It has been hypothesized that life will form and evolve whenever the energetic, chemical, and geological conditions are met. Organic molecules, typical for life as we know it, are relatively easily formed and can be polymerized into larger molecules under the right chemical conditions [1
]. Urey and Miller revealed the abiotic formation of amino acids and related compounds in their famous 1953 experiment [3
]. This experiment was recently repeated with modern analytical techniques, with similar results [4
]. In another experiment, Levy et al.
] found that amino acids were produced abiotically in a frozen NH4
CH solution while Martins et al.
] showed the formation of several amino acids through a process called impact shock synthesis. Hydrocarbons can be synthesized by chemical reactions simulating high-pressure/temperature conditions (Fischer-Tropsch reactions) [7
], and nucleobases have been shown to form under simulated prebiotic conditions [10
], with formamide (HCONH2
) as the precursor molecule, which may have been available on the early Earth [12
]. These findings, also taking into account the detection of a wide variety of biologically relevant molecules in meteorites [13
], suggest that the building blocks of life as we know it are abundant throughout our solar system, which has intensified the search for life beyond Earth. However, when searching for molecular traces of life on other worlds, such as Mars, great care must be taken to distinguish between biotic and abiotic origin. Biologically produced or altered molecules hence have been termed biomarkers
and typically possess specific signatures that link them to biotic origin. Current and future life detection missions to Mars express a strong focus towards detecting those biomarkers as evidence for extant or extinct life [14
Early conditions on Mars, during the first billion years after planetary formation, may have allowed life to develop and remnants of it could have been preserved within protected niches [15
]. Current conditions on Mars include extreme aridity, freezing temperatures (average −60 °C) and high UV-flux, and are damaging to living organisms and their organic molecules, decreasing the chances for life to be present [16
]. Extreme environments on Earth are useful for astrobiologists since they often display environmental and geological parallels with current and past Martian conditions. Investigating those environments, and the effects they impose on life on Earth and the preservation of its associated biomarkers, has already contributed greatly to the search for life on Mars and other planets. Even in the most extreme environments on modern-day Earth, life forms are identified [18
]. Recent findings [20
] reveal that life can thrive in environments we thought previously uninhabitable, suggesting we have not yet encountered the limits of life on our own planet. Of special interest are subsurface environments where life has been cut off from sunlight, but instead manages to thrive solely on chemosynthesis, as observed in Movile Cave, Romania [23
]. The identification of ancient biomarkers on Earth [24
] suggests strong preservation potential for a subset of biomarkers under the right conditions, however large differences between preservation potential exist among biomarkers (see Figure 1
for a schematic representation of biomarker stability). Such observations provide important information on which biomarkers to look for and where to look for them on Mars.
The preservation potential of several biomarkers in Ka (thousand years) to Ga (billion years). Modified from Martins et al.
The preservation potential of several biomarkers in Ka (thousand years) to Ga (billion years). Modified from Martins et al.
The success of life detection depends, besides the obvious necessity of an organic inventory, on a trade-off between specificity, sensitivity and extraction efficiency of the applied techniques. Techniques in the field are continuously improving and molecules can be detected in the range between parts per billion and part per trillion (ppb-ppt) [29
]. This sensitivity is important since organic molecules are often strongly adsorbed to mineral surfaces, decreasing extraction and, thus, the chance for detection considerably [30
]. However, minerals are considered major targets for future missions to Mars due to their importance in biological processes. Polymerization of small molecules (e.g., amino acids) occurs on mineral surfaces [32
], linking them to the origin of life [33
]. Minerals also have nutritional value for microorganisms and it is becoming increasingly clear that specific minerals are selected by distinct microbial populations due to the absence/presence of certain trace elements [34
]. In addition, the organics-preserving effects observed for minerals makes them prime targets for the search for life on Mars [36
]. It is therefore of great importance that extraction of biomarkers from their environmental context is as efficient as possible.
