Here, we present abundant mineralised cell envelope structures fossilised within a goethite-rich vein structure that cross-cuts a weathering BIF6. These indisputable microbial fossils provide a superb opportunity to understand potential mineralogical biosignatures preserved during microbial fossilisation by authigenic minerals. Rock fissures, fractures and pore spaces present ideal locations for the preservation of microorganisms as elements in solution percolate throughout the weathering profile. The multivalent oxidation states of iron and its low solubility in circumneutral pH environments make iron-rich regions an attractive target for microbial fossilisation in near-surface environments, both on Earth and potentially Mars.
Cell envelope structures reduce the stability of cations in solution, altering the mineral phases that would otherwise have precipitated abiotically13. If these biominerals resist recrystallisation, unique mineral phase distributions may be preserved as mineralogical biosignatures3,14. In this study, crystal orientation and crystal size of the microbially-influenced iron oxide minerals appears to be altered by the microbial cell envelopes (Figs. 1 and 2) but no clear mineralogical biosignatures were preserved (Figs. 5–7). Although characterisation techniques used here are not exhaustive, there was no clear mineralogical difference between iron oxides that precipitated around the cell envelope and neighbouring iron oxide minerals in the matrix (Figs. 6–7), despite the apparent enrichment of aluminium associated with the cell envelope (Fig. 3). Therefore, the organic-mineral complexes that resist recrystallisation during the initial stages of mineral formation15 do not appear to be preserved in million-year-old fossils. Given the low-temperature formation and alteration of the iron oxide minerals9, the aliphatic hydrocarbon moieties preserved with the microfossils are likely to be replaced during low-temperature metasomatism by iron and aluminium in solution. Levett et al.6 demonstrated that organic biosignatures associated with younger (ca. 2 Ma) permineralised microfossils in the overlying duricrust are completely replaced, likely due to the increased cycling of iron oxide minerals that occurs near the surface16.
In this study, mineral precipitates associated with cell envelope structures are extremely fine-grained compared with the post-death intracellular mineral precipitates (Figs. 6–7). Cations binding to active sites on the cell envelope17 appears to create multiple mineral nucleation sites, restricting crystal growth. In contrast, post-death mineral precipitates within the intracellular voids have fewer mineral nucleation sites and, therefore, are allowed to grow in a less restricted manner. Consistent with Cosmidis, et al.18, intracellular mineral precipitates always grow from the cell envelope inward to fill the intracellular void.
The 3D characterisation of iron oxide encrusted microbial fossils, provides an important opportunity to produce orientated reconstructions useful in the search for fossilised bacteria or biofilm (Video 1). Though FIB-SEM was used in this study, synchrotron-based nanotomography now offers non-destructive 3D reconstructions with submicron spatial resolution19. These technical developments offer unparalleled opportunities to understand the mechanisms that contribute to microbial fossilisation. The 100 nm resolution of the 3D reconstruction produced for multiple sections of each microbial fossil. The microfossils examined here are typically cocci-shaped and approximate 1 μm in diameter. Sarcina-like multicellular packet structures (for example, Supplementary Fig. S3) are never observed in the fossilised biofilm characterised here; however, rarely, paired cells are preserved that share a cytoplasm in a single section (Fig. 2). These textures provide evidence that the fossilised microorganisms presented here may be able to replicate during the initial stages of biomineralisation as has been previously postulated 20,21,22.
Microorganisms fossilised by authigenic mineral nucleation, rather than the binding of sediments within the biofilm23, provide valuable insights into the environmental conditions in which the living microorganism existed. These biogenic minerals may also provide information on influence of the microorganisms on their surrounding environment. Previous experiments have demonstrated the precipitation of lepidocrocite associated with neutrophilic iron-oxidising microorganisms24 and nitrate-dependent iron-oxidising bacteria25. Given the microfossil structures in this study are generally cocci-shaped, they are unlikely to represent sheath structures of classic Leptothrix-type neutrophilic iron-oxidising bacteria26, though filaments do exist (see Video 1; 38–40 s, top right-hand corner). In addition, the low nitrogen availability in these environments27 suggests that microfossils are also unlikely to represent nitrate-dependant iron oxidisers. The apparent binding of aluminium with cell envelope structures, indicates that these microorganisms were likely to have been preserved by the passive nucleation of minerals on the cells’ surfaces14,28. The formation of lepidocrocite together with goethite (Fig. 5) suggests that the pH was between 5–7. Lepidocrocite forms preferentially to goethite under slightly slower oxidation rates of iron29, indicating reduced partial pressures of oxygen in pore spaces below the surface compared with atmospheric conditions.
