(**Figure 2D**). The increased level of released Ni ions was also detected in S-MRS grown cultures. The elevated levels of Sr ions were measured in culture supernatants of M. sedula grown on P-MRS, S-MRS, and MRS07/52, while increased soluble Zn was represented in JSC 1A and MRS07/52 grown cultures. The drop in P concentrations was detected in leachate solutions of all the tested MRS. The decrease of total iron observed in M. sedula cultures grown on JSC 1A, P-MRS, and S-MRS could be possibly attributed to the formation of insoluble iron oxyhydroxides which form precipitated mineral phase and therefore are not included in the samples of leachate solution used for the ICP-OES analysis. This phenomenon of 'lost iron' has been already previously reported in case of microbial cultivation on extraterrestrial material (Gronstal et al., 2009).

# SEM-EDS Investigation of Solid Mineral Phase

To further study the interactions of M. sedula with the Martian regolith simulants, the surface of these minerals was examined for possible alterations upon M. sedula growth. Inspection by SEM of solid mineral phase in cultures of M. sedula grown on synthetic Martian regolith revealed the presence of sphere-like particles of variable size (0.3–3 µm). These microhemispheroids were represented in cultures of M. sedula grown on JSC 1A, P-MRS, and S-MRS and absent in the corresponding abiotic controls (**Figure 3**). The results of SEM-EDS analysis indicate that these hemispheroidal morphologies are mainly composed of oxygen, aluminum and chlorine and with no or low carbon content (**Table 4** and Supplementary Figure 4). The solid mineral phase withdrawn from S-MRS grown cultures of M. sedula was especially enriched with aluminum/chlorine containing microspheroids, while both biotransformed JSC 1A and P-MRS had a minor occurrence of these particles. **Figure 3C** and Supplementary Figure 4 show budding microspheroids represented on biotransformed surface of S-MRS. SEM assisted investigations of a solid phase of biotransformed MRS07/52 showed the biofilm layer distributed over the mineral surface (**Figure 3G**). Such a deposited biofilm layer was absent in the corresponding abiotic control of MRS07/52 incubated in the growth medium at 73◦C but without M. sedula (**Figure 3H**).

### EPR Investigation of Solid Mineral Phase

Electron Paramagnetic Resonance measurements were performed to (1) identify paramagnetic species of manganese and iron in the different MRSs samples and to (2) investigate the impact of M. sedula on MRSs with a possible effect on the oxidative state of manganese and iron. The four different MRSs mineral mixtures were incubated in cultivation medium in the presence (biotic) or absence (abiotic) of M. sedula. The spectra

DAPI staining of the same field (blue). (C,F,I,L) MiL-FISH images of cells (green) after hybridization with the specific oligonucleotide probe targeting M. sedula. Cultures of M. sedula were examined with MiL-FISH conducted after Schimak et al. (2015) after 21 days of cultivation with JSC Mars 1A (A–C), P-MRS (D–F), S-MRS (G–I), and MRS07/52 (J–L).

of untreated (raw) MRSs were investigated as well. The abiotic samples of JSC 1A and P-MRS contained Mn2+ as evident by a prominent signal at g = 2.4 with a broad linewidth of >1200 G (**Figure 4** and Supplementary Tables 1, 2) typical for abiotic Mn2+ samples (Kim et al., 2011; Ivarsson et al., 2015). For JSC 1A, the biotic sample revealed an almost complete decline of the Mn2+ signal, while for P-MRS an alteration of the g = 2.4 signal in the biotic sample resulted to the appearance of a broad signal with g = 3.3, indicating dipolar interactions of mixed ionic states (Mn3+ and Mn4+) (Ivarsson et al., 2015). In biotic and abiotic samples of JSC 1A and P-MRS, Fe3+ could be identified by the characteristic g = 4.3 and g = 9 signals. A high spin d5 configuration of the Fe3+ can be inferred from the positions of these resonance signals. **Figure 4B** shows that the amplitude of high spin Fe3+ g = 4.3 signal is slightly lower in biotic P-MRS than in the corresponding abiotic sample, while the signals g = 11.5 and g = 9.9 measured at 90 and 293 K, respectively, are clearly diminished after M. sedula cultivation. Independent of the incubation conditions, the EPR spectra of all four MRSs samples recorded at 90 K revealed small signals with g-values in the range of 2.0 and 2.7, which were assigned to Fe3+ with low spin electron configuration. No significant changes were detected between the biotic and abiotic samples measured at 90 K with regard to the Fe3+ in the low spin state. In opposite, a continuously narrowing resonance signal g = 2 was recorded at 293 K in JSC 1A samples after cultivation

with M. sedula. The linewidth (1H) remained unaltered for raw and abiotic JSC 1A (732 G) and decreased to 600 G after the cultivation with M. sedula (**Figure 4** and Supplementary Table 2).

The EPR spectrum of the abiotic S-MRS sample exhibits a prominent resonance at g = 3.2 with a broad linewidth of 2061 G, which is absent in the EPR spectra of all the other MRSs samples measured at 90 K. This specific resonance is obtained from

samples containing tetrahedral Mn2+ species (Xu et al., 1999). A pronounced decrease of the g = 3.2 resonance signal could be demonstrated for the biotic sample of S-MRS compared to the corresponding abiotic sample. In MRS07/52, Fe3+ species in the low and high spin state were identified based on the characteristic EPR signals as mentioned above. No manganese

could be detected in MRS07/52. The minor difference in biotic and abiotic MRS07/52 samples occurred at the area of low spin Fe3+, which is even more detectable in spectra recorded at 293 K (**Figure 4D**). The slightly increased amplitude of high spin Fe3+ signal along with the shift of g-value from 8.7 for abiotic to 9 for biotic samples was also observed at 90 K. The strong resonance line at g = 4.1 which can be assigned to Fe3+ located in orthorhombic positions (Polikreti and Maniatis,

recorded at 90 and 293 K are grouped in Supplementary Table 1. The corresponding EPR Linewidths (1H) values are provided in Supplementary

Table 2.

2002) is not altered in biotic and abiotic MRS07/52 samples (**Figure 4D**).

# DISCUSSION

fmicb-08-01918 October 5, 2017 Time: 15:18 # 9

Evolutionally diversified metal-solubilizing microorganisms with their fascinating metabolic pathways have developed an exquisite set of capabilities for manipulating minerals by dissolving them to access useful metals. We tested the ability of M. sedula to grow chemolithoautotrophically in four different types of synthetic Martian regolith. As growth was detected in all four MRS, we concluded that M. sedula is capable of chemolithotrophic growth using the synthetic Martian regolith as the sole energy source. Further, our results indicated that M. sedula is able to solubilize metals from the synthetic Martian regolith (e.g., S, K, Ca, Na, Mg, Si, Mn, Fe, Zn, Ni, and Sr) into the growth medium (leachate solution). Interestingly, in all four tested MRSs, the amount of detected phosphorus decreased in the biogenic samples after 21 days of cultivation, possibly indicating phosphorous consumption by M. sedula in order to maintain the growing population of the cells, which can explain a decreased amount of P in leachate solutions (**Figure 2**). The observed metabolic activity coupled to the release of free soluble metals from the synthetic Martian regolith can certainly pave the way to asteroid biomining, launching the biologically assisted exploitation of raw materials from asteroids, meteors and other celestial bodies.

However, insufficient evidence exists to confidently identify which metal/or metals in all tested MRSs serve as electron donors utilized by M. sedula to satisfy its bioenergetics needs. Due to its versatile metal-oxidizing capacities (Huber et al., 1989; Auernik and Kelly, 2010a,b; Maezato et al., 2012; Mukherjee et al., 2012; Wheaton et al., 2016), the fact that M. sedula can potentially respire on a combination of different elements represented in MRSs cannot be ruled out. It would seem beneficial for M. sedula to attach preferentially to those minerals containing useable substrates such as reduced iron or sulfur compounds. Fe2+ as a constitutive element is represented in variety of minerals, including olivine [(Mg, Fe2+)2SiO4] (11% in JSC 1A, 2% in P-MRS, 15% in S-MRS), and siderite [Fe(CO3), 5% in P-MRS], or bound to the smectite clay chamosite [(Fe2+,Mg)5Al2Si3O10(OH)8, 20% in P-MRS] (**Table 2**). The comparison of the obtained EPR spectra of raw and abiotic samples (**Figure 4**, green and blue lines, correspondingly) with the mineralogy and chemical composition of all MRSs we tested suggested that the recorded signals are most likely due to Mn2+ and Fe3+ ions. The obvious alteration of the width of EPR signals g = 2 detected in JSC 1A samples at 293 K after M. sedula growth might well reflect the oxidation of Fe2+ into Fe3+ (Presciutti et al., 2005; Mangueira et al., 2011). Interestingly, no 1H alteration of g = 2 signal was observed for abiotically treated JSC 1A compared to raw JSC 1A material (**Figure 4A** and Supplementary Table 2). The slight increase of the signal amplitude of high spin Fe3+ g = 4.1 and g = 9 signals along with the appearance of g = 2.5 signal in biotic samples of MRS07/52 suggest the accumulation of Fe3+ in mineral phase, which also speaks on the account of Fe2+ oxidation mediated by M. sedula (**Figure 4D**). Interestingly, MRS07/52 is the only simulant among all tested MRSs, where Fe ions were detected to be released from after 21 days of cultivation with M. sedula. In biotic samples of Mn-bearing MRSs (JSC 1A, P-MRS, and S-MRS) the increased signals of mixed ionic states Mn3+ and Mn4+ and the decrease of Mn2+ in tetrahedral positions were observed pointing to a plausible redox couple for M. sedula respiration. The accumulation of redox heterogeneous Mn species may occur in the mineral phase due to M. sedulas oxidative metabolism, analogously to microbial mediated sulfur oxidation with the wide variety of redox heterogeneous intermediate sulfur compounds (Schippers et al., 1996) and serve as a fingerprint of chemolithoautotrophic life. However, to which extent Mn2+ is oxidized to Mn3+ and Mn4+ by M. sedula cannot be elucidated by EPR.

The signal g∼=4 assigned for high spin energy Fe3+ is well preserved within JSC 1A, P-MRS, and MRS07/52 abiotic and biotic samples, with only small variations in amplitude. This situation indicates that the Fe3+ remained associated to orthorhombic positions in the structure of mineral precipices upon M. sedula growth. Such an observation is also in line with EPR-characterization of iron-bearing multi-mineral materials under the oxidative conditions of firing temperatures, suggesting that a biologically mediated oxidative effect does not differ from abiotic physical–chemical oxidation in this spectral area. However, changes were detected at signals g = 9 (**Figures 4A,B,D** and Supplementary Table 1) originating from high-spin rhombic Fe3+ centers, supporting the microbial mediated alteration of Fe3+ complexes in axial symmetry.

The different composition of mineral phases of MRSs has to be taken into account when assigning a certain resonance signal and the formation of different environments in response to abiotic and biologically mediated oxidation. Nevertheless, the EPR data should be supported by other observations such as synchrotron assisted X-ray absorption techniques, to obtain a deeper insight into the microbial mediated mineralogical alterations.

The development of the microspheroids described here that overgrow mineral surface of Martian regolith simulants was one of the features mediated by M. sedula. Most of the microspheroids were observed in the range of 0,3 to 3 µm in size, with the majority in between 0,5 and 1 µm. Frequently, the microspheroids were characterized by very low carbon content, which did not exceed the background carbon level in surrounding mineral surface according to our EDS analysis and suggested the non-cellular nature of these morphologies (Supplementary Figure 4). Chlorine and aluminum content of the microspheroids has been constantly detected by EDS analysis, with chlorine represented solely in microspheroid structures and absent in the mineral background (Supplementary Figure 4 and **Table 4**). These microspheroid structures tend to cluster into aggregates, which overgrow the mineral surfaces while forming assemblages on underlying structures (**Figure 3E** and Supplementary Figure 4).

Occurring as single particles (**Figures 3A,B**), these microstructures expose correctly shaped hemispherical surface. When grouped into aggregates, overgrowing the surface of S-MRS and competing with each other in restricted size area,

microspheroids expose overlapping conjoined boundary sides, which results in a distorted hemispherical morphology and "overcrowded" appearance (**Figure 3C**). The neoformed opaline microhemispheroids of similar morphology were previously described as a part of possibly microbial mediated diagenesis of marine sediments (Monty et al., 1991). Massively deposited chlorine/aluminum microspheroids can be inferred to be selfassembled aggregated clusters that form new nuclei and mineral intermediates as a part of new mineral formation process biologically mediated by M. sedula. This fine-scale morphological signature along with leaching of elements as the signs of metabolic activity may serve as indication of chemolithotrophic life in extreme environments.

# CONCLUSION

The mineralogical composition of the synthetic Martian regolith analogs supports the chemolithotrophic growth of M. sedula, when Martian regolith simulants are used as the sole energy sources. Acquisition of Fe2+ and/or Mn2+ from these simulants seems to satisfy the bioenergetic needs of M. sedula. The obtained results highlight metallophilic life in extreme environments and reveal unique fingerprints of life in the extreme conditions.

# AUTHOR CONTRIBUTIONS

DK, MS, MP, OS, AB, and TM performed experiments. DK and MP planned, performed, and interpreted EPR experiments; all authors provided editorial input. All authors made substantial contributions to the acquisition, analysis, and interpretation of

### REFERENCES

data described in this perspective. All authors critically reviewed the report and approved the final version.

# FUNDING

This work was supported by the Austrian Science Fund (FWF) through an Elise-Richter Research fellowship V333 "Ironand Sulfur-oxidizing Machinery of the bioleaching Archaeon Metallosphaera sedula". OS was supported by COST Action STSM fellowship: "Understanding the metal oxidation machinery in archeon M. sedula" Nr. TD1308.

