Enceladus: Astrobiology Revisited

Abstract

Astrobiology research seeks to understand how life begins and evolves, and to determine whether life exist elsewhere in the universe. The discovery of diverse ocean worlds has significantly expanded the number of planetary bodies in the Solar System that could potentially contain life. Of the recognized ocean worlds, Saturn's moon Enceladus stands out because it appears to meet all requirements to sustain life. For that reason, robotic mission concepts are being developed to determine whether Enceladus' ocean is inhabited. The theory of organic chemical evolution (OCE) represents an ideal framework to guide this exploration strategy, articulating investigations and associated measurements of organic matter in the subsurface ocean. Within this reference frame, the immediate priority with the lowest science risk would be to understand molecular and structural properties of bulk organic matter in the ocean, and search for metabolic precursors and biochemical building blocks, both free and bound. This could be supplemented with “high-risk, high-reward” searches for functional polymers, catalytic activity, and cell-like objects with traits indicative of evolutionary adaptations. The theory of OCE provides a robust scientific foundation for the astrobiological exploration of ocean worlds, fostering a productive path to discovery with lower mission risk that could be implemented with existing technology. Strong synergies between astrobiology and Earth-bound research could ensue from this exploration strategy particularly in the context of terrestrial analog studies and laboratory simulations.

Plain Language Summary

There is a diversity of “ocean worlds” in our Solar System, which are of great scientific interest. Enceladus, a small moon of Saturn, has a global subsurface ocean that could sustain life and contains complex organic matter. To further understand the biological potential of Enceladus, and other ocean worlds, we need to consider how abiotic and prebiotic chemistry in Enceladus's ocean might play a role in the origin of life. The theory of organic chemical evolution provides the ideal framework to address this question. The top priority would be to study the organic inventory in the ocean, and to search for the basic building blocks of life, as well as simple compounds involved in metabolic processes. Next, we should search for complex polymers and cell-like structures with traits suggesting Darwinian evolution. This exploration strategy is a solid foundation for discovery and can be done with current technology, which lowers the risk and complexity of spaceflight missions.

Key Points

• Enceladus is one of the most compelling destinations in the solar system for exobiology exploration

• The theory of organic chemical evolution provides a framework for the continued and systematic exploration of Enceladus and other ocean worlds

• With this exploration framework biotic, abiotic, and prebiotic scenarios are all possible outcomes with profound implications

1 Introduction

The term “exobiology” was coined in the late 1950s in reference to the possibility of “cosmic microbiology” and “lunar biology,” and potential threats of the incipient space race to those life forms (Lederberg, 1960). In the ensuing decade, exobiology research emphasized microbial adaptations to extreme environments and the development of devices that could detect microorganisms on another planet (DeVincenzi, 1984), leading to the eventual launch of the first extraterrestrial biology experiments on the Mars Viking landers (Klein, 1979).

The focus of exobiology shifted in the 1980s toward understanding the origin of life on Earth, including the role of the physical and chemical environment, and the nature and extent of organic chemistry throughout the cosmos (DeVincenzi, 1984; Klein, 1986; NASA, 1988). The exploration of other solar system bodies remained an important aspect of the program, with the goal of understanding the extent to which prebiological evolution has proceeded outside the Earth (NASA, 1988).

Scientific discoveries in the mid 1990s catalyzed the establishment of a new program in astrobiology at NASA, which incorporated the existing exobiology program. The new astrobiology program framed its research around three fundamental questions: (a) How does life begin and evolve? (b) Does life exist elsewhere in the universe? (c) What is life's future on Earth and beyond? (NASA, 1988). These questions were adapted by the science community as guide for a new field of study (Des Marais et al., 2008).

In the coming decades, the exploration of ocean worlds—planetary bodies with a current liquid ocean (Hendrix et al., 2019) offers a tremendous opportunity to advance the field of astrobiology to significant new heights. To date, there are six confirmed ocean worlds other than Earth (Callisto, Ganymede, Europa, Enceladus, Titan, and Mimas) (Hendrix et al., 2019; Lainey et al., 2024). Each ocean world represents an opportunity to investigate protracted OCE on a planetary scale independently from terrestrial biology, consistent with the tenets and aspirations of the original exobiology program. In this paper, we highlight Saturn's moon Enceladus as one of the most interesting and immediate astrobiology targets in the solar system, based on previous studies conducted by the Cassini spacecraft.

