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Project Progress

1. Biosignatures in prebiotic materials and catalysts

Postdoctoral Fellow James Cleaves, Co-Investigators George Cody, Marilyn Fogel, and Robert Hazen, and others have been studying the analysis of chromatographed hydrolyzed and unhydrolyzed products, as well as the structure of polymeric fractions. HCN polymerizes spontaneously in water to produce a variety of heterocycles, amino acids, hydroxy acids, and other molecules of biological interest. In the presence of HCHO, a more diverse suite of compounds is produced. Comets and meteorites appear to contain similar chemical compounds, and it is possible that analogous chemistry takes place on these bodies. Additionally, the organics in meteorites have unusual isotopic compositions that undoubtedly give some clues to their chemical history.

The composition of comet Hale-Bopp, for example, is dominated by a small number of volatile compounds, including water, HCHO, HCN, and NH₃. Many of the other compounds present (e.g., CH₄, CO₂, CH₃OH) are not especially reactive in aqueous solution. It is noteworthy that the Murchison meteorite contains a host of organic compounds including an insoluble polymer and a suite of amino and hydroxy acids, as well as nitrogen heterocycles that are suggested to have been formed by the aqueous alteration of the parent bodies, giving rise to these species via Strecker-like chemistry and cyanide polymerization, among other proposed mechanisms.

HCHO and HCN are also two of the principal products of high-energy electric discharge reactions acting on gas mixtures, so it was of interest for Cleaves and his team to examine the effect of added HCHO on the structure of the soluble polymer, the nature of the low-molecular-weight products, and the isotopic fractionation that occurs during the reaction.

Upon mixing of the reagents a rapid and noticeable color change occurred, followed by the precipitation of insoluble material. The addition of HCHO to the reaction resulted in much lower production of insoluble polymer. There is some similarity between the low molecular weight and polymeric composition of the products of HCN polymerizations and the products found in the Murchison meteorite. CHNO analysis revealed that as the fraction of HCHO increases, the soluble polymer becomes both O and C enriched, and N and H depleted. There is a discontinuity in the compositional trends beginning at concentrations of ~ 0.2 M HCHO.

The ¹⁸O, ²H, ¹³C, and ¹⁵N fractionation of the non-volatile soluble polymer was studied as a function of mole fraction HCHO. Results suggest that most of the O incorporated into the polymers is from water, while most of the H is from HCHO. Little of the starting NH₃ is incorporated into the soluble polymer, and the polymer is enriched in ¹⁵N and ¹²C. There is a marked increase in the yield and chemical diversity of amino acids upon addition of HCHO to the mixtures.

2. Isotopic biosignatures of Earth’s early biosphere

The causes of atmospheric oxygenation in the Paleoproterozoic may be related to increased levels of primary productivity. Environmental consequences of these changes included increased abundance of seawater nitrate, which opened new niches to denitrifying organisms. In an attempt to study these possible changes, Postdoctoral Fellow Dominic Papineau, Co-Investigators Andrew Steele and Fogel, and others studied the C and N isotope compositions, trace elements, and REE abundances of carbonaceous shales from the Paleoproterozoic Aravalli Supergroup, India, which occur stratigraphically above the oldest phosphogenic event around 2.0 Ga. The group’s samples of carbonaceous shales represent various metamorphic grades and were found to have ranges of δ¹⁵N values and C/N ratios between +1‰ (C/N = 6) and +27‰ (C/N > 250). To evaluate the maturity and crystal structure of this carbonaceous material, Papineau and others performed laser Raman spectroscopy and O and H abundance measurements on insoluble organic matter from these shales. Carbonaceous shales from the Rama and Umra sub-basins have δ¹³C values between -13 and -18‰, δ¹⁵N values between +5 and +11‰, C/N below 56, and acid-insoluble organic matter with high H/C and O/C ratios.

These results suggest high productivity and/or evaporative conditions along with denitrification during deposition. Black shales from Udaipur and Amberi have high C contents up to 8%wt, δ¹³C values around -30‰, large ranges of δ¹⁵N values between +1 and +19‰, C/N between 7 and 260, and acid-insoluble carbonaceous material with Raman spectra and H/C and O/C typical for graphite. These δ¹³C values were significantly altered during metamorphism, and the C/N and N-isotope data indicate a progressive loss of ¹⁴N during metamorphism. Some of these shales also have relatively elevated amounts of V (near 100 ppm) that suggest euxinic conditions during deposition. Another group of shales from Udaipur, Lakarwas, and Ghasiar have δ¹³C values between -20 to -26‰ and a large range of δ¹⁵N values between +3 and +20‰ (C/N from 3 to 305). Some of these data are consistent with some metamorphic overprints, especially for shales metamorphosed at upper greenschist facies (high C/N). Combined with V/(V+Ni) ratios typically above 0.6 and often >0.85, these data suggest redox-stratifed seawater with active denitrification during sedimentation. The group’s interpretation of high primary productivity in some localities of the lower Aravalli transgression is consistent with associated shallow-marine stromatolitic phosphorites that have heavy δ¹³C_org values and dolomites enriched in ¹³C.

