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Project Progress

6. Molecular and Isotopic Biosignatures

1. Preservation of Molecular Biosignatures

Steele and Postdoctoral Fellow Marc Fries used the new WiTec Raman imaging system to begin the examination of in situ carbon formation in a variety of samples, including Precambrian rocks and samples from a Mars analog site in Svalbard. Carbonate globules contained within carbonate-cemented breccia are distinct from carbonate globules within olivine-rich mantle xenoliths from the same area of Svalbard. The team studied the mineralogy and morphology of these mantle xenoliths in an attempt to understand the conditions of formation of these structures and how they may relate to carbonate globules studied from this site previously as well as to the morphologically similar carbonate globules found in Martian meteorite ALH84001.

An interesting observation from these data is the detection of hematite and magnetite within distinct zones of the carbonate globules and the occurrence of C in conjunction with magnetite. Such a zonation is predicted from studies of the stability fields of hematite, magnetite, siderite, and graphite at 0.1-GPa pressure in a CO-rich mixture of CO₂ and CO over a range of temperatures and oxygen fugacities. Interestingly, the compositions of the carbonate globules seem to follow the trends expected from thermal decomposition of siderite and production of magnetite and polyaromatic hydrocarbons (PAHs) as a mechanism to explain the presence of PAHs and magnetite in ALH84001. However, no secondary shock can have occurred to influence formation of these compounds in terrestrial samples. On the basis of zonation patterns, carbon and magnetite therefore most likely formed during the precipitation of carbonate from a CO₂-rich hydrothermal fluid percolating through the mantle.

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Hazen collaborated with Nora Noffke (Old Dominion University) in studies of purported microbial mat structures from the 3.2-billion-year-old Moodies Group, Barberton Greenstone Belt, South Africa, which features rocks from one of Earth’s oldest known siliciclastic tidal marine environments. A combination of geological, chemical, and morphological features, including the shallow marine setting, thin mat-like distribution of carbon, negative carbon isotope values (δ¹³C ~ —21), trapping and binding of sediment grains, roll-up structures, desiccation features, and distinctive wrinkle structures are similar to those of modern microbial mats. These diagnostic biosignature features, some of which are recognizable in the field from a considerable distance, might represent potential targets in the search for life on Mars.

Understanding the role of life in the formation of early Proterozoic sedimentary manganese deposits is important for unraveling the history of the Earth during its transition from an anoxic to an oxic atmosphere. To recognize potential biomarkers in ancient deposits, Doctoral Student Rachel Schelbel is leading an experimental study simulating the degradation of microorganisms exposed to Mn(II) and SiO₂. Degradation of Bacillus subtilis and Escherichia coli is tracked by the break-down of a short-lived biomarker (DNA) and a long-lived biomarker (phospholipid fatty acids and their derivative n-alkanes). Comparison samples were collected from the early Proterozoic Kalahari Manganese Field of South Africa. Study procedures include measurements of carbon isotopes and remnant organic carbon and microscopic examination of samples.

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Steele and Doctoral Student Maia Schweizer studied the 400-million-year-old Rhynie Chert from Scotland, famous for its exquisite preservation of the Earth’s earliest land plants in hot spring deposits. Material from this fossiliferous chert has been characterized using confocal Raman imaging. Embedded fossil plant materials were examined in transmission using thin sections as well as embedded material exposed on the surface of thick sections. For surface analysis, surface-enhanced Raman spectroscopy (SERS) was employed to amplify spectra and enhance the signatures of latent post-organic compounds. These observations will be compared with those of other chert samples, such as the 3,465-million-year-old Apex chert, as part of a study to characterize the state of biosignatures in a series of progressively older cherts.

Vicenzi worked on the problem of discerning bona fide fossilization products, entombed and mineralized microorganisms, from abiologically derived materials. This task can be particularly vexing in ancient sediments that have suffered some level of metamorphism and metasomatism. The difficulty in identifying the origin of such terrestrial specimens highlights the daunting task facing those who seek to determine the biogenicity of features within specimens returned from Mars and elsewhere. To address this important paleobiological issue, Vicenzi and his group examined mineralized bacterial sheaths produced by Fe-oxidizers within a modern iron seep environment. Three chemical imaging techniques were used to characterize specimens: Raman spectroscopy, energy dispersive X-ray microanalysis, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The complexity of such a “simple” specimen is particularly evident in the distribution of molecular species (Figure 3). Geochemical traces of the departed bacteria can be seen along the walls of the interior of sheaths as seen in the C₂H and C₂ images. The sheath exteriors, by contrast, are enriched in (Ca,Fe) phosphate and Cl; this coating is assumed to be related to inorganic precipitation.