We propose an exploration strategy for Enceladus that focuses on testing specific aspects of the theory of organic chemical evolution (OCE). The discovery of life on Enceladus is one possible outcome of this exploration effort, but abiotic and prebiotic scenarios should also be considered and given significant intellectual value, as they could reveal important clues regarding the conditions in which life originated on our planet. The study of OCE at Enceladus, and other ocean worlds, would likely catalyze strong synergies between astrobiology and earth sciences.

2 The Enceladus Opportunity

In 2005, the Cassini spacecraft discovered a large plume of ice and gas emanating from four giant fractures on the South Polar Terrain of Enceladus (Figure 1) (Porco et al., 2006). The plume source appears to be a subsurface ocean of liquid water (Iess et al., 2014; Postberg et al., 2009; Thomas et al., 2016). Plume materials erupt as diffuse “curtains” of water vapor punctured by localized “jets” of gas and ice grains approximately 0.1–10 μm in size (Porco et al., 2014; Spitale et al., 2015). Cassini's Cosmic Dust analyzer (CDA) detected three types of ice grains in the plume: pure water-ice grains (Type I), organic-rich grains (Type II), and salt-rich grains (Type III). Type I grains likely form from vapor condensation inside the tiger stripe fissures. Type II and Type III grains likely represent frozen ocean spray (Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018; Postberg et al., 2023).

Figure 1

A plume of gas and ice grains emanates from the South Polar Terrain on Enceladus. The plume source is a global ocean of liquid water beneath the moon's icy crust. Plume materials erupt as diffuse “curtains” of water vapor punctured by ∼100 localized “jets” of gas and ice grains primarily 0.1–10 μm in size (Porco et al., 2006; Spitale et al., 2015). Small ice grains escape Enceladus' gravity and populate Saturn's E-ring, but larger, slower grains are ejected to an altitude of 30–50 km and then fall back (Goldstein et al., 2018). Image Credit: NASA/JPL-Caltech/Space Science Institute.

Analyses by the Cassini spacecraft of plume gases and ice suggest that the Enceladus ocean meets the requirements to sustain life (Cable et al., 2020; Waite et al., 2017), including the presence of bioessential elements (Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018; Postberg et al., 2023; Waite et al., 2017); geochemical conditions (temperature, pH, salinity) within the tolerance range of terrestrial organisms (Glein et al., 2015; Postberg et al., 2009; Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018; Taubner et al., 2018); and a redox pair (H₂ and CO₂) that chemoautotrophic life could use as an energy source (Hoehler, 2022; Waite et al., 2017).

Plume materials are also organic-rich. Besides CH₄, Cassini's Ion Neutral Mass Spectrometer (INMS) detected HCN, methanol, formaldehyde, and small hydrocarbons (Peter et al., 2023; Waite et al., 2009). These volatile compounds likely are fragments of larger molecules generated during high-velocity impacts of Type II ice grains with instrument walls (Postberg, Clark, et al., 2018, Postberg et al., 2023). Type II ice grains contain up to tens of millimolar abundance of low-mass organic cations <60 Da in size (Khawaja et al., 2019; Postberg, Clark, et al., 2018, Postberg et al., 2023). A fraction of Type II ice grains also contains concentrated macromolecular organic compounds up to at least ∼2,100 Da in size, with prominent peaks at 200–300, 800–1,100, and 1,600–1,800 Da (Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018; Postberg et al., 2023). The spectral properties of this macromolecular material are characteristic of hydrocarbons with functional groups that contain O and probably N (Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018; Postberg et al., 2023).