# ACKNOWLEDGMENTS

We warmly thank R. Moeller (German Aerospace Center (DLR), Cologne) for providing the synthetic Martian regolith analogs used in this study. We would like to acknowledge T. Hofmann and W. Obermaier (University of Vienna, Department of Environmental Geosciences) for help with ICP-OES and Dr. Pelin Yilmaz for ARB 16S rRNA probe design. The support of Stephan Puchegger (University of Vienna, Physics Faculty Center for Nano Structure Research) with electron microscopy investigations is greatly appreciated. TM would also like to thank Frances Westall for fruitful discussions.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2017.01918/full#supplementary-material

Mössbauer, and magnetic properties of nanophase and other iron oxides in Hawaiian palagonitic soil PN-9. Geochim. Cosmochim. Acta 57, 4597–4609. doi: 10.1016/0016-7037(93)90185-Y

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Kölbl, Pignitter, Somoza, Schimak, Strbak, Blazevic and Milojevic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Determination of Geochemical Bio-Signatures in Mars-Like Basaltic Environments

Karen Olsson-Francis 1 \*, Victoria K. Pearson2 , Elisabeth D. Steer 2, 3 and Susanne P. Schwenzer 1

1 School of Environment, Earth and Ecosystem Sciences, Open University, Milton Keynes, United Kingdom, 2 School of Physical Sciences, Open University, Milton Keynes, United Kingdom, 3 Nanoscale and Microscale Research Centre, University of Nottingham, Nottingham, United Kingdom

#### Edited by:

Philippe M. Oger, UMR CNRS 5240 Institut National des Sciences Appliquées, France

#### Reviewed by:

Aude Picard, Harvard University, United States Jennifer Ronholm, McGill University, Canada

\*Correspondence: Karen Olsson-Francis<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 11 May 2017 Accepted: 17 August 2017 Published: 08 September 2017

#### Citation:

Olsson-Francis K, Pearson VK, Steer ED and Schwenzer SP (2017) Determination of Geochemical Bio-Signatures in Mars-Like Basaltic Environments. Front. Microbiol. 8:1668. doi: 10.3389/fmicb.2017.01668 Bio-signatures play a central role in determining whether life existed on early Mars. Using a terrestrial basalt as a compositional analog for the martian surface, we applied a combination of experimental microbiology and thermochemical modeling techniques to identify potential geochemical bio-signatures for life on early Mars. Laboratory experiments were used to determine the short-term effects of biota on the dissolution of terrestrial basalt, and the formation of secondary alteration minerals. The chemoorganoheterotrophic bacterium, Burkholderia sp. strain B\_33, was grown in a minimal growth medium with and without terrestrial basalt as the sole nutrient source. No growth was detected in the absence of the basalt. In the presence of basalt, during exponential growth, the pH decreased rapidly from pH 7.0 to 3.6 and then gradually increased to a steady-state of equilibrium of between 6.8 and 7.1. Microbial growth coincided with an increase in key elements in the growth medium (Si, K, Ca, Mg, and Fe). Experimental results were compared with theoretical thermochemical modeling to predict growth of secondary alteration minerals, which can be used as bio-signatures, over a geological timescale. We thermochemically modeled the dissolution of the basalt (in the absence of biota) in very dilute brine at 25◦C, 1 bar; the pH was buffered by the mineral dissolution and precipitation reactions. Preliminary results suggested that at the water to rock ratio of 1 × 107 , zeolite, hematite, chlorite, kaolinite, and apatite formed abiotically. The biotic weathering processes were modeled by varying the pH conditions within the model to adjust for biologic influence. The results suggested that, for a basaltic system, the microbially-mediated dissolution of basalt would result in "simpler" secondary alteration, consisting of Fe-hydroxide and kaolinite, under conditions where the abiotic system would also form chlorite. The results from this study demonstrate that, by using laboratory-based experiments and thermochemical modeling, it is possible to identify secondary alteration minerals that could potentially be used to distinguish between abiotic and biotic weathering processes on early Mars. This work will contribute to the interpretation of data from past, present, and future life detection missions to Mars.

Keywords: bio-signatures, Mars, life detection, thermochemical modeling, microbial weathering, basalt

# INTRODUCTION

The surface of present-day Mars is cold, dry, highly oxidized, and exposed to ultraviolet (UV) and ionizing radiation. These conditions are considered inhospitable; yet early Mars may have had surface conditions more conducive to life, with a possible warmer climate and a denser atmosphere that could provide protection from UV and cosmic radiation (Molina-Cuberos et al., 2001; Bibring et al., 2006; Fairen et al., 2009; Tian et al., 2009). Geological, geochemical, and geomorphological observations made by orbiting spacecraft have provided definitive evidence of more clement and less oxidizing conditions in early martian history (e.g., Carr and Head, 2010; Mangold et al., 2012). Globally and locally, large ancient fluvial systems (including channels and fans) are observed, which may have been habitable (e.g., Malin and Edgett, 2003; Irwin et al., 2005; Mangold et al., 2012; Williams et al., 2013; Fassett and Head, 2015). On Mars' surface, Curiosity has detected complex lake bed stratigraphy, which are reported to contain nitrogen and carbon compounds, including complex organic molecules (Andeer et al., 2013; Williams et al., 2013; Vaniman et al., 2014; Grotzinger et al., 2015; Stern et al., 2015). As a second, independent, strand of evidence for a more hospitable past, the NASA Mars Exploration Rover Opportunity also found evidence for an impact-generated hydrothermal system at Endeavour crater (Arvidson et al., 2014; Fox et al., 2016); impactgenerated hydrothermal systems could have provided habitable conditions even during periods of very cold climate (Abramov and Kring, 2005; Schwenzer and Kring, 2009). Mineralogical and geochemical observations of clay minerals, and the detection of carbonates and sulfates, reveal a complex set of environmental conditions ranging from weathering to evaporation, and from cold surface to elevated subsurface temperature conditions, occurring in complex succession (Filiberto and Schwenzer, 2013; Arvidson et al., 2014; Grotzinger et al., 2014, 2015).

On Mars, the surface rocks are dominated by basaltic compositions, but range from ultramafic to basaltic to potentially even more evolved compositions (Nyquist et al., 2001; Christensen et al., 2005; Filiberto, 2008; Treiman and Filiberto, 2015; Morris et al., 2016; Sautter et al., 2016; Mangold et al., 2017). Alteration is known to range from open-system (with and without acidic influence) to closed system conditions, and as a consequence, the resultant rock chemistry may or may not reflect the basaltic source rocks; the products of weathering can be influenced by location, environmental conditions, geological time, and permeability of the rocks at the time of weathering (see Schwenzer and Kring, 2009; Ehlmann et al., 2013; Bridges et al., 2015; Carter et al., 2015; Zolotov and Mironenko, 2016 for example of the wide variety of alteration conditions found on Mars to date). Extensive laboratory based work has demonstrated that microorganisms can use olivinepyroxene-plagioclase-bearing rocks as a source of bio-essential elements and can enhance the weathering rates (e.g., Berthelin and Belgy, 1979; Vandevivere et al., 1994; Barker et al., 1998; Rogers et al., 1998; Kalinowski et al., 2000; Liermann et al., 2000; Bennett et al., 2001; Welch et al., 2002; Uroz et al., 2009).

The mechanisms employed by microorganisms in basaltic environments are variable and range from the production of excess protons, to the production of low molecular weight organic acids and siderophores (highly specific Fe(III) ligands), and, in some cases, and the production of extracellular polysaccharides and enzymes (Welch and Ullman, 1993; Vandevivere et al., 1994; Barker et al., 1998; Bennett et al., 2001; Wu et al., 2007; Olsson-Francis et al., 2015). These mechanisms may occur in the bulk phase or in local microenvironments, where concentrations would be much higher (Bennett et al., 2001). For example, in terrestrial systems, solutions of organic acids in concentrations comparable to, or slightly higher than, ground water show an increase in dissolution rates of less than one magnitude. In contrast, in local microenvironments the concentration of acid may be much higher (Banfield et al., 1999). If life had once existed on early Mars, it may have left a record of these processes within the martian rock record (Banfield et al., 2001) and recognizing microbially-induced minerals, which could have potentially formed in an early Mars environment, would identify important bio-signatures for use in future life detection efforts.

To ensure unequivocal identification of bio-signatures, comparison with abiotic processes is necessary. Terrestrial abiotic basalt weathering has been extensively studied and characterized using field observations, laboratory-based experiments and thermochemical modeling (e.g., Gislason and Eugster, 1987a,b; Oelkers and Schott, 2001; Wolff-Boenisch et al., 2006). In contrast, to date, stand-alone laboratory experiments have been the only way to investigate the influence that biota has on basalt dissolution and the growth and evolution of secondary alteration minerals (e.g., Wu et al., 2007; Olsson-Francis et al., 2012, 2015). From these experiments alone it is difficult to predict what would happen over years-long or even geological time scales, for example, on early Mars, when the rock is fully dissolved or, more likely, subject to the effects of incongruent dissolution. In these circumstances, the formation of amorphous and leached layers, and secondary mineral precipitation, may occur, influencing the availability of cations for use in biological metabolism.

Themochemical modeling is a powerful tool that aids the prediction of mineral assemblage formation by assessing reaction pathways and studying the formation of secondary minerals in a gas-fluid-rock system (e.g., Reed, 1982; Kühn, 2004) and this has been applied to studies of secondary mineral formation and fluid compositions on Mars (Bridges and Schwenzer, 2012; Filiberto and Schwenzer, 2013; Schwenzer and Kring, 2013; Bridges et al., 2015). In biotic experiments, geochemical models such as Geochemist's workbench are routinely used to characterize mineral precipitation during laboratory-based experiments (e.g., Orcutt et al., 2011; Olsson-Francis et al., 2012), and to characterize redox reactions in water-rock environments (Posth et al., 2008). However, there have been limited, if any, studies that have combined laboratory-based experiments and thermochemical modeling to compare biotically and abiotically generated secondary alteration mineralization over geological time scales.

In this paper, we present preliminary work carried out to investigate the feasibility of coupling microbial dissolution experiments with thermochemical modeling in order to identify mineralogical bio-signatures that could be used as evidence of life on early Mars. The experiments invoke chemoorganoheterotrophic metabolism as a plausible metabolism on early Mars because of the availability of suitable energy sources (electron donors) (Cockell, 2014; Westall et al., 2015). For example, organic carbon at Gale Crater has been predicted to be between 800 and 2,400 ppm. Even if 1% or less of this carbon was bio-available on early Mars this could sustain a chemoorganoheterotrophic community of 105 cells/g of sediment (Sutter et al., 2016). Further, if life had evolved on early Mars, either by photosynthesis or chemolithotrophy, their organic remnants could be used as an electron donor for this metabolism. Finally, chemoorganoheterotrophic metabolism is known to enhance silicate dissolution, predominantly by the production of excess protons and organic acids (Wu et al., 2007; Olsson-Francis et al., 2015).

For microorganisms that only utilize minerals as essential micronutrients and macronutrients (i.e., they are not respired), acidification is the most common, and straightforward, mechanism for silicate dissolution and is generally universal (for review see Uroz et al., 2009). Our understanding of basaltic weathering is predominately based on aerobic metabolism (e.g., Welch and Ullman, 1993; Vandevivere et al., 1994; Blake and Walter, 1996; Drever and Stillings, 1997). On Mars, aerobic metabolism may have been feasible, Curiosity data indicates that there is sufficient molecular oxygen (∼1,450 ppm; Mahaffy et al., 2013) in the martian atmosphere to support aerobic activity (King, 2015). In addition, inorganic alteration reactions that are expected to happen due to water-rock interactions have been shown to shift the redox environment to more oxidizing conditions (e.g., Bridges and Schwenzer, 2012). Therefore, traditional experiments in anoxic conditions (e.g., Schirmack et al., 2014, 2015) are complemented by the experiments in this study, conducted in a more oxidizing milieu.

Step by step titrations were modeled to investigate the effect that microbially-mediated acidification has on the aqueous environment and the precipitation of secondary alteration minerals during microbial-mediated basalt weathering. In parallel, abiotic alteration at different pH are investigated to understand the effects of biota by contrasting the two systems. Secondary alteration minerals uniquely produced by microbialmediated weathering could be used as a geochemical biosignature for life. The findings of this study are important for further developing geochemical models that could be used to predict bio-signatures for life on early Mars.

#### MATERIALS AND METHODS

#### Water to Rock Ratios

For this study, it is critically important to clearly define three different water to rock ratios: we use subscripts E (experiment), D (dissolved), and M (model) to denote the exact meaning of W/R in each context. (W/R)E is the experimental water to rock ratio, simply referring to the ratio of the amount of rock and the amount of water weighed out and mixed for the experiment. This is distinct from (W/R)D, which is the amount of rock actually dissolved during the experiment. These two water to rock ratios are different, because, even though dissolution of rock in water is slow, a very small amount of rock will dissolve during the experiment. We assess (W/R)D by looking at the most soluble elements in the fluids resulting from the experiment. (W/R)M is the water to rock ratio used in the model, and assumes complete dissolution of all rock. We compared the (W/R)D and (W/R)M values at the concentration where the most soluble elements are similar.

## Characterization and Preparation of Mars Analog Rock

Basalt from Skye was purchased from Richard Tayler minerals (United Kingdom) as an analog for martian basalts. A polished thin section of the rock was prepared for microprobe analysis. The remaining rock was broken with a hammer and pieces devoid of visible weathering were ground using a Tema swing mill, for 8 min. The crushed rock was sieved to select for a fraction size between 250 and 500 µm. Fine particles were removed by ultrasonication in MiliQ water (Olsson-Francis et al., 2010, 2012). The rock was then dried for 24 h, at 80◦C. The specific surface area of the ground rock samples was measured using multi-point BET (Brunauer, Emmett and Teller analysis at Imperial College London) with N2 and yielded a surface area of 0.976 m2 g −1 .

