BIOMARKERS AS TRACERS FOR LIFE ON EARLY EARTH AND MARS 

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BERND R. T. SIMONEIT1, R. E. SUMMONS2 and L. L. JAHNKE3

Petroleum and Environmental Geochemistry Group, College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331, U.S.A.; 2 Australian Geological Survey Organisation, GPO Box 378, Canberra, ACT 2601, Australia; 3 Planetary Biology Branch, NASA Ames Research Center, Moffett Field, CA 94035, U.S.A. (Received 12 November 1996)

Abstract. Biomarkers in geological samples are products derived from biochemical (natural product) precursors by reductive and oxidative processes (e.g., cholestanes from cholesterol). Generally, lipids, pigments and biomembranes are preserved best over longer geological times and labile compounds such as amino acids, sugars, etc. are useful biomarkers for recent times. Thus, the detailed characterization of biomarker compositions permits the assessment of the major contributing species of extinct and/or extant life. In the case of the early Earth, work has progressed to elucidate molecular structure and carbon isotopic signals preserved in ancient sedimentary rocks. In addition, the combination of bacterial biochemistry with the organic geochemistry of contemporary and ancient hydrothermal ecosystems permits the modeling of the nature, behavior and preservation potential of primitive microbial communities. This approach uses combined molecular and isotopic analyses to characterize lipids produced by cultured bacteria (representative of ancient strains) and to test a variety of culture conditions which affect their biosynthesis. On considering Mars, the biomarkers from lipids and biopolymers would be expected to be preserved best if life flourished there during its early history (3.5–4 109 yr ago). Both oxidized and reduced products would be expected. This is based on the inferred occurrence of hydrothermal activity during that time with the concomitant preservation of biochemically-derived organic matter. Both known biomarkers (i.e., as elucidated for early terrestrial samples and for primitive terrestrial microbiota) and novel, potentially unknown compounds should be characterized.



1. Introduction The NASA-Exbiology Program began support for the initial research on chemical evolution in the early 1960s. This became a vigorous program utilizing the contemporary state-of-the-science expertise and instrumentation for analyses of organic matter and provided the impetus for the advancement of organic geochemistry. Carbonaceous chondrites and ancient sediments (1–3  109 yr) were analyzed for biogenic organic residues (molecular fossils), such as lipid, amino acid and pigment compounds. This new research field, i.e. chemical evolution as applied to exobiology, was eloquently summarized for example by Calvin (1969) and in a broader context by Mason (1992). It had the goal to ultimately examine lunar samples for biogenic/prebiotic organic matter, but none was found. Also, organic analyses were performed to test for contemporary life on the Viking Mission to  Presented in part at the International Society for the Study of the Origin of Life Meeting, Orleans, France, July 1996.

Origins of Life and Evolution of the Biosphere 28: 475–483, 1998. c 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Mars. The results were inconclusive. Over the past 25 years organic geochemistry has expanded into a myriad of research areas and has built a vast data base on biomarkers indicative of biochemistry. This expertise is now ready to address the search for tracers of prebiotic organic chemistry and of extinct and extant life on Mars. 1.1. BIOMARKERS Biomarkers (also termed chemical fossils or molecular markers) are organic indicator compounds (or tracers) which are useful in correlations of the genetic sources of lipidic or bituminous matter (e.g., Brassell, 1992; Johns, 1986; Simoneit, 1978,1986, and references therein). Amino acids and other labile biochemicals have been used for elucidating biological activity in the more recent geological record (e.g., Abelson, 1954; Mitterer, 1993). Such biomarker molecules have definitive chemical structures, which can be related either directly or indirectly through a set of diagenetic alterations to biogenic sources, and they cannot be synthesized by abiogenic processes. The utility of biomarkers as indicators of biogenic, paleoenvironmental and geochemical processes on Earth has been widely accepted (e.g., Brassell, 1992; Imbus and McKirdy, 1993; Johns, 1986; Mackenzie et al., 1982; Mitterer, 1993; Simoneit, 1997; Simoneit et al., 1986, and references therein). Some commonly used examples are listed in Table I. An example of a biomarker product-precursor compound group, the hopanoids, is illustrated in Figure 1, showing both the reductive and oxidative diagenesispreservation pathways observed in the geosphere (e.g., Simoneit, 1994). The hopanoids are the most extensively documented cyclic biomarkers for bacteria. The same precursor-product relationship, both reductive and oxidative, was illustrated earlier for steroids (Simoneit, 1995). It is obvious that one precursor natural product can yield many derivatives as chemical fossils in a geological record, and that all these specific products can be correlated back to their precursor. Biomarkers are found in volatile, liquid and solid carbonaceous matter and their changes over geological time have also been elucidated (e.g., Summons, 1993, and references therein). Detailed characterization of biomarker mixtures in terms of sources and degree of alteration permits the assessment of: (a) extant life and major contributing species, and (b) extinct life, major contributing species and geo/hydrothermal alteration. In the case of Mars the biomarkers should be based on carbon biochemistry and those derived from lipids and biopolymers would be expected to be preserved best if life evolved there during its early history (3–4  109 yr ago). Both oxidized and reduced products would be expected. The most versatile and specific methods available now for biomarker characterization are summarized next.

