Texture of Microbial Sediments Revealed by Cryo-Scanning Electron Microscopy

TEXTURE OF MICROBIAL SEDIMENTS REVEALED BY CRYO-SCANNING ELECTRON MICROSCOPY

CHRISTIAN OEFARGEI. 2, JEAN TRICHETI, ANNE-MARIE JAUNE'fl, MICHEL ROBER'fl, JANE TRIBBLE4, AND FRANCIS J. SANSONE4 I Laboratoire de Geologie de la Matiere Organique, Unite de Recherche Associee 724 du Centre National de 10 Recherche Scientifique, Universite d'Orleans, BP 6759, 45067 Orleans Cedex 2, France 2 Ecole Superieure de I'Energie et des Materiaux, Universite d'OrLeans, 45072 Orleans CMex 2, France 3 Station de Science du Sol, Institut National de la Recherche Agronomique, 78026 Versailles Cedex, France 4 Department of Oceanagraphy, University of Hawaii at Manoa, 1000 Pope Road, Honolulu, Hawaii 96822, U.S.A.

ABSTRACT: Textures of modern lacustrine stromatolites on Kiritimati organic) parts is distorted upon thin-section preparation. There have been (Line Islands, Central Pacific Ocean), and of buried layers in the stro few electron microscope studies of the detailed organomineral texture; sig matolitic carbonate sediments from French Polynesian atoll lakes (leonificant examples are Horodyski and Vonder Haar (1975), Monty and Har para), have been studied using cryo-scanning electron microscopy die (1976), Krumbein and Cohen (1977), Golubic (1983), and Braithwaite (SEM equipped with a freeze-drying sample preparation device). This et aJ. (I989). study confirms that microscopic three-dimensional organic networks Results of such electron microscope studies of organomineral texture built through reorganization of polysaccharide fibers inherited from have already been reported for the superficial layers of modern lacustrine sheaths of dead cyanobacteria, and from other extracellular polymer stromatolites (kopara) in French Polynesian atolls (Defarge et al. I994a). secretions, are common components of microbial sediments, of which Observations of these gelatinous sediments have been possible only by use they may form the framework. In addition to this role in sediment of a cryo-SEM, that is, a scanning electron microscope equipped with a cohesion and formation of microstructure, the organic framework ap freeze-drying preparation device that does not disturb the three-dimensional pears to be involved in carbonate precipitation within the stromatolites, micro-organization of such highly hydrated, organic-rich samples upon de through chemical (nucleating), steric, and hydrodynamical controls. hydration (Oefarge et al. 1993). We have used the same technique to study The role of the dead organic constituents of the stromatolites is not other modern microbial sediments (lacustrine stromatolites from Kiritimati, only confirmed in micrometer-size crystal precipitation but extended Line Islands, Central Pacific Ocean), and buried examples of French Pol to the post mortem internal mineralization of cyanobacterial filaments, ynesian atoll lacustrine stromatolites. Textures under cryo-SEM of the mi and to the formation of peloids that evolve into spherulites. Bacterial, crobial sediments studied are described in this paper, and the petrogenetic including nannobacterial, carbonate bodies, and carbonate-impregnat processes that might be responsible for their formation are discussed. ed cyanobacterial sheaths are also shown to form in the stromatolites studied. All these carbonate precipitation processes may cooperate in MATERlALS AND METHODS lithification of a given sediment. The French Polynesian stromatolites (Oefarge et al. 1994a) are flat de posits, several tens of centimeters thick, that overlie the bottoms of most INTRODUCTION shallow « 2 mdepth) lakes and ponds on the rims of atolls in the Tuamotu Archipelago and Society Islands (intertropical Pacific area with humid cli Organosedimentary deposits formed by benthic microbial communities mate). The salinity of the lake waters fluctuates between that of fresh and are found today in various environments that include intertidal to supratidal fully saline water (Oefarge et al. I994a). Laminae consist of alternating settings, hypersaline regions, alkaline lakes, streams, and thermal springs organic-rich and calcium-carbonate-rich laminae, which are millimeters (Awramik 1984). The principal microbes involved in their formation are thick. The carbonates are essentially high-Mg calcite (7-19 mole % cyanobacteria, bacteria, and eukaryotic algae (Riding 1991 a). The precip MgC03) with minor percentages of aragonite « 22% of total mineral. itates that form within these deposits are mainly calcium carbonate minerals fraction, when present; Oefarge et al. 1994a; Oefarge, unpublished data). (Awramik 1984), which are generally associated with trapped detrital par The microbes are principally filamentous cyanobacteria, generally domi ticles that are dominant in environments where carbonate precipitation is nated by Phormidium spp., that live within a 1-3 cm thick layer at the reduced, such as in marine settings (Riding 1991 a), or with evaporites in sediment-water interface; these cyanobacteria are usually accompanied by hypersaline settings. coccoid species, and are underlain by living purple photosynthetic bacteria Since Walcott (1914) compared the calcareous concretions formed by (06farge et al. 1994a). The underlying sediment, whose organic fraction is cyanobacteria in fresh-water settings to Proterozoic stromatolites from the essentially composed of remains of extracellular polymer secretions (EPS; Belt Mountains of Montana, the conviction that such laminated sedimentary i.e., the sheaths and dispersed mucilage) of dead cyanobacteria, hosts a growth structures (referred to as Stromatolith by Kalkowsky in 1908), and vertical succession of living anaerobic bacteria, dominated by sulfate re other assimilated structures like thrombolites (characterized by a mesos ducers and, in the deeper levels, methanogens (Oefarge et al. I994a). L0 copic clotted fabric) or dendrolites (macroscopic dendritic fabric), have cation maps of the atolls and lakes studied, aerial and plan views of the been built by benthic microbial communities has been accepted by most lakes, and photographs of the microbial sediments are given in Ricard et specialists (Burne and Moore 1987; Riding 1991 a). Hence, modern micro' al. (1985), Oefarge et al. (1994a), and Oefarge et al. (1994b). Lake HI2 is bial sediments are principally studied in order to document composition or located at 140°51' Wand 18°10' Son Hao Atoll, Lake M3 at 138°55' W structural features, or petrogenetic processes, which might be extrapolated and 21 °52' S on Moruroa Aton, and Lake R2 at 147°47' Wand 14°56' S to the past. on Rangiroa Atoll. When the lakes were studied, HI2 was fully saline (33 Texture, i.e., the size, shape, and mutual arrangement of the constituent 41%0), M3-was brackish (10-20%0), and R2 was fresh to brackish (0-15%0). elements at the smaller scale of microbial sedimentary bodies, is a petro In some lakes (e.g., Lake R2), these superfidal stromatolitic layers lie logical feature that may serve to characterize and compare modern and upon buried ones (which are 12 cm thick in Lake R2) that have thinner fossil examples. Little work, however, has been done on details of either . organic laminae and are richer in carbonates than the surface layers (71 mineralogic or organic textures of modern microbial sediments. Most stud 86% dry weight vs. 30-67%). The compositions of the carbonate (high-Mg ies have been made at the scale of the light (polarizing) microscope, with calcite with 12-16 mole % MgC03, and less than 21% aragonite) and the risk, moreover, that the structure of the non-indurated (in particular, organic fractions are similar to those of the upper layer (Oefarge et al.

