By Ana Menchaca, Biochemistry and Molecular Biology ‘20

Author’s Note: As a biochemistry major who is interested in pursuing astrobiology research, I initially wrote this literature review for an assignment in my Writing in Biology course. Methods of life detection and what we know about life is a field in which we still have much to discover and explore, given Earth as our only example, and I hope to be involved in this exploration myself in the future.

Abstract

Ocean worlds, such as Enceladus, Saturn’s largest moon, provide intriguing environments and the potential for life as we continue to explore the Solar System. Organic compounds have been discovered in plumes erupting from the moon during flybys and point towards the presence of amino acids and other precursors of life. The data collected from these flybys, in turn, has been used to calculate the theoretical amounts of amino acids present in the oceans of Enceladus. While this data is intriguing, it relies on a limited definition of life, based on organisms and macromolecules that have only been observed on Earth. Other methods, including using nucleic acids or nanopores for detection, have been proposed. Nucleic acids utilize binding to identify a broad spectrum of compounds, while nanopores utilize the measurement of ionic flow. These alternative methods allow for a broader spectrum of compound detection than terran-based methods, creating the potential to detect unfamiliar kinds of life. Research into more holistic detection should continue.

Keywords: astrobiology, life detection, planetary exploration, biosignatures

Introduction

The search for life elsewhere in the Solar System is becoming increasingly relevant, and more importantly, feasible. Icy moons, such as Europa, Titan, and Enceladus, have been identified as holding the greatest potential for extraterrestrial life within the Solar System [1]. Europa and Enceladus, with seas below icy crusts, have geysers with unidentified fluctuations, along with evidence of tidal warming and geologic activity [2]. The Cassini spacecraft identified these geysers on Enceladus during flyby in 2006, spouting from four specific fractures on the surface of the moon [3]. Analysis of the vapors produced show that they mainly consist of water, along with CO2, N2, CO, CH4, salts, other organic compounds, and silica particulates [3, 4]. This points towards evidence of hydrothermal activity, the movement of heated water, which has the potential to provide necessary energy for life [4]. Additionally, the discovery of volatile aliphatic hydrocarbons in these plumes potentially indicate some degree of organic evolution within the seas of Enceladus [4].

However, there is no consensus yet on how to detect and identify life [1, 2]. Some scientists propose looking for life based on the shared ancestry hypothesis, which proposes all life shares the same genetic ancestry [2]. Others propose there is a potential for extraterrestrial life to present variations from terran life that we may neither be able to recognize nor detect with our current methods of biochemical detection [5]. Experimentally, the potential for nucleic acids based on different backbones has already been identified [5]. Here, we examine the range of proposed methods for identifying extraterrestrial life. 

Proposed theories and methods based on current knowledge of life

Collection of amino acids

Current data collected from Enceladus’ plumes presents organic compounds that provide potential evidence of amino acid synthesis taking place in the oceans of the moon. Steel et al. used the thermal flux at the moon’s South Polar Terrain (SPT) to predict the hydrogen produced by hydrothermal activity. The predicted rates of production ranged between 0.63 and 33.8 mol/s of H2, and from there, amino acid production rates were estimated to be between 8.4 and 449.4 mmol/s [4]. Annual biomass production was also modelled in these calculations and estimated at 4 · 104  to 2 · 106 kg/year, compared to 1014 kg/year on Earth. These estimates, however, are dependent on the environment being an abiotic, steady state ocean; the actual production rates could be different if there is life present in Enceladus’ ocean [4]. 

While this limits our predictions of Enceladus’ true environment, it still provides a basis that can be extrapolated for use in the design of modules to be sent out. One such module that has been proposed is the Enceladus Organic Analyzer, which is designed to analyze amino acids through chain length variations [3]. To properly collect and analyze the amino acids proposed to be in Enceladus’ oceans, there are several requirements. The sample must be collected from the subsurface ocean with minimal degradation, isomerization, racimerization, and contamination of biological molecules and amino acids [3]. A collection chamber made of aluminum has been modeled, designed to reduce the thermal heating caused by collection of samples, in order to best preserve them. If the moon contains bacteria as postulated, this design will lyse and kill collected cells through either heat or shock but release their more stable chemical components for analysis [3]. This depends highly upon current knowledge as a starting place, focusing with a limited scope on amino acid and cell identification. Another such method using cell identification is digital holographic microscopy. 

Digital holographic microscopy

The development and improvements of microscopy, while beneficial, depend heavily on the assumption that life in the same form as terran cells will be found. Investigators propose digital holographic microscopy (DHM) as a more efficient alternative over traditional light microscopy [1]. This technology produces a 100-fold improvement in the depth of field and is able to monitor both intensity and phase of images. However, even with the increase in resolution, differentiation of cells and cell-shaped structures is difficult, even before taking into account potential differences in extraterrestrial life. Refraction, an emerging field, was able to differentiate experimentally between crystalline structures and cells in the study’s Arctic samples. While the technology can be miniaturized and discriminate between cells and minerals, it depends highly on actual capture of a sufficient number of cells from plumes. This experimental data was obtained using dye-less techniques, which still function in the context of organisms without DNA or RNA, and refraction with the potential to differentiate structures [1]. DHM is both useful for detection of cells based on collected data and for the potential discovery of organisms without nucleic acids as we know them. 