Here, we review current approaches of sensitive life detection. Special attention will be given to a range of biomolecules indicative for life, such as DNA, amino acids, lipids, and their diagenetic breakdown products, and how useful they are as biomarkers based on their general properties, like preservation potential, specificity, and extractability.
We describe recent findings of terrestrial life in extreme environments and how these results may help in determining the most promising landing sites on Mars for future life detection missions. We have included the analysis of carbonaceous meteorites since they represent a unique extraterrestrial source of carbon compounds, which may have seeded planets in our solar system with organics through impact events. We also describe techniques currently in use for life detection on Earth and Mars and how techniques in development may improve sensitivity and efficiency of life detection. We conclude with a discussion concerning the implications for life detection on future planetary missions to Mars.
4. Techniques Currently in Use for Biomarker Detection: How to Look Here?
Life detection techniques generally comprise the extraction of biomolecules followed by an analytical component, such as High Performance Liquid Chromatography (HPLC) or Gas Chromatography (GC) coupled to Mass Spectrometry (MS) or fluorometric detection. Microarrays and immunoassays are also used to identify biomarkers. While genomic material can be multiplied using the Polymerase Chain Reaction (PCR), many other biomolecules are not prone to such multiplication techniques and thus need to be concentrated for detection and identification. Biomolecules or biota can be freely present in water reservoirs but can also be intercalated in the matrix of minerals, which obstructs efficient extraction [30
Efficient extraction of biomarkers from these minerals is the crucial step in detection because many minerals show high potential for the long-term preservation of biomolecules [157
]. In order to minimize biases in extraction due to soil particle size, a universal sample preparation method, which may include crushing, grinding or milling of the sample, has been proposed by Beaty et al.
One approach to enhance biomarker extraction is acid digestion, aimed at dissolving the entrapping mineral, and releasing the biomolecules from salt deposits like sulfates or other evaporate minerals [161
]. These sulfate-rich evaporates may be especially important with regard to the detection of life since microbial sulfate reduction is implicated in rocks that contain the oldest known traces of life dating 3.5 billion years back [162
]. Hydrofluoric acid has also been used to dissolve minerals and increase extraction yield, but was shown to have destructive effects on more fragile biomolecules such as DNA [30
]. However, other biomarkers may be more resistant to such an aggressive method, as was observed for proteins that were recovered from minerals by a similar approach [163
More commonly, a wide range of solvents is used to extract organics from their environment. Aqueous polar solvents are used to extract the polar substances (e.g., amino acids, DNA) and organic solvents are used to extract the non-polar organics (e.g., hydrocarbons, lipids). Solvents can be adjusted to improve extraction. Direito et al.
] used a hot phosphate-rich ethanolic buffer in the cell lysing step during DNA extraction from soil samples, reasoning that the phosphate would compete with the phosphate backbone of DNA for chemical binding to the soil matrix. This resulted in up to a hundredfold higher recovery of DNA. Such mechanism-based improvement of extraction could potentially also be used to enhance extraction of other biomarkers.
The use of surfactants in combination with aqueous solvents to apply combined solving power for polar and non-polar compounds is another good example of optimization of extraction/detection techniques [164
]. Surfactants are amphiphilic molecules, which makes them capable to interact with hydrophilic as well as with lipophilic compounds [167
]. The possibility to obtain both polar and non-polar molecules in one extraction is a large advantage over other extraction techniques and the exclusion of aggressive solvents like methanol or acetonitrile permits coupling to sensitive antibody-based detection techniques, as was planned for the Life Marker Chip (LMC), which was initially intended for ESA’s ExoMars mission [164
]. However, the extraction yield of hydrophobic molecules by surfactant-based aqueous solvents only displayed about one third of the extraction rates observed when using organic solvents such as methanol-based solvents [165
An alternative and attractive approach for the extraction of biomarkers with different polarities from a sample is offered by “subcritical water extraction”. This technique uses water as an extraction solvent at temperatures between 100 °C and 374 °C, while maintaining it liquid under high pressure (up to 22 MPa). These conditions dramatically alter the dielectric constant ε (i.e.
