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Figure 7. Simulated RBS spectrum superimposed on the acquired spectrum (rugged line) from the point-analysis of the film-like feature (Fig. 6c). Each acquired RBS spectrum represents the total number of backscattered alpha-particles (y-axis) as a function of the energy (x-axis). Note: The diagram is slightly truncated on the x-axis (starts at 200 KeV). The energy of the backscattered alpha particles depends on the cross section of the nuclei from which the alpha particles scattered, and the depth in the sample at which the scattering event took place. The simulated total spectrum (indicated "sum sectrum") in the figure was generated by summing the simulated spectral contributions of Ca, C, and O in the sample (see lables in the figure). The dashed line indicates the shape of the simulated C curve of the spectrum from the point analysis of the adjacent calcite grain (Fig. 6b). The difference in height of the dashed versus solid curve of the simulated C spectra corresponds to a concentration difference of about three times that relative to the amount of C in an adjacent carbonate grain.

height of the dashed versus solid curve of the simulated individual C spectra corresponds to a concentration difference of about three times.

Possible biogenic features with similar morphological traits as those shown in Figs. 4b-e, 5, and 6 were also found along calcite/pyrite grain boundaries, as shown in Figs. 4f and 8. A pyritized biogenic-like structure was found wrapped around and along the edge of an aggregate of vein-filling pyrite grains. Finegrained calcite crystals, which appear in petrographic thin section to have precipitated before the pyrite (Fig. 3c) grains that filled the remaining pore space, were removed during the etching process. The absence of any of the biogenic-like features in the non-etched specimens indicates that the biogenic-like features were embedded within the calcite precipitates that formed during post-impact hydrothermal activity (Figs. 9 and 10).

Figure 8. A pyritized biogenic-like structure. (a) The film-like feature is wrapped around and along the edge of an aggregate of vein-filling pyrite grains. Calcite removed during etching around the pyrite aggregate revealed that the curved film-like feature was not introduced into the sample preparation procedure prior to etching. White rectangles mark the areas shown in Fig. 8b, c. (b) SEM micrograph reveals that the film-like feature is partly embedded in the aggregate pyrite matrix. (c) Detail that shows an elliptical hole in the film-like feature, which indicates that it must have been flexible enough to tear and contract slightly prior to mineralization.

Figure 8. A pyritized biogenic-like structure. (a) The film-like feature is wrapped around and along the edge of an aggregate of vein-filling pyrite grains. Calcite removed during etching around the pyrite aggregate revealed that the curved film-like feature was not introduced into the sample preparation procedure prior to etching. White rectangles mark the areas shown in Fig. 8b, c. (b) SEM micrograph reveals that the film-like feature is partly embedded in the aggregate pyrite matrix. (c) Detail that shows an elliptical hole in the film-like feature, which indicates that it must have been flexible enough to tear and contract slightly prior to mineralization.

Figure 9. Series of SEM images that illustrate the effects of the etching protocols used in this study. (a, c) Non-etched specimen vs. (b, d) EDTA-etched split of the investigated calcite vein at different magnifications. The images are coupled in pairs (a & b, c & d) to show the effects of the EDTA-etching. (a) Freshly sawn calcite crystal surface of a non-etched split of the calcite vein. (b) EDTA-etched split of the calcite vein. Image shows an etched calcite crystal similar to the non-etched surface shown in Fig. 9a. Note how the etching is more severe in fractures and along edges, which creates a distinct etched topography. (c) Detail of non-etched area shown in Fig. 9a. (d) Detail of EDTA-etched area shown in Fig. 9b. Note how the EDTA-etching has caused the "crumbly" appearance of the surface in contrast to the crystal surface shown in Fig. 9c.

Figure 9. Series of SEM images that illustrate the effects of the etching protocols used in this study. (a, c) Non-etched specimen vs. (b, d) EDTA-etched split of the investigated calcite vein at different magnifications. The images are coupled in pairs (a & b, c & d) to show the effects of the EDTA-etching. (a) Freshly sawn calcite crystal surface of a non-etched split of the calcite vein. (b) EDTA-etched split of the calcite vein. Image shows an etched calcite crystal similar to the non-etched surface shown in Fig. 9a. Note how the etching is more severe in fractures and along edges, which creates a distinct etched topography. (c) Detail of non-etched area shown in Fig. 9a. (d) Detail of EDTA-etched area shown in Fig. 9b. Note how the EDTA-etching has caused the "crumbly" appearance of the surface in contrast to the crystal surface shown in Fig. 9c.

5. Discussion

The biogenic-like features, revealed by etching the hydrothermal calcite that filled an impact-induced fracture in sedimentary limestone of the Siljan structure, were found to consist of bundled or isolated "threads" (Fig. 5) and curved films (Figs. 6a and 8). The diameters of the threads vary, especially where they bifurcate near the attachment points. The thickness of a single thread rarely exceeds 0.3 |im, and some threads (not shown) extend several tens of micrometers between attachment points. These features were not visible with an optical microscope, which could be due to their small diameter, the lack of sufficient contrast between the partly calcified biogenic-like remnants and the limestone matrix, and the absence of extensive alteration of the organic matter, which could have enhanced its visibility in standard petrographic thin sections.