Cassini's payload instruments were not designed to identify individual organic compounds. The CDA relied on hypervelocity impact ionization to fragment molecules (Srama et al., 2004), and the INMS measured bulk gas composition (neutral and positive ions) after electron impact ionization in the instrument (Waite et al., 2004). Important source-diagnostic information encoded in the structure and chemical composition of parent molecules was lost during those measurements. Fitting algorithms are often used to model the composition of bulk measurements (e.g., Goesmann et al., 2015), but overlap in mass spectral signatures prevents their direct detection, and accurate identification of their molecular structure. Without chemical separation or higher resolution mass spectrometry with fragmentation, identification of compound structures is not possible, and organic sources are difficult to constrain.

The origin of organic matter in the Enceladus ocean is one of the most pressing and exciting questions in astrobiology. Astrobiological exploration often emphasizes the search for biosignatures—objects, substances, and/or patterns whose origin specifically requires a biological agent (Des Marais et al., 2008). However, a narrow focus on biosignatures in the case of Enceladus would carry significant scientific risk because it presupposes an origin of life in the subsurface ocean. This assumption is unsupported because we do not know where or how life originated on Earth, or how likely it is for life to originate even under favorable conditions, on Earth or elsewhere. A search for biosignatures also carries the risk of “confirmation bias,” by favoring data or interpretations that confirm the discovery of life while ignoring abiotic or prebiotic scenarios. Indeed, abiotic processes can lead to complex organic chemical systems that exhibit life-like properties, blurring the distinction between non-life and life (Barge et al., 2022). Low production rates, low accumulated abundances, dilution, and degradation over time can render biosignatures undetectable by existing instruments, which increases the probability of false negative results. Lastly, a focus on biosignatures also carries programmatic risk because it defines success as a binary proposition: either evidence of life is discovered, or the mission has failed to achieve its objective (J. Green et al., 2021).

We advocate instead for a more thorough, comprehensive, and impartial strategy framed around the theory of OCE. Rather than asking the question “is Enceladus inhabited?” we ask the question “what is the extent of OCE in Enceladus' ocean?”, which addresses abiotic, prebiotic and biological scenarios. The theory of OCE provides the framework for exploration, through progressive and systematic investigations that combine general, inclusive, and lower risk measurements with targeted, stringent, and higher risk ones (Table 1).

Table 1. The Theory of Organic Chemical Evolution as a Framework for the Exploration of Enceladus, and Other Ocean Worlds

	Theme	Investigation	Example measurements
	Organic synthesis	Bulk organic inventory	-Molecular structure and chemical composition of bulk organic matter -Diversity of functional groups -Molecular Assembly Index
		Primordial compounds involved in early stages of abiotic organic synthesis and possibly carbon and energy metabolism	-Presence of methane; acetate; formate; formaldehyde; pyruvate, etc.
		Biochemical building blocks	-Abundance distributions of monomers -Isomeric ratios of monomers -Molecular structure
	Polymerization	Polymers with repeating subunits	-Presence of oligopeptides; saccharides; nucleotides -Structural patterns in polymers
	Functionality	Replication Catalysis	-Presence of polyelectrolytes -Hydrolysis
	Evolution	Cell-like structures Metabolic activity Cell behavior	-Chemistry/Shape/Form of cell-like structures -Motility -Size/number of cell-like structures -Carbon fixation; respiration

• Note. Each stage in OCE is a research theme that encompasses a set of investigations and associated measurements. Multiple working hypotheses can already be formulated during the early stages of OCE (gray) to determine the origin, biotic or abiotic, of organic matter in Enceladus' ocean (see Table 2).

3 Organic Chemical Evolution: Knowledge and Gaps

The theory of OCE states that life is the result of a series of prebiotic chemical steps whereby complex chemical compounds and molecular assemblages originate from reactions between simpler molecules, and eventually combine to form the first cell (Figure 2).