Petrological analysis was carried out on thin sections of the sample using reflective and transmitted microscopy and a FEI Quanta 3D dual beam scanning electron microscope (SEM) fitted with an Oxford Instruments 80 mm X-Max energy dispersive X-ray spectrometer (EDS), which was operated with an accelerating voltage of 20 kV and a 10–15 mm working distance. The major elemental composition of the basalt was obtained using an ARL 8,420+ dual goniometer wavelength dispersive X-ray Fluorescence (XRF) spectrometer (Applied Research Laboratories, Ecublens, Switzerland). Mineral analysis was obtained from a Cameca SX100 electron microprobe (EMPA) at The Open University using a spot size of 10 µm, accelerated voltage of 20 kV and a beam current of 20 nA.

#### Basalt Mineralogy

The sample was an altered amygdaloidal basalt from the Skye Tertiary Province. The amygdales were filled with zeolites, which were removed from the sample before experimentation commenced. The sample was fine grained with a groundmass of augite and plagioclase with an accessory phase of Ti-spinel. Much of the original mineralogy had been altered at low temperature to form clays and the general mineralogy was characterized as: 20– 25% augite, 30% plagioclase, 50% clays. The amount of Ti-spinel was <1%. The plagioclase was present as laths up to 1 mm in size; the augite crystals were largely anhedral and below 0.5 mm in size, as shown in **Figure 1**. Anhedral grains of Ti-spinel below 100 µm occured throughout the sample. The size and shape of Tispinel crystals was prohibitive of EMPA analysis. Representative analyses of the minerals present are detailed in **Table 1**.

The clays present in the sample were vermiculite and saponite. These are similar in composition to clays found by the Curiosity rover on Mars by XRD (Vaniman et al., 2014) and to those modeled by Bridges et al. (2015) to be present in Gale Crater. As a comparison, analyses from this study have been plotted with clay

experiment. Augite, plagioclase, clay, and Ti-spinel are observed. Image width is ∼1.5 mm.

TABLE 1 | Representative analyses of minerals in the basalt (wt %).

compositions modeled to exist on Mars by Bridges et al. (2015) and with clays measured in the martian Lafayette meteorite by Hicks et al. (2014). **Figure 2** shows that the vermiculites and saponite identified in this study form a tight cluster that overlaps with the martian clays as well as the clays modeled here. However, the range of terrestrial clays extends beyond the narrow range of isochemical alteration, showing more Si and Al rich endmembers and clays depleted in those two elements. The latter could be due to an oxidizing reaction causing microcrystalline Fe-oxides and hydroxides intermixed with the clay. The mixture of clay and pristine mineral remnants makes this basalt a proxy for the recently discovered "mudstones" (Vaniman et al., 2014), of basaltic origin, on Mars.

## Model Bacterium

The microbiological experiments were conducted using the bacterium Burkholderia sp. strain B\_33, which has previously been isolated from nutrient limiting soil, in Snowdonia National Park, United Kingdom (Olsson-Francis et al., 2015). A Burkholderia sp. was selected because long-term experiments in nutrient poor soils have shown a correlation between the abundance of this genus and specific minerals, such as plagioclase and a mix of phlogopite and quartz (Uroz et al., 2009; Lepleux et al., 2012). It also exhibits chemoorganoheterotrophy, a plausible metabolism for life on past Mars.

Routine growth was carried out in a modified minimal medium, which contained the following (mg L−1 ): 10 of FeCl3, 150 of MgSO4•6H2O, 20 of CaCl2; 20 of KCl, 65 of NH4Cl, 100 of NaNO3, 70 of K2HPO4, 60 of KH2PO4, 20 of glucose. All of the reagents, except the MgSO4•6H2O and the FeCl3 were added and the pH was adjusted to pH 7.0. After the medium was autoclaved (20 min at 121◦C), filtered sterilized MgSO4•6H2O and FeCl3 were added (this did not alter the pH).

# Basalt Dissolution Experiment

The dissolution experiments were carried out in batch culture. The growth medium contained the following: 2 g L−1 of glucose, 0.06 g L−1 of NH4Cl, and 200 g L−1 of basalt. Twenty grams of the crushed basalt (0.5–1 mm) was placed in an acid-washed 125 ml glass Erlenmeyer culture flask and autoclaved at 121◦C for 15 min. Two g L−1 of glucose was added ensure that growth was not limited by the amount of carbon. One hundred milliliters of autoclaved liquid medium was added to the flask and the pH was adjusted to pH 7.0 with filtered sterilized 10 mM NaOH. The experiments were conducted with a water to rock (W/R)E ratio of 100/20 (BET surface area of 0.976 m2 g −1 ).

Prior to inoculating the flasks, cells were washed to remove any excess growth medium. Ten milliliters of exponentially grown cells were harvested by centrifugation at 4,000 × g, for 5 min. The cell pellet was washed three times with sterilized 50 mM Tris buffer (pH 7.0) and re-suspended to a final cell density of 107–108 cell mL−1 . A 0.5% inoculum was used to inoculate the flasks. The flasks were incubated, without shaking, at 25◦C for 28 days. Each of the experiments were carried out in triplicate. The biotic experiments were designated B1, B2, and B3, and the abiotic controls were designated C1, C2, and C3. Abiotic controls were prepared in an identical manner to the biotic flasks.

At day 1, 4, 7, 11, 14, 21, and 28, 5.5 mL aliquots were aseptically removed from the flask and transferred to a 10 mL acid-washed bottle. Each aliquot was immediately processed as follows: 4.5 mL was passed through a 0.2 µm nylon syringe filter and acidified with concentrated HNO3 (final 5% acid) for elemental concentration measurements and 1 mL was left unfiltered and used to measure microbial growth and pH.

# Measuring Bacterial Growth

Cells were stained with the nucleic acid-binding dye SYBR Green I DNA (0.1% w/v stock; Life Technologies, Paisley, UK) and 1 mL of culture was filtered through a 0.2µm black polycarbonate filter and then washed with 100µL of sterile dd H2O. The cells on the filter were enumerated using a Leica DMRP microscope equipped with epifluorescence, as previously described (Summers et al., 2013). The growth rate constant (k) for the log phase of growth was determined (Pirt, 1978).

### Siderophore Production

To determine the ability of the isolates to produce siderophores, the Chrome Azurol S liquid assay (CAS) was used (Schwyn and Neilands, 1987). As a control we used Cupriavidus metallidurans CH34, which has previously been shown to produce siderophores

under iron limiting conditions (Olsson-Francis et al., 2010). The detection of siderophores was quantified and defined, as previously described (Payne, 1994). The isolates were grown in the minimal medium, without iron (i.e., they were iron-limited), and siderophore production was measured in stationary stage cells.

# Chemical Analyses

The pH was measured using an Orion 3-Star Thermo Scientific bench top meter with an uncertainty of 0.01 pH unit. The total elemental concentration of dissolved elements in the growth medium was measured using Inductively Coupled Plasma– Mass Spectrometry (ICP-MS; Agilent 7500s ICP-MS with New Wave 213 laser system). Detection limits for the ICP-MS were as follows: Ca (39.89 nmol L−1 ), Mg (2.81 nmol L−1 ), P (236.64 nmol L−1 ), Fe (41.83 nmol L−1 ), Na (7.59 nmol L−1 ), Si (236.63 nmol L−1 ), and Al (2.03 nmol L−1 ). Each of the measurements were conducted in triplicate and the mean was reported; the standard deviation was <5%. The data was corrected for the decrease in fluid volume and the loss of elemental mass during sampling, as previously described (Wu et al., 2007). Glucose concentration was measured using the Amplex red glucose kit (Invitrogen). The absorbance was measured at 595 nm and compared with a calibration curve of known glucose concentrations. Oxalate was measured using an oxalate oxidase assay (Trinity Biotech), at 590 nm, as per manufacturer's instructions.

# FEG-SEM Analysis

After 28 d, rocks were removed from the flasks for Field Emission Gun (FEG)-SEM analysis. The rocks were air dried and carbon coated (15–20 nm thickness) on aluminum stubs. The surface of the rocks was examined using a using a FEG-SEM with an EDS detector (ZEISS Supra; 55-VP; Zeiss Microimaging, Göttingen, Germany), which was operated with an accelerating voltage of 2–15 kV and a 7–10 mm working distance.

### Elemental Release Rates

The kinetics of elemental release were calculated using the linear release rate (R l i ), as previously described (Wu et al., 2007). Any significant difference between the biotic and abiotic dissolution kinetics were identified using a Student's t-test.

## Elemental Uptake

The intracellular elemental concentration was measured at day 28. Twenty milliliters of liquid culture was aseptically taken from the biotic experiments and centrifuged at 13,000 × g for 20 min. The resulting cell pellet was washed three times in sterilized 0.5% HNO3. The cells were washed to ensure that no elements from the experimental solution were analyzed; however, we cannot rule out that elements that were loosely bound to the cells were not lost during this step. The pellet was dried at 80◦C overnight, and digested in 1 mL concentrated HNO3 (final 5% acid) and the resulting solution was analyzed by ICP-MS, as previously described (Olsson-Francis et al., 2012).

# Thermochemical Modeling

In order to assess the inorganic reaction pathways possible in the rock-fluid system, we used thermochemical modeling, specifically the code CHIM-XPT (previously CHILLER Reed and Spycher, 2006; Reed et al., 2010). This code has been used extensively in terrestrial basaltic environments (e.g., Reed, 1982) and applied to basaltic rocks of martian compositions (e.g., Debraal et al., 1993; Schwenzer and Kring, 2009; Filiberto and Schwenzer, 2013; Bridges et al., 2015; Schwenzer et al., 2016). We carried out stepwise titration simulations from the initial fluid to a (W/R)M of 1 to assess dissolution of the rock in the fluid without biotic activity, starting at a very high (W/R)M of over 108 , modeling titration to a low (W/R)M of 1. We report data from (W/R)M of 1 Mio ensuring that constant conditions independent of any starting mineral choices had been reached at this point. Input data included the rock composition as derived by XRF, and the fluid composition of the minimal growth medium (**Table 2**). Note that the fluid composition is summarized in **Table 2** as one species per element, but elements will be partitioned between several species as relevant to the pH and Eh conditions in the fluid during the modeling. For example, Fe is partitioned between Fe2+ and Fe3+ species according to redox conditions in the respective modeling step, and compound species, e.g., FeOH or FeCl-bearing ions, are also considered important in this respect. Overall, a set of ∼80 different ionic species are typically used to represent the fluid chemistry in each calculation iteration within the modeling steps. We model at 1 bar and 25◦C, which closely mimics the pressure and temperature conditions of the growth experiment and investigate three model experiments: In the first experiment, pH varies, and is treated as a free parameter. In two subsequent experiments, pH is set to 7 and 4, respectively, to simulate the conditions during the stationary and exponential phases in the growth experiment. The Cl-anion is used for charge balance, which is especially important for the pH 4 model, because additional Cl− is required to set the initial pH.

In the model, we assumed complete dissolution of the rock into solution, which is a simplification many codes make (for an explanation and overview of the diversity of

Species in the fluid are summarized in the table as one species, but during the modeling were partitioned into several dissolved species (for details see text).

thermochemical rate modeling see Kühn, 2004; Ganguly, 2008; Rimstidt, 2014). To account for a more complex natural system, e.g., varying dissolution rates of the minerals in the rock and/or incongruent dissolution of minerals, the input parameters were varied systematically and with close reference to available thermodynamic data, and experimental and natural observations and data. This results in a parameter space that can be reduced to the most likely approximation of the natural system.

# RESULTS

#### Bacterial Growth

The bacterium, Burkholderia sp. strain B\_33 was able to grow in the minimal medium with basalt as the sole source of bio-essential elements. No growth was detected in the growth medium without basalt. The bacterium utilized the glucose within the growth medium as a source of carbon and within 7 days the concentration of glucose decreased to ∼20%, as shown in **Figure 3A.** As the bacterium utilized the glucose, the metabolite oxalate was produced. The concentration of oxalate in the growth medium increased steadily until day 7 and then plateaued (**Figure 3B**). There was no significant difference between the triplicate flasks.

The initial cell counts, immediately after inoculation, were between 9.23 × 104 (B3) and 2.92 × 105 (B2) cell mL−1 . Within 7 days, the number of cells had increased to a maximum of between 1.53 × 108 (B2) and 2.82 × 108 (B3) cell mL−1 (exponential growth) and then remained relatively steady until the end of the experiment (stationary phase), as shown in **Figure 4A**. Coinciding with exponential growth, the pH decreased rapidly and at day 4 the pH was between 3.5 (B2) and 3.6 (B3) (**Figure 4B**). As the cells reached late exponential stage the pH values gradually increased to a steady-state equilibrium of between pH 6.8 and 7.1. In contrast, the pH in the abiotic controls remained near neutral pH (between pH 6.6 and 7.1) throughout the experiment (**Figure 4B**). No cells were observed in the abiotic controls.

## Siderophore Production

Burkholderia sp. strain B\_33 was screened for siderophore production using the Chrome Azurol S assay. In the minimal

medium, without iron, siderophores were produced. The amount varied between 1.20 and 2.10 µmol L−1 EDTA equivalent. However, when basalt was added to the growth medium, siderophores were not detected (data not shown).