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Tuross, now at Harvard University, developed an antibody for life-detection work. She also determined the N isotopic composition of various chemical fractions of the Green River Shale, a type material for organic geochemistry that has a range of molecular and anatomical fossils preserved in its matrix.

2. Stable Isotope Biosignatures

Fogel and colleagues collected a suite of samples from the Mars analog site in Svalbard for elemental and isotopic analyses with the goal of determining whether a biological signal can be detected above a background of abiogenic C in basalts, carbonates, and other surface rocks. Lichens, terrestrial higher plants, and photosynthetic microorganisms from thermal springs were also analyzed to provide signatures of the extant biological sources. The team found rocks with three potentially distinct pools of C: (1) biogenic photosynthetic organic matter, (2) high-temperature C with a mantle origin often associated with olivine crack surfaces and fluid/gas inclusions within the olivines, and (3) abiogenic C associated with cooling of volcanic gases during breccia formation. The xenolithic carbon representing the second pool of C was most likely not contaminated by biological material, because of the morphological provenance of C with specific mineral assemblages known to promote abiogenic organic C formation. Only the third pool of C has a distinct δ¹³C signature in the organic fraction.

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All of the rocks analyzed showed some trace of organic C and had an average δ¹³C value of —27.5‰, close to the value of —26.6‰ for plants and photosynthetic microbes. The most likely explanation of these results is that the majority of these rocks contained traces of modern biological C, because the soils and weathered specimens had a low C content (just above 0.2 wt ) compared with the high C content of plants (32). Inorganic carbonate, mostly calcium carbonate or dolomite, has δ¹³C of +0.7‰ and is associated with thermal spring carbonate precipitation.

δ¹³C analyses were also carried out on silicate minerals from spinel lherzolite xenoliths and carbonate globules occurring as inclusions in and on grain boundaries of xenolith minerals. Powdered xenoliths contained both inorganic carbonates (δ¹³C = —5‰) and organic matter (δ¹³C = —29.4‰) in the proportion of 4:1. Individual large-scale olivine crystals had less inorganic carbon (δ¹³C = —20‰) than the xenoliths as a whole and organic carbon with δ¹³C of —26‰ in the proportion of 2:1. Molecular biological evidence shows that the bioload in these rocks is at the limit of detection, thereby confirming an abiogenic source for this xenolith C. A third pool of C that we discovered was located in interior regions of volcanic breccia at Sverrefjell volcano. The inorganic carbon δ¹³C = —1.1‰, whereas the organic carbon (0.2% of the total rock) inside of the breccia itself had a δ¹³C = —12.1‰.

Fogel initiated study of interactions between the biological N cycle in plants, microbes, endolithic communities, and animals, and the geochemical N cycle in igneous and sedimentary rocks. The δ¹⁵N of the plants, lichens, and microbes on Svalbard were of course the result of biological processes. Plants that were directly fertilized by animal wastes had δ¹⁵N up to 14 ‰, whereas as those that obtained their N via atmospheric NH₄ transported 5 km downwind from massive seabird colonies had δ¹⁵N as low as —14 ‰. Such extremes in isotopic composition, for the same species and types of plants, can be related directly to biological N cycling.

Ubiquitous Mg-carbonate deposits and dolomite-magnesite globules associated with the volcanic eruptive centers all show unusual ¹⁸O-depletion with δ¹⁸O values between —27 and —34‰ indicating precipitation from solutions of glacial origin. Combined C and O isotope signatures are matched only by reported values for cryogenic carbonates formed during slow freezing of glacial melt water. The abundant carbonates in Svalbard carbonate rocks evidently originated by cryogenic precipitation from meteoric to mantle-derived fluids following volcanic eruptions under extremely cold ambient conditions. The unusual dolomite-magnesite globules occurring in Svalbard rocks and on Mars may be typical end products of cryogenic carbonate precipitation from hydrous fluids.