Figure 2

Schematic representation of the main steps in the theory of organic chemical evolution (OCE). In Stage 1, simple inorganic precursors exploit free energy to form small organic molecules. In Stage 2, reactions between small organic and inorganic molecules and minerals generates biochemical building blocks. In Stage 3, biochemical building blocks undergo condensation reactions and form small organic polymers. In Stage 4, organic polymers become structurally complex and display emergent autocatalytic properties such as the ability to self-replicate or to sustain cyclic networks reminiscent of metabolic cycles. In Stage 5, a self-copying organic polymer would emerge that can make (inexact) copies of itself and is susceptible to chemical alterations (mutations). Compartmentalization may be a prerequisite for the emergence of autocatalysis and evolution by natural selection (e.g., Deamer et al., 2002), leading to the first cells and the first cell lineages. In stage 6, competition for resources between genetic lineages can cause an explosion of forms and functions, and different cell lineages that slowly or rapidly diverge from each other. Applicable to every stage of OCE, organic materials, particularly more functionalized compounds, may encounter environmental instability after formation, making them susceptible to alteration.

The linear sequence outlined in Figure 2 is an oversimplification. In nature, OCE is an iterative, cyclic process that takes place in vast multicomponent mixtures, with myriads of reactants and products across a broad chemical space of molecular compositions and structures (Guttenberg et al., 2017). Indeed, one of the great mysteries of the origin of life is how a limited subset of molecular building blocks and functional polymers crucial to life can become enriched or segregated from this “messy chemistry.”

For all its predictive power, the theory of OCE still has significant gaps because we lack a well-preserved record of the prebiotic world from where terrestrial life originated. For example, several fundamentally different groups of ideas have been proposed to explain the mechanisms that drive chemical complexity toward self-replication (Figure 2; Stages 3–5): (a) replicator first theories (e.g., Higgs & Lehman, 2015); (b) metabolism-first theories (e.g., Martin et al., 2008); and (c) membrane-first theories (e.g., Deamer et al., 2002).

Consensus is also lacking regarding the types of environments that can support abiogenesis, with most scenarios falling in one of two generic groups: (a) subaerial aquatic environments that undergo wet-dry cycles (e.g., Damer & Deamer, 2020); and (b) submarine hydrothermal vents (e.g., Martin & Russell, 2007). Shallow-sea hydrothermal vents represent a third option that integrates both endmembers (Barge & Price, 2022), while other models advocate for cold or subfreezing environments (Miyakawa et al., 2002).

More specific aspects of OCE also remain puzzling. It is unclear how biochemistry's chiral asymmetry can arise from complex and largely racemic prebiotic chemical mixtures. Some have argued for a slight chiral bias in the early stages of OCE that is amplified over time leading to the emergence of homochiral polymers (Bonner, 1998). Others have argued for the emergence of chiral bias after the origin of life due to natural selection (M. M. Green & Jain, 2010; Konstantinov & Konstantinova, 2022).

Progress has been made to fill these knowledge gaps through laboratory studies (Beyazay et al., 2023; Cody et al., 2000; Huber & Wächtershäuser, 1997; Hudson et al., 2020; McCollom et al., 1999; Miller & Urey, 1959; Moser et al., 1968; Varma et al., 2018) and through analyses of astromaterials (e.g., Burton et al., 2012; Cooper & Rios, 2016; Ehrenfreund et al., 2001; Elsila et al., 2016; Glavin et al., 2020; Pizzarello et al., 2006; Sandford et al., 2006). This progress invites hopes that chemical evolution from non-life to life may eventually be recapitulated as a single continuous process.

However, most laboratory studies of OCE focus on replicating the steps that led to the formation of compounds observed in extant biochemistry. The possibility remains that other prebiotic organic compounds may have existed and played important roles in the origin and early evolution of life, but were not incorporated into biochemistry, or were part of incipient biochemistries that eventually were replaced or became extinct (Bartel & Unrau, 1999; Eschenmoser, 2007). Because laboratory approaches are necessarily limited in complexity and scope, they cannot recreate the chemical complexity of environments with protracted liquid water activity, chemical disequilibrium, and constant flux of reactants, or the timescales needed to drive OCE from simple inorganic precursors to protocells (Deamer et al., 2022; Pascal et al., 2013).

On the other hand, analyses of astromaterials, particularly chondrites, shed light on the earliest stages of OCE (Figure 2; Stages 1–3), and on aqueous and radiolytic alteration that cause condensation reactions and the formation of insoluble heteropolymeric substances (e.g., Alexander et al., 2017). However, the chemical composition of meteorites, comets, and asteroids is unlikely to provide new insights on the late stages of OCE, which are critical to understand the origin and early evolution of life (Figure 1; Stages 4–6).

