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The question of whether there is or was life on Mars has been one of the most pivotal since Schiaparellis' telescopic observations of the red planet. With the advent of the space age, this question can be addressed directly by exploring the surface of Mars and by bringing samples to Earth for analysis. The latter, however, is not free of problems. Life can be found virtually everywhere on Earth. Hence the potential for contaminating the Mars samples and compromising their scientific integrity is not negligible. Conversely, if life is present in samples from Mars, this may represent a potential source of extraterrestrial biological contamination for Earth. A range of measures and policies, collectively termed 'planetary protection', are employed to minimise risks and thereby prevent undesirable consequences for the terrestrial biosphere. This report documents discussions and conclusions from a workshop held in 2012, which followed a public conference focused on current capabilities for performing life-detection studies on Mars samples. The workshop focused on the evaluation of Mars samples that would maximise scientific productivity and inform decision making in the context of planetary protection. Workshop participants developed a strong consensus that the same measurements could be employed to effectively inform both science and planetary protection, when applied in the context of two competing hypotheses: 1) that there is no detectable life in the samples; or 2) that there is martian life in the samples. Participants then outlined a sequence for sample processing and defined analytical methods that would test these hypotheses. They also identified critical developments to enable the analysis of samples from Mars. © 2014 The Committee on Space Research (COSPAR).

Viruses are the most numerically abundant biological entities on Earth. As ubiquitous replicators of molecular information and agents of community change, viruses have potent effects on the life on Earth, and may play a critical role in human spaceflight, for life-detection missions to other planetary bodies and planetary protection. However, major knowledge gaps constrain our understanding of the Earth's virosphere: (1) the role viruses play in biogeochemical cycles, (2) the origin(s) of viruses and (3) the involvement of viruses in the evolution, distribution and persistence of life. As viruses are the only replicators that span all known types of nucleic acids, an expanded experimental and theoretical toolbox built for Earth's viruses will be pivotal for detecting and understanding life on Earth and beyond. Only by filling in these knowledge and technical gaps we will obtain an inclusive assessment of how to distinguish and detect life on other planetary surfaces. Meanwhile, space exploration requires life-support systems for the needs of humans, plants and their microbial inhabitants. Viral effects on microbes and plants are essential for Earth's biosphere and human health, but virus-host interactions in spaceflight are poorly understood. Viral relationships with their hosts respond to environmental changes in complex ways which are difficult to predict by extrapolating from Earth-based proxies. These relationships should be studied in space to fully understand how spaceflight will modulate viral impacts on human health and life-support systems, including microbiomes. In this review, we address key questions that must be examined to incorporate viruses into Earth system models, life-support systems and life detection. Tackling these questions will benefit our efforts to develop planetary protection protocols and further our understanding of viruses in astrobiology., Competing Interests: Conflict of interest. The authors report no conflict of interest.

Nearly half a century ago, two papers postulated the likelihood of lunar lava tube caves using mathematical models. Today, armed with an array of orbiting and fly-by satellites and survey instrumentation, we have now acquired cave data across our solar system-including the identification of potential cave entrances on the Moon, Mars, and at least nine other planetary bodies. These discoveries gave rise to the study of planetary caves. To help advance this field, we leveraged the expertise of an interdisciplinary group to identify a strategy to explore caves beyond Earth. Focusing primarily on astrobiology, the cave environment, geology, robotics, instrumentation, and human exploration, our goal was to produce a framework to guide this subdiscipline through at least the next decade. To do this, we first assembled a list of 198 science and engineering questions. Then, through a series of social surveys, 114 scientists and engineers winnowed down the list to the top 53 highest priority questions. This exercise resulted in identifying emerging and crucial research areas that require robust development to ultimately support a robotic mission to a planetary cave-principally the Moon and/or Mars. With the necessary financial investment and institutional support, the research and technological development required to achieve these necessary advancements over the next decade are attainable. Subsequently, we will be positioned to robotically examine lunar caves and search for evidence of life within Martian caves; in turn, this will set the stage for human exploration and potential habitation of both the lunar and Martian subsurface., Competing Interests: The authors declare no conflicts of interest relevant to this study., (© 2022. The Authors.)