Figure 10. Series of SEM images that illustrate the effects of the etching protocols used in this study (continued from Fig. 9). (a, c) Non-etched specimen vs. (b, d) EDTA-etched split of the investigated calcite vein at different magnifications. The images are coupled in pairs (a & b, c & d) to show the effects of EDTA-etching of the calcite. (a) Detail of the non-etched split that shows an unusual surface texture on a calcite precipitate. Scale bar equals 1 |m. (b) EDTA-etched equivalent of the area shown in Fig. 10a. The original appearance of the precipitates shown in Fig. 10a is still present in the EDTA-etched sample, but it is evident that the etching has "attacked" the unusual precipitates, which results in their shorter and "thicker" appearance. Scale bar equals 1 |im. (c) Pyrite rosette on a freshly sawn non-etched surface of the calcite vein. Scale bar equals 20 |im. (d) Pyrite rosette of an EDTA-etched split of the calcite vein. The etching has exposed the pyrite rosette by removing the surrounding calcite. Note that the etching did not produce any visible artifacts on the surfaces of the pyrite rosette. Scale bar equals 20 | m.

Figure 10. Series of SEM images that illustrate the effects of the etching protocols used in this study (continued from Fig. 9). (a, c) Non-etched specimen vs. (b, d) EDTA-etched split of the investigated calcite vein at different magnifications. The images are coupled in pairs (a & b, c & d) to show the effects of EDTA-etching of the calcite. (a) Detail of the non-etched split that shows an unusual surface texture on a calcite precipitate. Scale bar equals 1 |m. (b) EDTA-etched equivalent of the area shown in Fig. 10a. The original appearance of the precipitates shown in Fig. 10a is still present in the EDTA-etched sample, but it is evident that the etching has "attacked" the unusual precipitates, which results in their shorter and "thicker" appearance. Scale bar equals 1 |im. (c) Pyrite rosette on a freshly sawn non-etched surface of the calcite vein. Scale bar equals 20 |im. (d) Pyrite rosette of an EDTA-etched split of the calcite vein. The etching has exposed the pyrite rosette by removing the surrounding calcite. Note that the etching did not produce any visible artifacts on the surfaces of the pyrite rosette. Scale bar equals 20 | m.

As shown in Figs. 6a and 8, the curved films were flexible and attached to grains at discrete positions, and they appear to have contracted somewhat prior to mineralization. The curved film shown in Fig. 6a stretches nearly 30 |im between attachment points and is partly embedded in the calcite grain on the left. The lower right-hand side of the film is draped over underlying crystals and elongate objects, and wrapped up around the underside of the objects below. Though we found that the film was partly mineralized, the elliptical shape of two holes within it indicates that it must have been flexible enough to tear and contract slightly prior to mineralization. Similarly, the pyritized sheet-like structure shown in Fig. 8 has elliptical holes (Fig. 8c), which indicates the same type of flexibility as the feature shown in Fig. 6a. The pyritized sheet-like structure also wraps around the edge of a pyrite crystal in a flexible manner.

The embedded nature of several of the features indicates that they are not artifacts of the etching procedure, which is also supported by the presence of fully pyritized films (Fig. 4f) and threads (not shown). In addition, the distinct attachment points on mineral surfaces indicate that they were not passively embedded in the mineral matrix, but rather represent remnants of actively attached microbial extracellular polymeric substances (EPS). However, though it is likely that the organisms that produced the thread-like biofilm remnants lived within the hydrothermal system, the possibility exists that such objects were flushed along with the fluid from other parts of the system. The authors are not aware of any examples of allocthonous organic remains that display the morphological characteristics of the film-like structures with distinct attachment points revealed only by etching.

Several of the morphological attributes of the film-like structures match those of modern partly mineralized hyperthermophilic biofilms that form on sinter surfaces and among debris that accumulates at the bottom of active, near-boiling hot spring pools and outflow channels (Fig. 11). These remnants of modern hyperthermophilic biofilms reveal the flexible nature of the biofilm matrix, which is stretched and contracted between attachment points (Fig. 11a). In some cases, as shown in Fig. 11b, distinct threads that serve to attach the film to the substratum bifurcate and appear to splay into bundles of threads, which ultimately connect to the curved sheet-like biofim matrix remnant.

Demonstrating the biogenicity of ancient biofilm-like objects requires linking morphological (e.g., cell remnants and extracellular polymeric substances) and chemical (e.g., isotopes, biomarkers, biominerals) evidence indicative of microorganisms or microbial activity (Cady, 2001; Cady et al., 2003). Evidence of microbial activity can include the presence of extracellular polymeric substances (EPS) that form when organisms attach to surfaces during the process of biofilm formation (Westall et al., 2000, 2006; Westall and Southam, 2006). Hyperthermophilic biofilms develop on actively accreting mineral surfaces in modern hot springs and display key features that are likely characteristic of subsurface biofilms. The flexible nature of the extracellular matrix of the biofilms allows for resistance to even the most deleterious effects of fluid movement (Fig. 11c). We have observed in modern hot spring settings that the extracellular matrix of hyperthermophilic biofilms is much more recalcitrant than the microbial inhabitants that excreted the EPS, especially during the earliest stages of mineralization, as evidenced by the common occurrence of EPS remnants sans microbial cells in modern hot spring deposits (Fig. 11).

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