Applicable to every stage of OCE, organic materials, particularly more functionalized compounds, may encounter environmental instability after formation, making them susceptible to alteration. As a result, alteration products often overprint those most diagnostic of each evolutionary stage (see Figure 2), complicating environmental records. Such organic transformations can result from multiple diagenetic processes and events occurring immediately after organic synthesis and over geological time. Alteration processes may also differ spatially due to varying conditions; products may be autochthonous or allochthonous in nature dependent on transport mechanisms; and mixing of different pools of products is possible. Furthermore, geological materials ranging from mineral surfaces to ocean aggregates or hydrothermal vent precipitates may catalyze reactions, physically partition organic matter within their inorganic structures, and shield chemicals from oxidants or reductants by physical isolation.

Alteration processes can significantly change the inventory of compounds and their accessibility to reactions, and may be constructive and destructive agents in the continuum of OCE, contributing to “messy chemistry” associated with prebiotic chemistry (Guttenberg et al., 2017). Hiatus in alteration processes is only possible with physical and chemical stability, which is rare for dynamic systems.

The exploration of Enceladus is an opportunity to investigate protracted OCE, including alteration processes, on a planetary scale, circumventing many challenges and limitations of laboratory research and studies of astromaterials. We focus our analysis on Enceladus because it is the best studied ocean world to date. However, each ocean world is a unique environment with geological and geochemical idiosyncrasies, and can provide important insights into OCE on planetary systems.

4 The Exploration of Enceladus in the Context of Organic Chemical Evolution

Within the framework of the theory of OCE, the most immediate goal for the astrobiological exploration of Enceladus with the lowest scientific risk would be to search for compounds characteristic of the early stages of OCE (Figure 2, Stages 1–3), particularly in the context of hydrothermal vent models for the origin of life (e.g., Martin & Russell, 2007). According to those models, inorganic compounds such as H₂, H₂O, CO₂, and NH₃, all of which were detected by Cassini, are precursors of small organic molecules such a CH₄, HCN, and CH₃OH (Rimmer & Shorttle, 2019). Notably, CH₄ was also observed by Cassini's INMS (Waite et al., 2009, 2017), and HCN has been tentatively detected based on statistical models of INMS spectra (Peter et al., 2023).

Hydrothermal vent models further predict the formation of metabolic precursors such as acetate and formate, and of biochemical building blocks such as amino acids and small carboxylic acids, through mineral-catalyzed geochemical reactions on vent walls (Beyazay et al., 2023; Hudson et al., 2020; McCollom & Seewald, 2007; Simoneit, 2004). According to these models, biochemical building blocks would then undergo condensation reactions and form small organic polymers such as peptides and lipids. Indeed, oligomerization of amino acids has been shown to occur under various hydrothermal conditions (e.g., Imai et al., 1999).

The search for simple metabolic precursors and biochemical building blocks, either in free form or bound in small polymers, represents a logical follow-up to Cassini, and would be an important juxtaposition to chemical analyses of other astromaterials. Relevant biochemical building blocks to target include amino acids, oligopeptides, sugars, nucleobases and nucleotides, short- and long-chain carboxylic acids, and more chemically functionalized lipids (Figure 2). The presence of biochemical building blocks in ocean samples would allow testing multiple hypotheses for the origin of organic matter in the subsurface ocean. This is because these compounds represent a chemical “sweet spot” where products of abiotic and biotic synthesis overlap (Figure 2). Under abiotic conditions, these building blocks comprise mainly smaller, simpler molecules whose origin can be explained by kinetic and thermodynamic principles alone, invoking simple inorganic precursors. However, in the presence of life these building blocks show a higher degree of functionality, selectivity, and complexity that points to evolutionary adaptations. These distinctions allow to differentiate biotic from abiotic sources of organic matter (Davila & McKay, 2014; Dorn et al., 2011; Glavin et al., 2020; Lovelock, 1973; Marshall et al., 2021; McKay, 2010; Summons et al., 2008) (Table 2).

