Exploring the microbial biotransformation of extraterrestrial material on nanometer scale

Exploration of microbial-meteorite redox interactions highlights the possibility of bioprocessing of extraterrestrial metal resources and reveals specific microbial fingerprints left on extraterrestrial material. In the present study, we provide our observations on a microbial-meteorite nanoscale interface of the metal respiring thermoacidophile Metallosphaera sedula. M. sedula colonizes the stony meteorite Northwest Africa 1172 (NWA 1172; an H5 ordinary chondrite) and releases free soluble metals, with Ni ions as the most solubilized. We show the redox route of Ni ions, originating from the metallic Ni° of the meteorite grains and leading to released soluble Ni2+. Nanoscale resolution ultrastructural studies of meteorite grown M. sedula coupled to electron energy loss spectroscopy (EELS) points to the redox processing of Fe-bearing meteorite material. Our investigations validate the ability of M. sedula to perform the biotransformation of meteorite minerals, unravel microbial fingerprints left on meteorite material, and provide the next step towards an understanding of meteorite biogeochemistry. Our findings will serve in defining mineralogical and morphological criteria for the identification of metal-containing microfossils.


Results and
) 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 ore 9 . 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 × 10 8 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 × 10 8 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 abundance 12 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 percent 13 ) might also reflect the superior growth rate of M. sedula over refractory and densely packed chalcopyrite.
As a result of tight biogeochemical interactions, by means of its metal oxidizing machinery [14][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 hexahydrate 17 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 heptahydrate 18 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).
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 oxidases 10,14-16 . Apart from these multi-copper oxidases involved in iron and sulfur oxidative respiration network, An efficient Cu transportation and sequestration system was described in M. sedula 19,20 . 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 systems 21 and laboratory investigations of artificial microbial silicification [22][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 conditions 25 . 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  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 Fe 2+ species (dotted line at Fig. 3, bottom panel). Fe 2+ detected on the cell surface (Fe L 3 -edge at ~708 eV, Fig. 3, bottom panel) may primarily originate due to the leaching of Fe 2+ from the meteorite. This spectrum also shows the minor presence of Fe 3+ species (a shoulder of Fe L 3 -edge at ~710 eV) which can be explained by extracellular Fe 2+ oxidation performed by a membrane bound iron oxidizing machinery of M. sedula, which is encoded by genes of the fox cluster 10,14-16 . A microbially replenished Fe 3+ supply (in addition to abiotic Fe 3+ ) ensures further meteorite oxidation as well as access to metals for the cells. The Fe 3+ 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 Fe 3+ on the solid mineral enables the mobilization of metals from the solid matrix 26 , 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 Fe 3+ species (solid line at Fig. 3, bottom panel, Fe L 3 -edge at ~710 eV), which can be explained by accomplished Fe 2+ 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 Fe 3+ . Similar cell surface encrustation, but with tungsten crystalline nanolayers, we have previously shown for M. sedula grown on tungsten-bearing terrestrial materials 27,28 . 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.

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 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 www.nature.com/scientificreports www.nature.com/scientificreports/ meteorites provided metal components as suitable energy substrates to maintain the bacterial growth and chemolithotrophic metabolism 5,6 . 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. ferrooxidans 5 .
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,  www.nature.com/scientificreports www.nature.com/scientificreports/ 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 weathering 5 and closely related species Sulfolobus acidocaldarius, the only other member of the Sulfolobales for which such measurements are available 29 .
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 Fe 3+ species in high and low spin states in biogenic samples were identified via EPR. Accumulation of Fe 3+ in the biogenic sample at 90 K (Fig. 5a, red line) was observed and might be the consequence of extensive Fe 2+ oxidation of the minerals mediated by M. sedula. After cultivation with M. sedula, the biogenic sample shows a prominent sharp high spin Fe 3+ signal with a g-value of 4.35 and a low spin Fe 3+ signal at g-value 2 (Fig. 5a). Compared to the raw meteorite material, the appearance of a prominent high spin Fe 3+ signal (a sharp peak with a g-value of 4.35) along with a low spin Fe 3+ signal at g-value 2 in the biogenic sample may indicate an increase in Fe 3+ species due to biooxidative activity of M. sedula. These characteristic sharp high spin and low spin Fe 3+ 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 www.nature.com/scientificreports www.nature.com/scientificreports/ (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-MRS 11 . This abiotic peak at g-value 3.20 is not detectable anymore in biogenic samples of NWA 1172 (Fig. 5a, red curve) and S-MRS 11 . While the distinct high spin Fe 3+ signal is not detectable in the biogenic sample when measured at 273 K (Fig. 5b), the low spin Fe 3+ signal at g-value 2 appears after the cultivation showing us the presence of mineral transforming iron-oxidizing microorganisms.