Methane oxidizing microorganisms (methanotrophs) are ubiquitous in the environment and represent a major sink for the greenhouse gas methane (CH 4 ). Recent studies have demonstrated methanotrophs are abundant and contribute to CH 4 dynamics in caves. However, very little is known about what controls the distribution and abundance of methanotrophs in subterranean ecosystems. Here, we report a survey of soils collected from > 20 caves in North America to elucidate the factors shaping cave methanotroph communities. Using 16S rRNA sequencing, we recovered methanotrophs from nearly all (98%) of the samples, including cave sites where CH 4 concentrations were at or below detection limits (≤0.3 ppmv). We identified a core methanotroph community among caves comprised of high-affinity methanotrophs. Although associated with local-scale mineralogy, methanotroph composition did not systematically vary between the entrances and interior of caves, where CH 4 concentrations varied. We also observed methanotrophs are able to disperse readily between cave systems showing these organisms have low barriers to dispersal. Lastly, the relative abundance of methanotrophs was positively correlated with cave-air CH 4 concentrations, suggesting these microorganisms contribute to CH 4 flux in subterranean ecosystems. IMPORTANCE Recent observations have shown the atmospheric greenhouse gas methane (CH 4 ) is consumed by microorganisms (methanotrophs) in caves at rates comparable to CH 4 oxidation in surface soils. Caves are abundant in karst landscapes that comprise 14% of Earth's land surface area, and therefore may represent a potentially important, but overlooked, CH 4 sink. We sampled cave soils to gain a better understand the community composition and structure of cave methanotrophs. Our results show the members of the USC- γ clade are dominant in cave communities and can easily disperse through the environment, methanotroph relative abundance was correlated with local scale mineralogy of soils, and the relative abundance of methanotrophs was positively correlated with CH 4 concentrations in cave air.

Lava caves, tubes, and fumaroles in Hawai'i present a range of volcanic, oligotrophic environments from different lava flows and host unexpectedly high levels of bacterial diversity. These features provide an opportunity to study the ecological drivers that structure bacterial community diversity and assemblies in volcanic ecosystems and compare the older, more stable environments of lava tubes, to the more variable and extreme conditions of younger, geothermally active caves and fumaroles. Using 16S rRNA amplicon-based sequencing methods, we investigated the phylogenetic distinctness and diversity and identified microbial interactions and consortia through co-occurrence networks in 70 samples from lava tubes, geothermal lava caves, and fumaroles on the island of Hawai'i. Our data illustrate that lava caves and geothermal sites harbor unique microbial communities, with very little overlap between caves or sites. We also found that older lava tubes (500-800 yrs old) hosted greater phylogenetic diversity (Faith's PD) than sites that were either geothermally active or younger (<400 yrs old). Geothermally active sites had a greater number of interactions and complexity than lava tubes. Average phylogenetic distinctness, a measure of the phylogenetic relatedness of a community, was higher than would be expected if communities were structured at random. This suggests that bacterial communities of Hawaiian volcanic environments are phylogenetically over-dispersed and that competitive exclusion is the main driver in structuring these communities. This was supported by network analyses that found that taxa (Class level) co-occurred with more distantly related organisms than close relatives, particularly in geothermal sites. Network "hubs" (taxa of potentially higher ecological importance) were not the most abundant taxa in either geothermal sites or lava tubes and were identified as unknown families or genera of the phyla, Chloroflexi and Acidobacteria. These results highlight the need for further study on the ecological role of microbes in caves through targeted culturing methods, metagenomics, and long-read sequence technologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Prescott, Zamkovaya, Donachie, Northup, Medley, Monsalve, Saw, Decho, Chain and Boston.)

Ancient Venus and Earth may have been similar in crucial ways for the development of life, such as liquid water oceans, land-ocean interfaces, favorable chemical ingredients, and energy pathways. If life ever developed on, or was transported to, early Venus from elsewhere, it might have thrived, expanded, and then survived the changes that have led to an inhospitable surface on Venus today. The Venus cloud layer may provide a refugium for extant life that persisted from an earlier more habitable surface environment. We introduce the Venus Life Equation (VLE)-a theory and evidence-based approach to calculate the probability of extant life on Venus, L, using three primary factors of life: Origination, Robustness, and Continuity, or L  =  O  ·  R  ·  C. We evaluate each of these factors using our current understanding of Earth and Venus environmental conditions from the Archean to the present. We find that the probability of origination of life on Venus would be similar to that of Earth, and argue that the other factors should be nonzero, comparable with other promising astrobiological targets in the solar system. The VLE also identifies poorly understood aspects of Venus that can be addressed by direct observations with future exploration missions.