Table 2. Measurements of Bulk Organic Matter and of Biochemical Building Blocks Such as Amino Acids and Carbocyclic Acids Allow to Test Multiple Working Hypotheses Regarding the Sources, Biotic or Abiotic, of Organic Matter in the Enceladus Ocean

	Hypotheses	Measurement	Proposed criteria
Organic Inventory	Abiotic ocean	-Molecular assembly index (Marshall et al., 2021)	-Organic mixtures with high molecular assembly values (e.g., MA > 15) are rare or absent^a.
	Biotic ocean		-Organic mixtures with high molecular assembly values (e.g., MA > 15) are common and diverse^a.
Amino acids	Abiotic ocean	-Abundance distribution (Dorn et al., 2011) -Enantiomeric ratios (Glavin et al., 2020) -Isotopic composition (Glavin et al., 2020) -Degree of polymerization	-Smaller amino acids (e.g., Gly; Ala; AiB) dominate. -Equal proportions of L/D-enantiomers. -Enriched in heavy isotopes (D, 13C, and 15N) -Peptides are rare or absent. If present, they contain 2 subunits (dipeptides) -Peptides made primarily of Gly subunits
	Biotic ocean		-Most or all amino acids present in similar abundances. -Amino acids of the same enantiomeric form (L or D) predominate. -Depleted in D, ^13C, and ^15N, relative to the inorganic substrate -Peptides are present and contain ≥3 subunits -Peptides made of diverse amino acid subunits
Carboxylic acids	Abiotic ocean	-Abundance distribution (Lovelock, 1973) -Molecular structure (Summons et al., 2008)	-Long-chain carboxylic acids (>10C) are rare and do not display a modal distribution -Long-chain carboxylic acids do not display periodic patterns in abundance
	Biotic ocean		-Long-chain carboxylic acids (>10C) predominate and display a modal distribution with few dominant carbon sizes. -Long-chain carboxylic acids display characteristic structural patterns (e.g., odd-vs-even)

• Note. The proposed criteria to validate each hypothesis assumes a dominant biotic or abiotic endmember. If Enceladus had a mixture of biotic and abiotic chemistry this would likely lead to intermediate scenarios were only some biotic criteria would be fulfilled (see text for further details).
• ^a The molecular assembly index is a proxy for the number of joining operations (e.g., formation of chemical bonds) needed to generate a product (e.g., a molecular structure). A MA threshold of 15 to differentiate biotic from abiotic organic mixtures is based on the analysis of a relatively small set of samples, and therefore this threshold value may be subject to revisions.

The search for metabolic precursors and biochemical building blocks could be supplemented with chemical and structural measurements of bulk organic matter (organic compound inventory), including the types and diversity of functional groups, and the presence of heteroatoms (O, S, P, N). Detailed analyses of the bulk organic fraction would provide chemical context to understand organic sources and possible alteration processes in the subsurface ocean.

A null result is an inherent risk in space exploration. The absence in ocean samples of metabolic precursors and biochemical building blocks could reflect a lack of sources—biotic and abiotic—or point to processes in the ocean that segregate, alter, or destroy organic matter. Both would place important constraints on the theory of OCE and challenge theories for the origin of life at submarine hydrothermal vents. However, the risk of a null result appears to be low in the case of Enceladus because conditions in the subsurface ocean are seemingly favorable for the preservation of organic matter (low temperature; low radiation; anoxia), and the Cassini spacecraft already detected organic compounds across a broad range of molecular sizes, with inferred organic abundances in individual plume ice particles up to tens of mmol levels (Khawaja et al., 2019). A small fraction of plume ice grains analyzed by Cassini's CDA showed an abundant signature at 18 amu ([NH₄]⁺ within the water ion cluster) and an unspecified organic peak between 26 and 31 amu, upon impact with the instrument (Khawaja et al., 2019), which could be indicative of amines, amides, or nitriles (Khawaja et al., 2019), and are also consistent with the fragmentation patterns of amino acids (Klenner et al., 2020). Larger mass fragments detected by Cassini's CDA suggest aliphatic hydrocarbons with >100 amu (Postberg et al., 2023; Postberg, Clark, et al., 2018; Postberg, Khawaja, et al., 2018) that are consistent with lipid-like components.

