccation and precipitation of a suite of evaporite minerals on its surface, and (4) final impregnation and coating by hydothermal silica.

Many non-photosynthetic biological mechanisms can lead to calcification, e.g., chemosynthetic systems around cold seeps [START_REF] Barbieri | Microbial fabrics from Neogene cold seep carbonates, Northern Apeninne, Italy[END_REF][START_REF] Power | The hydromagnesite playas of Atlin, British Columbia: A biogeochemical model for CO 2 sequestration[END_REF].

Sulphur/sulphate reducing microbial activity and methane oxidation can also lead to carbonate precipitation, as can photosynthesis (the latter only under particular environmental conditions of low dissolved carbon but high Ca; Arp et al., 2001). Carbonate precipitation can also occur through abiological CO 2 degassing and evaporation, for example in travertine. For the JCMB, calcification of the lower layer of the biofilm, below the evaporative surface, suggests the importance of the biosphere in carbonate formation where autotrophy-generated

Supplementary


Biodétérioration d'une matrice cimentaire par les champignons : influence du vieillissement accéléré sur le développement fongique. Biodeterioration of cementeous matrix by fungi: influence of accelerated weathering on

Keywords: ciment, Alternaria alternata, Aspergillus niger, biodétérioration, test accéléré

fungal development.

