Color Catalogue of Life in Ice: Surface Biosignatures on Icy Worlds

1. Introduction

More than 4,000 planets orbiting other stars have been detected to date (exoplanets.nasa.gov, September 2020), with dozens of Earth-sized planets orbiting in the temperate zone of their stars (Kane et al.,2016; Berger et al.,2018) that would allow for liquid water on the surface of an Earth-like planet. Upcoming ground-based Extremely Large Telescopes as well as the James Webb Space Telescope can search for signs of life on extrasolar planets (Ben-Ami et al.,2018; Serindag and Snellen, 2019). However, the detection of a planet in the temperate zone does not guarantee its habitability (e.g., Kaltenegger, 2017). Detailed characterization requires observations of the planet's or moon's spectrum to assess its atmosphere and surface properties. In addition to atmospheric biosignature pairs like O₂ and CH₄ or O₃ and CH₄ (Lederberg, 1965; Lovelock, 1965), several studies explored whether surface features could indicate life in the spectrum of an exoplanet (e.g., Seager et al.,2005; Schwieterman et al.,2015; O'Malley-James and Kaltenegger, 2018, 2019). Future large space-based telescope designs such as the Large Ultraviolet Optical Infrared Surveyor (LUVOIR; e.g., Kouveliotou et al.,2014) and the Habitable Exoplanet Observatory (HabEx; e.g., Mennesson et al.,2016) are being formulated to explore biosignatures in the atmosphere and on the surface of exoplanets.

Biological pigments dominate many diverse landscapes on Earth and are present in a wide range of organisms (e.g., Hegde and Kaltenegger, 2013; Hegde et al.,2015; Schwieterman et al.,2015). Pigments of microbes present in green algae blooms, pink “watermelon” snow, red saltern crystallizer ponds, and heterogeneous microbial mats are signs of life on Earth's surface that can be detected from orbiting satellites (e.g., Oren and Rodriguez-Valera, 2001; Lutz et al.,2016; Williamson et al.,2020) as well as space missions like the Galileo probe (Sagan et al.,1993). Biological pigments on the surface of Earth could have indicated life for up to 2 billion years (O'Malley-James and Kaltenegger, 2018, 2019).

Pigmented organisms have unique reflectance signatures to search for on exoplanets orbiting other stars (e.g., Hegde et al.,2015; Kaltenegger, 2017; Fujii et al.,2018). A previous color catalogue of life by some of the authors of this study (Hegde et al., published in 2015) established a database of reflectivity spectra for a diverse set of 137 microorganisms from different environments as it would be seen on an exoplanet (Hegde et al.,2015). However, the database did not include any microorganisms from frozen environments.

A great abundance of pigmented microbes found in the highly diverse ice microbial community (Vincent et al.,1993, 2004)—isolated from the Arctic and Antarctic, which are icy terrestrial analogues (Martins et al.,2017)—suggests that icy worlds orbiting other stars as well as icy moons in low-radiation environments in the Solar System may provide promising targets for the search for life. Life may have even started on Earth in ice—the so-called “cold origin of life” hypothesis (e.g., Price, 2007).

Ice is widely considered an extreme habitat for life. Besides extreme dryness and low temperatures, radiation can be excessive in this environment. Ice acts as a selective agent against organisms that are not efficient in photooxidative protection (Lemoine and Schoefs, 2010). Hence, pigmented organisms have been reported to be dominant in the microbiota of icy environments (e.g., Cottrell and Kirchman, 2009; Marizcurrena et al.,2019).

Most of these pigments have a role in photosynthesis (e.g., chlorophylls and carotenoids). Photosynthetic pigments absorb and channel part of the incoming light as usable energy for organisms. Photosynthesis turns inorganic carbon into biomass with light as the energy source and molecules such as H₂, H₂S, Fe²⁺, or H₂O as electron donors. Photosynthetic pigments become oxidized while capturing light photons, and redirect this photochemical energy (Bassham and Calvin, 1960; Cohen et al.,1986).