I994a). The maximum 14C age measured in 1982 for the organic matter cluding critical-point drying (Lock et al. 1984; Nicholson et al. 1987; from the deeper levels of a deposit was 1390 ::!: 60 yr (Defarge et al. Thompson et al. 1990; Merz 1992; Pedley 1992; Westall and Rince 1994). I994a). Sample collapse was also observed when the sediments of this study were Stromatolites in Kiritimati (formerly known as Christmas Island, in the examined under conventional SEM, or under the recently developed low dry equatorial area of the Pacific Ocean) are found on the bottoms of many vacuum (or "environmental") SEM (Defarge, unpublished results). of the ca. 500 shallow lakes and ponds that occupy approximately 25% of the large surface area (ca. 360 km2) of this nearly filled atoll (Schoonmaker RESULTS et aI. 1985). The salinity of the water bodies ranges from that of fresh water to more than 300%0 (Schoonmaker et aI. 1985). The stromatolitic The photic bilayer of the microbial deposits, which is mainly composed layers of the lake sediments are as much as 30 cm thick, and consist of of live cyanobacteria and photosynthetic, bacteria, and which forms only alternating organic-rich and mineral-rich flat laminae, which are millimeters the topmost centimeters of sedimentary accumulations of ca. 50 cm to more to centimeters thick (Defarge et aI. 1993). The principal mineral compo than I m thickness, is not discussed here. Below this layer, the sediments nents are calcium carbonates, which are accompanied, in hypersaline set are composed of two dominant components: (I) organic matter, essentially tings, by gypsum and minor halite (Schoonmaker et aI. 1985; Defarge et derived from remains of cyanobacteria that have lived at the sediment aI. 1993). Only the organomineral texture of the stromatolitic layers that water interface, and (2) carbonate grains. are devoid of evaporite minerals is discussed in this paper. Location maps of Kiritimati and of lakes and ponds, and pictures of sediments, are given Organic Matter in Schoonmaker et aI. (1985) and Defarge et aI. (1993). Hypersaline Lake Under cryo-SEM the bulk of the organic matter in stromatolites of Kir 22 is located at 157°20.0' W and 1°58.0' N, Hypersaline Lake 23 at itimati and in buried layers of the French Polynesian atoll lacustrine stro 157°21.5' Wand 1°53.3' N, and Brackish Lake 30 at 157°21.3' Wand matolites (kopara) appears, as in the superficial layers of the latter stro 1°52.7' N. matolites (Defarge et al. I994a), to consist of a three-dimensional network Short vertical cores of the sediments « 10 cm long) were carefully of membranous walls enclosing alveoli (Fig. I). In the hydrated state, the collected in large cylindrical vessels and immediately placed at 0-4°C in alveoli are subspherical, with diameters ranging from a few tens of nano iced coolers, then refrigerators, until samples were prepared for micros meters to a few micrometers. With decreasing water content, they undergo copy. Most were covered with glutaraldehyde (4% by volume in water) to horizontal flattening (Fig. IC; water content of this kopara buried layer was stop biological activity. Control samples that were only refrigerated showed 74-80% wet weight, vs. 78-95% for the other, superficial ones shown in no textural difference under cryo-SEM from samples of the same sediment Figure 1), and they collapse upon complete desiccation (Defarge, unpub maintained in glutaraldehyde. lished results). The walls are a few nanometers to about one micrometer Two types of cryo-SEM were used: a Jeol JSM 35 equipped with a thick. They have been shown to be made basically of polysaccharide fibers Cryoscan device, and a Philips 525 M equipped with an Oxford System derived from the sheaths of dead cyanobacteria (Defarge et aI. I994a). This Cryotrans CT 1500 (and a Link Analytical PCXA-1186 EDS). Cryo-SEMs polysaccharide gel is the framework of both organic and carbonate laminae are scanning electron microscopes equipped with a freeze-drying sample in the stromatolites studied (see all figures in this paper), down to the preparation system that eliminates the component water through extremely deepest layers of the deposits (e.g., down to ca. I m depth, in Lake 30 of rapid (a few milliseconds) freezing (cryofixation), followed by sublimation Kiritimati). under vacuum within the microscope chamber. Cryofixation of the samples The other organic components of the sediments are minor in abundance was performed by immersion in liquid Freon cooled by liquid nitrogen at in comparison to the three-dimensional network. They consist of living -160°C (Cryoscan) or in nitrogen slush at -210°C (Cryotrans). The cry bacteria, empty cyanobacterial filaments that are incorporated in the net ofixation step transforms the water to ice whose crystalline domain size work, and occasional remains or products of various eukaryotic microbes, does not exceed 10-100 nm, and thus, does not modify the three-dimen animals, and vascular plants (Defarge et al. 1994a) scattered within the sional organization of the sample at the observational scale. Any biological polysaccharide framework. or chemical activity is also instantaneously stopped. Ice sublimation, which is obtained by progressively raising the temperature to about -80°C, com CarbolUltes pletes stabilization of the microstructure of the sample in a state closest to its natural one. The carbonates that form the subhorizontal mineral laminae of the stro The freeze-drying technique is widely accepted as the gentlest SEM matolites can be distinguished, on the basis of shapes and formation pro preparation method for delicate, highly hydrated biological samples, and is cesses, into several categories. Most are precipitated in situ and are asso preferable to standard drying or critical-point drying (Steinbrecht and MUll ciated with minor reef-derived and other marine biogenic grains (essentially er 1987). In particular, the three-dimensional structure of microbial EPS coral fragments, with molluscan shells, foraminifera tests, echinoderm skel was confirmed only after this technique was developed (Decho 1990). Vi etal parts, etc.), which are deposited in the lakes by incursions of sediment sualization of interactions between EPS and associated microscopic mineral laden seawater or by wind. The detrital particles appear entrapped within particles is also facilitated by cryo-SEM (Robert and Schmit 1982; Chenu the organic network. The precipitates can be distinguished into true biom and Jaunet 1992). inerals (formed through encapsulation of live bacteria or sheath impreg Shrinkage of the EPS-dominated organic constituents of modem micro nation of live cyanobacteria), post mortem cyanobacterial carbonates, or bial, or microbe-inhabited, sediments under conventional SEM has fre ganominerals (Trichet and Defarge 1994), and peloids and spherulites. quently been noted whatever the sample dehydration technique used, in Bacterial Biominerals.-Aggregates of micrometer-size carbonate