In this BIF weathering profile, many cells contribute to mineral nucleation by the passive interaction of cations with the net negative cell envelope28. Even amongst the cells that contribute to biomineralisation30, few are likely to achieve a state of ‘fossilisation’, whereby they are preserved in the geological record. Extensive mineralisation is required to achieve microbial fossilisation and preservation. Based on earlier findings6, aluminium binding irreversibly with cell envelope structures appears to play an important role in the preservation of organic biosignatures; however, synchrotron-based FT-IR analysis could not resolve aluminium-organic complexes.
The preservation of microorganisms in the geological record is rare. As such, an abundance of well-preserved microfossils in any environment requires careful consideration. To resist the breakdown of cellular components, particularly cell envelope structures and potentially EPS, rapid and extensive mineralisation is required. The influence of water in microbial fossilisation within the lithosphere is also likely to be critical. While fine-grained, generally amorphous iron oxide precipitates readily nucleate on cell envelope structures in iron-rich aqueous environments28, for example, 2-line ferrihydrite31; these cells are unlikely to be fossilised within water saturated environments. Therefore, following this initial stage of biomineralisation during a cells exposure to cation-rich solutions, periods of drying appear to be imperative for fossilisation. During dehydration, any remaining ions in solution (in this case, predominately iron and aluminium), would be concentrated, accelerating additional mineral nucleation on the cells’ surfaces. Alternating wet and dry periods may be required to promote additional mineralisation. In this scenario, additional metals in solution would be provided during wet periods, which may allow for the recrystallisation of existing iron oxide minerals and the additional precipitation of new iron oxide minerals32. During drying periods, newly mineralised microorganisms may be committed to the geological record, contributing to preservation of relatively large microfossil clusters.
The organic compounds associated with the cell envelopes are likely to be preserved by the electrostatic-driven nucleation of aluminium and iron oxide minerals within relatively oxidising environments6,7,28. As the microfossils are continuously exposed to aluminium and iron-rich solutions, the mineralised cell envelopes appears to act as a filter; iron is allowed into the cell whereas aluminium is enriched around the cell envelope6,33. The structure of the cell envelop appears to restrict aluminium transfer into the cell33, possibly even after cell death. Aluminium may also continue to be enriched around the cell envelope as it preferentially precipitates with existing aluminium-substituted iron oxide minerals that have previously nucleated on the cells’ surfaces29. In this way, even after all the organic components of the cell envelope are replaced and the cell has been completely permineralised, aluminium enrichment around the cells may help to preserve the bacteriomorphic structure within the geologic record6,34.
Appropriate sample preparation for high-resolution analytical work is critical. Many techniques require a polished surface to spatially resolve distinctions between minerals influenced by microorganisms compared with ‘abiotic’ mineral precipitates. To study microfossils, sample preparation using a FIB-SEM offers a number of benefits including, targeted preparation of localised regions of interest without introducing organic contaminants. As a destructive sample preparation technique, great care and skill is required when preparing microfossil lamella using a FIB-SEM; however, this sample preparation technique is highly versatile10. Samples can be made thin enough to be analysed using transmission X-ray and infrared sources but also robust enough for NanoSIMS, a destructive secondary ion technique. Therefore, FIB-SEM sample preparation allows for highly targeted, polished sample preparation and for correlation between several different analytical datasets, as demonstrated in this study. Ultrathin samples (~100 nm thick) can also be prepared for high-resolution transmission electron microscopy (HR-TEM) and scanning transmission X-ray microscopy.
The redistribution of elements that contribute to near-surface microbial fossilisation in rock pore spaces and fissures is fundamental to targeting drill regions for the identification of microfossils on samples from Mars. Organic biosignatures and abundant microbial fossils preserved in iron-rich environments highlights the potential to target iron-rich regions on the Martian surface for the search of potential microbial biosignatures. In depth characterisation of indisputable microbial fossils combining a suite of nano- and microscale analytical techniques sets important benchmarks for the identification of biosignatures within the geological record35. Additional studies of natural microbial fossils in a variety of environments is required to understand potential biosignatures preserved in different environments and aim to develop new robust biosignatures, for example, mineralogical biosignatures.