On the other hand, a positive result would represent the first detection of metabolic precursors or biochemical building blocks in a habitable environment beyond Earth; an important validation of the theory or OCE and a positive test of hydrothermal vent models for the origin of life. One possible outcome from these investigations is the discovery of biochemical building blocks that only meet abiotic criteria, akin for example, to meteoritic or cometary materials (see Table 2). This would point to a habitable subsurface ocean that is otherwise sterile. The exploration of a sterile subsurface ocean that can seemingly support life would have significant scientific value as it would represent a control habitat to study protracted and planetary scale geochemical processes in the absence of life (Cockell et al., 2012). Concomitant analyses of the bulk organic inventory could reveal other prebiotic organic compounds that may have existed and played important roles in the origin and early evolution of life on Earth, but were not incorporated into biochemistry, or were part of incipient biochemistries that eventually were replaced or became extinct (Bartel & Unrau, 1999; Eschenmoser, 2007).

Another possible outcome is the discovery of biochemical building blocks that meet biotic criteria (see Table 2). A preponderance of data consistent with biotic sources of biochemical building blocks could already be considered a successful life detection experiment. However, a mixed scenario where biotic and abiotic sources coexist is also possible. This could point to a prebiotic state, slowly evolving toward life, or a biosphere that is too tenuous to entirely dominate OCE. Such a mixed scenario could be disambiguated with a search for more specific—and higher risk—indicators of advanced OCE (Figure 2; Stage 4–6) including, but not limited to, polyelectrolytes with a repeating charge; catalytic activity; and cell-like objects (Benner, 2017; Georgiou et al., 2023; Nadeau et al., 2016).

It has been argued that self-replicating polymers in aquatic environments (Figure 2; Stage 4) will by necessity be polyelectrolytes with a repeating charge (Benner & Hutter, 2002). A repeating negative charge on the backbone of a linear polymer addresses important biochemical requirements, such as solubility in water, and supports Darwinian evolution by permitting random chemical changes (e.g., replacement of nucleobases) without significantly altering the chemical properties of the polymer (Benner, 2017). Polyelectrolytes can be detected with current sequencing technologies that are insensitive to the actual chemistry of the polymer (Carr et al., 2020), and therefore can be used to search for life even if the details of its biochemistry are unknown.

Catalytic activity has also been proposed as a universal functional marker of life in ocean worlds, as it is required to speed up organic reactions to sustain biological function, growth and replication (Georgiou et al., 2023). Catalytic activity is likely to manifest already in a prebiotic state, and is an inherent part of the postulated evolutionary processes leading to the emergence of life (Nowak & Ohtsuki, 2008), and can be measured directly by means of nutrient uptake and waste release, or by quantifying the rates of certain chemical reactions such as hydrolysis (Georgiou et al., 2023).

Finally, cellular organization is likely to be a universal trait of life, and the detection of cells in samples from the Enceladus ocean would be difficult to refute as a successful life detection experiment. However, recognizing cells in natural samples can be challenging based on structural or morphological grounds alone (García-Ruiz et al., 2003). Additional measurements of motility (Nadeau et al., 2016) or a combination of morphological and chemical analyses (e.g., Hand et al., 2022) are necessary orthogonal lines of evidence that allow to discriminate true cells from abiotic mimics.

The search for polyelectrolytes, catalytic activity, or cells in the Enceladus ocean would be a paradigm shift, and likely usher a new era of astrobiology research, paving the way for studies of comparative biology on a cosmic scale. However, these investigations are especially risky in the case of Enceladus where energy availability could limit biological abundance, diversity, and productivity (Chyba & Phillips, 2001; Ray et al., 2021). Estimates of the potential biomass that could be generated in the Enceladus ocean vary by several orders of magnitude based on the types and fluxes of energy sources considered, but could be lower than the most biologically lean environments on Earth (Ray et al., 2021). Based on the biomass content in low-productivity regions of Earth's ocean, a search for polyelectrolytes and measurements of catalytic activity could require significant volumes of sample (MacKenzie et al., 2021) and a search for cell-like objects would have to contend with the possibility of extremely low cell densities (Ray et al., 2021), all of which may pose technical challenges for sample acquisition and handling.