FIG. l.-Cryo-SEM micrographs of the organic framework of the sediments. All examples are three-dimensional networks of membranous walls enclosing alveoli. A) Fully saline Lake H12, Hao Atoll, Tuamotu Archipelago. Superficial stromatolitic layer. B) Brackish Lake M3, Moruroa Atoll, Tuamotu Archipelago. Superficial stromatolitic layer. C) Fresh-water Lake R2, Rangiroa Atoll, Tuamotu Archipelago. Buried stromatolitic layer. Some wall thickening (see the arrowed example) may be due to locally incomplete ice sublimation. D) Hypersaline Lake 22, Kiritimati. Superficial stromatolitic layer. Same local wall thickening as in C. E) Brackish Lake 30, Kiritimati. Superficial stromatolitic layer. F) Hypersaline Lake 23, Kiritimati. Superficial stromatolitic layer. MICROBIAL SEDIMENT TEXTURE REVEALED BY CRYO-SEM 937 938 C. DEFARGE ET AL.

FIG. 2.-Cryo-SEM micrographs of the bacterial carbonates formed within the sediments. A through C are from the buried stromatolitic layer of fresh-water Lake R2, Rangiroa Atoll. D is from a buried stromatolitic layer of brackish Lake 30, Kiritimati. A) The carbonates are found in rounded microzones within the organic network. The arrowed area is magnified in B. B) The microzones consist of carbonate aggregates principally composed of rods, a few micrometers long, and spheres whose diameter is under 0.5 fLm and which probably originated as nannobacteria. C) View showing, from left to right, the organic network, aragonite rods and a dumbbell (arrow), and aragonite-entombed nannobacteria (small spheres). D) Magnesian calcite rods, spheres, dumbbells, and "rosette" (arrow), all of which are evocative of bacterial precipitates, and organic network membranes. spheres, rods, dumbbells, and "rosettes", which are evocative of encap Cyanobacterial Biominerals.-These biominerals are present as car sulated bacteria (see Castanier et a!. 1989; Buczynski and Chafetz 1991; bonate-impregnated tubular sheaths (Fig. 3), which is a common mineral Chafetz and Buczynski 1992), are found in the sediments studied (Fig. 2). ization mode for filamentous cyanobacteria (Riding 1991 b). They are rare They are present within rounded microzones, generally 10-100 J.Lm wide, in the sediments studied, and are absent from most sites. They have been that infill several contiguous alveoli of the organic network (Fig. 2A). The found to be the dominant component of the mineral fraction only in one carbonate bodies frequently have a bimodal size distribution, with rods a lamina, < 0.5 cm thick (in the buried stromatolitic layer of Lake R2, on few micrometers to 10 J.Lm long associated with spheres whose diameter Rangiroa), whose filamentous structure was visible to the naked eye. They is under 0.5 J.Lm (Fig. 2B, C). Both the shapes and the size distribution of are present as 15-30 J.Lm broad tubes, with magnesian calcite walls 2-10 the individual bodies, and the clustering in space of the aggregates, are J.Lm thick (Fig. 3). The tubes appear to be bound to the surrounding network typical of bacterial precipitates (Buczynski and Chafetz 1991; Folk 1993); by organic membranes inherited from the sheaths (Fig. 3A); the network the tiniest spheres were probably nannobacteria (dwarfed forms), whose in this lamina is likely built from the material derived from the nonminer natural naked size is 0.05-0.2 J.Lm (Folk 1993). The bodies are composed alized part of the sheath (see Figure 3B) and from the surrounding muci of aragonite or magnesian calcite, the specific mineralogy of which appears lage. Mineralized coccoid cyanobacteria have not been observed in these to depend on the site: e.g., aragonite is dominant in fresh-water Lake R2 sediments. of Rangiroa Atoll, in the Tuamotu Archipelago (Fig. 2A-C), whereas mag Post Mortem Cyanobacterial Carbonates.-Cyanobactelial filaments nesian calcite dominates in brackish Lake 30 of Kiritimati (Fig. 2D). These that have undergone post mortem mineralization have also been observed bacterial precipitates are major components of the buried layers of the in the buried stromatolitic layer of fresh-water Lake R2. They are present sediments, whereas they are rare within the superficial ones in spite of the within a < 0.5 cm thick lamina that has numerous weakly degraded cy presence of numerous live bacteria. anobacterial filaments (Fig. 4A). The filament remains are incorporated in MICROBIAL SEDIMENT TEXTURE REVEALED BY CRYO·SEM 939

FIG. 3.----{:ryo-SEM micrographs of the calcified filamentous cyanobacteria formed rIG. 4.-Cryo-SEM micrographs of the post mortem cyanobacterial carbonates within the sediments. A) View of a lamina in the buried stromatolitic layer from fresh formed within the sediments. A) View of a lamina in the buried stromatolitic layer water Lake R2 sediment, which shows numerous magnesian-calcite-impregnated cy from fresh-water Lake R2 sediment. The lamina consists of dead, weakly degraded anobacterial sheaths incorporated into the organic network. All trichome loci (central cyanobacterial filaments incorporated into the organicnetwork. Some filaments have cavities of the filaments) are uncalcified; the only exception (white arrow) appears to enclosed organic membranes (examples are indicated by white arrows), whereas be filled with scattered grains rather than carbonate-impregnated. The black-arrowed others are almost entirely filled with aragonite (black arrows). The aragonite-filled specimens are magnified in B (thin arrow) and C (thick arrow). B) Close-up of a filaments indicated by the straight, and curved, black arrows are magnified in B and transversely cut calcified sheath. The surrounding organic membranes probably derive C, respectively. B, C) Transversally cut filaments or U-curved filament (B), and from the outer, non mineralized part of the sheath. C) Close-up of a longitudinally cut obliquely cut filament (C), in which aragonite precipitation took place only after the calcified sheath whose carbonate content has been partly scattered (center of the mi filament cavity had been filled with organic membranes (arrows), i.e., after the tri crograph). Note another, shrunken, calcified sheath (arrow). chome died or had abandoned the sheath. 940 C. DEFARGE ET AL.