conclusions
Our investigations provide the further step towards extending our knowledge of meteorite biogeochemistry. Heterogeneous iron species Fe 2+ /Fe 3+ with dominant Fe 2+ are represented on the cell surface, indicating active Fe redox processing at the cell surface of M. sedula and suggesting Fe oxidative solubilization of meteorite material. M. sedula releases free solubilized metals (Fig. 1g) and is capable of intracellular and cell surface incorporation of metals when grown on meteorite material (Figs. 2, 3, and 4). Meteorite biotransformation mediated by M. sedula leads to nickel solubilization (Fig. 1g-i) with the formation of nickel sulfate (NiSO 4 )-bearing material (Fig. 1h,i). Such nickel enrichment/NiSO 4 deposition can be considered as a metabolic signature resulted from microbial leaching activity of meteorite minerals. The heavily metal encrusted cell remnants described in the present study and its fine structural details may constitute relevant biosignatures to be looked for in the geological record, if they are not destructed during diagenetic or metamorphic processes and are intact during different stages of fossilization. Our nanoanalytical spectroscopy investigations of heavily mineralized M. sedula cells grown on the NWA 1172 meteorite revealed the enrichment of Cu, Fe, O, and Si in the amorphous crust with a mixture of P, Ni, S and Al, which may likely correspond to the amorphous product Cu x Fe y O z (SPNiAl)-SiO 2 formed on the late biomineralization stages of cells encrustation. Our work provides a deeper insight into the biologically-mediated processing of extraterrestrial material and implications for natural samples, representing a special interest for space exploration missions. Cultivation of M. sedula was performed as described before 11 in 1 L glassblower modified Schott-bottle bioreactors (Duran DWK Life Sciences GmbH, Wertheim/Main, Germany), equipped with a thermocouple connected to a heating and magnetic stirring plate (IKA RCT Standard/IKA C-MAG HS10, Lab Logistics Group GmbH, Meckenheim, Germany) for temperature and agitation control. The bioreactors were equipped with three 10 mL graduated glass pipettes, permitting carbon dioxide and air gassing (with the gas flow of 9 mL min −1 , adjusted to five bubbles s -1 by using 8 mm valves (Serto, Frauenfeld, Switzerland)) and sampling of culture, respectively. The graduated pipettes used for gassing were connected by silicon tubing to sterile 0.2 µm filters (Millex-FG Vent filter unit, Millipore, Billerica, USA). The graduated pipettes used for sampling were equipped with a Luer-lock system in order to permit sampling with sterile syringes (Soft-Ject, Henke Sass Wolf, Tuttlingen, Germany). The offgas was forced to exit via a water-cooled condenser (Ochs GmbH, Bovenden, Germany). For the cultivations of M. sedula at 73 °C the temperature inside the bioreactors was controlled by electronic thermocouple via the heating and magnetic stirring plates. For chemolithoautotrophic growth cultures were supplemented with 10 g/ liter either chalcopyrite (provided by E. Libowitzky from the mineral collection of the Department of Mineralogy www.nature.com/scientificreports www.nature.com/scientificreports/ and Crystallography, University of Vienna) or NWA 1172 meteorite (provided by the NHM, Vienna). The minerals were ground and temperature sterilized at 180 °C in a heating chamber for a minimum of 24 hours prior to autoclavation at 121 °C for 20 min. When intact, rectangular slabs of NWA 1172 were used for fermentation, the aforementioned two steps sterilization procedure was applied, too. Abiotic controls consisting of culture media supplemented with sterilized NWA 1172 without M. sedula cells were included in the experiments. Growth of cells was monitored by phase contrast/epifluorescence microscopy and metal release; doubling times (in h) during exponential phase growth were calculated from the slopes of the growth curves based on time required for 2-fold increase of cell number. For the visualization of cells wiggling on solid particles they were stained by a modified "DAPI" (4′-6′-Diamidino-2-phenylindole) procedure 30 , observed and recorded with ProgRes ® MF cool camera (Jenoptik) mounted on Nikon eclipse 50i microscope, equipped with F36-500 Bandpass Filterset (ex, 377/50 nm; em, 447/60 nm). Hybridization (MiL-FISH). M. sedula cells were fixed in 2% paraformaldehyde (PFA) at room temperature for 1 hour, washed three times in distilled water, centrifuged at 10,000 rpm and stored in 50:50, ethanol:PBS (phosphate buffer saline). A 16 S rRNA phylotype specific probe for M. sedula was designed with the software package ARB 31   Preparation and characterization of NWA 1172 meteorite samples. A partially fusion crusted NWA 1172 meteorite sample with fresh broken surfaces was selected for the current study ( Supplementary  Fig. 1). This sample was selected because it represents a very pristine example of an H5 type ordinary chondrite. Even it is not an observed fall meteorite, it is classified as W0, thus corresponding to the least weathered (or more pristine) type of meteorite (i.e., with no visible oxidation of metal or troilite; see, e.g., Wlotzka, 1993) 32 . The classification as W0 was further confirmed during our preliminary optical microscope observations (i.e., thin sections of this meteorite).