, the polarity) of water molecules: water at ambient temperature and pressure has a ε = 79 while increasing the water’s temperature to 250 °C and 5 Mpa yields ε = 27 [168
], which is similar to ethanol at 25 °C and 0.1 MPa. By changing temperature and pressure it is possible to extract a range of polar as well as non-polar molecules with high efficiency [169
], while the eventual extracts will be dissolved in water, making follow up applications less dependent on the solvent type, which would be appropriate for antibody based assays like the LMC.
The principle of a microarray/immunoassay approach shows great prospect in the field of life detection since this approach allows for screening many biomarkers at once by aiming for a multitude of generic molecular structures or, if preferred, very specific structures or sequences [166
]. Another major advantage of using an antibody based detection instrument is that samples do not need to be heated prior to analysis. This eliminates the danger of perchlorates, present in the Martian soil, destroying, or altering organic matter under heated conditions. The SOLID instruments (Signs of Life Detector), of which SOLID3 is the latest, are microarray-based instruments designed for astrobiological purposes and can detect a wide range of molecular compounds ranging from peptides to whole cells and spores [172
]. Extraction of biomolecules from the soil is conducted by using a water-based extraction buffer containing 0.1% surfactant (Tween 20), combined with sonication steps [172
]. Relatively cheap reproduction of microchips and the possibility of reuse by washing with an eluting solvent makes them very suitable for astrobiology missions, with solvent carrying capacity being the major limiting step. However, no extensive testing of the functionality of antibodies in open space has been done to our knowledge and the effects of such an environment on the functioning of antibodies needs to be investigated.
Other approaches for the detection and identification of biomarkers are often based on the separation of molecules by their binding and solving behavior with respect to a stationary adsorbing phase (column) and mobile solving phase (solvent). This is the basic principle of separation techniques such as High Performance Liquid Chromatography (HPLC) [174
]. Due to differences in binding and solving characteristics, some molecules are retained longer on the column than others, providing a means of separation and thus identification. Detection most often occurs via UV-visible light, fluorescence-detection or mass spectroscopy [175
], where in the latter case the mass-to-charge ratio of ionized molecule fragments is determined as a means of identification, resulting in very high sensitivity [176
]. To improve separation and fluorescence detection, derivatization methods have been developed, which are often used in amino acid analyses [177
] and can also improve separation of amino acid enantiomers [179
]. Gas chromatography is also based on separation of compounds by retention to a stationary phase but utilizes a carrier gas instead of a liquid mobile phase, and the column is contained in an oven to regulate the temperature of the gasses, of which the vapor pressure is used to determine the concentration of gaseous analytes [181
Methods for the identification of microbial communities are often based on in situ
extraction of genomic DNA from samples rather than by a culture based approach [182
]. As a consequence, the yield of genomic material is relatively low, and an underestimation of the microbial diversity can occur through interspecies differences in extractability or proneness to amplification techniques [184
]. There are several approaches to perform the cell lysing step, but currently the bead-beating methods are considered to give most satisfying results [185
]. After extraction, DNA can be amplified by PCR [49
]. The most used approach in identifying microbial communities is to screen for specific gene segments, such as 16S ribosomal RNA (rRNA) gene segments [39
], which is present in all terrestrial microorganisms but expresses slight differences in their sequence per species, by which they can be related to other species. An approach aiming to amplify conserved terrestrial sequences may however not be adequate in the search for extraterrestrial life [39
]. Even on Earth, screening for the 16S rRNA gene does not detect all organisms [186
]. Whole genome amplification (WGA) may be a better approach if the goal is to detect unknown life that utilizes DNA-like molecules. Microbial communities have been identified with this technique that would not have been identifiable with a specific primer based approach [187
]. The use of primer libraries, to cover a wide variety of amplifiable DNA gene sequences, would also increase the chance of identifying otherwise undetected species. Furthermore, with the arrival of next generation sequencing techniques it has become possible to obtain huge amounts of sequences in a massive parallel reaction in a fraction of the time (and costs) that Sanger-based sequencing would require [188
]. A perquisite for detection of extraterrestrial DNA however would still be a partial overlapping evolution, or an identical genesis of DNA-based life, of which the former possibility seems more likely than the latter due to the complexity of the molecule.
