Chemolithotrophic growth of M. sedula on NWA 1172

M. sedula was capable of autotrophic growth on stony meteorite NWA 1172, utilizing metals trapped within it as the sole energy source. Scanning electron microscopy (SEM) investigations (Fig. 1a,b and Supplementary Fig. 2) and multi-labeled fluorescence in situ hybridization (MiL-FISH) with an M. sedula-specific ribosomal RNA-targeted probe (Fig. 1c–e, Supplementary Fig. 3, and Supplementary Table 1) indicate an active colonization of the meteorite material by M. sedula. As reported earlier, the round to slightly irregular coccoid cells of M. sedula possess pilus-like structures and can be motile when grown on the metal ore9. When grown in the presence of NWA 1172, cells of M. sedula were characterized by intensive vivid motility (Supplementary Movie 1). In comparison to chalcopyrite, M. sedula had a superior growth rate on the stony meteorite (Fig. 1f). Grown in the presence of chalcopyrite, cells of M. sedula displayed motility as well (Supplementary Movie 2). Meteorite-grown M. sedula reached stationary phase at 163 h (maximum density 18.5 ± 2 × 108 cells/mL, a mean generation time of 8 h), while chalcopyrite-grown cells were still in exponential phase at that time point, and reached the stationary phase after 300 h (maximum density 15.1 ± 5 × 108 cells/mL, a mean generation time of 21.4 h) (Fig. 1f). This difference reflects the beneficial contribution of NWA 1172 as the sole electron donor suggesting its preferential nature as energy source for M. sedula. NWA 1172 is a chondrite meteorite with relatively high iron abundance12 and a wide range of other metal elements (Supplementary Fig. 1). Those might be used by M. sedula as an energy source to satisfy its bioenergetic needs and as specific metabolic/enzymatic cofactors, providing more optimal constitutive and/or structural elements for enzymatic machinery. In addition, copper sulfides, such as chalcopyrite, dictate certain inevitable issues in respect with metal mobilizing due to their refractory characteristics. Hence, the porosity of NWA 1172 (typically of a few percent13) might also reflect the superior growth rate of M. sedula over refractory and densely packed chalcopyrite.

Figure 1 Biotransformation of the chondrite meteorite NWA 1172 by M. sedula. (a) Scanning electron microscopy (SEM) image of fragments of the chondrite meteorite NWA 1172 bioprocessed by M. sedula. (b) SEM image showing M. sedula cells colonizing the surface of the meteorite particles. (c–e) Multi-Labeled-Fluorescence in situ Hybridization (MiL-FISH) of M. sedula cells grown on NWA 1172 as the sole energy sources: (c) MiL-FISH images of cells (green) after hybridization with the specific oligonucleotide probe targeting M. sedula; (d) DAPI staining of the same field (blue); (e) Overlaid epifluorescence image, showing overlap of the specific oligonucleotide probe targeting M. sedula with DAPI signals. Arrows indicate cells of M. sedula. Scale bar, 2 µm. (f) Growth curves of autotrophic cultures of M. sedula cultivated at 73 °C on NWA 1172 (red) and chalcopyrite (blue). Legends represent the corresponding type of energy source. (g) Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis of released metal ions in the supernatant of M. sedula cultures grown on NWA 1172 as the sole energy source. Samples were taken at “0” time point (red), from late exponentially growing cultures of M. sedula (purple), and from corresponding abiotic controls (blue and grey, respectively). (h) Single crystals of nickel sulfate hexahydrate and magnesium sulfate heptahydrate were obtained after recrystallization of the crystalline material (α) shown in Supplementary Fig. 4. (i) Atomic structures of NiSO 4 × 6 H 2 O and MgSO 4 × 7 H 2 O from crystals in (h) as investigated with single crystal X-ray diffraction, with the unit cell for each structure represented. Crystal water has been removed for clarity. Legend: Ni(H 2 O) 6 , red octahedra; Mg(H 2 O) 6 , blue octahedra; SO 4 2−, yellow tetrahedra. Points and error bars show the mean and error-represented standard deviation, respectively, of n = 3 biological replicates. If not visible, error bars are smaller than symbols. Full size image

As a result of tight biogeochemical interactions, by means of its metal oxidizing machinery14,15,16, M. sedula released free soluble metals (Ni, Si, Co, Mn, and K) into the cultivation medium (Fig. 1g). Ni and Si were predominantly released, reaching 0.55 and 0.17 g/L, respectively, with lesser contributions from Co, Mn, and K ions (Fig. 1g). Dehydration-crystallization and X-ray diffraction experiments (Supplementary Fig. 4) revealed the chemical speciation of the released Ni: nickel sulfate hexahydrate17 in the tetragonal space group P 4 1 2 1 2 (a = 6.8 Å, b = 6.8 Å, c = 18.29999, α = β = γ = 90°) and magnesium sulfate heptahydrate18 in the orthorhombic space group P 2 1 2 1 2 1 (a = 11.868 Å, b = 11.996 Å, c = 6.857 Å, α = β = γ = 90°), suggesting +2 oxidation state of extracted Ni ions and the occurrence of nickel sulfate in the leachate solution (Fig. 1i).