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Table I Some examples of biomarkers characteristic of life Compound classa

Common biotic sourceb

Analytical detection method

1. Labile compounds to assess contemporary or recent life processes Amino acids/peptides all life GC, Py-GC Sugars/polysaccharides flora (some fauna) GC Nucleotides, bases, etc. all life specific methods Unsaturated lipid compounds all life GC, GC-MS 2. Lipid/bitumen compounds to assess biosynthesis in prior geological times: Aliphatic hydrocarbons ubiquitous/not specific GC, GC-MS, GC-IRMS Isoprenoids biogenic GC, GC-MS, GC-IRMS Steroids flora/fauna GC-MS, GC-IRMS Triterpenoids flora/microbes GC-MS, GC-IRMS Diterpenoids flora/microbes GC-MS, GC-IRMS Pigments flora/microbes HPLC-MS, HPLC-IRMS Biopolymers flora/microbes Py-GC-MS Novel and unknown biomarkers – GC-MS, Py-GC-MS a

Labile markers are stable for briefer geological periods after diagenetic preservation. Group 2 is stable for longer geologic periods. b Slash indicates that a distinction is possible.

The analytical methods commonly used for the structural characterization of biomarkers have made vast advances over the past 25 yr. They are gas chromatography (GC), GC-mass spectrometry (MS), pyrolysis (Py)-GC-MS, with the most recent addition of GC-isotope ratio mass spectrometry (IRMS) (Table I). GC at ultra high resolution is attained with flexible fused silica or metal capillary columns for the analytical range from C1 !C100 and sensitivities as low as pg levels. Various detectors (FID, ECD, NPD, etc.) make GC more versatile for fingerprinting and coupling pyrolysis provides a means to analyze macromolecular organic matter. Nominal (low) resolution mass spectrometry coupled to GC is used for most compound elucidation. Some applications use high pressure liquid chromatography (HPLC) as inlet to MS. A myriad of reference standards have been synthesized or certified and there are numerous MS library files. Typical instrument sensitivities are in the low ng range. High resolution MS coupled with GC is another option for special cases, although with slightly lower sensitivity. The new method of GC-IRMS is gaining rapid acceptance for determining the stable carbon isotope compositions of individual compounds in mixtures (e.g., Hayes et al., 1990; Schoell et al., 1992; Simoneit et al., 1993, 1995). Such data complement the compound source assessments based on chemical structures. The sensitivity of GC-IRMS is approximately the same as GC-MS. The arsenal of

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Figure 1. Alteration scheme for hopanoids by reduction to the geo-hopanes, by oxidation to aromatic hydrocarbon derivatives, and by reaction with sulfur to thio-aromatic products.

ancillary instrumental methods can be extended if needed and includes for example ICP-MS, GC-FTIR, and FT-NMR. Methods for the analysis of labile biomarkers (e.g., amino acids, carbohydrates) are designed specifically for that purpose but can include the application of instrumentation described above.

2. Biomarkers of Bacterial Ecosystems The biomarker concept will be illustrated here with bacterial lipids which are informative about the development and evolution of oxygenic photosynthesis and aerobiosis, because these processes are at the heart of the modern carbon cycle and sustain complex life (Summons et al., 1996). Bacterial mats from modern