Introduction

De nombreux matériaux de construction sont sujets à une colonisation par les microorganismes. Cette colonisation est responsable de changements de la coloration, et des propriétés chimiques et/ou physique du matériau. Depuis la fin des années 60, ces changements ont été regroupés sous les termes de « biodégradation » ou « biodétérioration ». Ce dernier terme semble être utilisé principalement en relation avec l'altération du matériau [1]. Le degré de colonisation n'est pas seulement lié aux conditions environnementales, mais aussi aux propriétés intrinsèques du matériau utilisé qui déterminent sa bioréceptivité, c'est-àdire sa susceptibilité à être colonisé par les organismes vivants [1,2]. Le rôle précis des caractéristiques des matériaux de construction dans la colonisation n'est pas totalement compris, à l'exception de l'acidité, dont l'influence sur la composition taxonomique des micro-organismes colonisateurs est bien connue [1]. Les infections biologiques et l'intensité du processus de détérioration sont fortement influencées par la disponibilité en eau [3]. Pour la colonisation et le développement d'une communauté microbienne sur un matériau organique ou inorganique, la composition du matériau utilisé et son état de conservation, les conditions environnementales et climatiques favorables (température, humidité, pluie, exposition au soleil, polluants organiques et inorganiques) peuvent créer de bonnes conditions, parfois même optimales, pour le développement des organismes en surface (détérioration esthétique), et/ou jusqu'à des couches plus profondes (détérioration physique/mécanique et chimique) [START_REF] Urzi | Biodeterioration of cultural heritage in Italy : State of art, ADRIANE workshop[END_REF]. Généralement, la colonisation microbienne des matériaux de construction commence avec le développement du biofilm et des bactéries. Les biofilms microbiens sont des assemblages de cellules microbiennes encastrées dans une matrice complexe de substances polymériques complexes : les exo polysaccharides (EPS). En fait, les EPS constituent un véritable ciment qui permet le développement microbien et l'adhésion des micro-organismes au substrat. Elles protègent les cellules de la déshydratation, et représentent un accumulateur de carbone organique. Elles jouent un rôle important dans la formation du biofilm. Les biofilms microbiens se développent pratiquement sur tous les substrats inorganiques, altérant la couleur originale et l'aspect par le développement d'une population avec des pigments variés, en piégeant la poussière, la suie et la matière particulaire dans les EPS. Généralement, la pollution environnementale représente une source de nutriments, augmentant considérablement la croissance du biofilm microbien et ainsi le processus de détérioration [START_REF] Tomaselli | Biodeterioration processes in inorganic substrata[END_REF]. Ensuite peuvent se développer les algues et cyanobactéries, ainsi que les champignons. Les premiers sont responsables principalement d'une détérioration esthétique, alors que les champignons peuvent être à l'origine d'une détérioration chimique et physique, en plus de la détérioration esthétique. Les dommages principaux dérivés de l'activité métabolique des organismes sont associés aux mécanismes physiques, chimiques et esthétiques, tandis que l'intensité des dommages est directement corrélée aux type et dimensions de l'organisme impliqué, au type de matériau et à son état de conservation, aux conditions environnementales, à l'exposition, au type et niveau de la pollution atmosphérique [START_REF] Tiano | Biodegradation of cultural heritage: Decay mechanisms and control methods[END_REF]. Nous nous intéressons dans cette étude, plus particulièrement à la biodétérioration intervenant bien après la formation du biofilm bactérien, nous ne parlerons donc pas de biodétérioration causée par les bactéries. L'action chimique est imputable à la production de divers métabolites, qui agissent par assimilation ou dissimilation. Lors des processus d'assimilation, les constituants du matériau sont utilisés comme nutriment après avoir été réduits par diverses enzymes extracellulaires qui facilitent la pénétration des hyphes dans le matériau [START_REF] Sand | [END_REF]. Les processus de dissimilation sont liés à la production d'acides organiques et de pigments. Les acides organiques sont capables de réagir avec le substrat par dissolution des cations ou par chélation des ions métalliques. Le dioxyde de carbone produit par tous les organismes aérobies, notamment les moisissures lors de la respiration, joue également un rôle dans la dégradation des supports. En effet, à partir d'un certain taux d'humidité, ce gaz peut se transformer en acide carbonique, capable de dissoudre le calcium et le magnésium pour former des bicarbonates de calcium et de magnésium hydrosolubles [3,[START_REF] Boissier | Etude et compréhension des phénomènes environnementaux régissant la colonisation des produits de construction par les aérosols fongiques : Application à l'hygiène des environnements intérieurs[END_REF][START_REF] Sand | [END_REF]. Outre l'action directe de ces acides, leur production favorise la croissance d'espèces fongiques acidophiles qui pourront poursuivre la dégradation du support [START_REF] Garg | Conservation, Preservation and Restoration: Traditions, Trends and Techniques[END_REF]. La biodétérioration physique résulte de la pression qu'exercent les micro-organismes sur le matériau pendant leur croissance. C'est le cas des hyphes pour les champignons, ou des racines pour des systèmes plus complexes comme les mousses ou les lichens [3,[START_REF] Sand | [END_REF]. Les travaux antérieurs portent essentiellement sur la biodétérioration esthétique, très peu s'intéressent aux mécanismes. Ainsi, les champignons permettent d'étudier les différents aspects de la biodétérioration : esthétique, chimique et physique. Afin de mieux comprendre ces mécanismes, nous mettons au point un test accéléré de laboratoire, qui nous permette de décrire les mécanismes de la biodétérioration d'une matrice cimentaire par les champignons. La première souche sélectionnée est Alternaria alternata, choisie pour plusieurs raisons: (i) Tout d'abord elle a été fréquemment isolée lors de prélèvements sur sites. (ii) Elle a la particularité de synthétiser de la mélanine. Leur pigmentation sombre (mélanine et mycosporines) les protège des irradiations, des températures extrêmes et d'une possible dessiccation [START_REF] Zhdanova | [END_REF]. Ces pigments contribuent considérablement à la survie de ces champignons aux conditions extrêmes [11]. (iii) Son mécanisme d'action est essentiellement physique par la pénétration en profondeur des hyphes [12]. De plus, c'est un champignon très difficile à enlever du support après colonisation. La seconde souche retenue dans le cadre de notre étude est Aspergillus niger. (i) Elle est également mentionnée à plusieurs reprises dans les travaux de la littérature. (ii) C'est un champignon acidogénique, c'est à dire qu'il produit divers acides organiques et aura donc un mode d'action essentiellement chimique. Ces deux souches sont intéressantes dans la mesure où elles vont nous permettre d'étudier deux modes d'action différents (physique et chimique) de la biodétérioration. Cependant, il faut souligner un point important : ces deux souches ont été sélectionnées parmi les souches isolées et identifiées à partir de prélèvements sur sites. (i) Leur présence n'implique pas qu'elles soient responsables de la biodétérioration (outre esthétique). (ii) Sur site, les souches vont être prélevées stérilement pour être cultivées en laboratoire afin de les isoler et de les identifier. En laboratoire, seules les souches cultivables et à croissance rapide vont être isolées et identifiées, au détriment des souches à croissance lente ou non cultivables. Il est reconnu que probablement plus de 90% des micro-organismes présents sur les matériaux des biens culturels dégradés ne sont pas identifiés. Ceci a une importance majeure, car la plupart des travaux de restauration utilisent les biocides conventionnels qui ne conviennent probablement pas aux communautés microbiennes complexes et inconnues qui se développent sur et sous les surfaces [START_REF] Saiz-Jimenez | Presentation of COALITION[END_REF]. Concernant le choix de la matrice support, nous travaillons avec une pâte de ciment. En effet, le ciment constitue le matériau d'enrobage de nombreux matériaux de construction (mortier technique, béton…), il joue donc un rôle important dans l'intégrité structurale de ces matériaux. Le ciment est un matériau basique (pH~13) ; ce qui n'est pas favorable à la croissance des champignons. Pour favoriser le développement fongique, il faut diminuer l'alcalinité de notre support, donc le vieillir artificiellement. Nous avons choisi d'étudier deux modes de vieillissement accéléré dans cette étude : la carbonatation et la carbonatation couplée à la lixiviation.