While chlorophylls are virtually universal among oxygenic photoautotrophs (Lubitz et al.,2019), carotenoids are widespread among the three domains of life, including non-photosynthetic organisms (e.g., heterotrophs) (Yabuzaki, 2017). Due to their hydrophobic nature, they are immersed in biological membranes, playing a role in the modulation of membrane fluidity to survive under low-temperature conditions (Seel et al.,2020). Carotenoids also behave as light-absorbing chromophores, playing important roles in photooxidative protection (Mathews and Sistrom, 1959). Radiation is deleterious to biomolecules, such as DNA and proteins, and creates reactive oxygen species, which are major oxidative stress agents. Carotenoids quench photosensitizers and singlet oxygen, consequently becoming oxidized and dissipating excess energy and oxidative power, protecting other sensitive molecules. Their color depends on chemical modifications of the original molecule determined by the number of conjugated double bonds within the hydrocarbon backbone (Armstrong, 1997). As a result of oxidation upon radiation stress, carotenoids change the color of organisms, turning them yellow, orange, pink or ultimately red (Latowski et al.,2011). Thus, the presence of carotenoids in icy biota is a strategy of adaptation to the extreme cold temperatures and to the high radiation (Dieser et al.,2010)—analogues to conditions of extraterrestrial icy environments (Martins et al., 2017).

A reference database of biota in icy environments on Earth is a critical tool to enable our search for life on the surface of icy exoplanets and exomoons. Thus, we created such a reference catalogue for life in ice by measuring the reflection spectra of 80 colorful microorganisms, isolated from ice and water below the ice, collected in the Canadian subarctic, to identify potential signs of life on icy worlds.

The ice and water come from the mildly briny Hudson Bay and the Great Whale River (freshwater) that floods into the bay. The isolated microorganisms used in this study were previously identified and include 77 bacteria, 1 yeast (all heterotrophs), and 2 algae (photosynthetic). Our database provides a crucial tool to detect and identify potential signatures of life on icy planets and moons orbiting other stars.

2. Methods

We collected the 80 microorganisms from ice and water at Kuujjuarapik, Canadian subarctic, in collaboration with Université Laval during the winter of 2019. The microorganisms belong to the Culture Collection of psychrotolerant and psychrophilic subarctic strains of the University of Lisbon, Portugal (78 isolates from Instituto Superior Técnico and 2 isolates from Instituto Superior de Agronomia). From the 80 microorganisms, 66 were isolated from the Hudson Bay (salinity approx. 1%) and 14 from the Great Whale River (freshwater). No particular pigment dominated the landscape on those sampling days, allowing for a less challenging cultivation of a more diverse group of pigmented microorganisms afterword. They arrived at Zinder lab at the Department of Microbiology at Cornell University on solid media and were stored at 4°C before transfer and growth. All hemispherical reflectance measurements were performed at Philpot's lab at the School of Civil and Environmental Engineering at Cornell University. Both labs are part of the interdisciplinary Carl Sagan Institute at Cornell University.

2.1. Sample preparation

We grew bacteria and fungi in Reasoner's 2A broth (R2A) and microalgae in Tris-Acetate-Phosphate (TAP) liquid media at 18–20°C. The heterotrophic cultures were grown aerobically up to a stationary phase, except for phototrophic cultures that were grown under 16 h light/8 h dark cycles by using white light. All cultures were grown at room temperature. Depending on the isolate, the time required for growth varied from about 24 h to 1 week. All procedures during the culturing process occurred under sterile conditions. Pure sample cultures were transferred to 50 mL Falcon centrifuge tubes until filtration.