the surrounding network (Fig. 4). Transverse cross sections of the cylin drical carbonate bodies are 10-20 f.Lm in diameter. These precipitates are distinguished from the cyanobacterial magnesian calcite described above because: (I) they are aragonite; (2) they infill the entire filament cavity, including the trichome locus (compare Figures 4B and 3B); and (3) they formed only after the trichome was dead, or had abandoned the sheath, and the cavity had been filled with membranes derived from the cell wall or the sheath (see Figure 4B, C, arrows), whereas cyanobacterial calcification begins during the life of the microbe (Riding 1991 b). Organominerals.-Organominerals have been defined as natural min erals whose precipitation is mediated by sedimentary organic substrates, by analogy with biominerals, whose formation is mediated by living organic matrices (Trichet and Defarge 1994). Such precipitates have been shown to be the dominant minerals in the superficial layers of the French Poly nesian atoll lacustrine stromatolites (Defarge et al. 1994a), and they are also found in the other stromatolitic layers studied (Fig. 5). They range from micrometer-size individual crystals to aggregates of high-Mg calcite

(7-19 mole % MgC03) that are enclosed within alveoli and supported by walls of the three-dimensional organic network (Fig. 5). Individual crystals are commonly clusters of parallel subcrystals that are oriented perpendic ular to the network walls that support them (Fig. 5C). Peloids and Spherulites.-Other carbonate precipitates observed in the sediments, in particular, in the superficial stromatolitic layers of the French Polynesian atoll sediments in lakes HI2 (Hao Atoll, Tuamotu Archipelago) and R2, can be grouped under the descriptive term peloids. They are spher ical to elliptical grains of magnesian calcite, 50-150 f.Lm in diameter and up to 650 f.Lm long, that display a radially oriented internal structure ar ranged around one or more centers (Fig. 6). Some are true spherulites, the radial crystals projecting into the very center of the grain (Fig. 6A, B). The others have a fine-grained nucleus surrounded by a more coarsely crystal line radial cortex, generally 5-15 f.Lm wide (Fig. 6C-F). The carbonates composing the nuclei are micrometer-size clusters of parallel crystals sim ilar to those associated with the three-dimensional organic network in the sediments (compare Figures 6F and 5C). These clusters also appear to be closely associated with organic membranes resembling the network walls (Fig. 6F, arrows) that seem to be present throughout the grains (Fig. 6E, F). Carbonates extracted from the same layer of Lake HI2 sediment, and washed by sodium hypochlorite (20% by volume in water) for several days, contain significant concentrations of organic matter (total organic carbon ranges from 1.57 to 2.09% dry weight; Defarge, unpublished results). Such organic remains can also be seen enclosed within the true spherulites (Fig. 6B, arrow). The intimate association of the peloids with the organic frame work of the sediments indicates that they have grown within it.

DISCUSSION Organic Framework Cryo-SEM studies of the superficial layers of French Polynesian atoll lacustrine stromatolites had revealed that microscopic, three-dimensional organic networks derived from microbe EPS may form the framework of deposits of benthic microbial communities (Defarge and Trichet 1984). The