Matériel et méthodes

Matrice d'étude

Nous utilisons des éprouvettes en ciment blanc CEM I 52,5 R, élaborées avec un rapport massique eau/ciment de 0,55. Les éprouvettes sont de forme parallélépipédique : 1 x 2,5 x 8,5 cm 3 . Les éprouvettes sont démoulées après 24h et conservées 28 jours à 100% d'humidité relative à température ambiante. Les éprouvettes sont stérilisées par rayonnements ionisants (rayon Gamma) au Commissariat à l'Energie Atomique (CEA) de Cadarache, après 28 jours de conservation ou après carbonatation ou lixiviation.

Vieillissement accéléré de la matrice

Essai de carbonatation accéléré

L'essai de carbonatation est réalisé dans une enceinte avec un balayage de CO 2 pur. L'humidité relative est maintenue à 65±5% par une solution saline de K 2 CO 3 (RP Normapur). Afin de suivre l'évolution de la profondeur de carbonatation, des échantillons sont cassés à intervalles de temps réguliers, et caractérisés à l'aide d'une solution de phénolphtaléine. La phénolphtaléine est incolore pour des pH inférieurs à 9,2 environ, et se colore en violet au dessus de cette valeur [START_REF]Measurement of hardened concrete carbonation depth[END_REF]. Certaines éprouvettes sont séchées 24h à 40°C avant carbonatation.

Lixiviation

La lixiviation des éprouvettes carbonatées est réalisée dans des bacs de 10 litres. La solution lixiviante (eau déminéralisée) est remplacée en continu. Le débit est de 7 ml/min. Trois éprouvettes carbonatées sont placées dans le réacteur. Le pH est suivi en continu. Par soucis de simplicité, nous appellerons par la suite « éprouvettes non vieillies » les éprouvettes obtenues après 28 jours de conservation à 100% d'humidité relative, « éprouvettes carbonatées » les éprouvettes ayant subies une carbonatation après 28 jours de conservations, « éprouvettes carbonatées puis lixiviées » les éprouvettes qui ont été carbonatées après 28 jours de conservation puis lixiviées.

Diffraction des Rayons X

La diffractométrie des rayons X est réalisée sur un appareil Bruker D8 advance. On utilise la raie Kα1 du cuivre (λ=0.15406 nm). Le dispositif comprend une anticathode de cuivre et un détecteur ponctuel. Le temps de comptage est de 5 secondes et le pas du goniomètre de 0.04°. L'analyse se fait sur une plage en 2θ allant de 5 à 59°.

Champignons

Milieux de culture

Alternaria alternata Fries von keissler est une souche de collection DSMZ. Les cultures sont réalisées sur milieu V 8 agar (CaCO 3 3g/l, Agar 20g/l, jus V8 200ml/l), et sur Czapeck-Dox modifié pour favoriser la sporulation (poudre de cellulose 30g/l, KNO 3 3 g/l, MgSO 4 ,7 H 2 O 500 mg/l, KCl 500 mg/l, FeSO 4 , 7 H 2 O 10 mg/l, K 2 HPO 4 1 g/l, agar 20 g/l). La souche Aspergillus niger DSM n°823 est issue de la collection DSMZ. La souche est cultivée sur le milieu Potatoes Dextrose Agar (Potatoes Dextrose Agar broth 39 g/l).

Le pH est ajusté à 7,2 par ajout de NaOH ou HCl 0,1M. Les milieux sont autoclavés 20 minutes à 121°C.