Biovermiculations are uniquely patterned organic rich sediment formations found on the walls of caves and other subterranean environments. These distinctive worm-like features are the combined result of physical and biological processes. The diverse microbial communities that inhabit biovermiculations may corrode the host rock, form secondary minerals, and produce biofilms that stabilize the sediment matrix, thus altering cave surfaces and contributing to the formation of these wall deposits. In this study, we incubated basalt, limestone, and monzonite rock billets in biovermiculation mixed natural community enrichments for 468-604 days, and used scanning electron microscopy (SEM) to assess surface textures and biofilms that developed over the course of the experiment. We observed alteration of rock billet surfaces associated with biofilms and microbial filaments, particularly etch pits and other corrosion features in olivine and other silicates, calcite dissolution textures, and the formation of secondary minerals including phosphates, clays, and iron oxides. We identified twelve distinct biofilm morphotypes that varied based on rock type and the drying method used in sample preparation. These corrosion features and microbial structures inform potential biological mechanisms for the alteration of cave walls, and provide insight into possible small-scale macroscopically visible biosignatures that could augment the utility of biovermiculations and similarly patterned deposits for astrobiology and life detection applications.

Microbial life permeates Earth's critical zone and has likely inhabited nearly all our planet's surface and near subsurface since before the beginning of the sedimentary rock record. Given the vast time that Earth has been teeming with life, do astrobiologists truly understand what geological features untouched by biological processes would look like? In the search for extraterrestrial life in the Universe, it is critical to determine what constitutes a biosignature across multiple scales, and how this compares with "abiosignatures" formed by nonliving processes. Developing standards for abiotic and biotic characteristics would provide quantitative metrics for comparison across different data types and observational time frames. The evidence for life detection falls into three categories of biosignatures: (1) substances, such as elemental abundances, isotopes, molecules, allotropes, enantiomers, minerals, and their associated properties; (2) objects that are physical features such as mats, fossils including trace-fossils and microbialites (stromatolites), and concretions; and (3) patterns, such as physical three-dimensional or conceptual n -dimensional relationships of physical or chemical phenomena, including patterns of intermolecular abundances of organic homologues, and patterns of stable isotopic abundances between and within compounds. Five key challenges that warrant future exploration by the astrobiology community include the following: (1) examining phenomena at the "right" spatial scales because biosignatures may elude us if not examined with the appropriate instrumentation or modeling approach at that specific scale; (2) identifying the precise context across multiple spatial and temporal scales to understand how tangible biosignatures may or may not be preserved; (3) increasing capability to mine big data sets to reveal relationships, for example, how Earth's mineral diversity may have evolved in conjunction with life; (4) leveraging cyberinfrastructure for data management of biosignature types, characteristics, and classifications; and (5) using three-dimensional to n -D representations of biotic and abiotic models overlain on multiple overlapping spatial and temporal relationships to provide new insights.

We present the case for the presence of complex organic molecules, such as amino acids and nucleobases, formed by abiotic processes on the surface and in near-subsurface regions of Pluto. Pluto's surface is tinted with a range of non-ice substances with colors ranging from light yellow to red to dark brown; the colors match those of laboratory organic residues called tholins. Tholins are broadly characterized as complex, macromolecular organic solids consisting of a network of aromatic structures connected by aliphatic bridging units ( e.g., Imanaka et al., 2004; Materese et al., 2014, 2015). The synthesis of tholins in planetary atmospheres and in surface ices has been explored in numerous laboratory experiments, and both gas- and solid-phase varieties are found on Pluto. A third variety of tholins, exposed at a site of tectonic surface fracturing called Virgil Fossae, appears to have come from a reservoir in the subsurface. Eruptions of tholin-laden liquid H 2 O from a subsurface aqueous repository appear to have covered portions of Virgil Fossae and its surroundings with a uniquely colored deposit (D.P. Cruikshank, personal communication) that is geographically correlated with an exposure of H 2 O ice that includes spectroscopically detected NH 3 (C.M. Dalle Ore, personal communication). The subsurface organic material could have been derived from presolar or solar nebula processes, or might have formed in situ . Photolysis and radiolysis of a mixture of ices relevant to Pluto's surface composition (N 2 , CH 4 , CO) have produced strongly colored, complex organics with a significant aromatic content having a high degree of nitrogen substitution similar to the aromatic heterocycles pyrimidine and purine (Materese et al., 2014, 2015; Cruikshank et al., 2016). Experiments with pyrimidines and purines frozen in H 2 O-NH 3 ice resulted in the formation of numerous nucleobases, including the biologically relevant guanine, cytosine, adenine, uracil, and thymine (Materese et al., 2017). The red material associated with the H 2 O ice may contain nucleobases resulting from energetic processing on Pluto's surface or in the interior. Some other Kuiper Belt objects also exhibit red colors similar to those found on Pluto and may therefore carry similar inventories of complex organic materials. The widespread and ubiquitous nature of similarly complex organic materials observed in a variety of astronomical settings drives the need for additional laboratory and modeling efforts to explain the origin and evolution of organic molecules. Pluto observations reveal complex organics on a small body that remains close to its place of origin in the outermost regions of the Solar System.

