Variable and Large Losses of Diagnostic Biomarkers After Simulated Cosmic Radiation Exposure in Clay- and Carbonate-Rich Mars Analog Samples.

Mars has been exposed to ionizing radiation for several billion years, and as part of the search for life on the Red Planet, it is crucial to understand the impact of radiation on biosignature preservation. Several NASA and ESA missions are looking for evidence of ancient life in samples collected at depths shallow enough that they have been impacted by galactic cosmic rays (GCRs). In this study, we exposed a diverse set of Mars analog samples to 0.9 Megagray (MGy) of gamma radiation to mimic 15 million years of exposure on the Martian surface. We measured no significant impact of GCRs on the total organic carbon (TOC) and bulk stable C isotopes in samples with initial TOC concentration > 0.1 wt. %; however, diagnostic molecular biosignatures presented a wide range of degradation that didn't correlate to factors like mineralogy, TOC, water content, and surface area. Exposure dating suggests that the surface of Gale crater has been irradiated at more than five times our dose, yet using this relatively low dose and "best-case scenario" geologically recalcitrant biomarkers, large and variable losses were nevertheless evident. Our results empasize the importance of selecting sampling sites at depth or recently exposed at the Martian surface.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Survival strategies of an anoxic microbial ecosystem in Lake Untersee, a potential analog for Enceladus.

Lake Untersee located in Eastern Antarctica, is a perennially ice-covered lake. At the bottom of its southern basin lies 20 m of anoxic, methane rich, stratified water, making it a good analog for Enceladus, a moon of Saturn. Here we present the first metagenomic study of this basin and detail the community composition and functional potential of the microbial communities at 92 m, 99 m depths and within the anoxic sediment. A diverse and well-populated microbial community was found, presenting the potential for Enceladus to have a diverse and abundant community. We also explored methanogenesis, sulfur metabolism, and nitrogen metabolism, given the potential presence of these compounds on Enceladus. We found an abundance of these pathways offering a variety of metabolic strategies. Additionally, the extreme conditions of the anoxic basin make it optimal for testing spaceflight technology and life detection methods for future Enceladus exploration.

Draft Genome Sequence from a Putative New Genus and Species in the Family M1A02 within the Phylum Planctomycetes, Isolated from Benthic Pinnacle Mats in Lake Untersee, Antarctica.

Here, we report the draft genome sequence for a new putative genus and species in the family M1A02 within the order Phycisphaerales. Isolated from the metagenome of a benthic pinnacle-shaped mat in the Antarctic Lake Untersee, the members of this family have been found in biofilms and freshwater environments.

The Grayness of the Origin of Life.

In the search for life beyond Earth, distinguishing the living from the non-living is paramount. However, this distinction is often elusive, as the origin of life is likely a stepwise evolutionary process, not a singular event. Regardless of the favored origin of life model, an inherent "grayness" blurs the theorized threshold defining life. Here, we explore the ambiguities between the biotic and the abiotic at the origin of life. The role of grayness extends into later transitions as well. By recognizing the limitations posed by grayness, life detection researchers will be better able to develop methods sensitive to prebiotic chemical systems and life with alternative biochemistries.

Fatty Acid Preservation in Modern and Relict Hot-Spring Deposits in Iceland, with Implications for Organics Detection on Mars.

Hydrothermal spring deposits host unique microbial ecosystems and have the capacity to preserve microbial communities as biosignatures within siliceous sinter layers. This quality makes terrestrial hot springs appealing natural laboratories to study the preservation of both organic and morphologic biosignatures. The discovery of hydrothermal deposits on Mars has called attention to these hot springs as Mars-analog environments, driving forward the study of biosignature preservation in these settings to help prepare future missions targeting the recovery of biosignatures from martian hot-spring deposits. This study quantifies the fatty acid load in three Icelandic hot-spring deposits ranging from modern and inactive to relict. Samples were collected from both the surface and 2-18 cm in depth to approximate the drilling capabilities of current and upcoming Mars rovers. To determine the preservation potential of organics in siliceous sinter deposits, fatty acid analyses were performed with pyrolysis-gas chromatography-mass spectrometry (GC-MS) utilizing thermochemolysis with tetramethylammonium hydroxide (TMAH). This technique is available on both current and upcoming Mars rovers. Results reveal that fatty acids are often degraded in the subsurface relative to surface samples but are preserved and detectable with the TMAH pyrolysis-GC-MS method. Hot-spring mid-to-distal aprons are often the best texturally and geomorphically definable feature in older, degraded terrestrial sinter systems and are therefore most readily detectable on Mars from orbital images. These findings have implications for the detection of organics in martian hydrothermal systems as they suggest that organics might be detectable on Mars in relatively recent hot-spring deposits, but preservation likely deteriorates over geological timescales. Rovers with thermochemolysis pyrolysis-GC-MS instrumentation may be able to detect fatty acids in hot-spring deposits if the organics are relatively young; therefore, martian landing site and sample selection are of paramount importance in the search for organics on Mars.