FIG. 5.-Cryo-SEM micrographs of the magnesian calcite organominerals formed within the sediments. A) Fresh-water Lake R2, superficial stromatolitic layer. The carbonates are intimately associated with the three-dimensional organic network. B) Brackish Lake 30, buried stromatolitic layer. The texture is essentially the same as that of the Lake R2 superficial stromatolitic layer shown in A. The carbonates appear to have precipitated on the organic network walls. C) Brackish Lake M3, superficial stromatolitic layer. The individual carbonate grains conunonly appear as clusters of parallel crystals that have grown perpendicular to the surface of their supporting organic network wall. MICROBIAL SEDIMENT TEXTURE REVEALED BY CRYO-SEM 941 cryo-SEM observations reported here confirm this result for buried layers environments at the surface of Earth (Table 3). They are produced by mi of stromatolites from the same fresh-water to fully saline lakes (Figs. 1C, crobes of the same phyla as those forming benthic organosedimentary de 2A), and for superficial (Fig. ID-F) and buried (Figs. 20, 5B) stromatolitic posits (diverse bacteria, cyanobacteria, diatoms, and other eukaryotic algae) layers of sediments forming in brackish to hypersaline conditions on Kir and, occasionally, by associated invertebrates such as polychaetes, and also itimati. by fungi, corals, and plant roots (Table 3). Their reticular micro-organi Parts or remnants of microscopic, three-dimensional organic networks zation has also been shown in the case of fungi (Ruel and Joseleau 1991; can also be observed in other deposits of benthic microbial communities Chenu and Jaunet 1992) and (garlic) roots (Gessa and Deiana 1992). In (Table I). These occurrences cover most modern environments where such particular, scleroglucan, a fungal exopolysaccharide, has a structure similar deposits are encountered (Table I). These organic networks have been to bacterial xanthan (Chenu and Jaunet 1992). shown to consist of, or to be derived from, EPS of the microbes forming Thus, polysaccharide organic frameworks may be important not only in the deposits: filamentous or coccoid cyanobacteria, or bacteria (Table I). organosedimentary deposits of benthic microbial communities but also in Such networks can also be recognized in fossil stromatolites: a spongy all life-inhabited sediments at the surface of Earth. The role of such con texture similar to that of modern deep-water deposits of Beggiatoaceae stituent polymers in sediment cohesion has frequently been noted in the (filamentous sulfur-oxidizing bacteria) has been described by Williams case of benthic organosedimentary deposits (e.g., Neumann et aI. 1970), (1984) from the Miocene Monterey Formation, California, where bacterial and it is responsible for their occurrence within the sedimentary environ sheath remains are still present in abundance; the organic framework of ment. A similar cohesive role has been demonstrated for sands: resistance stromatoporoid stromatolites from the Silurian in NE Gotland, Sweden to erosion of fine quartz sand has been shown to be enhanced in vitro by (Kazmierczak and Krumbein 1983), and from the Upper Devonian in cen bacterial exopo1ymers even in the absence of the living bacteria that pro tral Poland (Kazmierczak et al. 1985, see their figure 3b), is comparable duced them (Dade et al. 1990); the in situ stabilization of intertidal sandy with that of modem coccoid cyanobacterial stromatolites referred to in sediments by diatom exopolysaccharides persists after the microbes have Table I (Horodyski and Vonder Haar 1975; Kazmierczak and Krumbein been killed (Vos et al. 1988). Our results produce evidence of the cohesive 1983). effect of a three-dimensional polysaccharide network derived from micro The production of copious amounts of mucilage by the constituent or bial secretions in all stromatolite examples studied (see all figures in this ganisms is a common feature of deposits of benthic microbial communities. paper). Significant examples of such secretions have been described in all major environments where they form (Table 2). In addition to various species of Processes of Carbonate Formation filamentous and coccoid cyanobacteria, and bacteria, the producers include diatoms and other eukaryotic algae; participation of organic mucus pro The historical evolution of theories on mineral deposition involved in duced by tube-building invertebrates has also been noted (Table 2). Abun stromatolite formation has been reviewed often (e.g., see Monty 1977). It dant mucilaginous or cyanobacterial sheath remains have also been rec is characterized by a succession of controversies, the first of which centered ognized in buried layers of similar sediments (Horodyski et al. 1977; Park on the origin of stromatolites by bioprecipitation (e.g., Ka1kowsky 1908; 1977; Kazmierczak and Krumbein 1983; Castenholz 1984, his figure 5; Walcott 1914) versus by inorganic precipitation. After Black (1933) Casanova and Hillaire-Marcel 1993). Mucilage or sheath remains are prob claimed that stromatolites form through trapping and binding of detrital ably also major components in older laminae of other deposits whose sur mineral grains by cyanobacterial filaments, the main dispute involved ar face consists of filamentous cyanobacteria and photosynthetic bacteria, and guments for the detrital versus in situ precipitation origin of mineral con whose consistency is gelatinous or rubbery (see Javor and Castenholz 1981, stituents. The intervention of both processes is now acknowledged (Burne their figure 4; Castenholz 1984, his figure 3). Greater resistance to degra and Moore 1987; Riding 1991a). Discrimination of biologically int1uenced dation of cyanobacterial or bacterial sheath constituents relative to the cells precipitation (i.e., induced by the activity of the microbe in its environ has commonly been noted both in nature and laboratory experiments (Hor ment), and "skeletal precipitation" (i.e., biomineralization producing a spe odyski and Vonder Haar 1975; Horodyski et al. 1977; Kazmierczak and cific mineralized structure in close association with the living cell) has also Krumbein 1983; Williams 1984; Chalansonnet et al. 1988). The organic been debated (see Burne and Moore 1987). The participation of bacteria, texture of all sediments referred to in this paragraph is unstudied. frequently demonstrated in modem deposits of benthic microbial commu EPS are essentially microfibrillar and polysaccharide in composition (De nities, has recently been strongly restated by Chafetz and Buczynski (1992). cho 1990). The organization of the dispersed exopolysaccharide microfi Studies by two of us (Defarge and Trichet 1984, 1990, 1994) on the brils in networks which appear to be three-dimensional when observed French Polynesian atoll lacustrine stromatolites have revealed an additional under SEM, and which form gels in solution, seems universal, having been type of in situ precipitation process involved in microbial sediment calci shown for various bacteria, both in vitro (Fletcher and Floodgate 1973; fication: precipitation controlled by the three-dimensional organic frame Morris et al. 1977; Mutaftschiev et aI. 1982; Robert and Schmit 1982; work of the sediments. This process is also important in stromatolite cal Chenu and Jaunet 1992) and in nature (LaRock and Ehrlich 1975; Paerl cification on Kiritimati (Fig. 5B; see also Defarge et al. 1993 for those 1975), for cyanobacteria (Leppard et aI. 1977), and for diatoms (Paterson stromatolites whose mineral fraction contains gypsum and halite in addition 1989). In the case of xanthans (exopolysaccharides of the Gram-negative to carbonates). We (Trichet and Defarge 1994) proposed to name this type bacteria genus Xanthomonas), it has been argued that this reticular organ of precipitation organomineralization because of both its strong similarities ization is promoted through the alignment of polysaccharide chains in rigid with, and its distinction from, the biomineralization processes, in which the rod-like units stabilized by intramolecular noncovalent bonding, and then formation of minerals occurs by living organisms (see Addadi and Weiner by the association of these macromolecular units (Morris et al. 1977). 1992). The organic walls act as a matrix for calcification, initiating and Thus, microscopic, three-dimensional organic networks derived from supporting mineral growth (Fig. 5B, C). Individual crystals, when they can EPS of the constituent organisms might commonly form the framework of be unequivocally observed, appear to have grown perpendicular to the sup stromatolites and other deposits of benthic microbial communities. This porting organic surface (Fig. 5C). Crystal nucleation is probably initiated hypothesis (Defarge and Trichet 1990; Defarge et al. 1993, 1994a) is re at acidic sites of the polysaccharide framework of the sediments, which inforced by the results presented here. All similar deposits should be stud include those of uronic, aspartic, and glutamic acids, and have been shown ied by cryo-SEM for the presence of such networks. to fix various divalent cations in vitro (Disnar 1981). The network alveoli EPS not only are cardinal constituents of organosedimentary deposits of that enclose the precipitates probably function as confining spaces favoring benthic microbial communities but also are found more generally in most supersaturation within, then precipitation from, their inner solutions (Fig. 942 C. DEFARGE ET AL. MICROBIAL SEDIMENT TEXTURE REVEALED BY CRYO-SEM 943

TABLE I.-Occurrences of microscopic. three-dimensional organic networks in modem deposits of benthic microbial communities