Conditions de culture

La souche Alternaria alternata est cultivée à 24±1°C, et à une humidité relative de 90±5%. Certaines cultures sont exposées à la lumière de néons horticoles 10h/j pour favoriser la sporulation. La souche Aspergillus niger est cultivée à 24±1°C, et une humidité relative de 75±5%.

Inoculation

Résultats et discussion

Caractérisation de la matrice après vieillissement

Test à la phénolphtaléine -essai de carbonatation accéléré

Durant l'essai de carbonatation, l'humidité relative est maintenue à 60±5% par une solution saline de K 2 CO 3 , ce qui permet une carbonatation optimale. En effet, la vitesse de carbonatation est maximale pour des humidités relatives comprises entre 50 et 65% [START_REF] Chaussadent | [END_REF]. Après 48 heures de carbonatation, le test à la phénolphtaléine révèle une zone carbonatée de moins de 1 millimètre pour les éprouvettes non séchée. En revanche, les éprouvettes séchées au préalable à l'étuve, ont été carbonatées sur une épaisseur de 2-3 millimètres (Fig. 2). Pour l'éprouvette non vieillie, nous identifions, essentiellement la portlandite, l'ettringite et du gypse en proportions moindres, et quelques traces de calcite (Fig 4a). En revanche le diffractogramme obtenu pour l'éprouvette carbonatée (Fig. 4b) est plus complexe. Nous identifions toujours la portlandite et l'ettringite en proportions moins importantes que précedemment, et beaucoup plus de carbonate de calcium sous forme de calcite, aragonite et vatérite. Enfin, pour l'éprouvette carbonatée puis lixiviée (Fig. 4c), nous observons seulement les pics caractéristiques du carbonate de calcium sous forme de calcite et aragonite. De plus, le pH de la solution lixiviante mesuré en fin de lixiviation est de l'ordre de 8,5. Ce pH est typique du pH des phases carbonatées.

3.1.4. Discussion D'après ces résultats, il apparaît que sur les premiers millimètres, la matrice a profondément changé après vieillissement. On note une fissuration et une rugosité plus importante après lixiviation (Fig. 3). D'autre part, ces premiers millimètres après lixiviation sont constitués essentiellement de gels de silice et d'alumine (identification par ATD-TG, résultats non montrés), et de phases carbonatées (DRX), ce qui est très différent chimiquement du coeur de la matrice. Le réservoir d'alcalinité de la matrice sur ces premiers millimètres a considérablement diminué, ce qui se traduit par une diminution du pH, qui devrait offrir ainsi de meilleures conditions pour le développement fongique.

Inoculation -Incubation

Essai d'inoculation avec Alternaria alternata

Nous n'observons aucun développement de la souche pour toute la durée du test sur toutes les éprouvettes imbibées de milieu nutritif liquide. Concernant les éprouvettes recouvertes d'un film de gélose, aucun développement n'est observé sur les éprouvettes non vieillies et carbonatées. En revanche, après 10 jours d'incubation nous observons un développement sur les éprouvettes carbonatées puis lixiviées (Fig. 5).

Essai d'inoculation avec Aspergillus niger

Concernant les éprouvettes non vieillies, nous n'observons aucun développement, quel que soit le mode d'inoculation (Fig. 6). Pour les éprouvettes carbonatées, aucune croissance n'est observée sur les éprouvettes imbibées de milieu nutritif liquide, mais nous notons un développement de la souche dès 10 jours d'incubation sur 2/3 des éprouvettes recouvertes d'un film de gélose. Le résultat le plus intéressant est obtenu pour les éprouvettes carbonatées puis lixiviées. Nous notons un développement d'Aspergillus niger sur 1/3 des éprouvettes imbibées à 10 jours d'incubation. La croissance de la souche est bien amorcée sur toutes les éprouvettes recouvertes d'un film de gélose (Fig. 7). L'absence de croissance sur les échantillons non vieillis confirme que le réservoir d'alcalinité de la matrice est un paramètre clé pour la mise au point du test de biodétérioration. Ces résultats doivent être confirmés après 3 mois d'incubation.