#### Basalt Dissolution

Dissolution was measured by the concentration of key elements (Si, K, Ca, P, Mg, and Fe) within the growth medium. All of the values were corrected for the decrease in fluid volume and the loss of elemental mass during sampling. Without the basalt, the concentrations of Si, K, Ca, Mg, Na, Fe, and P in the growth medium were 0.034, 0.024, 0.021, 0.038, 0.018, and 0.01 µmol L −1 , respectively. **Figure 5** shows the elemental concentrations in the dissolution experiments as a function of time. The presence of Burkholderia sp. strain B\_33 enhanced the release of Si, Ca, Mg, Al, K, Na, P, and Fe into the growth medium. In general, the concentration increased linearly until day 4–11 (depending on the element) and then they either reached or began to approach steady-state conditions.

The initial linear release rates were calculated to compare the dissolution kinetics in the abiotic and biotic flasks, as demonstrated in **Table 3**. The presence of Burkholderia sp. strain B\_33 had a significant (p < 0.05) effect on the release rates of Si, Ca, Mg, Al, and Fe (there was no significant effect on the release of P, Na, and K). For example, for B1, the R l i value for Ca was approximately eight-fold higher than the abiotic controls. Plotting the logR l i values for Mg, Ca, Si, and K against the average pH of the initial growth phase (day 1–7, which corresponds to the period used to determine the R l i values) suggests that the release rates were dependent on pH (**Figure 6**).

### Cellular Element Uptake

The intracellular elemental concentration was measured in bacteria collected from day 28. The values in **Table 4** are reported as the elemental concentration per cell. This data was used to determine if elemental uptake was significant with respect to the solute concentrations at day 28. The values reported in **Table 3** were multiplied by the number of cells measured at day 28 and the values were corrected for the decrease in fluid volume during sampling, as previously described (Wu et al., 2007; Olsson-Francis et al., 2012). The intracellular elemental concentrations were <1% of the final fluid concentrations.

#### Secondary Alteration Minerals

The surface of the basaltic rock was examined for secondary alteration products with FEG-SEM and EDS analysis. Where possible, EDS analysis was performed on areas of the rock where secondary alteration was visible on the surface (**Figure 7**) i.e., where there was a positive relief and evident morphological differences to the crystalline basalt and the clay minerals already contained within the basalt. However, there was limited evidence of alteration on any mineral surfaces; mineral grains

from the biotic and abiotic flasks showed predominantly pristine surfaces with the exception of dissolution pits and an amorphous silicate layer on the surface of the rocks in the biotic flasks. **Figure 7** also shows small pockets of alteration products in the biotic flasks, which are remarkably diverse (**Table 5**).

The mineral assemblage labeled "spectrum 20" is dominated by Si, with minor Al, Na, Fe, S, and Cl. Dividing this analysis

( ), C2 ( ), C3 ( )]. The values reported are the means of three independent experiments, and the standard error associated with these determinations is shown.

into element-oxides results in a "high total," >100%. Even if it is assumed that Cl is more likely to be bound in CaCl, and all Fe is FeS, the total remains above 100%. Inspection of **Figure 7** and knowledge from our models (see below) allows us to assume that kaolinite [Al2Si2O5(OH)4] might be present, and if this accounts for all of the Al, the "high total" is reduced further (**Table 5**). Therefore, it is likely that the alteration assemblage on "spectrum 20" contains silica and kaolinite with minor FeS and NaCl. Alternatively, instead of NaCl, some minor amount of carbonate could take up the excess cations, but is considered unlikely in this partitioning exercise because of the Si-dominance of the precipitate and the amounts of Cl in solution.

The adjacent alteration mineral assemblage has a different composition, since it is even more dominated by Si and Fe, has around 4% Cl, P, and Al, and minor Na and S. The total, after partitioning into oxides, is only slightly above 100%, but is within 2% error of 100% if all Na is assumed to be NaCl and all S is FeS. According to our models, the remaining Fe could be in nontronite [(CaO0.5,Na)0.3Fe3+ 2 (Si,Al)4O10(OH)2·nH2O]. This analysis also has P (**Table 5**), which could explain the high Cl; there is significantly Cl more than would be taken by NaCl suggesting the presence of another phase that could take both of these elements. We considered Cl-apatite, which also occurs in our models of this system. Unfortunately, Ca was not evident in our analyses. But with the very small grain size used in the experiments, precision of such results is limited. We therefore note the similarity of some of those results with the models, but do not wish to over-interpret them (which is why we have not balanced the amount of kaolinite to result in a 100% analysis total for point 20 in **Table 5**).

Any differences between the mineralogy before and after the experiment were below the detection limits for such changes with XRD, which is readily explained by the very small amounts of dissolution and volume of alteration phases observed.

#### Thermochemical Models—Three Cases

A set of thermochemical models was used to simulate rock dissolution without bacteria (with pH as free parameter), and to better understand the reactions at the pH conditions invoked by the presence of bacteria during the growth phase (pH 4 and 7; see Section Materials and Methods). We have shown that our model runs within a range of 1,000 > (W/R)M > 2 Mio, but focus our results and discussion on the range between 100,000 and 1 Mio. This water to rock ratio range is considered to be representative of all (W/R)D observed in the biotic experiments. Note, that the overall amounts of precipitated minerals in all modeled scenarios are very small and are typically in the order of 0.1–0.3 mg L−1 solution at (W/R)M of 1 Mio and reach ∼1 g at (W/R)M of 1,000. Variation in the amount of precipitated phases results from the varying solubility of species under different conditions, but also from structurally bound water in the precipitating phases. Further, we use "quartz" for the precipitation of any of the SiO2 polymorphs: quartz, amorphous silica, and chalcedony.

#### Abiotic Modeling—pH as Free Parameter

If pH is not held constant in the modeling run, it is calculated together with the other parameters from the dissolution and precipitation processes in the system. The fluid invoked has a neutral pH, which is maintained in the very initial dissolution steps but over the course of the titration changes to alkaline (**Figure 8**). At (W/R)M of 1 Mio, a mixture of diaspore [α-AlO(OH)] and goethite [FeO(OH)] precipitate. With increasing rock dissolution, Al is no longer precipitated as hydroxide but instead consumed by sheet silicate formation. Chlorite [(Mg,Fe2+,Mn)5Al(AlSi3O10)(OH)8] and kaolinite form alongside goethite, while diaspore is no longer part of the precipitate. Phosphorus is precipitated as chlorapatite [Ca5(PO4)3Cl] as soon as the concentrations of Ca and P are sufficiently high in the fluid for to reach the saturation of apatite. With further dissolution, zeolite [mainly stilbite, NaCa4(Si27Al9)O72·28(H2O)] formation sets in, resulting in a zeolite–hematite–chlorite–kaolinite–apatite assemblage at (W/R)M of 100,000. With further dissolution, the smectite-group silicate non-tronite is added to the assemblage. As shown above, some of those minerals have been observed in the biotic alteration assemblages, which supports the model results. To observe the full alteration assemblage or fully quantify it using mineralogical methods rather than chemistry, a longer experimental run with more progressive alteration mineral formation would be needed, because quantities are very low. In theory, ∼0.05 g of kaolinite would be expected to form at a W/R of 1,000 per 1 g of dissolved basalt in the model, translating to very small amounts of material expected in the experiment. Given the total amount of basalt in the experiment (20 g) and the very limited dissolution observed, we expect alteration phases to be in the ng range, distributed over 20 g of basaltic grains.

#### Biotic Simulation—pH Buffered at pH 7

If pH is held constant at pH 7, the resulting precipitates look somewhat similar to the abiotic model with pH as a free parameter, except chlorite does not form at all, and quartz formation begins at around (W/R)M of 100,000 (**Figure 9**). In detail, at (W/R)M of >1 Mio, diaspore and goethite precipitate. With increasing rock dissolution Al is no longer precipitated as hydroxide but increasingly consumed by the formation of kaolinite, which forms alongside goethite. In contrast to the previous model, the smectite-group silicate nontronite is added to the assemblage next, together with chlorapatite, while the relative abundance of goethite decreases and eventually

TABLE 4 | Chemical composition of bacterial cells after 28 days.

B.D is below the detection limits.

becomes unstable. With further increasing dissolution, zeolite (mainly stilbite) formation begins. In summary, many of the same minerals form in both experiments, but the absence of chlorite and the formation of nontronite are important differences between the assemblages that form under the different conditions.

### Biotic Simulation—pH Buffered at pH 4

If pH is held constant at pH 4, which is the pH during exponential growth, the resulting precipitates have a much simpler composition than in the neutral to alkaline parameter space (**Figure 10**). In detail, at (W/R)M of >1 Mio, only goethite precipitates while Al stays in solution. Al-precipitation begins with the formation of kaolinite, which forms alongside goethite. In contrast to the previous model, apatite does not form at any (W/R)M, but the smectite-group silicate nontronite is added to the assemblage at (W/R)M below 100,000. Again, the relative abundance of goethite sharply decreases and goethite finally becomes unstable at which point quartz forms. Zeolites do not form at low pH.

# DISCUSSION

## Microbially-Mediated Basalt Dissolution

Traditionally, martian analog studies have predominantly focused on chemolithoautotrophic microorganisms from extreme environments (e.g., Fernández-Remolar et al., 2004). However, these lack relevance to the large ancient fluvial systems, which are known to exist on Mars (e.g., Malin and Edgett, 2003; Irwin et al., 2005; Mangold et al., 2012; Williams et al., 2013; Fassett and Head, 2015). Although, chemolithoautotrophic metabolism may have occurred within these fluvial systems, more recent data suggests that chemoorganoheterotrophy was also feasible (Mahaffy et al., 2013; Sutter et al., 2016). Based on the data from Curiosity and our extensive understanding of their role in mineral weathering a chemoorganoheterophic microorganisms (e.g., see review Uroz et al., 2009) was selected for this preliminary study.

Consistent with previous studies, microbial growth enhanced the rate of basalt dissolution in batch culture experiments (e.g., Berthelin and Belgy, 1979; Welch and Ullman, 1993; Wu et al., 2007; Olsson-Francis and Cockell, 2010). The results suggest that, specifically, acidification of the experimental fluid had a positive effect on basalt dissolution, also in agreement with prior studies (e.g., Gislason and Eugster, 1987a,b; Oelkers and Schott, 2001). Acidification is likely to have been caused by the presence of both excess protons and organic acids produced as a by-product of

FIGURE 7 | FEG-SEM micrograph of a typical secondary minerals produced in the biological flasks after 28 days. No secondary minerals were observed in the abiotic controls.

chemoorganoheterophic metabolism (Welch and Ullman, 1993; Vandevivere et al., 1994; Blake and Walter, 1996; Drever and Stillings, 1997). Although, it is difficult to distinguish between organic ligand and proton-mediated dissolution, organic acids have been shown to increase the rate of plagioclase dissolution up to 10 times that achieved in the presence of inorganic acids at the same pH (Welch and Ullman, 1993). At acidic pH, organic acids are more likely to be in the protonated form and thus the proton enhanced pathways of weathering are more favorable, consistent with abiotic inorganic aqueous systems (Buffle, 1990).

Burkholderia sp. strain B\_33, produced siderophores in the minimal medium, which contained limited iron [the concentration was below the detection limits of the analysis (1µmol L−1 )]. However, siderophores were not detected in the medium with the basalt. This is in agreement with previous studies, which were unable to measure siderophore production in the presence of silicate rocks, under laboratory conditions (Frey et al., 2010; Olsson-Francis et al., 2010, 2015). The Fe released from the Fe-bearing minerals (augite, clay) in the rock during dissolution may have inhibited siderophore production.

TABLE 5 | EDS analysis of two alteration mineral assemblages (refer to spectra 20 and 21 as shown in Figure 7).

aAll Cl is calculated as NaCl, all Fe as FeS, and the remainder of the S as SO2.

bAll Na is calculated as NaCl with the remaining Cl as element. This analysis has significant P, which could form apatite and host Cl.

c In addition to the partitioning in a, Al is calculated as kaolinite. N.D is not detected.

Data are given as measured weight % of element converted to oxides, or partitioned into more likely phases, for example, NaCl (for details see text). Zero refers to partitioning results, where an element is calculated as a different species and therefore zero by calculation.

In this study, the basalt contained both augite (8.83% FeO) and vermiculite (20.78% Fe), and ICP-MS data demonstrated that after 1 day the concentration of Fe in the medium was between 3.2 and 5.8µmol L−1 . Based on previous work, this is sufficient to inhibit siderophore expression (Olsson-Francis et al., 2010).

#### Thermochemical Modeling of Microbial Dissolution

The focus of the thermochemical modeling was the initial stage of the weathering experiments, where supersaturation and the likelihood of secondary phase precipitation was lower; this also reflects the fact that microbial and inorganic weathering in the natural environment often happens in an open system or at least a system open to fluid and the most soluble elements. To compare the experimental and model results, the amount of actual dissolved basalt (W/R)D in the experimental dissolution experiments needed to be deduced. This value was directly comparable to the model water to rock ratio, (W/R)M (**Figure 8B**). The (W/R)D represented by the vertical lines in **Figure 8B** was calculated based on the element K, because it is not incorporated into any of the expected mineral precipitates and the intracellular concentration was <2%. Mass balance calculations based on K demonstrate that very little rock dissolution happened over the course of the experiment in either the abiotic [(W/R)D value of ∼877,000] or biotic [(W/R)D value of 230,000] experiments. The elemental abundances of the major cations, in the abiotic experiment, were compared with the model at the (W/R)D value of 230,000 [the (W/R)D value determined under biotic conditions].