Lipid Biomarkers in Ephemeral Acid Salt Lake Mudflat/Sandflat Sediments: Implications for Mars.

Sedimentary strata on Mars often contain a mix of sulfates, iron oxides, chlorides, and phyllosilicates, a mineral assemblage that is unique on Earth to acid brine environments. To help characterize the astrobiological potential of depositional environments with similar minerals present, samples from four naturally occurring acidic salt lakes and adjacent mudflats/sandflats in the vicinity of Norseman, Western Australia, were collected and analyzed. Lipid biomarkers were extracted and quantified, revealing biomarkers from vascular plants alongside trace microbial lipids. The resilience of lipids from dead organic material in these acid saline sediments through the pervasive stages of early diagenesis lends support to the idea that sulfates, in tandem with phyllosilicates and iron oxides, could be a viable target for biomarkers on Mars. To fully understand the astrobiological potential of these depositional environments, additional investigations of organic preservation in ancient acidic saline sedimentary environments are needed.

Life and its traces in Antarctica's McMurdo Dry Valley paleolakes: a survey of preservation.

The extremely cold and arid conditions of Antarctica make it uniquely positioned to investigate fundamental questions regarding the persistence of life in extreme environments. Within the McMurdo Dry Valleys and surrounding mountain ranges are multiple ancient relict lakes, paleolakes, with lacustrine deposits spanning from thousands to millions of years in age. Here we present data from light microscopy, scanning electron microscopy, electron dispersive x-ray spectroscopy, and radiocarbon dating to catalog the remarkable range of life preserved within these deposits. This includes intact microbes and nanobacteria-sized cocci, CaCO3 precipitations consistent with biogenic calcium, previously undescribed net-like structures, possible dormant spores, and long-extinct yet exquisitely preserved non-vascular plants. These images provide an important reference for further microbiome investigations of Antarctic paleolake samples. In addition, these findings may provide a visual reference for the use of subsurface groundwater microbial communities as an analog for paleolake subsurface water on planets such as Mars.

Draft Genome Sequence from a Putative New Genus and Species in the Family Methanoregulaceae Isolated from the Anoxic Basin of Lake Untersee in East Antarctica.

Here, we report the draft genome sequence for a new putative genus and species in the Methanoregulaceae family, whose members are generally slow-growing rod-shaped or coccoid methanogenic archaea. The information on this sediment-dwelling organism sheds light on the prokaryotes inhabiting isolated, deep, and extremely cold methane-rich environments.

Recovery of Fatty Acids from Mineralogic Mars Analogs by TMAH Thermochemolysis for the Sample Analysis at Mars Wet Chemistry Experiment on the Curiosity Rover.

The Mars Curiosity rover carries a diverse instrument payload to characterize habitable environments in the sedimentary layers of Aeolis Mons. One of these instruments is Sample Analysis at Mars (SAM), which contains a mass spectrometer that is capable of detecting organic compounds via pyrolysis gas chromatography mass spectrometry (py-GC-MS). To identify polar organic molecules, the SAM instrument carries the thermochemolysis reagent tetramethylammonium hydroxide (TMAH) in methanol (hereafter referred to as TMAH). TMAH can liberate fatty acids bound in macromolecules or chemically bound monomers associated with mineral phases and make these organics detectable via gas chromatography mass spectrometry (GC-MS) by methylation. Fatty acids, a type of carboxylic acid that contains a carboxyl functional group, are of particular interest given their presence in both biotic and abiotic materials. This work represents the first analyses of a suite of Mars-analog samples using the TMAH experiment under select SAM-like conditions. Samples analyzed include iron oxyhydroxides and iron oxyhydroxysulfates, a mixture of iron oxides/oxyhydroxides and clays, iron sulfide, siliceous sinter, carbonates, and shale. The TMAH experiments produced detectable signals under SAM-like pyrolysis conditions when organics were present either at high concentrations or in geologically modern systems. Although only a few analog samples exhibited a high abundance and variety of fatty acid methyl esters (FAMEs), FAMEs were detected in the majority of analog samples tested. When utilized, the TMAH thermochemolysis experiment on SAM could be an opportunity to detect organic molecules bound in macromolecules on Mars. The detection of a FAME profile is of great astrobiological interest, as it could provide information regarding the source of martian organic material detected by SAM.