Discussion

Ces résultats soulignent bien l'importance de l'étape d'inoculation. Nous voulons obtenir un développement rapide du champignon sur les éprouvettes, pour cela les conditions initiales de croissance sont primordiales. Nous constatons que le développement fongique est très bien amorcé lorsque les éprouvettes sont recouvertes d'un film de gélose, contrairement à celles imbibées de milieu nutritif liquide. Nous pouvons supposer qu'avec un film de gélose les nutriments nécessaires au bon développement des souches restent concentrés sur toute la surface de l'éprouvette. En revanche, il faut s'assurer que le film de gélose ne constitue pas un artefact, et que le développement se poursuit une fois la gélose consommée. Le test d'incubation avec deux souches différentes nous montre l'importance du pH de la matrice pour le test. Bien que des souches telles qu'Aspergillus niger sont moins sensibles au pH, elles ne peuvent pas se développer sur des surfaces très basiques (pH~13) tel que le ciment. Nous pouvons supposer que la biodétérioration par Aspergillus niger sera plus rapide qu'avec Alternaria alternata, étant donné qu'il se développe plus rapidement, et que la détérioration chimique par la production d'acides organique nécessite moins de temps pour être observée que la détérioration physique.

Conclusion

L'objectif de cette étude est de mettre au point un essai accéléré de laboratoire en vue d'avoir une meilleure compréhension des mécanismes de biodétérioration. L'étape la plus délicate est d'obtenir à l'échelle du laboratoire (quelques mois) les effets, en termes de vieillissement de la matrice et de développement fongique, observés au bout de plusieurs années en environnement naturel. Les tests d'incubation réalisés avec les éprouvettes vieillies et non vieillies (Fig. 67) soulignent bien l'importance du réservoir alcalin sur le développement fongique. Nous notons que plus le réservoir alcalin de la matrice s'appauvrit, plus le développement est important. Concernant notre essai de laboratoire accéléré, dans nos conditions expérimentales, le protocole le plus adapté est donc le suivant :

(i) vieillissement accéléré : conservation des éprouvettes en ciment 28 jours à 100% d'humidité relative. Concernant l'essai de carbonatation, les éprouvettes sont séchées 24h à 40°C, puis placées dans l'enceinte à 65% d'humidité relative, avec un balayage de CO 2 pur, pendant 48 heures. Les éprouvettes sont ensuite lixiviées à l'eau déminéralisée 28 jours. Trois éprouvettes sont immergées par bac de 10 litres, avec un renouvellement continu de l'eau à un débit de 7ml/min. (ii) inoculation : les éprouvettes sont recouvertes par un fin film de milieu nutritif gélosé, et ensemencées par une suspension de spores.

  Deux méthodes d'inoculation sont testées. Les éprouvettes sont disposées dans des boîtes stériles (Fig 1). Dans la première méthode, les éprouvettes sont recouvertes d'un fin film de milieu nutritif solide (milieu gélosé) ; Dans la seconde méthode, les éprouvettes sont imbibées de milieu nutritif liquide. Des éprouvettes non vieillies, carbonatées et carbonatées puis lixiviées sont inoculées par une suspension de spores. Les boîtes sont incubées dans une chambre de culture à 24±1°C, et 90±5% ou 75±5% HR, respectivement pour Alternaria alternata et Aspergillus niger.

Figure 1 :

 1 Figure 1 : Dispositif expérimental de l'inoculation des éprouvettes

Figure 2 :a 3 . 1 . 2 .Figure 3 :Figure 4 :

 231234 Figure 2 : Révélation de la zone carbonatée (en blanc) par le test à la phénolphtaléine. (a) éprouvette non séchée. (b) éprouvette séchée b

  Après 3 mois d'incubation, nous n'observons pas de croissance supplémentaire sur les éprouvettes, la culture n'évolue plus. Ce test d'incubation sur une courte durée met en évidence 2 points importants : (i) tout d'abord, nous notons que le développement n'est possible que sur les éprouvettes carbonatées puis lixiviées, ce qui signifie que la quantité d'élément alcalin de la matrice est un paramètre clé pour la mise au point de notre test accéléré de biodétérioration. (ii) Ensuite, la méthode d'inoculation (sur éprouvettes imbibées ou recouvertes d'un film de gélose) est fondamentale pour amorcer le développement.

Figure 5 :Figure 6 :Figure 7 :

 567 Figure 5 : Test d'inoculation d'éprouvettes carbonatées puis lixiviées recouvertes d'un film de gélose pour Alternaria alternata : (a) à 10 jours, (b) à 3 mois d'incubation