2.2. Spectrometer system

We used an ASD FieldSpec 4 Spectrometer, which covered the wavelength range from 350–2500 nm at intervals of 1 nm and an ASD integrating sphere to measure the reflectance of our samples, as described in the work of Hegde et al. (2015). Bidirectional reflectance measurements at a single viewing angle often result in a poor approximation of the albedo, especially if, for a given biomass, the sample has high bidirectional reflectance distribution function anisotropy (Kriebel, 1978; Hegde et al.,2015). For exoplanets, remote observations will be disk-integrated; hence disk-integrated reflectance measurements that use hemispherical geometry are needed for more realistic surface albedo modelling of Earth-like exoplanets.

2.3. Sample measurements

We deposited cultures on a 25 mm plain white mixed cellulose ester filter (0.45 μm) using a 10 mL syringe and a filtration system as described previously (Hegde et al.,2015). Cells were homogeneously layered when deposited onto the filter substrate (Hegde et al.,2015), and the saturation limit was reached at about 20 mL of cell suspension for most cultures. The sample was then used to acquire high-resolution hemispherical reflectance measurements by using the spectrometer system described before. The same sample was measured at two different times: immediately after being deposited on the filter (fresh) and after 1 week (dry). Translucent cultures were measured as biological control. Unused filters, filters with only culture media, and filters with only water were measured as experimental controls.

2.4. Microscopy

The micrographs were obtained by analyzing 2 μL of fresh cell suspension of each sample fixed on agar slides through a Nikon Eclipse E600 microscope. Micrographs of cells were taken under a bright-field with phase-contrast microscopy at 400 × magnification for all samples and 1000 × (using immersion oil) for all bacteria.

3. Results

The 80 pigmented microorganisms were previously isolated from ice and water below the ice; collected at Kuujjuarapik, Canadian subarctic; and identified by rRNA gene-based taxonomic affiliation. Seventy-seven isolates are bacteria belonging to the phyla Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. Three are eukaryotes: fungi from the division Basidiomycota and microalgae from the phylum Chlorophyta.

Figure 1 shows an example of the diversity in color among six samples from the 80 isolates: Sphingomonas sp. (bright and dark orange), Microbacterium sp. (yellow), Arthrobacter sp. (pink), Chlorophyta algae (green), and Bacillus sp. (white), along with their photomicrographs.

Figure 2 shows the diversity in color signatures of pigmented biota isolated from an icy environment on Earth: we group different organisms by their colors—yellow, orange, pink, green, or white. Unpigmented translucent organisms (clear) act as a biological control for uncolored pigments/unpigmented biota in our measurements. The colors of the pigments have the strongest influence on the reflectance spectra in the wavelength range between 0.35 and 0.7 μm (see the “zoom” panels in Fig. 2). The blue dotted line in each graph shows the experimental control spectra, that is, a filter containing only culture media. Fresh samples are shown in Fig. 2A and dry samples in Fig. 2B. The reflection spectra of the collected biota changed with water content: Figure 2A (left) shows the reflection spectra shortly after depositing the fresh organisms on the filter, representing a hydrated sample (fresh). Figure 2B shows the dry reflection spectra after 1 week of deposition of the organisms on the filter (dry).

Fresh samples showed a weaker reflection than dry samples. Indeed, strong water absorption features can be seen around 1.45 and 1.95 μm in the fresh sample reflection spectra in Fig. 2A. Fresh samples correspond to life on a planet like Earth, with available liquid water.

Dry samples provided a stronger reflection of the pigment for all samples, because the water, which reduces the reflectance, had been evaporated. The red edge peaks at a slightly higher wavelength for some dry samples than it does for fresh ones: for carotenoids this agrees with a known strategy to endure oxidation (Vítek et al.,2017).

Translucent samples (clear) showed a similar reflection spectrum to the experimental control (only culture media) for both fresh and dry samples.

Related organisms (same taxa) can have very different colors and thus different corresponding reflection spectra (see Fig. 2). Moreover, yellow, orange, and red carotenoids are produced by a wide range of organisms: photosynthetic and non-photosynthetic bacteria as well as eukaryotes such as algae, fungi, and plants (Armstrong, 1997). Although specific colors can be abundant in specific phyla, they are not exclusive of any phylum or genus, as discussed below (see Fig. 3). The collection of ice microorganisms shows neither a color relation between heterotrophic (non-photosynthetic) organisms from the same phylum nor a relation between their genus and specific color signatures.

