The Microbial Community of a Terrestrial Anoxic Inter-Tidal Zone: A Model for Laboratory-Based Studies of Potentially Habitable Ancient Lacustrine Systems on Mars

Evidence indicates that Gale crater on Mars harboured a fluvio-lacustrine environment that was subjected to physio-chemical variations such as changes in redox conditions and evaporation with salinity changes, over time. Microbial communities from terrestrial environmental analogues sites are important for studying such potential habitability environments on early Mars, especially in laboratory-based simulation experiments. Traditionally, such studies have predominantly focused on microorganisms from extreme terrestrial environments. These are applicable to a range of Martian environments; however, they lack relevance to the lacustrine systems. In this study, we characterise an anoxic inter-tidal zone as a terrestrial analogue for the Gale crater lake system according to its chemical and physical properties, and its microbiological community. The sub-surface inter-tidal environment of the River Dee estuary, United Kingdom (53°21'015.40" N, 3°10'024.95" W) was selected and compared with available data from Early Hesperian-time Gale crater, and temperature, redox, and pH were similar. Compared to subsurface ‘groundwater’-type fluids invoked for the Gale subsurface, salinity was higher at the River Dee site, which are more comparable to increases in salinity that likely occurred as the Gale crater lake evolved. Similarities in clay abundance indicated similar access to, specifically, the bio-essential elements Mg, Fe and K. The River Dee microbial community consisted of taxa that were known to have members that could utilise chemolithoautotrophic and chemoorganoheterotrophic metabolism and such a mixed metabolic capability would potentially have been feasible on Mars. Microorganisms isolated from the site were able to grow under environment conditions that, based on mineralogical data, were similar to that of the Gale crater’s aqueous environment at Yellowknife Bay. Thus, the results from this study suggest that the microbial community from an anoxic inter-tidal zone is a plausible terrestrial analogue for studying habitability of fluvio-lacustrine systems on early Mars, using laboratory-based simulation experiments

Molecular, isotopic and <i>in situ</i> analytical approaches to the study of meteoritic organic material

Organic materials isolated from carbonaceous meteorites provide us with a record of pre-biotic chemistry in the early Solar System. Molecular, isotopic and in situ studies of these materials suggest that a number of extraterrestrial environments have contributed to the inventory of organic matter in the early Solar System including interstellar space, the Solar nebula and meteorite parent bodies. There are several difficulties that have to be overcome in the study of the organic constituents of meteorites. Contamination by terrestrial biogenic organic matter is an ever-present concern and a wide variety of contaminant molecules have been isolated and identified including essential plant oils, derived from either biological sources or common cleaning products, and aliphatic hydrocarbons, most probably derived from petroleum-derived pollutants. Only 25% of the organic matter in carbonaceous chondrites is amenable to extraction with organic solvents; the remainder is present as a complex macromolecular aromatic network that has required the development of analytical approaches that can yield structural and isotopic information on this highly complex material. Stable isotopic studies have been of paramount importance in understanding the origins of meteoritic organic matter and have provided evidence for the incorporation of interstellar molecules within meteoritic material. Extending isotopic studies to the molecular level is yielding new insights into both the sources of meteoritic organic matter and the processes that have modified it. Organic matter in meteorites is intimately associated with silicate minerals and the in situ examination of the relationships between organic and inorganic components is crucial to our understanding of the role of asteroidal processes in the modification of organic matter and, in particular, the role of water as both a solvent and a reactant on meteorite parent bodies

Viable metabolisms in a simulated martian environments

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

Microbial growth in simulated martian environments

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

A Study of the Microbial Community at the Interface between Granite Bedrock and Soil Using a Culture-Independent and Culture-Dependent Approach