Fogel, Steele, Doctoral Student Maia Schweizer, and Collaborator Jan Toporski studied the carbon and nitrogen isotopic compositions of autolithified soft tissues from 49-Ma vertebrate fossils from the Messel Formation in Germany. Messel’s fossil flora and fauna are diverse and exquisitely preserved. Insects and plants are preserved with original material intact. Stable isotope results have been compared with previous trophic reconstructions based on gut contents and coprolites and also with related modern organisms. Different tissues within fish specimens are isotopically distinct, with intraorganism fractionations similar to those observed in modern organisms. Stable isotope signatures also clearly reflect the feeding patterns of fossil organisms. Primary producers are associated with low δ¹⁵N values, and consumers are enriched by +2‰ or more in δ¹⁵N relative to these values depending on their diets. Messel fossil organisms represent several trophic levels in each of two trophic webs, one aquatic and one terrestrial. Both trophic systems include primary producers (terrigenous and aquatic plants), primary consumers (insects), and higher consumers (carnivores such as fish, crocodiles, and frogs). δ¹³C values for these organisms trace carbon sources and indicate widespread omnivory in both low- and high-trophic-level consumers.

Postdoctoral Fellow Jennifer Eigenbrode worked on several tasks aimed at distinguishing sources of organic molecules (i.e., geochemical vs. biological, modern vs. ancient), particularly when present at trace levels. Ultimately, her studies should provide insight into the evolution of carbon cycling on Earth and offer analytical and interpretive strategies for similar studies on other planets. In collaboration with Roger Summons (MIT), she completed new organic geochemical analyses of extracts from late Archean (2.7-2.6 Ga) shale and carbonate rocks from Western Australia. Those analyses revealed relationships among lipid biomarkers, carbon isotopes, and rock types similar to those documented earlier. The expanding isotopic and molecular dataset from these units support syngenicity between fossil lipids and their host rocks and indicates biological cycling of oxygen approximately 400 My before the steep rise of atmospheric oxygen. Because fossil designation for some of the alkane biomarkers identified in these Precambrian rocks is uncertain, samples were prepared for additional analysis (to be carried out collaboratively with Fogel) that will target more recent biological signatures and compound-specific isotopic compositions. Isolation of DNA from these rock samples (by Eigenbrode and Steele) produced negative results. Eigenbrode also carried out organic geochemical analysis of hydrothermally altered mantle peridotite rocks, associated carbonate, and biota from Svalbard and Cedar Springs, California. In these systems, organic molecules thought to have been produced by geochemical processes provide a unique environment for geo-biological interaction. Collaborators on this last project, in addition to Fogel and Steele, include Hans Amundsen (University of Oslo) and Ken Nealson (USC).

As an early step in her analysis of biogenic and abiogenic hydrocarbon gases, Postdoctoral Fellow Penny Morrill initiated a calibration for stable carbon isotope measurements of hydrocarbon gases. Gas samples were obtained from Jug Bay, a fresh-water marsh where gas bubbles are frequently observed. The samples were analyzed for their gas compositions and carbon isotope ratios. As anticipated, the isotope signatures were typical of biological hydrocarbons. Experiments have been initiated to determine the carbon isotope fractionation between silicate minerals and a C-O-H gas phase at ranges of temperature and pressure appropriate to the mantle and crust. A preliminary experiment using silver oxalate (a source of carbon dioxide) and water heated to 1600°C and 1.5 GPa yielded a small amount of CO₂ gas, with a δ¹³C value of —3.1 ‰. The initial δ¹³C of the silver oxalate was approximately —2 ‰, which suggests that there was little to no isotopic fractionation between the initial substrate and CO₂. A subsequent experiment using the same starting materials heated to 1150°C suggested a large carbon isotopic fractionation. Isotopic equilibrium will be tested by conducting time studies with experiments at a given temperature, pressure, and hydrogen fugacity, over pressures between 0.5 and 2 GPa and temperatures between 1000°C and >1500°C and over durations varying between minutes and several days using starting materials having a grain size on the order of 1 mm. Experiments have been initiated to create abiogenic hydrocarbons under temperatures and pressures experienced at deep-sea hydrothermal vents. Formic acid is used as the source of dissolved CO₂, water is used as a source of hydrogen, and a variety of metals are being used as catalysts. A preliminary experiment using nickel as the catalyst yielded gaseous CO, CO₂, and CH₄. The methane had a δ¹³C value of —39.6 ‰.