The search for life and habitable environments on other Solar System bodies is a major motivator for planetary exploration. Due to the difficulty and significance of detecting extant or extinct extraterrestrial life in situ, several independent measurements from multiple instrument techniques will bolster the community's confidence in making any such claim. We demonstrate the detection of subsurface biosignatures using a suite of instrument techniques including IR reflectance spectroscopy, laser-induced breakdown spectroscopy, and scanning electron microscopy/energy dispersive X-ray spectroscopy. We focus our measurements on subterranean calcium carbonate field samples, whose biosignatures are analogous to those that might be expected on some high-interest astrobiology targets. In this work, we discuss the feasibility and advantages of using each of the aforementioned instrument techniques for the in situ search for biosignatures and present results on the autonomous characterization of biosignatures using multivariate statistical analysis techniques. Key Words: Biosignature suites-Caves-Mars-Life detection. Astrobiology 17, 1203-1218.

A committee of the Mars Exploration Program Analysis Group (MEPAG) has reviewed and updated the description of Special Regions on Mars as places where terrestrial organisms might replicate (per the COSPAR Planetary Protection Policy). This review and update was conducted by an international team (SR-SAG2) drawn from both the biological science and Mars exploration communities, focused on understanding when and where Special Regions could occur. The study applied recently available data about martian environments and about terrestrial organisms, building on a previous analysis of Mars Special Regions (2006) undertaken by a similar team. Since then, a new body of highly relevant information has been generated from the Mars Reconnaissance Orbiter (launched in 2005) and Phoenix (2007) and data from Mars Express and the twin Mars Exploration Rovers (all 2003). Results have also been gleaned from the Mars Science Laboratory (launched in 2011). In addition to Mars data, there is a considerable body of new data regarding the known environmental limits to life on Earth-including the potential for terrestrial microbial life to survive and replicate under martian environmental conditions. The SR-SAG2 analysis has included an examination of new Mars models relevant to natural environmental variation in water activity and temperature; a review and reconsideration of the current parameters used to define Special Regions; and updated maps and descriptions of the martian environments recommended for treatment as "Uncertain" or "Special" as natural features or those potentially formed by the influence of future landed spacecraft. Significant changes in our knowledge of the capabilities of terrestrial organisms and the existence of possibly habitable martian environments have led to a new appreciation of where Mars Special Regions may be identified and protected. The SR-SAG also considered the impact of Special Regions on potential future human missions to Mars, both as locations of potential resources and as places that should not be inadvertently contaminated by human activity.

Many copper water lines for municipal drinking water in Santa Fe, New Mexico USA, have developed pinhole leaks. The pitting matches the description of Type I pitting of copper, which has historically been attributed to water chemistry and to contaminants on the copper tubing surface. However, more recent studies attribute copper pitting to microbial induced corrosion (MIC). In order to test for microbes, the copper tubing was fixed in hexamethyldisilazane (HMDS), then the tops of the corrosion mounds were broken open, and the interior of the corrosion pits were examined with scanning electron microscopy (SEM). The analysis found that microbes resembling actinobacteria were deep inside the pits and wedged between the crystallographic planes of the corroded copper grains. The presence of actinobacteria confirms the possibility that the cause of this pitting corrosion was MIC. This observation provides better understanding and new methods for preventing the pitting of copper tubing in municipal water.

Lava caves contain a wealth of yellow, white, pink, tan, and gold-colored microbial mats; but in addition to these clearly biological mats, there are many secondary mineral deposits that are nonbiological in appearance. Secondary mineral deposits examined include an amorphous copper-silicate deposit (Hawai'i) that is blue-green in color and contains reticulated and fuzzy filament morphologies. In the Azores, lava tubes contain iron-oxide formations, a soft ooze-like coating, and pink hexagons on basaltic glass, while gold-colored deposits are found in lava caves in New Mexico and Hawai'i. A combination of scanning electron microscopy (SEM) and molecular techniques was used to analyze these communities. Molecular analyses of the microbial mats and secondary mineral deposits revealed a community that contains 14 phyla of bacteria across three locations: the Azores, New Mexico, and Hawai'i. Similarities exist between bacterial phyla found in microbial mats and secondary minerals, but marked differences also occur, such as the lack of Actinobacteria in two-thirds of the secondary mineral deposits. The discovery that such deposits contain abundant life can help guide our detection of life on extraterrestrial bodies.