Expanding outside the current knowledge of life

Detection using nucleic acids
Other experiments and proposals, while not explicitly targeting life outside the current perceptions, propose a more holistic collection of data. This carries the potential of identifying life outside our current scope, as opposed to focusing directly on known amino acids and cells. Using a broader concept of nucleic acids as a means of detection and identification is one such method. 

Oligonucleotides, through forming secondary and tertiary structures, have specificity and affinity to a wide variety of molecules, both organic and inorganic [2]. Even at a length of only 15 base pairs and within complex mixtures, these molecules can bind to what is being analyzed, or the analytes. Systematic evolution of ligands by exponential enrichment (SELEX) is a process that can identify oligonucleotides that bind very specifically to analytes. However, this method proposes the use of low affinity and low specificity nucleic acids that are typically discarded in this process. Unlike antibodies, this method requires no prior knowledge of the surface attributes or the three-dimensional structure of the molecule that is being bound. Through accumulating a wide range of binding sequences and statistical analysis, a vast number of compounds can be collected and environmental variations identified. Additionally, this method posits that the optimal means of capturing sequences is through proximity ligation assay (PLA), a technique currently used in scientific fields. PLA purifies the binding species based on ligation and amplification, producing a lower background than sieving, which separates based on size. It is also capable of capturing a vast range of sequences and structures, including inorganic, organic, or polymeric molecules [2], and thus is more capable of providing holistic results.

Nanopore-based sensing

Nanopore-based sensing, presented as an alternative to current methods, detects and analyzes genetic information carriers in watery systems without making assumptions about its chemical composition [5]. This system relies upon the restrictions placed on these sorts of compounds within watery systems, as the repeating charge of backbones keeps polymer strands from folding and favors solubility in water. A nanopore is a hole with a diameter of a few nanometers, surrounded by an insulating membrane within two chambers containing an electrolyte solution. Due to its diameter, only single-stranded DNA can pass through the nanopore, allowing for slow movement and characteristic signals that produce data clearly distinguishable from other molecular data. This method can detect and analyze molecules by measuring the ionic flow across the membrane. While biological nanopores are able to detect and resolve individual terran bases, nonbiological, solid-state nanopores provide the same function, avoiding the limitations of detecting terran molecules that may be present in biological nanopores. Graphene, with its crystalline form, can have its membrane adjusted to only accommodate one nucleotide at a time or can be sculpted to produce varying sizes of nanopores. This could allow for the detection of other polymers with chemical and sterical properties that vary from currently known polymers. This approach has the potential to analyze a broad range of molecules without any assumptions regarding the external structure’s outside charge and linearity. Few identified nonbiological polymers are structured this way, so any data picked up by nanopores would be significant [5].

There are, however, limitations to this approach. Nucleic acids have high electroporation speeds, making it necessary to find methods of slowing these speeds down for accuracy [5]. Electroporation uses an electrical charge to make the cell membrane more permeable. Potential methods include control through physical factors, such as temperature, salinity, and viscosity. Conditions of collection on other planets also pose the problem of extreme dilution of the target molecules, which depends on a large number of variables [5]. 

Conclusion

Radiation and stability are major concerns in moving forward with any sort of data collection from extraterrestrial worlds. Mechanisms and samples are potentially open to the detrimental effects of extreme vacuum and solar radiation [5]. These problems should be addressed in conjunction with the technology actually being used for analysis to produce the most beneficial results. Some of these issues have been addressed to some extent, such as using microfluidics for collection because they are unaffected by the vacuum of space due to their own internal surface tension [3]. However, these problems need to be explored further in all cases to ensure that each method can function in uncontrolled or non-terran environments.

The presented data indicates potential for the existence of amino acids in these environments. Even though prediction and detection of these amino acids seems a logical step forward, the development of further, broader technology for life detection should also be pursued. Current knowledge is limited by the qualities of terran life; while that is a well-supported starting point, methods that leave open the potential of deviation from this point may allow for the detection of otherwise overlooked forms of life. Moving forward, it seems only logical to combine these methods that can detect both the known and the unknown, allowing scientists to gather the widest possible array of data in future missions, especially on promising worlds like Enceladus. 

References

1. Bedrossian M, Lindensmith C, Nadeau JL. 2016. Digital Holographic Microscopy, a Method for Detection of Microorganisms in Plume Samples from Enceladus and Other Icy Worlds. Astrobiology 17(9):913–925.
2. Johnson SS, Anslyn EV, Graham HV, Mahaffy PR, Ellington AD. 2018. Fingerprinting Non-Terran Biosignatures. Astrobiology 18(7):915–922.
3. Mathies RA, Razu ME, Kim J, Stockton AM, Turin P, Butterworth A. 2016. Feasibility of Detecting Bioorganic Compounds in Enceladus Plumes with the Enceladus Organic Analyzer. Astrobiology 17(9):902–912.
4. Steel EL, Davila A, Mckay CP. 2017. Abiotic and Biotic Formation of Amino Acids in the Enceladus Ocean. Astrobiology 17(9):862–875. 
5. Rezzonico F. 2014. Nanopore-Based Instruments as Biosensors for Future Planetary Missions. Astrobiology 14(4):344–351.