Hazen and Collaborator Nora Noffke studied the extensive microbial mats that colonize sandy tidal flats along the coasts of today’s Earth. The microbenthos (mainly cyanobacteria) respond to the prevailing physical sediment dynamics by biostabilization, baffling, and trapping, as well as binding. This biotic-physical interaction gives rise to characteristic microbially induced sedimentary structures (MISS) that differ greatly from both purely physical structures and from stromatolites. Actualistic studies of the MISS on modern tidal flats have been shown to be the key for understanding equivalent fossil structures. Fossil MISS occurs in tidal and shelf sandstones of all Earth ages. However, until now the fossil record of Archean MISS has been spotty, and relatively few specimens have been found.

The team’s study location displays a unique assemblage, with a multitude of exceptionally preserved MISS in the 2.9-Ga-old Pongola Supergroup, South Africa. The “Nhlazatse Section” includes structures such as “erosional remnants and pockets,” “multidirected ripple marks,” “polygonal oscillation cracks,” and “gas domes.” Concomitant optical and geochemical analyses support the biogenicity of microscopic textures such as filamentous laminae or “oriented grains.” Textures resembling filaments are lined by iron oxide and hydroxides, as well as clay minerals. They contain organic matter, whose isotopic composition is consistent with carbon of biological origin.

The ancient tidal flats of the Nhlazatse Section record four microbial mat facies that occur in modern tidal settings as well. The group distinguishes endobenthic and epibenthic microbial mats, including planar, tufted, and spongy subtypes. Each microbial mat facies is characterized by a distinct set of MISS and relates to a typical tidal zone. The microbial mats are preserved in situ, and the structures they caused are consistent with similar features constructed today by benthic cyanobacteria. However, the team does not exclude other mat-constructing microorganisms to have formed the structures in the Archean tidal flats.

3. Stable isotope biosignatures of high-temperature macromolecular carbon

Collaborator Penny Morrill and coauthors have shown that gaseous hydrocarbons can also be formed by abiogenic chemical reactions (e.g., photochemistry, hydrothermal reactions), and they documented the possible abiogenic origin of hydrocarbons at a site of active serpentinization, collected from ultrabasic reducing springs flowing through serpentine and peridotite rocks in northern California.

Morrill, Fogel, and Collaborator Bjorn Mysen worked on determining the carbon content and isotopic fractionation between CO₂ and the C included in diopside minerals and melts. They began collaborating with Elizo Nakamura and others at the Institute for the Study of the Earth’s Interior in Misasa, Japan. In follow-up to this work, Steele and Fogel analyzed chemical and temperature-resistant macromolecular carbon from Martian meteorites and peridotite xenoliths collected in Svalbard. Their method included HCl washing to remove carbonates followed by combusting at 550°C for two hours to remove any traces of organic materials originating from terrestrial or surface contamination (in the case of the xenoliths), and their results can be compared with those of step-combustion instruments previously used for Martian carbon experiments.

Co-Investigator Conel Alexander, along with Fogel, continued to measure the stable isotopic composition of H, O, N, and S in insoluble meteoritic organic matter. Both have extended the number and types of meteorites analyzed.

4. Biosignatures formed by the cycling of elements by biota, communities, and ecosystems

Fogel and colleagues from the Arctic Mars Analog Svalbard Expedition (AMASE) studied nitrogen cycling in an Arctic volcanic ecosystem. As part of a study on Mars analog environments, the biogeochemistry of Sverrefjellet Volcano, Bocfjorden, Svalbard, was documented and compared with those of surrounding glacial, thermal spring, and sedimentary environments (Figure 2). An understanding of how nitrogen might be distributed in a landscape that had extinct or very cold-adapted, slow-growing extant organisms should be useful for detecting unknown life forms. From high elevations (900 m) to the base of the volcano (sea level), soil and rock ammonium concentrations were uniformly low, typically less than 1-3 micrograms per gram of rock or soil. In weathered volcanic soils, reduced nitrogen concentrations were higher and oxidized nitrogen concentrations lower. The opposite was found in a weathered Devonian sedimentary soil. Plants and lichens growing on volcanic soils have an unusually wide range in N isotopic compositions, from —5 to +12 ‰, a range rarely measured in temperate ecosystems.

