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Nitrate-dependent Fe2+ oxidation (NDFO) is a microbially mediated process observed in many anaerobic, low-nutrient (oligotrophic) neutral–alkaline environments on Earth, which describes oxidation of Fe2+ to Fe3+ in tandem with microbial nitrate reduction. Evidence suggests that similar environments existed on Mars during the Noachian epoch (4.1–3.7 Ga) and in periodic, localised environments more recently, indicating that NDFO metabolism could have played a role in a potential early martian biosphere. In this paper, three NDFO microorganisms, Acidovorax sp. strain BoFeN1, Pseudogulbenkiania sp. strain 2002 and Paracoccus sp. strain KS1, were assessed for their ability to grow oligotrophically in simulated martian brines and in a minimal medium with olivine as a solid Fe2+ source. These simulant-derived media were developed from modelled fluids based on the geochemistry of Mars sample locations at Rocknest (contemporary Mars soil), Paso Robles (sulphur-rich soil), Haematite Slope (haematite-rich soil) and a Shergottite meteorite (common basalt). The Shergottite medium was able to support growth of all three organisms, while the contemporary Mars medium supported growth of Acidovorax sp. strain BoFeN1 and Pseudogulbenkiania sp. strain 2002; however, growth was not accompanied by significant Fe2+ oxidation. Each of the strains was also able to grow in oligotrophic minimal media with olivine as the sole Fe2+ source. Biomineralised cells of Pseudogulbenkiania sp. strain 2002 were identified on the surface of the olivine, representing a potential biosignature for NDFO microorganisms in martian samples. The results suggest that NDFO microorganisms could have thrived in early martian groundwaters under oligotrophic conditions, depending on the local lithology. This can guide missions in identifying palaeoenvironments of interest for biosignature detection. Indeed, biomineralised cells identified on the olivine surface provide a previously unexplored mechanism for the preservation of morphological biosignatures in the martian geological record.

The surface of Mars cannot sustain liquid water today, but there is evidence water was present during the Noachian era. The transition of the martian climate from the wet Noachian to the dry Hesperian would have resulted in saline and sulfur rich surface waters . Terrestrial analogue environments that possess a chemistry like these proposed waters can be used to develop an understanding of organisms that could have persisted under such conditions. Here we present the chemistry and microbiome of the analogue environment Colour Peak, a sulfidic and saline spring system located within the Canadian High Arctic.\ud \ud In this study, molecular and geochemical techniques were used to investigate the sediment of the Colour Peak springs. Nucleic acids were extracted from the microbes in the sediments and the microbiome was characterised by the amplification and sequencing of 16S rRNA gene amplicons. The elemental composition of the fluids and sediment was determined by ICP-OES and compared with brines determined from the chemistry of the “Rocknest” sand sample at Yellowknife Bay, Gale Crater (Mars) by thermochemical modelling. Gibbs energy values were calculated from this fluid chemistry to identify potentially viable metabolisms. \ud \ud Analysis of the chemistries of the Colour Peak fluids confirmed a chemical composition like the thermochemically modelled fluid, with this justifying the classification of Colour Peak as an appropriate analogue environment to investigate the habitability of former martian aqueous environments. 16S rRNA gene profiling of the Colour Peak microbial community revealed it was dominated by bacteria associated with oxidation of reduced sulfur species and carbon dioxide fixation. Gibbs energy values calculated using the chemistry of the modelled martian fluid demonstrated that the oxidation of reduced sulfur species was also viable in this chemical environment under aerobic and anaerobic conditions. These results demonstrate that microbial sulfide oxidation is thermodynamically viable using both modelled and environmental proxies for former martian aqueous environments. \ud \ud This study highlights that metabolisms utilising the oxidation of reduced sulfur species could have been thermodynamically viable in ancient martian aqueous environments. Further work is needed to assess this proposed viability and the potential for unambiguous biosignature formation.