Reference Environment Location Designation of the Organic Network Origin of the Organic Network Williams 1984 Deep-waler continental margin Eastern Pacific, off Central Peru "spongy network" Filament and sheath remnants of sulfur-oxidizing bacte ria of the family Beggialoaceae Reitner 1993 Shallow (6-10 m depth) underwater caves Lizard Island Group, Great Banier Reef "thin collagenous fiber network". Jts two-dimensional Mucilage of unidentified bacteria of fringing reefs aspect under TEM (Plate 611-3) is similar to that of the organic network of the French Polynesian atoll lacustrine stromatolites (see figure 5b in Dtfarge et aI. 19940; figures 3 and 4 Defarge and Trichet 1994) Golubic and Wide range of envirorunents, subtidal to 4 locations in the western subtropical At "three-dimensional reticular framework" Predominantly (90%) empty sheaths of filamentous cy Focke 1978 intertidal, exposed to protected: turbu laOlic and the Caribbean area: (I) Make anobacteria of the species Phormidium hendersonij lent subtidal (8-10 m depth) environ Do coral reef, Bermuda Islands; (2) Howe ment of a coralgal reef; subtidal (2-3 m Boca Jewfish, Bonaire Island, Nether depth) and intertidal zones of an ex lands Antilles; (3) Andros Island, Baha tremely protected shallow embayment; mas; (4) Spanish Harbor Key, Aorida protected to moderately exposed lower inlertidal environmenlS Golubic 1983 Hypersaline intertidal zone Hamelin Pool, Shark Bay, Western "three-dimensional network of polysaccharide fibers" EPS of coccoid cyanobacteria of the species EnUiphys Australia aUs major Ercegovic Horodyski and Pennanently submerged area of a hyper Laguna Grande, Laguna Mormona com Occurrence of a three-dimensional organic network can Resistant sheath material of coccoid cyanobacteria of Vonder Haar saline (up to 90%.) coastal pond plex, Baja California, Mexico be inferred from SEM micrographs shown in Figure the genus Enlophysalis 1975 8G,H Kazmierczak and Hypersaline (49-330%,) coastal pond Sabkha Gavish, southern Sinai, western "spiderweb-like pattern" Resistant EPS material of coccoid cyanobacteria of the Krumbein 1983 shore of the Gulf of Elat order Pleurocapsales Casanova 1986 Alkaline thermal waters Lake Bogoria, Kenya, East African Rift "polygonal organic network" that is interpreted as a Unidentified bacteria System "bacterial colony". However, it resembles (Fig. 4E, F) the organic frameworks presented here (Fig. I)

5), All these processes are similar to those involved in biomineralization and its associated organominerals (see figure fin Defarge and Trichet 1984; (Addadi and Weiner 1992), but they act outside of living organisms and figure 33 in Ricard et al. 1985), In addition, the main arguments advanced under the control of inherited (dead) organic substrates. Bacterial partici by Chafetz and Buczynski (1992) to support their hypothesis of widespread pation in local supersaturation within the network alveoli is likely (Defarge bacterial carbonates in modem deposits of benthic microbial communities et al. 1994a), but the resulting precipitates do not have bacterial carbonate may be used as well in favor of the ubiquity of organomineralization: shapes (compare Figures 2 and 5). lithification in most of these sediments does not occur at the surface, but As for the occurrence of exopolysaccharide-derived three-dimensional rather below the photic zone, where it is associated with decomposed or networks. it is also our contention that such organomineralization processes ganic matter of dead cyanobacteria, are commonly involved in microbial sediment calcification at the surface These results do not mean, however, that bacterial carbonates (encap of Earth. For example, Reitner (1993) cited evidence of nonbiological ma sulated bacteria) are not cardinal constituents of microbial sediments at the trix ("bacterial mucus")-mediated precipitation in the micritic calcification surface of Earth. The observations presented here (Fig. 2) also support the of modem microbial sediments from reef caves. The three-dimensional or findings of Chafetz and Buczynski (1992) on the importance of bacterial ganic networks documented by Horodyski and Vonder Haar (l975) and carbonates. Moreover. they appear to extend the occurrence and the role Golubic (l983) are entombed in calcium carbonate precipitates, whereas in carbonate formation of nannobacteria, from hot-springs travertines, and that shown by Casanova (1986) is the framework of calcite crystals. Pre ooids in normal marine tropical environments or evaporitic lakes (Folk cipitation of magnesian calcite has also been documented within the mu 1993), to modem lacustrine stromatolites forming in fresh to brackish-water cilaginous secretions of benthic microbial communities in all types of la conditions (Fig, 2B, C). Two types of bacterial encapsulation processes are custrine environments in a fresh-water marsh (Monty 1972; Monty and observed in samples from fresh-water Lake R2. Bacterial carbonates are Hardie 1976); the magnesian calcite crystals are distinctly coarser (2-8 encountered in the buried stromatolitic layer of this lake (Fig. 2A--e) where jJ-m) than those associated with the cyanobacterial sheaths (:S: I jJ-m), their encapsulation seems to have resulted from prolonged emergence of which might be encapsulated bacteria according to the micrographs shown the stromatolite body leading to evaporative concentration of porewaters by Monty and Hardie (1976), Occurrences of in situ calcium carbonate (Defarge, unpublished results), They are absent from the superficial stro precipitates have also been reported (Dalrymple 1965; Javor and Casten matolitic layer, but have recently (1991) appeared at its surface in associ holz 1981; Braithwaite et al. 1989) in older organic material of other ben ation with natural desalinization (down to < 1,2%0) of the lake (Defarge thic microbial deposits whose exopolysaccharide-derived nature is either et al, 1994b). The former bacterial carbonates are aragonite, whereas the noted directly or can be inferred. Moreover, the appearance under polarized latter are magnesian calcite, light, including the birefringence of the organic matter, of buried laminae Desalinization of Lake R2 is also responsible for significant development of the sediments documented by Dalrymple (1965, his figures 6 and 7) is of biomineralized (magnesian calcite) filamentous cyanobacteria of the spe the same as that seen in thin sections of stromatolites studied from the cies Scytonerrw mirabile at the surface of the sediment (Defarge et al. French Polynesian atoll lakes, which consist of the polysaccharide network 1994b). This top lamina is similar to the magnesian-calcite-impregnated

FIG. 6,-Cryo-SEM micrographs of the magnesian calcite peloids formed within the sediments. Fully saline Lake H12, superficial stromatolitic layer. A, B) Some of the peloids are spherulites, B is a closer view of the right spherulite in A, showing that the crystals are grouped in radial sectors, and that organic network walls have been enclosed within the growing carbonate body (arrow), C-F) Views of the other peloids, Differentiation of a cortex and a nucleus is conspicuous in D, which provides a higher magnification of the upper right quarter of the peloid in C. E is a closer view of the arrowed part of the cortex seen in D, showing thaI the crystals resemble the radially oriented spherulite constituents (compare with B). F is a closer view of part of the peloid nucleus in C, showing the enclosed organic membranes (see the arrowed examples) and that the individual carbonate grains are similar to the magnesian calcite organominerals found elsewhere in the sediments (compare with Figure SC). 944 C. DEFARGE ET AI.