In summary, the availability of a large variety of approaches is advantageous to life detection. Combining various techniques, for example different extraction and detection techniques on the same sample allows us to obtain robust and comprehensive results. Detection and correlation of a variety of biomarker classes would induce more diagnostic power than the characterization of just a single biomarker class. For example, Lester et al.
] combined total organic carbon (TOC) load and phospholipid fatty acids (PLFA) analysis data with DNA profiles. Such combined approaches can greatly increase our knowledge of the interactions between biota, biomarkers and the environment.
5. Current Instrumentation on Mars Life Detection Missions: How to Look There?
Current and future Mars missions will search for past and present life with the help of instruments that can identify traces of life. Organic molecules; microfossils or isotopic data indicating microbial activity in rocks are all tracers for biological processes. Life detection instruments are optimized to detect robust biomarkers such as (homochiral) amino acids and small hydrocarbons [14
]. The Sample Analysis at Mars (SAM) instrument on the NASA Curiosity rover is currently operating on Mars. SAM is equipped with three instruments that aim to detect organic compounds: A Quadrupole Mass Spectrometer (QMS); a Gas Chromatograph (GC); and a Tunable Laser Spectrometer (TLS). GC-MS analysis can be performed by combining the two instruments, which facilitates separation on the GC column and subsequent identification of organic compounds by the MS component. The TLS gathers isotope ratios for carbon and oxygen from carbon dioxide and can measure trace levels of methane and its carbon isotope [189
]. Other instruments, which include CheMin (Chemistry and Mineralogy) and RAD (Radiation Assessment Detector), are also incorporated in the Curiosity rover and can identify mineral types by X-ray diffraction or measure radiation, respectively. A recent discovery of Curiosity rover is the identification of an environment at Gale crater, which was once an aqueous environment with neutral pH, low salinity and variable redox states for both iron and sulfur, capable of sustaining life on a chemolitoautotrophic basis [190
]. Water was also detected, bound within the amorphous soil components [191
The Pasteur payload for the joint ESA-Roscosmos Exomars mission in 2018 has been revised several times. The Mars Organic Molecular Analyzer (MOMA) is the most powerful organics-detection payload instrument, which can detect molecules in the range of ppb-ppt [29
]. This instrument uses a laser to combust organic materials, after which the resulting products will be separated by GC and identified with ion-trap coupled mass spectroscopy [192
]. The laser used in the GC is also able to combust compounds ranging from volatile to non-volatile compounds, thereby increasing the detection range. A GC column is specifically designed for detection of homochirality in amino acids. Another important function embedded in MOMA is the direct derivatization of organic compounds with labile hydrogen groups (e.g., amino acids, nucleobases) to stabilize them and improve separation detection sensitivity. A wide range of biomarkers can be detected, increasing the chance of finding traces of life (if ever present). By implementing thermal volatilization for the GC, a drawback of MOMA could be the reaction induced by a combination of organics and perchlorates under heating, resulting in the production of chlorohydrocarbons [102
]. Other instruments on the ExoMars payload include a Raman spectrometer, an infrared imaging spectrometer. Spectrometric detection techniques like Raman spectroscopy are non-destructive and independent of consumables like extraction solvents and rinsing buffers. Another advantage is that sample processing is not required. However, most promising is that it can measure biological and geological signatures simultaneously. These characteristics make Raman spectroscopy ideal for robotic remote field missions [193
], and thus a valuable asset for astrobiology, especially as a tool complementary to more analytical instruments like GC or microarrays. Several instruments have been de-scoped, such as the LMC and the Urey instrument. The latter is equipped with a Subcritical Water Extractor (SCWE) and its main goal is to determine biotic versus
abiotic origin of detected molecules, such as amino acids and nucleobases but also targets PAHs [194
6. Conclusions and Looking forward
We have provided an overview of biomarkers and techniques relevant for life detection and what knowledge can be obtained by investigating extreme environments on Earth as a template for Mars. A large variety of biomarkers are available for tracking past or present life. Although life uses and produces many different biomolecules, not all are suitable as diagnostic tools. Preservation potential and extractability of a molecule are parameters that must be taken into account. In addition, the original concentration as well as the spatial distribution of biomarkers will influence the chances of detection. Furthermore, a distinction must be made between extant and extinct life. Most of the molecular compounds that are typical for extant life (DNA, carbohydrates, ATP, proteins) have a relatively short lifetime and, thus, degrade fast outside the protected confinements of a cell, especially when exposed to UV radiation or oxidizing conditions present on Mars. Robust biomarkers would form a more appropriate target for extinct life.