Nanoanalytical spectroscopy of meteorite-microbial interface

Observations of ultra-thin sections of M. sedula cells grown on NWA 1172 revealed round-shaped, irregular cocci with a diameter around 1 µm (Fig. 2). These irregular coccoid morphologies were characterized by the presence of electron-dense dark areas along the cell envelope and extensive dark accumulations in the cytosol (Fig. 2). Elemental ultrastructural analysis of M. sedula grown on NWA 1172 was performed to investigate metal acquisition patterns of this archaeon, i.e., enabling us to verify the content and localization of metals in M. sedula (Fig. 2). The following observations were made using energy-dispersive X-ray spectroscopy (EDS) analysis performed in scanning transmission electron microscopy (STEM) mode: (1) the elemental maps show abundant C, O, N, S, Cu, P, Fe, Al, Co, and K content of M. sedula cells; (2) Cu, K, Cl, Fe, Al, and P signals were localized both on the cell surface and intracellularly; (3) C, O, and N were evenly distributed giving strong intracellular signals which likely arose from organic content (e.g., proteins and carbohydrates) present in M. sedula cells; (4) Si accumulations produced strong intracellular signals, which correspond to the dark electron dense areas of the TEM image (intracellular deposits); and (5) Co and K were evenly represented inside the cell; (Fig. 2, Supplementary Fig. 5).

Figure 2 Elemental ultrastructural analysis of M. sedula grown on NWA 1172. The annular dark field (ADF) scanning transmission electron microscopy (STEM) image of a cell of M. sedula used for the EDS spectrum image acquisition and corresponding extracted carbon (C), oxygen (O), nitrogen (N), copper (Cu), sulfur (S), potassium (K), chlorine (Cl), iron (Fe), aluminum (Al), phosphorus (P), cobalt (Co), silicon (Si), and uranium (U) elemental maps. Scale bar, 0.5 µm. Full size image

The pronounced membrane-bound signal of Cu reflects the great diversity and active content of multi-copper oxidases widely distributed in M. sedula branched respiratory chains within a diverse number of terminal oxidases10,14,15,16. Apart from these multi-copper oxidases involved in iron and sulfur oxidative respiration network, membrane-associated specific Cu-transporters might also implement in Cu binding observed in Fig. 2 (Cu map). An efficient Cu transportation and sequestration system was described in M. sedula19,20. Incorporation of Fe and S may naturally speak on the account that these elements are potentially used as energy sources in oxidation process coupled to electron breathing (Fig. 2, Fe and S maps). Intracellular Si accumulations in form of bulk deposits comprise 5.9% of the overall elemental content, and suggest the beginning of silicification of M. sedula during growth on meteorite material (Fig. 2, Si map and Supplementary Table 2). Silicification processes have been shown for various microbial systems, including in situ studies in modern silicifying hydrothermal systems21 and laboratory investigations of artificial microbial silicification22,23,24, highlighting the potential of silica matrices for the morphological preservation of putative microfossils.

TEM analysis revealed the presence of extracellular vesicle-like morphologies (encapsulated round-shaped particles with an average diameter of 200 nm; Supplementary Fig. 6a). The monolayers of M. sedula vesicles have been shown to catalyze iron oxidation and solubilization of mineralized copper from chalcopyrite under the energy-limited lithoautotrophic conditions25. Apart from metal transformation, synthesis and secretion of membrane vesicles might serve as a general mechanism of extracellular metal sequestration by binding with chelating agents, e.g., proteins, and enzymatic detoxification of the metal to a less toxic form. The observed vesicles, formed during the growth of M. sedula, may be capable of mineral oxidation, contributing to metal biotransformation and potentially metal immobilization from NWA 1172. Our EDS analysis performed in STEM mode showed that these carbonaceous and oxygenated vesicles harbor Si, Ni, Cu, Fe, Cl, and Al (Supplementary Fig. 6a), which reflects their ability to immobilize metals from the meteorite material.