TABLE 2.-Examples of copious mucilage production in modem deposits of benthic microbial communities

Reference Environment Location Mucilage Producers Neumann el aI. 1970 Subtidal (shallow warer) carbonare sellings Several locations in the Rock Harbour Cays Diverse filamentous (Lyngbya sp., Microcoleus sp., Schizo/hru sp.) and area, Abaco, Bahamas coccoid (Arnu:yslis marina) cyanobacteria, diatoms (Amphora terror;s, Nituchia doslenum, ThallJssiolhrix woodii). and tube-building invent brares (polychaeres, copepods, lanaids, and tunicales) Awramik and Riding 1988 Hypersaline sublidal zone Hamelin Pool, Shark Bay, Wesrem Auslralia Predominantly diatoms (in particular, Mas/ogloia sp.) Gunalilaka 1975 Inrerlidal zone of a c1aslic (noncarbonare). Mannar Lagoon, northwest Ceylon Diverse filamentous and coccoid cyanobacteria (in particular, Microcoleus Iropical, hypersaline (38-120%,), lagoonal chtonoplas/es, Lyngbya sp .. and Schizo/hrix sp.), rhodophyta (essential lidal flat ly Porphyridium sp.), chlorophyta and diatoms Nicholson el al. 1987 Salt marsh Great Sippewisseu Sail Marsh. Cape Cod, Diverse green sulfur bacteria (in particular, Prosthecochloris aestuaril), Massachuseus purple sulfur bacteria, filamenlous cyanobacteria (in particular, Lyngbya nes/uarii. OscillalOria spp., and Pharmidium spp.), and diatoms (in particular, Novicula spp.) Grey el aI. 1990 Saline (39-53%0) coastal lake Lake Thelis, soulhwestern Auslra!ia Coccoid cyanobacreria (principally Gloeocapsa sp.). and diverse diatoms Monly 1972 Shallow fresh-waler lake Wilmo Lake, Andros Island, Bah3l11JlS Diverse cyanobacteria and diatoms Thompson et aI. 1990 Meromictic. hardwarer, marl lake Green Lake, Green Lakes Stale Park, Fayene Predominantly coccoid cyanobacreria of the genus Synechococcus ville, New York Casrenholz 1984 Hot springs Yellowstone National Park Thermophilic cyanobacreria (e.g.. species of the genus Phormidium) sheath lamina observed within the buried layer of Lake R2 sediment (Fig. stromatolite body resulting in evaporative porewater concentration (see 3). The two occurrences might have had similar causes. The cyanobacteria above). This would explain the formation of aragonite instead of magnesian that were present prior to desalinization at the surface of the sediment calcite, which is precipitated both within the cyanobacterial sheaths and on (essentially Phormidium crossbyanum) remained uncalcified. The devel the network walls. However, studies of bacterial calcification by Buczynski opment of cyanobacterial biocalcification is linked with the appearance of and Chafetz (1991) have also shown that a high precipitation rate alone a new species, that is, S. mirabile (Defarge et al. I994b). can explain the formation of aragonite rather than calcite in a given water In contrast to sheath impregnation, which begins during the life of the environment. microbe, filling of the cyanobacteria with aragonite (Fig. 4) takes place Mediation of the organic framework of the sediments is also evident in only after the filament has lost its living trichome after death and subse the carbonate precipitation that produces peloids (Fig. 6). Peloid nuclei quent decomposition, or because it has abandoned the sheath. Aragonite consist of organominerals whose associated membranes were probably part filled cyanobacterial remains are found in the same lamina with emptied of the three-dimensional polysaccharide network (Fig. 6D-F). Only sub filaments that illustrate the transition stage between the loss of the trichome sequent growth of the cortex around existing organominerals can explain and the internal aragonite filling of the organic envelope (Fig. 4A). Mineral the formation of these peloids; we have found no argument in favor of formation appears to be controlled, at least morphologically, by the organic precipitation of such large spheroidal coronas prior to their internal filling remains, with the envelope itself restricting growth and expansion of crys by organominerals. The radially disposed crystals of the cortex probably tals (Fig. 4B, C). Chemical control is also likely; a similar calcification of precipitated from pore solutions concentrated by diffusion from the alveoli dead filaments of the chlorophyte genus OSlreobium has been shown by as they closed around organomineral-rich microzones upon sediment com Kobluk and Risk (1977) to begin on, and perpendicular to, the internal paction. Closure of alveoli devoid of organominerals has been observed surface of the empty envelope, and then to fill the interior. This process under conventional SEM in desiccated samples whose organominerals involves an organic matrix, which promotes crystal nucleation and growth, maintained porosity within their enveloping alveoli (Defarge, unpublished and a delineated space, which favors supersaturation and limits growth and results). Examples of alveolar closure can also be found around the peloids expansion of the crystals. It is, thus, similar to biomineralization (Addadi (Fig. 6C). Continuation of centripetal cortex crystal growth may produce and Weiner 1992) but is mediated by dead microbe relics; it is an inter spherulites (Fig. 6A, B), whose shapes and sizes are similar to those of the mediate step in the gradation from biomineralization to micrite organomin other peloids (compare Figure 6A and B with Figure 6C-E). These results eralization associated with the three-dimensional network of the sediments. appear to confirm and specify the organic-matrix-mediated precipitation The association in the same layer of Lake R2 of these post mortem process proposed by Andrews (1986) and Reitner (1993) as the initiator of cyanobacterial carbonates with encapsulated bacteria, which are also com the formation of carbonate spherulites and other peloids in microbial sed posed of aragonite, suggests a similar origin, that is, the emergence of the iments and rocks. The Bathonian calcite spherulites shown by Andrews