The abundance of K indicated that dissolution in both the biotic and abiotic experiments was higher than expected from the mass balance calculated from the model. In fact, comparing the measured K abundances with fluid composition of the model indicates that the actual (W/R)M in the biotic experiment would lie in the range of 2,000–2,500. Fe and Ca concentrations predicted by the model are in the range of the measured values, which indicates their precipitation in the minerals and only a small uptake by the bacteria. In contrast, Na, Al, and Si are higher in solution in the experiment than in the model, which indicates inhibition of precipitation. This could occur due to utilization of bioessential elements required for precipitation, such as oxygen or phosphate, or complexation of the ions by inorganic or organic species produced by the bacterium. The former would reduce the concentration of another component of the precipitating phase, preventing solubility limits to be reached. This shows that, despite the similarities in detected alteration mineral assemblages and the modeled mineral assemblage [compare **Figure 7** and **Table 5** (experiment) to **Figures 8**–**10** (model)], biological activity changes precipitation characteristics significantly.

The analog rock used here was a naturally occurring basalt, which included clay minerals. This is in accordance with the "mudstones" found by the Curiosity rover at Gale Crater (Vaniman et al., 2014). The observed alteration minerals detectable through their position on the surface (**Figure 7**) agree with the modeled minerals, especially for the low-pH case, where kaolinite forms.

#### Bio-Signatures and Life Detection

Field studies have suggested a variety of bio-signatures of past life can be found in basaltic material on Earth. These

basalt at 25◦C with pH as a free parameter. The diagram shows the formation of diaspore, goethite, kaolinite, Cl-apatite, chlorite, zeolite, and nontronite. (B) Secondary minerals predicted from the dissolution of analog basalt at 25◦C with pH as a free parameter [at a higher resolution than (A)]. The (W/R)D, represented by the vertical lines, was calculated based on the element potassium because it is not incorporated into any of the expected mineral precipitates and the intracellular concentration was <2%.

include morphological fossils in basaltic amygdales and veins (e.g., Schumann et al., 2004; Cavalazzi, 2007), evidence of isotope fractionation in minerals, such as carbonates and sulfides (Demény and Harangi, 1996; Rouxel et al., 2008), or bio-alteration textures, such as potential biogenic tubulars or pitting (Thoreseth et al., 1991; Fisk et al., 1998). Analyses of the Columbia River basalts revealed organic structures, which were interpreted to be bacteria, intermingled with secondary

FIGURE 9 | Secondary minerals predicted from the dissolution of analog basalt at 25◦C, pH fixed at 7. The mineral precipitates can be divided into three principal assemblages. At low W/R (below about 10,000) zeolites become increasingly important and below about 7,000 are the dominant species. Between about W/R = 10,000 and 900,000, clay minerals (kaolinite and nontronite) are dominating, whereby kaolinite is more prominent at the higher W/R end of that bracket and nontronite at the lower W/R range. At the highest W/R (over 1 Mio) the assemblage contains hydroxides (diaspore and goethite) only.

FIGURE 10 | Secondary minerals predicted from the dissolution of analog basalt at 25◦C, pH fixed at 4. The precipitated secondary minerals can be grouped in two assemblages: at the highest W/R goethite is the sole precipitate. At lower W/R clays dominate, whereby kaolinte predominantly precipitates at the hither W/R compared to nontronite. Note that quartz starts to precipitate at W/R below about 50,000, but never becomes the dominating species.

iron oxyhydroxides and ferrous smectites, which suggested that the secondary minerals were caused by microbial activity (McKinley and Stevens, 2000). This study has similarly focused on determining the secondary alteration minerals that could form due to microbially-mediated dissolution of basalt in a Mars-like environment.

A small number of laboratory studies have investigated microbially-mediated basaltic weathering. For example, McKinley and Stevens (2000) studied geochemical modeling in sea water. They showed saturation with respect to Fe(OH)2.7Cl0.3, goethite (α-FeOOH), lepidocrocite (δ-FeOOH) schwertmannite (Fe8O8(OH)5.9(SO4)1.05), and K-jarosite (KFe3(SO4)2(OH)6 (Daughney et al., 2004). This is broadly consistent with our findings at the highest water to rock ratio (i.e., the formation of kaolinite, and goethite/lepidocrocite), but we note that our system did not contain enough K to form the jarosite modeled by McKinley and Stevens (2000).

Our findings are important for planning and interpreting investigations carried out by rover-based instrumentation currently active on Mars (e.g., CheMin and MastCam and ChemCam passive spectral investigations on Curiosity) and for the selection of future landing sites, e.g., Mawrth Vallis and Oxia Planum currently under discussion for the ESA ExoMars rover (http://exploration.esa.int/mars/53845 landing-site/). Potentially habitable environments may reveal secondary alteration minerals that require the origins (abiotic or biotic) to be unequivocally determined; the combination of thermochemical modeling with simulation studies enables the identification of mineral assemblages that can be used as potential indicators of biotic origin.

Although, the secondary mineral yield was not large enough for detection by XRD in our weeks-to-month long experiments, in a natural martian environment, subsequent generations of microbes would enhance this yield, enabling detection by spacecraft instrumentation.

# CONCLUSION

In this study, a novel approach was applied combining both laboratory-based experiments and thermochemical modeling to investigate the feasibility of identifying mineralogical biosignatures that can be used as evidence of life on early Mars. The modeled basalt–fluid reactions resulted in secondary mineral precipitates, which are comparable with the alteration mineralogy found in the rock after the dissolution experiments and the thermochemical models. This confirms the viability of this methodological approach.

The bacterium-enhanced rock dissolution by acidification was likely to be caused by both organic and inorganic acids. The bulk pH of the growth medium may simulate the microenvironment in the natural environment, which is thought to contain concentrated acids and solutes. Comparison of a carefully selected indicator element (K) in the model allowed the deduction of the actual (W/R)D. This comparison was used to follow individual element paths to understand how secondary mineral precipitation would differ in biotic and abiotic systems over geological time scales. This approach suggests that biologically-mediated secondary alteration is expected to be "simpler," consisting of Fe-hydroxide and kaolinite whereas the abiotic system would form chlorite in addition to Fehydroxide and kaolinite. This difference is due to differences in chemical diversity and mineralogical complexity; kaolinite has a 1:1 structure containing only Si, Al, and O, whereas chlorite, as a clay mineral, has a 2:1 structure with Si, Al, and O and additional cations (Fe, Mg, Ca). This is important, because most planetary exploration—in terms of spatial coverage—is from orbit using spectral investigations. Those techniques are capable of distinguishing the different groups of clays, but not necessarily subtle variations within a group that might be indicative of the presence or absence of life.

Therefore, due to the different mineral formation pathways utilized in abiotic and biotic systems, deciphering the chemical and mineralogical details necessary for identifying the presence of life is expected to be a task for rover-based investigation. Small sample size, detailed investigations, such as those possible with the rover-based instrumentation currently active on Mars (e.g., CheMin and MastCam and ChemCam passive spectral investigations on MSL), can make use of the fact that, under bacterially-mediated conditions, a less mineralogically and less chemically diverse precipitate is expected. Therefore, this study highlights the differences to look for when investigating a habitable environment for the presence of past life.

Future work will utilize a fresh basalt to minimize the difficulties that the presence of clay minerals had on the model system, e.g., from recent eruptions on Hawaii or Iceland, with polished surfaces singling out grains for preand post-experiment mineralogical investigations. The analysis will include instrumentation relevant to past and present life detection missions, including XRD, FTIR, and Raman, to determine whether bio-signatures are detectable. Furthermore, organic molecules will be included in the thermochemical modeling, for example using the program SOLMINEQ88, to investigate whether they inhibit precipitation (Kharaka et al., 1988). This work will further help to specify and quantify the secondary mineralogy that has the potential to serve as inorganic, radiation, and desiccation resistant biosignatures detectable by instruments such as the CheMin Instrument (Blake et al., 2012) on the Mars Exploration rover Curiosity (Blake et al., 2012).

# AUTHOR CONTRIBUTIONS

KOF: Designed the lab experiment, carried out the microbiology analyses, and wrote the manuscript. VP: Carried out the FEG-SEM work. ES: Carried out the geochemical analyses. SS: Ran the models.

# ACKNOWLEDGMENTS

This work was supported by a personal fellowship awarded by the UK Space Agency. We would like to thank Gordon Imlach for the support he provided with the FEG-SEM analysis.

## REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Olsson-Francis, Pearson, Steer and Schwenzer. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Cellular Responses of the Lichen Circinaria gyrosa in Mars-Like Conditions

Rosa de la Torre Noetzel 1 \* † , Ana Z. Miller 2†, José M. de la Rosa2 , Claudia Pacelli 3 , Silvano Onofri 3 , Leopoldo García Sancho4 , Beatriz Cubero2 , Andreas Lorek 5 , David Wolter 5 and Jean P. de Vera5

1 Departamento de Observación de la Tierra, Instituto Nacional de Técnica Aeroespacial, Madrid, Spain, 2 Instituto de Recursos Naturales y Agrobiología de Sevilla, Consejo Superior de Investigaciones Científicas, Sevilla, Spain, 3 Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy, 4 Departamento de Biología Vegetal II, Universidad Complutense, Madrid, Spain, 5 German Aerospace Center (DLR) Berlin, Institute of Planetary Research, Berlin, Germany

Lichens are extremely resistant organisms that colonize harsh climatic areas, some of

#### Edited by:

Masahiro Ito, Toyo University, Japan

#### Reviewed by:

Henrik R. Nilsson, University of Gothenburg, Sweden Lyle Whyte, McGill University, Canada

\*Correspondence:

Rosa de la Torre Noetzel<EMAIL_ADDRESS>
† These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 12 September 2017 Accepted: 09 February 2018 Published: 05 March 2018

#### Citation:

de la Torre Noetzel R, Miller AZ, de la Rosa JM, Pacelli C, Onofri S, García Sancho L, Cubero B, Lorek A, Wolter D and de Vera JP (2018) Cellular Responses of the Lichen Circinaria gyrosa in Mars-Like Conditions. Front. Microbiol. 9:308. doi: 10.3389/fmicb.2018.00308 them defined as "Mars-analog sites." There still remain many unsolved questions as to how lichens survive under such extreme conditions. Several studies have been performed to test the resistance of various lichen species under space and in simulated Mars-like conditions. The results led to the proposal that Circinaria gyrosa (Lecanoromycetes, Ascomycota) is one of the most durable astrobiological model lichens. However, although C. gyrosa has been exposed to Mars-like environmental conditions while in a latent state, it has not been exposed in its physiologically active mode. We hypothesize that the astrobiological test system "Circinaria gyrosa," could be able to be physiologically active and to survive under Mars-like conditions in a simulation chamber, based on previous studies performed at dessicated-dormant stage under simulated Mars-like conditions, that showed a complete recover of the PSII activity (Sánchez et al., 2012). Epifluorescence and confocal laser scanning microscopy (CLSM) showed that living algal cells were more abundant in samples exposed to niche conditions, which simulated the conditions in micro-fissures and micro-caves close to the surface that have limited scattered or time-dependent light exposure, than in samples exposed to full UV radiation. The medulla was not structurally affected, suggesting that the niche exposure conditions did not disturb the lichen thalli structure and morphology as revealed by field emission scanning electron microscopy (FESEM). In addition, changes in the lichen thalli chemical composition were determined by analytical pyrolysis. The chromatograms resulting from analytical pyrolysis at 500◦C revealed that lichen samples exposed to niche conditions and full UV radiation consisted primarily of glycosidic compounds, lipids, and sterols, which are typical constituents of the cell walls. However, specific differences could be detected and used as markers of the UV-induced damage to the lichen membranes. Based on its viability responses after rehydration, our study shows that the test lichen survived the 30-day incubation in the Mars chamber particularly under niche conditions. However, the photobiont was not able to photosynthesize under the Mars-like conditions, which indicates that the surface of Mars is not a habitable place for C. gyrosa.

Keywords: Mars environment, extremotolerance, lichens, Circinaria gyrosa, photosynthetic activity, analytical pyrolysis

# INTRODUCTION

Lichens are structurally complex organisms resulting from a symbiotic relationship of algae, also known as cyanobacteria (photobiont), and ascomycetes, also known as basidiomycetes (mycobiont). Some lichens may also be composed of a third partner consisting of a specific basidiomycete yeast, which forms the cortex of the lichen thallus (Spribille et al., 2016). When exposed to environmental stress (such as extreme temperatures, desiccation, and UV radiation), lichens develop photoprotective mechanisms, including light scattering, radiation screening, thermal dissipation, antioxidant defense, membrane repair, and macromolecular production (Nguyen et al., 2013).

Lichens may produce several secondary metabolites as adaptive mechanisms for growing in harsh living conditions. Direct gas chromatography (GC)–mass spectroscopy (MS) analysis of a solvent extract is not straightforward because of non-volatile residues, which remain undetected because they are trapped in the chromatographic system (Stojanovic et al., 2011). Thus, we propose direct analyses of the samples by analytical pyrolysis (Py-GC/MS), which is defined as the thermochemical decomposition of organic materials at elevated temperatures in the absence of oxygen. The pyrolysis products (pyrolysates) are amenable to chromatographic separation, which in combination with an MS detector (GC/MS), provide a fingerprint for the molecular structure, including complex mixtures of macromolecular substances (De la Rosa et al., 2009). Pyrolytic techniques have additional well-known advantages such as the requirement of small sample sizes and minimal, if any, sample preparation. Thus, Py-GC/MS has become an important tool for analytical characterization of a wide spectrum of complex carbonaceous matrices including fossil organic matter, algal and vascular plants, sediments, and urban dust (Lee et al., 2005; De la Rosa et al., 2008; Pereira de Oliveira et al., 2011; Braovaca et al., 2016). This technique has already been successfully used to determine changes in lichens' chemical composition under different environmental conditions (MacGillivray and Helleur, 2001). It is based on the fact that lichens often react to stress factors by changing their acids as the result of a defense- or stressinduced metabolism. Rikkinen (1995) studied lichens' response to UV light. It has been reported that lichens under harmful stress condition will alter their composition, either as a chemical defense or in response to damage (Treshow and Anderson, 1989; Richardson, 1992).