Notably, TEM observations of cross-sections of M. sedula cells revealed the coexistence of cells in different stages of biomineralization: cells with not fully mineralized cell envelope (Fig. 2) and at more advanced steps of mineral formation as heavily encrusted mineralized cell remnants (Fig. 3 and Supplementary Figs. 7, 8). Elemental ultrastructural analysis of heavily encrusted cell remnants showed that the crust layer of variable thickness (up to 250 nm) was composed of Fe, Cu, Si, Al, Ni, S, C, N, O, and P (Supplementary Table 3). Further comparative electron energy loss spectra (EELS) analysis uncovered the spatial distribution and fine structure of iron species in heavily encrusted cell remains and on the cell surface of M. sedula (Fig. 3, bottom panel). The EELS measurements, acquired locally (point analysis with a beam diameter of 1 Å) in STEM mode, demonstrate that the cell surface of M. sedula is bearing a mixed valence iron distribution with dominant Fe2+ species (dotted line at Fig. 3, bottom panel). Fe2+ detected on the cell surface (Fe L 3 -edge at ~708 eV, Fig. 3, bottom panel) may primarily originate due to the leaching of Fe2+ from the meteorite. This spectrum also shows the minor presence of Fe3+ species (a shoulder of Fe L 3 -edge at ~710 eV) which can be explained by extracellular Fe2+ oxidation performed by a membrane bound iron oxidizing machinery of M. sedula, which is encoded by genes of the fox cluster10,14,15,16. A microbially replenished Fe3+ supply (in addition to abiotic Fe3+) ensures further meteorite oxidation as well as access to metals for the cells. The Fe3+ species detected on the cell surface of M. sedula can effectively function as an oxidizing agent for meteorite at the cell-meteorite interface. In addition to the direct M. sedula mediated biooxidation of metals, the involvement of abiotic factors may facilitate the process of metals mobilization from NWA 1172, representing an indirect mechanism of metal solubilization. Abiotic oxidizing attack of Fe3+ on the solid mineral enables the mobilization of metals from the solid matrix26, suggesting that both direct and indirect mechanisms contribute to the dissolution of metals mediated by M. sedula. The Fe L 2,3- edges from heavily encrusted cell remnants show the predominant presence of Fe3+ species (solid line at Fig. 3, bottom panel, Fe L 3 -edge at ~710 eV), which can be explained by accomplished Fe2+ oxidation followed by cell encrustation and entombment in the mineralized form of a mixture of different amorphous iron oxides/hydroxides with the predominant form of Fe3+. Similar cell surface encrustation, but with tungsten crystalline nanolayers, we have previously shown for M. sedula grown on tungsten-bearing terrestrial materials27,28. In the case of our study with NWA 1172, TEM observations show that the encrusted cell remnants and iron bearing accumulations on the cell surface of M. sedula have an amorphous structure. Consequently, further studies were directed at spectral and mineralogical analysis of the meteorite surface after the exposure to M. sedula.

Figure 3 Elemental ultrastructural analysis of M. sedula empty envelopes encrusted during growth on NWA 1172 and corresponding Fe L 2,3 -edge core electron energy loss (EEL) spectra. The high angular annular dark field (HAADF) scanning transmission electron microscopy (STEM) image of a heavily encrusted cell remnants of M. sedula used for the EDS spectrum image acquisition and corresponding carbon (C), copper (Cu), phosphorus (P), iron (Fe) oxygen (O), nickel (Ni), sulfur (S), and nitrogen (N) elemental maps. Corresponding Fe L 2,3 -edge core electron energy loss (EEL) spectra acquired from the S-layer of M. sedula cells depicted in Supplementary Fig. 8a (shown as dotted line) and from the crust in Supplementary Fig. 8c (shown as solid line) are provided at the bottom panel. Full size image

Mineralogical and geochemical analyses of microbial fingerprints left on NWA 1172

Investigations of the biologically mediated alteration of the meteorite surface by cultivation of M. sedula with slab fragments of NWA 1172 indicated the presence of globular iron-rich aggregates (with a size ranging from <0.5 to 2 μm for single globules) along with areas of crystalline iron oxides as a specific microbial alteration left upon M. sedula growth (Fig. 4a,b,d). Similar types of globular iron-rich aggregates were observed on the surface of the meteorite abiotically exposed to M. sedula medium at 73 °C, illustrating that they are the result of abiotic, i.e., chemically induced rusting processes (Fig. 4c). EDS analysis of these globular aggregates in chemically changed and biologically altered meteorite surfaces showed that they are dominated by Fe and P with a variable amount of S and Si (Fig. 4e). However, the iron oxides (as determined using EDS), which appear as branched porous network represented on Fig. 4b,d,f, occurred only along the meteorite surface exposed to M. sedula, suggesting that it is solely of biogenic origin. In a few previously published studies, iron meteorites and carbonaceous chondrite meteorites provided metal components as suitable energy substrates to maintain the bacterial growth and chemolithotrophic metabolism5,6. Similar near-spherical Fe-, P-, and S-containing aggregates along with needle-like crystals of iron oxides were observed during iron meteorite weathering by the iron-oxidizing acidophilic bacteria A. ferrooxidans5.