3. Scanning Transmission X-ray Microscopy and In Situ Chemical Analysis of Organic Fossils

Cody and colleagues have applied molecular spectroscopy and spectrometry to the analysis of ancient organic fossils as part of three specific tasks. First, together with Andy Czaja (UCLA), Cody utilized ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy and pyrolysis gas chromotagraphy mass spectrometry (GC-MS) to explore the chemical transformations that accompany the conversion of modern organic matter contained within ferns into the complex organic matter detected in 50-Ma fossil ferns of identical type. Second, in collaboration with C. Kevin Boyce, a former NAI-NRC Postdoctoral Associate now at the University of Chicago, Cody applied pyrolysis GC-MS to characterize the preserved biomass of Devonian (~ 400-Ma) organisms. A comparison of pyrolysis GC-MS data (Figure 5) from a well-characterized albeit enigmatic fossil of Prototaxites with similarly ancient organic matter from an organic-rich sediment (Devonian coal) indicates that the distribution of molecular constituents from the fossil and the coal are quite different even as the specific types of molecular compounds detected reveal that both the Prototaxites and coal samples have undergone significant thermochemical evolution. The third task utilizes scanning transmission X-ray microscopy (STXM) to study ancient kerogens from the Hammersley Basin, Western Australia. Together with Postdoctoral Fellow Jennifer Eigenbrode, Cody began the process of preparing ultra-thin sections of proterozoic kerogens for in situ STXM analysis this fall.

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4. Studies of Organic Compounds at the Kamchatka Hydrothermal Region

Organic species on the prebiotic Earth would serve as monomers for the synthesis of more complex molecules, such as peptides and oligonucleotides, and to form membranous boundary structures. However, for such species to accumulate in concentrations sufficient to support synthetic reactions, there must have been a source providing the material at a rate equal to or greater than they were removed by degradation, adsorption, or burial. Deamer last year initiated an experiment in the Kamchatka geothermal region with the aim of determining how a set of pertinent organic compounds behave in such a setting. Situated in the far northeastern portion of Russia, the Kamchatka Peninsula spans a region approximately 400 by 1200 kilometers. Like Yellowstone National Park in Wyoming, Kamchatka represents a modern analog for hydrothermal ecosystems that have been active throughout most of Earth’s history. One such site in the region was chosen as a model system to investigate both degradative reactions and possible synthetic reactions driven by condensation. To a small (ten-liter) boiling pond Deamer’s group added four amino acids and four nucleobases, and they included phosphorus as phosphate, a fatty acid as a model amphiphile, and glycerol. Specific goals included learning the fate of a mixture of organic compounds in a natural setting (i.e., assuming that there was a source of organic compounds on the prebiotic Earth, at what rate would such compounds undergo degradation and thereby be lost as potential reactants for synthetic processes?) and assessing the synthetic reactions that are possible under such conditions.

The basic observation of the experiment was that amino acids, nucleobases, and phosphate are removed as solutes with half-times measured on time scales from minutes to a few hours. Only cytosine and myristic acid could still be detected after nine days. The loss was not due simply to flow through the pond, because one of the amino acids – glycine – and one of the nucleobases – cytosine – had half times much longer than the other solutes. Nor was the loss due to simple acid-catalyzed degradation reactions, because a laboratory simulation showed that the solutes were all stable for at least two hours. The disappearance of phosphate on the same time scale offers one possible clue. Phosphate is very stable and could not undergo degradative reactions in the same way that organic compounds might. Nor could it precipitate as calcium phosphate (apatite) because there was little calcium ion in the water and the low pH of 3.1 allows calcium phosphate to remain in solution. This inference suggests that the phosphate and organic solutes were lost from solution by adsorption to mineral surfaces of clays and then transported and deposited in the layers of clay at the borders of the puddle. When clay samples were analyzed, virtually all of the added components could be released from the clay by treatment with a dilute sodium hydroxide solution.

The conclusion of the first season of work was that the organic compounds required for the origin of life in a natural setting would have had a variety of possible fates other than those possible in a laboratory setting, in which pure compounds react in glass containers. To understand these processes, it will be necessary to repeat these experiments in a variety of natural environments that are potential models for sites associated with the origin of life. One such repetition has just been completed in a geothermal area of Iceland, and the samples are being analyzed.