The martian surface cannot sustain liquid water today, but there is evidence water was present during the Noachian era. The transition of the martian climate into the Hesperian would have resulted in saline and sulfuric waters. Terrestrial analogue environments that possess a chemistry like these proposed waters can be used to develop an understanding of organisms that could have persisted. Here we present the chemistry and microbiome of Colour Peak, a sulfidic and saline spring system located within the Canadian High Arctic. \ud Nucleic acids were extracted from the microbes in the sediments and the microbiome was characterised by the amplification and sequencing of 16S rRNA gene amplicons. The elemental composition of the fluids and sediment was determined by ICP-OES and compared with brines determined from the chemistry of the “Rocknest” sample at Yellowknife Bay, Gale Crater (Mars) by thermochemical modelling. Gibbs energy values were calculated from this fluid chemistry to identify potentially viable metabolisms. \ud Analysis of the chemistries of the Colour Peak fluids confirmed a composition like the thermochemically modelled fluid, providing justification for the classification of Colour Peak as an appropriate analogue environment to investigate the habitability of former martian waters. Profiling of the Colour Peak microbial community revealed domination by bacteria associated with oxidation of reduced sulfur species and carbon dioxide fixation. Gibbs energy values calculated using the modelled martian fluid chemistry demonstrated that oxidation of reduced sulfur species was also viable in this chemical environment under aerobic and anaerobic conditions. These results demonstrate microbial sulfide oxidation is thermodynamically viable using both modelled and environmental proxies for former martian aqueous environments. \ud This study highlights that metabolisms utilising the oxidation of reduced sulfur species could have been thermodynamically viable in ancient martian aqueous environments. Further work is needed to test this viability and the subsequent potential for biosignature formation.

Habitability of martian waters would have been partially determined by the chemistry arising from interactions with martian lithologies. In this study, the habitability of groundwater chemistries (based on basaltic, iron- and sulfur-enriched lithologies) and the resulting variation in biosignatures was investigated, with microbes from anaerobic estuarine sediment used as an inoculum. The microbial community was monitored by cell counts and 16S rRNA gene profiling. Changes in fluid and precipitate chemistries were measured using ICP-OES and IC, with changes over geological timescales modelled using CHIM-XPT. The fluid chemistries were shown to be habitable, with distinct patterns in cell abundance and growth phases between the chemistries. However, the same genera dominated (Acetobacterium, Desulfovibrio and Desulfosporomusa) regardless of the initial fluid chemistry. In the biotic test group, changes in fluid chemistry were the same in the three chemistries, with an enhanced concentration of aluminium and iron and the removal of sulfate. However, geochemical modelling of the fluids under abiotic conditions over geological timescales revealed similar changes to those in the biotic test groups. Therefore, these samples require further analysis to assess whether we can identify any potentially unambiguous biosignatures that could develop between geologically distinct sites.

Microbes have multiple ways of producing energy. Which of these methods are possible depends on the chemistry of the environment the microbes are in (e.g. not enough of a metal or too much salt), with only specific methods working in certain environments. The same would be true of any waters that might continue to exist on Mars. To narrow down which methods of producing energy would be theoretically possible we simulated martian waters using a collection of minerals that are chemically similar to the chemistry measured by the Mars rover Curiosity in a crater on Mars. We added mud from an estuary to the simulated martian water and identified which microbes were able to grow. We then repeatedly transferred the growing microbes to fresh “martian” water to dilute out the nutrients from the mud. Over time we observed that most of the microbes from the mud have been lost but a few specific microbes were growing well. From this we hope to investigate changes in the chemistry of the water that happen because of these microbes, to try and identify specific chemistries that can be looked for by the future rover missions on Mars seeking evidence of life.

In this study, four new simulants have been developed, and their associated fluid chemistries have been derived for use in a series of microbiological simulation experiments. These experiments will determine if aqueous environments on Mars, past or present, could potentially support microbial life and identify any key geochemical biosignatures that may arise as a result of that life.