Multi-labelled-fluorescence in Situ
After cutting off the fusion crusted parts of the sample, rectangular slabs with dimensions of about 40 × 23 × 10 mm and about 30 g each were prepared (the rest of the sample was used for the preparation of polished thin sections and for the experiments with crushed material). One of the two large surfaces of all the prepared slabs was then polished, the other five faces staying "raw" (i.e., cut surfaces).
After completion of the experiments, the slabs were withdrawn from 1 L glassblower modified Schott-bottle bioreactors and characterized with SEM. SEM characterization was conducted at the Natural History Museum (Vienna, Austria) using a JEOL JSM 6610-LV with the operating conditions of 15 kV accelerating voltage and 0.1 nA beam current.
Scanning electron microscopy. Cells of M. sedula harvested at stationary phase were prepared for electron microscopy by fixing in a solution of 1 vol.% glutaraldehyde in Na-Cacodylate buffer. Samples were dehydrated in a graded series of ethanol solutions and dried chemically using Hexamethyldisilazan (HMDS). Fixed samples were mounted on aluminum stubs, sputter-coated with Au, and examined at the University of Vienna with a Zeiss Supra 55 VP scanning electron microscope operated at 5 kV. transmission electron microscopy. Cells of M. sedula harvested at stationary phase were primary fixed at 4 °C in a 1 M Na-Cacodylate buffer containing 2.5% glutaraldehyde. After primary fixation cells were post-fixed for 2 h in 1% OsO 4 . Cells were washed three times (2 × 0.1 M Na-cacodylate, 1x dH 2 O) and subsequently dehydrated by a gradual ethanol series (30%, 50%, 70%, 90%, abs.), each step with an incubation time of 30 minutes. Cells were spun down after each dehydration step for another 30 minutes and resuspended for the following ethanol treatment. Samples were embedded in Spurr Low Viscosity Resin (Electron Microscopy Sciences, United