TABLE 3.-Environments where significant EPS occurrences have been observed

Environment Location (when precised) EPS Producers Reference Marine waler column and bOltom sediments. coral reefs. Bacteria, diatoms, and other microalgae, corals, and Decho 1990 and deep-sea hydrothermal venl systems polychaetes Subtidal, nearshore zone of an estuary (pebbly sands) Poquonock River estuary, Groton, Conneclicut Bacteria, cyanobacteria. or diatoms Frankel and Mead 1973 Inrerlidal seagrass beds (Zostera Capricorni Aschers), and a Moreton Bay, Queensland, Auslra!ia Bacteria Moriarty and Hayward 1982 coral reef Sandy mudflats Bay of Bourgneuf, Vendee, France Diatoms Westall and Rinc6 1994 Sandy shoals of a mesolidal estuary Galgeplaal and Roggenplaal shoals, Oosrer· Bacreria and diatoms (in partiCUlar, Navicula spp.) Vos el aI. 1988 schelde esluary, The Netherlands Submerged surfaces in boreal rivers Muskeg and Sleepbank rivers, north-easrem Bacteria, cyanobacteria, and dialoms (primarily CamphoneLock et al. 1984 Alberta, Canada ma sp.) Fresh-water (phytoherm) reefs Hererorrophic bacreria, filamentous cyanobacreria (principal Pedley 1992 ly Pharmidium sp. and Schizo/hrix sp.), and pennale diatoms Lake water column and bottom sediments Sl. George Lake and Lake Ontario, Ontario, Bacleria, cyanobacreria, and eukaryolic microalgae (in par Leppard et aI. 1977 Canada ticular, chlorophyta) Soils Bacteria, fungi, and planl roots FOSler 1988 MICROBIAL SEDIMENT TEXTURE REVEALED BY CRYO-SEM 945

(1986) are very similar to those documented here (e.g., compare Figure 6A cation; intervention of similar carbonate grain aggregation processes in the and B with Andrews's figure 5B, C). The cores of most of Reitner' s (1993) stromatolites studied here have been observed (Defarge, unpublished re peloids consist of closely associated magnesian calcite crystals and "bac sults). The precipitation of gypsum and halite within deposits of the cya terial mucus" from which bacterial cells are absent; they are surrounded nobacterium Entophysalis major has been convincingly shown by Braith by a dentate rim of coarser magnesian calcite crystals (Reimer 1993). Such waite and Whitton (1987) to occur under the physicochemical control of an organic-matrix-mediated precipitation process might also be responsible cyanobacterial mucilage. The importance of the metal-binding ability of for the formation of the great majority of magnesian calcite marine peloids dead bacterial cell walls and EPS, thereby allowing in situ growth of me studied by Chafetz (1986). In his examples, the "cloudy" cores of the tallic minerals, has been notably supported by in vitro experiments using 3 peloids do not exhibit any bacterial cell but are accompanied, as are Reit U022+ and Sc + ions and Bacillus subtilis specimens. These experiments ner's (1993) examples, by other peloids with cores rich in recognizable have shown, moreover, that metal retention by the dead cells is consider bacterial rods. These observations do not mean, however, that none of these ably higher than that effected by the same cells during their life (Urrutia peloids has formed through calcification of the microbes during their life. Mera et al. 1992). The metal-fixation capacity of the dead organic constit In fact, spheroidal carbonate bodies, a few tens of micrometers wide, com uents of stromatolites such as those studied here has been demonstrated posed of radially oriented crystals, have been shown to grow in vitro around (Disnar 1981). living bacteria (Castanier et al. 1989; BuczynSki and Chafetz 1991). Ex The existence of mineral deposition processes controlled by dead organic amples resembling those documented by Buczynski and Chafetz (1991, components is not exclusive of the involvement of living organisms in their figure IE-H) have been observed under conventional SEM in buried lithification of microbial sediments. Microbes not only participate in pore layers from Kiritimati lacustrine stromatolites (Defarge, unpublished re water supersaturation, leading to carbonate precipitation within the stro sults). The coexistence of the two types of peloids seems analogous to that matolites studied, but they are the object of individual processes of biomin of micrite organominerals and bacterial biominerals in the sediments stud eral formation. These carbonate biomineralization processes include encap ied (see above). In addition to its role in formation of peloid cores, the sulation of bacteria, including nannobacteria, and sheath impregnation of three-dimensional organic network of the sediments also appears to be in filamentous cyanobacteria. In addition, calcium carbonate wall-impregnated volved in cortex precipitation through concentration and expulsion of the oogonia have been observed using transmission electron microscopy in a pore solutions. This centripetal growth contrasts with the centrifugal growth French Polynesian atoll lake stromatolite whose living surface was rich in generally assumed for natural spherulites and other peloid cortices (Reitner chlorophytes (Defarge and Trichet 1994). All types of in situ formation of 1993). carbonate biominerals and organominerals may coexist in a given sediment, as in that of Lake R2: e.g., Figures 2C, 3B, 4B, and 5A, and the above CONCLUSIONS cited observations of peloids under conventional SEM.

The results presented here emphasize the cryo-SEM as an indispensable ACKNOWLEDGMENTS tool for studying the natural texture of modern microbial deposits and other microbe-inhabited sediments. Its application to the stromatolites studied has We thank all those who participated in this project, in particular 1 Berrier and M. Mosnier-Arnou for providing the former cryo-SEM micrographs of these sedi revealed the natural, three-dimensional reticular organizational texture of ments. B. Le Berre assisted in certain cryo-SEM investigations. H. Couratier and their main organic constituent (Fig. I), and the mutual arrangement of this her team made the photographic prints. L.A. Chambers, C. Chenu, M.U.E. Merz, organic network and the different mineral components (Figs. 2-6). The and loP. Perthuisot provided helpful comments on the results or on the manuscript. organomineral formation process of micrite grains and of peloid cores can Reviews by SJ. Mazzullo and C.D. Burke significantly improved the paper. This be proved currently only by cryo-SEM observation of the spatial relation research has been supported by the Compagnie Fran~aise des Petroles-Total, the ships between the carbonate crystals and the organic substrates (Figs. 5C, Service Mixte de ContrOie Biologique (Monthlery, France), and the Etablissement pour la Valorisation des Activites Aquacoles et Maritimes du Territoire de la Po 6F). Iynesie Fran~aise. The organic fraction consists of a dead, microscopic, polysaccharide This paper is a contribution to the International Geological Correlation Pro based, three-dimensional network of membranous walls derived from mi gramme (IGCP) Project Number 380 "Biosedimentology of Microbial Buildups". crobial EPS, and plays a wide range of roles in stromatolite petrogenesis. The first is in development of sediment cohesion and microstructure, the REFERENCES organic network being the framework of each stromatolite. 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