Assembly of Bacterial Genome Sequences from Metagenomes of Spacecraft Assembly Cleanrooms

Characterizing the microbiome of spacecraft assembly cleanrooms is important for planetary protection. We report two bacterial metagenome-assembled genomes (MAGs) reconstructed from metagenomes produced from cleanroom samples from the Kennedy Space Center’s Payload Hazardous Servicing Facility (KSC-PHSF) during the handling of the Phoenix spacecraft. Characterization of these MAGs will enable identification of the strategies underpinning their survival

Western Sahara salt plains as a potential novel Mars analogue

The identification of novel terrestrial sites that are analogous for other planetary bodies is an active area of research within astrobiology, because of the logistical and financial difficulties in obtaining extraterrestrial samples for analysis. Characterisation of potential analogue sites is undertaken to assess how accurately they represent a specific extraterrestrial environment. Analysing their physicochemical conditions and microbial communities are key components of these studies to understand what metabolisms would be viable in such environments. One such novel analogue environment is the salt plains of Western Sahara. Western Sahara is one of the driest regions on Earth. It is located on the northwest coast of West Africa and is characterised by high UV exposure, low annual precipitation and water activity, subsurface water and high annual temperatures. These features make Western Sahara a potential analogue site for Mars during the Noachian-Hesperian transition period (3.5 – 3.8 Ga), when the atmosphere began to thin and surface water started evaporating (Warner et al., 2010), similar to other terrestrial deserts, such as the Atacama Desert and the McMurdo Dry Valleys. The hypersalinity, aridity and high UV radiation levels of the Western Sahara salt plains would also be appropriate to study whether dissimilatory sulfur metabolisms would be viable in a Noachian-Hesperian Mars analogue environment. Dissimilatory sulfur cycling refers to the use of inorganic sulfur compounds for energy conservation and it has been recognised as a metabolic strategy of interest for putative martian life (Macey et al., 2020). On Earth, evidence from stable sulfur isotope fractionation has suggested this metabolism emerged early in the history of life (~3.5 Ga). During this period, the conditions on Mars were predicted as being more habitable than present-day, with an active magnetic field, thicker atmosphere and liquid water on the surface. In this study, molecular and geochemical techniques were used to give first insights into the potential of the Western Sahara salt plains to serve as an analogue of Mars during the Noachian-Hesperian transition period. The microbiology was investigated through cultivation-independent and culture-dependent analyses of salt crystals, sediment and water samples obtained at three sites near Llaayoune (Fig. 1). The chemical nature of the samples was analysed through ion chromatography (IC) and inductively coupled plasma - optical emission spectrometry (ICP-OES). The geochemical characterisation confirmed the high salinity of the samples and identified that sodium, potassium, magnesium and sulfur were the most enriched elements within all samples. Cultivation-dependent work resulted in the enrichment of a wide range of metabolic strategies from the samples including aerobic heterotrophs, phototrophs and sulfate-reducers. The enrichments from the salt were dominated by strains of Bacillus, whereas sulfate-reducing strains of Clostridium were isolated from the sediment samples. Microscope analysis of phototroph-selective media also indicated that algae and Cyanobacteria were successfully enriched from the samples. 16S rRNA amplicon sequencing results will also be presented to gain further in-depth understanding of the microbial community composition. Additionally, results from quantitative polymerase chain reaction (qPCR) experiment targeting sox and dsr genes will be presented to identify the abundance of genes specific for dissimilatory sulfur metabolisms within the samples. Preliminary data shows that sulfur cycling is occurring in Western Sahara salt plains. Future characterisation of this environment will involve metagenomic analysis of the samples and genome sequencing of the isolates to identify the key metabolisms underpinning the survival and viability of the microbial community. Comparative studies with other Mars analogue environments will then be undertaken to identify metabolisms that may have been thermodynamically viable in ancient martian aqueous environments

Functional potential of microbial communities within the Western Sahara salt plains and implications for life on early Mars

Mars is the most studied astrobiological target, with extensive geochemical and morphological data collected via satellite and lander/rover missions. These data indicate that liquid water, bio-essential elements, and possible energy sources such as compounds in different oxidation states (e.g., sulfates and sulfides) were present on the surface of early Mars, thereby rendering it potentially habitable for terrestrial-like life. An understanding of relevant geochemical and biological processes on early Mars can be developed through chemically-relevant analogue environments on Earth. For example, the Western Sahara salt plains are an analogue for salt-rich Noachian/Hesperian-aged terrain, representing a period when atmospheric loss resulted in widespread surface water evaporation and concentration of salt phases within fluids. In this study, salt crystals, water and sediment were collected from the Western Sahara salt plains. To identify the suitability of the study site as an analogue for early Mars, the chemistry of the samples was studied by ion chromatography (IC) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The microbiomes of the samples were characterised through 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing of extracted DNA. Enrichments with liquid Postgate media were set up to identify metabolically-active sulfate reducing microbes. The chemical analyses showed that sodium and chloride ions were most prevalent in the water and salt crystal samples, suggesting that halite was the dominant salt phase. Notably, there was a high concentration of sulfate ions, ~1.5 – 2 g/L, supporting the relevance of the site as a Mars analogue for sulfate-rich salt deposits on Mars. 16S rRNA gene amplicon sequencing showed that moderately halophilic and extremely halophilic Bacteria and Archaea were most abundant across all sample types. Sequences belonging to sulfate reducing bacteria (SRB) and sulfur oxidising bacteria (SOB) were identified in the sediment, suggesting that active microbial sulfur cycling is present. Metagenomic analysis revealed that the functional capacity of the salt microbiome is different from the functional capacity of the sediment microbiome. Specifically, genes associated with nitrogen cycling were highly abundant in the salt crystal samples, whereas genes associated with sulfur cycling and methanogenesis/methanotrophy were abundant in the sediment samples. Moreover, high-quality metagenome assembled genomes (MAGs) of the dominant taxa and sulfur cycling microbes were assembled from the salt and sediment samples, allowing the reconstruction of metabolic pathways that might be active at the study site. Finally, microbial enrichments from the environmental samples successfully isolated sulfate reducing bacteria from the sediment, indicating that microbial sulfur cycling is active in the site. The results of this study add to the existing knowledge of microbial life in hypersaline environments and showcase a halite-rich Mars analogue environment with high sulfate content and the potential for biogeochemical sulfur cycling. Future work will focus on full genome sequencing of the isolated sulfate reducing species and isolation of the sulfur oxidising species identified in the sequencing data