Calcite cave pool precipitates often display a variety of potential biosignatures from the macroscopic to the submicroscopic. A fossil cave pool in Cottonwood Cave, New Mexico, exhibits older stalactites and stalagmites that are completely coated in brown, laminated calcitic crust that extends down as pool fingers and u-loops. The pool fingers and u-loops are mainly micrite to clotted micrite, some recrystallized to microspar, with some isopachous spar layers. Micrite, particularly clotted micrite, is usually interpreted by carbonate workers as microbial in origin. Scanning electron microscopy examination of etched pool fingers, u-loops, and the brown crust revealed abundant calcified microbial filaments and biofilm. Energy dispersive X-ray analysis showed that these features have excess carbon, above that found in pure calcite. Independent carbon analysis indicated that these same samples contain up to 0.2% organic carbon. Since pool fingers hang down but form underwater, we hypothesize they are biogenic with hanging microbial filaments or biofilm acting as nuclei for calcite precipitation. Because of the abundance of micrite and fossil filaments, we further hypothesize that these pendant features formed during a period of plentiful nutrients and active hydrological activity when the pool was literally dripping with microbial slime. Although each of these lines of evidence could be interpreted in other ways, their combined weight strongly suggests the cave pool precipitates in Cottonwood Cave are biogenic. These investigations can be used to help inform extraterrestrial life-detection studies.

Lechuguilla Cave is an ancient, deep, oligotrophic subterranean environment that contains an abundance of low-density ferromanganese deposits, the origin of which is uncertain. To assess the possibility that biotic factors may be involved in the production of these deposits and to investigate the nature of the microbial community in these materials, we carried out culture-independent, small subunit ribosomal RNA (SSU rRNA) sequence-based studies from two sites and from manganese and iron enrichment cultures inoculated with ferromanganese deposits from Lechuguilla and Spider Caves. Sequence analysis showed the presence of some organisms whose closest relatives are known iron- and manganese-oxidizing/reducing bacteria, including Hyphomicrobium, Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea. The dominant clone types in one site grouped with mesophilic Archaea in both the Crenarchaeota and Euryarchaeota. The second site was dominated almost entirely by lactobacilli. Other clone sequences were most closely related to those of nitrite-oxidizing bacteria, nitrogen-fixing bacteria, actinomycetes and beta- and gamma-Proteobacteria. Geochemical analyses showed a fourfold enrichment of oxidized iron and manganese from bedrock to darkest ferromanganese deposits. These data support our hypothesis that microorganisms may contribute to the formation of manganese and iron oxide-rich deposits and a diverse microbial community is present in these unusual secondary mineral formations.

Caves have been used in the ancient past as shelter or habitat by many organisms (including humans). Since antiquity, humans have explored caves for the minerals they contain and sometimes for ceremonial purposes. Over the past century, caves have become the target of increasing exploration, scientific research, and recreation. The use of caves on extraterrestrial bodies for human habitation has been suggested by several investigators. Lunar lava tube bases received early attention because lava tubes were clearly visible in lunar images from the Apollo Era. More recently, Mars Observer Camera data has shown us clear evidence of large tubes visible in a number of volcanic regions on Mars. The budding field of cave geomicrobiology has direct application to questions about subsurface life on other planets. Caves contain many unusual organisms making their living from unlikely materials like manganese, iron, and sulfur. This makes caves and other subsurface habitats prime targets for astrobiological missions to Mars and possibly other bodies. We present the results of a completed Phase I and on-going Phase II NASA Institute for Advanced Concepts (NIAC) study that intensively examines the possibilities of using extraterrestrial caves as both a resource for human explorers and as a highly promising scientific target for both robotic and future human missions to Mars and beyond.