Several studies have been performed to test the response of various lichen species to harsh conditions in space and Marslike conditions on board space missions. Space is an extremely hostile environment, which is characterized by a high vacuum (10−7–10−4 Pa), an intense field of ionizing radiation of solar and galactic origin, unfiltered solar ultraviolet (UV) radiation, and extreme temperatures (−120 to +120◦C). Among the tested organisms, bacterial spores of Bacillus subtilis (Firmicutes, Bacillales) (Horneck et al., 1984, 2001; Horneck, 1993; Rettberg et al., 2002), the lichens Rhizocarpon geographicum, Xanthoria elegans (Sancho et al., 2007), and Circinaria gyrosa (De la Torre et al., 2010), and adults and eggs of the tardigrades Richtersius coronifer and Milnesium tardigradum (Jönsson et al., 2008) turned out to be the most resistant to these conditions. About 70% of the B. subtilis spores survived 2,107 days in space on board the National Aeronautics and Space Administration Long Duration Exposure Facility after being shielded from solar UV (Horneck et al., 1994). However, direct exposure to the solar extra-terrestrial UV radiation, as experienced in space, reduced their survival by orders of magnitude. So far, lichens are the only organisms that were able to survive under space conditions, which included solar extraterrestrial UV radiation as tested during the 2-week flight of BIOPAN-5 and -6 (Sancho et al., 2007; De los Ríos et al., 2009; De la Torre et al., 2010). Other studies were performed to concurrently test the response of lichen species under space and Mars-simulated conditions on board two space missions, EXPOSE-E and -R2 (Onofri et al., 2012; 2014–2016 mission), on the International Space Station. Laboratory studies using a simulated Mars environment and space-relevant ionizing radiation have also been conducted (Sánchez et al., 2012; De la Torre et al., 2017; Meeßen et al., 2017). The results showed that the species most resistant to space and Mars conditions was the vagrant lichen species C. gyrosa in a latent state. Circinaria gyrosa (formerly Aspicilia fruticulosa, Sohrabi et al., 2013) is a vagrant fruticose lichen with a crustose growth form. It does not grow while attached to a substrate. It is from the steppes and shrub lands with outliers in Southwest Asia and the Mediterranean area of Spain (Sohrabi et al., 2013). This lichen is characterized by a coralloid thallus with dichotomous branching and a compact internal structure (Sancho et al., 2000; Meeßen et al., 2013). Externally, it is limited by a thick cortex that provides efficient shielding against the hostile parameters of outer space (Sancho et al., 2007) and planetary conditions such as those seen on Mars. Yet, physiologically active C. gyrosa has not been exposed to Mars' environmental conditions. For this reason, our aim was to perform a first-time multidisciplinary assessment of C. gyrosa's viability and morphological changes after exposure to the Mars conditions. Lichen viability was assessed by fluorescence microscopy as chlorophyll-induced red autofluorescence is used as an indicator of cell viability such as seen with intact chlorophyll molecules, which display red fluorescence in living cells. This method has been used to assess terrestrial phototrophic microorganisms' and marine phytoplanktons' viability in addition to a plant's ability to tolerate stress factors (Hense et al., 2008; Osticioli et al., 2013) since the red color of photosynthetic cells under UV light (Gausla and Ustvedt, 2003) is related to optimal photosynthetic activity that produces colored light or absence of light in cases of cell damage. Recently, this technique has been introduced in the field of astrobiology for examination of the viability of lichens exposed to simulated space conditions in order to help to distinguish intact cells from cells injured by high-dose ionizing radiation (De la Torre et al., 2017). In addition, changes in the chemical composition of the lichen thalli were determined by analytical pyrolysis (Py-GC/MS).

# MATERIALS AND METHODS

### Sample Preparation and Irradiation

We chose the lichen species C. gyrosa (Sohrabi et al., 2013) for the Mars simulation experiment. It is characterized by a coralloid thallus with dichotomous branching and a compact internal structure (Sancho et al., 2000; Meeßen et al., 2013). All samples were collected from clay soils of the Central Spain steppic highlands, which are characterized by extreme insolation, high temperature contrasts, and arid summers (Crespo and Barreno, 1978). In the presence of these extremely harsh environmental conditions, C. gyrosa will enter a desiccated state in the form of a metabolic suspension or stasis (known as cryptobiosis or latent state), in which the cells of the lichen symbionts are dehydrated, and most biochemical activity ceases.

Replicate samples of C. gyrosa with 10 mm diameter were used and classified into two groups: (i) the first was composed of eight exemplars for exposure to simulated Mars environmental conditions (Cg1–Cg8; **Figure 1**), and (ii) the second, also formed with eight samples, was used as laboratory control (Cg9–Cg16). Samples for the Mars simulation experiment were again classified into two sets, each composed of four samples: (i) the first set was used for niche-led conditions (Cg1–Cg4), and (ii) the second set included samples that were exposed to full UV radiation (Cg5– Cg8). Before the Mars experiment, all samples were activated (as described below), and the photosynthetic activity was measured.

# Description of Experiment Equipment

The experiment was carried out at the Mars Simulation Facility (MSF) of the DLR Institute of Planetary Research in Berlin (Lorek and Koncz, 2013, **Figure 2A**). The main part of the MSF is a climate chamber (CC) with inside dimensions of 80 cm height, 60 cm depth, and 50 cm width. The experiment was performed in an experimental chamber (EC), a vacuum sealed stainless steel chamber with a volume of 10.3 L inside the CC (**Figure 2B**). The

simulation experiment. (B) After Mars simulation experiment.

EC has electrical connectors, connectors for gas in and output, four fibers for xenon light, and one fiber for photosynthetic activity measurements obtained with a photosynthesis yield analyzer (MiniPAM, Walz GmbH, Germany) as described by Sancho et al. (2007). Inside the chamber is a turntable with eight sample holders, a LED unit for the illumination of one sample with visible light and UV-radiation, and two humidity sensors close to the samples (approximately 1 cm) calibrated for the Martian atmosphere, each equipped with a Pt100-sensor. The gas flow through the EC is generated by a gas mixing system, which can control up to five gases and EC humidification. The pressure inside is controlled by a membrane vacuum pump. The radiation dose is measured with an X92-optometer and a RCH-106-4 probe (Gigahertz-Optik GmbH, Germany) at wavelengths ranging from 250 to 400 nm.

# Experimental Conditions

Over the complete experimental time course, the EC had a continuous gas flow of 20 L h−1 (at 101,325 Pa). The gas consisted of 95% CO2 and 5% air (4% N2, 1% O2). The pressure inside the EC was approximately 750 Pa, the temperature varied diurnally between −50◦C (night) and 20◦C (day), whereas the relative humidity (with respect to ice) ranged between approximately 75% (night) and near 0% (day). **Table 1** summarizes the experimental conditions used in the chamber in comparison with environmental conditions on the surface of Mars.

Four samples of C. gyrosa were illuminated with xenon light (full UV-radiation), and four samples were exposed to nicheled conditions. The LED lamp produces photosynthetic active radiation (PAR) and is used in addition to scattered UV-radiation produced by the xenon lamp. This allowed us to approach as closely as possible the conditions that could be faced in microfissures and micro-caves (so-called micro-niches) in rocks and the Martian soil. In these fissures and caves, only a short exposure period of scattered or direct light for a few minutes depending on the angle of the sun could be measured (Schuerger et al., 2003; de Vera et al., 2014). The LED unit was active for 16 h and switched off 8 h daily to simulate the Sun's diurnal cycle. During the same time period, the xenon-lamp was switched on and off with the exception that on weekends the UVlamp remained off because only manual operation was possible. The resulting average radiation doses were 5.19 KJ m−2 for niche-led conditions (samples 1–4) and 127.52 KJ m−2 for full UV-radiation (samples 5–8). **Figure 3** shows the environmental conditions.

# Chlorophyll a Fluorescence Analysis

To assess photosynthetic activity of the lichen photobiont after a 4-week exposure to simulated Mars conditions, the maximum quantum yield (QY) calculated as Fv/Fm (Schreiber et al., 1994), of the photosystem II (PS II) was measured in pre-activated samples by chlorophyll a fluorescence analysis. Reactivation of samples was performed for 72 h in a growth chamber at 10 ◦C and 100 µmol m−2 s −1 photosynthetic photon flux density (PPFD) for a daily 12 h photoperiod and moistened twice a day with mineral water. These conditions mimicked the environmental conditions accompanying high water availability corresponding

TABLE 1 | Experimental conditions used in the Mars Simulation Facility (MSF) of the DLR Institute of Planetary Research in Berlin in comparison to Mars surface conditions.

to physiological activity such as early morning dew formation, low temperatures, and low light conditions (Lange et al., 1970). Chlorophyll a fluorescence of re-activated samples was measured with a photosynthesis yield analyzer (Mini-PAM, Walz GmbH, Germany) as described by Sancho et al. (2007).

# Epifluorescence and Confocal Laser Scanning Microscopy

Changes in cell viability and metabolic activity of C. gyrosa exposed to simulated Mars conditions were assessed by epifluorescence and confocal laser scanning microscopy (CLSM). Before observation, the lichen samples were rehydrated with mineral water and reactivated for 12 h at 10◦C. Subsequently, samples were incubated for an additional period of 24 h in a Sanyo MLR-351 plant growth chamber under environmental parameters consisting of 21◦C, 60–70% relative humidity, and 12 h dark/12 h illumination with 250 µmol m−2 s −1 photosynthetically active radiation. Thin sections (80– 100µm) of re-activated lichen samples were prepared with a vibratome and immediately visualized using a Zeiss Apotome epifluorescence microscope (Zeiss, Hamburg, Germany) with a HXP 120 lamp and an Axiocam 506 digital camera. The cube filters for epifluorescence were 38 HE GFP (excitation 450– 490 nm and emission 500–550 nm) and cube 50 Cy5 (excitation 625–655 nm and emission 665–715 nm).

Thin sections from re-activated thallus samples for CLSM were stained with 25µM of the fluorescent dye FUN-1 R (Molecular Probes, Oregon, USA) in 2% glucose and 10 mM HEPES buffer (pH 7.0) and incubated at 25◦C for 3 h. FUNstained samples were then observed using a Zeiss LSM 7 DUO Confocal Microscope (Zeiss GmbH, Germany) with a 10X EC Plan-Neofluar objective. Images were acquired at eight bits with 1,024 × 1,024 pixels format using the 488 nm line of an argon ML laser as the only excitation source and a mean beam splitter of 488/561. Emission bands for each channel were 505–550 nm (for green), 581–620 nm (for red), and 672–758 nm (for blue). Image stacks were taken through the z axis at 4 µm intervals through the whole section and analyzed with ZEN 2011 software (Zeiss GmbH, Germany). Images were obtained by combining the three fluorescence signals.

# Field Emission Scanning Electron Microscopy

To accurately assess thallus anatomy and morphological changes in C. gyrosa samples exposed to simulated Mars conditions, field emission scanning electron microscopy (FESEM) with energy dispersive X-ray spectroscopy (EDS) was conducted. Before FESEM examinations, thin sections of the reactivated thallus samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate-buffer (pH 7.4) at 4◦C for 2 h, washed three times in cacodylate-buffer for 5 min per wash period, and post-fixed in 1% osmium tetroxide for 1 h at 4◦C. The samples were

then dehydrated by subsequent serial dilutions in ethanol and acetone and then dried in a critical point drying device (Leica EM CPD300) at 34.5◦C. Finally, the fixed lichen samples were sputter-coated with a thin gold film and observed with an FEI Teneo FESEM (FEI Company, Eindhoven, The Netherlands) using the secondary electron detection mode with an acceleration voltage of 5 kV for ultra-high resolution images and 10 kV for elemental microanalysis and mapping.

# DNA-Based Analyses

Polymerase chain reaction (PCR) analyses were performed to verify deoxyribonucleic acid (DNA) damage by both random amplification of polymorphic DNA (RAPD) fingerprinting and single gene amplifications. DNA was extracted from rehydrated lichen samples using the Nucleospin Plant kit (Macherey-Nagel, Düren, Germany) following the protocol optimized for fungi. Amplification of the internal transcribed spacer (ITS) regions and the large and small subunits ribosomal RNA genes were performed using BioMix (BioLine GmbH, Luckenwalde, Germany) after adding 5 pmol of each primer and 20 ng of template DNA at final volume of 25 µl. The amplification was carried out using a MyCycler Thermal Cycler (Bio-Rad Laboratories GmbH, Munich, Germany) equipped with a heated lid. Conditions for rDNA regions amplification and primer sequences are reported in **Tables 2**, **3**, respectively. For the mycobiont, the RAPD protocol of Selbmann et al. (2011) was applied. For RAPD of the C. gyrosa photobiont, the OPA 13 primer (**Table 3**) was used and conditions for amplification included several steps: (i) a denaturation step at 94◦C for 4 min; (ii) denaturation at 96◦C for 30 s; (iii) annealing at 49◦C for 60 s; and (iv) extension at 72◦C for 30 s. The final three steps were repeated 40 times with a final extension at 72◦C for 6 min. Band intensity was measured and compared using Image J software, version 1.45 (Schneider et al., 2012).

# Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS)

Chemical analyses of lichen thalli were carried out by direct pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) using a double-shot pyrolyser (Frontier Laboratories, model 2020i) attached to a GC/MS Agilent 6890N as described in Miller et al. (2016). One milligram each of C. gyrosa samples from the control, niche-led conditions, and exposed-to-full UV sets were placed in small crucible capsules and introduced into a preheated micro-furnace at 500◦C for 1 min. The volatile pyrolysates were then directly injected into the GC/MS for analysis. The gas chromatograph was equipped with a HP-5ms-UI, low polar-fused silica (5%-phenyl-methylpolysiloxane) capillary column of 30 m × 250µm × 0.25µm film thickness. The oven temperature was held at 50◦C for 1 min and then increased to 100◦C at 30◦C min−1 , from 100 to 300◦C at 10◦C min−1 , and then stabilized at 300◦C for 10 min. The carrier gas was helium at a controlled flow of 1 ml min−1 . The detector was an Agilent 5973 mass selective detector, and mass spectra were acquired at 240◦C under electron impact (70 eV of energy). Masses were scanned from m/z 40–600. Each sample was pyrolyzed twice without perceptible differences between the chromatograms of the same sample. Compound assignment was achieved via comparison with already published data or stored in digital libraries (NIST and Wiley libraries).

#### TABLE 3 | Primer sequences.

#### RESULTS AND DISCUSSION

#### Mars-Like Conditions

Before examining and analyzing the results, it has to be clarified that Mars-like conditions in reference to our present work means that we cannot simulate the reduced Martian gravity and direct exposure to heavy radiation particles in the laboratory; these conditions are expected to happen on the surface of Mars because of the lack of a protective global magnetic field; however, by simulating Mars-like niche conditions we are approaching conditions with reduced radiation (that could be expected in micro-fissures and micro-caves close to the surface of Mars) or reduced sunlight intensity caused by dust in the atmosphere.

#### PSII Activity During the Exposure to Simulated Mars-Like Conditions

Photosynthetic activity measurements were executed in 30 min steps. The yield-value dropped from 0.35 down to approximately 0 for all samples within the first hour of the experiment. Over the remaining experimental period, the photosynthetic activity of all samples ranged from very low to non-existent (**Figure 4**). Although the photosynthetic light reaction was measured before and after the experiment, it can be concluded that C. gyrosa was not photosynthetically active during the Mars exposure experiment. The lichen therefore coped with Mars-like conditions by entering a dormant stage and would not be able to survive on Mars in the long-term in the absence of photosynthetic activity.

#### PSII Activity Before and After the Exposure to Simulated Mars-Like Condition0073

The PSII was measured as the maximum QY for the different set of samples before exposure to simulated Mars-like conditions and showed a homogeneous tendency without significant differences between them (**Figure 5**). However, after exposure to the Mars-like environment, the mean values showed differences over a range of 20% for both sample sets, including the control set (Cg9–Cg12, **Figure 5**). These differences were higher than those of the second control set (Cg13–Cg16) with 0.5%.

These results indicate that a simulated Mars-like environment with attenuated niche-like conditions and full impact of Mars UV radiation (full UV) did not significantly affect the photosynthetic activity of the re-activated C. gyrosa. In other words, the photosynthetic activity of the lichen thalli recovered efficiently after re-activation (rehydration after exposure to Mars-like conditions).

#### Morphology and Cell Viability After Exposure to Simulated Mars-Like Conditions

Chlorophyll autofluorescence monitored by epifluorescence microscopy for evaluating cell viability of the photobiont showed slight differences in the red light emission (chlorophyll autofluorescence) among the three sets of samples (**Figure 6**). The C. gyrosa thalli exposed to niche-led conditions exhibited red fluorescence similar to the control samples (**Figures 6A,B**). In samples fully exposed to UV radiation, algal cells were less abundant, and almost no autofluorescence was observed in zones occupied by algal clusters (**Figure 6C**). This indicates an irradiation-induced degradation of chlorophyll or an inefficient reactivation of lichen physiological processes after rehydration. However, minimal red fluorescence was recorded in the algal layer (**Figure 6D**).

The FUN-1 stain was used for detecting the presence of metabolically active fungal cells by CLSM due to intracellular processing of the vacuolar dye (red fluorescence). Algal cells could also be observed and identified by recording their autofluorescence signal in the blue channel (**Figure 7**). CLSM analyses of the lichen photobiont were in line with the epifluorescence-monitored chlorophyll autofluorescence. Living algal cells (with intense blue fluorescence) were more abundant

the Mars simulation experiment. No effective photosynthetic activity was measured during the simulation.

in the control (**Figure 7A**) and samples exposed to nicheled conditions (**Figure 7B**) than to full UV (**Figure 7C**). The latter samples showed clear asymmetry of the algal layer in the thin lichen cross-sections as intense chlorophyll autofluorescence was intermixed with faded blue fluorescent cells (**Figure 7C**). Concerning the mycobiont, similar levels of metabolization of the FUN-1 stain yielding green fluorescent hyphae with clear red fluorescent vacuoles were observed in the control and irradiated samples, particularly in the proximity of the algal layer, suggesting metabolically active fungal cells (**Figures 7A,B**). Green fluorescent hyphae without red structures were particularly observed in the innermost medullary part of the lichen, pointing to a lack of viability in these regions.

FESEM of the thin lichen sections used for assessing thallus structural changes and damage to cell walls revealed differences among the three set of samples (**Figure 8**). The control sample showed the characteristic C. gyrosa thallus structure with the algal layer composed of photobiont cells arranged in clusters (**Figure 8A**) and a medulla with loosely interwoven fungal hyphae (**Figure 8B**). Both control and niche-led samples showed good anatomical preservation of the algal clusters in addition to the individual cells (**Figure 8C**). In addition, the medulla did not seem to be structurally affected and consisted of interlaced hyphae with numerous voids (**Figure 8D**), indicating that the niche-like exposure conditions did not disturb either the lichen thallus structure or the morphology. Interestingly, FESEM examinations revealed highly mineral-encrusted hyphae in all samples, predominantly in the inner medullary structure (**Figure 8E**). According to crystal shape and EDS analyses, this mineral is consistent with whewellite (calcium oxalate monohydrate), an insoluble organic salt secreted by the cells for several purposes, including calcium regulation, protection, and structural strengthening (Giordani et al., 2003). Whewellite crystals are widespread among organisms and have been reported on stone surfaces that were densely colonized by lichens (Pereira de Oliveira et al., 2011). Extracellularly deposited whewellite-crystals were also observed by Meeßen et al. (2013),

Böttger et al. (2014), and De la Torre et al. (2017) on C. gyrosa medullary hyphae.

On the other hand, the asymmetry observed by CLSM relating to the numbers and distribution of algal cells throughout the lichen thalli of the full UV exposed samples was clearly detected by FESEM. **Figure 8F** shows part of a lichen thallus with disrupted photobiont cells in the zones occupied by algal clusters, whereas other parts of the same thin section showed wellpreserved algal cells (**Figure 8G**). The loose inner medulla of the niche-led samples (**Figure 8C**) contrasted with the tightly packed fungal cells of the full UV exposed samples conglutinated with extracellular cementing substances, particularly in the proximity of the algal layer (**Figure 8H**). Taken together, these data indicate that C. gyrosa samples exposed to full UV radiation show a slight delay in physiological activity recovery after rehydration in comparison to the lichen thalli exposed to niche-led conditions. In a review by Holzinger and Karsten (2013) about desiccation tolerance of aeroterrestrial algae, it was reported that green algae rapidly reduced photosynthetic activities during desiccation but recover quickly after rehydration. Similarly, Aubert et al. (2007) showed that samples of the lichen X. elegans in high mountain environments instantly recovered respiration after hydration and both photobiont and mycobiont cells did not suffer irreversible desiccation-induced damage.

### DNA Integrity Evaluation

DNA integrity was assessed by amplifying three different genes both for the mycobiont and photobiont and also by using a fingerprinting analysis to analyze whole genome integrity. PCRstop assays are a powerful method for evaluating DNA damage since lesions block the progression of DNA polymerase (Kumar et al., 2004; Trombert et al., 2007), and long amplicons have a higher likelihood of undergoing DNA damage than short PCR amplicons (Rudi et al., 2010). In addition, in genomic PCR fingerprinting that amplifies short genomic DNA portions, PCR band intensity mainly decreased in the highest molecular weight fragments in single-gene PCR (Atienzar et al., 2002; Atienzar and Jha, 2006). Amplicons were obtained both for ITS and large subunit (LSU) regions of the mycobiont (**Figure 9A**). These approaches have been applied to dried Cryomyces antarcticus colonies exposed to UV irradiation (Selbmann et al., 2011) and then successfully applied under both simulated space- and Mars conditions-, ionizing radiation-, and alpha particles-exposed samples of the same fungus (Onofri pers. com.; Pacelli et al., 2017a,b,c). A reduction of amplicon intensity was observed with increasing genetic marker length. Nevertheless, DNA was still intact and perfectly detectable at the highest molecular weight band (2,000 bases). Single gene amplifications of photobiont DNA (**Figure 9B**) revealed that DNA was amplified at both 1,000 and 2,000 bases. We could not obtain a PCR product (band) for the longest length tested (3,000 bases).

RAPD profiles of samples under Mars-like conditions were identical to those of both the mycobiont and photobiont since no band disappearance was observed; the whole genome was conserved (**Figure 10**). For DNA analyses, the photobiont was the most affected by Martian conditions, showing a reduction

FIGURE 7 | radiation (C). The FUN-1 stain yielding green fluorescent hyphae with red fluorescent vacuoles represent metabolically active hyphae, which are particularly observed in the proximity of the algal clusters. Blue cells represent chlorophyll autofluorescence. Green fluorescent hyphae without red structures, indicating lack of viability, are observed in the innermost medullary part of the lichen fully exposed to UV radiation (C).

up to 50% for the maximum gene length (**Figure 10B**), while the mycobiont DNA was perfectly detectable at every examined gene length (**Figure 10A**). Notably, in our experiments DNA could be easily extracted and detected even after being exposed to Mars-like conditions. The low temperatures in space was shown to reduce DNA breaks by half compared to ambient Earth temperatures (Lindahl, 1993), and the dry space environment was shown to preserve DNA (Lyon et al., 2010). Furthermore, it has been already reported that the low temperatures and dry conditions on Mars may preserve DNA in a better way in the long-term than do the conditions on Earth (Kanavarioti and Mancinelli, 1990; Sephton, 2010). Other studies on C. antarcticus DNA damage after exposure to the Martian atmosphere similarly demonstrated that fungal DNA was not affected and remained perfectly detectable by PCR (Pacelli et al., 2017b).

# Analytical Pyrolysis (Py-GC/MS)

The GC/MS total ion chromatograms (TIC) of the pyrolysates of the three sets of samples are shown in **Figure 11**. They were very similar and consisted mainly of furanes, furfural, and methyl derivatives of these compounds. They were polysaccharide-derived xyloses and hexoses which constituted about 95% of the lichen molecular composition according to Schellekens et al. (2009). These compounds were also found by analytical pyrolysis to be common products of metabolic lichen thalli activities (Saiz-Jimenez et al., 1991). The most abundant compound of the three TICs was levoglucosan (peak 25, **Figure 11**), which is a six carbon ring structure formed from the carbohydrate (such as starch or cellulose) pyrolysis. One of the differences observed in the chromatograms was the presence of L-arabinopyranose in the full UV-irradiated lichens (peak 26, **Figure 11C**). This was a furan-related aldopentose, which is found in nature as a component of biopolymers such as hemicellulose. Neophytadiene, a marker of chlorophyll in algae and cyanobacteria (Saiz-Jimenez et al., 1990), was present in all of the samples (peak 27, **Figure 11**). Its relative abundance decreased in the fully UVirradiated lichens (**Figure 11C**); nevertheless, in the latter case, the peak was partially masked by L-arabinopyranose (peak 26).

A series of sterols and terpenes were identified in all the TICs (from min 22 to 28). Our results revealed a mixture of C28−30 sterols (peaks 37–41 and 43–46, **Figure 11**), which included ergosterol acetates, ergostanol, stigmastan-3,5-dien, and βsitosterol. They were plant cell membrane components and were lichen secondary metabolites, which have been widely studied due to their medical and pharmaceutical applications. Ergosterol is regarded as the typical membrane constituent of fungi and serves as a bioregulator of membrane fluidity, asymmetry, and

integrity. Sufficient ergosterol content is necessary for fungal cell growth and normal membrane function. The TIC of the niche-led samples showed a relative increase in the presence of sterols (**Figure 11B**), while a clear decrease is observed for the full UV-irradiated samples (**Figure 11C**). Taking into account that the fluidity of biological membranes is influenced by the amount of sterols (Russel, 1989), this result could indicate surface damage to the membrane resulting from high UV radiation doses. Furthermore, the decrease in the relative abundance of β-sitosterol of the full UV-irradiated samples (**Figure 11C**) could be due to its dehydration and subsequent transformation into stigmasta-3,5-diene (León-Camacho et al., 2004). In line with the previous findings, D-α-Tocopherol (vitamin E; Peak 42), a key lipid-soluble antioxidant in membranes (Munne-Bosch and Alegre, 2002), was found solely in the control sample (**Figure 11A**). Fatty acids (FAs) are homologous and typically

FIGURE 10 | RAPD profile of C. gyrosa mycobiont (A) and photobiont (B). Samples correspondence as reported in Figure 9.

found in the molecular composition of lichens. Nevertheless, only pentanoic, palmitic, and linoleic acids are detected in the TICs of the lichens (peaks 2, 28, and 29, respectively). The low FA abundance was due to the incompatibility with the apolar columns used in GC and to secondary reactions that occur at the elevated temperatures applied during the pyrolysis (Dignac et al., 2006). A few nitrogen (N)-containing compounds were detected among the pyrolisates (peaks 23, 30, and 34; **Figure 11**), which were probably derived from peptides. Their decline in the full UV-irradiated lichens could indicate the use of N-precursors to sustain the synthesis of N-containing compounds under stress conditions. These results show that analytical pyrolysis is a valuable tool for assessing alterations in lichens' chemical composition. For instance, specific compounds could be used as biomarkers of cell membrane damage. There are several main advantages of analytical pyrolysis use in for astrobiology: (i) easy sample preparation (drying and milling); (ii) fast analysis time; (iii) very high reproducibility; and (iv) only small amount of sample is required (0.5– 5 mg).