For example, Brevundimonas and Sphingomonas, both Alphaproteobacteria, have very different spectra in the visible range. Pedobacter and Rhodococcus, from Bacteroidetes and Actinobacteria, respectively, are more uniform, likely because of the few representative isolates of these genera in our database (Fig. 3). Almost all these genera share the same colors. These are expected results from colors given by the presence of carotenoids. These traits are not produced as primary metabolites and may even be passed through lateral transfer events between unrelated organisms, as will be discussed later. The spectral features of carotenoid pigments are broad indisputable signs of life, but they cannot be associated with a specific organism.

4. Discussion

4.1. Carotenoids as potential biosignatures in icy extraterrestrial environments

We found no relation between the phylum of the tested heterotrophic organisms and their color signature as shown in Fig. 3 (see also discussion in Hegde et al.,2015). Carotenoids are widespread pigments among the three domains of life, and their color is highly dependent on interactions with the ecosystem. It has been suggested that the evolutionary path of microbial carotenoids appears to be more weblike instead of tree-like due to the occurrence of extensive lateral gene transfer, gene loss, and gene duplication events (Klassen, 2010). Lateral gene transfer can occur between individuals of different taxa, which means that the gene—or gene cluster underlying pigment biosynthesis—will not exclusively be passed through its hereditary line (vertically) but will instead be lineage-unspecific and feature in unrelated places of the evolutionary tree (being more relatable with a web). Carotenoids are signatures of life not limited to a small taxonomic group of organisms or specific populations of both photosynthetic and non-photosynthetic groups.

These molecules cover different physiological roles in the overall adaptation of all types of cells to conditions found in extreme cold environments, such as low temperature, radiation, photooxidation, lack of resources, and dryness. The functions of carotenoids include energy dissipation, antimicrobial activities, and regulation of membrane fluidity (Lemoine and Schoefs, 2010; Seel et al.,2020). These physiological functions would be useful for life beyond Earth as well.

Carotenoids dominate some of Earth's landscapes, like salty red lakes. Pink and red carotenoids present in lakes and in snow have been detected by astronauts on the International Space Station (https://eol.jsc.nasa.gov), as well as spectrometers flying at high altitudes, such as the Airborne Visible InfraRed Imaging Spectrometer (AVIRIS) (Painter et al.,2001; Serrano et al.,2002; Dalton et al.,2009), and NASA's Airborne Snow Observatory (ASO) (Carey et al.,2018).

Future large space-based telescopes designs like the Habitable Exoplanet Observatory (HabEx; e.g., Mennesson et al., 2016) and the Large Ultraviolet Optical Infrared Surveyor (LUVOIR; e.g., Kouveliotou et al.,2014) could search for blooms of microbial pigments on exoplanets to detect carotenoids.

4.2. Dry samples reflect stronger: Implications for searching for life

Dried samples show more intense colors and a higher reflectance overall than fresh samples in our studies, indicating that water absorption reduces the reflectivity of pigments. In our study, fresh samples (Fig. 2A) have higher water content than dry samples, which are dried for 1 week (Fig. 2B). Water evaporates while the pigments remain on the filter. The water absorption features also decrease for dry samples, shown in Fig. 2 at about 1.5 and 2.0 μm with a slight dependence on the specific pigment (see also Dalton et al.,2003; Hegde et al.,2015; Ball, 2017). A recent study in which Raman spectroscopy was used on carotenoids also shows higher pigment signal intensities on dried cells (Baqué et al.,2020), which is consistent with our results. This increase in reflectance of dried cells is likely due to the change in the relative index of refraction at the water-cell interface as the cell dries. The relative index of refraction at the water-cell interface is relatively small and would tend to enhance the absorption into the cell. When the water evaporates, the higher relative index of refraction would lead to increased reflectance at the surface (and less absorption). A similar phenomenon has been described for sand particles as the water evaporated (Tian and Philpot, 2018). This is also the reason why a geologist will wet the surface of a rock to better see the mineral colors: with water on the surface, more light penetrates and is partially absorbed, and the light reflected from the interior (still at the surface) is richer in color (Lekner and Dorf, 1988).