Introduction: The surface of Mars cannot sus-tain liquid water today, but there is evidence for the extended presence of liquid water during the Noa-chian era [1-2]. The transition of the martian cli-mate from the wet Noachian to the dry, late Hespe-rian would have resulted in saline and sulfur-rich surface waters [1-4]. Terrestrial analogue environ-ments that possess a similar chemistry to these pro-posed waters can be used to develop an understand-ing of the diversity of organisms that could have persisted under such conditions. Combining this with laboratory simulation experiments, which ena-ble a greater level of accuracy regarding the chemi-cal environment, allows for concepts regarding di-versity and function to be developed. \ud Here we present the chemistry and microbial community of the highly reducing sediment of the springs of Colour Peak, a sulfidic and saline spring system located within the Canadian High Arctic [2]. We also present details of the viability of this mi-crobial community when grown in defined, simulat-ed martian fluid chemistries based on the chemistry of Rocknest at Gale crater in combination with ba-saltic and iron enriched martian simulants.\ud Methodology: In this study, the elemental com-position of the fluids and sediment porewater of Colour Peak was determined by ICP-OES. This data was compared with a range of fluid chemistries, including those from other analogue environments and martian brines, the composition of which were determined based on the chemistry of the “Rock-nest” sand sample at Yellowknife Bay, Gale crater (Mars) by thermochemical modelling [5]. The fluid chemistry derived from the thermochemical model-ling was used to calculate Gibbs energy values to identify metabolic pathways that could be energeti-cally feasible. Molecular techniques were also used to investigate the microbial community of the sedi-ment of the Colour Peak Springs. Both DNA and RNA were extracted from the microbes in the sedi-ment using a novel extraction technique that was developed to overcome issues associated with low biomass and the high concentrations of inhibitory substances (e.g. salt and sulfide). The microbial community was characterised by the amplification and sequencing of 16S rRNA gene amplicons pro-duced from the extracted nucleic acids. The ability of the microbial community to grow under defined martian chemical conditions was tested using the modelled fluid chemistry in combination with sim-ulants representative of either a general martian chemistry (OUCM-1, also based on the chemistry of Rocknest) or an iron enriched chemistry (OUHR-1, based on the chemistry of Haematite Slope) [6]. Enrichments were incubated at 10 °C for 28 days with a headspace of H2/CO2 (80:20) at one bar pres-sure. Growth was monitored using microscopy with Live/Dead staining and the enriched communities were characterised by sequencing of 16S rRNA gene amplicons produced from the extracted DNA. \ud Results and Discussion: Analysis of the chemis-tries of the Colour Peak fluids confirmed a chemical composition that was similar to the thermochemi-cally modelled fluid derived from in-situ measure-ments of the chemistry of Gale crater sediments (Figure 1). This similarity in elemental composition confirms the classification of Colour Peak as an appropriate analogue environment to investigate the habitability of former martian aqueous environ-ments. 16S rRNA gene profiling of the Colour Peak mi-crobial community (Figure 2) revealed it was domi-nated by bacteria associated with the oxidation of reduced sulfur species and the fixation of carbon dioxide (autotrophy). Gibbs energy values demon-strated that the oxidation of reduced sulfur species was a viable metabolism in this chemical environ-ment under both oxic (using modern day concentra-tions of oxygen in the martian atmosphere [7]) and anoxic (denitrification-enabled [8]) conditions. The enrichments performed under simulated martian chemical conditions confirmed that the Colour Peak Spring sediment contained microbes that were via-ble under reconstructed martian chemical condi-tions. However, whilst sulfide oxidising bacteria were viable in the enrichments, sulfate reducing bacteria dominated the enriched communities.\ud In the unenriched community, non-autotrophic, fermentative bacteria were also detected as being active. Given the low concentration of carbon in the sediment and the persistence of bacteria that are dependent on an exogenous supply of organic car-bon, the community profile suggests that the sulfur oxidising bacteria may be driving primary produc-tion in this environment. The simulation experi-ments support the possible role of primary produc-ers supporting the persistence of additional diversi-ty, with the enrichments comprising clades of bacte-ria associated with autotrophy and additional het-erotrophic bacteria. The potential for the auto-trophic sulfur-cycling bacteria to enable the surviv-al of heterotrophic bacteria within the sediment has implications for the viability of metabolisms on Mars, since syntrophy may facilitate a greater di-versity of metabolisms.\ud Conclusion: This study highlights the potential role of oxidation of reduced sulfur species as a me-tabolism on Mars using either oxygen or nitrate as an electron acceptor. This needs further characteri-sation with regards to its viability in a martian con-text. It also shows the importance of community dynamics and the role of syntrophy when consider-ing the viability of metabolisms under terrestrial and martian chemical conditions.\ud Acknowledgments: We would like to acknowledge Hugo Moors from the Belgian Nuclear Research Center for his advice on handling nucleic acids extracted from saline environments. We would like to thank Gordon Osinski from Western University, Ontario for leading the sampling trip to Axel Heiberg island in 2017. We would like to acknowledge funding from the Science and Technology Facilities Council, Leverhulme Trust for funding and the Polar Continental Shelf Program (Natural Resources Cana-da) for logistical field support in Nunavut.