Figure 4 Alteration of the surface of the chondrite meteorite NWA 1172 slabs mediated by M. sedula. (a) Secondary electron (SE) image of a NWA 1172 slab cultivated with M. sedula at 73 °C. (b) Magnified SE image of a NWA 1172 slab cultivated with M. sedula at 73 °C. (c) SE image of abiotically exposed slab of NWA 1172 to the cultivation medium at 73 °C. (d) Magnified area of SE image of NWA 1172 slab cultivated with M. sedula at 73 °C. (e) EDS spectra of globular structures (marked with red A) that form iron oxides aggregates, containing mainly Fe and P. (f) EDS spectra of branched network of crystalline iron oxides (marked with red b). Arrows indicate the areas where crystalline iron oxides occur. Full size image

Micro X-ray diffraction (µXRD) data were acquired on a bioprocessed NWA 1172 slab fragment after the cultivation with M. sedula, and the following secondary oxides/alteration minerals were characterized: goethite, lepidocrocite, ferrihydrite, hematite, and maghemite (i.e., similar work conducted on an unprocessed meteorite slab shows that none of these minerals were originally present in the meteorite) (Supplementary Fig. 9). The most-abundant primary minerals in ordinary chondrites are olivine and enstatite (+/− plagioclase). These primary minerals are visible on every pattern in µXRD analysis performed in this study, as X-rays penetrate through the surface layer with microbial alterations. Occasionally, primary kamacite, troilite, and magnetite were also detected. Our µXRD measurements are in agreement with previously reported biogenic crystalline ferric iron (oxy)hydroxides, such as goethite and lepidocrocite for the iron oxidizing acidophile Acidithiobacillus ferrooxidans during iron meteorite weathering5 and closely related species Sulfolobus acidocaldarius, the only other member of the Sulfolobales for which such measurements are available29.

Electron Paramagnetic Resonance (EPR) measurements were performed to (1) identify paramagnetic species in NWA 1172 and to (2) investigate the impact of M. sedula on NWA 1172 with a possible effect on the oxidation state of paramagnetic species. Measurements were performed at 90 K (Fig. 5a) and 273 K (Fig. 5b) to increase the chance for unambiguous iron detection. Iron oxides are the predominant component (51%) of the chondrite NWA 1172 (Supplementary Fig. 1) and iron mixed paramagnetic Fe3+ species in high and low spin states in biogenic samples were identified via EPR. Accumulation of Fe3+ in the biogenic sample at 90 K (Fig. 5a, red line) was observed and might be the consequence of extensive Fe2+ oxidation of the minerals mediated by M. sedula. After cultivation with M. sedula, the biogenic sample shows a prominent sharp high spin Fe3+ signal with a g-value of 4.35 and a low spin Fe3+ signal at g-value 2 (Fig. 5a). Compared to the raw meteorite material, the appearance of a prominent high spin Fe3+ signal (a sharp peak with a g-value of 4.35) along with a low spin Fe3+ signal at g-value 2 in the biogenic sample may indicate an increase in Fe3+ species due to biooxidative activity of M. sedula. These characteristic sharp high spin and low spin Fe3+ signals are not represented in abiotically treated NWA 1172 (Fig. 5). The abiotic spectrum is characterized by a signal with a shifted g-value 3.20 and broad linewidth (ΔH = 2010 G), which might refer to multiple ionic mixtures. Interestingly, the spectral features of abiotically treated NWA 1172 closely resemble those of abiotically treated Martian regolith simulant S-MRS11. This abiotic peak at g-value 3.20 is not detectable anymore in biogenic samples of NWA 1172 (Fig. 5a, red curve) and S-MRS11. While the distinct high spin Fe3+ signal is not detectable in the biogenic sample when measured at 273 K (Fig. 5b), the low spin Fe3+ signal at g-value 2 appears after the cultivation showing us the presence of mineral transforming iron-oxidizing microorganisms.