The dissolution of minerals plays an important role in the formation of soils and sediments. In nutrient limiting soils, minerals constitute a major reservoir of bio-essential cations. Of particular interest is granite as it is the major rock type of the continental land mass. Although certain bacteria have been shown to enhance weathering of granite-forming minerals, little is known about the dissolution of granite, at the whole rock scale, and the microbial community involved. In this study, both culture-independent and culture-dependent approaches were used to study the bacterial community at the interface between granite bedrock and nutrient limiting soil in Dartmoor National Park, United Kingdom. High throughput sequencing demonstrated that over 70% of the bacterial population consisted of the bacterial classes Bacilli, Beta-proteobacteria and Gamma-proteobacteria. Bacteria belonging to the genera Serratia, Pseudomonas, Bacillus, Paenibacillus, Chromobacterium and Burkholderia were isolated from the sample site. All of the isolates were able to grow in a minimal growth medium, which contained glucose and ammonium chloride, with granite as the sole source of bio-essential elements. Sixty six percent of the isolates significantly enhanced basalt dissolution (p < 0.05). Dissolution of Si, K, Ca and Mg correlated with production of oxalic acid and acidification. The results of this study suggest that microorganisms in nutrient limiting soils can enhance the rate of granite dissolution, which is an important part of the biogeochemical cycle

Testing the habitability of distinct simulated martian environments

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

A culture-independent and culture-dependent study of the bacteria community from the bedrock soil interface

In nutrient limited soils, minerals constitute a major reservoir of bio-essential elements. Consequently, the release of nutritive elements during weathering is crucial. Bacteria have been shown to enhance weathering rates; however, there has been limited work that has focused on the bacterial weathering of bedrock or parent rock, which are the major sources of minerals, in nutrient limiting soils. In this study, both a culture-independent and culture-dependent approach was used to study the bacterial community at the interface between basaltic bedrock and nutrient limiting soil in Cadiar Idris region of Snowdonia National Park, United Kingdom. High throughput sequencing method, Ion Torrent, was used to characterise the bacterial community, which generated over 250,000 sequences. Taxonomical assignment demonstrated that approximately 50% (125,000 sequences) of the community consisted of the orders Actinomycetales, Burkholderiales, Clostridales, Bacillales, Rhizobiales and Acidobacterium, with unclassified sequences representing 44% ± 1.46% (110,000 ± 3650). Bacteria belonging to the genera Serratia, Pseudomonas, Bacillus, Paenibacillus, Chromobacterium, Janthinobacterium, Burkholderia and Arthrobacter, were isolated from the sample site. All of the isolates were able to grow in a minimal growth medium, which contained glucose, ammonium chloride with basalt as the sole source of bio-essential elements. Seventy percent of the isolates significantly enhanced basalt dissolution (p < 0.05). The rate of dissolution correlated to the production of oxalic acid and acidification of the growth medium. The findings of this work suggest that at the interface between bedrock and soil heterotrophic members of the bacterial community can enhance weathering, an essential part of biogeochemical cycling in nutrient limiting soil

Colour Peak:An analogue environment for the waters of late Noachian Mars

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. 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. 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. 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

Geochemical bio-signatures in Martian analogue basaltic environments using laboratory experiments and thermochemical modelling

The identification of geochemical bio-signatures is important for assessing whether life existed on early Mars. In this paper, experimental microbiology and thermochemical modelling were combined to identify potential inorganic bio-signatures for life detection on early Mars. An analogue mixed microbial community from an analogue terrestrial fluvio-lacustrine environment similar to an ancient lacustrine system at Gale Crater was used to study microbial dissolution of a basalt regolith simulant and the formation of bio-signatures over a short time frame (1°month) at 14°C, 2 bar. Microbial growth influenced element dissolution (Mg, Fe, Mn, Ca and K) and the formation of morphologies and Fe-Si amorphous layers on mineral surfaces. Thermochemical models were performed at 14°C, 2 bar; the results were compared with experimental data to predict bio-signatures that would occur over geological timescales. The pH was varied to simulate abiotic and biotic experimental conditions. Model results suggest that, at water to rock ratios of 100 to 38, a less complex secondary mineral assemblage forms during biotic dissolution compared to abiotic weathering. Carbonates, quartz, pyrite and hydroxyapatite form under biotic conditions, whereas in the abiotic system magnetite and phyllosilicates would also precipitate. These results could be used to distinguish between abiotic and biotic basalt weathering processes, aiding the interpretation of data from Mars exploration missions

Oligotrophic Growth of Nitrate-Dependent Fe ²⁺ -Oxidising Microorganisms Under Simulated Early Martian Conditions

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 Fe3+ 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 Fe3+ oxidation. Each of the strains was also able to grow in oligotrophic minimal media with olivine as the sole Fe3+ 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