If extraterrestrial life exists, it likely also makes use of membrane compartmentalization [55
]. Lipids are therefore considered high priority targets [14
]. Amino acid enantiomeric excesses are often used as proof for biotic origin [77
]. However, the uncovering of abiotic processes that could lead to homochirality in amino acid mixtures has made enantiomeric excesses lose some of its diagnostic power for biotic origin. Combined approaches may be needed to explain enantiomeric excess rather than using it as a diagnostic means by itself. Targeting of nucleic acids with non-specific amplification techniques such as WGA may reveal the existence of alternative forms of DNA [195
], and the development of next generation sequencing techniques will most likely have a large impact on the search for extraterrestrial life. Scanning for “hypothetical biopolymers” by using synthetically created molecular probes could potentially reveal the presence of alternative nucleic acids.
Investigation of extreme environments on Earth has shown us that microbial life tends to prefer relatively sheltered places. Life and biomolecules accumulate in these environments that offer protection against desiccation, UV radiation and other degrading effects. If there is, or was life on Mars, one might expect the same. This implies a strong focus towards localizing potentially protective environments on Mars. Rock and ice formations, caves and subsurface brine systems would be good candidates to look for traces of life. The role of clay-rich minerals in these local areas is important since they offer the desired protection and can function as a catalyst for chemical reactions. Aiming for such clay-rich formations is, therefore, given consideration in current life detection strategies. However, the adsorbing properties of clay rich minerals in turn may hinder efficient extraction of biomolecules. An important strategy for future life detection techniques should be the optimization of biomolecule extraction from clay-rich minerals. A good approach is to combine solvents with a competitive binding molecule that would free biomolecules from the mineral surfaces. Switching between polar and non-polar solvents, the addition of surfactants or using pressurized solvent extractions are other approaches to increase extraction rates and should be further optimized.
The identification of extracted compounds can be conducted by many different techniques. Since many biomarkers demand specific procedures to be identified it is a complex endeavor to analyze soils for their contents with a single procedure. Separation and detection in techniques, such as HPLC or GC, often require special derivatization protocols that are not universally applicable to different biomolecules. A future goal would be to develop a “standard life detection package” for use in astrobiological missions. Immunoassays and microarrays may be of specific use in this context. These assays can identify thousands of compounds by binding or hybridizing to specifically created antibodies or DNA probes (lock-and-key mechanism). Immunoassays, specifically designed to detect a range of biomolecules indicative for extant or extinct live could facilitate an easy read-out for sample analysis. Examples of such approaches include the LMC and the SOLID instruments discussed above and are gaining in popularity. Improving compatibilities between antibodies and the more aggressive organic solvents may be necessary in order to make this assay more effective for the detection of non-polar compounds, such as PAHs, lipids and pigments, which are more efficiently extracted with organic solvents. The concentration of methanol has been shown to effect the formation of immune complexes [196
]. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic molecular imprinted polymers can be selectively produced to have higher chemical and physical stability, which would be a major advantage if a more hazardous extraction solvent is to be used [197
It remains important for future planetary missions to search for robust biomarkers in regions with high organic preservation potential. The optimization of extraction methods that could extract polar and non-polar molecules from clay-rich mineral samples is equally important. A special focus to advance techniques that can identify many different biomarkers at once would be a major contribution to future planetary missions.