Active microbial sulfur cycling in the Western Sahara salt plains and implications for life on early Mars

The Western Sahara salt plains are a potential analogue of early Mars. Within astrobiology, defining the physicochemical limits of terrestrial life is essential for precisely constraining the possibility of finding evidence of extant or extinct life in the Solar System. Mars is the most studied astrobiological target with extensive geochemical and morphological data collected via satellite and lander/rover missions. These data indicate that liquid water, bio-essential elements, and possible energy source such as sulfur compounds in different oxidation states (sulfides and sulfates) were present on the surface of early Mars, thereby rendering it plausibly habitable for terrestrial-like life. An understanding of relevant geochemical and biological processes on early Mars can be developed through chemically-relevant analogue environments on Earth. In this study, samples of salt crystals, water and sediment were collected from the Western Sahara salt plains. To identify the suitability of the study site as an analogue for early Mars, the chemistry of the samples was studied by ion chromatography (IC) and inductively coupled plasma optical emission spectroscopy (ICP-OES). The microbiomes of the samples were characterised through 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing of extracted DNA. Enrichments were set up to identify metabolically-active microbes within the collected samples. The chemical analyses showed that sodium and chloride ions were most prevalent in the water and salt crystal samples, confirming that halite was the dominant salt phase. Notably, there was a high concentration of sulfate ions, supporting the relevance of the site as a Mars analogue. The taxonomic diversity of the microbiomes determined through 16S rRNA gene amplicon sequencing showed that moderately halophilic and extremely halophilic Bacteria and Archaea were most abundant across all sample types. High-quality metagenome assembled genomes (MAGs) of the dominant taxa and a sulfur-cycling microbe were assembled from the salt and sediment samples, allowing the reconstruction of metabolic pathways that might be active at the study site. Finally, microbial enrichments from the environmental samples successfully isolated a range of halophilic heterotrophs from all sample types and sulfate-reducing bacteria from the sediment, indicating that microbial sulfur cycling is active in the site. The results of this study add to the existing knowledge of microbial life in hypersaline environments and showcase a Mars analogue environment with active biogeochemical sulfur cycling. Future work will focus on isolation and genome sequencing of sulfur cycling microbes identified in the sequencing dat

First insights into the chemistry and microbial community composition of the Western Sahara salt plains, a potential Mars analogue

Microorganisms that use inorganic sulfur compounds for energy conservation are dissimilatory sulfur cyclers. There is a great diversity among the metabolic strategies and biochemical pathways that these microbes use, which results in the production of sulfur compounds in varied chemical forms and oxidation states. The high abundance of sulfur cycling microbes in nature has made them an important contributor to the evolution of the biogeochemical sulfur cycle on Earth. The identification of mineral assemblages on the surface of Mars and within martian meteorites that contain sulfur species in different redox states has made dissimilatory sulfur cycling metabolisms of interest for possible extinct or extant life on Mars. A viable strategy to understand whether putative martian life could have been sustained through dissimilatory sulfur cycling is to study the prevalence of sulfur cycling microbes in Mars analogue environments. One such analogue environment is the Western Sahara salt plains, which are hypersaline, arid environments with high levels of UV exposure, which we propose as a suitable analogue for the Noachian-Hesperian transition period on Mars, characterised with thinning atmosphere and evaporation of surface water. In this study, molecular and geochemical techniques were used to give the first insights into the Western Sahara salt plains. The microbiology was investigated through cultivation-independent and culture-dependent analyses of salt crystals, sediment and water samples obtained at three sites near Llaayoune. The chemical nature of the samples was analysed through ion chromatography (IC) and inductively coupled plasma - optical emission spectrometry (ICP-OES). The geochemical characterisation confirmed the high salinity of the samples and identified that sodium, potassium, magnesium and sulfur were the most enriched elements within all samples. 16S rRNA gene amplicon sequencing of the samples identified a high relative abundance of sulfate reducing bacteria (SRB), Cyanobacteria, and Bacillus. To complement the 16S rRNA gene amplicon sequencing, enrichments were established to isolate aerobic heterotrophs, phototrophs and SRBs. The enrichments from the salt were dominated by strains of Bacillus, whereas sulfate-reducing strains of Clostridium were isolated from the sediment samples. Microscopic analysis of phototroph-selective media also indicated that algae and Cyanobacteria were successfully enriched from the samples. The preliminary analysis has confirmed that there is active sulfur-cycling occurring in the environment. Future work will involve metagenomic analysis of the samples and genome sequencing of the isolates to identify the key metabolisms underpinning the survival and viability of the microbial community. Comparative studies with other Mars analogue environments will then be undertaken to identify metabolisms that may have been thermodynamically viable in ancient martian aqueous environments