Earth's subsurface offers one of the best possible sites to search for microbial life and the characteristic lithologies that life leaves behind. The subterrain may be equally valuable for astrobiology. Where surface conditions are particularly hostile, like on Mars, the subsurface may offer the only habitat for extant lifeforms and access to recognizable biosignatures. We have identified numerous unequivocally biogenic macroscopic, microscopic, and chemical/geochemical cave biosignatures. However, to be especially useful for astrobiology, we are looking for suites of characteristics. Ideally, "biosignature suites" should be both macroscopically and microscopically detectable, independently verifiable by nonmorphological means, and as independent as possible of specific details of life chemistries--demanding (and sometimes conflicting) criteria. Working in fragile, legally protected environments, we developed noninvasive and minimal impact techniques for life and biosignature detection/characterization analogous to Planetary Protection Protocols. Our difficult field conditions have shared limitations common to extraterrestrial robotic and human missions. Thus, the cave/subsurface astrobiology model addresses the most important goals from both scientific and operational points of view. We present details of cave biosignature suites involving manganese and iron oxides, calcite, and sulfur minerals. Suites include morphological fossils, mineral-coated filaments, living microbial mats and preserved biofabrics, 13C and 34S values consistent with microbial metabolism, genetic data, unusual elemental abundances and ratios, and crystallographic mineral forms.

We have reexamined the question of extant microbial life on Mars in light of the most recent information about the planet and recently discovered nonphotosynthetic microbial ecosystems on Earth--deep sea hydrothermal vent communities and deep subsurface aquifer communities. On Mars, protected subsurface niches associated with hydrothermal activity could have continued to support life even after surface conditions became inhospitable. Geochemical evidence from the SNC meteorites and geomorphological evidence for recent volcanism suggest that such habitats could persist to the present time. There are also extensive geological features that attest to the ubiquitous nature of volcano-ground ice interactions on Mars. We suggest a possible deep subsurface microbial ecology similar to those discovered to depths of several kilometers below the surface of the Earth. We focus on anaerobic systems utilizing CO2 as the primary source of carbon. Liquid water could be provided by the heat of geothermal or volcanic activity melting permafrost or other subsurface water sources. Gases from volcanic activity deep in the planet could provide reducing power (as CH4, H2, or H2S) percolating up from below and enabling the development of a microbial community based upon chemolithoautotrophy. We suggest a methanogen (or acetogen) and sulfur-based microbial ecology as one possible basis for microbial primary production. Our hypothetical ecosystem is neither supported, nor excluded, by current observations of Mars. Tests for such a subsurface system involve locating active geothermal areas associated with ground ice or detecting trace quantities of reduced atmospheric gases that would leak from such a system.

In this paper, we show that a wide variety of common soil bacteria are able to obtain their carbon and energy needs from tholin (a class of complex organic heteropolymers thought to be widely distributed through the solar system; in this case tholin was produced by passage of electrical discharge through a mixture of methane, ammonia, and water vapor). We have isolated aerobic, anaerobic, and facultatively anaerobic bacteria which are able to use tholin as a sole carbon source. Organisms which metabolize tholin represent a variety of bacterial genera including Clostridium, Pseudomonas, Bacillus, Acinetobacter, Paracoccus, Alcaligenes, Micrococcus, Corynebacterium, Aerobacter, Arthrobacter, Flavobacterium, and Actinomyces. Aerobic tholin-using bacteria were first isolated from soils containing unusual or sparse carbon sources. Some of these organisms were found to be facultatively anaerobic. Strictly anaerobic tholin-using bacteria were isolated from both carbon-rich and carbon-poor anaerobic lake muds. In addition, both aerobic and anaerobic tholin-using bacteria were isolated from common soil collected outside the laboratory building. Some, but not all, of the strains that were able to obtain carbon from tholin were also able to obtain their nitrogen requirements from tholin. Bacteria isolated from common soils were tested for their ability to obtain carbon from the water-soluble fraction, the ethanol-soluble fraction, and the water/ethanol-insoluble fraction of the tholin. Of the 3.5 x 10(7) bacteria isolated per gram of common soils, 1.7, 0.5, and 0.2%, respectively, were able to obtain their carbon requirements from the water-soluble fraction, the ethanol-soluble fraction and the water/ethanol-insoluble fraction of the tholin. The palatability of tholins to modern microbes may have implications for the early evolution of microbial life on Earth. Tholins may have formed the base of the food chain for an early heterotrophic biosphere before the evolution of autotrophy on the early Earth. Where tholins are present on other planets, they could possibly be metabolized by contaminant microorganisms transported to these bodies via spacecraft. Thus, the presence of tholins should be taken into account when evaluating the planetary quarantine requirements for probes to other planets.