The type of pigment determines the chemical relationship with water; for example, carotenoids are hydrophobic and chlorophylls are partially hydrophobic. Thus, the influence of water on the reflectivity of the pigment depends on the type of cell, organism, and pigment. Fresh samples are representative of life on Earth, where liquid water is readily available for most biota. However, our results show that dry samples would make detection of pigment-biosignatures easier. Icy worlds would provide such dry environments.

The samples used in this study are not expected to have an abundance of liquid water available in their natural environment in the Arctic. This is expected to help the organisms adapt to the high photooxidative stress they are subjected to for being in ice. Thus, dry microbial cells tend to be more resilient to high-radiation environments, making them interesting targets to search for in dryer and highly irradiated planets (Billi et al.,2019). The closest potentially habitable worlds outside our solar system orbit a different kind of star than our sun—smaller red dwarf stars. Such stars can flare frequently, bombarding their planets with biologically damaging high-energy UV radiation, placing planetary atmospheres at risk of erosion and bringing the habitability of these worlds into question (e.g., Scalo et al.,2007; Tarter et al.,2007; Segura et al.,2010; Shields et al.,2016; Kaltenegger, 2017; Tilley et al.,2019). However, the surface UV flux of these worlds is unknown. First models of the surface UV environment for the four closest potentially habitable exoplanets—Proxima-b, TRAPPIST-1e, Ross-128b, and LHS-1140b, assuming different atmospheric compositions, from Earth analog to eroded and anoxic atmospheres—show that surface UV radiation remains below early Earth levels, even during flares (O'Malley-James and Kaltenegger, 2019). But even high UV radiation on the surface of rocky exoplanets circling active M stars may not limit extraterrestrial life evolution if carotenoid-like pigments are developed for photooxidative protection. In addition, while liquid water is widely considered the “matrix of life” (Ball, 2017), discussions on other solvents such as methane are becoming more pertinent, informing ideas on the evolution of alternative non-water-based “weird life” (e.g., Stevenson et al.,2015).

We speculate that on other planets biota could use similar pigments to the ones used on Earth, independent of the solvent. For dry planets and planets with solvents other than water, biopigments could provide stronger signatures than on worlds with abundant water, like Earth.

4.3. Our solar system and the search for life

The search for life in our solar system focuses on a dry planet—Mars—as well as three cold moons—Titan, Enceladus, and Europa. The color catalogue for biota in ice also provides insights for the search for life closer to home.

The idea that the building blocks of life (dos Santos et al.,2016; Laurent et al.,2019) or even dried cells may be preserved (Bryce et al.,2015; Baqué et al.,2016) can be expanded to currently dry planets like Mars (Baqué et al.,2020). For example, one of our samples—Bacillus safensis—has also been isolated from the spacecraft Mars Odyssey orbiter (Satomi et al.,2006) and could have survived the extreme radiation environment in space. The same bacterium has also been described in a study as growing better on the International Space Station than on Earth (Coil et al.,2016). This type of bacterium forms spores, which are inactive dried latent cell forms with increased resistance to almost any type of stress. Thus, the dry samples of our color catalogue of life in ice can provide insights into what spectral features missions to Mars could look for.