#### CONCLUSIONS

Our multidisciplinary approach allowed us to assess the viability, structure, DNA integrity, and chemical composition of the lichen C. gyrosa after exposure to simulated Mars conditions. Although the lichen samples did not have the capability to be photosynthetically active during the 30-day

exposure to the Mars-like environment, their photosynthetic activity efficiently recovered after re-activation of the lichen thalli. However, microscopic examination revealed a slight delay in the recovery of C. gyrosa after exposure to full UV conditions in comparison to the lichen thalli exposed to niche-led conditions. Good anatomical preservation of the algal clusters and fungal hyphae from the medulla indicated that the niche-led exposure conditions did not disturb either lichen thalli structure or morphology. No DNA damage was observed in the fungal component, while the amplicon for the algal component disappeared after exposure to simulated Mars conditions, suggesting that the photobiont is the most affected component after exposure to simulated conditions. Analytical pyrolysis revealed a similar composition for the UV- and non-irradiated lichens, which were dominated by the typical cell wall constituents. Nevertheless, the changes in the relative abundance of specific sterols in the lichens exposed to full UV conditions indicate that the UV irradiation induced injury to the lichen membranes.

We showed by experimental means that the surface of Mars would be harmful for the tested lichen C. gyrosa; therefore, the surface of Mars would not be a habitable place for C. gyrosa. In contrast, niches on Mars close to the surface, in which the organism is protected against UV-radiation, might be retreat areas for this lichen. It means that we have to clearly differentiate between the Martian surface (not habitable), the subsurface (potentially habitable), and niches near the surface (partially habitable in fissures, cracks, and micro-caves in rocks, de Vera et al., 2014).

#### AUTHOR CONTRIBUTIONS

RT: coordinated the complete study, and determined the chlorophyll a-fluorescence. AM: provided several analysismethods: CLMS (Confocal-Laser-Scanning Microscopy), Epifluorescence and FESEM/Field Emission Scanning Microscopy), and TEM (Transmission Electron Microscopy). JR: provided the analytical pyrolysis (Py-GC/MS) methodology. CP: provided the DNA integrity evaluation (RAPD and PCR methods). SO: coordinated and supervised the DNA integrity evaluation (RAPD and PCR methods). LG: selected the lichen sample (field campaigns) and supervised the writing of the manuscript. BC: provided the CLMS analysis. AL: provided the technical control of the Mars Simulation Chamber at DLR-Berlin. DW: prepared the biological samples for the simulation test and evaluated the results. JV: coordinated and supervised the Mars simulation test.

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors acknowledge the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, project SUBLIMAS ESP2015-69810-R). AM and JR thank the MINECO for the Juan de la Cierva–Incorporación (IJCI-2014-20443) and Ramón y Cajal (RYC-2014-16338) post-doctoral contracts, respectively. The authors are grateful to Dr. Ronald Charles Wolf for English improvement. CP and SO thank ASI grant BIOMEX-MCF n. 2013-063-R.0.

White, T. J., Bruns, T., Lee, S. J. W. T., and Taylor, J. L. (1990). "Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics," in PCR Protocols: A Guide to Methods and Applications, eds M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (San Diego, CA: Academic Press), 315–322.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 de la Torre Noetzel, Miller, de la Rosa, Pacelli, Onofri, García Sancho, Cubero, Lorek, Wolter and de Vera. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Silicates Eroded under Simulated Martian Conditions Effectively Kill Bacteria—A Challenge for Life on Mars

Ebbe N. Bak 1 \*, Michael G. Larsen1 , Ralf Moeller 2 , Silas B. Nissen1 , Lasse R. Jensen1 , Per Nørnberg1 , Svend J. K. Jensen3 and Kai Finster 1, 4

1 Department of Bioscience, Aarhus University, Aarhus, Denmark, 2 Space Microbiology Research Group, Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany, 3 Department of Chemistry, Aarhus University, Aarhus, Denmark, 4 Stellar Astrophysics Center, Department of Physics and Astronomy, Aarhus University, Aarhus, Denmark

The habitability of Mars is determined by the physical and chemical environment. The

#### Edited by:

Daniela Billi, Università degli Studi di Roma Tor Vergata, Italy

#### Reviewed by:

Aude Picard, Harvard University, United States Timothy Ferdelman, Max Planck Institute for Marine Microbiology, Germany

> \*Correspondence: Ebbe N. Bak<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 30 June 2017 Accepted: 23 August 2017 Published: 12 September 2017

#### Citation:

Bak EN, Larsen MG, Moeller R, Nissen SB, Jensen LR, Nørnberg P, Jensen SJK and Finster K (2017) Silicates Eroded under Simulated Martian Conditions Effectively Kill Bacteria—A Challenge for Life on Mars. Front. Microbiol. 8:1709. doi: 10.3389/fmicb.2017.01709 effect of low water availability, temperature, low atmospheric pressure and strong UV radiation has been extensively studied in relation to the survival of microorganisms. In addition to these stress factors, it was recently found that silicates exposed to simulated saltation in a Mars-like atmosphere can lead to a production of reactive oxygen species. Here, we have investigated the stress effect induced by quartz and basalt abraded in Mars-like atmospheres by examining the survivability of the three microbial model organisms Pseudomonas putida, Bacillus subtilis, and Deinococcus radiodurans upon exposure to the abraded silicates. We found that abraded basalt that had not been in contact with oxygen after abrasion killed more than 99% of the vegetative cells while endospores were largely unaffected. Exposure of the basalt samples to oxygen after abrasion led to a significant reduction in the stress effect. Abraded quartz was generally less toxic than abraded basalt. We suggest that the stress effect of abraded silicates may be caused by a production of reactive oxygen species and enhanced by transition metal ions in the basalt leading to hydroxyl radicals through Fenton-like reactions. The low survivability of the usually highly resistant D. radiodurans indicates that the effect of abraded silicates, as is ubiquitous on the Martian surface, would limit the habitability of Mars as well as the risk of forward contamination. Furthermore, the reactivity of abraded silicates could have implications for future manned missions, although the lower effect of abraded silicates exposed to oxygen suggests that the effects would be reduced in human habitats.

Keywords: habitability, erosion, reactive oxygen species, forward contamination, stress factors, saltation, toxicity, microorganisms

### INTRODUCTION

An in-depth understanding of the interaction between the Martian surface environment and living cells is essential for assessment of the habitability of Mars, the risk of forward contamination and the hazards associated with manned missions. The Martian surface temperatures vary from −150◦ to 20◦C, and the surface pressure is usually 4–10 mbar (Millour et al., 2015). Consequently, the Martian surface is extremely arid and even if liquid water is present, the water activity is likely too low to support life (Martin-Torres et al., 2015; Ojha et al., 2015). While such an arid, cold, low-pressure environment often is not detrimental for desiccation tolerant, dormant cells and spores, the radiation on the Martian surface can be highly damaging (Hansen et al., 2009; Johnson et al., 2011). The solar irradiation flux on Mars is about 40% of the irradiation on Earth, but, the thin atmosphere and the sparse ozone layer (Lefevre et al., 2008) result in a substantially higher UVB and UVC radiation than on Earth (Cockell et al., 2000). Schuerger et al. (2003) demonstrated that 15 min of exposure to Mars-like UV radiation sterilized monolayers of Bacillus subtilis spores. The effect of UV radiation is, however, reduced dramatically when cells and spores are shielded by a thin layer of dust or a few layers of dead cells (Mancinelli and Klovstad, 2000; Diaz and Schulze-Makuch, 2006; de La Vega et al., 2007; Paulino-Lima et al., 2010). The flux of ionizing radiation from the sun and galactic cosmic rays are three orders of magnitude higher on the Martian surface as compared to the surface of Earth due to the thin Martian atmosphere and the absence of a global magnetic field (Hassler et al., 2014). Nevertheless, ionizing radiation from the sun only poses a minor challenge for B. subtilis and D. radiodurans, and a cover that would protect against UV radiation would also shield from the charged particles in the solar wind (Tuleta et al., 2005; Paulino-Lima et al., 2011). Protection against galactic cosmic rays, mainly high-energy particles, requires much thicker shielding. However, the flux to the Martian surface would only cause a half-life of B. subtilis spores within the order of thousands of years (Moeller et al., 2010; Hassler et al., 2014). In summary, the Martian surface does not generally allow life to proliferate, but may not pose an immediate threat to dormant cells.

The Martian soil is dominated by silicate minerals (Bish et al., 2013) primarily produced by physical abrasion of olivine basalts (Morris et al., 2004; Gunnlaugsson et al., 2009). The soil and dust particles are produced by meteor impacts and by winddriven saltation through repetitive low-energy collisions (Kok et al., 2012). A recent study showed that silicates exposed to simulated wind-driven saltation in a Mars-like atmosphere could lead to a production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) (Bak et al., 2017). A production of ROS from abraded silicates may explain the oxidative capabilities of the Martian soil as observed by the Viking Biological Experiments (Klein, 1978) and may cause oxidative stress for living cells. Based on these observations, we hypothesized that wind abraded silicates on Mars may pose an additional stress factor for living cells.

To test our hypothesis, we conducted a series of experiments in which cell suspensions of Pseudomonas putida, B. subtilis and D. radiodurans were exposed to quartz and basalt samples abraded by simulated wind-driven saltation in Marslike atmospheres. The exposure experiments were conducted without exposing the abraded material to oxygen to simulate the in situ conditions for indigenous organisms and a scenario of forward contamination. Furthermore, we investigated the effect of secondary exposure to air to simulate the environment inside a human habitat. Pseudomonas putida is a common soil bacterium, which can withstand some level of oxidative stress (Kim and Park, 2014). The endospores of B. subtilis and cells of D. radiodurans have been reported to be highly resistant against a range of stress factors including desiccation, chemical oxidative stress (Melly et al., 2002; Slade and Radman, 2011) and ionizing radiation (Anderson et al., 1956; Moeller et al., 2008). Furthermore, they have been used to study the effects of exposure to a Mars-like environment including the impact of UV and ionizing radiation (Diaz and Schulze-Makuch, 2006; de La Vega et al., 2007; Moeller et al., 2010; Kerney and Schuerger, 2011). The resistance of B. subtilis to abraded silicates was further investigated by testing a range of mutant strains lacking specific spore components, which previously have been found to be related to resistance against oxidizing agents (Setlow, 2014). Bacillus sp. spores are of particular interest and concern, as they are common contaminants in cleanrooms used for assembly of spacecraft (Puleo et al., 1977; La Duc et al., 2009; Smith et al., 2017) and thus likely agents of forward contamination.

# MATERIALS AND METHODS

### Simulated Saltation of Silicates

Quartz sand samples were prepared by sieving commercially available quartz (Merck, Cat. No. 1.07536) to obtain the 125– 1,000µm fraction. The quartz sand was washed and dried to remove small particles and finally divided into 10 g aliquots using a Fritsch Rotary cone samples divider. The same procedure was used to produce 10 g basalt aliquots based on olivine basalt that was collected in Gufunes on Iceland (64◦ 08′ 22.18′′N, 21◦ 47′ 21.27′′W) and crushed. The mineral composition of the same batch of crushed basalt was reported in Bak et al. (2017). The samples were transferred to about 20 cm long and 3 cm wide quartz ampoules with rounded ends (**Figure 1A**). One end of the ampoules was extended by a narrow quartz inlet tube that allowed connection to a vacuum system. The ampoules were evacuated to <0.12 mbar and filled with 8 ± 0.2 mbar of a Mars-like atmosphere composed of 95% CO2 (>99.9% purity, AGA, Denmark) and 5% of a custom made gas mixture (>99% purity, Air Liquid, Denmark) to give a final composition of 95% CO2, 3% N2, 1.75% Ar, 0.15% O2 and 0.1% CO, which is similar to the composition of the Martian atmosphere (Mahaffy et al., 2013). The pressure was monitored with a Pfeiffer TPR 265 Pirani gauge for p < 1 mbar and a Pfeiffer APR 250 Pirani gauge for p > 1 mbar. The ampoules were sealed by melting off the narrow inlet tubes. Wind-driven saltation was simulated by mounting the ampoules in a turning wheel running at 30 rpm (**Figure 1B**). This set-up caused the silicate samples to fall from one end of the ampoules to the other once per second, which mimics the repeated low energy impacts that are characteristic for saltation (Merrison, 2012). The samples were tumbled for 63 days, which led to a considerable abrasion of the material resulting in an increase of the specific surface area of 3.44 and 0.76 m2 g −1 for the quartz and the basalt samples, respectively (Bak et al., 2017). Some of the abraded samples were suspended in 10 ml of water followed by overnight drying at 60◦C, which has been shown to neutralize the potential for production of ROS (Bak et al., 2017).

(C) Media inoculated with cell cultures. (D) Abraded silicate samples mixed with washed cell cultures. (E) Agar plates with colonies of the tested bacteria.

These samples were used as negative controls and are hereafter

# Preparation of Cell Suspensions

referred to as inactivated samples.