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hot springs (e.g., Yellowstone National Park) have lipid distributions with striking similarities to preserved hydrocarbons from the Proterozoic and Archean. Points of resemblance include abundant, low molecular weight, linear and simple branched alkanes, acyclic isoprenoids such as phytenes, phytol and carotenoids (e.g., Dobson et al., 1988; Shiea et al., 1990; Ward et al., 1989; Zeng et al., 1992a,b). Polycyclic isoprenoids such as steroids and hopanoids are generally undetectable or occur at very low levels. Differences are mainly that modern systems reflect the living microbiota, rich in functionalized lipids such as glycerol esters and ethers, fatty acids, alcohols and wax esters, whereas fossil assemblages contain mostly saturated hydrocarbon skeletons. In order to obtain more information about lipids produced by extant bacteria with affinities to geologically ancient groups, various specific organisms are under study. Of these, the cyanobacterium Phormidium luridum was chosen as an example for its relationship to conophyton stromatolite-forming cyanobacteria and for its ability to produce a range of hydrocarbon skeletons which are prevalent in geologically old bitumens. These biomarkers are illustrated in Figure 2 and include methylalkanes and dimethylalkanes, hydrocarbons which are particularly abundant in Proterozoic and Archean sediments and in recent cyanobacterial mats (e.g., Robinson and Eglinton, 1990; Shiea et al., 1990; Kenig et al., 1995). It is noteworthy that other Phormidium species occur widely in hot spring mat communities and are especially prominent in the modern conophytons found there (Summons et al., 1996). In contrast to terrestrial hydrothermal systems, submarine systems have a better potential for long-term preservation of organic matter due to thermal alteration, migration, and preservation processes of organic products under a higher pressure cover of water, and subsequent burial in the sediment pile. Based on this, the dual aspects of biota-biomarker relationships and the longer-term fate of the organic matter are known. Hydrothermal alteration of immature organic detritus, generally under strongly reducing conditions, occurs in such high temperature and rapid fluid flow regimes (Simoneit, 1990). Pressure in the marine systems (1 to >3 km water depth) maintains the fluid state. The agent of thermal alteration and mass transfer, hot circulating water (temperature range from warm to >400 C), is responsible for molecular alterations which are primarily reductive. The ‘reduced’ nutrients, such as hydrogen, hydrogen sulfide, methane and higher hydrocarbons, emanating with the fluids from hydrothermal vent systems support an ecosystem with biota carrying out chemosynthesis for biomass production and respiration (e.g., Childress, 1988; Jones, 1985; Laubier, 1988; Simoneit, 1990). The lipid and biomarker compositions of these organisms are just beginning to be elucidated (e.g., Brault et al., 1989; Comita et al., 1984; Rieley et al., 1995). Extrapolation of biomarker evolution in hydrothermal environments to ancient ecosystems will become more definitive as more molecular data are reported. Hydrothermal alteration of organic matter in contact with hot fluids progresses from reductive to more oxidative reactions as the temperature increases. Reduction is strongly mediated by metal sulfides and other catalytic surfaces, with the hydro-

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Figure 2. Geologically significant lipids and saturated hydrocarbon counterparts isolated from Phormidium luridum.

gen being derived from both water and organic matter (Leif, 1993). Oxidation of organic matter in the system is enhanced by the presence of sulfur and sulfate, yielding hydrogen sulfide (Leif et al., 1992). At very high temperatures, organic matter is partly destroyed and partly converted to aromatics (e.g., PAH). These

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thermogenic products are more soluble in the ambient hot fluid which rapidly moves away from the hot zone by convection. Hydrothermal products, including biomarkers, are then trapped and preserved in interstitial fractures and voids or in fluid inclusions (e.g., Peter et al., 1990). This type of hydrothermal activity may have also occurred on Mars during its early history (Jakosky and Jones, 1994; Squyres and Kasting, 1994). The possibility of life on Mars in its early history and the search for extant life have recently been reviewed (McKay et al., 1992; Klein et al., 1992) and evidence for possible relic biogenic activity in a Martian meteorite has been presented (McKay et al., 1996).

3. Expectations and Recommendations The characterization of the early record of biomarkers and carbon isotope systematics should continue for Precambrian (Archean) sediments of Earth. In the case of Mars, biomarkers as hydrocarbons from lipids and biopolymers are expected to be preserved best if: (a) life evolved during its early history (3 to 4  109 yr ago), (b) life was based on carbon biochemistry, (c) water was abundant, and (d) tectonic and geothermal stress was mild over the intervening geological time. Both labile/polar and lipidic biomarkers are of utility to assess whether life processes are currently active or were active in the recent geologic past. Ideal samples for biomarker screening would be sediments, rocks and soils cored to various depths (preferably into unoxidized and unweathered facies) from a selection of locales on Mars. Both molecular and carbon isotopic characterization should be carried out. Sample sizes of 0.1–1 g are adequate, especially if a total organic carbon analysis reveals values of >0.1%. The search for evidence of life on Mars should include a strong program of biomarker characterization, in the broad sense including both labile/polar and more stable lipidic/aliphatic components, as confirming indicators for biochemistry analogous as we know it from Earth history. A proponent should become involved to synthesize and review the existing data, be part of mission planning, and eventually participate in returned sample analysis.

Acknowledgements Financial support from the U.S. National Aeronautics and Space Administration to L.L. Jahnke and to B.R.T. Simoneit (Grant NAGW-4172 to BRTS) is gratefully acknowledged.

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