The exploration strategy outlined in this paper could be implemented with a flagship-class mission (MacKenzie et al., 2021) or it could require a multigenerational approach. The latter is not new in planetary exploration, as best exemplified by the armada of orbiters, landers and rovers that have explored Mars since the mid 1970s, and the more than 30-year succession of astrophysics observatories (i.e., Hubble, James Webb Space Telescope, and future ROMAN telescope). Multigenerational strategies can take advantage of lessons learned from previous missions and of new technology development.

5 Synergies With Earth-Bound Research

The exploration strategy outlined in this paper ought to stimulate strong synergies with Earth-bound research, particularly regarding the early stages of OCE. For example, studies in terrestrial environments analogous to Enceladus (e.g., McDermott et al., 2015; Proskurowski et al., 2008), and time-dependent laboratory simulations of varying geochemical parameters (e.g., pH, iron redox state, chemical concentrations) under Enceladus-like conditions could provide clues to understand early stages of OCE in the Enceladus ocean, and the resulting organic product distributions (Barge et al., 2020). In that context, mineral-catalyzed reactions in terrestrial hydrothermal systems of the type envisioned at Enceladus, such as hydrothermal vents at Lost City (Kelley et al., 2001), have been shown to generate molecules observed in metabolic cycles (e.g., in the acetyl-CoA pathway) (Beyazay et al., 2023; Huber & Wächtershäuser, 1997; Hudson et al., 2020; Varma et al., 2018), as well as biochemical building blocks such as amino acids and small carboxylic acids (McCollom & Seewald, 2007; Ménez et al., 2018).

Laboratory simulations have also shown that oligopeptides can form by heating aqueous solutions of amino acids to temperatures of 200–250°C (Imai et al., 1999), but only at amino acid concentrations that are not prebiotically plausible (Cleaves et al., 2009; Danger et al., 2012). Di- and tripeptides have also been synthesized in the laboratory by mixing CO and H₂S with a slurry of Ni-S and Fe-S particles at 100°C (Huber & Wächtershäuser, 1997), which may replicate conditions in Enceladus' ocean floor. Oligomerization of amino acids under various hydrothermal conditions. Laboratory simulations can also shed light on the fate of organic mixtures exposed to hydrothermal conditions in the presence of serpentinite mineral assemblages (Salter et al., 2022; Tan et al., 2023). These types of studies help define requirements for spacecraft missions and provide important context for the interpretation of spacecraft data.

6 Concluding Remarks

Saturn's moon Enceladus represents one of the most exciting astrobiology opportunities in the solar system, given the presence of complex organic matter in an environment that could support life. A robust framework is now needed to foster a productive path for the astrobiological exploration of Enceladus with lower mission risk and which could be implemented with existing technology. The theory of OCE provides such a framework, which could be articulated as a set of investigations and associated measurements that minimize risk and maximize science return. The primary focus would be on the early stages of OCE, targeting compounds that are expected to be present in the ocean whether life exists there or not. Detection of these compounds, and measurements of certain “source-diagnostic” molecular properties, would provide a strong foundation to search for more complex organic compounds that display emergent biochemical properties, as well as cell-like objects and evidence of evolutionary adaptations. In this strategy, the discovery of extraterrestrial life is one possible experimental outcome, but abiotic and prebiotic scenarios are also considered and given significant intellectual value. Strong synergies between exobiology and Earth-bound research could ensue from this exploration strategy.

Acknowledgments

AD's support for this research was provided by NASA's Planetary Science Division Research Program, through ISFM work package Center for Life Detection at NASA Ames Research Center. JE's support was provided by NASA Goddard Space Flight Center's Solar System Exploration Division Strategic Science funding. We thank Chris McKay, Andrej Grubisic, and Dale T. Andersen for helpful comments and suggestions during the preparation of the manuscript, and three anonymous reviewers whose comments and suggestions helped improve the original submission.

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Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.