Earth is the only known place with surface blooms of microbial pigments. A key issue when considering the search for biological pigments in our solar system is the radiation environment. All biological pigments presented in this article are found in icy environments on Earth, which are sheltered by Earth's atmosphere and magnetic field. Biological pigments disintegrate rapidly in high-radiation environments. For example, UVA radiation causes a significant drop in productivity of ice algae communities (McMinn et al.,1999). Thus, finding biological pigments on the surface of cold moons without an atmosphere, especially in strong radiation environments like that of Europa, is unlikely. Nevertheless, searching for signatures of traces of biological pigments (Martins et al.,2013; d'Ischia et al.,2021) on the icy near-subsurface (Nordheim et al.,2018) and in ice particles in jets of geysers could provide an alternative way to search for life on icy moons. The high success of Earth-orbiting spectrometers on detecting microbial pigments (Painter et al.,2001; Serrano et al.,2002; Dalton et al.,2009; Carey et al.,2018) serves as inspiration for high-resolution imaging spectrometers exploring the icy moons in the Solar System, such as the Mapping Imaging Spectrometer for Europa (MISE) that will fly on the NASA Europa Clipper in late 2024 to explore the potential signatures of extremophiles implanted in the ice surface (Chen et al.,1997; Carey et al.,2018), as well as the Europa Lander Stereo Spectral Imaging Experiment (ELSSIE) (Murchie et al.,2020). Antarctic microbes forming complex communities have also been successfully identified from their carotenoid-like pigmentation by low-altitude unmanned aerial vehicles carrying spectrometers on Earth (Levy et al.,2020), which are a similar concept to future small missions such as Titan's Dragonfly.

5. Conclusion

The unequivocal proof of the existence of extraterrestrial life is one of the most thought-provoking events humanity could experience. To enable the search for life on the surface of icy exoplanets and moons, we developed the first catalogue of microbial life in ice. We measured the reflection for a diverse range of pigmented microorganisms isolated from the subarctic—an analogue for those extraterrestrial environments. Similar biota could become the dominant life-form on icy worlds orbiting other stars. Life might have even originated in such icy environments.

Here, we present the first database of surface reflection for biota from icy environments to provide a tool for upcoming space- and ground-based telescopes that will search for life in the cosmos. While our color catalogue of life in ice has been developed as a guide to search for life on icy planets and moons orbiting other stars, it also holds the key to expand the search for life in the Solar System.

For the first time in human history, we have the tools to search for life in the universe. Icy environments on Earth show a surprisingly wide diversity of life and might have even provided the environment for life to originate. The color catalogue of life on Earth's subarctic will serve as the guide to search for surface life on icy worlds to discern whether we are alone in the cosmos.

Author Contributions

LK, ZM, and LFC designed the research. LFC and JM performed the experimental work and data analysis. LFC prepared samples. RC, MGE, SZ, WP, WFV, JC, and ZM contributed with samples, reagents, and analytical tools. LFC wrote the manuscript with support from JM, LK, and ZM. All authors provided critical feedback and helped shape the research, analysis, and manuscript.

Funding Statement

LFC was funded by the MIT-Portugal Program (MPP) through FCT - Fundação para a Ciência e Tecnologia with the scholarship PD/BD/139840/2018. LK and JM acknowledge support from the Carl Sagan Institute and the Brinson Foundation. ZM acknowledges support by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), and by Portuguese funds through FCT - Fundação para a Ciência e Tecnologia in the framework of the project POCI-01-0145-FEDER-029932 (PTDC/FIS-AST/29932/2017). Centro de Química Estrutural acknowledges the financial support of FCT- Fundação para a Ciência e Tecnologia (UIDB/00100/2020). Institute for Bioengineering and Biosciences acknowledges the financial support of FCT - Fundação para a Ciência e Tecnologia (UIDB/04565/2020). LEAF Unit (Linking Landscape, Environment, Agriculture and Food) acknowledges the financial support from FCT (UID/AGR/04129/2020). This research is framed within the College on Polar and Extreme Environments (Polar2E) of the University of Lisbon and received support from the project PERMAMERC funded by the Portuguese Polar Program (PROPOLAR).

Competing Interest Statement

The authors declare no conflict of interest.

Supplementary Material

Supplementary Table S1

Associate Editor: Sara Seager