Nitrogen contents and isotopic compositions of volcanic soils and rocks were strongly influenced by the presence or absence of terrestrial herbivores or marine avifauna with higher concentrations of N and elevated N isotopic compositions occurring as patches in areas immediately influenced by reindeer, Arctic fox (Alopex lagopus), and marine birds. Because of the extreme conditions in this area, ephemeral deposition of herbivore feces results in direct and immediate N pulses into the ecosystem. The lateral extent and distribution of marine-derived nitrogen was measured on a landscape scale surrounding an active fox den. Nitrogen was tracked from the bones of marine birds to soil to vegetation. Because of extreme cold, slow biological rates, and nitrogen cycling, a mosaic of N patterns develops on the landscape scale.

5. Molecular and isotopic biosignatures in sediments from impact structures

Postdoctoral Fellow Mihaela Glamoclija and colleagues have combined microbial paleontology and molecular biology methods to study the Eyreville B drill core from the 35.3-million-year-old Chesapeake Bay impact structure. The investigated sample is a pyrite vein collected from the depth interval 1,353.81 to 1,353.89 m, located within the biotite-granite megablock sequence. The megablock belongs to pre-impact beds that were disrupted by the impact event. The search for inorganic (mineral) biosignatures revealed the presence of micron-size rod morphologies of anatase (TiO₂) embedded in chlorite coatings on pyrite grains.

Neither the Acridine Orange microbial probe nor DNA extractions followed by PCR amplifications showed the presence of DNA or RNA at the location of anatase rods, implying the absence of viable cells in the investigated area. A Nile Red microbial probe revealed the presence of lipids in the rods. Because most of the lipids are resistant over geologic time spans, they are good biomarkers and an indicator of biogenicity for these possibly 35-million-year-old microbial fossils. The mineral assemblage suggests that rod morphologies are associated with low-temperature (<100°C) hydrothermal alteration that involved aqueous fluids (Figure 3). The temporal constraints on the anatase fossils are still uncertain, because both pre-impact alteration of the granite and post-impact heating may have provided identical conditions for anatase precipitation and microbial preservation. Raman spectroscopy has proved to be a valuable tool for measuring mineral phases and organic phases in astrobiologically interesting rocks.

As part of this work Glamocjlia and Fogel have begun the analysis of total carbon in samples collected from the meteoritic impact that formed the Chesapeake Bay. This work is in its early stages and will continue in the coming year. Glamocjlia hopes to be able to distinguish carbon from recently living organisms from organic carbon that originated from sedimentary organic matter prior to impact.

6. Scanning transmission X-ray microscopy and in situ chemical analysis of organic fossils

Scanning transmission X-ray microscopy (STXM) employing X-ray absorption near-edge spectroscopy (XANES) on the carbon, nitrogen, and oxygen k edges as well micro-Raman spectroscopy were utilized for molecular characterization of diagenetically altered biological macromolecules that constitute type II-S kerogen from Neogene sediments of the Shinjo Basin, Japan. These sediments are known to be oil source rocks from 3 to 8 Ma ago, and the intense oil generation occurred in the lower part of the sequence. Fourteen kerogens extracted from sediment samples of the 1600-m-thick sequence were analyzed to reveal the relationship between molecular structural characteristics of the kerogen and sedimentary diagenesis. This study allowed Cody, Postdoctoral Fellow Hikaru Yabuta, and others to follow in detail the molecular structural changes that transform biological materials subjected to temperature, pressure, and time into so-called geo-polymeric matter.

7. Biosignatures in nearby planetary systems

Upcoming Mars missions (e.g., Mars Science Laboratory, ExoMars, Astrobiology Field Laboratory or equivalent, and Mars Sample Return) will search for evidence of extant and fossil microbial habitats and the potential for future habitation. Steele, Collaborator Jennifer Eigenbrode, and Fogel will be involved in several aspects of Mars Science Laboratory, in particular the Sample Analysis on Mars (SAM) instrument. Understanding the distribution and composition of reduced carbon (or organic carbon) is critical for unraveling the Martian carbon cycling, potential for life, and possible biosignature record. Reduced carbon may be produced from biological, geochemical, or interstellar processes; however, evidence for reduced carbon on Mars is lacking with the exception of atmospheric methane. In contrast, abundant atmospheric carbon dioxide may reflect surface oxidation of reduced carbon and accumulation over geological timescales. This suggests that there is an undetected or lost pool of reduced carbon — a pool that may host molecular biosignatures, a characteristic of extant or extinct habitability. Steele and his group will evaluate factors influencing the preservation potential for organic molecules in rocks on Earth and Mars and draw from examples from organic molecules in samples (sulfates, basalts, ancient shales) from Mars-analog settings to show how the distribution of organics and structural patterns will aid Martian habitability studies.