Radiation Tolerance of Nanopore Sequencing Technology for Life Detection on Mars and Europa.

The search for life beyond Earth is a key motivator in space exploration. Informational polymers, like DNA and RNA, are key biosignatures for life as we know it. The MinION is a miniature DNA sequencer based on versatile nanopore technology that could be implemented on future planetary missions. A critical unanswered question is whether the MinION and its protein-based nanopores can withstand increased radiation exposure outside Earth's shielding magnetic field. We evaluated the effects of ionizing radiation on the MinION platform - including flow cells, reagents, and hardware - and discovered limited performance loss when exposed to ionizing doses comparable to a mission to Mars. Targets with harsher radiation environments, like Europa, would require improved radiation resistance via additional shielding or design refinements.

Microbial communities and their predicted metabolic functions in a desiccating acid salt lake.

The waters of Lake Magic in Western Australia are among the most geochemically extreme on Earth. This ephemeral saline lake is characterized by pH as low as 1.6 salinity as high as 32% total dissolved solids, and unusually complex geochemistry, including extremely high concentrations of aluminum, silica, and iron. We examined the microbial composition and putative function in this extreme acid brine environment by analyzing lake water, groundwater, and sediment samples collected during the austral summer near peak evapoconcentration. Our results reveal that the lake water metagenome, surprisingly, was comprised of mostly eukaryote sequences, particularly fungi and to a lesser extent, green algae. Groundwater and sediment samples were dominated by acidophilic Firmicutes, with eukaryotic community members only detected at low abundances. The lake water bacterial community was less diverse than that in groundwater and sediment, and was overwhelmingly represented by a single OTU affiliated with Salinisphaera. Pathways associated with halotolerance were found in the metagenomes, as were genes associated with biosynthesis of protective carotenoids. During periods of complete desiccation of the lake, we hypothesize that dormancy and entrapment in fluid inclusions in halite crystals may increase long-term survival, leading to the resilience of complex eukaryotes in this extreme environment.

Insights from the metagenome of an acid salt lake: the role of biology in an extreme depositional environment.

The extremely acidic brine lakes of the Yilgarn Craton of Western Australia are home to some of the most biologically challenging waters on Earth. In this study, we employed metagenomic shotgun sequencing to generate a microbial profile of the depositional environment associated with the sulfur-rich sediments of one such lake. Of the 1.5 M high-quality reads generated, 0.25 M were mapped to protein features, which in turn provide new insights into the metabolic function of this community. In particular, 45 diverse genes associated with sulfur metabolism were identified, the majority of which were linked to either the conversion of sulfate to adenylylsulfate and the subsequent production of sulfide from sulfite or the oxidation of sulfide, elemental sulfur, and thiosulfate via the sulfur oxidation (Sox) system. This is the first metagenomic study of an acidic, hypersaline depositional environment, and we present evidence for a surprisingly high level of microbial diversity. Our findings also illuminate the possibility that we may be meaningfully underestimating the effects of biology on the chemistry of these sulfur-rich sediments, thereby influencing our understanding of past geobiological conditions that may have been present on Earth as well as early Mars.

The most conserved genome segments for life detection on Earth and other planets.

On Earth, very simple but powerful methods to detect and classify broad taxa of life by the polymerase chain reaction (PCR) are now standard practice. Using DNA primers corresponding to the 16S ribosomal RNA gene, one can survey a sample from any environment for its microbial inhabitants. Due to massive meteoritic exchange between Earth and Mars (as well as other planets), a reasonable case can be made for life on Mars or other planets to be related to life on Earth. In this case, the supremely sensitive technologies used to study life on Earth, including in extreme environments, can be applied to the search for life on other planets. Though the 16S gene has become the standard for life detection on Earth, no genome comparisons have established that the ribosomal genes are, in fact, the most conserved DNA segments across the kingdoms of life. We present here a computational comparison of full genomes from 13 diverse organisms from the Archaea, Bacteria, and Eucarya to identify genetic sequences conserved across the widest divisions of life. Our results identify the 16S and 23S ribosomal RNA genes as well as other universally conserved nucleotide sequences in genes encoding particular classes of transfer RNAs and within the nucleotide binding domains of ABC transporters as the most conserved DNA sequence segments across phylogeny. This set of sequences defines a core set of DNA regions that have changed the least over billions of years of evolution and provides a means to identify and classify divergent life, including ancestrally related life on other planets.

Ancient bacteria show evidence of DNA repair.

Recent claims of cultivable ancient bacteria within sealed environments highlight our limited understanding of the mechanisms behind long-term cell survival. It remains unclear how dormancy, a favored explanation for extended cellular persistence, can cope with spontaneous genomic decay over geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long-term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability.