Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

Geochemical properties of blue carbon sediments through an elevation gradient: study of an anthropogenically impacted coastal lagoon.

Global research is showing that coastal blue carbon ecosystems are vulnerable to climate change driven threats including accelerated sea-level rise and prolonged periods of drought. Furthermore, direct anthropogenic impacts present immediate threats through deterioration of coastal water quality, land reclamation, long-term impact to sediment biogeochemical cycling. These threats will invariably alter the future efficacy of carbon (C) sequestration processes and it is imperative that currently existing blue carbon habitats be protected. Knowledge of underlying biogeochemical, physical and hydrological interactions occurring in functioning blue carbon habitats is essential for developing strategies to mitigate threats, and promote conditions to optimise C sequestration/storage. In this current work, we investigated how sediment geochemistry (0-10 cm depth) responds to elevation, an edaphic factor driven by long-term hydrological regimes consequently exerting control over particle sedimentation rates and vegetation succession. This study was performed in an anthropogenically impacted blue carbon habitat along a coastal ecotone encompassing an elevation gradient transect from intertidal sediments (un-vegetated and covered daily by tidal water), through vegetated salt marsh sediments (periodically covered by spring tides and flooding events), on Bull Island, Dublin Bay. We determined the quantity and distributions of bulk geochemical characteristics in sediments through the elevation gradient, including total organic carbon (TOC), total nitrogen (TN), total metals, silt, clay, and also, 16 individual polyaromatic hydrocarbon's (PAH's) as an indication of anthropogenic input. Elevation measurements for sample sites were determined on this gradient using a LiDAR scanner accompanied by an IGI inertial measurement unit (IMU) on board a light aircraft. Considering the gradient from the Tidal mud zone (T), through the low-mid marsh (M) to the most elevated upper marsh (H), there were significant differences between all zones for many measured environmental variables. The results of significance testing using Kruskal-Wallis analysis revealed, that %C, %N, PAH (µg/g), Mn (mg/kg), TOC:NH4 + and pH are significantly different between all zones on the elevation gradient. The highest values for all these variables exists (excluding pH which followed a reverse trend) in zone H, decreasing in zone M and lowest in the un-vegetated zone T. TC content is 16 fold higher overall in vegetated (3.43 -21.84%) than uninhabited (0.21-0.56%) sediments. TN was over 50 times higher (0.24-1.76%), more specifically increasing in % mass on approach to the upper salt marsh with distance from the tidal flats sediments zone T (0.002-0.05%). Clay and silt distributions were greatest in vegetated sediments, increasing in % content towards upper marsh zones The retention of water, metals, PAHs, mud, chloride ions, NH4 +, PO4 3- and SO4 2- increased with elevated C concentrations, concurrently where pH significantly decreased. Sediments were categorized with respect to PAH contamination where all SM samples were placed in the high polluted category. The results highlight the ability of Blue C sediments to immobilise increasing levels of C, N, and metals, and PAH with over time and with both lateral and vertical expansion. This study provides a valuable data set for an anthropogenically impacted blue carbon habitat predicted to suffer from sea-level rise and exponential urban development. Graphical abstract: Summarized results from this study demonstrating the geochemical changes through an elevation gradient, with a transect encompassing intertidal sediments through supratidal salt marsh sediments within Bull Island's blue carbon lagoon zones. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-00974-0.

Biogeochemical properties of blue carbon sediments influence the distribution and monomer composition of bacterial polyhydroxyalkanoates (PHA).

Coastal wetlands are highly efficient 'blue carbon' sinks which contribute to mitigating climate change through the long-term removal of atmospheric CO2 and capture of carbon (C). Microorganisms are integral to C sequestration in blue carbon sediments and face a myriad of natural and anthropogenic pressures yet their adaptive responses are poorly understood. One such response in bacteria is the alteration of biomass lipids, specifically through the accumulation of polyhydroxyalkanoates (PHAs) and alteration of membrane phospholipid fatty acids (PLFA). PHAs are highly reduced bacterial storage polymers that increase bacterial fitness in changing environments. In this study, we investigated the distribution of microbial PHA, PLFA profiles, community structure and response to changes in sediment geochemistry along an elevation gradient from intertidal to vegetated supratidal sediments. We found highest PHA accumulation, monomer diversity and expression of lipid stress indices in elevated and vegetated sediments where C, nitrogen (N), PAH and heavy metals increased, and pH was significantly lower. This was accompanied by a reduction in bacterial diversity and a shift to higher abundances of microbial community members favouring complex C degradation. Results presented here describe a connection between bacterial PHA accumulation, membrane lipid adaptation, microbial community composition and polluted C rich sediments. Graphical Abstract: Geochemical, microbiological and polyhydroxyalkanoate (PHA) gradient in a blue carbon zone. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-01008-5.

A prebiotic basis for ATP as the universal energy currency.

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

Spontaneous assembly of redox-active iron-sulfur clusters at low concentrations of cysteine.

Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO2 fixation. FeS clusters have structures similar to the unit-cell of FeS minerals such as greigite, found in hydrothermal systems linked with the origin of life. However, the prebiotic pathway from mineral surfaces to biological clusters is unknown. Here we show that FeS clusters form spontaneously through interactions of inorganic Fe2+/Fe3+ and S2- with micromolar concentrations of the amino acid cysteine in water at alkaline pH. Bicarbonate ions stabilize the clusters and even promote cluster formation alone at concentrations >10 mM, probably through salting-out effects. We demonstrate robust, concentration-dependent formation of [4Fe4S], [2Fe2S] and mononuclear iron clusters using UV-Vis spectroscopy, 57Fe-Mössbauer spectroscopy and 1H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO2 reduction could have formed spontaneously from monomers at the origin of life.

Promotion of protocell self-assembly from mixed amphiphiles at the origin of life.

Vesicles formed from single-chain amphiphiles (SCAs) such as fatty acids probably played an important role in the origin of life. A major criticism of the hypothesis that life arose in an early ocean hydrothermal environment is that hot temperatures, large pH gradients, high salinity and abundant divalent cations should preclude vesicle formation. However, these arguments are based on model vesicles using 1-3 SCAs, even though Fischer-Tropsch-type synthesis under hydrothermal conditions produces a wide array of fatty acids and 1-alkanols, including abundant C10-C15 compounds. Here, we show that mixtures of these C10-C15 SCAs form vesicles in aqueous solutions between pH ~6.5 and >12 at modern seawater concentrations of NaCl, Mg2+ and Ca2+. Adding C10 isoprenoids improves vesicle stability even further. Vesicles form most readily at temperatures of ~70 °C and require salinity and strongly alkaline conditions to self-assemble. Thus, alkaline hydrothermal conditions not only permit protocell formation at the origin of life but actively favour it.

Isoprenoids enhance the stability of fatty acid membranes at the emergence of life potentially leading to an early lipid divide.

Two key problems concern cell membranes during the emergence and early evolution of life: what was their initial composition, and why did the membranes of archaea and bacteria diverge? The composition of the first cell membranes could shed light on the most likely environment for the emergence of life. The opposing stereochemistry of modern lipid glycerol-phosphate headgroups in bacteria and archaea suggests that early membranes were composed of single chain amphiphiles, perhaps both fatty acids and isoprenoids. We investigated the effect of adding isoprenoids to fatty acid membranes using a combination of UV-visible spectroscopy, confocal microscopy and transmission electron microscopy. We tested the stability of these membranes across a pH range and under different concentrations of ionic species relevant to oceanic hydrothermal environments, including Na2+, Cl-, Mg2+, Ca2+, HC O 3 - , Fe3+, Fe2+ and S2-. We also tested the assembly of vesicles in the presence of Fe particles and FeS precipitates. We found that isoprenoids enhance the stability of membranes in the presence of salts but require 30-fold higher concentrations for membrane formation. Intriguingly, isoprenoids strongly inhibit the tendency of vesicles to aggregate together in the presence of either Fe particles or FeS precipitates. These striking physical differences in the stability and aggregation of protocells may have shaped the divergence of bacteria and archaea in early hydrothermal environments.

Possible mechanisms of CO2 reduction by H2 via prebiotic vectorial electrochemistry.

Methanogens are putatively ancestral autotrophs that reduce CO2 with H2 to form biomass using a membrane-bound, proton-motive Fe(Ni)S protein called the energy-converting hydrogenase (Ech). At the origin of life, geologically sustained H+ gradients across inorganic barriers containing Fe(Ni)S minerals could theoretically have driven CO2 reduction by H2 through vectorial chemistry in a similar way to Ech. pH modulation of the redox potentials of H2, CO2 and Fe(Ni)S minerals could in principle enable an otherwise endergonic reaction. Here, we analyse whether vectorial electrochemistry can facilitate the reduction of CO2 by H2 under alkaline hydrothermal conditions using a microfluidic reactor. We present pilot data showing that steep pH gradients of approximately 5 pH units can be sustained over greater than 5 h across Fe(Ni)S barriers, with H+-flux across the barrier about two million-fold faster than OH--flux. This high flux produces a calculated 3-pH unit-gradient (equating to 180 mV) across single approximately 25-nm Fe(Ni)S nanocrystals, which is close to that required to reduce CO2. However, the poor solubility of H2 at atmospheric pressure limits CO2 reduction by H2, explaining why organic synthesis has so far proved elusive in our reactor. Higher H2 concentration will be needed in future to facilitate CO2 reduction through prebiotic vectorial electrochemistry.

Large perturbations in CO2 flux and subsequent chemosynthesis are induced in agricultural soil by the addition of elemental sulfur.

The microbial contribution to soil organic matter has been shown to be much larger than previously thought and thus it plays a major role in carbon cycling. Among soil microorganisms, chemoautotrophs can fix CO2 without sunlight and can glean energy through the oxidation of reduced elements such as sulfur. Here we show that the addition of sulfur to soil results in an initial surge in production of CO2 through microbial respiration, followed by an order of magnitude increase in the capture of carbon from the atmosphere as elemental sulfur is oxidised to sulfate. Thiobacillus spp., take advantage of specific conditions to become the dominant chemoautotrophic group that consumes CO2. We discern the direct incorporation of atmospheric carbon into soil carbohydrate, protein and aliphatic compounds and differentiate these from existing biomass. These results suggest that chemoautotrophs can play a large role in carbon cycling and that this carbon is heavily influenced by land management practises.

Iron catalysis at the origin of life.

Iron-sulphur proteins are ancient and drive fundamental processes in cells, notably electron transfer and CO2 fixation. Iron-sulphur minerals with equivalent structures could have played a key role in the origin of life. However, the 'iron-sulphur world' hypothesis has had a mixed reception, with questions raised especially about the feasibility of a pyrites-pulled reverse Krebs cycle. Phylogenetics suggests that the earliest cells drove carbon and energy metabolism via the acetyl CoA pathway, which is also replete in Fe(Ni)S proteins. Deep differences between bacteria and archaea in this pathway obscure the ancestral state. These differences make sense if early cells depended on natural proton gradients in alkaline hydrothermal vents. If so, the acetyl CoA pathway diverged with the origins of active ion pumping, and ancestral CO2 fixation might have been equivalent to methanogens, which depend on a membrane-bound NiFe hydrogenase, energy converting hydrogenase. This uses the proton-motive force to reduce ferredoxin, thence CO2 . The mechanism suggests that pH could modulate reduction potential at the active site of the enzyme, facilitating the difficult reduction of CO2 by H2 . This mechanism could be generalised under abiotic conditions so that steep pH differences across semi-conducting Fe(Ni)S barriers drives not just the first steps of CO2 fixation to C1 and C2 organics such as CO, CH3 SH and CH3 COSH, but a series of similar carbonylation and hydrogenation reactions to form longer chain carboxylic acids such as pyruvate, oxaloacetate and α-ketoglutarate, as in the incomplete reverse Krebs cycle found in methanogens. We suggest that the closure of a complete reverse Krebs cycle, by regenerating acetyl CoA directly, displaced the acetyl CoA pathway from many modern groups. A later reliance on acetyl CoA and ATP eliminated the need for the proton-motive force to drive most steps of the reverse Krebs cycle. © 2017 IUBMB Life, 69(6):373-381, 2017.

The occurrence of PAHs and faecal sterols in Dublin Bay and their influence on sedimentary microbial communities.

The source, concentration, and potential impact of sewage discharge and incomplete organic matter (OM) combustion on sedimentary microbial populations were assessed in Dublin Bay, Ireland. Polycyclic aromatic hydrocarbons (PAHs) and faecal steroids were investigated in 30 surface sediment stations in the bay. Phospholipid fatty acid (PLFA) content at each station was used to identify and quantify the broad microbial groups present and the impact of particle size, total organic carbon (%TOC), total hydrogen (%H) and total nitrogen (%N) was also considered. Faecal sterols were found to be highest in areas with historical point sources of sewage discharge. PAH distribution was more strongly associated with areas of deposition containing high %silt and %clay content, suggesting that PAHs are from diffuse sources such as rainwater run-off and atmospheric deposition. The PAHs ranged from 12 to 3072ng/g, with 10 stations exceeding the suggested effect range low (ERL) for PAHs in marine sediments. PAH isomer pair ratios and sterol ratios were used to determine the source and extent of pollution. PLFAs were not impacted by sediment type or water depth but were strongly correlated to, and influenced by PAH and sewage levels. Certain biomarkers such as 10Me16:0, i17:0 and a17:0 were closely associated with PAH polluted sediments, while 16:1ω9, 16:1ω7c, Cy17:0, 18:1ω6, i16:0 and 15:0 all have strong positive correlations with faecal sterols. Overall, the results show that sedimentary microbial communities are impacted by anthropogenic pollution.

A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance.

The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography--mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life.